A Pathogenomic Approach towards Characterising the

South African Population of Puccinia striiformis f. sp. tritici,

the Causal Agent of Wheat Stripe Rust

Hester Josina van Schalkwyk

Thesis submitted in fulfilment of the requirements for the degree

Doctor of Philosophy

University of the Free State

Bloemfontein
South Africa

Department of Plant Sciences (Plant Pathology and Plant Breeding)

Faculty of Natural and Agricultural Sciences

January, 2018

Promoter:
Dr R Prins
Department of Plant Sciences, University of the Free State and CenGen (Pty) Ltd

Co-promoters:
Dr DGO Saunders
John Innes Centre, Norwich, United Kingdom
Dr LA Boyd
National Institute of Agricultural Botany, Cambridge, United Kingdom
Prof. ZA Pretorius
Department of Plant Sciences, University of the Free State
Declaration

I, Hester Josina van Schalkwyk, declare this thesis hereby submitted by me for the
degree Doctor of Philosophy at the University of the Free State is my own independent
work and has not previously been submitted by me to another university for any
degree.

I cede copyright of this thesis in favour of the University of the Free State.

Hester Josina van Schalkwyk Date

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Dedicated to Mrs Marlize Huisamen (née Vivier),

my high school biology teacher who first taught me about DNA
and nurtured my curiosity about living things.
Acknowledgements

I would like to express my sincere gratitude to my mentors and the funding

bodies that supported me during my PhD.

This work was funded by the Biotechnology and Biological Sciences Research

Council (BBSRC), the Department for International Development and (through

a grant to BBSRC) the Bill & Melinda Gates Foundation, under the Sustainable

Crop Production Research for International Development (SCPRID) programme,

a joint initiative with the Department of Biotechnology of the Government of

India’s Ministry of Science and Technology. Two SCPRID grants supported this

study: (BB/J011525/1) to Dr L Boyd, Dr R Prins and Prof. ZA Pretorius, and

(BB/J012017/1) to Dr Cristobal Uauy. Additional support were received from

The Monsanto Beachell-Borlaug International Scholars Program (MBBISP) and

the Winter Cereal Trust (WCT), South Africa, through PhD scholarships.

The contributions of my supervisors go far beyond what I can summarise in a

paragraph, nonetheless, a special thank you for the unique role they each played

during my PhD. I thank Dr Diane Saunders and Dr Renée Prins for creating

environments with nearly unlimited resources where I could work. I thank Dr

Lesley Boyd for intense supervision while I was preparing my thesis, and Prof.

Zakkie Pretorius for mentoring me in the art and science of rust pathology. I

thank Dr Prins for her vision for the project and allowing me to change to this

project that I so enjoyed working on. I would also like to thank Dr Cristobal Uauy

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for being instrumental in arranging my placement in the Saunders lab.

I thank the following people for their involvement in obtaining the sequencing

datasets: Historical South African isolates were obtained from Zakkie Pretorius.

Historical East African isolates were obtained from Mogens Hovmøller. The Pak-

istan isolates were obtained from Sajid Ali. Samples of the recent South African

Pst population were obtained from Driecus Lesch, Tarekegn Terefe, Zakkie Pre-

torius and Willem Boshoff (lost in transit). Renée Prins was instrumental in the

preparatory work and shipment of the South African isolates for sequencing.

Recent East African isolates were obtained from David Hodson (Ethiopia, 2014)

and Ruth Wanyera (Kenya, 2014). Existing datasets of Pst isolates were obtained

from Diane Saunders.

What fantastic opportunities to work at CenGen (Pty) Ltd, Earlham Institute,

John Innes Centre, and the University of the Free State, during my PhD! A

special mention to the following people for support in and out of the lab. Debbie

Snyman performed qPCR assays and gel electrophoresis towards this project.

Zakkie Pretorius multiplied the historical South African Pst urediniospores for

sequencing and mentored me in inoculation and scoring of the infection assays

on the differential wheat set seedlings. Sarah Holdgate for providing the United

Kingdom (UK) differential wheat lines and informative discussions regarding Pst

in the UK and UK wheat cultivars. Elsabet Wessels, Debbie Snyman, Jens Mains

and Clare Lewis mentored me in specific molecular genetic procedures. I thank

Philippa Borril and Oluwaseyi Shorinola for advice on RT-qPCR data analysis,

and Albor Dobón for help with the planning of the time course experiment. I

also thank Antoine Persoons for valuable discussions in population genetics and

advice on sections of this thesis and my fellow PhD students in the Saunders lab,

especially Pilar Corredor-Moreno and Vanessa Bueno-Sancho, for always being

ready to advise me on the newest updates in data handling or Norwich BioScience

Institutes (NBI) cluster computing. Also, thank you to the Computing NBI
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Helpdesk staff, especially Tom Betteridge and Mohamed Imram, for computer

support. I thank Sadie Geldenhuys for administrative support at UFS, Lizaan

Rademeyr for great practical advice on best practices in laboratory record keeping,

Carel van Heerden for input in the early days of the project and Anelda van der

Walt for initial bioinformatics training. I thank Cari van Schalkwyk for advice on

statistical analysis. I thank Prof. Ed Runge and the MBBISP panel for the very

special ongoing experience of being an MBBISP scholar.

Thank you to every friend that ran, walked, climbed mountains, or performed

some strange hobby with me. That helped to keep me going through the hard

times. George, for your rock-solid support and your immense contribution to

tailoring my skill set, thank you. I thank my family for all their love and support

along the way. Thank you, dad, for reminding me that I am a finisher, and mum,

for your consistent positivity, enthusiasm, and encouragement that runs through

my life like a golden thread.

Abstract

Stripe (yellow) rust caused by the fungus Puccinia striiformis Westend. f. sp. tritici

(Pst) is a major disease of wheat prevalent in most areas where wheat is culti-

vated across the globe. It can completely destroy a crop if left untreated. The

Pst fungus develops feeding structures that form a close relationship with the

host tissue where it facilitates extraction of water and nutrients from the plant,

while manipulating the host for its own benefit using effector proteins. This

parasitic behaviour reduces yield and grain quality, leading to the propagation of

numerous Pst spores, spreading infection. In South Africa stripe rust was first

detected in 1996 with the initial pathotype being designated 6E16A-. Thereafter,

three more Pst pathotypes were detected in subsequent years (6E22A- in 1998,

7E22A- in 2001 and 6E22A+ in 2005), gaining virulence in a stepwise manner by

overcoming additional resistance genes one by one. However, the source of the

original pathotype and the current genetic diversity of the Pst population within

South Africa remain open questions.

To get a better understanding of the South African Pst pathotypes and how

they relate to Pst pathotypes globally, the historical population was described

using a recently developed “field pathogenomics” approach. High-resolution,

next-generation sequencing data utilised in this method aided in determining the

genomic relationships between the four historical pathotypes and investigating

their potential origin. Historic South African isolates representing the four identi-

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fied pathotypes were re-sequenced, and their comparison with isolates from the

United Kingdom, France, Pakistan, Ethiopia, Eritrea and Kenya revealed that the

closest relatives of the historical South African isolates were a group of isolates

from East Africa.

We further described polymorphisms in the South African Pst population

that supported the existing hypothesis of stepwise evolution. Through applying

pairwise comparisons between polymorphic sites across isolates, 27 potential

effector proteins that could be instrumental in the stepwise virulence gain, were

identified. To study the role these candidates may play during the infection

processes in different pathotypes, gene expression profiling was conducted using

RT-qPCR. Preliminary patterns of up- or down-regulation of these effectors be-

tween time points, over a time course of compatible interactions, were described.

Furthermore, infected wheat tissues collected from locations across South Africa

during the 2013, 2014 and 2015 cropping seasons, were sequenced. The “field

pathogenomics” method, using RNA-Seq, was applied to compare the historic

Pst isolates with the recent population. This analysis indicated the possibility of a

novel introduction of Pst into South Africa in recent years, possibly between 2011

and 2013. Pathotyping of selected Pst isolates on supplementary wheat tester

genotypes revealed novel variation in infection types that has not been described

previously.

This study provides a high resolution, genomic view of the historical and

prevailing Pst populations and adds valuable information to the potential origin

and adaptation of stripe rust in South Africa. The research outcomes provide

a genomic base for further investigation of candidate effector genes and the

possible recent novel incursion of a pathotype group also seen in Europe, East

Africa and New Zealand into South Africa.

Keywords: effector, origin, plant pathology, population genomics, virulence

Contents

Declaration ii

Acknowledgements iv

Abstract vii

List of Figures xv

List of Tables xix

List of Abbreviations xxi

1 General Introduction 1
1.1 Socio-economic importance of wheat . . . . . . . . . . . . . . . . . 2
1.2 Wheat cultivation in South Africa . . . . . . . . . . . . . . . . . . . 2
1.3 Wheat rusts reduce yields . . . . . . . . . . . . . . . . . . . . . . . 4
1.4 Motivation for this study . . . . . . . . . . . . . . . . . . . . . . . . 6
1.5 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.6 Thesis outline and approaches . . . . . . . . . . . . . . . . . . . . . 7

2 The Wheat Rusts: Life Histories, Host Response Mechanisms and Ge-
nomic Resources 9
2.1 The rusts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.1.1 Filamentous plant pathogens . . . . . . . . . . . . . . . . . 9
2.1.2 Rusts and their primary host . . . . . . . . . . . . . . . . . 11
2.1.3 The alternative host . . . . . . . . . . . . . . . . . . . . . . . 12
2.1.4 Global distribution of stripe rust . . . . . . . . . . . . . . . 13
2.1.5 Favourable conditions for wheat rusts . . . . . . . . . . . . 13
2.1.6 Infection cycle of Puccinia rusts . . . . . . . . . . . . . . . . 15
2.1.7 The stripe rust infection process on wheat . . . . . . . . . . 19
2.2 Combating wheat stripe rust . . . . . . . . . . . . . . . . . . . . . . 21

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CONTENTS x

2.3 Plant defence mechanisms . . . . . . . . . . . . . . . . . . . . . . . 22

2.3.1 Host-pathogen interaction . . . . . . . . . . . . . . . . . . . 23
2.3.2 Other sources of resistance . . . . . . . . . . . . . . . . . . 26
2.4 The Pst genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.4.1 Genomic variation . . . . . . . . . . . . . . . . . . . . . . . 26
2.4.2 Rust genomics . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.4.3 Challenges in bioinformatics . . . . . . . . . . . . . . . . . 31
2.4.4 Effector identification . . . . . . . . . . . . . . . . . . . . . 32

3 General Materials and Methods 35

3.1 Preparation and collection of materials . . . . . . . . . . . . . . . . 35
3.1.1 Inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.1.2 Protocol for sampling infected wheat tissue . . . . . . . . . 36
3.2 Nucleic acid extraction and quantification . . . . . . . . . . . . . . 37
3.2.1 Genomic DNA extraction . . . . . . . . . . . . . . . . . . . 37
3.2.2 RNA extraction . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.2.3 DNA and RNA quantification . . . . . . . . . . . . . . . . . 38
3.3 Next-generation sequencing and data analysis . . . . . . . . . . . 39
3.3.1 Library preparation . . . . . . . . . . . . . . . . . . . . . . . 39
3.3.2 Genomic DNA sequencing . . . . . . . . . . . . . . . . . . 39
3.3.3 RNA sequencing . . . . . . . . . . . . . . . . . . . . . . . . 40
3.3.4 Bioinformatics pipeline . . . . . . . . . . . . . . . . . . . . 40
3.3.5 Clustering analysis . . . . . . . . . . . . . . . . . . . . . . . 42

4 Origin of the South African Pst Pathotypes 48

4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.1.1 Wheat stripe rust in South Africa . . . . . . . . . . . . . . . 48
4.1.2 Pst population diversity . . . . . . . . . . . . . . . . . . . . 52
4.1.3 Molecular markers and Pst . . . . . . . . . . . . . . . . . . 53
4.1.4 Next-generation sequence analyses of South African Pst . 55
4.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.2.1 Data description . . . . . . . . . . . . . . . . . . . . . . . . 56
4.2.2 Sample preparation for DNA extraction . . . . . . . . . . . 57
4.2.3 Genomic DNA extraction and quantification . . . . . . . . 59
4.2.4 Sequencing and mapping . . . . . . . . . . . . . . . . . . . 59
4.2.5 Phylogenetic analysis . . . . . . . . . . . . . . . . . . . . . 60
4.2.6 Population structure analysis . . . . . . . . . . . . . . . . . 60
CONTENTS xi

4.2.7 Genetic diversity assessment . . . . . . . . . . . . . . . . . 60

4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
4.3.1 Re-sequencing of South African Pst pathotypes . . . . . . . 61
4.3.2 Purity assessment of samples . . . . . . . . . . . . . . . . . 62
4.3.3 Clustering analyses . . . . . . . . . . . . . . . . . . . . . . . 62
4.3.4 Phylogenetic analysis . . . . . . . . . . . . . . . . . . . . . 62
4.3.5 Population structure analysis . . . . . . . . . . . . . . . . . 64
4.3.6 Population differentiation . . . . . . . . . . . . . . . . . . . 71
4.3.7 Genetic diversity within and between population clusters 71
4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

5 Analyses of Polymorphisms in Historical South African Pst Isolates in

Search of Candidate Effector Genes 79
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5.1.1 The importance of Pst variability . . . . . . . . . . . . . . . 81
5.1.2 Mutations—causes, types and effects . . . . . . . . . . . . 82
5.1.3 Genomic approaches used to identify effectors . . . . . . . 85
5.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . 86
5.2.1 SNP analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
5.2.2 Positive selection . . . . . . . . . . . . . . . . . . . . . . . . 87
5.2.3 Presence-absence analysis . . . . . . . . . . . . . . . . . . . 87
5.2.4 Comparisons of nonsynonymous SNP sites between isolates 88
5.2.5 Multiple sequence alignments to visualise biallelic SNPs . 88
5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
5.3.1 SNP identification in the genomes of the historical South
African isolates . . . . . . . . . . . . . . . . . . . . . . . . . 89
5.3.2 Assessment of polymorphisms to detect positive selection 93
5.3.3 Presence or absence of genes . . . . . . . . . . . . . . . . . 98
5.3.4 Investigation of candidate genes that are likely to experi-
ence evolutionary changes . . . . . . . . . . . . . . . . . . . 105
5.3.5 Candidate effectors with sequence polymorphisms between
the South African isolates . . . . . . . . . . . . . . . . . . . 106
5.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.4.1 Polymorphic sites . . . . . . . . . . . . . . . . . . . . . . . . 108
5.4.2 STOP codons . . . . . . . . . . . . . . . . . . . . . . . . . . 110
5.4.3 Transitions and transversions at specific codon positions . 111
CONTENTS xii

5.4.4 Stepwise mutations . . . . . . . . . . . . . . . . . . . . . . . 112

5.4.5 Positive selection . . . . . . . . . . . . . . . . . . . . . . . . 112
5.4.6 Presence-absence analysis . . . . . . . . . . . . . . . . . . . 113
5.4.7 Nonsynonymous polymorphisms . . . . . . . . . . . . . . 114
5.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

6 Gene Expression Analysis of Candidate Effectors Identified in South African

Pst Isolates 115
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
6.1.1 Regulation of gene expression in eukaryotes . . . . . . . . 116
6.1.2 Quantification of gene expression . . . . . . . . . . . . . . 117
6.1.3 Candidate effector features . . . . . . . . . . . . . . . . . . 118
6.1.4 Gene transcription analysis . . . . . . . . . . . . . . . . . . 118
6.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
6.2.1 Inoculation and sampling . . . . . . . . . . . . . . . . . . . 120
6.2.2 Tissue disruption and RNA extraction . . . . . . . . . . . . 122
6.2.3 RNA quality control and quantification . . . . . . . . . . . 123
6.2.4 Complementary DNA synthesis . . . . . . . . . . . . . . . 123
6.2.5 Primer design . . . . . . . . . . . . . . . . . . . . . . . . . . 123
6.2.6 PCR plate setup . . . . . . . . . . . . . . . . . . . . . . . . . 124
6.2.7 Quantitative real-time polymerase chain reaction . . . . . 126
6.2.8 Reference gene selection . . . . . . . . . . . . . . . . . . . . 127
6.2.9 Efficiency determination of primers . . . . . . . . . . . . . 127
6.2.10 Statistical evaluation of the data . . . . . . . . . . . . . . . 129
6.2.11 Linear mixed effect analysis . . . . . . . . . . . . . . . . . . 129
6.2.12 Relative expression of Pst candidate effector genes . . . . . 130
6.2.13 Assessment of genes . . . . . . . . . . . . . . . . . . . . . . 131
6.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
6.3.1 RNA yield, RNA quality scores and cDNA yield . . . . . . 131
6.3.2 Primer design . . . . . . . . . . . . . . . . . . . . . . . . . . 132
6.3.3 Efficiency determination of primers . . . . . . . . . . . . . 134
6.3.4 Statistical analysis of the relative expression of nine Pst
candidate effector genes . . . . . . . . . . . . . . . . . . . . 134
6.3.5 Expression profiles of candidate genes . . . . . . . . . . . . 139
6.3.6 Gene validation using revised gene models and transcript
data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
6.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
CONTENTS xiii

6.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

7 Analysis of the Current Stripe Rust Threat in South Africa 145

7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
7.1.1 Pst virulence since 2005 . . . . . . . . . . . . . . . . . . . . 145
7.1.2 Global reports on Pst population shifts . . . . . . . . . . . 146
7.1.3 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
7.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . 149
7.2.1 Stripe rust samples used in RNA sequencing analyses . . . 149
7.2.2 Transcriptome sequencing of stripe rust infected wheat leaves151
7.2.3 Pst pathotype determination . . . . . . . . . . . . . . . . . 152
7.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
7.3.1 Clustering analysis using RNA-Seq and whole genome
sequencing data . . . . . . . . . . . . . . . . . . . . . . . . . 153
7.3.2 Seedling Pst pathotype testing . . . . . . . . . . . . . . . . 162
7.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
7.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

8 General Discussion 173

8.1 The historical South African Pst population . . . . . . . . . . . . . 173
8.2 Candidate effector identification and evaluation . . . . . . . . . . 175
8.3 The recent South African Pst population . . . . . . . . . . . . . . . 177
8.4 Future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
8.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

Appendices 181

A The Origin of the South African Pst Pathotypes 181

B Analyses of Polymorphisms in Historical South African Pst Isolates in

Search of Candidate Effector Genes 183
B.1 Genes present in the PST130 reference genome but absent in the
four historical South African Pst isolates . . . . . . . . . . . . . . . 183
B.2 Annotations of genes homologous to identified PST130 genes . . 185
B.3 Nonsynonymous polymorphisms in candidate genes . . . . . . . 193

C Gene Expression Analysis of Candidate Effectors Identified in South African

Pst Isolates 222
C.1 Candidate gene inspection . . . . . . . . . . . . . . . . . . . . . . . 223
CONTENTS xiv

C.2 Additional figures of statistical analyses . . . . . . . . . . . . . . . 232

C.3 Variability in RT-qPCR . . . . . . . . . . . . . . . . . . . . . . . . . 239
C.3.1 Variation in the application of treatments to biological repli-
cates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
C.3.2 Variation introduced by the RNA extraction process . . . . 240
C.3.3 Variation introduced by the reverse transcription process 241
C.3.4 Variation introduced by RT-qPCR . . . . . . . . . . . . . . 242
C.3.5 Variation introduced by primers . . . . . . . . . . . . . . . 242
C.3.6 Choice of reference genes . . . . . . . . . . . . . . . . . . . 244
C.3.7 Results of efficiency corrected relative gene expression . . 245

D Analysis of the Current Stripe Rust Threat in South Africa 248

Bibliography 254
List of Figures

1.1 Area harvested, production and yield statistics for South African
wheat cultivation between 1990 and 2017. . . . . . . . . . . . . . . 4

2.1 The phylogenetic relationship of plant pathogenic ascomycetes,

basidiomycetes and oomycetes following Neighbour Joining anal-
ysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2 Taxonomic classification of the wheat rusts. . . . . . . . . . . . . . 11
2.3 Global distribution of Puccinia striiformis f. sp. tritici, before and
after 2000. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.4 Spore stages and the infection cycle of Pst. . . . . . . . . . . . . . . 16
2.5 A stripe rust uredinium pustule. . . . . . . . . . . . . . . . . . . . 16
2.6 Illustration of the infection process of Pst. . . . . . . . . . . . . . . 19
2.7 Illustration of a filamentous plant pathogen haustorium. . . . . . 20
2.8 The five main classes of plant disease resistant proteins. . . . . . . 25

4.1 Locations of the original detections of South African Pst pathotypes. 49

4.2 Temperature and rainfall measured in 1996 during the wheat-
growing season in the Western Cape, compared to the 11 year
mean. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.3 Schematic illustration of the increase of Pst virulence in South Africa. 52
4.4 Pathotype identification tests of South African Pst pathotypes. . . 52
4.5 Read frequency graphs from heterokaryotic SNP sites for SA1–SA4. 63
4.6 The phylogenetic relationship between the South African Pst iso-
lates and European, Asian and East African isolates. . . . . . . . . 65
4.7 Evaluation of the number of population clusters following STRUC-
TURE analyses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4.8 Bar charts representing STRUCTURE population clusters. . . . . . 67
4.9 Discriminant analysis of principal components analysis of 48 Pst
isolates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.10 Bar charts representing DAPC population structure analysis. . . . 70
xv
LIST OF FIGURES xvi

4.11 Genetic diversity assessed between 10 population clusters. . . . . 72

5.1 Nucleotide changes that introduced stop codons. . . . . . . . . . . 92

5.2 Distribution of stop codons accross all genes per isolate. . . . . . . 92
5.3 Percentage frequency matrices of transitions and transversions at
monoallelic SNP sites. . . . . . . . . . . . . . . . . . . . . . . . . . 94
5.4 Percentage occurrence matrices of transitions and transversions at
biallelic SNP sites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
5.5 Codon positions of nucleotide changes at homokaryotic SNP sites. 96
5.6 Codon positions of nucleotide changes at heterokaryotic SNP sites. 97
5.7 Presence-absence analysis. . . . . . . . . . . . . . . . . . . . . . . . 103
5.8 Nonsynonymous SNPs in the gene space of the four South African
isolates increase over time and with increasing virulence. . . . . . 106
5.9 Translated sequence alignment of gene PST130_00285. . . . . . . . 107
5.10 Over- and underestimates of SNP sites. . . . . . . . . . . . . . . . 109

6.1 Experimental setup for the infection time course experiment. . . . 121
6.2 Plate layouts for RT-qPCR assays. . . . . . . . . . . . . . . . . . . . 125
6.3 Linear regression showing estimated efficiency of primers. . . . . 135
6.4 Relative gene expression of nine candidate effector genes. . . . . . 138

7.1 Prevalence of Pst in South Africa between 2008 and 2016. . . . . . 147
7.2 Locations of Pst collections between 2013 and 2015. . . . . . . . . 151
7.3 Phylogenetic tree displaying the relationship between Pst isolates. 155
7.4 Relative distance maximum likelihood phylogenetic tree. . . . . . 156
7.5 Evaluation of number of population clusters following STRUC-
TURE analyses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
7.6 STRUCTURE histogram plots of population clusters. . . . . . . . 159
7.7 Discriminant analysis of principal components analysis of Pst iso-
lates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
7.8 Histogram plots indicating population structure as inferred by
DAPC analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
7.9 Measurements of genetic diversity by FST calculation of pairs of
population groups. . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
7.10 Infection type comparisons between one historical and one recent
Pst isolate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
7.11 Number of international tourist arrivals in South Africa between
1995 and 2014. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
LIST OF FIGURES xvii

A.1 Read frequency graphs for East African isolates analysed in Chapter 4182

B.1 Translated sequence alignment of gene PST130_02001. . . . . . . . 193

B.2 Translated sequence alignment of gene PST130_02118. . . . . . . . 194
B.3 Translated sequence alignment of gene PST130_02403. . . . . . . . 195
B.4 Translated sequence alignment of gene PST130_05023. . . . . . . . 196
B.5 Translated sequence alignment of gene PST130_05454. . . . . . . . 197
B.6 Translated sequence alignment of gene PST130_05944. . . . . . . . 198
B.7 Translated sequence alignment of gene PST130_06503. . . . . . . . 199
B.8 Translated sequence alignment of gene PST130_06558. . . . . . . . 200
B.9 Translated sequence alignment of gene PST130_07448. . . . . . . . 201
B.10 Translated sequence alignment of gene PST130_07513. . . . . . . . 202
B.11 Translated sequence alignment of gene PST130_07564. . . . . . . . 203
B.12 Translated sequence alignment of gene PST130_08031. . . . . . . . 204
B.13 Translated sequence alignment of gene PST130_08984. . . . . . . . 205
B.14 Translated sequence alignment of gene PST130_09018. . . . . . . . 206
B.15 Translated sequence alignment of gene PST130_09275. . . . . . . . 207
B.16 Translated sequence alignment of gene PST130_10286. . . . . . . . 208
B.17 Translated sequence alignment of gene PST130_12487. . . . . . . . 209
B.18 Translated sequence alignment of gene PST130_12491. . . . . . . . 210
B.19 Translated sequence alignment of gene PST130_12956. . . . . . . . 211
B.20 Translated sequence alignment of gene PST130_13969. . . . . . . . 212
B.21 Translated sequence alignment of gene PST130_14091. . . . . . . . 213
B.22 Translated sequence alignment of gene PST130_14831. . . . . . . . 214
B.23 Translated sequence alignment of gene PST130_16778. . . . . . . . 215
B.24 Translated sequence alignment of gene PST130_17605. . . . . . . . 216
B.25 Translated sequence alignment of gene PST130_17605. . . . . . . . 217
B.26 Translated sequence alignment of gene PST130_07579. . . . . . . . 218
B.27 PST130_07579 continued from previous page. . . . . . . . . . . . . 219
B.28 Translated sequence alignment of gene PST130_15131. . . . . . . . 220
B.29 PST130_15131 continued from previous page. . . . . . . . . . . . . 221

C.1 Nonsynonymous polymorphisms and primer design of the candi-

date effector gene PST130_02001 in SA1 and SA4. . . . . . . . . . 223
C.2 Nonsynonymous polymorphisms and primer design of the candi-
date effector gene PST130_02403 in SA1 and SA4. . . . . . . . . . 224
LIST OF FIGURES xviii

C.3 Nonsynonymous polymorphisms and primer design of the candi-

date effector gene PST130_05023 in SA1 and SA4. . . . . . . . . . 225
C.4 Nonsynonymous polymorphisms and primer design of the candi-
date effector gene PST130_06503 in SA1 and SA4. . . . . . . . . . 226
C.5 Nonsynonymous polymorphisms and primer design of the candi-
date effector gene PST130_07513 in SA1 and SA4. . . . . . . . . . 227
C.6 Nonsynonymous polymorphisms and primer design of the candi-
date effector gene PST130_09725 in SA1 and SA4. . . . . . . . . . 228
C.7 Nonsynonymous polymorphisms and primer design of the candi-
date effector gene PST130_12487 in SA1 and SA4. . . . . . . . . . 229
C.8 Nonsynonymous polymorphisms and primer design of the candi-
date effector gene PST130_12491 in SA1 and SA4. . . . . . . . . . 230
C.9 Nonsynonymous polymorphisms and primer design of the candi-
date effector gene PST130_12956 in SA1 and SA4. . . . . . . . . . 231
C.10 Graphical tests for normality and equal variances of the residuals
and random intercepts. . . . . . . . . . . . . . . . . . . . . . . . . . 233
C.11 Gene and isolate specific tests for equal variances after the model
was fitted to the relative gene expression values. . . . . . . . . . . 234
C.12 Gene and isolate specific tests for equal variances after the model
was fitted to the relative gene expression values. . . . . . . . . . . 235
C.13 Graphical tests for normality and equal variances of the residuals
and random intercepts following a log10 transformation. . . . . . 236
C.14 Gene and isolate specific normal probability plots of the residuals
after the model was fitted to the log10 transformed relative gene
expression values. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
C.15 Gene and isolate specific tests for equal variances after the model
was fitted to the log10 transformed relative gene expression values. 238
C.16 High inter-run variability in relative expression patterns. . . . . . 246
C.17 The Pfaffl method of relative gene expression shows the relative
gene expression of SA1 to SA4. . . . . . . . . . . . . . . . . . . . . 247

D.1 Read frequency graphs from heterokaryotic SNP sites for the recent
South African field isolates. . . . . . . . . . . . . . . . . . . . . . . 249
D.2 Read frequency graphs from heterokaryotic SNP sites for the recent
East African field isolates. . . . . . . . . . . . . . . . . . . . . . . . 250
D.3 Circular relative distance maximum likelihood phylogenetic tree. 251
List of Tables

1.1 Domestic grain consumption of the three highest consumed grains

worldwide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1 Whole genome sequencing projects using next- and third-generation

sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

4.1 Global isolates included in the clustering and genetic diversity

analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.2 Historical isolates used in re-sequencing and an infection time
course experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.3 Statistics of read alignment of the historical South African isolates
to the PST130 reference genome . . . . . . . . . . . . . . . . . . . . 63

5.1 Homokaryotic and heterokaryotic SNPs in the South African isolates 90

5.2 The number of SNPs identified in coding regions of the four South
African Pst isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
5.3 Polymorphic genes with positive dN values indicating nonsynony-
mous changes in isolate pairwise comparisons . . . . . . . . . . . 99
5.4 Polymorphic genes with positive dS values indicating synony-
mous changes in isolate pairwise comparisons . . . . . . . . . . . 99
5.5 Number of absent genes in the four South African Pst pathotypes 100
5.6 Potential orthologs of genes absent in all four of the South African
isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
5.7 The number of potential paralogs identified in genes absent in all
four South African isolates . . . . . . . . . . . . . . . . . . . . . . . 102
5.8 Potential paralogs of genes absent in the four South African isolates102
5.9 Potential orthologs of genes absent in three or less of the South
African isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
5.10 Number of potential paralogs in PST130 . . . . . . . . . . . . . . . 104

xix
LIST OF TABLES xx

5.11 Paralogs of genes that only occurred in one of the South African
isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

6.1 Effector features of the identified candidate effectors . . . . . . . . 119

6.2 Summary statistics describing RNA yield, integrity and cDNA
yield as required in the MIQE guidelines . . . . . . . . . . . . . . . 132
6.3 Primer and amplicon specifications for Pst candidate effector gene
identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
6.4 Significance of the factor “Time Point” in the linear mixed model
for those genes where it was significant . . . . . . . . . . . . . . . 139
6.5 Multiple comparisons between time points for each gene that
showed significant difference in expression over the time series . 140

7.1 Wheat differential lines used at Agricultural Research Council,

Small Grain, South Africa . . . . . . . . . . . . . . . . . . . . . . . 146
7.2 African isolates collected between 2013 and 2015 . . . . . . . . . . 150
7.3 Infection type scores used to assess Pst infection on wheat seedlings153

B.1 PST130 genes (211) that were absent in all four historical South
African isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

D.1 Differential testing of South African Pst isolates previously defined

as pathotype 6E16A- on an extended set of wheat seedling testers 252
D.2 Differential testing of South African Pst isolates previously defined
as pathotype 6E22A+ on an extended set of wheat seedling testers 253
List of Abbreviations

30 three prime
50 five prime
A adenine
ABI Applied Biosystems Integrated
ADP Adenosine diphosphate
ACTB β-Actin
AFLP Amplified Fragment Length Polymorphism
ANOVA analysis of variance
ARC-SG Agricultural Research Council, Small Grain
ARF ADP ribosylation factors
ATP Adenosine triphosphate
Avr avirulence
BAC bacterial artificial chromosome
BAM binary alignment map
BBSRC Biotechnology and Biological Sciences Research Council
BGRI Borlaug Global Rust Initiative
BIC bayesian information criterion
bp base pairs
C cytosine
CAF Central Analytical Facilities
cDNA complementary DNA
CEC Crop Estimate Committee
CIMMYT International Maize and Wheat Improvement Center
CTAB cetyltrimethylammonium bromide
CVEGE clonal variation in effector gene expression
DA discriminant analysis

xxi
LIST OF ABBREVIATIONS xxii

DAPC discriminant analysis of principal components

DNA deoxyribonucleic acid
dpi days post inoculation
ds double stranded
dsDNA double stranded DNA
EMS ethyl methanesulfonate
EST expressed sequence tag
ETI effector-triggered immunity
FIR flanking intergenic regions
G guanine
GAPs GTPase activating proteins
GAPDH glyceraldehyde 3-phosphate dehydrogenase
gDNA genomic DNA
gene virus induced
GTR general time reversible
HCD hypersensitive cell death
HIGS host-induced gene silencing
HMC haustorial mother cell
IH infection hyphae
IP infection peg
kbp kilo base pairs
Lr wheat leaf rust resistance gene designation
MAMPs microbe-associated molecular patterns
MAS marker assisted selection
MBBISP Monsanto Beachell-Borlaug International Scholars Program
Mbp mega base pairs
MCMC Markov Chain Monte Carlo
miRNAs microRNAs
mRNA messenger RNA
MSL Molecular marker Service Laboratory
NB-LRR nucleotide-binding site (NBS)-leucine-rich repeat (LRR) proteins
NBI Norwich BioScience Institutes
NGS next-generation sequencing
NLS nuclear-localisation signal
LIST OF ABBREVIATIONS xxiii

NMD nonsense-mediated mRNA decay

NTC non template control
oligo-dTs thymine oligonucleotides
PAMPs pathogen-associated molecular patterns
PCA principal component analysis
Pgt Puccinia graminis f. sp. tritici
PI phosphoinositide
PR pathogen-related
PRRs pathogen receptor proteins
Pst Puccinia striiformis f. sp. tritici
Pt Puccinia triticina
PTI PAMP triggered immunity
qPCR quantitative or real time PCR
R resistance or resistant
RAxML randomized axelerated maximum likelihood
RIN RNA integrity number
RNA ribonucleic acid
RNA-Seq RNA sequencing
ROS reactive oxygen species
RT reverse transcriptase
S susceptible (as in Avocet S)
SAGL South African Grain Laboratory
SAM sequence alignment map
SCAR sequence-characterised amplified region
SCPRID Sustainable Crop Production Research for International Development
SCR small and cysteine rich
siRNA small interfering RNA
SNP single nucleotide polymorphism
SNPs polymorphisms
Sr wheat stem rust resistance gene designation
ss single strand
SSV substomatal vesicle
T thymine
tRNA transfer RNA
LIST OF ABBREVIATIONS xxiv

TUBB β-Tubulin
UK United Kingdom
UKCPVS UK Cereal Pathogen Virulence Survey
USA United States of America
UTRs untranscribed regions
UV ultraviolet
VIGS virus induced gene silencing
WC wheat control
WCT Winter Cereal Trust
Yr wheat stripe rust resistance gene designation
Z12 Zadoks growth stage 12

Mathematical notation
CT threshold cycle
FT fluorescence threshold
R2 Pearson correlation coefficient
Chapter 1

General Introduction

W HEAT IS A STAPLE CROP in many countries around the globe, including South

Africa. In most areas of wheat cultivation, one or more of the three rust diseases

have the potential to severely compromise yields (Kolmer, 2005; Huerta-Espino

et al., 2011; Shaw and Osborne, 2011; Dean et al., 2012; Beddow et al., 2015).

Rusts are specialised in infecting wheat and maintain an obligatory parasitic

symbiosis with susceptible hosts throughout their life cycles, using resources

predestined for plant growth, maintenance, and grain development to ultimately

produce multitudes of spores (Chen, 2005). The continuously growing demand

for wheat requires careful consideration of mechanisms to address host resistance

to manage these crippling diseases. Management strategies aim to increase crop

yields and reduce quantities of inoculum. Smaller rust population sizes reduce

the potential of the fungus to gain new pathogenicity through evolutionary

machineries such as mutation and somatic and sexual recombination (Hovmøller

and Justesen, 2007a; Jin et al., 2010; Zhao et al., 2013; Jiao et al., 2017).

1
CHAPTER 1: GENERAL INTRODUCTION 2

1.1 Socio-economic importance of wheat

Bread wheat, Triticum aestivum L., is an important food source making up 20 % of

global calories and protein intake (Shiferaw et al., 2014). Recent estimates placed

domestic consumption at 736.86 million tons for the 2016/2017 market year (FAS

USDA, 2017). The three most prominent staple grains—wheat, maize and rice—

are under heavy pressure for increased yields to secure food for the growing

world population (Table 1.1; FAS USDA, 2017). By mid-2017, the estimated global

population size was 7.6 billion people and predictions estimate increases of up to

9.8 billion by 2050, with a further increase to 11.2 billion by 2100 (United Nations,

2017). The growing population places increased pressure on crop production

as a primary source of human nutrition, animal feed and bio-fuel (Edgerton,

2009). Yield improvements of roughly 2.4 % per year are needed to be able to

meet the target of doubling global crop production, but currently global average

rates are failing to reach this target (Ray et al., 2013). Other sectors also rapidly

out-compete the agricultural sector for land, adding to the pressure to produce

enough food for the growing population, while acreage continues to diminish.

1.2 Wheat cultivation in South Africa

Wheat was brought to South Africa by the Dutch settlers in 1652 and drought,

wind, and disease challenged early wheat production (Du Plessis, 1933), as is still

the case today (FAS USDA, 2016). Currently, wheat is the most planted winter

cereal crop in South Africa, ranking second to maize for overall crop size (SAGL,

2012) and consumption (FAS USDA, 2017) in the country.

South Africa is the largest consumer of wheat in Sub-Saharan Africa, and

population growth and urbanisation will likely continue to increase the demand

(ITA USDC, 2017). Most of the crop is cultivated on dry land and grown in
CHAPTER 1: GENERAL INTRODUCTION 3

Table 1.1: Domestic grain consumption of the three highest consumed grains worldwide,
in million tons, as recorded for 2016/17 (FAS USDA, 2017)

Grain World South Africa

Rice 478.46 0.83
Wheat 736.86 3.40
Maize 1 053.85 11.70

the winter-rainfall areas of the Western Cape where there is a Mediterranean

climate. Here wheat is planted from mid-April until mid-June, and harvested

from October to December. In the Eastern Free State, a summer-rainfall area,

wheat is sown from June until August and harvested between November and

January. Irrigated wheat cultivation is practised in the Northern Cape, using

water from the Orange River (Van Niekerk, 2001; SAGL, 2012).

The trend over the last 20 years indicates a reduction in wheat cultivation

(Figure 1.1). This is driven by considerable annual production fluctuations,

caused by unpredictable weather patterns and declining profit margins for wheat

(AgriOrbit, 2017). The decrease in wheat cultivation—in favour of other, more

climate tolerant and often higher value crops such as maize, canola and soybeans—

increases the dependence on imports to meet the growing wheat demand in South

Africa (ITA USDC, 2017).

The prominent grain industry in South Africa contributes more than 30 % of

the total gross value of agricultural production in the country (DAFF, 2015). On

average, 63 % of the total demand over the past 10 years was produced domesti-

cally, while the remainder was imported (DAFF, 2016). In 2017, 1.8 million tons of

wheat were imported (IndexMundi, 2017). South Africa exported 0.2 million tons

in 2017 (IndexMundi, 2017). Most exports are destined for neighbouring coun-

tries, Zambia, and Mauritius (FAS USDA, 2016).

The increase in yield, despite the reduction in planted area (Figure 1.1), can

be attributed to improved agronomic practices and the development of better

CHAPTER 1: GENERAL INTRODUCTION 4

WHEAT CULTIVATION IN SOUTH AFRICA SINCE 1990

2700.0
3000 4

2500.5

2450.0
2427.0
2348.6
3.5

2133.0

2130.0
2105.0
2500

2005.0
1975.3

1968.5

1958.0

1909.5
1905.0

1905.0

1870.0
1870.0
3

1832.2

1770.0

1750.0
1702.4

1687.5

1680.0
2000
Thousand ha or ton

1550.6

1540.0
2.5

1457.0
1434.0

1430.0
1382.3
1363.2
1316.1

1293.8

t/ha
1500 2
1064.8
1039.5

973.5
941.1
934.0

830.0
805.0
1.5

764.8
748.0

748.0
747.3

745.0
718.0
1000

642.5
632.0

604.7
558.1

511.2

508.4
505.5

496.4
482.2
476.6
1
500
0.5

0 0
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
1
17 7

19 /01
19 /92
19 /93
19 /94
19 /95
19 /96
19 /97
19 /98
19 /99
20 /00
20 /01
20 /02
20 /03
20 /04
20 /05
20 /06
20 /07
20 /08
20 /09
20 /10
20 /11
20 /12
20 /13
20 /14
20 /15
20 /16

8*
20 6/1
/1
90
19

Production years

Area (ha) Production (t) Yield (t/ha) Linear (Area (ha)) Linear (Yield (t/ha))

Figure 1.1: Area harvested, production and yield statistics for South African wheat
cultivation between 1990 and 2017 (adapted from Production Reports - Crop
Estimate Committee (CEC), GRAIN SA, 2017).

varieties. Together and in parallel with global efforts, local research has assisted

South African wheat breeders by improving yields, bread making quality, and

pest and disease resistance of South African wheat varieties (Smit et al., 2010).

1.3 Wheat rusts reduce yields

Some of the extra demand for wheat has been met by continuing genetic im-

provement, which leads to the development of high-yielding varieties, but the

protection of crops from diseases remains critical to support the higher produc-

tion requirements (Edgerton, 2009). The wheat rusts—leaf (brown) rust, stem

(black) rust and stripe (yellow) rust—occur in most wheat-growing areas around

the world and cause widespread disease which is detrimental for yields (Kolmer,

2005; Dean et al., 2012). Rust infection cripples all components of the host, whilst

robbing the plant of water and nutrients (Panstruga and Dodds, 2009; Chen et al.,
CHAPTER 1: GENERAL INTRODUCTION 5

2015). Rusts further reduce water content in the host through compromising the

epidermis. This allows increased water evaporation and renders the plant an

easy target for secondary attack by other pests and diseases (Bockus and Wiese,

2010; Malinovsky et al., 2014). Rust infection ultimately results in the death of

photosynthetic tissues (Chen et al., 2015). Together, water and green tissue loss

decrease the ability of the plant to trap solar energy through photosynthesis for

growth and production of grain (Bockus and Wiese, 2010; Chen et al., 2015).

The occurrence of rust on wheat in South Africa was documented in reports

dating back to 1726 (Du Plessis, 1933) and today all three rusts occur in South

Africa (Pretorius et al., 2007). Pretorius et al. (2007) explain early record-keeping

of rust occurrence in South Africa: Improved records became available as struc-

tured pathotyping of stem rust started in 1920, and in 1960 regular surveys were

introduced. Leaf rust pathotypes were first described in 1937 but were not closely

monitored until the 1980s after new pathotypes caused significant yield losses.

In contrast, no early official disease reports could be found for stripe rust.

In 1996, however, it was seen on spring wheat in the Western Cape and sur-

veys throughout the growing season revealed stripe rust infections in most of the

winter-rainfall wheat cultivating areas. Irrigated wheat in the Northern Cape was

also under attack (Pretorius et al., 1997). Mean yield losses attributed to wheat

rusts in South Africa were estimated to be between 35 and 65 % (Pretorius et al.,

2007). Given the global and local importance of wheat, the detrimental effects of

rust pathogens, and the constant emergence of new pathotypes of the pathogen,

researchers need to continually monitor the changing rust populations, while

searching for new ways and sources of resistance to protect wheat (McIntosh

et al., 1995).
CHAPTER 1: GENERAL INTRODUCTION 6

1.4 Motivation for this study

The foliar disease stripe rust, caused by the biotrophic fungus Puccinia stri-

iformis Westend. f. sp. tritici (Pst), results in major yield losses annually around the

globe (Hovmøller et al., 2010). Growing resistant host varieties has reduced the

impact of stripe rust (Hovmøller et al., 2016). However, knowledge of increased

aggressiveness and shifts in Pst populations (Milus et al., 2009; Rodriguez-Algaba

et al., 2014; Hubbard et al., 2015; Hovmøller et al., 2016; Bueno-Sancho et al.,

2017) encourages investigation of this pathogen and how it is actively evolving

in different geographical areas.

At the start of this project, little was known about the genetic diversity of

stripe rust in South Africa. Previous work had genotyped South African Pst

pathotypes using amplified fragment length polymorphism (AFLP) markers

(Hovmøller et al., 2008) and more recently microsatellite markers (Ali et al., 2014;

Visser et al., 2016). However, these limited marker systems do not provide a

comprehensive genetic picture of the changes that can occur in a Pst population.

The four pathotypes of Pst found in South Africa suggest a clonal lineage, which

has evolved within South Africa since its original introduction in 1996 (Visser

et al., 2016). In this study, next-generation sequencing and advanced bioinfor-

matic tools were used to answer a number of questions regarding the origins

of Pst and the evolution of the Pst population within South Africa. Through an

examination of candidate effector genes, the study also aimed to facilitate a better

understanding of the biological interaction between wheat and Pst.

1.5 Objectives

Stripe rust first appeared as a significant field disease in South Africa in 1996

(Pretorius et al., 1997). Since its introduction, four distinct pathotypes of Pst have
CHAPTER 1: GENERAL INTRODUCTION 7

been detected and pathologically confirmed, the last being identified in 2005 (ZA

Pretorius, unpublished data). This well-defined and presumed clonal population

of Pst formed an ideal population to study the genetic evolution of Pst within

a defined geographical region, addressing the hypothesis of a stepwise gain of

virulence within the four South African pathotypes.

The availability of next-generation sequencing datasets of Pst isolates from

locations in East Africa, South Asia and Europe also allowed a comparative

approach to determine where the Pst introduction in 1996 may have originated.

In addition, the genome sequences obtained for each of the four historical South

African Pst isolates were used to identify candidate effector proteins that may

be associated with avirulence. Lastly, a survey of Pst isolates in 2013, 2014, and

2015 within South Africa was undertaken to compare current field isolates to the

four historical isolates to assess the stability of Pst populations across cropping

seasons.

1.6 Thesis outline and approaches

Background information concerning Pst can be found in Chapter 2, while detailed

methodology is described in Chapter 3 or the relevant research chapters. Five

approaches were undertaken to characterise the Pst population in South Africa,

which are presented across four research chapters. Firstly, the genomes of the

four historical South African pathotypes were sequenced using Illumina next-

generation sequencing. In Chapter 4, this data was analysed using phylogenetic

and statistical clustering analyses to assess the relationship and genetic diversity

between isolates and to hypothesise a potential origin of the Pst incursion in South

Africa in 1996. To further describe the differences between the four South African

pathotypes, comparative genomics analyses were performed, as presented in

Chapter 5, by investigating signatures of positive selection, as well as the presence

CHAPTER 1: GENERAL INTRODUCTION 8

or absence of genes and polymorphisms in genic regions. Chapter 6 reports an RT-

qPCR approach that was used to assess candidate effectors showing differential

gene expression between different Pst pathotypes. To compare the more recent

field population of Pst with the historical South African isolates and to describe

the evolutionary dynamics in the Pst population within South Africa, Pst-infected

wheat leaf tissues from the 2013–2015 seasons were collected and sequenced

using an RNA sequencing (RNA-Seq) approach. Pathotyping of a selection of the

2013–2015 field isolates was conducted to link their genotypes to their pathotypes

and identify any isolates with profiles distinct to those previously identified in

South Africa. This research on the recent population is discussed in Chapter 7. A

final discussion of the findings and last remarks for future research conclude the

thesis in Chapter 8.
Chapter 2

The Wheat Rusts: Life Histories,

Host Response Mechanisms and
Genomic Resources

2.1 The rusts

2.1.1 Filamentous plant pathogens

F ILAMENTOUS PLANT PATHOGENS are highly specialised and include a wide va-

riety of fungi and oomycetes (Wang et al., 2017). Ascomycota and Basidiomycota

are both phyla in the fungi kingdom. The oomycetes include an array of plant

pathogens that share many morphological characteristics with fungi, although be-

ing distantly related. A representation of the phylogenetic relationships between

a number of plant pathogens are illustrated in Figure 2.1 (Fernández-Ortuño

et al., 2007). Many of these pathogens have a similar infection process, using

haustoria to maintain close interaction with the host (Dodds et al., 2009). There

exist thousands of different rust species in the Pucciniales order, of which about

4000 species belong to the genus Puccinia (Hawksworth et al., 1995; Kirk et al.,

2008). 9
 Ascomycetes
Blumeria graminis f. sp. tritici Podosphaera fusca Mycosphaerella fijiensis 0.1
Powdery mildew Powdery mildew Black Tsigatoka
(wheat) (melons) (Banana leaf-spot)
Venturia inaequalis
Botrytis cinerea Leotiomycetes Apple scab
Broad host range Dothideomycetes
(viticulture: botrytis bunch rot)
(horticulture: grey mould)
Verticillium dahliae
Verticillium wilt
Oomycetes (Broad host range)
Sordariomycetes
Phomopsis viticola
Downy mildew Podospora anserina
10

(Grapevine) Model fungus

Phytophthora infestans Magnaporthe grisea

Rice blast
Late blight
(Rice and other cereals)
(Potato and tomato and
some other nightshades)
Fusarium oxysporum
Broad host range
Uromyces appendiculatus
Bean rust
Puccinia graminis f. sp. tritici
Basidiomycetes Wheat stem rust Bootstrap > 80%

Figure 2.1: The phylogenetic relationship of plant pathogenic ascomycetes, basidiomycetes and oomycetes following Neighbour Joining
analysis (adapted from Fernández-Ortuño et al., 2007). Bootstrap values are obtained from 1000 replications. The length of the
bar represents 0.1 substitutions per nucleotide. The tree was constructed using nucleotide sequences of nuclear ribosomal DNA
internal transcribed spacer regions.
CHAPTER 2: WHEAT RUSTS 11

2.1.2 Rusts and their primary host

Rusts are a group of fungi that are harmful to a wide variety of plants with high

socio-economic importance such as cereals, legumes, fruit trees, sugarcane, coffee

and trees (See taxonomic classification in Figure 2.2; Kirk et al., 2008).

Kingdom: Fungi
Subkingdom: Dikarya
Phylum: Basidiomycota
Class: Pucciniomycete
Order: Pucciniales
Family: Pucciniaceae
Genus: Puccinia
P. striiformis
Species: P. graminis
P. triticina

Figure 2.2: Taxonomic classification of the wheat rusts (Chen, 2005; Kirk et al., 2008).

Within Puccinia (P.) species, different formae speciales (f. sp.) describe spe-

cialisation towards specific grass hosts (Anikster, 1984; Wellings, 2007). To date

nine f. sp. have been defined (Chen et al., 2017). The three wheat rusts that

infect wheat are obligate biotrophs, requiring living plant tissues from which

they extract water and nutrients (Dean et al., 2012). Stem rust, also known as

black rust, occurs on the leaf and stem surface as oval-shaped brick-red pustules

that burst through the host tissue and is caused by the fungus P. graminis Pers. f.

sp. tritici, or Pgt (Schumann and Leonard, 2000). Leaf rust, also known as brown

rust, caused by P. triticina Erikss. (Pt) is the most common of the three rusts and

the orange to brown spores occur on the leaf surface in round lesions (Bolton

et al., 2008). Stripe rust mainly forms yellow to orange lines as pustules occur

along leaf veins of adult plants, but it can also infect other parts of the plant such

as leaf sheaths, glumes and awns. Stripe rust of wheat, also known as yellow

rust, is caused by P. striiformis Westend. f. sp. tritici (Pst; Roelfs and Hettel, 1992).
CHAPTER 2: WHEAT RUSTS 12

Each f. s. is further divided into races, strains, or pathotypes (Wellings, 2007),

where the ability to infect the host plant depends on the avirulence genes carried

by the Pst isolate and the resistance genes present in the host plant genotype

(Chen, 2005). In the present study, the term “pathotype” is used throughout. To

further describe the differences in different rust genotypes, a set of wheat lines

with known resistances, is used in infection assays to determine the virulence

profile of the isolate. These host plant genotypes form a differential set and the

range of Pst infection phenotypes seen on each host plant genotype define the

pathotype of the Pst isolate (Allison and Isenbeck, 1930; Roelfs et al., 1992).

2.1.3 The alternative host

Besides the grass hosts, the rust fungi can also infect a second group of hosts. Pgt

has been known to infect alternative hosts Berberis L. (Jin et al., 2010; Zhao et al.,

2011) and Mahonia Nutt. (Wang and Chen, 2013), while Pt infects Thalictrum spp.

as alternative hosts (Bolton et al., 2008). Only recently has Berberis been confirmed

as an alternative host for Pst. Berberis spp. are not native to South Africa, but

are popular ornamentals, commonly stocked by nurseries and are becoming

invasive in the wild (Keet, 2015). In South Africa, cultivation of 24 species of

Berberidaceae, including 18 Berberis and 5 Mahonia have been reported (Glen,

2002). Among these are rust susceptible Berberis holstii, Berberis vulgaris, and

Berberis aristata (Keet, 2015), but Jin (2011) advised that many more susceptible

species could still be discovered. The sexual life cycle of rust fungi is completed

in the alternative host (Chen, 2005). Infection of the alternative host has not been

reported in South Africa. The rare occurrence thereof globally is fortunate, as

it limits the potential for sexual recombination that can lead to faster evolving

populations.
CHAPTER 2: WHEAT RUSTS 13

2.1.4 Global distribution of stripe rust

Stripe rust exists in most parts of the world where wheat is cultivated and

continues to spread (Figure 2.3). In recent years epidemics of stripe rust have

been seen in regions of the world where it did not previously occur (Chen, 2005;

Milus et al., 2006). In contrast with the other rusts, distant dispersal of Pst has

only recently been reported (Zadoks, 1961; Hovmøller et al., 2002; Justesen et al.,

2002; Hovmøller and Justesen, 2007b; Wellings, 2011). There is evidence that new

pathotypes of Pst are more aggressive and able to thrive at higher temperatures,

showing the ability of this fungus to adapt to new environments (Milus et al.,

2006; Markell and Milus, 2008). To date, aggressive pathotypes have not been

described in South Africa.

2.1.5 Favourable conditions for wheat rusts

The occurrence of stripe rust on wheat is dependent on climatic and environmen-

tal conditions. Compared to leaf and stem rust, stripe rust has lower temperature

optima, is prominent in cooler, high altitude and maritime regions and tends to

occur earlier in the growing season (Chen, 2005). Stripe rust urediniospore ger-

mination is most successful between 9 ◦C to 13 ◦C, while stem rust’s germination

optimum is higher at 15 ◦C to 24 ◦C (Roelfs et al., 1992) and leaf rust, the most ver-

satile and common, can infect the host in temperatures ranging between 10 ◦C to

25 ◦C (Bolton et al., 2008). Reports of adaptation to higher temperatures in newly

emerging Pst populations in North America (Milus et al., 2006) show that higher

temperatures, while suboptimal, is not insurmountable to Pst. Another study

suggests that with sufficient light intensity, high temperatures are not necessarily

inhibiting to Pst infection (de Vallavieille-Pope et al., 2002). However, Chen (2005)

reports that temperatures below −10 ◦C can kill the pathogen in infected leaves.

Free moisture in the form of rain or dew for 3 to 6 hours is essential for germi-
CHAPTER 2: WHEAT RUSTS 14

Global 1960–1999

Not recorded
Rare
Localised in some seasons
Localised in most seasons
Widespread in some seasons Global 2000–2012
Widespread in most seasons
N/A

Figure 2.3: Global distribution of Puccinia striiformis f. sp. tritici, before and after 2000
(from Beddow et al., 2015).
CHAPTER 2: WHEAT RUSTS 15

nation of Pst urediniospores (Roelfs et al., 1992; Chen, 2005). On the contrary, dry

weather and wind, towards the end of the growing season, are favourable for

pathogen survival, as dry spores stay viable for longer and are wind dispersed

(Zillinsky, 1983; Chen, 2005). Compared to moisture and temperature optima,

little work has been done on optimal light requirements during the rust life

cycle. There is some evidence that exposure of wheat seedlings to elevated light

intensities before inoculation with urediniospores increases infection success

(de Vallavieille-Pope et al., 2002). Conversely, compared to stem and leaf rust,

Pst urediniospores are sensitive to ultraviolet light, and excess exposure reduces

long-term viability (Roelfs et al., 1992).

2.1.6 Infection cycle of Puccinia rusts

The life cycles of the three wheat rusts are similar. In this section, the Pst life

cycle is described. There are five spore stages in the life cycle of Pst. Three of

these—urediniospores, teliospores and basidiospores—occur on wheat and the

remaining two—pycniospores and aeciospores—on the alternative host. This is

illustrated in Figure 2.4.

Very few cases of sexual reproduction have been reported, leaving the fungus

to almost completely rely on asexual reproduction (Jin et al., 2010; Zhao et al.,

2013; Chen et al., 2017). In areas where the sexual cycle takes place, aeciospores

are formed after infection of the alternative host (Chen, 2005). These spores

can infect wheat and result in pustules releasing urediniospores for reinfection.

In the majority of regions, where the grass host is the main or only host, only

urediniospores are available for host infection. Characteristic of this spore stage,

each spore carries two haploid nuclei. About two weeks after urediniospores

landed on a leaf and entered the leaf through the stoma, the newly produced,

yellow urediniospores erupt through the surface of the leaf (Figure 2.5).

The urediniospores are dispersed by wind, or the mechanical action resulting

CHAPTER 2: WHEAT RUSTS 16

Uredia Telia

Teliospore
Mini cycle of infection
by urediniospores 2n

Aeciospores infection Basidiospores

n+n Asexual stage on wheat
on wheat

Aeciospore Sexual stage on Berberis spp. n

Aecial-cup clusters
Aecial-cup bearing
aeciospores Pycnium

n+n
Pycniospores
Pycnial nectar

n n

Figure 2.4: Spore stages and the infection cycle of Pst. The mini cycle of (re)infection,
indicated with red arrows, is the primary source of inoculum for most stripe
rust outbreaks in wheat-growing areas worldwide. Only recently, the sex-
ual cycle, indicated with blue arrows, have been observed under natural
conditions in China (from Zheng et al., 2013).

Figure 2.5: A stripe rust uredinium pustule. Thousands of yellow spherical echinulated
spores, typically 28–34 µm in diameter (Zillinsky, 1983), erupts through the
wheat leaf surface (Photo: Kim Findley, John Innes Center, UK).
CHAPTER 2: WHEAT RUSTS 17

from raindrops falling onto leaves (Chen, 2005). This phase of Pst development

constitutes the asexual cycle. This cycle typically takes 12 to 14 days depending

on the isolate and environmental conditions (Chen et al., 2014), but Australian

studies confirmed a shorter life cycle in aggressive Pst pathotypes (Sharma, 2012).

The number of infection cycles the pathogen complete in a season determines the

severity of the epidemic (de Vallavieille-Pope et al., 2012).

Urediniospores can over summer on voluntary wheat plants and other sus-

ceptible grasses. Examples include the wild rye species, Secale L. strictum subsp.

africanum, seen in South Africa (Pretorius et al., 2007, 2015). Alternatively, towards

the end of the wheat-growing season, as the wheat plant undergoes senescence,

infection sites from some Pst isolates can form telia (Chen et al., 2014). The subepi-

dermal telia are present on both sides of the leaf blade and produce dark brown,

two-celled, oblong-clavate teliospores (Zillinsky, 1983; Chen, 2005; Chen et al.,

2014). Through karyogamy, the nuclei in each of the two cells of the teliospore

fuse, resulting in two diploid cells. The diploid nucleus in each cell undergoes

meiosis, and the two cells grow into a promycelium of four cells. This develops

into a basidium consisting of four cells, each of which releases a haploid basid-

iospore. These basidiospores can infect an alternative host, initiating the sexual

cycle (Chen et al., 2014).

The haploid basidiospores infect the alternative host and forms either pycnia

(female) or spermagonia (male) on the adaxial side of the leaf. These spore-

producing structures contain haploid reproductive structures. Rusts are het-

erothallic, and spermatia produce pycniospores (the male gametes), which are

transferred to pycnia to fertilise receptive hyphae, the female gamete (Rapilly,

1979). Dispersal of pycniospores can be facilitated by precipitation running down

the leaf, while the pycnia also produce nectar. It has been described in stem and

leaf rust that visiting insects that come into contact with the nectar can act as

vectors to spread the spermatia to other pycnia (Leonard and Szabo, 2005; Bolton
CHAPTER 2: WHEAT RUSTS 18

et al., 2008). After fertilisation, plasmogamy of compatible mating types develops

into a dikaryotic primordium, which matures into an aecium on the abaxial side

of the alternative host leaf. The aecium produces dikaryotic aeciospores that

can only infect the primary host (wheat), forming an urediospore-producing

uredium—the starting material for the roughly 14 day asexual cycle that contin-

ues on wheat throughout the growing season (Chen et al., 2014).

Currently, two factors are considered responsible for the rare occurrence of

sexual recombination. Firstly, contrasting to other rusts of wheat, teliospores do

not enter a dormant phase and readily germinate under prolonged dew condi-

tions (Chen et al., 2014). The time frame in which viable teliospores exist is thus

short. Secondly, germination of teliospores requires very specific environmental

conditions. The rare occurrence of alternative host infection by Pst testifies to the

fact that spore availability and lengthy periods of dew formation do not often

coincide. Such a natural occurrence has only been recorded twice, both times in

China (Zhao et al., 2011, 2013).

Although infection of the alternative host remains rare, these observations

explain the increased Pst population variation found in the Himalayan region,

compared to other regions (Ali et al., 2014). Barberry is also common in these

areas, further supporting the hypothesis of genetic recombination through sexual

reproduction in the Himalaya region (Ali et al., 2014). Additional evidence based

on AFLP and microsatellite markers illustrates the need for further investigation,

determining the importance of the sexual stage in Pst for the generation of genetic

variability (Mboup et al., 2009; Duan et al., 2010; Zheng et al., 2013). Fortunately,

in South Africa and most other wheat-growing areas where stripe rust occurs,

mutation and somatic hybridisation are believed to be the major sources of

variation, theoretically supporting slower evolution. However, in the absence of

the sexual cycle, somatic recombination can still contribute to variation leading

to the formation of new pathotypes, as described by Lei et al. (2017).

CHAPTER 2: WHEAT RUSTS 19

2.1.7 The stripe rust infection process on wheat

Wheat, as the primary host of Pst, provides water and photosynthates for uredio-

niospore production, maintaining the dominant asexual stage (Chen et al., 2014).

Throughout the wheat-growing season, it repetitively infects the crop while

cycling through clonal reproduction (Figure 2.6). Pst, as an obligate biotroph,

needs to maintain the integrity of the plant cells during this infection process.

Resources, predestined for plant growth and grain development, are diverted by

the fungus for hyphal growth and spore production. In resistant wheat varieties,

the evoking of a cellular hypersensitive response causes necrosis and chlorosis,

stopping pathogen development but further compromising the plant’s ability to

photosynthesise (Chen, 2005).

Figure 2.6: Illustration of the infection process of Pst (from Cantu et al., 2013). dpi,
days post inoculation; S, uredinospore; SV, substomatal vesicle; IH, invasive
hyphae; HM, haustorial mother cell; H, haustorium; P, pustule; G, guard cell.

With sufficient moisture on the leaf surface for the urediniospore to germinate,

the germ-tube grows across the leaf surface in search of a stoma through which it

enters the plant. Unlike Pgt and Pt, Pst does not produce a visible appressorium

(Niks, 1989). A substomatal vesicle (SSV) forms within the substomatal cavity

from which up to four infection hyphae (IH) develop (Figure 2.6). When an IH
CHAPTER 2: WHEAT RUSTS 20

reaches a mesophyll cell, the tip of the IH differentiates a haustorial mother cell

(HMC). An infection peg (IP) forms at the tip of the HMC that breaches the cell

wall of the plant mesophyll cell (Figure 2.7).

Spore or hypha
Plant extracellular space

Infection peg Neck band

Host cell wall

Extrahaustorial
Host plasmalemma membrane

Extrahaustorial
Effector with matrix
N-terminal Haustorium
secretion tag
Pathogen cell wall
Mature and plasmalemma
Secretory
effector
pathway

Exocytosis

Host cytoplasm
Endocytosis
?

Figure 2.7: Illustration of a filamentous plant pathogen haustorium. Three mem-

branes and the extra haustorial matrix separate the host cytoplasm and the
pathogen’s haustorium content. The pathogen cell wall and plasmalemma is
situated on the haustorium side. The modified host plasma membrane and
neck band seals off the haustorial matrix from the host cytoplasm. Effector
delivery is illustrated by the inset (from Panstruga and Dodds, 2009).

Some fungi use mechanical force aided by the turgor of the cell to breach

the cell wall, for example in Magnaporthe oryzae (Hebert) Barr, or enzymes as in

the case of Pgt (Duplessis et al., 2011), or a combination, as used by powdery

mildew (Pryce-Jones et al., 1999). A different set of enzymes has been found in

Pgt and other fungi, that likely plays a role in disguising the penetrating hyphae

by remodelling of the fungal cell wall (El Gueddari et al., 2002). However, it is

currently unknown how the Pst IP achieve cell wall penetration (Panstruga and

Dodds, 2009).

Having breached the plant cell wall, Pst needs to establish a compatible

association with the cell, keeping it alive while feeding. From the end of the
CHAPTER 2: WHEAT RUSTS 21

IP a haustorium develops that invaginates the plant cell membrane, causing

the plant cell membrane to envelope the haustorium (Figure 2.7; Panstruga and

Dodds, 2009). Three layers separate the content of the haustorium from the

cytosol of the plant cell: the haustorial plasmalemma, the haustorial wall and the

extrahaustorial membrane. The haustorial membrane and wall are surrounded

by a gel-like layer, called the extrahaustorial matrix (Panstruga and Dodds,

2009). The extrahaustorial membrane is likely derived from the plant cell plasma

membrane and is in contact with the cytoplasm of the plant cell (Szabo and

Bushnell, 2001).

Due to the biotrophic nature of cereal rust pathogens, it is mostly impossible to

culture the fungus artificially. As the multi-layered haustorium cannot be grown

in vitro (Panstruga and Dodds, 2009), the exact mechanisms of how transport

across the membranes is facilitated are currently not confirmed. The haustorium

has a dual function, allowing two-way traffic across the membranes (Mendgen

et al., 2000). It acts as a feeding structure to take up amino acids and sugars from

the host (Panstruga and Dodds, 2009), while at the same time delivering fungal

molecules to the plant that enable pathogenicity (Mendgen et al., 2000). Among

these are effector proteins that are delivered into the host cytosol and the apoplast,

altering plant processes to the advantage of the pathogen, while protecting itself

against the host defence systems (Kamoun, 2007; Rovenich et al., 2014; Petre et al.,

2016a). Once infection is established, long hyphae branch lengthwise within the

leaf, colonising a large area and causing the typical striped pattern of uredinia

seen on older plant leaves (Moldenhauer et al., 2006).

2.2 Combating wheat stripe rust

Agronomic management of stripe rust involves both the deployment of host resis-

tance and the application of fungicides. Multiple fungicide applications are often
CHAPTER 2: WHEAT RUSTS 22

required during the wheat-growing season, being costly, potentially problematic

to the environment, and not always 100 % effective, whereas the right combina-

tion of resistance genes can provide complete stripe rust resistance (Boshoff et al.,

2003). Despite treatment, significant losses have been recorded (Oerke and Dehne,

2004). Even with resistance breeding and chemical crop protection, yield losses of

14 % to 40 % have been reported (Flood, 2010). Increased success in protection of

foliage and ears can, however, be achieved when fungicide application is timed

correctly (Boshoff et al., 2003).

Genetic resistance in the host causes selection pressure on the pathogen

to overcome that resistance. Strategies to relieve selection pressure include

the rotational deployment of resistance genes, regional gene deployment and

pyramiding of resistance genes (Chen et al., 2017). Quantitative, polygenic

resistance is considered a better choice due to its potential durability and will be

discussed later in this chapter.

2.3 Plant defence mechanisms

Plants have passive and active defence mechanisms to protect them from biotic

stresses. A compatible interaction between a pathogen and its host is one where

the pathogen successfully infects and colonises the host. However, incompati-

ble interactions exist between some combinations of Pst pathotypes and wheat

genotypes. Different mechanisms contribute to the host being able to withstand

a pathogen attack.

Passively, preformed defence mechanisms include the composition of the

waxy layers, the cuticle being the first structural barrier to pathogen invasion. Fur-

ther passive defence is put in place by pre-formed antimicrobial proteins and sec-

ondary metabolites, including photoanticipins, inhibitors of essential pathogen

enzymatic activities, hydrolytic enzymes, lectins, and defensins (Selitrennikoff,

CHAPTER 2: WHEAT RUSTS 23

2001; Egorov et al., 2005; Coram et al., 2008). Passive defence is not pathogen

specific, in contrast with many active defence mechanisms that are induced by

the presence of the pathogen, and can be either specific or non-specific. Both

physical and chemical changes are seen. These include the deposition of callose,

cell wall cross-linking and the formation of papillae, changes in membrane per-

meability, production of reactive oxygen species (ROS), and the synthesis of a

whole range of pathogen-related (PR) proteins and secondary metabolites, such

as phytoalexins (Malinovsky et al., 2014).

2.3.1 Host-pathogen interaction

The active plant defence mechanisms require very specific pathogen recogni-

tion. For biotrophic pathogens, the current model of host-pathogen interac-

tions involves an initial general recognition of a potential pathogen, triggered

by plant recognition of conserved pathogen molecular motifs. The conserved

pathogen molecular motifs are referred to as pathogen-associated molecular

patterns (PAMPs) or microbe-associated molecular patterns (MAMPs), as de-

scribed by van der Hoorn and Kamoun (2008). These motifs are recognised by

transmembrane pathogen receptor proteins (PRRs).

Recognition of pathogen-associated patterns triggers defence responses, which

are collectively known as PAMP triggered immunity (PTI; Jones and Dangl, 2006).

Many pathogens are able to suppress the defence responses mounted by PTI,

leading to successful infection. Proteins, secreted by the pathogen into the plant,

facilitate the down-regulation of PTI defence. These proteins, generally consid-

ered to be small peptides able to cross membranes, are referred to as effectors

(Franceschetti et al., 2017). In addition to down-regulating host defence responses,

effectors also have an active role to play in pathogenicity, modifying the plant

cellular and molecular environment in such a way that it eventually supports

CHAPTER 2: WHEAT RUSTS 24

pathogen growth and reproduction (Rovenich et al., 2014).

The second stage of the host-pathogen interaction involves specific recogni-

tion by the plant of specific pathogen effector molecules (Jones and Dangl, 2006).

This second layer of defence is referred to as effector-triggered immunity (ETI).

This involves recognition of specific pathogen effectors, now termed an aviru-

lence (Avr) factor, by a receptor protein in the plant termed an R gene (Dangl and

Jones, 2001; van der Hoorn and Kamoun, 2008). Alternative models of indirect

associations, referred to as the guard and decoy models, have been described

(van der Hoorn and Kamoun, 2008). The direct relationship between R genes and

their corresponding Avr genes is known as the gene-for-gene concept described

by Flor (1956). This specific plant-isolate recognition enables the plant to trigger

a stronger defence response that restricts pathogen growth and reproduction,

with the strength of resistance differing with each R gene/Avr combination. ETI

is a specific host-pathogen interaction, depending on the presence of the R gene

in the plant genotype and the presence of the corresponding avirulence factor

in the pathogen isolate. The R gene/Avr interaction usually results in death of

the infected plant cell (and possibly also surrounding plant cells) in a reaction

known as hypersensitive cell death (HCD; Jones and Dangl, 2006).

The pathogen can evade R gene recognition by selection of mutations within

the avirulence effector factor that break the R gene/Avr interaction (Dodds and

Rathjen, 2010). When this occurs, the aviruelence factor is subsequently referred

to as a virulence factor. The continuing cycle of the pathotype-specific R gene/Avr

interaction breakdown is known in wheat disease breeding as the Boom-and-Bust

cycle (Knott, 1989; McDonald, 2004) and is one of the reasons why wheat breeders

are interested in characterising and using rust resistance genes that do not fit the

R gene/Avr model (see Section 2.3.2).

Five classes of proteins encoded by plant R genes have been modelled, and are

illustrated in Figure 2.8. The biggest class characteristically encode for nucleotide-
CHAPTER 2: WHEAT RUSTS 25

LRR

CC TIR

NB NB
• Cf-2 Kin Kin CC
• Cf-4
• Cf-5 • Pto • Xa21
LRR LRR • Cf-9 • FLS2 RPW8

NB-LRRs

Figure 2.8: The five main classes of plant disease resistant proteins (from Dangl and
Jones, 2001). Cytoplasmic nucleotide-binding site leucine-rich repeat proteins
are typically not membrane-associated and represent the largest class of
resistance proteins. Cf-X and Xa21 typically carry a large transmembrane
leucine-rich repeat region. The serine/threonine protein kinase is encoded
by the Pto gene, with possible membrane association through the N-terminal
myristoylation site. A putative N-terminal signal anchor is carried by the
RPW8 gene product. CC, coiled-coil domains; NB, nucleotide-binding site;
LRR, leucine-rich repeat; TIR, Toll and Interleukin-1 receptor type region;
Kin, kinase

binding site (NBS)-leucine-rich repeat (LRR) proteins (NB-LRR; Kolmer, 2005).

NB-LRRs are thought to be cytoplasmic and in contrast with the other four classes

of R proteins, Xa21 and Cf-X proteins contain transmembrane and extracellular

LRR domains, while the Pto gene product is membrane-associated with a cyto-

plasmic kinase. The RPW8 protein has a putative signal anchor at the N-terminus

(Dangl and Jones, 2001).

The expression of R gene resistance is usually qualitative and expressed at

all wheat growth stages (Dangl and Jones, 2001). The profile of R gene/Avr

interactions, tested on a set of wheat lines with known resistance, defines the
CHAPTER 2: WHEAT RUSTS 26

pathotype of any given Pst isolate. “Yr”, followed by a number, designate genes

that confer resistance to stripe rust (McIntosh, 1983).

2.3.2 Other sources of resistance

Other forms of stripe rust resistance have been characterised that are not pathotype-

specific. These forms of resistance have remained effective to all Pst isolates tested

and therefore are termed pathotype-non-specific resistance (Van der Plank, 1968).

These forms of resistance are usually quantitative, being partial in effect, ex-

pressed more strongly in mature wheat tissues and is therefore also termed adult

plant resistance (Simmonds, 1991; Parlevliet, 2002; Mallard et al., 2005). It can fur-

ther reduce the rate of disease progress, called slow-rusting, partial, or horizontal

resistance (Van der Plank, 1968).

2.4 The Pst genome

2.4.1 Genomic variation

When point mutations occur in genes, it can change an amino acid which in

turn can change the functionality and stability of the protein. If this has a no-

table impact on the phenotype, it will change the way in which the organism

interacts with its environment. Such a change will be under selection to either

eliminate it from the population or increase the frequency, depending on the

impact of the change on the reproductive ability of individuals with the particular

polymorphism.

The variation in Pst Avr genes has been evaluated for many years. Pathotype

(race) profiling is widely deployed and extremely informative. It has been prac-

tised for about 100 years (Thach et al., 2015) and changes in pathotype profiles

mostly support a clonal lineage for Pst. The ability to genotype isolates to support
CHAPTER 2: WHEAT RUSTS 27

pathotypes was a major addition to the development of rust population studies.

In the last 30 years the development of molecular markers, which have been used

to track global movement, has supported the hypotheses of these clonal popula-

tion structures. Deployment of molecular marker technologies for Pst genotyping

have included AFLP markers (Steele et al., 2001; Brown and Hovmøller, 2002;

Hovmøller et al., 2008; Mboup et al., 2009), and more recently microsatellite

markers (Mboup et al., 2009; Ali et al., 2014; Visser et al., 2016; Walter et al., 2016)

and sequence-characterised amplified region (SCAR) markers (Walter et al., 2016)

were implemented.

The so-called “genomics era” provides an even higher resolution view of

the diversity within and between Pst populations. The development of high

throughput sequencing techniques provides the opportunity to answer many

more questions about the ongoing evolutionary processes in Pst and just how far

this airborne pathogen can travel.

2.4.2 Rust genomics

Stem rust was the first of the wheat rusts to be sequenced, followed by leaf and

stripe rust. The Fungal Genome Initiative at the Broad Institute of Massachusetts

Institute of Technology and Harvard University was instrumental in sequencing

all three wheat rusts. Genomic research in Pst saw fast development as the

international community has published a high number of Pst next-generation

sequencing datasets.

Some of these resources have been applied specifically to develop represen-

tative draft reference sequences of Pst pathotypes from distinct pathotypes and

geographical areas. These are summarised in Table 2.1 and include the North

American isolates, PST130 (Cantu et al., 2011) and PST-78 (Cuomo et al., 2017), the

Chinese isolate, CY32 (Zheng et al., 2013) and the Indian isolates 46S 119 (Kiran
CHAPTER 2: WHEAT RUSTS 28

et al., 2017) and 38S102 (Aggarwal et al., unpublished). The Australian founder

pathotype, Pst 104E137A-, has recently been assembled using a combination of

next-generation Illumina sequencing and third generation sequencing, alterna-

tively termed long read sequencing, on the PacBio platform (Schwessinger et al.,

2018). Deployment of such advances in sequencing technology enables compari-

son of the dikaryotic nuclei in Pst to investigate the evolutionary machinery used

to drive the development of new Pst variation.

Puccinia graminis f. sp. tritici

The first rust reference genome and, to date, only Pgt reference, was sequenced

from the pathotype CRL 75-36-700-3 (Duplessis et al., 2011). The project was led

by the Szabo group at the USDA-ARS Cereal Disease Laboratory, University of

Minnesota, USA. In 2007 the first 7.88× draft of the genome sequence assembly

was released. It was updated in 2010 with a mitochondrial assembly and its

accompanying annotation data, and finally in 2011 with an RNA-Seq based

annotation. In addition to sequencing the genome, the shotgun fosmid library

was used to prepare a physical fingerprint map. To investigate gene expression

at various stages of Pgt development, complementary DNA (cDNA) libraries

were constructed for such tissues. The estimated genome size of Pgt is 80 mega

base pairs (Mbp). The outbreak of the highly virulent Pgt pathotype, Ug99,

prompted this research, resulting in the development of many useful markers for

pathotype-diagnostic tests since (Godfrey et al., 2010).

Puccinia triticina

Genome sequencing of the Pt isolate 1-1 was done using Fosmid-end and bacterial

artificial chromosome end (BAC-end) libraries and a hybrid of 454 and Applied

Biosystems Integrated (ABI) sequencing technologies, also known as Sanger

 Table 2.1: Whole genome sequencing projects using next- and third-generation sequencing. Genomes that were proposed as reference
sequences are listed exclusively. Various methodologies have been used for library construction, sequencing and assembly, with
varying results. These assemblies are invaluable tools that can be used to reveal genome characteristics of the three wheat rusts
(adapted from Kang, 2017 including Cantu et al., 2011, 2013; Cuomo et al., 2017; Schwessinger et al., 2018)

Wheat rust Genome Size Protein coding Secreted No. of contigs* Sequencing
Isolate % TE
pathogen (Mbp) genes proteins or scaffolds technology

Illumina Genome Analyzer II

P. striiformis PST130 Φ 64.8 18 149 1 088 *22 815 ∆ 17.8
sequencing

Fosmid-to-fosmid strategy by
P. striiformis CYR32 110.0 25 288 2 092 12 833 48.9 Illumina GA paired-end
sequencing
29

Roche 454 FLX and Illumina

P. striiformis PST-78 117.3 19 542 2 146 9 716 31.5 fosmid-end sequencing

P. striiformis 38S102 75.6 – – 996 – Illumina NextSeq 500

P. striiformis Pst-104E 79.8 15 303 – 996 53.7 PacBio RSII

Roche 454 FLX and Sanger

P. triticina 1-1 135.3 14 880 1 358 14 820 50.9 fosmid-end and BAC-end
sequencing
Sanger sequencing
P. graminis CRL 75-36-700-3 88.6 15 800 1 106 392 36.5 whole-genome shotgun strategy

Φ, 60 % of genome; TE, Transposable and repetitive elements; ∆ , Only transposable elements; *, indicate number of contigs if present,
otherwise number of scaffolds; –, not available; BAC, bacterial artificial chromosome
CHAPTER 2: WHEAT RUSTS 30

sequencing (Cuomo et al., 2017). Considerable advances in characterising Pt

genes and genomic variation was enabled through the assemblies of two more

genomes—the virulent pathotype, Race77, and an older avirulent pathotype,

Race106 (Kiran et al., 2016).

Puccinia striiformis f. sp. tritici

A number of draft sequences are now available for Pst. The PST130 isolate was

first identified in Oregon and Washington, USA, in 2007 (Chen et al., 2010). The

isolate was chosen to be sequenced for technical reasons and not because it was

biologically specifically interesting. Subsequent to genome assembly the PST130

genome has been continually investigated in the research group of Dr Diane

Saunders (JIC, UK). PST130 was used as reference genome in the present study

as the candidates association with this research group allowed building on and

making direct comparisons with previous work in the group.

CYR32 was sequenced as it was a highly prominent pathotype in China. This

work confirmed and further emphasised previous reports of high heterozygosity

between the two nuclei as a fosmid-to-fosmid sequencing strategy was applied

(Zheng et al., 2013). PST-78 was chosen to represent the Pst pathotypes virulent to

Yr8 and Yr9 that were first identified in 2000 (Cuomo et al., 2017). The isolate was

collected from the US Great Plains. Incorporating many sequencing platforms,

this multi-approach resulted in a high quality genome. Gene annotation was

done using transcriptome sequence data and de novo gene prediction (Cuomo

et al., 2017). The initial approximately 81× cover assembly of PST-78 was released

in 2012, with the RNA-Seq-based annotation containing 19 542 genes. The first

genome from an Indian Pst isolate was published in 2017 (Kiran et al., 2017). The

pathotype 46S 119 has virulence to Yr9 and emerged and recently spread into the

north-western plains of India. The 38S102 pathotype was first isolated from the
CHAPTER 2: WHEAT RUSTS 31

Neelgiri Hills in India in 1973 and also has avirulence to Yr9 (Aggarwal et al.,

unpublished). These isolates are interesting as many wheat varieties in the north-

west of India are protected by the Yr9 resistance gene (Kiran et al., 2017). The

long read assembly of the Australian pathotype, Pst 104E137A- (Schwessinger

et al., 2018), refined earlier conclusions on genetic diversity that were drawn from

short read assessments.

2.4.3 Challenges in bioinformatics

All rust genome sequencing projects have used urediniospores, the major spore

stage on wheat. The two nuclei of the dikaryotic urediniospore have been shown

to be highly heterozygous (Zheng et al., 2013). A large portion of all genomes

was repetitive content and transposable elements. The PST130 genome reference,

with 18 % transposable elements, was estimated to include only about 60 % of

the genome, although assembly of 95 % of the reads was possible. Highly similar

repetitive sequences would be assembled in common contigs, and it was esti-

mated that repetitive content that was misassembled could add an additional

10.6 Mbp to the genome size (Cantu et al., 2011). These repetitive sequences and

high density of transposable elements impede the principles assemblers use to

reconstruct a genome (Duplessis et al., 2011; Castanera et al., 2016).

Haplotype-phased genomes address this problem to some extent. The first

phased Pst sequencing effort, (Schwessinger et al., 2018), using long-read DNA

sequencing technology, demonstrated the nucleotide and structural differences

between the two haploid nuclei. It is expected that single consensus sequences,

as generated for all former Pst genome sequencing experiments, would be subop-

timal in their description of genome diversity and structure.

CHAPTER 2: WHEAT RUSTS 32

2.4.4 Effector identification

After assembly and gene annotation, the focus for plant pathogen research is

shifted to effector coding gene identification. Investigation of effector proteins is

crucial as these proteins are utilised by pathogens to alter biological and metabolic

processes in the host (Kamoun, 2007). Resources developed by earlier studies, as

the development of cDNA and expressed sequence tag (EST) libraries (Ling et al.,

2007; Zhang et al., 2008), and existing knowledge of known effector characteristics

of other pathogens, provide resources for the development of bioinformatic

pipelines. Using computational methods and gene discovery algorithms, these

pipelines facilitate rapid effector gene identification. High throughput sequencing

technologies and bioinformatics further relief the challenges of studying effectors

of obligate biotrophs by providing a platform to investigate complete transcripts

(Joly et al., 2010; Hacquard et al., 2011; Saunders et al., 2012).

Highly conserved motifs have been useful in identifying effector families,

such as the RXLR and LXFLAK motifs in oomycetes (Bozkurt et al., 2012). For

Pgt the [YFW]xC motif has been identified by Godfrey et al. (2010). However, the

characteristic of many of the rusts to rarely display conserved motifs known from

other plant pathogens makes effector prediction challenging (Hacquard et al.,

2011; Saunders et al., 2012; Lorrain et al., 2015). This constraint stresses the need

for functional validation that remains a limiting factor due to the relatively low

throughput of validation systems that can confirm the pathogen effector targets

in the host (Petre et al., 2016a).

Only a few such targets have been identified in hosts of filamentous plant

pathogens, among which the dothideomycete (Figure 2.1) Cladosporium fulvum

Cooke causing tomato leaf mold, the rice blast fungus Magnaporthe oryzae, the

potato blight fungus Phytophthora infestans (Mont.) de Bary and Ustilago maydis

from the class Ustilaginomycetes, causing corn smut (Rovenich et al., 2014). For
CHAPTER 2: WHEAT RUSTS 33

Blumeria graminis (DC.) Speer f. sp. hordei, the causal agent of powdery mildew

in barley, an ARF-GAP target protein was identified in the host (Rovenich et al.,

2014). Adenosine diphosphate (ADP) ribosylation factors (ARF) are important

for vesicle trafficking, while its activity is regulated by Guanosine triphosphatase

(GTPase) activating proteins (GAPs). The pathogen targets this protein com-

plex to interfere with the host’s trafficking of vesicles containing biochemical

molecules (Mandiyan et al., 1999). Association of pathogen genes with vesicle

trafficking in the host has also been proposed in Pst-wheat interaction using

RNA-Seq (Dobon et al., 2016).

Genomic resources enabled the use of yeast-two hybrid screens to identify

associations between Pst and wheat proteins (Lowe et al., 2011). Non-host model

plants were further proposed to characterise effector candidates, specifically

Nicotiana benthamiana Domin, as rust fungi hosts are difficult to manipulate

with molecular genetic techniques (Petre et al., 2015). This approach has been

instrumental in functional characterisation of a number of Pst effectors (Petre

et al., 2016a). The authors warn that although the leaf cell environment of

N. benthamiana is advantageous for protein interaction screens, compared to

expression in yeast, false negatives are common due to differences between

N. benthamiana and the host species (Petre et al., 2016b). A combination of the

two approaches can be followed (Liu et al., 2016). Other examples of functional

validation include transient expression assays and host-induced gene silencing

(HIGS) using RNA interference (Yin and Hulbert, 2015; Liu et al., 2016).

Recent successes in rust effector identification were achieved with the cloning

of the two stem rust effectors, AvrSr35 (Salcedo et al., 2017) and AvrSr50 (Chen

et al., 2017). Variation in AvrSr35 and loss of heterozygosity in AvrSr50 resulted

in the respective inability of Sr35 and Sr50 to recognise specific isolates of the

stem rust fungus, resulting in disease. The methodology that was implemented

could be transferable to other rust effector searches and is therefore noteworthy.

CHAPTER 2: WHEAT RUSTS 34

Candidates were obtained from comparative transcriptomic analysis between

wild type and mutant Pgt isolates. Validation of candidates included a whole

host of techniques including microscopy, transient expression in N. benthamiana

and N. tabacum and yeast-two-hybrid analyses. Transient expression in wheat

made use of transforming constructs into Escherichia coli (Migula) Castellani and

Chalmers and Agrobacterium tumefaciens (Smith and Townsend) Conn. strains.

Virus-mediated effector expression assays were also performed in wheat using

the barley stripe mosaic virus (Lee et al., 2012).

The present study is based on advances in Pst bioinformatics regarding Pst

next-generation sequencing and gene and effector annotations. Annotation pro-

cedures considered knowledge of the life history, molecular mechanisms, and

complementing computational biology resources in Pst and related filamentous

plant pathogens. Together, these techniques enabled the identification of genes

likely involved in distinct virulence profiles of South African Pst pathotypes. Ad-

ditional functional validation methods discussed in this review would add value

in future studies to further investigate the identified candidate effector genes.

Furthermore, genomic and transcriptomic Pst resources allowed predictions to be

made regarding the relatedness of different Pst isolates to one another, based on

genetic proximity when single nucleotide polymorphisms (SNPs) were evaluated

in population analyses. This provided valuable insights into the global preva-

lence of specific genetic groups to better understand their potential movement

and the risks it may involve.

Chapter 3

General Materials and Methods

3.1 Preparation and collection of materials

3.1.1 Inoculation

T HE FOLLOWING STANDARD Pst inoculation protocol, developed and performed

at the University of the Free State (UFS), South Africa, was performed to obtain

urediniospores for genomic DNA (gDNA) extraction used for next-generation

sequencing (NGS) and total RNA extraction of infected tissue used for analyses

of gene expression through RT-qPCR.

For multiplication of urediniospores for sequencing purposes (Chapter 4),

as well as the time course (Chapter 6), and infection assays (Chapter 7), the

wheat variety Morocco was used as a susceptible host. The time course itself was

performed on Avocet S (susceptible), and the infection assay varieties are listed

in Chapter 7. Seedlings were grown for seven days until two unfolded leaves

developed (Zadoks growth stage 12 (Z12); Zadoks et al., 1974). For initial multi-

plication, urediniospores, previously dried on silica gel and stored at −80 ◦C were

suspended in Soltrol® 130 Isoparaffinic Solvent oil (Chevron Phillips Chemical

Company, USA), at 5 mg/ml, upon retrieval from the freezer. Several rounds of

multiplication were performed for the sequencing experiment (see Chapter 4).

35
CHAPTER 3: GENERAL MATERIALS AND METHODS 36

Inoculations of the time course and the infection assays were done with fresh

spores harvested from initial multiplication. Seedlings of seven-day-old wheat

(Z12), grown in Mikskaar Professional Potting Soil 70 (Mikskaar, Estonia) in

10 cm diameter plastic pots, were lightly sprayed with the spore-oil suspension.

Inoculated plants were dried in a growth cabinet at 25 ◦C for about 45 minutes.

Custom-made incubation chambers (755 × 500 × 300 mm) made from galvanised

metal sheeting, with a 30 mm raised grid at the bottom, were filled with hot tap

water to just below the grid level. Seedlings were then placed on the grid, and

the chambers were immediately sealed to capture maximum water vapour and

maintain saturated conditions. The chambers were housed in a cold room at

11 ◦C, after which plants were incubated for 24 hours at 11 ◦C, in total darkness.

These conditions simulate high atmospheric moisture levels and low tempera-

tures resulting in dew formation, usually during night time, in natural conditions.

Next, inoculated plants were transferred to a growth chamber at 17 ◦C for 1.5

days, with a 14 hour day and 10 hour night cycle. Daylight was simulated with a

light intensity of 200 µmol/(m2 s). Plants were then moved to a glasshouse with

natural light and a day-night temperature cycle set to 20 ◦C (06:00–18:00) and

15 ◦C (18:00–06:00), respectively.

3.1.2 Protocol for sampling infected wheat tissue

Infected wheat leaf samples that were used for RNA-Seq discussed in Chapter 7

were collected in wheat fields in South Africa. For every sample, an area of

approximately 20 mm of the leaf covered in Pst pustules was cut into small

segments of roughly 7 mm and placed in a 5 ml tube with RNAlater® solution

(Thermo Fisher Scientific, USA), immediately after sampling from the wheat

plant. RNAlater® was used to preserve RNA integrity as advised by Taylor et al.

(2010). The same procedure was used to collect material from the time course for

gene expression analysis (Chapter 7).

CHAPTER 3: GENERAL MATERIALS AND METHODS 37

3.2 Nucleic acid extraction and quantification

3.2.1 Genomic DNA extraction

Genomic DNA was extracted from urediniospores using the cetyltrimethylammo-

nium bromide (CTAB) extraction method of Chen et al. (1993). Beforehand, CTAB

was heated to 65 ◦C, and 70 % ethanol was prepared and chilled at −20 ◦C. Spores

were frozen using liquid nitrogen and ground using a pestle and mortar. Silicon

dioxide (SiO2 ; Sigma-Aldrich, USA) was used to aid in tissue disruption, using

100 mg of spores with 600 mg of sand. The disrupted material was transferred

to a 15 ml Falcon tube. In a separate tube, 2 ml of pre-warmed CTAB buffer was

added to 5 µl Proteinase K 10 mg/ml), mixed, and incubated at 65 ◦C for 2 hours.

After incubation, 1 volume of chloroform:isoamylalcohol (24:1, v/v) was added

to the previous mixture and vigorously mixed followed by centrifugation at

12 000 g for 10 minutes. The aqueous, upper phase was transferred to a fresh tube,

and 20 µl of RNaseB 10 mg/ml was added after which samples were incubated

at room temperature ( 20 ◦C) for 1 hour. The chloroform step was repeated and

the supernatant transferred to a fresh tube again. Pre-chilled isopropanol was

added (1 volume), followed by gentle inversion to precipitate the gDNA. Samples

were incubated at −20 ◦C overnight. The next day, samples were centrifuged at

12 000 g for 10 minutes. The pellet was washed in 1 ml to 2 ml of the pre-chilled

70 % ethanol. The ethanol was decanted without disturbing the pellet, which

was subsequently allowed to dry at room temperature ( 20 ◦C) and dissolved

in 50 µl 1 % TE buffer [10 mM Tris-Cl (pH 8.0); 1 mM Ethylenediaminetetraacetic

acid (EDTA) (pH 8.0)].

3.2.2 RNA extraction

Total RNA was extracted from Pst inoculated leaf tissue, non-inoculated wheat

and germinated fungal spores using the RNeasy Plant Mini Kit (Qiagen, Ger-
CHAPTER 3: GENERAL MATERIALS AND METHODS 38

many) according to the manufacturer’s instructions. Tissue was disrupted with a

pestle and mortar. To promote tissue disruption, SiO2 was added to the mortar.

All instruments used, including the mortar and pestle and the spatula used to

scrape the homogenised tissue from the mortar, were washed with detergent,

ethanol, and RNase AWAY Decontamination Reagent (Thermo Fisher Scientific,

USA) between extractions. All instruments were cooled in liquid nitrogen or on

dry ice to prevent degradation of RNA due to ubiquitous RNase activity (Holland

et al., 2003). The dry mortar and pestle were placed on dry ice in a polystyrene

box, and further cooled with liquid nitrogen. Approximately 100 mg SiO2 was

added to the mortar with the liquid nitrogen before the leaf sample was added.

Forceps were used to move the preserved sample material from the tubes to

a clean paper towel, where samples were tapped dry to prevent the RNAlater

solution from forming ice crystals when the sample came into contact with the

liquid nitrogen. Samples were then placed in the mortar with liquid nitrogen and

SiO2 , followed by homogenisation of the sample into a fine powder. The ground

sample was scraped with a cooled spatula into a 2.2 ml safe-lock microcentrifuge

tube without allowing it to thaw. The tube with the ground sample was kept on

dry ice until extraction buffer was added.

The procedure was concluded followed the optional step in the protocol. To

prevent degradation RNase inhibitor (0.5 µl) was added to each sample. Aliquots

of 3 µl were prepared for RNA quantification and quality control. Extracted RNA

samples were stored at −80 ◦C.

3.2.3 DNA and RNA quantification

Extracted gDNA was quantified using the Qubit 2.0 Fluorometer (Invitrogen/

Thermo Fisher Scientific, USA). The rationale behind the method is that it detects

dyes that only fluoresce when bound to a specific substrate, in this case, double
CHAPTER 3: GENERAL MATERIALS AND METHODS 39

stranded (ds) DNA. The intensity of the fluorescence is indicative of the amount

of dsDNA in the sample (Simbolo et al., 2013). Assays were performed at room

temperature ( 20 ◦C) as recommended. The instrument was calibrated with the

Quant-iT dsDNA BR Assay according to the manufacturer’s instructions, and

DNA concentrations quantified for all samples.

The Agilent 2100 Bioanalyzer (Agilent Technologies, USA) was used to assess

the quality and quantity of the extracted RNA. The reaction kit was stored at

4 ◦C. A gel-dye mix was first prepared according to the manufacturer’s instruc-

tions. The quality of RNA samples was assessed within one to three days after

preparation and RNA was converted into cDNA within one to three days after an

aliquot passed the quality assessment. Aliquoting prevented multiple freezing

and thawing cycles, as this imposes a risk of degradation of RNA (Taylor et al.,

2010). RNA stocks were stored at −80 ◦C between extraction and being used for

cDNA synthesis.

3.3 Next-generation sequencing and data analysis

3.3.1 Library preparation

A sequencing library was prepared from raw extracted nucleic acids. DNA

fragmentation was followed by size selection and the addition of oligonucleotide

adapters to fragments, for the sequencer to process the library.

3.3.2 Genomic DNA sequencing

Libraries for gDNA sequencing were prepared by the Earlham Institute, UK, us-

ing the Illumina TruSeq DNA Sample Preparation Kit (Illumina, UK), according

to the manufacturer’s instructions. To assess library quality before sequencing, a

High Sensitivity DNA analysis assay was performed on the Agilent 2100 Bioana-
CHAPTER 3: GENERAL MATERIALS AND METHODS 40

lyzer. Quantification of libraries was conducted with the Qubit 2.0 Fluorometer.

One lane of the Illumina flow cell was used for a pool of 10 libraries diluted to

a concentration of 12.71 nM. Sequencing was performed on the Illumina HiSeq

2500 platform at the Earlham Institute, UK, where after adapter and multiplexing

barcode oligonucleotide sequences were removed. Upon receipt of the data, read

quality was assessed using FastQC software (version 0.10.1; Andrews, 2010).

3.3.3 RNA sequencing

Sequencing of messenger RNA (mRNA) extracted from Pst infected wheat sam-

ples was performed at Earlham Institute, UK. The mRNA was reversed tran-

scribed to cDNA. Sequencing libraries were prepared using the Illumina TruSeq

RNA Sample Preparation Kit (Illumina, UK). The RNA 6000 Nano kit was used

to assess the library quality on the Agilent 2100 Bioanalyzer. Libraries were

sequenced using the Illumina HiSeq 2500 platform, and adapters and barcodes

were removed from the resulting sequences.

3.3.4 Bioinformatics pipeline

Mapping of gDNA samples

The 100 bp Illumina paired end reads were filtered using a Perl script to discard

reads containing N calls where nucleotides could not be determined by the

sequencer (Cantu et al., 2013; Hubbard et al., 2015). After filtering, each gDNA

sample was independently aligned to the PST130 reference genome (Cantu et al.,

2011) implementing Burrows-Wheeler Alignment tool (BWA version 0.7.7; Li and

Durbin, 2009) with parameters set to the default setting.

CHAPTER 3: GENERAL MATERIALS AND METHODS 41

Mapping of cDNA (RNA-Seq) samples

Similar to the gDNA samples, the 100 bp Illumina paired end reads were filtered

to discard reads containing nucleotides that could not be determined by the

sequencer (Cantu et al., 2013; Hubbard et al., 2015). The alignment of cDNA

samples was carried out using the Bowtie alignment program (version 0.12.7;

Langmead et al., 2009) from the TopHat package (version 1.3.2; Trapnell et al.,

2012), again aligning to the PST130 reference genome (Cantu et al., 2011), using

the parameter –r set to 200 to accommodate the mate pair sequences with 50 bp

ends.

Identifying single nucleotide polymorphisms

Resulting sequence alignment map (SAM) format files from the gDNA and

RNA-Seq mapping, were converted to binary alignment map (BAM) format with

the software package SAMtools (version 0.1.19; Li et al., 2009). SAMtools sort,

SAMtools index and SAMtools mpileup were used to identify SNPs. Custom

Perl scripts were used to extract allele counts at each position of the genome. A

depth of coverage threshold was set for polymorphic sites, and gDNA SNPs with

a minimum depth of coverage of 10× were extracted, while for RNA-Seq data

minimum depth coverage of 20× were required.

Allele frequencies between 0.2 and 0.8 were classified as heterokaryotic sites,

whereas sites with allelic frequencies above 0.8 were classified as homokaryotic

sites (Cantu et al., 2013). SnpEff (version 3.6; Cingolani et al., 2012) was used to

annotate polymorphisms, to indicate whether they resulted in synonymous or

nonsynonymous substitutions, or whether a stop codon was gained or lost in

coding regions. SnpEff further displayed the codon position of polymorphisms.

Polymorphisms in intergenic regions were also indicated.

CHAPTER 3: GENERAL MATERIALS AND METHODS 42

Quality assessment of samples through sequence data

Each of the two haploid nuclei in the dikaryotic urediniospore is assumed to

contribute a maximum of one allele to each nucleotide site. A variant site is de-

scribed as a homokaryotic SNP when both alleles are identical, but different from

the reference PST130 nucleotide. A heterokaryotic SNP describes the situation

where two different alleles occur at the nucleotide site. These alleles may both be

different from the reference, or only one, while the other would be identical to

the reference (Hubbard et al., 2015).

To ensure that the genomic data was in each case derived from a single

genotype, the allelic distribution at heterokaryotic sites was assessed across

the genome. It is important to note that the reference genome is not phased.

Implications are discussed in Chapter 5. When a single genotype is present, it is

expected that the frequency plot, exhibiting both alleles at the heterokaryotic SNP

sites, will form a distribution with a mode of 0.5 due to the equal contribution of

both nuclei (Yoshida et al., 2013).

In this analysis, the number of heterokaryotic SNP sites were plotted on the

y-axis, and the proportion of alleles across reads at each site, ranging between

0 and 1, on the x-axis, as explained in the supplementary documents of Cantu

et al. (2013). Read frequency graphs of isolates unique to the current study are

summarised in Appendices A and D.

3.3.5 Clustering analysis

Clustering analyses are grouping algorithms that operate in such a way that

individuals placed in the same group are more similar compared to individuals

in other groups. Genomic and transcriptomic data were used for phylogenetic

clustering and population cluster analyses. As the transcriptomic data does

not include intergenic regions, only the coding regions of the gDNA samples
CHAPTER 3: GENERAL MATERIALS AND METHODS 43

were considered in this analysis. Brief descriptions of the different underlying

statistical and genetic models deployed in the analyses follow in the next sections.

Phylogenetic analysis

A “Randomized Axelerated Maximum Likelihood” (RAxML) phylogenetic ap-

proach was used to determine the genetic relationships between South African

Pst and to compare them to Pst isolates from other countries.

First a subset of sites in each gene in the PST130 gene models was used to

construct synthetic genes. Sites identical to the PST130 reference genome were

only included when a minimum of 2× depth of coverage was reached. Variant

sites were included when coverage depths of 10× for gDNA samples or 20×

for cDNA samples were reached. Introducing placeholders at sites where the

required depth of coverage was not achieved preserved codon positions. Then a

phylip file was prepared as input to RAxML software (version 8.0.20; Stamatakis,

2014) to construct the phylogenetic tree.

Accurate nucleotide substitution models are required in most phylogenetic

analyses as the rate of nucleotide substitution varies in molecular evolution (Jia

et al., 2014). To account for the fact that all sites do not evolve at an identical

rate, codon positions and the model used to determine phylogenetic clades were

considered. Due to the degeneracy of codons, there is redundancy in the genetic

code that can cause the occurrence of synonymous substitutions. Substitutions

at the third position are more often synonymous, and therefore less likely to

influence the phenotype and be a target for positive or negative selection, than at

the first and the second codon position. Nucleotide changes at the third codon

positions can, for this reason, be considered to evolve at a higher rate (Rambaut

and Grass, 1997). The third codon position further shows less nucleotide bias and

a more homogenous rate of evolution when compared to the first and second
CHAPTER 3: GENERAL MATERIALS AND METHODS 44

codon position (Bofkin and Goldman, 2006).

Nonsynonymous sites are not evolutionary neutral and, depending on the

effect of the resulting phenotype, can experience high levels of selection pressure

resulting in gene specific evolution. Phylogenetic trees derived from such data

can be misleading when convergent evolution of such genes in different popu-

lations are present. The phylip input file was therefore prepared containing the

third codon positions of synthetic genes to illustrate the evolutionary history of

the populations without being influenced by gene specific evolutionary devel-

opment. The third codon position of those synthetic genes that had a minimum

of 80 % breadth of coverage of the original reference gene length in at least 80 %

of isolates were included in the phylogenetic analysis to ensure that only genes

with high coverage were included.

In addition, the General Time Reversible (GTR) model of nucleotide substi-

tution under the Gamma (Γ) model of rate heterogeneity was selected for the

RAxML model parameter (–m GTRGAMMA). The GTR model parameters account

for unequal frequencies for the four nucleotides and the unique rate of each of the

possible six nucleotide substitutions. Furthermore, the Γ model uses a discrete Γ

distribution to assign different rates of heterogeneity to different sites (Stamatakis,

2014). Reproducibility was ensured by specifying an initialising value for the

pseudo-random number generator (–p 100) and the process was parallelised on

10 threads (–T 10). To demonstrate the reliability of the inferred tree, bootstrap-

ping was applied by generating 100 (–N 100) alternative runs on distinct starting

trees (–b 12345). Bootstrap values were added to the maximum likelihood tree

with the –f b parameter to generate the bipartition tree where after MEGA (ver-

sion 6.06; Tamura et al., 2013) was used to visualise the phylogenetic tree (Cantu

et al., 2013; Hubbard et al., 2015).

CHAPTER 3: GENERAL MATERIALS AND METHODS 45

Population structure analyses

Two methods were used to predict population structure: STRUCTURE (version

2.3.4; Pritchard et al., 2000) and Discriminant analysis of principal components

(DAPC; Jombart et al., 2010). STRUCTURE is a model-based approach, whereas

DAPC does not make any assumptions about the biological processes that influ-

enced and shaped the dataset. Both methods have limitations and benefits, and

these are discussed in the relevant research chapters. The same depths of cover-

age minima as for the phylogenetic tree were required: 10× coverage for gDNA

samples and 20× coverage for cDNA samples. The SNP data was prepared using

BEDTools (version 2.17.0; Quinlan and Hall, 2010) for variant site annotation in

SnpEff.

Sites where a synonymous substitution was introduced in at least one iso-

late were extracted. These, together with sites identical to the reference with at

least 2× coverage, were repositioned according to their position in the reference

genome. From these files, a data matrix was generated using a custom python

script. The software, STRUCTURE, was used to assign isolates to specific popula-

tion groups and to determine the number of these groups, or clusters (K), due to

genetic differentiation. For this analysis, nonsynonymous SNPs were excluded

as these sites are more likely involved in fitness traits and under selection and

STRUCTURE relies on neutral substitution models. Furthermore, different popu-

lations could have evolved convergently. Such similarity would falsely deduce

that individuals are related.

Analyses consisting of five independent runs for each value of K were carried

out. The “admixture” model was used, and each run was set to a burn-in period

of 110 000 iterations. Thereafter, 200 000 Markov Chain Monte Carlo (MCMC)

generations for each value of K, ranging from 1 to 15, were carried out. K values

were evaluated in two ways: the Evanno method (Evanno et al., 2005) and by
CHAPTER 3: GENERAL MATERIALS AND METHODS 46

calculating the log probability, referred to as LnP(D), of each K value (Pritchard

et al., 2000). STRUCTURE assumes a population that is under Hardy-Weinberg

equilibrium, and the Pst data does not fit this assumption. Therefore the multi-

variate DAPC analysis within the adegenet R software package (Jombart et al.,

2010), was carried out on the same dataset used with STRUCTURE. Principal

component analysis (PCA) summarised genetic variation in the dataset by re-

ducing the dataset to include only the most impactful loci. The lowest Bayesian

information criterion (BIC) suggested the optimum number of population clus-

ters (K), thereafter discriminant analysis (DA) was used to divide samples into

subgroups of population clusters.

Differentiation between and within population clusters

In a segregated population, individuals that aggregate into a subpopulation

tend to interbreed more than what is expected under random mating of the

whole population under Hardy-Weinberg equilibrium. When assessing a dataset,

groups with low levels of heterozygosity among individuals within groups allow

the identification of genetic structure in a global population from which biological

interpretations can be made. To quantify the variation between subpopulations,

the general reduction in heterozygosity HX is assessed by evaluating the observed

heterozygosity Hobs against the expected heterozygosity Hexp using the equation

Hexp − Hobs
HX = . (3.1)
Hexp

Three specific inbreeding coefficients need consideration to take into account

heterozygosity observed in individuals, subpopulations and the whole popula-

tion, substituting HX in Eq. (3.1) with H I , HS and HT , respectively.

CHAPTER 3: GENERAL MATERIALS AND METHODS 47

Reduction in heterozygosity that is due to the population structure can then

be evaluated using the so called “F-statistics”

HS − H I
FIS = ,
HS
H − HI
FIT = T ,
HT
H − HS
FST = T ,
HT

with the relationship

1 − FIT
FST = 1 − .
1 − FIS

The proportion of the genetic variance assigned to the differences between

subpopulations, evaluated in Section 4.2.7 and Section 7.3.1, were calculated

using GenePop (version 4.2; Rousset, 2008) to estimating Wright’s FST statistic

(Hubbard et al., 2015). The FST values varied from zero to one, where zero

indicated the absence of differentiation and one complete differentiation (Hartl

and Clark, 1998).

To assess the genetic diversity within each of the Pst population clusters

identified herein, the population diversity parameter theta (θ) was estimated in

Section 4.2.7 and Section 7.3.1. Theoretically, θ estimates genetic differentiation

amongst subpopulations depending on the number of reproducing individuals

in the population and the mutation rate. Different empirical approximations of θ

exist. In this study, Watterson’s theta, θ̂W , was reported as it takes into account the

number of segregating sites—SNPs in the current case—to estimate the mutation

rate of the population.

The degree of polymorphism between genes in individuals of a subpopula-

tion was calculated using DnaSP (version 5.10.1; Librado and Rozas, 2009) as

suggested by Hubbard et al. (2015).

Chapter 4

Origin of the South African Pst

Pathotypes

4.1 Introduction

4.1.1 Wheat stripe rust in South Africa

I N MOST WHEAT CULTIVATION REGIONS globally, Puccinia striiformis f. sp. tritici

prevails and is a threat to wheat production (Brown, 2003; Hovmøller et al., 2010;

Sharma-Poudyal et al., 2013). Wind dispersal of the asexual urediniospores en-

ables Pst to travel thousands of kilometres (Kolmer, 2005; Hovmøller et al., 2008;

Ali et al., 2014). Foreign incursions can become established in new geographical

regions, completely shifting the pathotype profile of the Pst population in a single

season. In addition to wind dispersal, Pst can be transmitted via anthropogenic

activities such as human travel. For instance, Wellings et al. (1987) considered that

the introduction of Pst into Australia in 1979 could easily have been facilitated

by human-assisted movement. With increases in global travel and freight move-

ment in recent years, multiple destinations are now within easy reach of many

pathogens in a single day (Parker and Gilbert, 2004), regardless of wind dispersal

patterns. In South Africa, the first verified identification and characterisation of

48
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 49

6E22A-
7E22A-
6E22A+

Free State

6E16A-

Western Cape

Figure 4.1: Locations of the original detections of South African Pst pathotypes. Stripe
rust was first detected near Moorreesburg in the Western Cape in 1996. It
occurred throughout the wheat breeding regions of the southwestern part of
South Africa during the season. The pathotype 6E16A- was designated. New
pathotypes (6E22A-, 7E22A- and 6E22A+) observed in following years were
first detected in the Eastern Free State and Lesotho.

stripe rust was in the Western Cape in 1996 (Figure 4.1; Pretorius et al., 1997),

making it a relatively new disease compared to leaf rust and stem rust that were

already recorded in the 1700s (Du Plessis, 1933).

Subsequent surveys in 1996 confirmed that the disease was well established

throughout the winter rainfall regions of the Western, Northern and Eastern

Cape (Pretorius et al., 1997). Traces were also found on irrigated wheat in sum-

mer rainfall regions. As stripe rust has a lower temperature optimum (Roelfs

and Hettel, 1992), the lengthy cool and wet conditions in the Western Cape in

1996 (Figure 4.2), likely contributed to the rapid spread and development of Pst

epidemics (Boshoff et al., 2002).

The first Pst pathotype was confirmed as pathotype 6E16A- through testing

of 32 Pst isolates on 17 standard stripe rust wheat differential lines and seven

supplementary tester lines with known resistance genes (Pretorius et al., 1997).
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 50

14

Min. temp. (°C)

12

10

26
Max. temp. (°C)

24

22

20

18

16

100

80
Rainfall (mm)

60

40

20
May

Sep

Nov
Aug
Jun
Apr

Oct
Jul

Month
11 year mean 1996

Figure 4.2: Temperature and rainfall measured in 1996 during April to November in the
Western Cape compared to the 11 year mean (from Boshoff et al., 2002). Max.
temp., maximum temperatures; Min. temp., minimum temperatures.
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 51

This pathotype was similar to the stripe rust pathotype 6E16 found in the Mediter-

ranean region in the 1970s (Wahl et al., 1984). A similar pathotype, 6E16 was also

detected in East and North Africa, the Middle East and Western Asia (Stubbs,

1988; Badebo et al., 1990; Pretorius et al., 1997). The “A-” added to the pathotype

name of the South African isolate expanded on the notation protocol developed

by Johnson et al. (1972) by adding testing for virulence to YrA, as described by

Wellings et al. (1988).

In 1998 another stripe rust epidemic occurred in South Africa, this time in

the Eastern Free State. The wheat varieties Hugenoot and Carina, that were

resistant to 6E16A-, were widely and severely affected (Boshoff and Pretorius,

1999). Frequent cases of severe Pst infection were observed, often colonising 100 %

of wheat leaves. Virulence tests on an expanded wheat differential set confirmed

a virulence gain for Yr25, defining a new pathotype, 6E22A- (Figure 4.3; Boshoff

and Pretorius, 1999). Pathotype 6E22 has since been reported in Iran in 2009 and

2010 (Elyasi-Gomari and Petrenkova, 2011).

In 2001 yet another new pathotype, 7E22A- (Figure 4.3), was detected on the

wheat variety Chinese 166 in trap nurseries in Makobateng, Lesotho (Pretorius

et al., 2007). This pathotype contained additional virulence to Yr1, but although

Lesotho neighbours the Eastern Free State, an important wheat cultivation area

in South Africa, the pathotype was not considered a threat to the South African

wheat industry, as Yr1 did not occur in local wheat varieties (Pretorius et al.,

2007).

In 2005 a fourth new pathotype, 6E22A+ (Figure 4.3), was detected near

Clocolan in the Eastern Free State. This pathotype was virulent to YrA, but

avirulent to Yr1 (Visser et al., 2016). The phenotypic characterisation of the four

Pst pathotypes is indicated in Figure 4.4.

CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 52

First detection: 6E16A- (1996) SA1

Virulent to: Yr2, Yr6, Yr7,Yr8, Yr11, Yr14, Yr17, Yr19

+Yr25

Virulence gain
6E22A- (1998) SA2

+Yr1
+YrA

7E22A- (2001) SA3

6E22A+ (2005) SA4

Figure 4.3: Schematic illustration of the increase of Pst virulence in South Africa. Gain of
virulence in South African Pst populations, based on traditional pathotype
analysis, between 1996 and 2016 (Pretorius et al., 1997; Boshoff et al., 2002;
Pretorius et al., 2007; ZA Pretorius, unpublished data). Pathotypes analysed
in this study that represent the identified pathotypes were named SA1—SA4.

4.1.2 Pst population diversity

Sufficient genetic diversity in a population increases the likelihood that some

individuals will have superior fitness in changing environmental conditions

(Hartl and Clark, 1998). Due to the stepwise gain in virulence together with

molecular evidence (Visser et al., 2016), Pst likely reproduces clonally in South

Africa. Factors that can increase genetic diversity in asexual Pst populations

are mutations and gene flow, and although not considered to occur frequently,

somatic recombination. Newly introduced alleles–that can be slightly deleterious,

neutral, or slightly advantageous–can stay in the population just by chance, called

genetic drift. When new alleles provide a fitness incentive, positive selection can

SA4
Pst pathotype

SA3 Resistant
SA2 Virulent

SA1
1
10
11
14
15
17
19

2
25
27
3a
4a
4b

5
6
7
8
9
A
le

Yr v

Yr II
or
Sd
Sp
Su
C
Yr

Yr

Yr
Yr
Yr
Yr
Yr

V
Yr

M
Yr
Yr
Yr
Yr
Yr
Yr

Yr
Yr
Yr
Yr
Yr

Yr

Yr
Yr
Yr
Yr

Resistance

Figure 4.4: Pathotype (race) identification tests of South African Pst pathotypes. Patho-
types were defined by compatibility with wheat hosts possessing indicated
sources of resistance (data from Visser et al., 2016).
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 53

fix such alleles in the population, while negative selection will remove deleterious

mutations. Such selective evolutionary forces can result in an erosion in genetic

diversity, dominating the direction of change in allele frequencies, which can, in

turn, be counteracted by balancing and diversifying selection to increase diversity

again (Hartl and Clark, 1998).

Allele frequencies in a population can be influenced by multiple biotic and

abiotic factors. Clustering analyses can be implemented to illustrate the genetic

relationship between individuals, define the number of populations, and assign

isolates within these populations. This population structure indicates the evolu-

tionary history through alleles present in samples (McDonald and Linde, 2002).

To quantify the genetic diversity between individuals and populations a wide

range of molecular markers have been developed and deployed over the past 37

years (Schlötterer, 2004).

4.1.3 Molecular markers and Pst

Molecular markers improved the traceability of Pst considerably, enabling refine-

ment of dispersal distance approximations and population dynamics. Population

studies based on AFLP molecular markers (Vos et al., 1995) were first applied

(Hovmøller et al., 2002). A widely inclusive population study, analysing isolates

from North America, Australia, Europe, Western and Central Asia, the Red Sea

Area, East Africa and South Africa provided the first genotyping information

for the South African pathotypes (Hovmøller et al., 2008). These 876 Pst isolates,

collected over a period of 30 years between 1975 and 2005, were pathotyped

on a set of 30 wheat differential lines including at least 17 stripe rust resistance

genes. A subset containing 151 of the collected isolates, which represented the

diversity with respect to virulence phenotypes, region, and sampling year, were

then genotyped using AFLP molecular markers (Hovmøller et al., 2008), identify-
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 54

ing presence-absence polymorphisms. This subset contained South African Pst

isolates representative of the pathotypes 6E16A-, 6E22A-, and 7E22A- that were

sampled between 1996 and 2001. The subset was screened with 117 informative

AFLP makers, however, these markers did not show any differentiation between

the South African isolates. This analysis indicated that the South African Pst

isolates were closely related to isolates detected in Central (sampled in 2003)

and Western Asia (sampled in 2005), and Southern Europe (sampled in 1997 and

1998).

Differential testing showed that 6E16A- is similar to the pathotype 6E16, also

called PstS3 (Hovmøller et al., 2016), that was identified in Southern Europe since

1985 (Enjalbert et al., 2005). In the south of France, a stable divergent subpopu-

lation was described using AFLP markers, also comparable to an Italian isolate

sampled in 1998 (Enjalbert et al., 2005). Pathotypes similar to the South African

pathotypes (Figure 4.3) have also repeatedly been detected in Northern Europe

since 2004 (Hovmøller et al., 2008). Ali et al. (2014) concluded similar results using

20 microsatellite markers (Vieira et al., 2016), identifying the Mediterranean re-

gion and Central Asia as the probable origin of the South African Pst pathotypes.

In these two studies, seven and six South African isolates were used, respectively.

Pathotype 6E22A+, detected in South Africa in 2005, was not included in these

analyses. Similar to AFLP markers, microsatellite markers reported low levels

of genetic diversity in the South African population and could not differentiate

between pathotypes.

Since 1996 characterisation of the Pst population in South Africa has largely

been carried out through traditional pathotype analysis methods (see Figure 7.1).

More recently 17 microsatellite markers were used to genetically characterise the

South African Pst pathotypes (Visser et al., 2016), confirming previous findings

of low genetic variability between pathotypes (Hovmøller et al., 2008; Ali et al.,

2014). These markers were however able to distinguish between the South
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 55

African pathotypes. Through network analysis Visser et al. (2016) proposed

seven hypothetical intermediates between the four South African pathotypes,

indicating a model for the establishment of Pst in South Africa.

4.1.4 Next-generation sequence analyses of South African Pst

Along with the cost and time limitations in the development of traditional marker

systems such as microsatellites and AFLPs, genotyping samples with traditional

marker panels—even with a large marker selection—will only provide a low

resolution view of the genetic diversity between samples (Davey et al., 2011).

This can be especially problematic when aiming to distinguish between samples

with low genetic variability. Next-generation sequencing relieved this limitation

of traditional molecular markers by facilitating the limitless identification of

markers in a multitude of samples (Davey et al., 2011). As is the case with AFLP

markers, another advantage is that no prior knowledge of the target is needed

(Naccache et al., 2014). The extensive datasets generated from this technology

across species’ genomes, enable searches for diversity at nucleotide level that tra-

ditional marker systems will never generate. It allows addressing of population

structure questions with a level of detail and improved accuracy that ordinary

markers have not achieved.

To add to the traditional pathology and marker work carried out on the

South African Pst pathotypes whole genome sequencing of four Pst isolates was

undertaken. These isolates represent the major pathotypes following the first

confirmed incursion of stripe rust into South Africa in 1996. Data from the four

representative isolates of the identified South African pathotypes, together with

available data from global isolates, were used to (i) re-evaluate the potential

origin of the South African pathotypes using a comparative genomics approach

and to (ii) assess the genetic diversity within the South African population. The
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 56

South African pathotypes identified between 1996 and 2005 will be referred to as

the historical South African population. Specific isolates analysed in this study

that represent the identified pathotypes were named SA1—-SA4.

4.2 Materials and methods

Work done by co-workers is indicated in the relevant sections. The methodology

followed the field pathogenomics approach described in (Hubbard et al., 2015).

See Chapter 3 for detailed descriptions.

4.2.1 Data description

Four isolates representing the four pathotypes observed in South Africa to date

have been sequenced in this study. Hubbard et al. (2015) reported an in-depth

analysis of the UK population comparing several UK Pst isolates, collected

between 1974 and 2013. A subset of the data used by Hubbard et al. (2015) was

included in the present study to draw comparisons between the South African

isolates and other available Pst datasets.

The UK Pst population in 2013 showed high diversity and differed to the

pre-2011 population Hubbard et al. (2015). Population genetic analysis defined

this 2013 population into four distinct genetic groups. Notable features of these

four groups were that UK Group II was detected on triticale and UK Groups I

and II were genetically less diverse compered to Groups III and IV.

Sequence data of the South African historical isolates, together with sequence

data of 44 other isolates including 32 isolates from Europe (Table 4.1) that were

sequenced and described before (Hubbard et al., 2015), five isolates from Pakistan

(Bueno-Sancho et al., 2017) and seven isolates from East Africa, including three

isolates from Ethiopia, two from Kenya and two from Eritrea, were used in

this chapter to determine the relationship of the South African isolates with the
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 57

available data from other wheat-growing areas where stripe rust occurs. The East

African isolates were obtained from Mogens Hovmøller. The isolate ET03b/10,

that was assigned to the pathotype group PstS2, and ET08/10 were included

in previous analysis by Ali et al. (2017). Isolates KE74217, KE89069 (V23) and

ET87094 are part of the Stubbs collection and were described by Thach et al. (2015,

2016).

4.2.2 Sample preparation for DNA extraction

The urediniospores used for extraction of gDNA were purified and multiplied

at UFS, South Africa. The isolates that were sequenced were representative of

the identified pathotypes. Table 4.2 lists the UFS stocks collection identities and

the collection date of the Pst isolates that were used for multiplication of the

urediniospore samples that were sequenced.

To obtain single pustule isolates for genome sequencing, seeds of the suscep-

tible wheat variety, Morocco, were planted and grown for seven days to the two

leaf stage (Z12; Zadoks et al., 1974). Urediniospores of the four pathotypes were

previously dried on silica gel and kept at −80 ◦C in storage. Inoculations were

performed where after plants were moved to a glasshouse with natural light and

a day—night temperature cycle set to 20 ◦C (06:00-18:00) and 15 ◦C (18:00-06:00),

respectively. When flecks appeared, all plants were cut away to leave only half a

leaf with a single infection site, the result of infection by a single spore. Due to

the systemic nature of the infection, the entire leaf segment eventually sporulated

from the single infection site. For each isolate urediniospores were collected

from one actively sporulating lesion and increased twice on Morocco seedlings

to produce several grams of spores. The final spore harvest was desiccated for

five days on silica gel and used to extract the DNA for sequencing. To maintain

isolate purity, multiplication of the different isolates were spatially or temporally

separated in the glasshouse.

CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 58

Table 4.1: Global isolates included in the clustering and genetic diversity analyses

Isolate Country of Year of Type of

Isolates References
number isolation isolation data

1 88.55S1 UK Pre 2011 gDNA Hubbard et al. (2015)

2 03/7 UK Pre 2011 gDNA Hubbard et al. (2015)
3 08/21 UK Pre 2011 gDNA Hubbard et al. (2015)
4 88.45SS UK Pre 2011 gDNA Hubbard et al. (2015)
5 78.66SS1 UK Pre 2011 gDNA Hubbard et al. (2015)
6 88.44SS3 UK Pre 2011 gDNA Hubbard et al. (2015)
7 J0085F France Pre 2011 gDNA Hubbard et al. (2015)
8 J01144Bm1 France Pre 2011 gDNA Hubbard et al. (2015)
9 J02-022 France Pre 2011 gDNA Hubbard et al. (2015)
10 J02055C France Pre 2011 gDNA Hubbard et al. (2015)
11 11/13 UK 2011 gDNA Hubbard et al. (2015)
12 11/75 UK 2011 gDNA DGO Saunders & S Holdgate
13 11/128 UK 2011 gDNA Hubbard et al. (2015)
14 11/140 UK 2011 gDNA Hubbard et al. (2015)
15 11/08 UK 2011 gDNA Hubbard et al. (2015)
16 11/08 UK 2011 RNA-Seq Hubbard et al. (2015)
17 13/19 UK 2013 RNA-Seq Hubbard et al. (2015)
18 13/15 UK 2013 RNA-Seq Hubbard et al. (2015)
19 13/123 UK 2013 RNA-Seq Hubbard et al. (2015)
20 13/27 UK 2013 RNA-Seq Hubbard et al. (2015)
21 CL1 UK 2013 RNA-Seq Hubbard et al. (2015)
22 T13/2 UK 2013 RNA-Seq Hubbard et al. (2015)
23 T13/3 UK 2013 RNA-Seq Hubbard et al. (2015)
24 T13/1 UK 2013 RNA-Seq Hubbard et al. (2015)
25 13/38 UK 2013 RNA-Seq Hubbard et al. (2015)
26 13/21 UK 2013 RNA-Seq Hubbard et al. (2015)
27 13/33 UK 2013 RNA-Seq Hubbard et al. (2015)
28 13/182 UK 2013 RNA-Seq Hubbard et al. (2015)
29 13/25 UK 2013 RNA-Seq Hubbard et al. (2015)
30 13/29 UK 2013 RNA-Seq Hubbard et al. (2015)
31 13/71 UK 2013 RNA-Seq Hubbard et al. (2015)
32 13/40 UK 2013 RNA-Seq Hubbard et al. (2015)
33 SA1 SA 1996 gDNA Pretorius et al. (1997)
34 SA2 SA 1998 gDNA Boshoff and Pretorius, (1999)
35 SA3 SA 2001 gDNA Pretorius et al. (2007)
36 SA4 SA 2005 gDNA Pretorius, (Unpublished)
37 KE74217 Kenya 1974 gDNA Thach et al. (2015; 2016)*
38 KE89069 Kenya 1989 gDNA Thach et al. (2015; 2016)*
39 ET87094 Ethiopia 1987 gDNA Thach et al. (2015; 2016)*
40 ET08/10 Ethiopia 2010 gDNA Ali et al. (2017)**
41 ET03b/10 Ethiopia 2010 gDNA Ali et al. (2017)**
42 ER179b/11 Eritrea 2011 gDNA Ali et al. (2017)**
43 ER181a/11 Eritrea 2011 gDNA Ali et al. (2017)**
44 Qld-1 Pakistan 2014 gDNA Bueno-Sancho et al. (2017)
45 Qld-2 Pakistan 2014 gDNA Bueno-Sancho et al. (2017)
46 ATR-1 Pakistan 2014 gDNA Bueno-Sancho et al. (2017)
47 ATR-2 Pakistan 2014 gDNA Bueno-Sancho et al. (2017)
48 ATR-3 Pakistan 2014 gDNA Bueno-Sancho et al. (2017)
*Isolates KE74217, KE89069, and ET87094 were provided by Aarhus University, Denmark, and
Plant Research International, Wageningen, The Netherlands, maintaining the Global Yellow Rust
Gene Bank of the late ir. RW Stubbs up to 25-01-2010. ** Provided by MS Hovmøller. Personal
communication with MS Hovmøller confirmed inclusion in the listed studies.
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 59

Table 4.2: Historical isolates used in re-sequencing and an infection time course experi-
ment (Chapter 6)

Pathotype First occurrence Alias Isolate ID Collection date

6E16A- 1996 SA1 Isolate 49 2003

6E22A- 1998 SA2 Isolate 3 2001
7E22A- 2001 SA3 Isolate 27 2004
6E22A+ 2005 SA4 Isolate 35 2011

4.2.3 Genomic DNA extraction and quantification

Genomic DNA was extracted from urediniospores using the CTAB extraction

method described by Chen et al. (1993) and quantified using the Qubit 2.0 Fluo-

rometer (Invitrogen/Thermo Fisher Scientific, USA).

4.2.4 Sequencing and mapping

Sequencing libraries were prepared, quality assessed, quantified and sequenced

by the Earlham Institute. Sequences containing missing data indicated with

“N” were discarded (Cantu et al., 2013; Hubbard et al., 2015). The 100 bp paired

end reads were aligned to the PST130 draft reference genome (Cantu et al.,

2011) using BWA (version 0.7.7; Li and Durbin, 2009) with default parameters

producing sequence alignment map (SAM) format files. SAMtools (version

0.1.19; Li et al., 2009) was used, to identify variant sites. SnpEff (version 3.6;

Cingolani et al., 2012) was used to identify whether homokaryotic SNPs resulted

in synonymous or nonsynonymous substitutions similar to the procedures in

Cantu et al. (2013). Based on the rationale explained in Yoshida et al. (2013),

the read frequency graph of each isolate was assessed to determine whether the

starting material could be considered uncontaminated containing predominantly

a single genotype (Cantu et al., 2013; Hubbard et al., 2015). Read frequency

graphs of other isolates used in this chapter that have not been published before

are displayed in Appendix A, Figure A.1.

CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 60

4.2.5 Phylogenetic analysis

A maximum likelihood phylogenetic approach was used to determine the genetic

relationships amongst the South African Pst isolates and to compare them with

isolates from elsewhere. Synthetic genes were prepared, and the third codon

positions of these genes were used to determine the phylogeny. Due to the

degeneracy of the genetic code, this will include mostly nucleotide changes

that do not result in amino acid changes resulting in more evolutionary neutral

positions. The RAxML software (version 8.0.20; Stamatakis, 2014) was used. One

hundred iterations of bootstrapping were performed to assess the reliability of

the maximum likelihood dendrograms (Cantu et al., 2013; Hubbard et al., 2015).

4.2.6 Population structure analysis

The genetic differentiation of the 48 isolates (Table 4.1) was assessed by two

population-clustering methods: (i) STRUCTURE (version 2.3.4; Pritchard et al.,

2000) was used to assign isolates to subpopulation clusters (K) based on genetic

differentiation at nearly neutral or neutral SNP sites, and (ii) Multivariate DAPC

within the Adegenet package (Jombart et al., 2010) was carried out in the R

environment on the same dataset as STRUCTURE.

4.2.7 Genetic diversity assessment

Inter-cluster variance

The SNP dataset used in STRUCTURE and DAPC analyses containing only bial-

lelic synonymous SNPs was converted to the applicable format for the program

Genepop (version 4.2.2; Rousset, 2008) using a Perl script. The dataset was split

into population clusters as differentiated by DAPC. The between population

differentiation was then determined by calculating the special case of Wright’s

CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 61

F-statistic (FST ) to describe the repartition of allelic frequency between subpopu-

lations.

Intra-cluster variance

Synthetic genes, containing SNP sites and sites identical to the reference that

passed the respective coverage thresholds, were used to quantify the genetic

diversity in subpopulations that were determined by clustering analysis. The

program DnaSP (version 5.10.1; Librado and Rozas, 2009) was used to compare

loci between individuals within each cluster. The average and standard deviation

of the Watterson theta estimate (θ̂W ) across all sites were calculated to obtain

the genetic diversity estimate within each cluster. A characteristic of DnaSP is

that it cannot differentiate between intra-individual (between haplotypes) and

inter-individual diversity (between isolates). It means that when the diversity of

a population is computed, it actually considers the haplotype diversity. Every

haplotype is considered as one “isolate”. Generally speaking, Pst contains two

haplotypes, therefore one can compute the diversity with only one isolate. This

was not the main focus of this analysis but was conducted on the isolate that was

on its own in a genetic group. Haplotype diversity in Pst is generally considered

to be high and was confirmed by the phased haplotype sequencing effort of

Schwessinger et al. (2018).

4.3 Results

4.3.1 Re-sequencing of South African Pst pathotypes

To investigate variation in the South African population, whole genome, next-

generation sequencing of four historical South African isolates (SA1–SA4) was

performed. More than 20 million reads were generated for each isolate using the
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 62

Illumina HiSeq2500 platform (Table 4.3). Reads were filtered and subsequently

mapped to the PST130 reference genome (Cantu et al., 2011). The average genome

depth of coverage across the PST130 genome for SA1–SA4 was between 25 and

39× (Table 4.3). All four alignments spanned 97 % of the breadth of the reference

genome with at least 2× coverage depth.

4.3.2 Purity assessment of samples

To assess whether the urediniospores used as starting material consisted of a

single genotype, allele frequencies for each of the historical South African isolates

were analysed. The resulting plots displayed clear peaks at 0.5 (Figure 4.5) and a

fairly bell-shaped distribution. Although a pattern such as seen in SA4 is more

desirable, SA1–SA3 still followed the expected trend that supports that samples

consisted predominantly of a single genotype.

4.3.3 Clustering analyses

Three methods of data clustering were implemented to infer population structure.

First, a maximum likelihood RAxML phylogenetic tree was generated, using the

third codon position of the synthetic genes. Next, STRUCTURE and DAPC were

used to assign isolates to population clusters.

4.3.4 Phylogenetic analysis

To determine the relationship of the historical South African Pst isolates to avail-

able isolates from the UK, France, Pakistan, Eritrea, Ethiopia and Kenya, phylo-

genetic analyses using available genomic and transcriptomic data from 48 Pst

isolates (Table 4.1) were carried out. To characterise the genetic relationship

between these isolates, a maximum likelihood approach was used. The third

codon position across 5844 predicted genes, including 2 437 462 sites, were used
 Table 4.3: Statistics of read alignment of the historical South African isolates to the PST130 reference genome. An average of 85.2 ± 4.0 % of
filtered reads mapped to the reference genome

Lab Total number Filtered Percent Number of Unmapped Average depth

Platform Pathotype
code of reads reads discarded reads aligned reads of coverage

SA1 Illumina Hi-Seq 6E16A- 23 031 402 22 827 102 0.89 % 20 131 984 2 695 118 30
SA2 Illumina Hi-Seq 6E22A- 22 628 648 22 433 194 0.86 % 16 490 301 5 942 893 25
SA3 Illumina Hi-Seq 7E22A- 26 876 262 26 637 762 0.89 % 23 960 896 2 676 866 36
SA4 Illumina Hi-Seq 6E22A+ 30 300 476 30 056 556 0.81 % 26 751 160 3 305 396 41
63

SA1 SA2 SA3 SA4

60000 60000 60000 60000

50000 50000 50000 50000

40000 40000 40000 40000

Count

Count

Count

Count
30000 30000 30000 30000

20000 20000 20000 20000

10000 10000 10000 10000

0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00
Frequency Frequency Frequency Frequency

Figure 4.5: Read frequency graphs from heterokaryotic SNP sites for SA1–SA4.
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 64

to generate the phylogenetic tree (Figure 4.6), including those sites in genes that

had 80 % breadth of coverage in 80 % of the isolates.

From the phylogenetic tree (Figure 4.6), it can be concluded that the South

African isolates a) are closely related to one another, and b) are most closely

related to isolates from Kenya and Ethiopia. This is indicative of either (i) south-

ward movement of inocula, with the South African pathotypes being derived

from East African isolates, or (ii) that the South African and the identified East

African isolates may share a common origin.

4.3.5 Population structure analysis

STRUCTURE

To assign individual Pst isolates to population groups the Bayesian model based

clustering method STRUCTURE (Pritchard et al., 2000) was applied to the 146 400

biallelic synonymous SNP sites that were identified across the 48 isolates.

The log probability plot in Figure 4.7(i) confirmed the optimum number of

population clusters as 4, with the graph reaching a plateau parallel to the x-axis

for 4 or more population clusters (Pritchard et al., 2000). The number of popula-

tion clusters was also evaluated using the Evanno method of population cluster

analysis (Evanno et al., 2005). This method, based on the second order derivation

of the maximum likelihood estimation of the model given a specific K, suggested

the population number K = 2 (Figure 4.7(ii)). From these two estimates of K,

STRUCTURE suggests the number of population clusters is either K = 2 or

K = 4. Figure 4.8 displays bar charts representing STRUCTURE population clus-

ters. To further assess population structure, STRUCTURE results were compared

to DAPC clustering that does not assume Hardy-Weinberg equilibrium.

 88.5SS1
88.45SS UK (pre-2011—WGS) Pakistan (2010—WGS) South Africa (WGS)
08/21
03/7 France (pre-2011—WGS) Kenya (Old—WGS) Bootstrap values > 80
11/140
J0085F UK & France (Pre-2011)
88.44SS3
UK (2011—WGS) Ethiopia (Old—WGS) Race: Warrior
78.6SS1
j02-022 UK (2011—RNA-Seq) Ethiopia (2010—WGS) Race: PstS2
J01144Bm1
J02055C UK (2013—RNA-Seq) Eritrea (2011—WGS)
11/128
UK (2013 - Group III)
13/33
13/21
13/182
T13/3
UK (2013 - Group II)
T13/1
CL1
T13/2
13/38 UK (2013 - Partially assigned to Group III : blue and Group IV: red)
13/40
13/27
13/19 UK (2013 - Cluster I)
13/15
13/123
11/08
11/08
13/29
13/25 UK (2013 - Group IV)
13/71
11/13
65

ATR-1
Qld-1
Qld-2 Pakistan (2010) East Africa (B) — (2001 to 2011)
ATR-2
ATR-3
×3 ET08/10
// ER179b/11
ER181a/11
SA3
SA4 South Africa — (2001 to 2011)
SA1
SA2
ET03b/10
KE89069 East Africa (A) — (1974 to 2010)
KE74217
ET87094 0.0007

Figure 4.6: The phylogenetic relationship between the South African Pst isolates and European, Asian and East African isolates. South
African Pst isolates are closely related to isolates from East Africa. RAxML non-routed phylogenetic analysis were performed
assessing four South African and 44 global Pst isolates using the third codon position of 5844 PST130 gene models. Only those
genes that had 80 % coverage in 80 % of the isolates were included, resulting in the inclusion of 2 437 462 sites to construct the
tree. Clades are supported by evaluation of 100 bootstrap iterations. Bootstrap values of greater than 80 are indicated with green
dots on applicable nodes.
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 66

● ● ● ● ● ● ● ● ● ● ● ●
●

−3200000
LnP(D)

−3600000

−4000000
●

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
K

(i) Log probability of data L(K ) as a function of K to identify the optimal

amount of clusters. The population structure of Pst inferred by
model based Bayesian cluster analysis of genome-wide SNP data
indicate the optimum number of clusters K = 4.

800 ●

600
Delta K

400
●
200 ●
●

0 ● ● ● ● ● ● ● ● ● ●

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
K

(ii) The Evanno method of inferring the number of STRUCTURE pop-

ulations (K) from the modal value of ∆K. A strong signal was
detected for K = 2 with where ∆K was at a maximum. ∆, Delta.

Figure 4.7: Evaluation of the number of population clusters following STRUCTURE

analyses.
 K2

K3

K4

K5

K6

K7

K8

K9

K10

K11

K12

67

K13

K14

K15

J01144Bm1

J02055C

J0085F

T13/2

T13/3

T13/1

j02-022

11/140

08/21

03/7

11/128

11/75

11/13

PK5

PK3

PK4

PK1

PK2

CL1

13/71

13/29

13/25

13/40

13/38

13/182

13/21

13/33

13/27

13/19

13/15

13/123

11/08

WYR 88.5SS1

WYR 78.6SS1

WYR88.45SS

11/08*

SA1

SA2

SA3

SA4

KE89069

KE74217

ET87094

ET03b/10

ET08/10

ER179b/11

ER181a/11

WYR88.44SS3

II IV III I
Old French
Old UK
UK
Pakistan
UK
South Africa
Kenya
Ethiopia
Eritrea

1996

1998

2001

2005

1989

1974

1987

2010

2010

2011

2011

Pre 2011
2011
2014
2013
2011

Figure 4.8: Bar charts representing STRUCTURE population clusters, with colour representing a group and each bar indicating the fraction
of sites assigned to a specific group representing estimated membership fractions for each individual isolate. The UK 2013
population is divided in subgroups: green (UK Cluster II), red (UK Cluster IV) blue (UK Cluster III) and pink (UK Cluster I)
as previously described by Cantu et al. (2013). Asterisk (*) indicates genomic data of isolate 11/08, while no asterisk indicates
RNA-Seq data for 11/08. K = 4 was proposed as the optimal population number (see Figure 4.7(i)). K2 to K15 indicate the
number of clusters individuals in the population were assigned to in each cluster number evaluation.
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 68

Discriminant analysis of principal components

The same 146 400 synonymous biallelic SNP sites were used as input for the

analysis. Genetic variation within and between population clusters was then

summarised using PCA. The elbow of the Bayesian Information Criterion (BIC)

curve formed at 6 and a minimum was observed at 10 (Figure 4.9(i)), indicating

the optimum number of clusters ranged between 6 and 10. Discriminant analysis

(DA) of eigenvalues was performed to assign individuals to population clusters.

The bar-plot in Figure 4.9(ii) represents the DA of eigenvalues for the main

principal components. The scatterplot (Figure 4.9(iii)) uses the first two principal

components (the y-axis and x-axis, respectively) of the DAPC of the synonymous

SNP sites. Each circle represents a single Pst isolate.

The non-parametric DAPC of the Pst isolates identified at most ten clusters

(K = 10), as supported by the BIC curve (Figure 4.9(i)). Some similarities between

the STRUCTURE groups and the DAPC groups can be seen (Figure 4.8 and

Figure 4.10). The elbow of the BIC curve suggests six populations (Figure 4.9(ii))

(Jombart et al., 2010). The bar charts corresponding to K = 6 has similarity

to the STRUCTURE bar chart for K = 4. Differences between STRUCTURE

and DAPC included that UK Cluster I was the fifth cluster to differentiate in

DAPC analysis, while the post 2011 UK clusters did not show clear differentiation

in the STRUCTURE analysis. Pakistan isolates differentiated at K = 4 in the

STRUCTURE analysis and only differentiated at K = 7 in the DAPC analysis. Due

to Pst predominantly reproducing asexually, specifically in regions where isolates

were obtained from, DAPC is more suitable for the specific dataset. Subsequent

analyses were based on DAPC results.

CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 69

Value of BIC Discriminant analysis eigenvalues

versus number of clusters

7000
450

6000
5000
440

F-statistic
4000
BIC
430

3000
2000
420

1000
410

0
5 10 15
Number of clusters Linear Discriminants

(i) Bayesian information criterion (ii) Discriminant analysis (DA) of

(BIC) curve. eigenvalues.

Cluster 4
Pakistan (2014)

Cluster 8
UK Cluster I

Cluster 1,2,3
UK & French
(Pre-2011 & 2011)
Cluster 5
UK Cluster I

Cluster 6
Cluster 9 UK Cluster III & IV
Cluster 10 East Africa &
East Africa South Africa
Cluster 5,6,7
(including PstS2) (UK 2013)

Cluster 7
UK Cluster I

Cluster 1 Cluster 6
Cluster 2 Cluster 7
Cluster 3 Cluster 8
Cluster 4 Cluster 9
Cluster 5 Cluster 10

(iii) Relative proximity of Pst population clusters.

Figure 4.9: Discriminant analysis of principal component (DAPC) analysis of 48 Pst iso-
lates. (i) Bayesian Information Criterion (BIC) curve suggesting the minimum
number of clusters (K) required to explain variation between pathotype clus-
ters to be between 6 and 10. The first nine eigenvalues components from
the DAPC analysis (ii), supported the maintenance of three discriminant
functions in the DAPC analysis indicated with red bars. (iii) DAPC for 48 Pst
isolates.
 K2!
K3!
K4!
K5!
K6!
K7!
K8!
K9!
K10!
K11!
K12!
70

K13!
K14!
K15!
J01144Bm1!

J02055C!
J0085F!

T13/2!
T13/3!
T13/1!
j02-022!

WYR 88.5SS1!
WYR 78.6SS1!
WYR88.45SS!
WYR88.44SS3!
11/140!
08/21!
03/7!
11/128!
11/75!
11/13!
PK5!
PK3!
PK4!
PK1!
PK2!

CL1!
13/71!
13/29!
13/25!
13/40!
13/38!
13/182!
13/21!
13/33!
13/27!
13/19!
13/15!
13/123!
11/08!
11/08*!
SA1!
SA2!
SA3!
SA4!
KE89069!
KE74217!
ET87094!
ET03b/10!
ET08/10!
ER179b/11!
ER181a/11!
Old French! Old UK! UK! Pakistan! UK! South Africa! Kenya! Ethiopia! Eritrea!

1996!
1998!
2001!
2005!
1989!
1974!
1987!
2010!
2010!
2011!
2011!
Pre 2011! 2011! 2014! 2013! 2011!

Figure 4.10: Bar charts represent DAPC population structure analysis, with each bar estimating the proportion ascription of each isolate to a
population cluster. UK clusters are indicated similar to Figure 4.8. Asterisk (*) indicates genomic data of isolate 11/08, while
no asterisk indicates RNA-Seq data for 11/08. K2 to K15 indicate the number of clusters individuals in the population were
assigned to in each cluster number evaluation.
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 71

4.3.6 Population differentiation

FST values were calculated using the software Genepop (version 4.2.2; Rousset,

2008) to show genetic differentiation between clusters. Pairwise comparisons

of biallelic SNP data were assessed for each group comparison. This analysis

quantifies the correlation of alleles within a subpopulation comparing to all

subpopulations. Some clusters were very similar and others more divergent with

FST values ranging between 0.08 and 0.86 across the 10 Pst clusters (Figure 4.11).

The biggest genetic differentiation (0.37 to 0.86) was seen when Group 10—East

Africa (B)—was compared to other groups. Comparison between Group 9 and

Group 7 showed the highest genetic differentiation involving the South African

isolates. Group 7 comprised of the UK Cluster I isolates.

In addition to calculating the FST values for the population groups as de-

fined by DAPC, this diversity statistic was calculated among the historical South

African isolates and isolates from East Africa that were co-arranged by the phy-

logenetic tree (Figure 4.6) and the clustering analysis (Figure 4.10). The high

similarity between these two groups (Group A: SA1-SA4 and Group B: KE89069,

KE74217, ET87094 and ET03b/10) was quantified by a very low FST of 0.08. In

contrast, a second group of East African isolates containing two isolates from

Eritrea and one Ethiopian isolate, was generally the most genetically diverse from

all other Pst isolates maintaining high FST values throughout all comparisons.

This genetic difference was also reflected by their position in the distantly related

clade in the phylogenetic analysis (East Africa (B); Figure 4.6).

4.3.7 Genetic diversity within and between population clusters

To estimate the genetic variation within the subpopulations the Watterson esti-

mator was used as described in Chapter 3. The Watterson estimator incorporates

the number of SNPs and the population size of each population cluster. The
 Group! 1! 2! 3! 4! 5! 6! 7! 8! 9! 10!

0.0031 #
1! "! "! "! "! "! "! "! "! "!
0.0041!
0.0003 #
2! 0.08! "! "! "! "! "! "! "! "!
0.0013!
0.0022 #
3! 0.18! 0.20! "! "! "! "! "! "! "!
0.0035!
0.0012 #
4! 0.32! 0.39! 0.16! "! "! "! "! "! "!
0.0021!
0.0006 #
5! 0.41! 0.61! 0.36! 0.33! "! "! "! "! "!
0.001!
0.0005 #
6! 0.39! 0.52! 0.23! 0.31! 0.21! "! "! "! "!
0.0008!
0.0002 #
7! 0.47! 0.74! 0.48! 0.46! 0.53! 0.38! "! "! "!
0.0009!
0.0042 #
8! 0.38! 0.59! 0.40! 0.45! 0.60! 0.49! 0.32! "! "!
0.0092!
72

0.002 #
9! 0.21! 0.27! 0.23! 0.26! 0.29! 0.39! 0.43! 0.35! "!
0.003!
0.0031 #
10! 0.39! 0.49! 0.57! 0.59! 0.78! 0.78! 0.86! 0.71! 0.37!
0.0055!

GROUPS! 1! 1! 1! 2! 2! 1! 1! 1! 1! 1! 1! 3! 3! 3! 4! 4! 4! 4! 4! 5! 5! 5! 5! 6! 6! 6! 6! 6! 6! 6! 6! 6! 7! 7! 7! 7! 8! 9! 9! 9! 9! 9! 9! 9! 9! 10! 10! 10!

WYR88.44SS3!
WYR 88.5SS1!
WYR 78.6SS1!
WYR88.45SS!
J01144Bm1!

ER179b/11!
ER181a/11!
ET03b/10!
KE89069!
KE74217!
ET87094!
J02055C!

ET08/10!
j02-022!

11/140!

11/128!

13/182!

13/123!
J0085F!

11/08*!
08/21!

11/75!
11/13!

13/71!
13/29!
13/25!
13/40!
13/38!

13/21!
13/33!
13/27!
13/19!
13/15!

11/08!
T13/2!
T13/3!
T13/1!
03/7!

SA1!
SA2!
SA3!
SA4!
PK5!
PK3!
PK4!
PK1!
PK2!

CL1!
ISOLATES!
ORIGIN! Old French! Old UK! UK! Pakistan! UK! South Africa! Kenya! Ethiopia! Eritrea!
COLLECTED! Pre 2011! 2011! 2014! 2013! 2011!

1996!
1998!
2001!
2005!
1989!
1974!
1987!
2010!
2010!
2011!
2011!
Figure 4.11: Genetic diversity assessed between 10 population clusters derived from DAPC analysis of biallelic SNP data. FST values are
indicated in the lower diagonal matrix, with the diversity in the groups indicated on the diagonal. Group 8 contains one isolate
indicating haplotype diversity in this isolate on the diagonal. Isolate information is displayed in the key. The East African
isolates in group 9 (purple) are referred to as East Africa I, while group 10 (red) is referred to as East Africa II in the text.
Asterisk (*) indicates genomic data of isolate 11/08, while no asterisk indicates RNA-Seq data for 11/08.
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 73

degree of polymorphism in the gene set of each subpopulation was calculated

by evaluating SNPs across isolates in a population cluster, gene-by-gene. Thetas

of different clusters, as shown on the diagonal of the matrix in Figure 4.11, can

subsequently be compared to assess the relative nucleotide diversity in the dif-

ferent clusters. This metric of Group 8 was calculated on a single isolate and

indicates haplotype diversity of this isolate. The highest intra-cluster variability

was computed for Groups 1 and 10 and the lowest for Group 7.

4.4 Discussion

To test prevalence and identify new pathotypes of Pst, surveys are routinely car-

ried out in South Africa by seasonal phenotyping of rust isolates on a differential

set of wheat lines that possess an array of rust resistance genes. Pathotype names,

such as 6E16A-, are based on such traditional pathology screens on differential

sets. In addition to the pathotype description pathologists often report the viru-

lence profile of specific isolates that show virulence to additional resistance genes

not represented in the differential set. These descriptions are complementary, but

not necessarily identical across all isolates of a specific pathotype. For example,

Ethiopian wheat varieties resistant to Pst isolates of pathotype 6E16A- and 6E22A-

from South Africa were susceptible to a 6E22 isolate from Germany (Hussein and

Pretorius, 2005; Denbel, 2014). Also, different isolates of the 0E0 Pst pathotype,

showing avirulence to all wheat genotypes with known Yr genes, were suggested

to be genetically different using microsatellite marker screens (Hovmøller et al.,

2016).

In addition to these phenotypic markers, genotyping, using molecular mark-

ers has aided in a more detailed description of Pst isolates. For instance, South

African pathotypes have been genotyped using AFLP markers (Hovmøller et al.,

2008) and phylogenetic analysis using these markers indicated that the South
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 74

African isolates were related to isolates from Western and Central Asia and South-

ern Europe. However, the seven isolates, belonging to the pathotype groups

6E16A-, 6E22A-, and 7E22A-, collected between 1996 and 2001 could not be differ-

entiated using AFLP markers. A subsequent study using microsatellite markers

that genotyped South African isolates collected between 1996 and 2004 also in-

dicated a close relationship with Central Asian and Mediterranean Pst isolates

(Ali et al., 2014). Only a single genotype was recorded for the six South African

samples tested. More recently the pathology characterisation of the virulence

profiles of the South African isolates has been complemented with genotype in-

formation from microsatellite markers. The diversity in these molecular markers

successfully distinguished the South African isolates (Visser et al., 2016). The

close relationship of the South African pathotypes and the stepwise development

of new pathotypes were confirmed through this analysis.

Further to this work, the current study implemented a next-generation sequen-

cing approach to determine the possible origin and characterise the genetic

relatedness of the four historical South African Pst pathotypes identified in 1996,

1998, 2001 and 2005, through investigation of isolates SA1–SA4. First, population

substructure was assessed based on allele frequencies at multiple loci of neutral

or nearly neutral alleles. After that, the FST was calculated to quantify genetic

variation between the predefined population clusters (Pritchard et al., 2000) and

the diversity amongst isolates in a group was assessed. Knowledge of population

structure is valuable in the study of emerging and re-emerging pathogens as it

reports the dynamics of subpopulations with distinct pathogenicity (Hubbard

et al., 2015). In this study, the Bayesian clustering method STRUCTURE (Pritchard

et al., 2000) and multivariate DAPC (Jombart et al., 2010) were used to identify

genetic clusters.

It is often hard to meet the assumptions analysis methods rely on. STRUC-

TURE is one of the most popular methods to infer population structure. It was
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 75

developed to be applied to various markers that are not closely linked, and

assumes Hardy-Weinberg equilibrium (Pritchard et al., 2000). The high marker

density obtained from re-sequencing data, together with the asexual reproduc-

tion of Pst, resulted in violation of this prerequisite, making STRUCTURE less

appropriate for analysing clonal populations. An additional shortcoming of

STRUCTURE is that the complex models include many parameters to estimate,

causing lengthy runtimes when assessing large data sets (Jombart et al., 2010),

as is the case with sequence data. In contrast, DAPC first transforms the data

using PCA to prepare the input variables to the DA to be uncorrelated principle

components. The DA then predicts a grouping variable using one or more of

the principle components. This approach is time efficient, and can easily be

applied to large re-sequencing datasets. In DAPC, like in STRUCTURE, K-means

clustering is run with different numbers of clusters (K). The clustering models

resulting from each chosen K can be assessed by their likelihood. DAPC uses

BIC to determine the model that fits the data best and by implication the number

of clusters (Jombart et al., 2010). After assessment of population structure, the

genetic differentiation between and within proposed clusters can be calculated to

quantify the diversity between and within groups.

In the pairwise comparisons of clusters, lower FST values indicate groups that

are closely related, while groups distant from each other have high FST values.

Phylogenetic and clustering analysis illustrated that from the isolates evaluated

in this study, the historical South African isolates were most closely related to

isolates from East Africa (A), also confirmed by the low FST of 0.08. Higher

genetic differentiation between East African and South African isolates (FST =

0.23) was previously reported using microsatellite markers (Ali et al., 2014). In the

present study, high differentiation was observed between East African isolates,

with an FST of 0.37 observed between Group 9 (containing East Africa (A)) and

group 10 (East Africa (B)). Group 9 and Group 10 included isolates from Ethiopia
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 76

sampled in 2010. This indicates high diversity in the Pst population in Ethiopia

and that the South African isolates is closely related to some of the East African

isolates, but different to others.

Diversity calculations amongst isolates assigned to groups using DAPC in-

dicated that groups 2, 5, 6 and 7 were less diverse by one order of magnitude

compared to groups 1, 3, 4, 9 and 10. Group 8 consists of a single isolate and the

diversity calculation represent the haplotype diversity for this isolate. This high

haplotype diversity is a characteristic of Pst. Schwessinger et al. (2018) describe

the haplotype diversity measured in Pst-104E higher than a number of plant

pathogens, including Puccinia coronata Corda f. sp. avenae, Zymoseptoria tritici

(Desm.) Quaedvl. & Crous and Verticillium dahliae Kleb., and associates this

diversity with long-term asexual reproduction.

One isolate in Group 10, ET08/10, has previously been assigned to the patho-

type PstS2 (Ali et al., 2017). This aggressive pathotype possibly originated in

East Africa and quickly spread to the Middle East, Australia, and Europe. In

aggressive pathotypes like PstS2 generation time is shortened and it is able to

infect in spite of relatively warm and dry climates (Hovmøller et al., 2008; Walter

et al., 2016).

From this analysis, it was concluded that the closest relatives of the South

African isolates were a group of isolates from East Africa. As the East African

isolates included historical isolates that date back to the 1970s and 1980s, this

result supports the hypothesis that inoculum could have moved southwards

from East Africa with subsequent introduction to South Africa. The East African

isolates showing high similarity to the South African isolates also included a

more recent isolate from 2010, indicating that the historical pathotypes are likely

still occurring in Ethiopia. Alongside these pathotypes, new pathotypes have

clearly developed, as reported for the aggressive pathotypes PstS1 and PstS2,

for example. Group 10 included two isolates from Eritrea that was sampled
CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 77

in 2011 and the PstS2 2010 isolate from Ethiopia. The historical South African

isolates showed significant differentiation from this group. Previous studies

that speculated about the origin of South African Pst excluded East Africa as a

possible origin based upon the diversity observed between South African and

Eritrean isolates (Hovmøller et al., 2008; Ali et al., 2014). These studies did not

include isolates from Ethiopia.

Apart from considering an incursion from East Africa, the South African

and some East African isolates could also share a similar origin. To assess their

relationship with isolates from Central and Western Asia and the Mediterranean,

suggested to be the origin of the South African isolates (Hovmøller et al., 2008;

Ali et al., 2014), the same resolution of variation assessment would be needed for

historical isolates from these regions. Currently, molecular marker work suggests

East African isolates to have originated from the Middle East (Ali et al., 2014) and

isolates sampled from this region, at different time points in the past, should also

be considered to unravel possible origins.

From this study, it cannot be confirmed that the South African isolate SA1 is

closely related to the 6E16 pathotype found in Southern and Northern Europe

(Enjalbert et al., 2005; Hovmøller et al., 2008). Although samples from the same

regions and possibly the same time frame were considered, the samples did not

overlap between the current study and the work of Enjalbert et al. (2005) and

Hovmøller et al. (2008).

4.5 Conclusion

Based on genomic analysis, this study confirms the association between the

South African and East African Pst populations previously proposed through

pathotype analysis (Pretorius et al., 1997; Boshoff et al., 2002; Pretorius et al.,

2007). In future, similar next-generation sequencing analysis of Central and

CHAPTER 4: THE ORIGIN OF SOUTH AFRICAN PST 78

Western Asian, Mediterranean and Middle Eastern isolates would fill in the

missing information to be able to draw parallels between the traditional marker

work and the next-generation sequencing data analysis included in this work.

From the samples analysed in this work, it was demonstrated that the South

African isolates are closely related to one another, which supports the findings

of the microsatellite marker work of Visser et al. (2016) that stepwise evolution

is likely responsible for the consecutive pathotypes. This hypothesis is further

assessed in Chapter 5 when polymorphisms in the South African isolates will

be analysed in search of the evolutionary changes that gave raise to subsequent

pathotypes of Pst in South Africa.

Chapter 5

Analyses of Polymorphisms in
Historical South African Pst
Isolates in Search of Candidate
Effector Genes

M ANY FILAMENTOUS PLANT PATHOGENS, such as Pst, use effector proteins to

manipulate their hosts (Kamoun, 2007). These proteins also put the pathogen

at risk of being recognised by the host via the resistance (R) proteins leading

to an incompatible interaction (Rovenich et al., 2014). A change in amino acid

sequences could lead to the host defence mechanisms not being able to recog-

nise the pathogen. This inability results in a compatible interaction where the

pathogen is virulent on host genotypes that were previously able to detect the at-

tack and restrict or stop infection. In this study, Pst isolates collected from a wide

geographical area were assessed using different clustering analysis methods to

assign isolates to population clusters (discussed in Chapter 4). It was concluded

that the historical South African isolates that were collected between 2001 and

79
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 80

2011 (Table 4.2) are closely related, while their closest relatives outside South

Africa are isolates from East Africa. In this chapter differences and similarities

among these South African isolates were further explored. In particular, to gain

an understanding of how the different pathotypes became established in the pop-

ulation. In accordance, a search for candidate genes that could be involved in the

specific virulence of individual isolates was conducted using three approaches: i)

polymorphisms in the genomes were evaluated to determine whether selection

pressure could be detected, ii) the presence or absence of selected genes and the

impact that such inclusion or exclusion could bring about was investigated and

iii) genes of interest with regards to virulence were identified through isolate

specific nonsynonymous polymorphisms in putative effector coding genes.

5.1 Introduction

To obtain nutrients from the host for its own development, Pst must grow in-

fection structures able to bridge host structural barriers, while simultaneously

trying to avoid recognition by the host’s molecular defence mechanisms (Garnica

et al., 2014). To achieve this, Pst, like other filamentous plant pathogens, makes

use of a diverse set of proteins called effector proteins which the pathogen uses

to manipulate host metabolism for its own advantage in cases where it can es-

cape the host’s ETI (see Section 2.3.1). These proteins have critical roles during

the infection process and fulfil specific tasks with accurate timing at particular

locations inside the host (Hogenhout et al., 2009; Stergiopoulos and de Wit, 2009).

Two major groups of effectors exist, namely apoplastic and cytoplasmic effec-

tors. Among the apoplastic effectors are toxins and cell wall degrading proteins,

which are important for necrotrophs, freeing up nutrients by degrading plant

tissues. For hemibiotrophic and biotrophic pathogens, a more subtle approach is

needed, in which the integrity of the host cell is preserved, allowing the pathogen
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 81

to obtain nutrients from living tissues. These groups of pathogens rely more on

intracellular effector proteins to modify the host cellular environment (Dou and

Zhou, 2012; Stotz et al., 2014). Biotrophic fungi, like rusts, make use of haustoria

to deliver fungal effectors into the plant’s living cells (Garnica et al., 2014). Some

genotypes of the host have the ability to recognise these cytoplasmic effector

proteins that activate ETI, triggering a cascade of defence processes that reduce

or completely halt ingress of the pathogen. Genetic changes within the plant,

or elimination or modification of effector genes by the pathogen, can prevent

recognition of pathogen invasion by the host’s defence system (Dodds et al.,

2006). This underpins what is commonly known as resistance gene mediated,

pathotype-specific resistance. This type of resistance leads to the classic “Boom-

and-Bust” cycle described for R-Avr interactions in phytopathology (McDonald,

2004).

5.1.1 The importance of Pst variability

Isolates with a pathotype that enables the pathogen to remain undetected, or

which is able to overcome plant defence systems, will become established in the

population. In addition to gene flow, genetic recombination and mutation can

introduce genetic variability within the population that enables Pst pathotypes to

continue to evolve and overcome host resistance.

Genetic recombination can occur during sexual reproduction in the form of

sexual recombination, or in asexual populations through somatic recombination.

Somatic recombination is believed to be rare in Pst (Little and Manners, 1969),

however recent evidence in the stem rust gene, AvrSr50 indicates somatic re-

combination as the mode of action to overcome Sr50 (Chen et al., 2017). This

illustrates the significance of somatic recombination to be responsible for new

variation. Sexual recombination in Pst requires an alternative host to wheat.

CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 82

Although Pst susceptible Berberis and Mahonia species have been found in South

Africa, providing the opportunity for sexual reproduction, infection by Pst has

not been observed in nature (Visser et al., 2016). The apparent stepwise changes

in virulence seen in South African Pst isolates further confirms the absence of

sexual recombination in South African Pst populations, suggesting that variation

in the Pst population in South Africa might be mostly due to mutations (Visser

et al., 2016).

5.1.2 Mutations—causes, types and effects

Mutations occur naturally due to errors in DNA replication (Griffiths et al.,

2015), spontaneous DNA lesions (Bienko et al., 2005) and by the action of mobile

elements within the genome, called transposons (Klug, 2012). The mutation rate

is the number of mutations that occur in a gene or organism in a given time

period. Natural mutations vary between genes within an organism and occur at

different rates across species (Drake et al., 1998; Scally, 2016). In general mutation

rates are low in most organisms, but this depends on evolutionary forces, the

life history of the organism and chance events (Drake et al., 1998). Agents called

mutagens can accelerate the rate of mutation. A wide variety of mutagens exist,

and they induce different types of mutations. Physical mutagens such as radiation

from the invisible light spectrum can cause chromosomal aberrations, including

chromosomal inversions, chromosomal arm deletions, duplications and repeat

expansions, for example, ultraviolet light can cause various types of mutations

with distinct properties for each wavelength component UVA (320—400 nm),

UVB (280—320 nm), and UVC (200—280 nm) (Pfeifer et al., 2005).

Some chemicals react directly with DNA, for example, ethyl methanesulfonate

(EMS) and sodium azide induce SNPs in the form of random point mutations

(Rao and Sears, 1964; Olsen et al., 1993). Mutagens can cause diseases such as
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 83

cancer in mammals (Ames, 1979), but are also used in functional genomic studies

to develop populations used in reverse genetic techniques such as targeting

induced local lesions in genomes, or TILLING (Henikoff et al., 2004). A common

challenge in these approaches is that many of the mutated individuals are in a

compromised condition, highlighting that beneficial mutations are rare. Most

mutations are either neutral or deleterious and not conserved in the population

(Kimura and Ohta, 1969).

In the absence of gene flow and genomic recombination, mutations are the

main source of genetic variation. Natural selection removes harmful mutations

from the population through a reduced ability of affected individuals to grow

and reproduce. A carrier of a beneficial mutation will have enhanced fitness traits

and therefore will be able to pass the mutation on to the next generation. Such

a mutation will likely become fixed in the population (Hartl and Clark, 1998).

Mutations that are passed on to the next generation increase gene polymorphisms,

for example, multiple alleles of the same gene in the species (Salemi et al., 2009).

In a deterministic model of evolution, changes in allele frequency depend on

fitness and selection, assuming an infinitely large population size. On the other

hand, the stochastic model acknowledges the influence of genetic drift, that

increases as the effective population size decreases. Depending on the phenotype

of the mutation—whether it is advantageous, neutral or deleterious—and the

effective population size, population evolution is more influenced by either drift

or natural selection (Salemi et al., 2009).

Polymorphisms outside coding regions are not usually under strong selection

pressure, however, depending on where SNPs occur in intron splice sites of pre-

mRNA, they may interfere with alternative splicing operations during or shortly

after transcription. This can lead to altered levels of mRNA, modified mRNA, or a

complete shift in the reading frame. Additionally, in coding regions, synonymous

SNPs can occasionally have functional consequences due to alterations in the

CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 84

structure and stability of the translated protein, but are generally considered

to maintain the integrity and function of the protein. Nonsynonymous SNPs,

however, result in amino acid changes, which can significantly change the protein.

These SNPs can have an effect on the function of the resulting protein and the

phenotype.

Mutations within genes

When a mutation results in a purine (nucleotides G and A) being substituted with

another purine, or a pyrimidine (nucleotides C and T) with another pyrimidine,

it is called a transition, while substitution of a purine with a pyrimidine, or vice

versa, is called a transversion (Salemi et al., 2009). Although there are twice

as many possible transversions mutations compared to transitions, transitions

are 10 times more common than transversions because of chemical and steric

properties (Klug, 2012; Griffiths et al., 2015).

Mutations do not always result in a functional change in the protein encoded

by the gene. A silent mutation or synonymous mutations describes a codon

change that does not alter the amino acid in the encoded protein due to degen-

eracy in the genetic code. Most synonymous mutations are considered to be

selectively neutral, but may alter RNA secondary structure and stability (Salemi

et al., 2009). In addition, tRNA molecules can vary in abundance, which is impor-

tant for the success of translation. Mutations in genic regions can be missense

or non-sense. Missense being single point mutations that result in amino acid

changes, while non-sense mutations introduce early stop codons that truncate

proteins. Some consider conservative missense mutations synonymous, as in

the case where similar chemical properties or structures are encoded by the new

amino acid, for instance, leucine and isoleucine that are both aliphatic. Nonsyn-

onymous mutations describe a mutation where the new codon specifies an amino
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 85

acid with different chemical properties from the amino acid it replaces.

In this study, silent mutations are regarded as synonymous mutations and

missense and non-sense mutations as nonsynonymous mutations (Miyata and

Yasunaga, 1980; Li et al., 1985; Nei and Gojobori, 1986). Mutations can also

interfere with gene expression if they occur in a promotor region of a gene or

at the splice site of an intron. Mutations in these regions of the gene are not

considered in this study.

5.1.3 Genomic approaches used to identify effectors

Effector annotation used in this chapter relied on the bioinformatics pipeline

developed by Saunders et al. (2012). The pipeline provides a basis for candidate

effector gene identification. It first clusters secreted proteins into protein families

and classifies and ranks these protein families for their likeliness to be effectors.

Using a modified version of this pipeline, Cantu et al. (2013) annotated the PST130

transcriptome, identifying genes encoding candidate effectors and ranking these

to generate a top 100 tribe list that contained high priority candidate effector

genes. Due to the biotrophic nature and the infection structures produced by Pst,

effector proteins are likely to be secreted. Therefore, at first, the pipeline screened

the predicted proteome for candidates with secreted signals. Markov clustering

was then used to group secreted and non-secreted proteins into protein families

using sequence similarity with secreted proteins. Thirdly, tribe annotation was

carried out based on sequence homology, after which a search for conserved

motifs was performed. Individual members of secreted protein families were

annotated based on features they share with known effectors. Through hierarchi-

cal clustering of tribes, a priority list was compiled for functional validation of

candidates that were most likely effectors.

In this chapter, the focus was on the investigation of SNPs found between
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 86

the genomes of the four historic South African Pst pathotypes, with specific

concentration on the protein coding regions of predicted effector genes to link

specific Pst virulence profiles with nucleotide polymorphisms within these effec-

tor genes. The effector feature annotations, ranking protein tribes according to

their probability of containing effectors, were used (Saunders et al., 2012; Cantu

et al., 2013).

5.2 Materials and methods

The genomes of four South African historical isolates, representing the four patho-

types found in South Africa, were sequenced, mapped to the PST130 reference

genome, and polymorphisms were identified, as described in Chapter 3.

5.2.1 SNP analysis

From the SAMtools mpileup files, with coverage information of each position,

Perl and Python scripts were used to find SNPs with at least 10× depth of

coverage and to identify homokaryotic and heterokaryotic SNPs (see Chapter 3).

SNP effect prediction

SnpEff software (version 3.6; Cingolani et al., 2012) was used to predict the

effects of the polymorphisms and to investigate the frequency of transitions and

transversions in the gene space. SnpEff distinguishes SNP location and type,

including characterisation of nonsynonymous and synonymous SNPs in coding

regions, which indicates introduced or lost stop codons, lost start codons and

changes in splice sites and introns. For this analysis, a bed format file of each

isolate’s SNP set was prepared using BEDTools (version 2.17.0; Quinlan and Hall,

2010) and the annotation information of the PST130 genome. The bed file was

converted into a SnpEff input file using a Perl script. The predicted effects of

SNPs in the gene space were evaluated with specific focus on the introduced stop
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 87

codons and synonymous and nonsynonymous polymorphisms. Codon positions

of SNP sites that introduced stop codons were evaluated, and the gene positions

where stop codons occurred were considered to evaluate any biases that could

indicate the effect on the resulting protein. The frequency of specific nucleotide

changes resulting in transitions and transversions were determined and further

evaluated to determine biases in codon positions for specific nucleotide changes.

5.2.2 Positive selection

The program Yn00 (Yang and Nielsen, 2000), which is part of the PAML package

(Yang, 2007), was used to assess genetic diversity through polymorphism and

positive selection analysis using the synthetic genes described in Chapter 3. A

pairwise comparison that yielded a nonsynonymous substitution rate or dN

value of more than zero indicated a polymorphic gene, while positive selection

was considered when a dN/dS value that indicates the rate of nonsynonymous

vs synonymous polymorphisms, also called the omega value, of more than one

was observed. Perl scripts were used to enable the automated use of Yn00 on the

PST130 gene set (Cantu et al., 2013).

5.2.3 Presence-absence analysis

Unique presence and absence of genes were investigated to identify possible asso-

ciations between specific genes and a gain in virulence in the four South African

isolates. The read coverage of each gene was calculated using BEDTools (version

2.17.0; Quinlan and Hall, 2010). Genes with zero coverage were considered ab-

sent from the specific isolate (Cantu et al., 2013). The nucleotide and amino acid

sequences of these genes were used to query publicly available databases using

the basic local alignment search tool (BLAST version 2.6.0; Altschul et al., 1997) to

find homologous genes in related species and orthologs in the PST130 reference

genome.
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 88

5.2.4 Comparisons of nonsynonymous SNP sites between isolates

An additional method to investigate polymorphisms across isolates was used.

Polymorphic sites predicted to cause nonsynonymous changes were identified

and nucleotides at these positions, across isolates, were compared in a pairwise

manner. The number of nucleotide sites at which a difference in nucleotides

between two isolates was observed was used as a distance statistic in an un-

weighted pair group method with arithmetic mean (UPGMA) tree, indicating the

relationship of isolates to one another in terms of the number of nonsynonymous

changes. The list of genes showing differences between each pairwise compari-

son was compared to the list of candidate effector genes and the list of secreted

proteins generated by Cantu et al. (2013). These lists were generated as described

in Section 5.1.

5.2.5 Multiple sequence alignments to visualise biallelic SNPs

A custom Python script was developed to visualise translated proteins of candi-

date genes indicating the presence of alternative amino acids due to nonsynony-

mous polymorphisms. Where coverage was lower than 2× at nonpolymorphic

sites or 10× at polymorphic sites, manual inspection of the genome was done

using Integrative Genomics Viewer (IGV version 2.3.91; Thorvaldsdóttir et al.,

2013). In cases where the low coverage sequence was the same as in the other

South African isolates, these nucleotide sequences were included in the figure,

but indicated with lighter shading. Blank spaces indicate isolates with no se-

quence information. Colours were assigned according to the “Clustal X Colour

Scheme” used in Jalview (Waterhouse et al., 2009), indicating specific categories.

CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 89

5.3 Results

5.3.1 SNP identification in the genomes of the historical South African

isolates

Polymorphism data provides information on how a population is evolving. After

filtering the Illumina paired end reads and independent mapping of each of the

four South African isolates to the PST130 draft reference genome (as described

in Chapter 3), SNPs were identified across the whole genome, using SAMtools

mpileup. Variant sites were only taken into account in cases where a coverage

depth of 10 reads or more was seen.

The four isolates displayed similar SNP frequencies with 0.62 ± 0.12 % of

the genomes containing polymorphisms when compared to the PST130 refer-

ence, resulting in an average rate of heterozygosity of 6.25 ± 1.15 SNPs/kbp.

Heterokaryotic SNPs were polymorphic to the reference, being biallelic or multi-

allelic, while homokaryotic SNPs were monoallelic. Heterokaryotic SNPs were

in the majority and averaged 92.96 ± 0.18 % of all variant sites across the four

isolates, with a SNP density of 5.81 ± 1.06 SNPs/kbp, a high number comparing

to the 1.51 SNPs/kbp found on Melampsora larici-populina Kleb., the sexually re-

producing poplar rust fungus (Persoons et al., 2014). The remaining 7.04 ± 0.18 %

of variant sites comprised of homokaryotic sites occurring at a frequency of

0.44 ± 0.09 SNPs/kbp (Table 5.1).

Determining the genetic impact of polymorphisms

Information regarding polymorphisms in genes can be used to determine the

impact of the variant on the resulting protein. Identifying the nature and location

of SNPs show how the pathogen changes on the genetic level, including changes

related to its pathogenicity phenotype. To determine the nature and genome

position of polymorphisms, the SNPs identified in SA1 to SA4 (Table 5.1) were
 Table 5.1: Homokaryotic and heterokaryotic SNPs in the South African isolates

Homokaryotic Heterokaryotic
Monoallelic Biallelic Biallelic Multiallelic
PST130 Total Total
% of
Isolate reference number SNPs/kb One alternative One alternative Two alternative Three or four
reference
sites of SNPs allele allele alleles alternative alleles
Number % SNPs/kbp Number Number Number Number % SNPs/kbp

SA1 64 782 816 378 259 0.58 5.84 25 975 6.87 0.40 351 719 228 337 352 284 93.13 5.44
SA2 64 782 816 324 200 0.50 5.00 22 788 7.03 0.35 300 839 211 362 301 412 92.97 4.65
SA3 64 782 816 414 489 0.64 6.40 28 853 6.96 0.45 384 958 275 403 385 636 93.04 5.95
SA4 64 782 816 501 728 0.77 7.74 36 588 7.29 0.56 464 344 334 462 465 140 92.71 7.18

Average 0.62 6.25 7.04 0.44 92.96 5.81

Standard deviation 0.12 1.15 0.18 0.09 0.18 1.06
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 91

annotated using SnpEff. Across the four isolates 29.93 ± 0.20 % of SNPs were

within genes, of which 52.74 % resulted in synonymous substitutions, while

47.26 % represented nonsynonymous substitutions. Loss or gain of start and stop

codons can also have major effects on translation, resulting in complete loss of

translation or truncated peptides. Table 5.2 describes the major predicted effects

of polymorphisms in genic regions in the four South African isolates.

Table 5.2: The number of SNPs identified in coding regions of the four South African Pst
isolates

Location of polymorphism
Isolate
Synonymous Nonsynonymous Stop
coding coding gained

SA1 58 868 52 499 3 347

SA2 50 008 44 829 2 933
SA3 59 595 53 481 3 380
SA4 71 140 64 278 3 992

Between about 3000 and 4000 SNPs resulted in stop codons (Table 5.2). The

three stop codons are TAA, TAG, and TGA. C to T mutations in the first codon

position often introduces stop codons in the gene space (Hane and Oliver, 2010).

In the second and third codon position, SNP sites where changes to an A or G

occur, are responsible for the introduction of stop codons. The majority (99.4 %)

of SNPs that introduced stop codons were biallelic/heterokaryotic. G to Y (C or

T) mutations occurred most frequently (29.2 %), followed by C to R (A or G) at

17.5 % at the second codon position and 14.7 % at the third codon position. Biases

in SNP type at codon positions were assessed in Figure 5.1. Patterns of nucleotide

changes were conserved between isolates.

To identify the impact of introduced stop codons, the gene positions where a

stop codon was introduced were evaluated for possible patterns in occurrence

(Figure 5.2). No distinct trend was observed, and it appears that stop codons are

introduced with no particular preference, randomly appearing in the gene.

CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 92

a) Monoallelic SNP sites introducing stop codons Isolate

SNP count SA1
10 SA2

5 SA3

SA4
2 3 1 2 3 1 2 3
C−A C−A C−T G−A G−A G−T T−A T−A
IUPAC
b) Biallelic SNP sites introducing stop codons
K G or T
900
SNP count

M A or C
600
R A or G
300
S G or C
0
1 1 2 3 2 3 2 3 1 1 1 2 3 2 3 1 2 3 2 3 2 3 W A or T
C−W
A−W

G−M

G−M
C−M

C−M

T−W

T−W
G−R

G−R

G−Y
C−R

C−R

G−K
C−S

C−S

C−Y
C−K

T−R

T−R
T−K

T−K
Y C or T
Nucleotide change at codon position

Figure 5.1: Nucleotide changes that introduced stop codons were highly conserved be-
tween isolates. A small number of monoallelic SNPs (0.6 %) were responsible
(a), but 99.4 % of stop codons were introduced at biallelic SNP positions (b).
Numbers indicate codon positions 1, 2 or 3. Nucleotide changes are indicated
underneath the codon position, the first nucleotice indicating the reference
nucleotide and the second, the polymorphism nucleotide(s).

SA1 SA2

75

50

25
Number of genes

Isolate
0 SA1
SA2
SA3 SA4
SA3

75 SA4

50

25

0
0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00
Proportion of gene retained

Figure 5.2: Distribution of introduced stop codons across all genes per isolate. The bar
charts show the number of genes with a specific gene proportion retained
after a stop codon was introduced.
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 93

Frequency of transitions and transversions at polymorphic sites

The SNPeff information was used to determine whether mutations represented

transversions or transitions. When considering the frequency at which transitions

occurred in comparison with transversions, more transitions than transversions

occurred at SNP sites, as expected. At synonymous SNP sites, C to T transitions

were most common, while A to G transitions occurred most frequently in non-

synonymous SNPs at homokaryotic sites. At homokaryotic SNP sites, where

synonymous substitutions were observed, a transition to transversion ratio of

2 : 1 was displayed, while a 3.5 : 1 ratio was observed for nonsynonymous

substitutions (Figures 5.3 and 5.4). Similar to the finding in Figure 5.1, Figures 5.5

and 5.6 indicated conserved patterns in the specific nucleotide changes at codon

positions 1, 2 and 3, respectively.

5.3.2 Assessment of polymorphisms to detect positive selection

This SNP data reveals information about how the population is evolving. Highly

polymorphic genes are more likely linked with improved fitness and being

under positive selection. The dN/dS statistic, which assesses the ratio of non-

synonymous polymorphisms to synonymous polymorphisms, was evaluated to

identify genes that are under selection. The term “dN” describes nonsynonymous

polymorphisms that replace an amino acid and “dS” describes synonymous poly-

morphisms where the amino acid remains unchanged. SNPs within all genes

annotated within the PST130 reference genome (18 023 genes) were compared

in a pairwise isolate analysis. It is commonly expected that synonymous sites

will evolve more neutrally and that changes in allele frequencies would be due

to random chance (genetic drift). In contrast, a polymorphism that affects fitness

will evolve more rapidly due to its selective advantage.

Synthetic, consensus genes were created for each isolate that incorporated

SNPs that had a 10× or higher coverage and where nonpolymorphic sites had
 +',",'#"-. 5"6'#"&78).#234
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Figure 5.3: Percentage frequency matrices of transitions and transversions at monoallelic SNP sites. In both synonymous and nonsynony-
mous substitutions, transitions were more frequent compared to transversions. Darker red indicates a higher percentage and
darker blue a higher standard deviation.
 8/%,%/3,76 1,2/3,$45063'()
734-45640 834!4564/ 934!4564- :34-4564/ ;34!45640 234/45640
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Figure 5.4: Percentage occurrence matrices of transitions and transversions at biallelic SNP sites. Biallelic SNP sites showed a high transition
frequency of 14 % to 15 % for C and T to Y (C or T), and 8.5 % for A and G to R (A or G) at synonymous sites. For nonsynonymous
sites transition occurrences were still fairly high with an average of 6.84 % across all possible transitions. However, transversion
occurrences were more frequent at 11.98 %. Darker red indicates a higher percentage and darker blue a higher standard deviation.
 Homokaryotic nonsynonymous SNPs

Isolate
SNP count

400
SA1

200 SA2

SA3

0 SA4
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
A−C A−G A−T C−A C−G C−T G−A G−C G−T T−A T−C T−G
IUPAC
Homokaryotic synonymous SNPs K G or T
800 M A or C
96

600 R A or G
SNP count

S G or C
400
W A or T
200
Y C or T

0
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
A−C A−G A−T C−A C−G C−T G−A G−C G−T T−A T−C T−G
Nucleotide change at codon position

Figure 5.5: Codon positions of nucleotide changes at homokaryotic SNP sites explained broadly in terms of transitions and transversion in
Figures 5.3 and 5.4.
 Heterokaryotic nonsynonynous SNPs
4000

3000
SNP count

Isolate
2000 SA1

SA2
1000
SA3
0
SA4
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
A−K A−M A−R A−S A−W A−Y C−K C−M C−R C−S C−W C−Y G−K G−M G−R G−S G−W G−Y T−K T−M T−R T−S T−W T−Y
IUPAC
Heterokaryotic synonymous SNPs K G or T
97

7500 M A or C

R A or G
SNP count

5000
S G or C

2500 W A or T

Y C or T
0
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
A−K A−M A−R A−S A−W A−Y C−K C−M C−R C−S C−W C−Y G−K G−M G−R G−S G−W G−Y T−K T−M T−R T−S T−W T−Y

Figure 5.6: Codon positions of nucleotide changes at heterokaryotic SNP sites explained broadly in terms of transitions and transversion in
Figures 5.3 and 5.4.
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 98

at least 2× coverage of the PST130 reference gene (see Section 3.3.5). Pairwise

isolate comparisons of each consensus gene were carried out using the YN00

program in the PAML package. Pairwise comparisons yielding positive dN

values indicated that the specific gene under investigation was polymorphic

between the two isolates. Alternatively, where positive dS values were obtained,

genes were considered to have evolved more neutrally.

No signals for positive selection were detected, as no dN/dS values, also

known as omega values, of greater than 1.0 were observed. Only seven genes

were given a positive dN value in the pairwise comparisons of the South African

isolates, while positive dS values were computed for two genes. There were

no genes in common and therefore all dN/dS values were undefined. These

nine genes (Tables 5.3 and 5.4) were not investigated further as they did not

display characteristics of genes coding for secreted proteins or putative effectors,

as identified in the lists reported by Cantu et al. (2013), and were therefore not

considered likely candidates for pathogenicity factors.

5.3.3 Presence or absence of genes

Elimination of an effector gene and its resulting protein could aid the pathogen to

escape host recognition. Similarly, specific genes may enhance the pathogenicity

and reproducibility of the pathogen. Therefore, in addition to point mutations,

inclusion or exclusion of entire genes was also assessed to look for associations of

genes with virulence phenotypes. After monitoring whether there were genes in

the PST130 reference genome that were not covered by read sequences from the

South African isolates, 211 genes were found to be absent in all four the South

African isolates. In addition, there were 36 genes that were absent in three or

fewer of the South Africa isolates, in different combinations, that were present in

the reference genome of PST130 (Table 5.5).

 Table 5.3: Polymorphic genes with positive dN values indicating nonsynonymous changes in isolate pairwise comparisons

Gene SA1 vs SA2 SA1 vs SA3 SA2 vs SA3 SA1 vs SA4 SA2 vs SA4 SA3 vs SA4

PST130_03694 0 0 0 0.000 9 ± 0.000 9 0.000 9 ± 0.000 9 0.000 9 ± 0.000 9

PST130_07979 0.000 2 ± 0.000 2 0 0.000 2 ± 0.000 2 0 0.000 2 ± 0.000 2 0
PST130_09146 0 0 0 0.001 3 ± 0.001 3 0.001 3 ± 0.001 3 0.001 3 ± 0.001 3
PST130_10326 0 0.001 3 ± 0.001 3 0.001 3 ± 0.001 3 0.001 3 ± 0.001 3 0.001 3 ± 0.001 3 0
PST130_10374 0.003 6 ± 0.003 6 0 0.003 6 ± 0.003 6 0 0.003 6 ± 0.003 6 0
PST130_11223 0 0 0 0.001 7 ± 0.001 7 0.001 7 ± 0.001 7 0.001 7 ± 0.001 7
PST130_17618 0 0 0 0.000 6 ± 0.000 6 0.000 6 ± 0.000 6 0.000 6 ± 0.000 6
99

Table 5.4: Polymorphic genes with positive dS values indicating synonymous changes in isolate pairwise comparisons

Gene SA1 vs SA2 SA1 vs SA3 SA2 vs SA3 SA1 vs SA4 SA2 vs SA4 SA3 vs SA4

PST130_00923 0.003 7 ± 0.003 7 0 0.003 7 ± 0.003 7 0 0.003 7 ± 0.003 7 0

PST130_04022 0 0 0 0.001 5 ± 0.001 5 0.001 5 ± 0.001 5 0.001 5 ± 0.001 5
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 100

Table 5.5: Number of absent genes in the four South African Pst pathotypes. This in-
cludes a total of 247 genes, where 211 genes were absent in all four isolates
and 36 genes that were absent in one to three isolates

Isolate Pathotype Number of absent genes

SA1 6E16A- 211 + 11

SA2 6E22A- 211 + 13
SA3 7E22A- 211 + 19
SA4 6E22A+ 211 + 18

Figure 5.7 displays genes that are absent in the South African isolates. Presence-

absence genes may be involved in virulence of the pathogen, however, none of

these genes was on the list of putative effector genes (Cantu et al., 2013). The

number of genes absent in a single isolate increased with the increase in virulence.

(See Appendix B, Table B.1, for gene names of the 211 genes that were absent in

all four South African isolates).

A BLAST search against the National Center for Biotechnology Information

(NCBI) non-redundant nucleotide databases using default parameters, revealed

homology in other plant pathogens for eight of the 211 genes absent in all South

African isolates (Table 5.6). Investigations of the functionality of the Pgt homologs

were undertaken, and characteristics are listed in Appendix B, Section B.2. As

redundancy often exists in genomes of filamentous plant pathogens (Dangl and

Jones, 2001) a BLAST search of the 211 genes against the PST130 transcriptome

was performed. Of the 211 genes, 152 had one or more potential paralogs within

the PST130 genome (Table 5.7).

 Table 5.6: Potential orthologs of genes absent in all four South African isolates. All orthologs identified were from fungi, besides one
ortholog from the oomycete, Albugo laibachii

PST130 PST130 Match

Homolog
gene length length

PST130_00159 Pgt isoleucyl-tRNA synthetase (PGTG_09131), mRNA 252 252

PST130_07080 Pgt hypothetical protein (PGTG_01952), mRNA 252 133
PST130_16763 Pgt hypothetical protein (PGTG_02128), mRNA 798 431
101

PST130_17182 Pgt hypothetical protein (PGTG_02971), mRNA 270 247

PST130_17354 Pgt hypothetical protein (PGTG_20899), mRNA 1 188 345
Pgt glycogen [starch] synthase (PGTG_07651), mRNA 1 188 562
PST130_17620 Pgt hypothetical protein (PGTG_15464), mRNA 141 136
PST130_17815 Pgt 1,3-beta-glucan synthase component FKS1 (PGTG_00125), mRNA 666 535
PST130_06262 Albugo laibachii Nc14, genomic contig CONTIG_2252_NC14_v4_941_117 210 175
Rhynchosporium orthosporum mitochondrion, complete genome 210 196
Rhynchosporium secalis mitochondrion, complete genome 210 196
Rhynchosporium commune mitochondrion, complete genome 210 196
Rhynchosporium agropyri mitochondrion, complete genome 210 196
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 102

Table 5.7: The number of potential paralogs identified in genes absent in all four South
African isolates

Number of Number of potential

genes paralogs in PST130

107 1
22 2
9 3
9 4
2 7
2 5
1 10

In the group of 211 genes that were absent in all four South African isolates,

only five genes were coding for secreted proteins according to the lists in Cantu

et al. (2013). They were PST130_01946, PST130_03059, PST130_03060, PST130_-

06608 and PST130_08220. These five genes returned no hits in a BLAST search

against NCBI non-redundant nucleotide databases. Two of the genes, PST130_-

01946 and PST130_03059, had potential paralogs within the PST130 transcriptome

with higher than 80 % identity and E-values lower than 0.01 (Table 5.8).

The PST130 paralogs identified in these BLAST hits did not appear in the

original list of 247 genes absent across the four South African isolates and there-

fore were present in the South African isolates. PST130_01946 had four paralogs,

while PST130_03059 had one paralog, highlighting the occurrence of redundancy

in the Pst genome that could be the result of duplication events.

Table 5.8: Potential paralogs of genes absent in the four South African isolates

qseqid sseqid % Identity Length Mismatch Gaps E-value Bit score

PST130_01946 PST130_00235 95.745 329 11 1 1.04E-15 527

PST130_10569 89.362 235 25 0 3.21E-80 296
PST130_08196 87.179 234 30 0 2.51E-71 267
PST130_11479 87.342 158 20 0 9.36E-46 182
PST130_03059 PST130_02767 92.958 142 9 1 7.54E-53 206
qseqid, query sequence ID; sseqid, subject sequence ID
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 103

PST130_01827
PST130_05182
PST130_03318
2
PST130_03983 PST130_04442
1 2

SA2, SA3

4
SA
PST130_03509

SA
1,
PST130_01450 PST130_08345

3,
SA
3

SA
PST130_10298 2 n two isolate PST130_14450
nt i

4
s 3
SA se SA
1 ab
A 2,
,S ,S
A3 A1

s
ne
S

Ge

Ge
nes
211 genes

absent in
PST130_00826
2 SA1 SA1, SA3, SA4 1 PST130_14553
olate
PST130_14554 absent in all 4 isolates
e is

thr
on

2 SA

ee
SA 1,
in

S
so

i
nt lat A2
se es ,S
A4
b
PST130_09396 Genes
a

SA
2
3

PST130_13177 3
SA

SA4

PST130_00111

2,
PST130_17608

SA
PST130_12299

3,
S A4
6
PST130_04061 PST130_07666 3 PST130_03002
9
PST130_12309 PST130_15299 PST130_13389
PST130_16907 PST130_17504 PST130_14325

PST130_00758 PST130_01120
PST130_01245 PST130_01754
PST130_04241 PST130_04996
PST130_10076 PST130_12228
PST130_16847

Figure 5.7: Presence-absence analysis revealed 211 genes absent in all four South African
isolates and an additional 36 genes absent in some isolates.

Of the 36 genes that were absent in three or less of the South African iso-

lates (Figure 5.7), three had highly similar nucleotide sequences in NCBI non-

redundant nucleotide databases with more than 80 % identity and E-values

smaller than 0.01 in BLAST searches (Table 5.9). PST130_00758, PST130_08345

(only present in SA4) and PST130_12299 (only present in SA3) had hits with

PGTG_02401, PGTG_03886 and PGTG_14583 respectively. However, these three

Pgt proteins are uncharacterised to date. Conserved domains of the Pgt proteins

are listed in Appendix B, Section B.2.

CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 104

Table 5.9: Potential orthologs of genes absent in three or less of the South African isolates

PST130 gene Homolog Species

PST130_00758 Pgt hypothetical protein (PGTG_02401) Fungi
PST130_08345 Pgt hypothetical protein (PGTG_03886) Fungi
PST130_12299 Pgt hypothetical protein (PGTG_14583) Fungi

Of the 36 genes absent in three or less of the isolates, nine genes were present

in only one of the South African isolates. These nine genes included three genes in

SA1: PST130_03002, PST130_13389 and PST130_14325, one in SA2: PST130_14553,

two in SA3: PST130_00111 and PST130_12299 and three genes in SA4: PST130_-

03509, PST130_08345 and PST130_14450. Notable BLAST hits for two of these

genes, PST130_12299 and PST130_08345, were obtained showing high similarity

with Pgt genes as shown in Table 5.9, where they were identified according to

their absence in one or more of the isolates. Conserved domains are listed in

Appendix B, Section B.2. Of these 36 genes absent in three or fewer isolates, 24

displayed potential paralogs in the PST130 genome (Table 5.10).

Table 5.10: Number of potential paralogs in PST130. Of the 36 genes that were absent in
three or less of the South African isolates, 24 had potential paralogs in the
PST130 genome. All potential paralog genes were present in all isolates

Number of Number of potential

genes paralogs in PST130

14 1
3 3
3 2
2 4
1 7
1 10

Two potential paralogs were identified in the PST130 genome for PST130_-

00111 (SA1) and one for PST130_03002 (SA1), PST130_14325 (SA1), PST130_12299

(SA3) and PST130_08345 (SA4) as summarised in Table 5.11.

To investigate possible functions of the present and absent genes, functional

CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 105

annotation of possible orthologs were assessed (see Appendix B, Section B.2).

Table 5.11: Paralogs of genes that only occurred in one of the South African isolates

qseqid sseqid % Identity Length Mismatch Gaps E-value Bit score

PST130_00111 PST130_12514 95.78 332 14 0 2.00E-156 536

PST130_15801 92.77 332 24 0 3.00E-140 481
PST130_03002 PST130_08845 89.36 235 23 2 2.00E-84 294
PST130_08345 PST130_11503 96.73 275 9 0 2.00E-133 459
PST130_12299 PST130_05481 96.21 396 15 0 0 649
PST130_14325 PST130_00979 95.93 246 10 0 1.00E-115 399
qseqid, query sequence ID; sseqid, subject sequence ID

5.3.4 Investigation of candidate genes that are likely to experience evolu-

tionary changes

By comparing heterokaryotic SNPs in the four South African isolates in a pairwise

manner, all genes with unique nonsynonymous changes in the four South African

isolates were identified. It was found that the number of genes with nonsyn-

onymous mutations increased with an increase in virulences as indicated by the

UPGMA dendrogram in Figure 5.8(a). This supports the previous hypothesis of

stepwise evolution, with each pathotype derived from the preceding pathotype

through single-step mutation events (Visser et al., 2016).

Nonsynonymous heterokaryotic biallelic SNPs that differed between isolates

(11 185 SNPs) were observed in 2689 genes. According to the gene annotation

of Cantu et al. (2013), 138 of these were predicted to encode secreted proteins

(613 SNPs), of which 27 were putative effector proteins (106 SNPs) that could

be involved in the specific virulence phenotypes of the four South African Pst

pathotypes. Figures 5.8 (b), (c) and (d) display the pairwise comparison of

isolates, with the number of genes that show nonsynonymous SNPs in each gene

set comparison, for example, proteomes, secretomes and effectomes.

CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 106

a) Distance tree b) Proteome c) Secretome

1200
SA3 53
900 SA3 1045

SA2 53 75
Distance

44 40 49
600

SA2 1095 1333 SA1

SA2

SA3

SA4
300

d) Effectome
SA1 912 924 1084 SA3 12
SA2 7 11
SA1 10 9 9
0
SA4

SA3

SA2

SA1

SA2

SA3

SA4

SA2

SA3

SA4
Figure 5.8: Nonsynonymous SNPs in the gene space of the four South African isolates
increase over time and with increasing virulence. The branch lengths of
the UPGMA distance tree (a) is derived from the distance matrix in (b) and
illustrates the progressive accumulation of genes with nonsynonymous mu-
tations over time as new pathotypes developed, given that the population
evolved stepwise through mutations. Heat maps indicate frequencies of
unique nonsynonymous substitutions in the Pst Proteomes (b), secretomes (c)
and effectomes (d). UPGMA, unweighted pair group method with arithmetic
mean.

5.3.5 Candidate effectors with sequence polymorphisms between the South

African isolates

After applying the three assessment methods (positive selection analysis, presence-

absence analysis and nonsynonymous polymorphism analysis) to the polymor-

phic datasets, only genes that were members of the top 100 ranking protein

families for effectors as described in Cantu et al. (2013), were considered for fur-

ther investigation to identify candidate genes that could explain gain-of-virulence.

The justification of selection of these 27 candidate genes (Figures 5.8), is shown in

Section 6.2, Table 6.1. As an example, Figure 5.9 illustrates five nonsynonymous

changes due to heterokaryotic SNPs in one of the 27 candidate genes, PST130_-

00285. Please consult the Appendix B, Section B.3, for changes in the remaining

26 genes.

A molecular analysis, focussed on a selection of the 27 polymorphic candidate

CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 107

SA1 M H L P F Y L I F L L I P L H G I G G V A H G P V G V E N G I H D L E S I K T L A L G N K
SA2 M H L P F Y L I F L L I P L H G I G G V A H G P V G V E N G I H D L E S I K T L A L G N K
45
SA3 M H L P F Y L I F IL L I P L H G I G G V A H G P V G V E N G I H D L E S I K T L A L G N K
SA4 M H L P F Y L I F IL L I P L H G I G G V A H G P V G V E N G I H D L E S I K T L A L G N K

E T G T M G E E A G D E L K L G P L E R T S S T Q N S I V E T N R V D L A N D D V D S E E
E T G T M G E E A G D E L K L G P L E R T S S T QR N S I V E T N R V D L A N D D V D S E E
46 90
E T G T M G E E A G D E L K L G P L E R T S S T QR N S I V E T N R V D L A N D D V D S E E
E T G T M G E E A G D E L K L G P L E R T S S T QR N S I V E T N R V D L A N D D V D S E E

A E E E A A L L I Y C L R E R E S M E T S L V Q S R T M T G R Q Q KR T L V K R G H S HN K K
A E E E A A L L I Y C L R E R E S M E T S L V Q S R T M T G R Q Q KR T L V K R G H S HN K K
91 135
A E E E A A L L I Y C L R E R E S M E T S L V Q S R T M T G R Q Q KR T L V K R G H S HN K K
A E E E A A L L I Y C L R E R E S M E T S L V Q S R T M T G R Q Q KR T L V K R G H S HN K K

C H K Y N G I P K R Q L W W L A A K S R L R Q A K H H T Q T H F Y R F S I W C R E M I A A
C H K Y N G I P K R Q L W W L A A K S R L R Q A K H H T Q T H F Y R F S I W C R E M I A A
136 180
C H K Y N G I P K R Q L W W L A A K S R L R Q A K H H T Q T H F Y R F S I W C R E M I A A
C H K Y N G I P K R Q L W W L A A K S R L R Q A K H H T Q T H F Y R F S I W C R E M I A A

L T S K S F W K L W K H K M R W A F F R K Y C L DY L P *
L T S K S F W K L W K H K M R W A F F R K Y C L D L P *
181 208
L T S K S F W K L W K H K M R W A F F R K Y C L D L P *
L T S K S F W K L W K H K M R W A F F R K Y C L DY L P *

Figure 5.9: Translated sequence alignment of gene PST130_00285. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The sig-
nal peptide, predicted using SignalP (version 2; Emanuelsson et al., 2007) is
indicated by the black box. Alternative amino acids resulting from nonsyn-
onymous SNPs at biallelic sites are indicated in the below diagonal triangles.
Please consult the appendix for the sequence alignments of the remaining
26 candidates. Colours were assigned according to the “Clustal X Colour
Scheme” used in Jalview (Waterhouse et al., 2009), categorising amino acid
profiles.

effector genes that were identified was the next step of investigation and is

reported in Chapter 6.

5.4 Discussion

The present study implemented the gene models developed for the PST130 draft

genome sequence (Cantu et al., 2011). These gene models have been further

assessed for various effector features to create a subset of genes that could likely

be involved in pathogenicity (Cantu et al., 2013).

In a clonal population, mutations are the main source of genetic variation. In

CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 108

this study, the focus was on point mutations causing SNPs—other DNA aber-

rations were not investigated. Characterisation of SNPs was undertaken to

understand how the pathogen changes at the genetic level to achieve changes

in its pathogenicity phenotype. SNPs that result in nonsynonymous amino acid

changes present an allelic pool of protein variation upon which selection pres-

sures can impact, leading to changes in allelic frequencies within the pathogen

population.

5.4.1 Polymorphic sites

SNP analysis showed a higher frequency of SNPs in isolate SA4 compared to iso-

lates SA1, SA2 and SA3. This is expected as the biggest time span between the col-

lection of these isolates was between SA3 and SA4 (seven years), while only one

to two years passed between collection of SA1 to SA3 and progressive accumula-

tion of mutations is expected over time (Salemi et al., 2009). The density at which

homokaryotic (0.44 ± 0.09 SNPs/kbp) and heterokaryotic (5.81 ± 1.06 SNPs/kbp)

SNPs occurred in the South African isolates mapped against the PST130 refer-

ence were comparable to SNP densities described by Cantu et al. (2013). The

authors investigated five isolates with distinct virulence profiles, two from the

UK and three from the USA. These displayed a homokaryotic SNP density of

0.41 ± 0.28 SNPs/kbp, and 5.29 ± 2.23 SNPs/kbp heterokaryotic SNP density

(Cantu et al., 2013). Using similar methods similar to Cantu et al. (2013), Kiran

et al. (2017) reported SNP densities of 1.90 ± 1.27 SNPs/kbp at homokaryotic

sites and 4.67 ± 1.17 SNPs/kbp at heterokaryotic sites for three Indian isolates

from different epidemiological regions, sequenced and mapped against each

other.

An average rate of heterozygosity of 6.25 ± 1.15 SNPs/kbp was computed in

the South African isolates. This is slightly higher when compared to the average
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 109

Identical sites Variant sites

Single (SNPs)
isolates

Reference genome

Mapped reads

Key: False positive SNP Indicate the allele that was retained
in the consensus reference sequence
False negative site

Figure 5.10: Over- and underestimates of SNP sites. Overestimation of heterokaryotic

SNP sites is indicated with a green star and underestimation of homokary-
otic SNP sites with a pink star. These misinterpretations occur due to un-
phased reference genomes (adapted from Cantu et al., 2013).

between PST-21 (USA), PST-43 (USA), PST130 (USA), PST-87/7 (UK) and PST-

08/21(UK) (5.70 ± 2.47 SNPs/kbp) (Cantu et al., 2013). Increased heterozygosity

was seen in intergenic regions compared to genic regions in the South African

isolates, as also reported by Cantu et al. (2013) and Cuomo et al. (2017). This is

expected as selection acts more strongly on coding regions.

Next-generation sequencing approaches for sequencing Pst have only re-

cently implemented long read information to produce phased genomes where

the genomes of the two haploid nuclei are separated (Schwessinger et al., 2018).

Due to this constraint, it is expected that homokaryotic SNPs will be underesti-

mated and heterokaryotic SNPs will be overestimated using short read assembly

reference genomes such as PST130 (Cantu et al., 2011), CY32 (Zheng et al., 2013),

PST-78 (Cuomo et al., 2017) and 46S 119 (Kiran et al., 2017).

Every position in the reference genome represents only one allele at that posi-

tion, although for genetic material present in both nuclei, two alleles (identical or

not) would be present in the genome (Figure 5.10). At nucleotide bases where

the reference would have two different alleles, such as heterozygous sites, only
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 110

one allele would be in the consensus reference sequence used to align reads in

re-sequencing. The mapped isolate identical to the biallelic reference site will

appear to be a heterokaryotic SNP site. For example, when the reference is a

heterokaryotic site (AT) and the mapped isolate is identical (AT) and the chosen

reference site is either A or T, it would indicate a polymorphism causing an over-

estimation of heterokaryotic SNP sites. It is however expected that heterokaryotic

SNPs will be in the majority as mutations are expected to be random and inde-

pendent between nuclei. True variant sites for single isolates that contain only

one genotype would have an allele frequency of one over all aligned reads at

monoallelic sites. When the consensus reference sequence contains the allele

at a biallelic site that is the same base in the mapped isolate in all alleles in the

mapped reads, it would not be known that the mapped isolate was not identical

to the reference genome. For example, when the reference is a heterokaryotic site

(AT) and the mapped isolate is homokaryotic (AA) and the chosen reference site

is A, it would underestimate homokaryotic sites. The availability of a high quality

phased reference genome (Schwessinger et al., 2018) allows the improvement of

accuracy of current polymorphism classification.

5.4.2 STOP codons

This study focused on polymorphisms in genic regions. SNP analysis revealed

the introduction of multiple stop codons. These stop codons appeared at similar

frequencies across genic sites in all four isolates. This is of interest as premature

stop codons can cause gain in virulence when it causes loss of an avirulence

effector function (Dong et al., 2015). The majority (99.4 %) of the SNP sites that

introduced stop codons were biallelic. This result will be interesting to re-evaluate

using a phased Pst genome to account for the overestimation in heterokaryotic

SNPs identified when using an unphased genome.

CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 111

5.4.3 Transitions and transversions at specific codon positions

A transition mutation does not alter the amino acid encoded by that codon, while

a transversion would incorporate a different amino acid into the peptide. Due to

the degeneracy of the genetic code, the third codon position can be changed for

12 of the 20 amino acids, without altering the amino acid. This is displayed in

the biallelic SNP data, where nonsynonymous biallelic SNP sites displayed more

transversions, while synonymous biallelic SNPs mainly displayed transitions.

At synonymous biallelic SNPs sites, transitions were most frequent with the

highest SNP frequencies in C ↔ T and G ↔ A mutations, while nonsynonymous

biallelic sites (excluding sites that induced stop codons) displayed higher number

of transversions, with C and T to R (A or G) and A and G to Y (C or T) occurring

most frequently. Transition:transversion biases occurred at different levels at

the three codon positions due to variability in physical constraints that in turn

caused variability in selection for or against a specific nucleotide change (Bofkin

and Goldman, 2006).

G to A and C to T changes have been described as the most frequent mutations

induced by long wave ultraviolet A (UVA) and short wave ultraviolet B (UVB)

irradiation in mouse embryo fibroblasts (Pfeifer et al., 2005). The exposure

of urediniospores to solar radiation and short wave ultraviolet (UV) light are

suggested to reduce viability (Sharp, 1967; Maddison and Manners, 1972). It

has also been hypothesised that the distance of dispersal of Pst is shorter in

comparison with Pgt and Pt, likely due to its sensitivity to UV light (Rapilly,

1979). Further investigation is needed to draw more parallels between the effect

UV irradiation has on mammalian cells, as explained by Pfeifer et al. (2005),

and urediniospores, or whether the phenomenon is mostly due to the stronger

selective pressure in favour of transitions compared to transversions (Bofkin and

Goldman, 2006). Nonetheless, multiple studies have shown the mutagenic effect
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 112

of UV light on urediniospores of Pst (Johnson, 1978; Cheng et al., 2014), while in

this study biases were observed in the frequency of nucleotide changes at specific

codon positions.

5.4.4 Stepwise mutations

It is hypothesised that the stepwise changes in virulence seen in South African

Pst pathotypes have resulted from mutations within a fairly static Pst population

(Visser et al., 2016). Establishment of new alleles in the population is due to the

unique combination of selection and genetic drift in the population (Salemi et al.,

2009). In the gene space, selection pressure acts on mutations that cause changes

in the function and stability of the gene or the resulting protein, ultimately

changing the manner in which the organism interacts with its environment.

Genotype frequencies depend on selection that is driven by fitness traits. Genes

that are highly polymorphic are thus likely to be involved in fitness traits that

enable the genotype to contribute to the next generation.

5.4.5 Positive selection

The YN00 software package was implemented to investigate the presence of sig-

natures of selection by comparing synonymous and nonsynonymous substitution

rates. The dN/dS statistics were computed. New alleles introduced by random

mutations that evolve neutrally will change in frequency in the population only

due to genetic drift and not because it has an effect on fitness. This is generally

expected for synonymous SNPs. In contrast, a nonsynonymous polymorphism

that affects fitness will evolve more rapidly.

Comparing synonymous and nonsynonymous substitution rates can reveal

whether a specific allele at a locus is under positive or negative selection. No

omega values greater than 1 were obtained in this analysis. The inability to
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 113

identify genes under selection could indicate that genes under strong selection

pressure in the South African isolates do not exist in the PST130 reference genome.

However, trade-offs exist between statistical robustness and power. It is known

that dN/dS methods often fail to detect signals of selection (Salemi et al., 2009).

The stringency of dN/dS methods could therefore fail to detect selection pressure

between the four clonally derived, and therefore relatively similar, pathotypes.

The McDonald-Kreitman test (McDonald and Kreitman, 1991) is often considered

more powerfull to detect positive selection. It compares dN/dS intra-species

against a sister species to remove the demographic background.

5.4.6 Presence-absence analysis

In addition, the South African pathotypes were compared on the basis of genes

that were uniquely present in, or absent from the South African pathotypes. This

method has shown changes in virulence in other pathogens (Bubić et al., 2004;

Yoshida et al., 2009; Gilroy et al., 2011).

Homology with genes of known functions was investigated to determine

whether genes could play a specific role in pathogenicity. BLAST searches in

public databases provided homology information for 11 genes. Gene ontology of

characterised identified genes suggested that these homologs were involved in

protein translation, sugar transport, metabolism and components of the fungal

cell wall. Postulated gene function did not indicate a role of the homologs in

host manipulation or the escape of host recognition, as expected for virulence

factors. Biological validation of suggested functionality is needed to draw clearer

conclusions.

BLAST searches against the PST130 transcriptome revealed putative paralogs

that could indicate functional redundancy for many of the genes shown as absent

from the South African isolates, where these paralogs could functionally replace
CHAPTER 5: EVOLUTION OF SOUTH AFRICAN PST 114

the absent gene. Such redundancy has been described as genetic buffering

(Dangl and Jones, 2001). However, genes that were absent or uniquely present

between pathotypes did not fit effector protein characterisation and were not in

the putative effector subset of Cantu et al. (2013). Therefore these genes were not

considered as candidate genes involved in pathogenicity dynamics.

5.4.7 Nonsynonymous polymorphisms

Lastly, pairwise comparisons of the South African pathotypes, evaluating non-

synonymous differences gene-by-gene, were performed similarly to Cantu et al.

(2013). A total of 2689 genes showed nonsynonymous differences in pairwise

comparisons between the four pathotypes. Of these genes, 138 carried a secretion

signal, of which 27 were in the subset of putative effector genes.

5.5 Conclusion

After characterisation of the polymorphisms across the genomes of the four South

African isolates, three methods were used to identify differences in the gene space

of the four South African pathotypes. Where applicable, results were compared

to lists containing genes that encode secreted proteins and putative effectors

(Cantu et al., 2013), to further narrow down the list of candidate genes. Of the

three methods followed, namely to search effector candidates with signatures of

positive selection, to evaluate the complete exclusion or the unique inclusion of

effector candidates and to evaluate nonsynonymous polymorphisms between

effector candidates between isolates, only the latter included genes that were

previously identified as effector candidates. Different methods exist for validation

of candidates, although limitations exist due to the biotrophic nature of Pst. One

example of validation is to test expression of genes at infection stages using time

course experiments. Candidate genes will be further investigated in Chapter 6.

Chapter 6

Gene Expression Analysis of

Candidate Effectors Identified in
South African Pst Isolates

6.1 Introduction

T HE OBLIGATE BIOTROPHIC NATURE of rust prevents in vitro functional valida-

tion. In addition, rust cereal hosts are difficult to transform, which makes in vivo

functional characterisation challenging (Petre et al., 2016b). While in planta stud-

ies have been undertaken using techniques as for example virus induced gene

silencing (VIGS), they are difficult and time consuming (Panwar and Bakkeren,

2017). Recent successes in stem rust effector identification are reviewed in Chap-

ter 2 Section 2.4.4. As an early step in functional validation, effector gene function

in the infection process can be predicted by evaluating gene expression at specific

developmental stages of the fungus (Wang et al., 2007, 2009; Sørensen et al., 2012;

Cantu et al., 2013). These gene expression levels can be evaluated using methods

including microarrays, transcriptome sequencing, and RT-qPCR that was used in

this chapter.

115
CHAPTER 6: GENE EXPRESSION ANALYSIS 116

6.1.1 Regulation of gene expression in eukaryotes

Gene expression differs throughout development, between cell types and in

response to different environmental stimuli. Regulation of gene expression is

an intricate, multi-stage process. Transcription regulatory processes occur in the

nucleus, while regulation of pre- and post-translation occur in the cytosol. This

ability to selectively express genes is essential for the development and survival

of a complex organism (Bustin and Nolan, 2004). For transcription factor proteins

to access genes and initiate transcription the chromatin must be remodelled

through a process of acetylation. Acetylation opens up nucleosomes and allows

transcription factor proteins access to gene promoter sites.

The transcription process is further regulated by the assembly and arrange-

ment of the transcriptional machinery enzymes that initiate transcription of RNA

from the DNA template. Processing of the pre-mRNA molecule prepares it as a

template for protein synthesis. A methylated cap is added to the 50 end soon after

transcription starts, while at the 30 end a poly-adenylated tail is added upon com-

pletion of transcription. Introns, if present, are then spliced from the pre-mRNA

molecule. Binding sites for microRNAs (miRNAs) and regulatory proteins are

often found in the 30 untranscribed regions (UTRs) that can down-regulate gene

expression or degrade the mRNA molecule.

Double stranded, small interfering RNA (siRNA) can also modulate gene

expression at the post-transcription stage. After maturation, the mRNA molecule

leaves the nucleus through a nuclear pore and enters the cytosol. Stable mRNA

molecules can now be translated into peptides. Post-translational modifications

may also be required to transform gene products into functionally active proteins

(Klug, 2012). These processes happen in various different organelles depending

on the protein, and determine whether a functional gene product is produced.

CHAPTER 6: GENE EXPRESSION ANALYSIS 117

6.1.2 Quantification of gene expression

Several approaches can be taken to assess the different stages of gene expression.

These include validating protein levels, transcription of genes and the effective-

ness of small interfering RNAs (siRNAs; Schmittgen and Livak, 2008). Different

methods have been developed for these multiple approaches (Speed, 2004; Mehta

et al., 2010). One such tool used to measure gene transcript levels is quantitative

or real time PCR (qPCR). The first form of qPCR was developed by Higuchi et al.

(1993). It measures the level of gene transcription by quantifying the amount of

a specific RNA (Schmittgen and Livak, 2008). Quantitative PCR is a powerful

tool, with its strength lying in its ability to detect DNA sequences with high

specificity, for a wide range of concentrations. In addition, qPCR also eliminates

downstream processing that is needed by some other assays using a camera that

can detect fluorescence (Higuchi et al., 1993). The fluorescent dye intercalates

with the double stranded DNA (dsDNA) as it is synthesised, so that, as dsDNA

accumulates the fluorescence increases. The rate at which the fluorescence in-

creases (kinetics) is directly proportional to the original amount of target cDNA.

The fluorescent signal is observed by the camera in the qPCR instrument at each

annealing/extension phase during thermocycling (Higuchi et al., 1993).

Different methods have been developed to study relative gene expression, e.g.

the comparative CT method, the simulated kinetic model (Livak and Schmittgen,

2001; Schmittgen and Livak, 2008) and the efficiency correction method (Pfaffl,

2001). The efficiency correction method of relative gene expression was used

for the analyses in this chapter. This method accounts for differences in the

efficiencies of the PCR reaction (see Section 6.2.9) when amplifying the target

regions of the test and reference genes, in contrast with the comparative CT

method (Livak and Schmittgen, 2001) that assumes equal amplification efficien-

cies between the two compared gene products. This is however only possible for
CHAPTER 6: GENE EXPRESSION ANALYSIS 118

small experiments, with a limited number of genes. Both the efficiency corrected

and simulated kinetic model approaches aim to improve the accuracy of the

comparative CT method. The simulated kinetic model is the best for studying

large numbers of genes (Schmittgen and Livak, 2008) as the efficiency correction

method is a relatively costly and time consuming process (VanGuilder et al.,

2008).

6.1.3 Candidate effector features

In Chapter 5, 27 candidate effector genes that displayed nonsynonymous SNPs

between the historical South African isolates were identified. These genes, based

on the PST130 gene models, were previously identified as putative effectors

(Cantu et al., 2013) using a modified version of the effector identification pipeline

developed by Saunders et al. (2012). For the 27 candidate effector proteins,

annotation and tribe rankings, as taken from Cantu et al. (2013) are listed in

Table 6.1. None of the 27 candidate genes had flanking intergenic regions (FIR) of

10 kbp (kilo base pairs) or more. Only PST130_05944 had a nuclear-localisation

signal (NLS) at amino acid position 238, and only PST130_07564 was classified as

a small and cysteine rich (SCR) protein.

6.1.4 Gene transcription analysis

In this chapter gene transcription is measured as an indication of gene expression,

although it is clear from the preceding explanations that many regulatory steps

need to be successfully completed to yield a functional protein. When gene

expression is studied under different conditions, or at different time points in a

developmental time series, spatial and temporal patterns of gene expression show

differential accumulation of gene products that are associated with treatment or

the specific stages of development (Tomancak et al., 2007). Ideally time points
 Table 6.1: Effector features of the identified candidate effectors. Identified candidate effectors were secreted proteins in tribes ranking within
the top 100 potential effector tribes as described by Cantu et al. (2013)

Length Similarity to No. of Expressed

Isolate pairs with Tribe Tribe Effector motifs PFAM Expressed
Gene ID (amino HESPs or repeat in infected
nonsynonymous substitutions no. ranking (amino acid position) mapping in Haust.
acids) fungal AVRs units material

PST130_06558 SA2 & SA3; SA3 & SA4 9 6 341 No 9 No No No

PST130_12487 SA1 & SA2; SA1 & SA3; SA1 & SA4; 31 7 197 No 0 No Yes Yes
SA2 & SA3; SA2 & SA4; SA3 & SA4
PST130_14091 SA1 & SA2; SA2 & SA4 11 14 167 No 0 Y/F/WxC(85);LIAR(32) Yes Yes Yes
PST130_17605 SA2 & SA4; SA3 & SA4 11 14 239 Yes 7 Y/F/WxC(103) No Yes Yes
PST130_05454 SA1 & SA2; SA2 & SA3; SA2 & SA4 68 15 266 No 0 Yes Yes No
PST130_09275 SA1 & SA2; SA1 & SA3; SA1 & SA4 134 16 210 Yes 0 Yes Yes No
PST130_12491 SA1 & SA4 8 17 182 No 13 No No No
PST130_05023 SA1 & SA4; SA3 & SA4 351 22 281 No 6 Yes Yes Yes
PST130_13969 SA3 & SA4 437 23 394 No 0 No Yes Yes
PST130_00285 SA1 & SA3; SA3 & SA4 317 25 207 No 0 Yes Yes Yes
PST130_14831 SA2 & SA4 596 31 139 No 0 No Yes Yes
PST130_10286 SA3 & SA4 54 33 254 No 0 LIAR(96) Yes Yes Yes
PST130_16778 SA3 & SA4 409 40 172 No 0 No Yes Yes
PST130_06503 SA1 & SA4; SA2 & SA3; SA3 & SA4 120 41 292 No 9 No Yes Yes
PST130_05944 SA2 & SA4; SA3 & SA4 320 49 318 No 0 LIAR(10) No Yes Yes
PST130_07579 SA2 & SA4 170 68 926 No 0 Yes Yes Yes
PST130_09018 SA1 & SA3 289 69 430 No 0 No Yes Yes
PST130_08031 SA1 & SA2; SA2 & SA4 162 77 206 No 0 LIAR(18) No Yes Yes
PST130_02403 SA1 & SA4; SA2 & SA3; SA3 & SA4 21 83 215 No 8 No Yes Yes
PST130_02001 SA1 & SA2; SA1 & SA3; SA1 & SA4; 65 84 148 No 0 Yes Yes Yes
SA2 & SA4
PST130_08984 SA2 & SA4 65 84 116 No 0 Yes Yes Yes
PST130_07564 SA1 & SA2; SA1 & SA3 482 86 145 No 10 No Yes Yes
PST130_15131 SA1 & SA2; SA1 & SA3; SA2 & SA3 186 87 546 No 2 No Yes Yes
PST130_02118 SA1 & SA2; SA2 & SA3; SA2 & SA4 92 88 187 No 0 Y/F/WxC(21) No Yes Yes
PST130_07513 SA1 & SA2; SA1 & SA3; SA1 & SA4 128 95 154 No 0 Yes No Yes
PST130_12956 SA1 & SA4 128 95 156 No 0 Yes Yes Yes
PST130_07448 SA1 & SA3; SA3 & SA4 192 100 191 No 0 Y/F/WxC(73) No Yes Yes

HESPs, Haustorial expressed secreted proteins; AVRs, proteins encoded by avirulence genes ;Haust, Haustorial library. PFAM, Protein family database.
Genes in boldface had nonsynonymous substitutions between SA1 and SA4 and their expressions were evaluated over a time series. Genes marked in
grey were also nonsynonymous between PST-87/7 and PST-08/21 (refer to Cantu et al., 2013). PST130_14091 also known as PST21_19014 and
PST130_13696 also known as PST21_18360.
CHAPTER 6: GENE EXPRESSION ANALYSIS 120

would be chosen that capture gene expression during early infection processes, at

various stages of haustorial and hyphae network development, and sporulation.

Comparisons were drawn from histological evaluation of Pst haustorial develop-

ment (Sørensen et al., 2012) and gene expression studies in seedlings (Wang et al.,

2007). Spore germination, formation of the substomatal vesicle, development

of infection hyphae, the formation of the haustorial mother cells, and haustoria

formation are all apparent within the first 24 hours after inoculation. Hyphae

and haustoria continue to develop in the host tissue until roughly 5 days post

inoculation (dpi). Sporogenous cells become visible at about 7 dpi. By 12 dpi to

14 dpi, depending on the experimental setup, visibly sporulating pustules are

usually apparent.

Two of the historical South African Pst isolates were further investigated for

gene expression using a selection of the 27 candidate effectors. The isolates that

were used are representatives of the first Pst pathotype detected in South Africa

in 1996: 6E16A- (SA1), and the most recent pathotype, 6E22A+ (SA4), that was

identified in 2005. These two isolates are the furthest apart in terms of time of

collection and pathogenicity as they differ in virulence for three Yr resistance

genes and were collected seven years apart (Table 4.2). They were chosen to

improve the chances of identifying a virulence-related effector candidate. This

chapter focuses on further investigation of these candidates using RT-qPCR gene

expression analysis. The nine genes selected were those polymorphic between

SA1 and SA4 (Table 6.1 boldface).

6.2 Methods

6.2.1 Inoculation and sampling

Seedlings of the stripe rust susceptible wheat variety, Avocet S, were inocu-

lated with urediniospores of the Pst South African pathotypes 6E16A- (SA1) and
CHAPTER 6: GENE EXPRESSION ANALYSIS 121

Tray 1 Tray 2 Tray 3

Isolate SA1 21 plants 21 plants 21 plants

126 samples evaluated

for gene expression of
nine genes, in triplicate.

Isolate SA4 21 plants 21 plants 21 plants

Figure 6.1: Experimental setup for the infection time course experiment.

6E22A+ (SA4) (see Section 3.1.1), using an inoculation concentration of 5 mg/ml.

As each seedling can only be sampled once, nine plants—subsequently referred

to as biological replicates—were sampled for each treatment (isolate SA1 or SA4)

at each time point (Figure 6.1) . The 63 plants (9 seedlings × 7 time points) were

equally divided between three trays (21 plants per tray). The three trays for each

of the treatments were inoculated independently, which introduced a blocking

variable to test reproducibility. Inoculated leaf samples were taken at 0, 1, 2, 3, 5,

9 and 12 dpi, taking three seedlings, per time point, from each of the three trays.

Samples were taken about 8 cm from the tip of each leaf, cut into shorter pieces

and immediately stored in the RNA stabilising agent, RNAlater (Thermo Fisher

Scientific, USA; Taylor et al., 2010). Scissors used to cut inoculated leaf samples

were wiped clean with ethanol between sample collections.

Fresh spores of both isolates were germinated and used as positive, fungal

controls. The germinated spore samples were prepared in a laminar flow cabinet.

Spores were sprinkled on a thin layer of autoclaved double distilled water in a

sterilised Petri dish, comparable to the method of Zhang et al. (2008), and kept

overnight in a dark room at 11 ◦C. After 8–12 hours a thick mat of intertwined

germination tubes was collected from the surface of the water with a spatula

and stored in RNAlater. The preserved samples in RNAlater were kept at room
CHAPTER 6: GENE EXPRESSION ANALYSIS 122

temperature for 20 days before RNA was extracted.

Caution was taken throughout the experiment to control and define condi-

tions to minimise external stimuli that could interfere with the sensitive process

of mRNA transcription (Taylor et al., 2010). A detailed explanation of the inocu-

lation protocol can be found in Chapter 3.

6.2.2 Tissue disruption and RNA extraction

Total RNA was extracted from the inoculated leaf tissue, non-inoculated wheat

and germinated fungal spore controls using the Qiagen RNeasy Plant Mini Kit

according to the manufacturer’s instructions. To minimise the time between

subsequent sampling events, the sample processing steps that follow were per-

formed on small batches of 12–24 samples as recommended by Taylor et al. (2010).

Tissue was disrupted using a mortar and pestle and the addition of extraction

sand (SiO2 ). All instruments used were washed with detergent, ethanol and

RNase AWAY decontamination reagent between samples, and cooled down in

liquid nitrogen, or on dry ice, to prevent degradation of RNA due to ubiquitous

RNases activity (Holland et al., 2003). The dry mortar and pestle were placed on

dry ice in a polystyrene box and was further cooled with liquid nitrogen. About

100 mg of extraction sand was added to each sample.

Forceps were used to move the preserved sample material from the tubes and

tapped dry on a clean paper towel to prevent the stabilising solution from forming

ice crystals when the sample comes in contact with liquid nitrogen. Samples

were then placed in the mortar, along with liquid nitrogen and extraction sand,

and homogenised into a fine powder. Without letting it thaw, the powder was

scraped with a cooled spatula into a 2.2 ml safe lock microcentrifuge tube. The

ground sample was kept on dry ice until extraction buffer was added.
CHAPTER 6: GENE EXPRESSION ANALYSIS 123

6.2.3 RNA quality control and quantification

Automated capillary-electrophoresis systems are popular for generating accurate

profiles for RNA quality assessment (Fleige and Pfaffl, 2006). The Agilent 2100

Bioanalyzer (Agilent Technologies, USA) was used to assess the quality and

quantity of the extracted RNA. The reaction kit was stored at 4 ◦C. A gel-dye mix

was first prepared according to the manufacturer’s instructions. The quality of

samples was assessed within 1 to 3 days after RNA extraction. RNA samples

were appropriately aliquoted to prevent multiple freezing and thawing steps that

impose the risk of RNA degradation (Taylor et al., 2010). RNA stocks were stored

at −80 ◦C.

6.2.4 Complementary DNA synthesis

The SuperScript IV First-Strand Synthesis System (Invitrogen/Thermo Fisher

Scientific, USA) was used for the conversion by reverse transcription of mRNA to

cDNA according to the manufacturer’s instructions. Excess RNA was removed

by adding 1 µl of E. coli RNase H to the synthesised cDNA. An aliquot of 3 µl

of cDNA was prepared and quantified on the Qubit 2.0 Fluorometer (Thermo

Fisher Scientific, USA) at the Central Analytical Facilities (CAF) at Stellenbosch

University, South Africa. cDNA was diluted to approximately 12.5 ng/µl for use

in PCR reactions, and cDNA was stored at −20 ◦C (Taylor et al., 2010).

6.2.5 Primer design

Primers for RT-qPCR were designed using the compiled Illumina sequences

obtained of the two Pst isolates, SA1 and SA4 respectively (Chapter 4). The

PrimerQuest Tool by Integrated DNA Technologies1 was used to design primers

for the nine Pst genes of interest. Primers were designed that would amplify the
1 http://eu.idtdna.com/scitools/Applications/RealTimePCR/
CHAPTER 6: GENE EXPRESSION ANALYSIS 124

respective gene from both SA1 and SA4, and produce gene amplicons between

84 bp to 129 bp in length (see Section 6.3.2), as the kinetics of the PCR reaction are

influenced by the length of the resulting amplicon.

The primer sequences were evaluated in NCBI BLAST (version 2.6.1; Altschul

et al., 1997) homology searches to ensure that they would not amplify sequences

within the wheat genome. The likelihood of the primers to form secondary

structures, such as primer dimers and hairpins were also assessed, and absence

of SNPs in primer sequences was confirmed (Derveaux et al., 2010). Primers were

manufactured by Integrated DNA Technologies, USA. Primers were empirically

tested for a negative result in a reaction with wheat template DNA and for

specificity to amplify the desired amplicon with Pst cDNA by evaluating the

melt curve of the RT-qPCR, followed by gel electrophoresis to confirm amplicon

length. Primer efficiencies were determined using CT (threshold cycle) of serial

dilutions (Derveaux et al., 2010).

6.2.6 PCR plate setup

Complementary DNA templates were used to evaluate transcription levels of

the nine Pst genes of interest. Only one target gene and the reference gene,

which is expected to be expressed constantly over the infection time course,

were evaluated on each PCR plate. The same isolates as used in sequencing in

Chapter 4 were used for inoculation. Three controls were included: two positive

controls—SA1 and SA4—in duplicate, a negative wheat control (WC) from the

same wheat variety, Avocet S, and a Non Template Control (NTC; Figure 6.2).

Quantitative PCRs of each cDNA sample were performed in triplicate for each

gene assay and time point measured in days post inoculation.

 rep 1 rep 2 rep 3 rep 1 rep 2 rep 3 rep 1 rep 2 rep 3 rep 1 rep 2 rep 3
cDNA 1 2 3 4 5 6 7 8 9 10 11 12

SA1: 0-3 dpi A SA1:0dpi SA1:0dpi SA1:0dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:3dpi SA1:3dpi SA1:3dpi
SA4: 0-3 dpi B SA4:0dpi SA4:0dpi SA4:0dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:3dpi SA4:3dpi SA4:3dpi

SA1: 0-3 dpi C SA1:0dpi SA1:0dpi SA1:0dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:3dpi SA1:3dpi SA1:3dpi
SA4: 0-3 dpi D SA4:0dpi SA4:0dpi SA4:0dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:3dpi SA4:3dpi SA4:3dpi

SA1: 5-12 dpi E SA1:5dpi SA1:5dpi SA1:5dpi SA1:9dpi SA1:9dpi SA1:9dpi SA1:12dpi SA1:12dpi SA1:12dpi SA1 rep 1 SA4 rep 1 WC
SA4: 5-12 dpi F SA4:5dpi SA4:5dpi SA4:5dpi SA4:9dpi SA4:9dpi SA4:9dpi SA4:12dpi SA4:12dpi SA4:12dpi SA1 rep 2 SA4 rep 2 NTC

SA1: 5-12 dpi G SA1:5dpi SA1:5dpi SA1:5dpi SA1:9dpi SA1:9dpi SA1:9dpi SA1:12dpi SA1:12dpi SA1:12dpi SA1 rep 1 SA4 rep 1 WC
SA4: 5-12 dpi H SA4:5dpi SA4:5dpi SA4:5dpi SA4:9dpi SA4:9dpi SA4:9dpi SA4:12dpi SA4:12dpi SA4:12dpi SA1 rep 2 SA4 rep 2 NTC

rep 1 rep 2 rep 3 rep 1 rep 2 rep 3 rep 1 rep 2 rep 3 rep 1 rep 2 rep 3
Primers 1 2 3 4 5 6 7 8 9 10 11 12
125

A SA1:0dpi SA1:0dpi SA1:0dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:3dpi SA1:3dpi SA1:3dpi
REF
B SA4:0dpi SA4:0dpi SA4:0dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:3dpi SA4:3dpi SA4:3dpi

C SA1:0dpi SA1:0dpi SA1:0dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:1dpi SA1:3dpi SA1:3dpi SA1:3dpi
GOI
D SA4:0dpi SA4:0dpi SA4:0dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:1dpi SA4:3dpi SA4:3dpi SA4:3dpi

E SA1:5dpi SA1:5dpi SA1:5dpi SA1:9dpi SA1:9dpi SA1:9dpi SA1:12dpi SA1:12dpi SA1:12dpi SA1 rep 1 SA4 rep 1 WC
REF
F SA4:5dpi SA4:5dpi SA4:5dpi SA4:9dpi SA4:9dpi SA4:9dpi SA4:12dpi SA4:12dpi SA4:12dpi SA1 rep 2 SA4 rep 2 NTC

G SA1:5dpi SA1:5dpi SA1:5dpi SA1:9dpi SA1:9dpi SA1:9dpi SA1:12dpi SA1:12dpi SA1:12dpi SA1 rep 1 SA4 rep 1 WC
GOI
H SA4:5dpi SA4:5dpi SA4:5dpi SA4:9dpi SA4:9dpi SA4:9dpi SA4:12dpi SA4:12dpi SA4:12dpi SA1 rep 2 SA4 rep 2 NTC

Figure 6.2: Plate layouts for RT-qPCR assays. Template cDNA layout: Plate layout for DNA of each biological replicate and gene assay.
Nine biological replicates were assessed for each gene assay. Primer layout: Plate layout for PCR reaction mix. Nine genes were
assessed in total. Each plate assessed transcript levels of one target, candidate Pst effector gene and one reference gene. REF,
reference gene; GOI, Gene of interest.
CHAPTER 6: GENE EXPRESSION ANALYSIS 126

6.2.7 Quantitative real-time polymerase chain reaction

Reactions were set up manually. Plates and accompanying seals were manu-

factured by Thermo Fisher Scientific, USA. Transcript levels of nine candidate

effector genes (Chapter 5; Cantu et al., 2013) were assessed using RT-qPCR. A

fully skirted, 96 well PCR plate was prepared with 2 µl (approximately 25 ng)

of template cDNA. The 8 µl reaction mix consisted of 2.4 µl of double distilled

water, 5 µl of BioRad Precision Melt Supermix and 3 pmol of each forward and

reverse primer. The plate with template cDNA was kept on an Eppendorf PCR

Cooler block (Sigma-Aldrich, USA) while the reaction mix was added. The plate

was sealed, briefly centrifuged and ran on the BioRad CFX96 Touch Real-Time

PCR System. The first part of the PCR program included the following steps:

An initiation step of 5 minutes at 95 ◦C, followed by 40 cycles of a 15 second

denaturation step at 95 ◦C, a 20 second primer annealing step at 60 ◦C and a 20

second primer extension step at 72 ◦C.

The second part of the PCR program was included to generate a dissociation

curve as an indication of the amplification specificity of the primers and to

evaluate the formation of primer dimers. High specificity was expected as

the fluorescent dye, EvaGreen, is known for its high sequence specificity, and

allowing a robust PCR with less PCR inhibition than SYBR Green I, due to its

thermal and hydrolytic stability (Mao et al., 2007). The following steps were

included in the program: 1 minute at 95 ◦C to denature the double stranded DNA

(dsDNA) with fluorescent intercalating dye to single strand DNA (ssDNA). No

fluorescence is expected after this step. To induce the formation of dsDNA, the

temperature was lowered for 10 seconds to 40 ◦C. A ramped step with a 0.2 ◦C/s

incremental increase of temperature starting at 60 ◦C and stopping at 90 ◦C was

used to denature the dsDNA incrementally. Fluorescence decreases as the dye

dissociates. A final cooling step of 10 seconds at 40 ◦C was added, where after

CHAPTER 6: GENE EXPRESSION ANALYSIS 127

the reaction was kept at 15 ◦C.

6.2.8 Reference gene selection

Examples of genes that are often used as internal references in qPCR are 18S

rRNA, 7S rRNA, U6 RNA, β-actin and glyceraldehyde 3-phosphate dehydroge-

nase (GAPDH; Schmittgen and Livak, 2008). Three genes were assessed for use

as standards of gene expression: P. striiformis elongation factor 1 (PST_EF1; Ling

et al., 2007), β-Actin (ACTB) and β-Tubulin (TUBB; Huang et al., 2012). Amplifi-

cation signals in the negative wheat control occurred in multiple qPCR reactions

with the primer pair for PST-EF1. However, no amplification with the wheat

DNA control was observed with the PST-ACTB and PST-TUBB primers. Both

genes would therefore be suitable to use as references. Due to limited wells on

the PCR plate, only one reference gene was used, and PST-TUBB was arbitrarily

chosen as the reference gene in this study.

6.2.9 Efficiency determination of primers

The BioRad2 Precision Melt Supermix contains hot-start iTaqTM DNA poly-

merase, dNTPs, MgCl2 , EvaGreen dye, enhancers and stabilisers. The poly-

merase enzyme is responsible for producing amplicons using primers, dNTPs,

and template cDNA, with the help of magnesium as a cofactor and optimal

temperature cycles. PCR efficiency describes the rate of action of the polymerase

and indicates the fold increase of the target DNA per thermocycle (Ruijter et al.,

2013). Full efficiency would mean that there is a 2-fold increase of amplicon with

every thermocycle during the exponential phase (Yuan et al., 2006). Efficiencies

between 90 % and 110 % are acceptable. Poorly calibrated pipettes are often the

reason for efficiencies to fall outside of this range. Additionally, low efficiency
2 http://www.bio-rad.com/webroot/web/pdf/lsr/literature/10022094.pdf
CHAPTER 6: GENE EXPRESSION ANALYSIS 128

can be caused by suboptimal temperatures, the presence of inhibitors or inactive

polymerase, poor primer design or amplicons with secondary structures, while

overly high efficiencies result from primer dimers or nonspecific amplicon ampli-

fication (Taylor et al., 2010). Efficiency is also not constant throughout the PCR

reaction, and low levels of DNA template can result in inaccurate determination

of efficiency (Karlen et al., 2007).

The efficiencies of primers were estimated by calculating the slope of the

standard curve of a serial dilution of template DNA. Two 2-fold serial dilutions

were made by adding RNase free water to the DNA sample, with PCR reactions

being done in duplicate. For each DNA concentration, in each dilution series, the

mean of the CT values of the two replicate PCRs was plotted against the base-10

logarithmic transformation of the dilution factor. The data was fitted to a linear

regression model and the Pearson correlation coefficient (R2 ) was assessed. The

amplification efficiency E is theoretically expected to be between 0 and 1, and

was calculated with

E = 10(−1/s) − 1, (6.1)

where the s is the gradient of the linear regression line (Kubista et al., 2006).

The obtained efficiencies were used in the efficiency corrected method to

obtain the expression pattern of each gene of interest. The relative expression, R,

of the candidate genes to the reference gene was first determined with

0
R = ECT/E0CT ,

where E and E0 are the efficiencies as calculated in Eq. (6.1) for the gene of interest

and reference gene, and CT and CT0 are the cycle threshold values for the gene

of interest and reference, respectively. The cycle threshold indicates the number

of cycles it took to reach the fluorescence threshold, FT . It is important that

CHAPTER 6: GENE EXPRESSION ANALYSIS 129

this threshold value is set to fall in the exponential phase of the amplification

process (Karlen et al., 2007). This is the earliest phase, with ample reagents, and

is followed by the linear phase as reagents decrease and finally reach the plateau

phase where reagents become depleted (Yuan et al., 2006). Default FT was used

for all PCR runs.

Transcript levels of the candidate genes were expressed as relative expression

to the reference gene P. striiformis β-tubulin (TUBB; Huang et al., 2012).

6.2.10 Statistical evaluation of the data

The treatments applied were SA1 and SA4 inoculations. These were applied

three times in three independent tray inoculations. For each tray inoculation,

three seedlings were prepared for each of the seven time point sampling efforts

(7 time points × 3 = 21 seedlings). The three treatment applications were used

as a grouping variable in a linear mixed model. Each of the nine biological

replicates per time point (3 plants × 3 trays) was assessed on a different plate.

Inter-plate variability was not corrected for. Intra-plate variability was addressed

by performing three technical PCR replications of each biological replicate (plant)

per plate (Schmittgen and Livak, 2008). Grubbs’ test (Grubbs, 1969) was applied to

identify outliers as suggested by Burns et al. (2005). The relative expression values

obtained by using the efficiency corrected method were statistically analysed.

One-way analyses of variance (ANOVAs) were performed to assess the vari-

ation within and between the groups of biological replicates at different time

points in each gene expression assay for both isolates, SA1 and SA4.

6.2.11 Linear mixed effect analysis

The R package, lme4, was used for statistical evaluation of the data (Bates et al.,

2014). To determine the relationship between the time that elapsed after inocula-
CHAPTER 6: GENE EXPRESSION ANALYSIS 130

tion and the relative expression of the candidate genes in each isolate, a linear

mixed model with random intercepts was fitted for the data generated for each

gene:

yij = β 0 + β 1 x Tij + β 2 x Iij + β 3 x Tij x Iij + b0j + eij , (6.2)

where β 0 is the fixed intercept; β 1 , β 2 , and β 3 are fixed effects for time, isolate, and

interaction, respectively; boj is a random intercept for each tray j; the x T and x I

terms are independent variables for time point and isolate, respectively; and eij is

error. The model was fitted, and assumptions that linear mixed models are based

on were assessed. These assumptions include equal variances, and normality of

the residuals and random intercepts. The tests were repeated, and re-evaluated

after a log10 transformation (Burns et al., 2005) of the relative expression values.

A likelihood ratio test of the full model against the model without the effect

(x I (Isolate), x T (Time Point), or x T x I (Isolate × Time Point)) in question were

performed (Winter, 2013) to assess which model fits the data best. A p-value

lower than 0.001 were considered statistically significant, providing evidence for

inclusion of the effect in the model. Such a high significance threshold was used

to account for the expected high variability in RT-qPCR data. Tukey multiple

comparison post-hoc tests were used to indicate where the significant differences

in effects were (Section 6.3.4).

6.2.12 Relative expression of Pst candidate effector genes

The Pst gene expression fold difference between the standardised expression

levels of SA1 and SA4 was estimated using the method proposed in Pfaffl (2001)

taking primer amplification efficiency into account:

0
R = E∆Ct (SA1−SA4) /E0∆Ct (SA1−SA4) ,
CHAPTER 6: GENE EXPRESSION ANALYSIS 131

where E and E0 are the efficiencies as calculated in Eq. (6.1) for the gene of interest

and reference gene, respectively, and ∆CT and ∆CT0 are the difference between

the two isolates (SA1 and SA4) in cycle threshold values for the gene of interest

and reference, respectively.

This method is similar to the 2−∆∆Ct method (Schmittgen and Livak, 2008)

for determining linearised values, with the difference that the 2−∆∆Ct method

assumes that primers have 100 % efficiency causing a two-fold increase of the

replicated amplicon in every thermocycle.

6.2.13 Assessment of genes

BLAST searches were performed to assess whether genes were present in both

the PST130 gene models and the revised gene models (Dobon et al., 2016). The

original PST130 gene discovery was done using the machine learning algorithm

geneid3 and Pgt gene annotations as training set, followed by filtering for trans-

posable elements (Cantu et al., 2011), while the revised annotation made use of

the 2013 UK Pst RNA-Seq data and the annotation tools cufflinks, trinity, stringtie

and portcullis (D Bunting, personal communication). BLAST searches of the nine

candidates against Pst transcript data sequenced from the 2013 UK Pst population

were used to evaluate the occurrence of alternative splicing.

6.3 Results

6.3.1 RNA yield, RNA quality scores and cDNA yield

The integrity of each RNA sample was evaluated on the Agilent 2100 Bioanalyzer

producing gel-like visuals, RNA integrity number (RIN) scores, RNA concentra-

tions, and ratios between ribosomal units. Summary statistics were performed on

the RNA yields, RIN scores and the reverse transcribed cDNA yields as required
3 http://genome.crg.es/software/geneid/
CHAPTER 6: GENE EXPRESSION ANALYSIS 132

Table 6.2: Summary statistics describing RNA yield, integrity and cDNA yield as re-
quired in the MIQE guidelines (Bustin et al., 2009). Yield was measured in
ng/µl

n Median IQR Mean SD

RNA_Yield 128 786.00 382.50 793.81 297.84
RIN 128 6.10 0.50 6.06 0.77
cDNA_Yield 128 151.50 137.00 178.26 90.98
RIN: RNA integrity number, n: number of samples, IQR: Inter-quartile range, SD:
standard deviation.

for reporting qPCR experiments (Table 6.2; Bustin et al., 2009). RIN scores had a

satisfactory mean of 6.06, while the respective means for total RNA and cDNA

were 786 and 151 ng/µl.

6.3.2 Primer design

Unique primers to each of the nine Pst candidate effector genes were designed

using PrimerQuest (Table 6.3). The NCBI databases were used in a BLAST

(version 2.6.1) search to test uniqueness of primers (Altschul et al., 1997). In no

case was a sequence similarity found that spanned 100 % of the primer length.

Primer lengths ranged from 19 to 23 nucleotides, and GC content was between 41

and 58 %. Amplicon size affects the number of amplicon copies at the threshold

fluorescence (Rutledge and Cote, 2003), so primers were designed to amplify

amplicons of identical size to ensure equal specificities in the two treatments (SA1

and SA4; Karlen et al., 2007). Amplicons were between 84 and 129 bp in length

(Table 6.3). Melting temperatures were optimised at 60 ◦C. Primers were tested

and dissociation curves were evaluated for specificity in the positive control Pst

cDNA. The negative control, wheat variety Avocet S gDNA and the NTCs did

not show any amplification. Further details on primer design, the location of

amplicons and the depth of coverage of the sequence data used to design primers

can be found in Appendix C, Figures C.1 to C.9.

 Table 6.3: Primer and amplicon specifications for Pst candidate effector gene identification

Gene Primer Primer Amplicon Amplicon GC

Efficiency %
name sequence length sequence length content

GTGGCCCTAGTGTACCAATTAT 22 GTGGCCCTAGTGTACCAATTATCTGGCATCAATGCCAACTCGATCGTCTCGCCTAAGCCCAACCAAA 50
PST130_02001 84 88
CTCTCCTGGATTGAGAGTTTGG 22 CTCTCAATCCAGGAGAG 50
CGAGGAACCCAAATATGCTAGT 22 CGAGGAACCCAAATATGCTAGTCCAAAATATGATSCGCCCTACGAGAAGACCCCTGATGAAGAGCCA 45
PST130_02403 122 107
GACGGTAGCCGTCTTTCTTT 20 AAATACTCGGCCCCAAGCTACGATTACAATCCACCAAAGAAAGACGGCTACCGTC 50
ACTTGGTACGGTGGACATTC 20 ACTTGGTACGGTGGACATTCGGCTGTGGCCAGGTTTTTGCGCCGCTTGGTTAATTACTTTCACCCAA 50
PST130_05023 97 97
CCTTGGCTTCCTTGCTCTTA 20 GAAAGATGAGTAAGAGCAAGGAAGCCAAGG 50
133

CAGCGGTGTCATTGCTTTAC 20 CAGCGGTGTCATTGCTTTACCTACTTCCAACCAAGCACAAATCGAAACTCGGGCCGAGAAGACCCGT 50
PST130_06503 98 107
TGTATTCGGAAGAGGCGTATTT 22 TCCAGCGACAAATACGCCTCTTCCGAATACA 41
GTACCGAGCAGGACGAATTATG 22 GTACCGAGCAGGACGAATTATGTGCCGAGCATTTACTTCCAAGTTACCCAACTCTCAAGGTGTTTT 50
PST130_07513 89 94
GTATACGGCCATCCTTCCATTT 22 CAAATGGAAGGATGGCCGTATAC 45
GAGCGAACTCAACCGCTAATA 21 GAGCGAACTCAACCGCTAATACCCCTGCTGCAAGTACTCCTGTCGCTAACACGACCTCCCCGACCCA 48
PST130_09275 92 101
CAGCCGTACCCGAGTTATATTT 22 ATCCACATCCTCCACTGGTGCACCA 45
CTACCATCATTAGACGGCACAT 22 CTACCATCATTAGACGGCACATTGTCGAATGCCCCATCACCTTCGTGGCAACTGACTATTGACAAT 45
PST130_12487 107 90
GCACTTGCTTCCACCATAAAC 21 GGTCAAATCAGGAACCGTAGGTTTATGGTGGAAGCAAGTGC 48
CAGAGCACTTCCGCCTTAC 19 CAATTTTCGAGAAGCGTGCCGAGACTGAAGGCACCGGAAAAGGTGAATCAAGCTCCCGCTCCTTAG 58
PST130_12491 90 90
CGAGAGGGCAATGTTGAGAA 20 GTGGCTGCAGCAACCAAGTTGGCC 50
TGTTTGCCCTAGCTTCTTCTATC 23 TGTTTGCCCTAGCTTCTTCTATCCATGCCGACGCAGGACTCAACCCCAATGACGCTCCAGATGACGT 43
PST130_12956 98 92
GGTGTCGAAGTTCTCTGATGTC 22 CATCGAATTGACATCAGAGAACTTCGACACC 50
CHAPTER 6: GENE EXPRESSION ANALYSIS 134

6.3.3 Efficiency determination of primers

Primer efficiency was evaluated using the standard curve method. The CT values

of a cDNA dilution series were plotted, with log10 dilution fold on the x-axis

and CT on the y-axis. A linear regression was fitted to the data and the Pearson

correlation coefficient (R2 ) calculated (Figure 6.3). This indicated how well the

data fitted a linear model, with R2 = 1 being a 100 % fit. A high R2 is needed

to accurately determine the efficiency of primers. It is recommended that the

efficiency of primers should be within 10 % of each other when a relative gene

amplification comparison is to be made. Less optimisation is required when the

efficiencies are taken into account as in the efficiency correction methodology

used in this work (Schmittgen and Livak, 2008). R2 values of greater than 0.95

were achieved for all Pst gene primers except for PST130_12491, which had a R2

value of 0.81.

6.3.4 Statistical analysis of the relative expression of nine Pst candidate

effector genes

Relative expression values were calculated using the method proposed in Pfaffl

(2001, See Section 6.2.12). To determine the relationship between the time that has

elapsed since Pst inoculation and the relative expression of the candidate genes in

each South African isolate, a linear model with mixed effects was fitted to the data

with “Gene” and “Time Point” and their interaction as fixed effects, and “Tray”

as a blocking variable or random intercept. This approach was taken as sampling

was not random as is expected in a simple linear model (Fitzmaurice et al., 2008).

The model explains the relationship between the independent and dependent

variables. An error term is used where the model does not fully represent the

data. It is expected that the three plants that were inoculated together, placed

in the same tray, will be more similar to each other. The mixed model therefore
 TUBB PST130_02001 PST130_02403 PST130_05023 PST130_06503

36

32

28

y = 27 + -3.1 ⋅ x, R 2 = 1, E = 111% y = 30 + -3.6 ⋅ x, R 2 = 0.99, E = 88% y = 28 + -3.2 ⋅ x, R 2 = 0.99, E = 107% y = 28 + -3.4 ⋅ x, R 2 = 0.99, E = 97% y = 24 + -3.2 ⋅ x, R 2 = 0.96, E = 107%
24
y

PST130_07513 PST130_09275 PST130_12487 PST130_12491 PST130_12956

135

36

32

28

y = 32 + -3.5 ⋅ x, R 2 = 0.96, E = 94% y = 27 + -3.3 ⋅ x, R 2 = 1, E = 101% y = 32 + -3.6 ⋅ x, R 2 = 0.98, E = 90% y = 32 + -3.6 ⋅ x, R 2 = 0.81, E = 90% y = 26 + -3.5 ⋅ x, R 2 = 1, E = 92%
24
-1.5 -1.0 -0.5 0.0 -1.5 -1.0 -0.5 0.0 -1.5 -1.0 -0.5 0.0 -1.5 -1.0 -0.5 0.0 -1.5 -1.0 -0.5 0.0
x

Figure 6.3: Linear regression figures indicate estimated efficiency of primers for nine Pst Candidate gene assays and the reference gene,
β-tubulin (TUBB). The threshold cycle number is indicated on the y-axis and plotted against the log10 dilution fold (x-axis). The
Pearson correlation coefficient, R2 , indicate how well the data fitted a linear model. Values over 0.95 are desired.
CHAPTER 6: GENE EXPRESSION ANALYSIS 136

reduces the error term by introducing the variable “Tray”. At every time point,

three samples (seedlings) were taken from each of the three trays. The same

sampling procedure was applied for both SA1 and SA4. The mixed model shown

in Equation 6.2 was at first fitted to the data.

Evaluations of the assumptions of linear mixed models were performed for

the relative expression dataset. The residuals of relative gene expression and the

random intercept (the grouping variable “Tray”) did not fit a normal distribution.

The residuals also did not scatter equally around the y = 0 horizontal line as

expected when variances are equal and showed clear fan-like patterns in some

cases. Appendix C, Figures C.10(i), (ii), and (iii) illustrate the graphical tests for

the whole dataset, while Figures C.11 and C.12 show assessments for each isolate

and gene.

Due to the use of the grouping variable, the normal probability plot of the

random intercepts was constructed from limited points as the intercepts per gene

only consisted of six data points at each time point, three per isolate. This data

was therefore only plotted for the whole dataset and not by gene.

The relative expression data did not follow a normal distribution and a log10

transformation was applied. Graphical tests for normality and equal variances of

the residuals were repeated. The log10 transformed data fitted the assumptions

of a linear mixed model considerably better and it was concluded to proceed

using the transformed data in the linear mixed model (Appendix C, Figures C.13,

C.14, and C.15). As equal variances and normality are assumed for the residuals

of the log10 transformed data, parametric tests can be applied. Variability of the

data across different trays was assessed by using a one-way ANOVA with “Trays”

as fixed effect on subsets of the data that included expression data of one isolate

and one gene at a specific time point (nine data points for each of the time points).

Time points 0 and 1 were excluded from this evaluation due to too many missing

values. This resulted in analysing the effect “Trays” on nine genes at five time
CHAPTER 6: GENE EXPRESSION ANALYSIS 137

points and was done for two isolates (90 ANOVAs). The effect of “Trays” over

these 90 cases were quantified.

The between group variance (between the three trays) was in only 15 % of

the cases more than the within groups variance (plants per tray). This showed

that there existed a high level of variability in the data. Such variation is often

accumulated from the multiple steps in RT-qPCR, described by some as a “fragile

assay” (Bustin and Nolan, 2004), due to its sensitivity to inevitably accumulate

technical noise. This result should be considered in further interpretation of the

data.

To assess the significance of the fixed effects (“Time Point” and “Isolate”) in

the model, likelihood ratio tests were performed on two linear mixed models,

one including the effect in question (“Time Point” or “Isolate”) and one without.

Because of the high variability in RT-qPCR data, a p-value was only considered

significant if it was smaller than 0.001. A significant p-value obtained indicated

that the fixed effect term was significant to include in the model. The factor

“Time Point” was significant for seven Pst genes (Table 6.4). For PST130_12956

and PST130_02403 the term “Time Point” was not significant. Figure 6.4 further

revealed relatively stable expression for PST130_12956, while PST130_02403

showed large error bars, especially at early time points. Variability in the data

makes it difficult to conclude a change in expression for PST130_02403. The

fixed effect “Isolate” was not statistically significant with any of the nine Pst

genes, both isolates displaying a similar expression profile across all time points

(Figure 6.4).

Multiple comparisons were done using the Tukey test to determine between

which time points significant differences in gene transcription occurred (Table 6.5).

As the term “Isolate” and the interaction term “Isolate × Time Point” were not

significant for any of the nine Pst genes, this showed that SA1 and SA4 have a

similar expression profile across all time points, for all genes (Figure 6.4).
CHAPTER 6: GENE EXPRESSION ANALYSIS 138

PST130_02001 PST130_02403 PST130_05023

-1
Relative Expression of Target Gene to Reference Gene

-2

-3

PST130_06503 PST130_07513 PST130_09275

1
Isolate
0 SA1

-1 SA4

-2

-3

PST130_12487 PST130_12491 PST130_12956

-1

-2

-3
0 1 2 3 5 9 12 0 1 2 3 5 9 12 0 1 2 3 5 9 12
Days Post Inoculation

Figure 6.4: Relative gene expression (log10 transformed) of nine candidate effector genes
expressed in the Pst isolates SA1 and SA4 measured at different time points
after inoculation. Significant changes in expression across the time series were
seen in all genes, except PST130_02403 and PST130_12956. PST130_06503 and
PST130_09275 showed the most dynamic expression patterns, while other
genes showed smaller differences in gene expression across time points. The
gene, β-tubulin, was used as reference gene.
CHAPTER 6: GENE EXPRESSION ANALYSIS 139

Table 6.4: Significance of the factor “Time Point” in the linear mixed model for those
genes where it was significant

Gene Chi-squared Df p-value

PST130_02001 22.542 6 0.000 965 4
PST130_05023 22.919 5 0.000 349 8
PST130_06503 113.71 6 < 2.2 × 10−16
PST130_07513 31.358 5 7.96 × 10−6
PST130_09275 173.93 6 < 2.2 × 10−16
PST130_12487 23.837 5 0.000 233 4
PST130_12491 27.644 5 4.27 × 10−5

6.3.5 Expression profiles of candidate genes

Significant changes in expression across the time series were seen in all genes, ex-

cept PST130_02403 and PST130_12956. PST130_06503 and PST130_09275 showed

similar and the most dynamic expression patterns. The remaining five genes

showed smaller differences in gene expression across time points. Expression pro-

files of PST130_02001 and PST130_05023 were comparable, while PST130_07513,

PST130_12491 and PST130_12487 followed a similar trend. (Compare Figure 2.6

that broadly illustrates the infection process and describes the physical processes

during the time course of infection in Pst.).

6.3.6 Gene validation using revised gene models and transcript data

The nine genes were assessed for alternative splicing using transcript data. The

quality of the PST130 gene models, specifically for the nine genes evaluated

were also assessed using improved PST130 gene models (Dobon et al., 2016).

PST130_07513 and PST130_12491 lacked high sequence similarity with predicted

genes in the revised gene models. The remaining seven gene sequences had high

(roughly 95 %) similarity and reasonable coverage with the revised predicted

genes. In four of the seven genes, PST130_02001, PST130_05023, PST130_06503

and PST130_09275, no evidence for alternative splicing was found. PST130_-

02001, PST130_05023, PST130_06503 and PST130_09275 are therefore most likely

CHAPTER 6: GENE EXPRESSION ANALYSIS 140

Table 6.5: Multiple comparisons between time points for each gene that showed signifi-
cant difference in expression over the time series. Differences with a p-value
of <0.001 were considered significant. From this data and Figure 6.4 it was
clear that PST130_06503 and PST130_09275 displayed a much more dynamic
expression pattern across time points compared to the other genes tested

Gene Time Point comparison z value Pr (> |z|)

PST130_02001 3-1 3.002 0.03673
12 - 1 3.77 0.00266
12 - 2 3.343 0.01265
12 - 9 3.076 0.02929
PST130_05023 5-1 3.613 0.00396
12 - 1 3.933 0.00113
12 - 2 3.242 0.01432
PST130_06503 3-0 6.876 < 0.001
5-0 9.337 < 0.001
9-0 9.008 < 0.001
12 - 0 3.532 0.0074
3-1 7.08 < 0.001
5-1 9.671 < 0.001
9-1 9.293 < 0.001
12 - 1 3.578 0.00616
3-2 5.257 < 0.001
5-2 8.409 < 0.001
9-2 7.963 < 0.001
5-3 3.153 0.02647
12 - 3 -4.403 < 0.001
12 - 5 -7.603 < 0.001
12 - 9 -7.137 < 0.001
PST130_09275 3-0 4.295 <0.001
5-0 6.541 <0.001
9-0 8.595 <0.001
12 - 0 3.305 0.0157
3-1 5.607 <0.001
5-1 8.297 <0.001
9-1 10.763 <0.001
12 - 1 4.466 <0.001
3-2 4.685 <0.001
5-2 8.064 <0.001
9-2 11.296 <0.001
12 - 2 3.335 0.0142
9-3 5.498 <0.001
12 - 5 -5.077 <0.001
12 - 9 -8.217 <0.001
PST130_12487 12 - 1 2.99 0.03029
12 - 2 4.44 < 0.001
12 - 3 3.45 0.00676
12 - 5 3.38 0.00852
12 - 9 3.53 0.00495
PST130_12491 12 - 1 4.158 < 0.001
12 - 2 3.864 0.00147
12 - 3 4.486 < 0.001
CHAPTER 6: GENE EXPRESSION ANALYSIS 141

correctly annotated and low risk sequences for alternative splicing. Significant

alternative splicing was revealed for PST130_02403. PST130_12487 displayed two

retained introns, while two overlapping genes in the new gene models mapped

to PST130_12956.

6.4 Discussion

Early time points yielded little fungal RNA due to the low Pst biomass in infected

wheat tissues. This was also the case in the RNA-Seq study of Dobon et al. (2016).

This is unfortunate as multiple effector proteins are known to be deployed during

the first 24 hours after inoculation. Consequently, amplification failed in samples

that were collected early after inoculation, mostly at 0 and 1 dpi, and occasionally

at 2 dpi, as the copy number of target sequences was not sufficiently high.

Statistical evaluation using a linear mixed model revealed that expression

patterns between the two isolates did not vary significantly. Differences in gene

expression across different time points were significant for most genes, with some

genes showing a dynamic expression pattern over the course of the time series.

However, considerable inter-plate variation was detected, and the relative gene

expression determination with efficiency correction did not correct for inter-plate

variability. One option of standardisation is to include a calibration sample in

multiple wells across all plates to correct for plate technical variation. Such

a sample can be prepared for each gene in the experiment to allow sufficient

quantities for all inter-plate comparisons.

The possibility of high biological variance in expression patterns of effectors

cannot be excluded. In the rice blast fungus Magnaporthe oryzae, clonal variation

in effector gene expression (CVEGE) has been suggested as a mechanism to

escape host recognition, a different suite of effector genes being expressed in

individual blast lesions (Mark Farman, University of Kentucky, personal commu-

CHAPTER 6: GENE EXPRESSION ANALYSIS 142

nication). If this was the case in Pst, different seedlings, or even infection sites on

a single seedling, inoculated with the same isolate might exhibit differences in

effector gene expression profiles. The discovery in M. oryzae establishes a new

paradigm for plant-microbe recognition wherein resistance involves detection

of deterministic Avr effectors which are layered over suites of effectors that are

variably expressed among individuals. Consequently, tracing the expression of

such effector genes in host-microbe interaction studies becomes a more difficult

proposition and would require a different approach to RT-qPCR analysis in whole

seedling leaves.

Pst gene expression early in the infection process, between 0 dpi and 1 to

2 dpi, needs further investigation to draw sound conclusions. For later time

points, PST130_05023 and PST130_02001 displayed a similar expression pattern,

showing an increase in expression early in the infection process that differed

between SA1 and SA4, although it was not statistically different, but had nearly

identical expression patterns at the later time points. This could indicate that

both these genes are functional in the same or co-occurring infection processes.

PST130_05023 was the only gene that was assessed in the current study as well

as in the RT-qPCR evaluation of Avocet S inoculated with PST-08/21 (Cantu

et al., 2013). In Cantu et al. (2013) it was found that PST130_05023 expression

peaked at sporulation (14 dpi), similar to the result in the current study, where

the expression peak was observed at sporulation (12 dpi).

The main differences in the evaluated gene expression profiles were between

5 and 9 dpi, and 9 and 12 dpi. Genes can be placed in three groups according to

their expression profiles.

Group 1 PST130_02001 and PST130_05023 shared an increase in expression up

to 3 dpi to 5 dpi, followed by a decrease in expression from 5 dpi to 9 dpi

and another increase from 9 dpi to 12 dpi. This could indicate that the gene
CHAPTER 6: GENE EXPRESSION ANALYSIS 143

is involved in the early establishment of the Pst colony, and then functional

again during the sporulation processes, such as the formation of vertical

hyphae and spores. These genes all contained a PFAM domain (PFAM,

Protein family database), and were expressed in both infected material and

haustoria (Cantu et al., 2013).

Group 2 PST130_07513, PST130_12491 and PST130_12487 exhibited an expres-

sion pattern of initial increase up to 3 dpi, followed by a relatively stable

expression, showing a slight increase all the way up to 12 dpi. This could

indicate some functionality during the early stages of colony establishment,

plus a constant requirement for the protein throughout the asexual lifecycle.

Group 3 PST130_06503 and PST130_09275 showed a similar expression profile.

A steep increase in gene expression was observed from 2 dpi to 5 dpi, with

maximum expression at 9 dpi falling off at 12 dpi. From the expression

profile, one can speculate that these genes have their main function in

establishment and maintenance of the Pst colony, and do not have a role

in sporulation. In Cantu et al. (2013) PST130_06503 was expressed in the

haustoria, while PST130_09275 was expressed in the infected material, but

not in the haustoria.

No statistically significant change was identified in PST130_02403 and PST130_-

12956 expression over the time course of this study. For PST130_02403 this could

be due to high variability in the data, as illustrated by the error bars at early time

points. Further investigation of the expression profile of this gene is needed to

draw conclusions. Variation in the data for PST130_12956 is smaller, and a fairly

stable expression across the infection process for this gene is concluded. Some

similarities can be drawn between the expression profiles of PST130_12956 and

genes in Group 1 in the previous paragraph.

The nine candidate effector genes were assessed for alternative splicing using

Pst transcript data. The genes were further verified by evaluating whether the
CHAPTER 6: GENE EXPRESSION ANALYSIS 144

candidate effector genes were included in both PST130 gene annotations. This

analysis revealed no evidence of alternative splicing for PST130_02001, PST130_-

05023, PST130_06503 and PST130_09275. High sequence similarity was also found

in the new gene models for these four genes. PST130_07513 and PST130_12491

did not have good hits in the new gene models and could have been misidentified,

in either attempt to predict genes. Although primers were not designed to amplify

fragments across splice sites, underestimation of gene expression could have

resulted in alternatively spliced genes if the exon containing the amplicon was

excluded during splicing.

In a functional study using heterologous expression screens in Nicotiana

benthamiana, accumulation patterns of PST130_05023 were observed in endomem-

branes that are suspended in the cytoplasm of leaf cells (Petre et al., 2016b).

6.5 Conclusion

Clear conclusions regarding gene expression could not be drawn from the RT-

qPCR experimental procedure applied in this chapter. Interesting questions arise

from the variability in the relative expression data. Future work addressing these

questions should involve the inclusion of different biological replicates in one

PCR run to investigate reproducibility. Other methods, such as RNA-Seq could

be explored, but as shown, does not address the problem of low fungal transcripts

at early time points.

In retrospect, it could be argued that the method would only work if genes

had no homologs and if they were absent from one of the isolates. If primers were

designed across SNP sites, they could have been more successful in displaying the

differences between the isolates for the nine candidate genes. Further discussion

on the qPCR experimental procedure outlining pitfalls and precautions taken is

included in Appendix C.3.

Chapter 7

Analysis of the Current Stripe Rust

Threat in South Africa

7.1 Introduction

7.1.1 Pst virulence since 2005

T HE FIRST DISCOVERY of Puccinia striiformis f. sp. tritici in South Africa was in

1996 (Pretorius et al., 1997), with three subsequent pathotypes that appeared to

have evolved in a clonal, stepwise manner (Visser et al., 2016). Previous analysis

that compared the virulence profiles of the historical and current Pst popula-

tions suggested that the population has stayed fairly consistent, with routine,

traditional pathology testing on wheat differential sets (Table 7.1) reporting no

additional virulences since 2005 (Agricultural Research Council, Small Grain

(ARC-SG), personal communication).

The prevalence of Pst pathotypes in South Africa during the growing seasons

of 2008 to 2016 is shown in Figure 7.1(i). Data was obtained from the South

African Pst virulence survey undertaken by ARC-SG, South Africa. The SA2

pathotype, 6E22A- (detected in 1998), and the SA4 pathotype, 6E22A+ (detected

in 2005), were present in all eight seasons. Pathotype 6E16A- (SA1), which

145
CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 146

Table 7.1: Wheat differential lines used at Agricultural Research Council, Small Grain,
Bethlehem, South Africa to identify Pst pathotypes. Standard world (1 to 7)
and European (10 to 17) differential sets are listed. Lines 9, 8 and 18, containing
resistance genes Yr5, Yr9 and YrA respectively, are used as supplemental lines

No Line/variety Yr gene
1 Chinese 166 1
2 Lee 7,22,23
3 Heines Kolben 2,6
4 Vilmorin 23 3a,4a
5 Moro 10,Mor
6 Strubes Dickkopf 25,Sd
7 Suwon 92/Omar Su,4
8 Clement 2,9,25,Cle
9 Triticum spelta 5
10 Hybrid 46 4b
11 Reichersberg 42 7,25
12 Heines Peko 2,6,25
13 Nord Desprez 3a,4a
14 Compair 8,19
15 Carstens V 25,32,Cv
16 Spaldings Prolific Sp,25
17 Heines VII 2,25,HVII
18 Avocet R A

was first detected in 1996, only occurred in samples collected in 2009 and 2011.

Figure 7.1(ii) displays the percentage of Pst samples, classified by pathotype,

collected between 2008 and 2012, and in 2016, and Figure 7.1(iii) shows the

corresponding sampling sites of each isolate by pathotype from 2008 to 2012.

Information about the number of samples collected per year per location could

not be obtained. The available survey data indicate that pathotype 6E22A+

was the most prevalent, followed by regular occurrence of 6E22A-, at a lower

frequency. It seems that 6E16A- has mostly been replaced by the 6E22 pathotypes,

with the pathotype 6E22A+, virulent to YrA, predominating.

7.1.2 Global reports on Pst population shifts

The dynamics and demographics of several Pst populations have been described.

Wellings (2007) described three reasons for a change in population demography

in clonal populations as seen in Australia. Firstly, increased pathogen virulence

CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 147

100
2016

2015

Samples collected in year (%)

2014 75

2013
Pathotype
Year

Absent 6E16A−
2012 50
Present 6E22A−
6E22A+
2011

2010 25

2009

2008
0
6E16A− 6E22A− 6E22A+ 2008 2009 2010 2011 2012 2016
Race Year

(i) South African Pst pathotypes ob- (ii) Percentage of Pst isolates, by specific
served between 2008 and 2016. pathotypes, found between 2008 and
2012, and 2016.

6E22A+
6E22A-
Limpopo
6E16A-

Mpumalanga
Gauteng
North West

KwaZulu-Natal

Northern Cape Lesotho

Free State

Eastern Cape

Western Cape

(iii) Collection sites and pathotypes of Pst isolates between 2008 to 2012
in South Africa.

Figure 7.1: Prevalence of Pst pathotypes in South Africa between 2008 and 2016. Data
was made available by the Agricultural Research Council, Small Grain (ARC-
SG) of South Africa (map adapted from SENSAKO’s oral presentation during
the Borlaug Global Rust Initiative (BGRI), New Delhi, 2013).
CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 148

through mutation and selection following resistance gene deployment are com-

mon mechanisms (Brown and Hovmøller, 2002; McDonald and Linde, 2002;

Milus et al., 2009; de Vallavieille-Pope et al., 2012). Secondly, exotic incursions

have been shown to occur over long distances, causing sudden unsuspected

epidemics and shifts in the pathogen population dynamics. This has included

Pst pathotypes with increased aggressiveness (Milus et al., 2009). The establish-

ment of such incursions seems to depend on the host population and possible

abiotic stressors (Wellings, 2007). Lastly, the survival of Pst mutations by genetic

drift, during unfavourable conditions, can totally change the following season’s

re-emerging population. Such population bottlenecks can lead to a severe shift in

allele frequencies.

Exotic incursions in the USA in 2000 and Australia in 2002 have shown

relatively homogeneous incursions suggesting that a single genotype of Pst

was introduced (Wellings, 2007; Milus et al., 2009; Hovmøller et al., 2016). In

Europe, a major population shift was seen in 2011 that included several Pst

pathotypes, some of which could infect the wheat variety, Warrior. Through

pathotyping in subsequent years, these newly introduced Pst pathotypes were

shown to be diverse. A method to rapidly genotype and compare field samples

was developed by Hubbard et al. (2015). Using next-generation sequencing

data, it was confirmed that the older UK Pst population was replaced by a new,

much more diverse population. UK and French Pst isolates pre-2011 were closely

related, with low genetic diversity, while isolates from 2011 and 2013 formed a

distinct, more diverse population. Isolates collected post-2011, included the Pst

pathotype virulent on the wheat variety Warrior and three more genetic groups.

Hubbard et al. (2015) also found historical and new Pst isolates with different

genetic profiles, but the same virulence profile. This radical population shift in

2011 was also confirmed by Hovmøller et al. (2016), and the authors suggested

that the two new pathotypes, “Warrior” and “Kranich” carried characteristics

that suggested that they might have originated from a sexual population possibly

from the near-Himalayan region in Asia.

CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 149

7.1.3 Objectives

Stripe rust is a global problem of increasing proportion (Hovmøller et al., 2010)

and migration of spores over long distances was repeatedly reported (Ali et al.,

2017). Furthermore, the existence of recombinant Pst populations increases the

risk for new variants appearing in each new season (Rodriguez-Algaba et al.,

2014). In Chapter 4, four historical South African Pst isolates were analysed in

context with other global isolates. In this chapter, changes seen in the current

field population of Pst in South Africa were characterised in context with the

global isolates examined in Chapter 4.

7.2 Materials and methods

7.2.1 Stripe rust samples used in RNA sequencing analyses

Field samples of stripe rust were collected in South Africa during the 2014 and

2015 wheat growing seasons. Twenty-five single lesion leaf samples of Pst-

infected wheat leaves were collected from various locations (Figure 7.2; Table 7.2).

In 2013, a Puccinia sample was collected on wild rye and found to be virulent on

wheat with the pathotype classification 6E16A- (Pretorius et al., 2015), similar

to SA1. This isolate was included in this analysis and named 13/SAZP1. Mi-

crosatellite markers have also been used to describe this isolate, also known as

Sutherland (Visser et al., 2016). In addition, four Pst isolates were collected from

Ethiopia and 14 isolates from Kenya during the 2014 growing season. All stripe

rust infected leaf samples were stored in RNA stabilising solution (RNAlater,

Life Technologies, UK). Selected samples (44) that passed quality assessments as

explained in Chapter 3 were included in the analysis.

CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 150

Table 7.2: African isolates collected between 2013 and 2015. Read frequency graphs of
these isolates are displayed in Appendix D, Figures D.1 and D.2

Isolate Isolates Country of Year Type

number (year/code) isolation collected of data

49 14/SADL1 South Africa 2014 RNA-Seq

50 14/SADL2 South Africa 2014 RNA-Seq
51 14/SADL3 South Africa 2014 RNA-Seq
52 14/SADL4 South Africa 2014 RNA-Seq
53 14/SADL5 South Africa 2014 RNA-Seq
54 14/SADL6 South Africa 2014 RNA-Seq
55 14/SATT1 South Africa 2014 RNA-Seq
56 14/SATT2 South Africa 2014 RNA-Seq
57 14/SATT3 South Africa 2014 RNA-Seq
58 14/SATT4 South Africa 2014 RNA-Seq
59 14/SATT5 South Africa 2014 RNA-Seq
60 13/SAZP1 South Africa 2013 RNA-Seq
61 14/SAZP2 South Africa 2014 RNA-Seq
62 14/SAZP3 South Africa 2014 RNA-Seq
63 15/SAZP1* South Africa 2015 RNA-Seq
64 15/SAZP2 South Africa 2015 RNA-Seq
65 15/SAZP3 South Africa 2015 RNA-Seq
66 15/SAZP4 South Africa 2015 RNA-Seq
67 15/SAZP5 South Africa 2015 RNA-Seq
68 15/SAZP6 South Africa 2015 RNA-Seq
. 69 15/SAZP7 South Africa 2015 RNA-Seq
70 15/SAZP8 South Africa 2015 RNA-Seq
71 15/SAZP9 South Africa 2015 RNA-Seq
72 15/SAZP10 South Africa 2015 RNA-Seq
73 15/SAZP11 South Africa 2015 RNA-Seq
74 15/SAZP12 South Africa 2015 RNA-Seq
75 14/ET2 Ethiopia 2014 RNA-Seq
76 14/ET3 Ethiopia 2014 RNA-Seq
77 14/ET4 Ethiopia 2014 RNA-Seq
78 14/ET5 Ethiopia 2014 RNA-Seq
79 14/K2 Kenya 2014 RNA-Seq
80 14/K4 Kenya 2014 RNA-Seq
81 14/K5 Kenya 2014 RNA-Seq
82 14/K6 Kenya 2014 RNA-Seq
83 14/K7 Kenya 2014 RNA-Seq
84 14/K8 Kenya 2014 RNA-Seq
85 14/K9 Kenya 2014 RNA-Seq
86 14/K10 Kenya 2014 RNA-Seq
87 14/K11 Kenya 2014 RNA-Seq
88 14/K12 Kenya 2014 RNA-Seq
89 14/K13 Kenya 2014 RNA-Seq
90 14/K14 Kenya 2014 RNA-Seq
91 14/K15 Kenya 2014 RNA-Seq
92 14/K16 Kenya 2014 RNA-Seq
*also known as Sutherland (Visser et al., 2016); 14/ET2-5 (Bueno-Sancho et al., 2017)
obtained from D Hodson; Kenyan field samples provided by DGO Saunders (14/K2-16)
obtained from R Wanyera; South African field samples collected by D Lesch (SADL), T
Terefe (SATT), and ZA Pretorius (SAZP).(15/SAZP2 was not used in the analyses due to
poor read frequency graph.)
CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 151

6E22A-
7E22A-
6E22A+ 2014

2014

2014

Free State

2013

6E16A-

2014
Western Cape

Figure 7.2: Locations of Pst collections between 2013 and 2015 for RNA sequencing and
historical isolate collection sites.

7.2.2 Transcriptome sequencing of stripe rust infected wheat leaves

Total RNA was extracted using the Qiagen RNeasy Mini kit (Qiagen, Germany).

RNA integrity and quantity were assessed using the Agilent 2100 Bioanalyzer

(Agilent Technologies, USA) as explained in Chapter 3. RNA was reverse tran-

scribed to cDNA using the Illumina TruSeq RNA sample preparation kit (Illumina,

UK). Transcriptome sequencing was perfomed on the Illumina HiSeq instrument

at the Earlham Institute, UK. Bowtie software (version 0.12.7; Langmead et al.,

2009) from the TopHat package (version 1.3.2; Trapnell et al., 2012) was used

to align the pair-end reads of each transcriptome independently to the PST130

reference genome (Cantu et al., 2011). Purity of isolates was confirmed using the

method described in Chapter 3. Phylogenetic and population structure analyses,

followed by FST calculations and the Watterson estimator of population diversity

(θ̂W ), were used to describe genetic variation in population clusters in a similar

manner to the methodology followed in Chapter 4 and described in Chapter 3.

These analyses were performed on the field isolates listed in Table 7.2 and the
CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 152

48 isolates in Chapter 4 in Table 4.1, resulting in the assessment of 92 isolates in

total.

7.2.3 Pst pathotype determination

Roelfs et al. (1992) explained that the infection types given in Table 7.3 “are often

refined by modifying characters as follows: = means uredinia at lower size limit

for the infection type; " means uredinia somewhat smaller than normal for the

infection type; + means uredinia somewhat larger than normal for the infection

type; ++ means uredinia at the upper size limit for the infection type; C means

more chlorosis than normal for the infection type; and N means more necrosis

than normal for the infection type.

Discrete infection types on a single leaf when infected with a single biotype are

separated by a comma (e.g., 4, ; or 2=, 2+ or 1,3C). A range of variation between

infection types is recorded by indicating the range, with the most prevalent

infection type listed first (e.g., 23 or ;1C or 31N) (Roelfs and Hettel, 1992).”

Fresh inoculum was prepared by inoculating seedlings of the susceptible

wheat variety Morocco. Four cultures were prepared: two cultures of the histori-

cal South African isolates, SA1 and SA4 and two more recently collected isolates,

13/SAZP1 and 15/SAZP4. The isolate 13/SAZP1 was previously tested and

identified to be pathotype 6A16A- (Pretorius et al., 2015), while 15/SAZP4 was

identified as 6E22A+ on the standard differential sets, using the scoring system

in Table 7.3 (ZA Pretorius, unpublished data).

An extended set of wheat differential lines were inoculated with each Pst

isolate after growing seedlings for 7 to 8 days as explained in Chapter 3. Infection

types were evaluated 21 days after inoculation and reported in Appendix D,

Tables D.1 and D.2 (UK differential lines were obtained from S Holdgate, National

Institute of Agricultural Botany (NIAB), UK and DGO Saunders, John Innes

Centre (JIC), UK).

CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 153

Table 7.3: Infection type scores used to assess Pst infection on wheat seedlings (adapted
from Roelfs et al., 1992 and McIntosh et al., 1995)

Host response (class) Infection typea Disease symptoms

Immune 0 No visible uredia

Very resistant ; Necrotic flecks
Resistant ;N Necrotic areas without sporulation
Resistant 1 Necrotic and chlorotic areas with re-
stricted sporulation
Moderately resistant 2 Moderate sporulation with necrosis
and chlorosis
Moderately susceptible 3 Sporulation with chlorosis
Susceptible 4 Abundant sporulation without chloro-
sis

7.3 Results

7.3.1 Clustering analysis using RNA-Seq and whole genome sequencing

data

To investigate the pathotype and genetic profile of the current Pst population in

South Africa, stripe rust infected wheat samples were collected from wheat fields

between 2013 and 2015 (Figure 7.2). The interaction transcriptomes of these Pst

infected wheat samples were sequenced along with similar field isolates from

Kenya and Ethiopia. Cluster analysis was carried out using SNP datasets to

assess the existence of population structure in the Pst population.

Phylogeny

A phylogenetic tree (Figure 7.3) was constructed using the randomized axelerated

maximum likelihood (RAxML) method as described in Section 3.3.5, to deter-

mine the genetic relationship among samples (Table 7.2). Isolates examined in

Chapter 4 (Table 4.1) were included in the analysis of the field samples. The tree
CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 154

illustrates a well-defined shift in the genetic structure of the South African Pst

population, with the recent samples collected between 2013 and 2015 clustering

distantly from earlier collected isolates. Field isolates from Ethiopia and Kenya

were more closely related to the historical East African and South African popu-

lations, while the South African field isolates clustered together with a group of

isolates found in the UK in 2013 on triticale, called UK Group II (Hubbard et al.,

2015). The relative distances tree was also constructed, (Figure 7.4) excluding

isolates from the East Africa (B) group in the interest of legibility of the figure.

The UK 2013 Group II isolates clusters distantly from the other 2013 UK isolates.

The 2013 - 2015 South African isolates cluster with these UK isolates, away from

the historical South African isolates.

Population structure analysis

To assess population structure, STRUCTURE software (version 2.3.4; Pritchard

et al., 2000) was applied to analyse a dataset of 112 180 synonymous biallelic

SNPs. Both the log probability plot (Figure 7.5(i)) from Pritchard et al. (2000) and

the plot of ∆ K (Figure 7.5(ii)), based on the method described by Evanno et al.

(2005), suggested that the population could be grouped into five subclusters.

The histogram plots of the data, with K estimated between 2 and 15 (Fig-

ure 7.6), describe each isolate’s cluster allocation given a certain number of

clusters (K). No additional information regarding population differentiation was

gained when K was increased above five.

STRUCTURE assumes that the population is under Hardy-Weinberg equilib-

rium: Equilibrium of allelic and genotypic frequency with infinite size population,

diploid and sexual reproducing species, no migration and panmixia (random

crossing among isolates). As some of these citeria are violated by our data (asex-

ual reproduction, small populations and no panmixia) the STRUCTURE result

 CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 155

UK & Fr
ance
(Pre
-201
1)

78.6SS1

88.45SS

88.5SS1

3
08/21

40

4SS
11/1
14/K

88.4
UK

85F
14/

03/7

C
6
14
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55
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13

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14 d-2
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14/K8

KE74217 ER179b/11

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ste
rn est
Fre
e St 14)-W
20
ate SA (
SA (

(EFS
SA (2

)
) - EFS

SA (2015) - KZN

2013

UK (2013)
0
14) -
SA (2015

)-W
KZN

Key
SA - Eastern Free State (2014) Kenya (Pre-1978) UK (Pre-2011) UK (2013) - Cluster I
Typical relative rainfall

SA - Eastern Free State (2015) Kenya (2014) France (Pre-2011) UK (2013) - Cluster II
SA - Western Cape (2013) Ethiopia (Pre-2011) UK (2011) UK (2013) - Cluster III
SA - Western Cape (2014) Ethiopia (2014) Pakistan (2014) UK (2013) - Cluster IV
SA - KwaZulu-Natal (2014) Eritrea (2011) Pathotypes Bootstrap value > 80
SA - KwaZulu-Natal (2015) Ethiopia (Pre-2011) Pathotyped in the
6E16A- 6E22A- 6E22A+
present study
SA (Pre-2012) Ethiopia (2014)

Figure 7.3: Phylogenetic tree displaying the relationship between Pst isolates. Samples
representative of older Pst populations and more recent populations were
compared. The maximum likelihood phylogenetic tree was obtained using
the RAxML method. The relationship between samples was determined using
those Pst genes that had 80 % breadth of coverage in 80 % of the samples.
This included 2597 genes and a total of 792 535 third codon sites. Only
the topology is indicated here, while Figure 7.4 displays relative distances.
Both dendrograms were visualised using MEGA software (version 6.06).
Asterisk (*) indicates genomic data of isolate 11/08, while 11/08 without an
asterisk indicates RNA-Seq data. RAxML, Randomized Axelerated Maximum
Likelihood; EFS, Eastern Free State; KZN, KwaZulu-Natal; WC, Western Cape
 UK (pre-2011—WGS) Pakistan (2010—WGS) UK (2013—RNA-Seq)
UK (2013)
France (pre-2011—WGS) Ethiopia (Old—WGS) South Africa (WGS)
(Group I)
UK (2011—WGS) Ethiopia (2014—RNA-Seq) South Africa (RNA-Seq)

UK (2013) UK (2013—RNA-Seq) Kenya (Old—WGS) Kenya (2014—RNA-Seq)

Pre-2011 UK & (Group IV)
French

UK (2013)
Pakistan (Group III)
UK (2013) South Africa
2013-2015

Etiopia (2014) UK (2013) UK 2013

(Group II)

Kenya (2014)
Ethiopia (2014) Kenya (2014)

Pre-2011 South Africa

Pre-2010 East Africa (A)
0.0001

Figure 7.4: Relative distance maximum likelihood phylogenetic tree describes the relative relationship between isolates described in
Figure 7.3 where branch lengths were ignored and only topology was considered. In this dendrogram, East Africa (B) was not
shown. Compare Appendix D, Figure D.3, that includes the East Africa (B) group.
CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 157

was compared with the non-parametric method DAPC (Jombart et al., 2010). The

biallelic synonymous SNP dataset used in the STRUCTURE analysis was used to

summarise genetic variance within and between populations by PCA. The BIC

graph (Figure 7.7(i)) illustrates an elbow at K = 7 to K = 8, while an absolute

minimum was observed at K = 11. This indicated that the optimum number of

population clusters falls between 7 and 11.

Individual isolates were assigned to population clusters by DA of eigenvalues

(Figure 7.7(ii)). According to the DA, the first two PCAs explained most of the

genetic variability seen in the data. The histogram plots (Figure 7.8) at different

values of K showed an increase in differentiation from K = 7 to K = 11. The

gain of differentiation from 10 to 11, shown in the South African isolates form

2014, is lost at K = 12. Taking this and the BIC graph into account, K = 10

was concluded to be the optimal estimate of population clusters. The first two

principal components of the DAPC analysis of the synonymous SNP sites are

shown in the scatter plot (Figure 7.7(iii)). The distances between groups are

representative of the relative differentiation between population groups, taking

the first two principle components into account.

CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 158

−2400000 ● ● ● ● ● ● ● ● ●
●
● ●
●
−2800000
●
LnP(D)

−3200000

−3600000

●
−4000000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
K

(i) Log probability of the data L(K ) as a function of K to estimate the optimal number
of population clusters as identified by STRUCTURE. The optimum number of
clusters (K) inferred by the model-based Bayesian cluster analysis of genome-wide
SNP data is 5.
●
10000

7500
Delta K

5000

2500
●

0 ● ● ● ● ● ● ● ● ● ● ●

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
K

(ii) The Evanno method of inferring the number of STRUCTURE populations (K)
from the modal value of ∆K. A strong signal was detected for K = 5 where ∆K
was at a maximum. ∆, Delta

Figure 7.5: Evaluation of number of population clusters following STRUCTURE analy-

ses.
 K2!
K3!
K4!
K5!
K6!
K7!
K8!
K9!
K10!
K11!
K12!
K13!
159

K14!
K15!

15/SAZP11

15/SAZP12
15/SAZP10

J01144Bm1

ER179b/11
ER181a/11
14/SADL3
14/SADL1
14/SADL2

14/SADL4

14/SADL5

14/SADL6
14/SATT2
14/SATT3
14/SATT5

14/SATT4

14/SATT1

ET03b/10
13/SAZP1

14/SAZP2

15/SAZP4
15/SAZP1

15/SAZP3
15/SAZP8

15/SAZP6
15/SAZP7

15/SAZP5

15/SAZP9

14/SAZP3

88.44SS3

ET08/10
KE74217
KE89069
ET87094
J02055C
88.5SS1

88.45SS
78.6SS1
14/K10
14/K11
14/K12
14/K13
14/K14
14/K15
14/K16

13/123

13/182

11/128

11/140
14/ET4
14/ET5

14/ET3
14/ET2
11/08*

J0085F

j02-022
14/K4
14/K5
14/K6
14/K7
14/K8
14/K9

14/K2

ATR-1

ATR-3
T13/1
T13/2
T13/3

13/19
13/15
11/08
13/27
13/71
13/40
13/29
13/25
13/38
13/21
13/33

11/13

08/21
Qld-2
Qld-1

03/7
SA1
SA2
SA3
SA4
CL1
!"#$%&'(")*++,& -./01&23(45%&'(")6,*6& 7!&6,**86,*9&)&:;/<0"(&=& 7!&6,**&
!"#$%&6,*>& -./01&23(45%&6,*9& 7!&6,*9&)&:;/<0"(&==& 7!&'(")6,**&
?014.@4%&'(")6,**& -./01&23(45%&6,*>& 7!&6,*9&)&:;/<0"(&===& A(%#5"&'(")6,**&
?014.@4%&6,*>& -./01&23(45%&6,*B& 7!&6,*9&)&:;/<0"(&=C& '%D4<0%#&6,*>&
?(40("%&6,**& &&

Figure 7.6: Histogram plots of population clustering with K between 2 and 15 as obtained from STRUCTURE analyses. Each bar represents
estimated membership fractions for each Pst isolate. No further differentiation was observed after K = 5. Asterisk (*) indicates
genomic data of isolate 11/08, while no asterisk indicates RNA-Seq data for 11/08. ATR-2 and 11/75 (Table 4.2) were not used in
this analysis.
CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 160

Value of BIC Discriminant analysis eigenvalues

versus number of clusters

10000 20000 30000 40000 50000 60000

820
800

F-statistic
BIC
780
760

0
0 10 20 30 40 50
Number of clusters Linear Discriminants

(i) Bayesian information criterion (ii) Discriminant analysis (DA) of

(BIC) curve. eigenvalues.

Cluster 1
Cluster 2
Cluster 3
Cluster 4
Eritrea & Cluster 5
Ethiopia (PstS2) Cluster 6
Pre-2010 Cluster 7
Cluster 8
Cluster 9
Cluster 10

Kenya 2014 South Africa & Ethiopia 2014

Pakistan 2014 SA 2013/2014

South Africa, Ethiopia & Kenya Pre-2012
UK Pre-2011 UK 2013 Group II
& 2011 SA 2014/2015
UK Pre-2011 Ethiopia 2014
Kenya 2014
UK 2013 Cluster I
UK 2013 Group III & Group IV

(iii) Relative proximity of Pst population clusters.

Figure 7.7: Discriminant analysis of principal components (DAPC) analysis of Pst isolates.
Panel (i) shows the Bayesian information criterion (BIC) curve suggesting
the minimum number of clusters (K) required to explain the variation be-
tween pathotype clusters. An elbow is observed at K = 7 and a minimum
at K = 11. From this result it can be derived that the optimal predicted
number of population clusters (K) for the dataset falls between 7 and 11.
Panel (ii) shows a bar plot representing discriminant analysis (DA) of eigen-
values for main principal component functions. This indicates that most
of the variation in the dataset can be explained by the first two principle
components. Panel (iii) shows a scatter plot indicating the relative proximity
of Pst population clusters following DAPC analysis.
 K2!
K3!
K4!
K5!
K6!
K7!
K8!
K9!
K10!
K11!
K12!
K13!
K14!
K15!

15/SAZP11

15/SAZP12
15/SAZP10

J01144Bm1

ER179b/11
ER181a/11
14/SADL3
14/SADL1
14/SADL2

14/SADL4

14/SADL5

14/SADL6
14/SATT2
14/SATT3
14/SATT5

14/SATT4

14/SATT1

ET03b/10
13/SAZP1

14/SAZP2

15/SAZP4
15/SAZP1

15/SAZP3
15/SAZP8

15/SAZP6
15/SAZP7

15/SAZP5

15/SAZP9

14/SAZP3

88.44SS3

ET08/10
KE74217
KE89069
ET87094
J02055C
88.5SS1

88.45SS
78.6SS1
14/K10
14/K11
14/K12
14/K13
14/K14
14/K15
14/K16

13/123

13/182

11/128

11/140
14/ET4
14/ET5

14/ET3
14/ET2
11/08*

J0085F

j02-022
161

14/K4
14/K5
14/K6
14/K7
14/K8
14/K9

14/K2

ATR-1

ATR-3
T13/1
T13/2
T13/3

13/19
13/15
11/08
13/27
13/71
13/40
13/29
13/25
13/38
13/21
13/33

11/13

08/21
Qld-2
Qld-1

03/7
SA1
SA2
SA3
SA4
CL1
!"#$%&'(")*++,& -./01&23(45%&'(")6,*6& 7!&6,**86,*9&)&:;/<0"(&=& 7!&6,**&
!"#$%&6,*>& -./01&23(45%&6,*9& 7!&6,*9&)&:;/<0"(&==& 7!&'(")6,**&
?014.@4%&'(")6,**& -./01&23(45%&6,*>& 7!&6,*9&)&:;/<0"(&===& A(%#5"&'(")6,**&
?014.@4%&6,*>& -./01&23(45%&6,*B& 7!&6,*9&)&:;/<0"(&=C& '%D4<0%#&6,*>&
?(40("%&6,**& &&

Figure 7.8: Histogram plots indicating population structure as inferred by DAPC analysis. Each bar indicates the group an isolate is assigned
to. Field samples from Africa, collected between 2013 and 2015 were assigned to three groups, coloured orange, light red and
red. The light red group contains South African isolates from 2014 and 2015, two Ethiopian isolates and one Kenyan isolate from
2014 and groups with the UK 2013 Cluster II, containing triticale field isolates. The red cluster contains field samples from 2014
collected in Kenya and South Africa, and one sample from 2013 that was collected from wild rye in South Africa. The orange
group differentiated earlier (K6) than the red (K8) and light red groups (K9). This small group contains field samples collected
in 2014 from Ethiopia and South Africa. From these three groups it is evident that the recent Pst population in South Africa
is fairly diverse and that South African isolates share similarities with the Kenyan and Ethiopian populations. The Ethiopian
population shows higher diversity compared to the Kenyan population. Asterisk (*) indicates genomic data of isolate 11/08,
while no asterisk indicates RNA-Seq data for 11/08. ATR-2 and 11/75 (Table 4.2) were not used in this analysis
CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 162

Differentiation within and between population clusters

Differentiation between groups was calculated through pairwise comparisons

of the 10 population clusters identified by the DAPC analysis. FST statistics

for all pairwise comparisons are indicated in Figure 7.9 in the lower diagonal

matrix. Highest FST values were observed in comparisons of Group 1 (≥ 0.37)

and Group 4 (≥ 0.58). These groups were also positioned most distantly from

the other eight groups in the DAPC scatter plot (Figure 7.7(iii)). Comparing the

diversity between the two groups resulted in a high FST of 0.8, further indicating

that the two groups differentiated distinctly from all other groups and from

each other. These high values of FST also confirmed the importance of asexual

reproduction known to increase the differentiation among populations by the

absence of genetic mixing. Group 1 contained an Ethiopian isolate, ET08/10,

previously identified as PstS2, as stipulated in Chapter 4 (Hovmøller et al., 2008;

Walter et al., 2016; Ali et al., 2017; M Hovmøller, personal communication). This

isolate grouped with two isolates from Eritrea collected in 2011. Group 4 included

two isolates from Ethiopia and one isolate from South Africa, all collected in 2014.

Group 4 was distinctly different to Groups 9 and 10, containing the remaining

South African and East African isolates collected from 2013 to 2015. Groups

9 and 10 had a low FST of 0.12, indicating that these two groups are closely

related. Besides the recent African field samples, Group 9 also contained samples

collected on triticale in the UK in 2013. Low variability within the three groups

(Groups 4, 9 and 10) that contained the post-2012 African samples was observed

as indicated on the matrix diagonal (Figure 7.9).

7.3.2 Seedling Pst pathotype testing

To compare the virulence profiles of the historical Pst isolates to isolates collected

from the field between 2013 and 2015, seedling inoculation tests were performed
 Group Isolate ID Group Isolate ID
Group 1 2 3 4 5 6 7 8 9 10
ET08/10 CL1
1 ER179b/11 T13/2
ER181a/11 T13/3
0.0031 03/7 T13/1
1
±0.0055 08/21 14/SADL4
88.45SS 14/SADL5
88.44SS3 14/SADL6
2
0.0005 J0085F 14/SATT1
2 0.39
±0.0008 J01144Bm1 14/SATT4
j02-022 15/SAZP1
11/140 15\SAZP3
9
0.0020 SA1 15/SAZP5
3 0.37 0.21 SA2 15/SAZP6
±0.0030 SA3 15/SAZP7
SA4 15/SAZP8
3
KE74217 15/SAZP9
0.0001
4 0.80 0.58 0.62 KE89069 15/SAZP10
±0.0004 ET87094 15/SAZP11
ET03b/10 15/SAZP12
14/SAZP3 14/ET4
0.0003 4
5 0.53 0.14 0.23 0.76 14/ET2 14/ET5
±0.0009 14/ET3 14/K2
J02055C 14/SADL1
5 11/13 14/SADL2
0.0012
6 0.59 0.32 0.26 0.79 0.20 11/128 14/SADL3
±0.0021 Qld-1 14/SATT2
Qld-2 14/SATT3
6
ATR-1 14/SATT5
0.0005 ATR-3 13/SAZP1
7 0.78 0.39 0.39 0.87 0.27 0.31
163

±0.0008 13/27 14/SAZP2

13/38 15/SAZP4
13/21 14/K4
0.0004 13/33
10
14/K5
8 0.82 0.45 0.42 0.91 0.48 0.47 0.41 7
±0.0013 13/182 14/K6
13/25 14/K7
13/29 14/K8
0.0001 13/71 14/K9
9 0.84 0.52 0.40 0.90 0.48 0.41 0.25 0.49 13/40 14/K10
±0.0004 11/08 14/K11
13/19 14/K12

0± 8 13/15 14/K13
10 0.85 0.52 0.41 0.92 0.50 0.45 0.32 0.51 0.12 13/123 14/K14
0.0001 11/08* 14/K15
14/K16

Figure 7.9: Measurements of genetic diversity by FST calculation of pairs of population groups indicated by the lower triangular matrix. The
Watterson estimator of population diversity is given on the diagonal of the matrix. Colours of subpopulations is as shown in the
DAPC population structure analysis bar plots (Figure 7.8). Comparisons with Group 4 (orange), and often Group 1, showed high
FST values indicating that these groups were genetically very different from the other samples. Asterisk (*) indicates genomic
data of isolate 11/08, while no asterisk indicates RNA-Seq data for 11/08.
CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 164

on an extended set of wheat differential lines. The wheat differential set contained

varieties with known Yr resistance genes, as well as unidentified sources of stripe

rust resistance. Seedlings of 56 wheat varieties were tested under controlled

environmental conditions with the historical South African isolates SA1 and

SA4 and two field isolates, 13/SAZP1 and 15/SAZP4, collected in 2013 and

2015, respectively. To determine whether the genetic variance displayed in the

phylogenetic analyses was linked to changes in the virulence profiles of these

isolates the differential set was expanded by including additional wheat lines

from the UK and Australia.

In the comparison between the isolates SA1 and 13/SAZP1 significant vari-

ability was not observed. The SA4 and the 2015 isolate, 15/SAZP4, displayed

slight differences in infection types, with most prominent differences after infec-

tion of the wheat varieties Monterey (;cn versus 2cn) and Heines VII (;1+cn versus

3c), and a smaller, but observable difference on Kranich (;cn versus 1cn), Solstice (;

versus ;c) and Selkirk (2cn versus 3=cn). These differences are visually displayed

in Figure 7.10. Detailed results of all infection assays are listed in Appendix D,

Tables D.1 and D.2.

7.4 Discussion

The field Pst population in South Africa was assessed at transcriptome level using

25 samples collected between 2013 and 2015. Along with these, four Pst isolates

collected in 2014 in Ethiopia and 14 isolates collected in Kenya in 2014 were also

evaluated.

Phylogenetic analysis placed the 2014 East African isolates in close proximity

to one another. Additionally, it revealed patterns of high similarity between the

field Pst population in South Africa, collected between 2013 and 2015, and the

UK Cluster II triticale field isolates (T13/1, T13/2, T13/3 and CL1) described
165

Figure 7.10: Infection type comparisons between one historical and one recent Pst isolate. Infection types of SA4, from the historical
population, and isolate 15/SAZP4 collected in 2015 are shown. Highly similar phenotypes were observed on wheat Warrior,
Vilmorin 23, Heines Peko, Reichersberg 42. Differences in UK testers, including Kranich, Monterey and Solstice, were observed.
The outcome of the remaining differential tests are summarised in Appendix D, Tables D.1 and D.2.
CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 166

by Hubbard et al. (2015). Isolates collected in the same region of South Africa

commonly clustered together. South African isolates from corresponding ge-

ographical and, by implication, climatic regions were often grouped together

in the phylogenetic tree. Although this result has to be further investigated to

draw further conclusions, pathotyped isolates with different virulence profiles

were positioned on different branches. Genotyping of the Pst isolates indicated

that a shift occurred in the South African Pst population, with the current Pst

population being clearly differentiated from the earlier isolates sampled before

2012, and assessed in Chapter 4. The unexpected genetic relationship with the

2013 UK isolates, found on triticale, suggests a potential recent incursion of Pst

into South Africa.

Results from the DAPC analysis mostly correlated with the phylogenetic

findings and revealed signs of population structure, with three distinct groups

containing field samples from South Africa. The historical South African isolates

were placed in a separate, fourth group. All three South African field sample

groups also included 2014 isolates from Kenya and/or Ethiopia. This supports

the hypothesis raised in Chapter 4 stating the potential exchange of inoculum

between South Africa and East Africa, although the East African isolates did

not show as close resemblance to the UK Cluster II isolates as the co-clustering

South African field isolates did (Figure 7.3). This indicates that inoculum was

somehow spread between these locations or could be derived from the same

progenitor. The South African and UK populations remained more similar, but

share similarity with the East African population.

Regarding the DAPC analysis outcome of the South African field population,

isolate 15/SAZP4, exhibiting a partially successful infection on Monterey that was

not seen in the compared earlier isolate (SA4), was placed in Group 10, that was

in addition to its high similarity to Group 9 (FST = 0.12), also very homogeneous

according to the differentiation calculation within groups (0.0000 ± 0.0001). Both

CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 167

Group 4 and Group 9 also containing South African field isolates, indicated low

diversity amongst isolates (0.0001 ± 0.0004). The DAPC clusters only differed

from the clades in the phylogenetic analysis by one isolate (14/SAZP3) when

K = 10 is considered. The placement of 14/SAZP3 together with two isolates

from Ethiopia, namely 14/ET2 and 14/ET3, forming Group 4 (orange) in the

DAPC analysis is noteworthy. In the phylogenetic tree, sample 14/ET2 is the

only isolate from the Ethiopian field isolates that show similarity to the East

Africa (B) group, which contains isolate ET08/10, that was identified to be of

the aggressive PstS2 type (Hovmøller et al., 2008; Walter et al., 2016; Ali et al.,

2017; M Hovmøller, personal communication). Grouping of these three isolates

was however not displayed in the inferred phylogeny where 14/SAZP3 grouped

with the other South African isolates. However, in the DAPC analysis, Group 4

differentiated early (K = 6) as displayed by the orange bars, compared to the red

group (K = 9), containing the rest of the East African and South African field

samples.

The high diversity that is shown when Group 4 (orange) is compared to

Group 9 (light red, FST value 0.90) and Group 10 (red, FST value 0.92) indicate

that Group 4, containing 14/SAZP3, differentiates considerably from Groups 9

and 10. This isolate, carrying the 6E16A- pathotype, was previously evaluated

using microsatellite markers, and differentiated from other South African 6E16A-

isolates (B Visser, personal communication). In previous infection assays, this

isolate had a typical 6E16A- pathotype. This isolate was not evaluated on the

extended differential set used in this study. This could be similar to the case

discussed by Hubbard et al. (2015), where phenotypically similar isolates were

genetically distinct and belonged to different populations. This further highlights

the importance of genotyping along with differential testing in seasonal surveys.

The genetic diversity between Groups 9 and 10 (FST value 0.12) was low in

comparison to their diversity with Group 4. Further investigation is needed to

CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 168

conclude that isolates related to the aggressive pathotype PstS2 are present in

South Africa.

Genetic change revealed by the phylogenetic and DAPC analyses in the South

African Pst post-2012 population does not support stepwise mutational adapta-

tion, but lead to speculations that an introduction of new Pst isolates occurred

after 2012. This introduction could have occurred either through natural means

where urediniospores could have been transferred by wind, or by human move-

ment. According to the virulence profiles of the South African field isolates,

pathological support for a new incursion is limited since new virulence has not

been described in routine surveys. Ali et al. (2014) describe how such surveys

can be biased as sampling is often done from wheat varieties that carry resis-

tance genes that have been overcome, and usually not from field isolates. After

evaluating some historical isolates with more recent phenotypic counterparts,

SA1 and 13/SAZP1 had near to identical seedling phenotypes on all the wheat

lines. Genotypic differences were however observed between SA1 and 13/SAZP1

using molecular marker analysis (Visser et al., 2016), as well as in this study.

Newly introduced Pst populations may also carry avirulences not inspected

in local differential sets, as seen in the differentiation in infection types between

SA4 and 15/SAZP3. The most notable difference between infection by these

two isolates were on Monterey. It is a winter wheat cultivar bred in the UK

by the company Senova. It is not known what stripe rust resistance genes are

present in this variety, but it shows moderate levels of resistance in the UK. For

instance, it was listed by the UK Cereal Pathogen Virulence Survey (UKCPVS)

as being “susceptible as an adult plant to one or more of the current stripe rust

pathotypes” and scored 7.3 on the stripe rust resistance rating in 2014, where

possible scores ranged from 1 to 9; 1 = highly susceptible, 9 = resistant (Hubbard

et al., 2014). Monterey has the pedigree Istabraq x Robigus. Robigus is fully

susceptible to all UK Pst pathotypes, whereas Istabraq has the pedigree Consort
CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 169

x Claire which both provide some resistance to UK Pst pathotypes, although

resistance in Claire has been eroded over the past few years. The aggressive

Pst Kranich pathotype was first detected in the UK on Monterey (S Holdgate,

personal communication). The wheat variety Kranich has the pedigree Heines-

2167-50/Heines-VII//Merlin/Deu. Small pustules were observed on Kranich

inoculated with 15/SAZP4, while the SA4 inoculation resulted in flecks only,

with signs of chlorotic and necrotic tissue. Taken together, this may indicate that a

source of stripe rust resistance from Heines VII, present in Kranich and Monterey

has become less effective towards Pst isolate 15/SAZP4.

The role that the host plays in shaping the characteristics of the pathogen has

not been addressed in this study. Wheat breeding in South Africa has generally

relied on selection for resistance in the field and information about stripe rust

resistance genes deployed in commercial wheat over the past 20 years is not

obtainable, as reviewed by Pretorius et al. (2007). Only as recent as 2012, marker

assisted selection (MAS) has been incorporated in breeding programmes with

the establishment of the Molecular marker Service Laboratory (MSL) for wheat

breeding in South Africa (Prins and Agenbag, 2013). In the past, germplasm from

the International Maize and Wheat Improvement Center (CIMMYT) has been

the origin of valuable resistance complexes. The presence of the slow rusting

complex Lr34/Yr18/Sr57, present in South African spring wheat cultivar, Kariega

(Ramburan et al., 2004; Prins et al., 2011), was likely introduced by a CIMMYT

source. The lack of structured molecular breeding efforts incorporating rust

resistance in the past makes it difficult to track specific selection pressures on Pst

imposed by host resistance. No connection could be made between the Monterey

and the South African germplasm.

The widely homogenous nature of the Pst population could be due to the

introduction of a relatively small amount of inoculum displaying the founder

effect, where genetic variation is lost when a small number of individuals es-
CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 170

tablish the population. Additionally or alternatively, a population bottleneck

could have occurred where a limited amount of genotypes, able to sustain for

example environmental conditions, survived from one wheat-growing season

to the next. Environmental factors, either directly, or indirectly through effects

on the host, can increase stress on the pathogen, acting as a force for adaptation.

Severe droughts have been experienced in both main wheat growing regions in

South Africa. This could have contributed to the low occurrence of stripe rust in

recent years. It is possible that these non-optimal conditions encountered by the

Pst population, during and between wheat seasons, may have also contributed

to a population bottleneck. The majority of the 2014 South African field samples

differentiated from the 2015 field samples, indicating that the population evolved

from one growing season to the next, or it could again indicate the influx of new

alleles into the population. The low occurrence of stripe rust in South Africa

could have led to a change in allele frequencies in the population, similar to the

“chance events” described by Wellings (2007). Relatively low numbers of spores

may survive during the non-crop seasons, possibly on alternative grass species

(Boshoff et al., 2002; Pretorius et al., 2015), resulting in such allele frequency shifts.

Anthropogenic movement in and out of South Africa has drastically increased

since the change in the country’s political system in 1994. Tourism and trade

act as passages for pathogens to travel long distances much more quickly and

frequently than through migration via animal vectors or storms (Anderson et al.,

2004). The increase in number of international arrivals indicated by the World

Tourism Organisation (Figure 7.111 ) demonstrates the increase in the potential

for exotic incursions by pathogens via human movement. Anderson et al. (2004)

considered this as the major driver of emerging infectious diseases.

1 https://data.worldbank.org/indicator/ST.INT.ARVL?contextual=default&end=2014&

locations=ZA&start=1995&view=chart
CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 171

11

● ● ●

●
●
9

● ●
Millions of arrivals

7 ●

●
●
●

● ●
● ●

5 ● ●

1995 2000 2005 2010 2014

Year

Figure 7.11: Number of international tourist arrivals in South Africa between 1995 and
2014.
CHAPTER 7: CURRENT PST THREAT IN SOUTH AFRICA 172

7.5 Conclusion

No new virulence profiles for stripe rust have been reported in routine surveys

in South Africa since 2005. In addition, no exclusive correlation could be seen

between the genotypic change observed in South African field isolates and their

virulence profiles, as shown in the SA1 vs 13/SAZP1 comparison in this study.

The 2015 field isolate 15/SAZP4 was found to be partially virulent on the UK

winter wheat cultivar, Monterey, but no change in virulence was seen with

isolate 13/SAZP1 on the extended differential wheat set. The differentiation from

SA1 that was observed in this study for 14/SAZP3, also carrying the 6E16A-

pathotype, was also characterised using microsatellite markers (Visser et al.,

2016). The microsatellite marker research supports the fact that a genetically

diverse population carries the pathotype 6E16A-. No evidence could be found

of common parentage between Monterey and South African wheat varieties

that could account for selection for virulence in South Africa to the stripe rust

resistance currently present in Monterey. Further investigation would be needed

to identify which source of resistance in Monterey was challenged by 15/SAZP4.

The data discussed in this chapter shows evidence of a definite change in

the South African Pst population between 2013 and 2015. It is likely that this is

due to an exotic incursion of Pst from outside South Africa. The Pst population

also showed an allele frequency change between 2014 and 2015. It is possible

that a population bottleneck, due to unfavourable environmental conditions, was

responsible for this shift. Further research is required to determine which scenario

has contributed to the changes in the Pst population in South Africa, including a

systematic collection of stripe rust infected wheat leaf samples throughout the

growing season, and wild grass between seasons.

Chapter 8

General Discussion

T HIS STUDY SET OUT to examine the genetic structure of the Pst population in

South Africa, with specific focus on the genetic variation related to pathotype vari-

ation. Previous descriptions made use of traditional pathotyping and molecular

marker technologies (Pretorius et al., 1997; Boshoff et al., 2002; Pretorius et al.,

2007; Ali et al., 2014; Hovmøller et al., 2008; Visser et al., 2016). In this study,

characterisation was undertaken using next-generation Illumina sequencing of

Pst genomes and transcriptomes, and bioinformatics analyses to extend our

knowledge of the South African Pst population and its evolutionary dynamics.

Specific interests included the origin of Pst introduced into South Africa, the

relationship between the four pathotypes identified so far, identification of effec-

tor coding genes possibly responsible for distinct virulences, and genomic and

pathological investigation of recent field Pst populations.

8.1 The historical South African Pst population

Phylogenetic and clustering analyses, supported by evaluation of the genetic

diversity, reinforced previous findings which stipulated that the historical Pst

population in South Africa, represented by the Pst isolates collected between 2001

173
CHAPTER 8: GENERAL DISCUSSION 174

and 2011, had a close relationship to each other despite their distinct differences

in virulence. Data from this study supports previous reports that the four patho-

types were derived from one another through stepwise evolution (Visser et al.,

2016).

Analysis of the relationship of the historical South African isolates with

available foreign isolates indicated a possible origin from Kenya and Ethiopia, or

a common progenitor from elsewhere. Significant diversity was observed in the

East African isolates, which formed two distinct groups, one closely related to

the South African isolates and one distant from all other isolates assessed in this

study. The East African isolates (Group A) that clustered with the historical South

African isolates contained three isolates collected in the 1970s and 1980s and one

isolate collected in 2010 (Figure 4.6 and 4.10). We therefore confirm associations

based on pathotype analysis that the South African Pst incursion of 1996 had a

high probability of originating from East Africa (Pretorius et al., 1997; Boshoff

et al., 2002; Pretorius et al., 2007).

These conclusions were supported by previous pathotype analyses that

showed the presence of 6E16A- in East Africa (Badebo et al., 1990). However,

similar pathotype designations may be shared between distinct isolates, for ex-

ample the Ethiopian wheat variety Et-13 A2 was resistant to 6E16A and 6E22A

isolates from South Africa, but susceptible to 6E22 isolates from Germany (Hus-

sein and Pretorius, 2005; Badebo et al., 2008; Denbel, 2014). Genetic evidence from

microsatellite marker analysis indicated 48 % similarity between South African

isolates and the Kenyan isolates KE 10/09 and KE 12/09 (Visser et al., 2016).

Differences could be due to virulence for Yr9 and Yr27 that is frequently observed

in East Africa but absent in South Africa1 . It was unfortunate that the present

study did not include isolate data from additional locations south of Kenya which

would have enabled the tracking of the putative southward spread of Pst into
1 http://rusttracker.cimmyt.org
CHAPTER 8: GENERAL DISCUSSION 175

South Africa. Earlier reports state that stripe rust is not a major problem in these

regions (Stubbs, 1985), however, analysis of samples from Rwanda and Tanzania

suggests that collections from more Southern African countries could be included

in on-going work to monitor gene flow (Ali et al., 2017).

Previous studies that included Pst isolates from Eritrea, indicated Central and

Western Asia, and the Mediterranean as possible origins of South African isolates

(Enjalbert et al., 2005; Hovmøller et al., 2008). Ali et al. (2014) further reported

that the South African isolates (collected between 1996 and 2004) grouped with

the older, aggressive group known as PstS3 often seen in Southern Europe. There

is agreement between these studies and the present study with regards to the

South African isolates not showing close relationships with isolates from Eritrea,

however, isolates from Ethiopia and Kenya were not included in these studies. It

would be interesting to assess more South African isolates collected between 1996

and 2011, and also to compare the South African isolates to Pst isolates from other

Eastern and Southern African countries, as well as Asian and Mediterranean

isolates, using the field pathogenomics approach as method of investigation.

Such analyses would be subject to the availability of historical samples, but

would enable inspection of the different hypotheses regarding the origin of South

African Pst.

8.2 Candidate effector identification and evaluation

Nonsynonymous polymorphism analysis aided in identifying candidate genes

possibly involved in virulence. The analysis relied on available effector gene

annotations and made use of the initial gene models developed for the PST130

reference genome. It is widely argued that high throughput effector gene an-

notation protocols are difficult to develop for the rusts as they do not exhibit

many of the common features that are known to be characteristic of other, more
CHAPTER 8: GENERAL DISCUSSION 176

thoroughly described pathogens (Dodds et al., 2009; Saunders et al., 2012). It

is therefore accepted that any computational protocol, despite its best efforts,

would likely misidentify some effector genes. New research findings and tools

allow constant refinement of gene predictions, as was the case for the PST130

reference, where gene annotations have been improved since the start of this

study.

To evaluate candidate effector gene expression during Pst infection, RT-qPCR

was used. This methodology has been used in a number of published studies,

but many of these lack detail on experimental procedures. It is often seen that

best practices, as advised by developers and supporters of the technology, are

not followed or not reported, misleading newcomers to the field. Greater efforts

are needed to ensure that published work using RT-qPCR follow The Minimum

Information for Publication of Quantitative Real-Time PCR Experiments (MIQE)

guidelines (Huggett et al., 2013).

In this study, the consistent expression patterns shown by the two South

African isolates across all genes indicated a low level of technical variation seen

between individual assays within a PCR plate. However, variation between

plates hindered the formulation of confident conclusions from these experiments.

In addition, evaluations of early time points were not informative using this

method due to low concentrations of fungal transcripts. Continued efforts are

needed to enable evaluation of gene expression from the moment of inoculation

up to around two dpi to capture expression profiles of genes involved in the early

processes of infection. Four candidate effector genes overlapped between this

study and time course evaluations of two UK Pst isolates (Cantu et al., 2013).

Future research should prioritise investigation of these four candidate genes.

As a start, heterologous expression screens in Nicotiana benthamiana could be

performed to add to the available information gained from this system about one

of the four candidates, PST130_05023 (Petre et al., 2016b).

CHAPTER 8: GENERAL DISCUSSION 177

8.3 The recent South African Pst population

Surprisingly, analysis of RNA-Seq data of recent field isolates indicated an allele

frequency shift in the South African Pst population. Previously this population

was thought of as fairly stable because of the lack of detection of additional

virulences between 2005 and 2015, when the last field isolates were sampled.

These field isolates showed a close relationship to UK Pst isolates collected

on triticale (UK Group II; Hubbard et al., 2015 and Bueno-Sancho et al., 2017).

Whether or not these UK isolates were able to infect wheat is not known as

they were not successfully cultured and have been lost (S Holdgate, personal

communication).

Compared to the 2013–2015 South African isolates, field isolates collected in

Kenya and Ethiopia in 2014 were more similar to the pre-2011 East African and

South African isolates, as indicated by the phylogenetic analysis. This analysis

used the third codon position of genes with 80 % breadth of coverage in 80 % of

isolates. DAPC clustering analysis used sites where a polymorphism resulting in

a synonymous substitution in at least one isolate was recorded. In this analysis,

the 2014 East African isolates did not group with the pre-2011 East African and

South African isolates, but with the recent South African and UK Group II isolates.

Two groups, namely Group 1—also described as East Africa (B)—indicated in

blue in Figure 7.7(iii), and Group 4, indicated in orange and containing three 2014

isolates, two from Ethiopia and one from South Africa, included in the dataset in

Chapter 7, showed high diversity, clustering away from the rest of the isolates

considered in the DAPC analyses. This diversity could result in the software

having difficulty to separate more similar isolates into population clusters. The

two results differ primarily in their indication of the closest relatives of the 2014

East African isolates. There is however consensus between the two analyses

with regards to the recent South African isolates, showing closer similarity to
CHAPTER 8: GENERAL DISCUSSION 178

the UK Group II isolates than the historical South African isolates. Comparative

re-evaluation of selected recent South African isolates to pre-2011 isolates on

an extended wheat differential set confirmed previous findings in two isolates.

The 6E16A- pathotype was confirmed in isolates SA1 and 13/SAZP1 with nearly

identical infection types. However, evaluation of SA4 and 15/SAZP4 revealed

diverging infection types.

Disagreement exists between studies regarding similarities in the European

and Ethiopian populations. Using virulence phenotyping together with AFLP,

microsatellite and SCAR marker information, Ali et al. (2017) described a diverse

Pst population with more than four pathotype groups in East Africa, collected

between 2009 and 2015, that were distinct from the assessed European isolates.

Among these East African isolates were samples from Ethiopia. In contrast,

support for a close relationship between the UK Group II isolates and Ethiopian

isolates from 2014 was reported where a number of Ethiopian isolates were

assigned to this group, along with isolates collected in Europe in 2014 (Bueno-

Sancho et al., 2017). The authors further revealed the assignment of historical Pst

isolates from New Zealand that were collected between 2006 and 2012, to this

group.

Taken together this data provides evidence that a new incursion may have

occurred in South Africa, possibly between 2011 and 2013, and the commonalities

with UK Group II Pst indicate the possible spread of this Pst group over vast

distances. These findings should alert the research and agricultural community

that the Pst population in South Africa could be more dynamic than is currently

thought to be the case. However, similar infection types in historical and recent

isolates tested on existing differentials gave rise to scepticism. Further investiga-

tion of East African and UK Group II Pst isolates is needed to support the current

findings and track the global movement of this group. Sequencing of field isolates

to monitor new incursions complementary to virulence profiling of Pst across

CHAPTER 8: GENERAL DISCUSSION 179

cropping seasons would be beneficial to facilitate comprehensive surveys. The

cost of implementing the field pathogenomics approach (Hubbard et al., 2015) is

unfortunately a major limiting factor to deployment of this technology in routine

pathotype surveys in South Africa.

8.4 Future work

Effective, long term rust resistance in wheat can be implemented by pyramiding

resistance genes. Ideally, breeders should combine major, R gene type and

APR genes. This relaxes selection pressure on the pathogen population that

can normally rapidly overcome singly deployed R genes. Understanding the

mechanisms of R genes and their corresponding Avr genes, as in the case of

the recently published AvrSr35 (Salcedo et al., 2017) and AvrSr50 (Chen et al.,

2017) studies, can help breeders to track high-risk pathotypes to help tailor

the deployment of resistance genes. Another approach would be to identify

the target “susceptibility” genes of Pst effectors, such as the barley powdery

mildew susceptibility gene Mlo. Mutations in Mlo created the recessive mlo

allele that has provided broad-spectrum resistance against the fungus Blumeria

graminis f. sp. hordei for many years (Büschges et al., 1997). Targeted mutation

breeding of Pst effector target genes in wheat, using DNA-editing technologies

such as CRISPR/Cas9 (Kim et al., 2018), could generate suites of mutant genes

conferring resistance to Pst. Identifying the mechanisms, both in the host and

the pathogen, that provide durable resistance is the aim of many future studies

(Harris et al., 2015). Advances in research that enable understanding of how

effectors function include protein interaction assays such as yeast-two-hybrid

screens, gene expression knock-downs, for example using virus-mediated host-

induced gene silencing and heterologous expression of effector genes in easily

transformed host plants such as N. benthamiana (Liu et al., 2016; Petre et al.,
CHAPTER 8: GENERAL DISCUSSION 180

2016b). Other delivery systems such as the type III secretion system in bacteria

have also been proposed to deliver specific proteins into host cells (Ma et al.,

2009; Upadhyaya et al., 2013). Using these technologies, refinement of Pst gene

annotations and the first available Pst haplotype-phased genome (Schwessinger

et al., 2018) all provide promising potential resources to further assess wheat-Pst

interactions in the search for long lasting resistance to improve wheat yields and

reduce the evolutionary potential of rust pathogens by reducing inoculum.

8.5 Conclusion

In conclusion, although there remains a significant gap in our understanding of

genes that are responsible for the virulence gain in the historical South African

population, this study showed that, contrary to conclusions from previous stud-

ies, novel genetic variation that has not been described previously, is indeed

present in the recent South African population. For the first time, according to

our knowledge, the Pst populations of Ethiopia, Kenya and South Africa were

linked using high-resolution genomic and transcriptomic data. This confirms

earlier associations between pathotypes from eastern Africa and South Africa and

verifies the risk for the introduction of more aggressive pathotypes into South

Africa. Further characterisation of isolates that are associated with the UK Group

II isolates, with specific focus on their pathogenicity, will aid in understanding the

risks involved in long distance movement of Pst and ultimately help producers

to decrease the incidence of disease and increase crop yields, which will in turn

relieve the pressure on global food production to meet rising demands.

Appendix A

The Origin of the South African Pst

Pathotypes

181
CHAPTER A: THE ORIGIN OF SOUTH AFRICAN PST 182

ER179b/11 ER181a/11 ET03b/10 ET08/10

15000 50000
15000
40000 20000
10000 10000 30000 15000
20000 10000
5000 5000
10000 5000
0 0 0 0
count

0.00
0.25
0.50
0.75
1.00
ET87094 KE74217 KE89069
50000 50000
40000 40000 40000
30000 30000 30000
20000 20000 20000
10000 10000 10000
0 0 0
0.00
0.25
0.50
0.75
1.00

0.00
0.25
0.50
0.75
1.00

0.00
0.25
0.50
0.75
1.00

frequency

Figure A.1: Read frequency graphs for East African isolates analysed in Chapter 4, that
have not been similarly assessed in published studies (Cantu et al., 2013;
Hubbard et al., 2015; Bueno-Sancho et al., 2017). See Table 4.1 for further
identification purposes.
Appendix B

Analyses of Polymorphisms in
Historical South African Pst
Isolates in Search of Candidate
Effector Genes

B.1 Genes present in the PST130 reference genome but ab-

sent in the four historical South African Pst isolates

183
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 184

Table B.1: PST130 genes (211) that were absent in all four historical South African isolates

PST130_00014 PST130_03142 PST130_08220 PST130_14020

PST130_00053 PST130_03351 PST130_08341 PST130_14034
PST130_00147 PST130_03414 PST130_08456 PST130_14069
PST130_00148 PST130_03415 PST130_08466 PST130_14429
PST130_00159 PST130_03429 PST130_08469 PST130_14430
PST130_00173 PST130_03543 PST130_08470 PST130_14605
PST130_00227 PST130_03607 PST130_08628 PST130_14606
PST130_00246 PST130_03762 PST130_08645 PST130_14653
PST130_00348 PST130_03775 PST130_08669 PST130_14781
PST130_00404 PST130_03798 PST130_08880 PST130_14925
PST130_00445 PST130_03847 PST130_08891 PST130_14963
PST130_00483 PST130_04103 PST130_09448 PST130_14964
PST130_00611 PST130_04396 PST130_10110 PST130_15027
PST130_00612 PST130_04591 PST130_10111 PST130_15648
PST130_00656 PST130_04612 PST130_10209 PST130_15841
PST130_00812 PST130_04613 PST130_10271 PST130_16094
PST130_00848 PST130_05005 PST130_11019 PST130_16216
PST130_00945 PST130_05050 PST130_11064 PST130_16356
PST130_00950 PST130_05150 PST130_11200 PST130_16357
PST130_00989 PST130_05183 PST130_11219 PST130_16435
PST130_01030 PST130_05199 PST130_11289 PST130_16508
PST130_01031 PST130_05303 PST130_11403 PST130_16509
PST130_01079 PST130_05357 PST130_11404 PST130_16568
PST130_01080 PST130_05569 PST130_11537 PST130_16737
PST130_01081 PST130_05640 PST130_11550 PST130_16763
PST130_01082 PST130_05683 PST130_11607 PST130_16764
PST130_01107 PST130_05804 PST130_11862 PST130_16830
PST130_01143 PST130_06069 PST130_11902 PST130_16914
PST130_01368 PST130_06079 PST130_11946 PST130_16963
PST130_01388 PST130_06120 PST130_11947 PST130_17078
PST130_01690 PST130_06121 PST130_11948 PST130_17111
PST130_01696 PST130_06122 PST130_12027 PST130_17182
PST130_01697 PST130_06123 PST130_12084 PST130_17218
PST130_01825 PST130_06147 PST130_12310 PST130_17238
PST130_01826 PST130_06262 PST130_12311 PST130_17253
PST130_01847 PST130_06356 PST130_12346 PST130_17316
PST130_01859 PST130_06479 PST130_12435 PST130_17354
PST130_01946 PST130_06533 PST130_12436 PST130_17435
PST130_02005 PST130_06608 PST130_12446 PST130_17515
PST130_02139 PST130_06609 PST130_12481 PST130_17560
PST130_02140 PST130_06687 PST130_12509 PST130_17599
PST130_02142 PST130_06741 PST130_12825 PST130_17620
PST130_02153 PST130_06775 PST130_12971 PST130_17812
PST130_02289 PST130_07080 PST130_12992 PST130_17815
PST130_02406 PST130_07081 PST130_13083 PST130_17898
PST130_02413 PST130_07180 PST130_13431 PST130_17956
PST130_02482 PST130_07220 PST130_13432 PST130_17990
PST130_02770 PST130_07285 PST130_13436 PST130_17991
PST130_02826 PST130_07330 PST130_13455 PST130_17992
PST130_03059 PST130_07486 PST130_13530 PST130_18018
PST130_03060 PST130_07943 PST130_13926 PST130_18083
PST130_03094 PST130_07959 PST130_13932 PST130_18108
PST130_03099 PST130_08034 PST130_13936
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 185

B.2 Annotations of genes homologous to identified PST130

genes

PST130_00159 Accession gi|403167846| ref|XM_003327549.2|

Homolog Pgt isoleucyl-tRNA synthetase (PGTG_09131)

UniProtKB/ TrEMBL ID E3KG82

Protein name Isoleucyl-tRNA synthetase

Associated Function or Cellular location

Enzyme involved in protein biosynthesis during translation. Present in cyto-

plasm.

GO terms

GO:0002161 (aminoacyl-tRNA editing activity), GO:0005524 (ATP binding), GO:0004822

(isoleucine-tRNA ligase activity), GO:0000049 (tRNA binding). GO:0006428

(isoleucyl-tRNA aminoacylation)

Conserved domains

PLN02882: aminoacyl-tRNA ligase; cd07961: Anticodon-binding domain of

archaeal, bacterial, and eukaryotic cytoplasmic isoleucyl tRNA synthetases;

cd00818: catalytic core domain of isoleucyl-tRNA synthetases

PST130_07080 Accession gi|403159121| ref|XM_003319730.2|

Homolog Pgt hypothetical protein (PGTG_01952)

UniProtKB/ TrEMBL ID E3JT80

Protein name Uncharacterised protein

Associated Function or Cellular location

Helicases are ATPase enzymes that catalyse the unwinding of double-stranded

nucleic acids. Involved in processes such as DNA replication, recombination,

and nucleotide excision repair, as well as RNA transcription and splicing.

CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 186

GO terms

GO:0009055 (electron transfer activity), GO:0016491 (oxidoreductase activity),

GO:0035091 (phosphatidylinositol binding), GO:0009061 (anaerobic respiration),

GO:0022900 (electron transport chain)

Conserved domains cd06869: The PX domain is a phosphoinositide (PI) bind-

ing module involved in targeting proteins to PI-enriched membranes. Diverse

functions such as cell signalling, vesicular trafficking, protein sorting, lipid mod-

ification, cell polarity and division, activation of T and B cells, and cell sur-

vival.; pfam12825: Domain of unknown function in PX-proteins.; pfam12828:

PX-associated

PST130_16763 Accession gi|403160602| ref|XM_003321038.2|

Homolog Pgt hypothetical protein (PGTG_02128)

UniProtKB/ TrEMBL ID E3JX92

Protein name Uncharacterised protein

Associated Function or Cellular location

Location associated with P-body and nucleolus. Cytoplasmic stress granule.

GO terms

GO:0005524 (ATP binding), GO:0004004 (ATP-dependent RNA helicase activity),

GO:0003676 (nucleic acid binding), GO:0033962 (cytoplasmic mRNA processing

body assembly), GO:0006417 (regulation of translation), GO:0010501 (RNA sec-

ondary structure unwinding)

Conserved domains

COG0513: Superfamily II DNA and RNA helicase [Replication, recombination

and repair], cd00079. Helicase superfamily c-terminal domain; cl21455. P-loop

containing Nucleoside Triphosphate Hydrolases. Involved in diverse cellular

functions
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 187

PST130_17182 Accession gi|403160450| ref|XM_003320901.2|

Homolog Pgt hypothetical protein (PGTG_02971)

UniProtKB/ TrEMBL ID E3JWV5

Protein name Uncharacterised protein

Associated Function or Cellular location

Location associated with cytoplasm, endoplasmic reticulum, membrane compo-

nent.

GO terms

GO:0016491 (oxidoreductase activity); GO:0016627 (oxidoreductase activity, act-

ing on the CH-CH group of donors); GO:0042761 (very long-chain fatty acid

biosynthetic process)

Conserved domains

PLN02560: enoyl-CoA reductase; cl00155: Ubiquitin homologs. Ubiquitin-

mediated proteolysis is part of the regulated turnover of proteins required for

controlling cell cycle progression. cl21511: The Saccharomyces cerevisiae Meyen ex

EC Hansen phospholipid methyltransferase (EC:2.1.1.16) has a broad substrate

specificity of unsaturated phospholipids.

PST130_17354 — A Accession gi|403161086| ref|XM_003890392.1|

Homolog Pgt hypothetical protein (PGTG_20899)

UniProtKB/ TrEMBL ID H6QPU7

Protein name Glycogen [starch] synthase

Associated Function or Cellular location

Enzyme that catalyse the transfer of glycosyl (sugar) residues to an acceptor, both

during degradation (cosubstrates= water or inorganic phosphate) and during

CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 188

biosynthesis of polysaccharides, glycoproteins and glycolipids.

GO terms

GO:0004373 (glycogen (starch) synthase activity); GO:0005978 (glycogen biosyn-

thetic process)

Conserved domains

cl10013: Glycosyltransferases catalyse the transfer of sugar moieties from acti-

vated donor molecules to specific acceptor molecules, forming glycosidic bonds.

PST130_17354 — B Accession gi|403166809| ref|XM_003326625.2|

Homolog Pgt glycogen [starch] synthase (PGTG_07651)

UniProtKB/ TrEMBL ID E3KCW8

Protein name Glycogen [starch] synthase

Associated Function or Cellular location —

GO terms

GO:0004373 (glycogen (starch) synthase activity); GO:0005978 (glycogen biosyn-

thetic process)

Conserved domains

cd03793: Glycogen synthase, catalyses the transfer of a glucose molecule from

UDP-glucose to a terminal branch of a glycogen molecule, a rate-limit step of

glycogen biosynthesis.; pfam05693: Glycogen synthase. It is the rate limiting

enzyme in the synthesis of the polysaccharide, and its activity is highly regulated

through phosphorylation at multiple sites and also by allosteric effectors, mainly

glucose 6-phosphate (G6P).

PST130_17620 Accession gi|403174779| ref|XM_003333656.2|

Homolog Pgt hypothetical protein (PGTG_15464)

CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 189

UniProtKB/ TrEMBL ID E3KYK8

Protein name Uncharacterised protein

Associated Function or Cellular location —

GO terms

GO:0003824(Catalysis of a biochemical reaction at physiological temperatures.);

GO:0009058 (The chemical reactions and pathways resulting in the formation of

substances; typically the energy-requiring part of metabolism in which simpler

substances are transformed into more complex ones.)

Conserved domains

cd00609: Aspartate aminotransferase family. This family belongs to pyridoxal

phosphate (PLP)-dependent aspartate aminotransferase superfamily (fold I). Pyri-

doxal phosphate combines with an alpha-amino acid to form a compound called a

Schiff base or aldimine intermediate, which depending on the reaction, is the sub-

strate in four kinds of reactions (1) transamination (movement of amino groups),

(2) racemisation (redistribution of enantiomers), (3) decarboxylation (removing

COOH groups), and (4) various side-chain reactions depending on the enzyme in-

volved.; COG0436: Amino acid transport and metabolism. linked to 3D-structure.

PST130_17815 Accession gi|403157775| ref|XM_003307127.2|

Homolog Pgt 1,3-beta-glucan synthase component FKS1 (PGTG_00125)

UniProtKB/ TrEMBL ID E3JR07

Protein name 1,3-beta-glucan synthase component FKS1

Associated Function or Cellular location

Component of the plasma membrane.

GO terms

GO:0003843 (1,3-beta-D-glucan synthase activity); GO:0006075 ((1->3)-beta-D-

glucan biosynthetic process).

CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 190

Conserved domains

pfam02364: 1,3-beta-glucan synthase component. 1,3-beta-glucan synthase EC:2.4.1.34

also known as callose synthase catalyses the formation of a beta-1,3-glucan poly-

mer that is a major component of the fungal cell wall/

PST130_00758 Accession gi|403160953| ref|XM_003321311.2|

Homolog Pgt hypothetical protein (PGTG_02401)

UniProtKB/ TrEMBL ID E3JY15

Protein name Uncharacterised protein

Associated Function or Cellular location

P-body: A focus in the cytoplasm where mRNAs may become inactivated by

decapping or some other mechanism. Protein and RNA localized to these foci

are involved in mRNA degradation, nonsense-mediated mRNA decay (NMD),

translational repression, and RNA-mediated gene silencing.

GO terms

GO:0003729 (mRNA binding) GO:0030371 (translation repressor activity - Antag-

onises ribosome-mediated translation of mRNA into a polypeptide) GO:0017148

(negative regulation of translation) GO:0000289 (nuclear-transcribed mRNA

poly(A) tail shortening).

Conserved domains

smart00454. Sterile alpha motif. Widespread domain in signalling and nuclear

proteins.; cl15755. SAM (Sterile alpha motif) is a module consisting of approx-

imately 70 amino acids. This domain is found in the Fungi/Metazoa group

and in a restricted number of bacteriaSAM domains have diverse functions and

locations. They can interact with proteins, RNAs and membrane lipids, contain

site of phosphorylation and/or kinase docking site, and play a role in protein

homo and hetero dimerisation/oligomerisation in processes ranging from signal

CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 191

transduction to regulation of transcription. Mutations in SAM domains have

been linked to several diseases.

PST130_08345 Accession gi|403162070| ref|XM_003322301.2|

Homolog Pgt hypothetical protein (PGTG_03886)

UniProtKB/ TrEMBL ID E3K0V5

Protein name Aconitate hydratase, mitochondrial

Associated Function or Cellular location

Associated with the mitochondrion. Protein which binds at least one iron atom,

or protein whose function is iron-dependent. Involved in metabolic processes

that result in cell growth.

GO terms

GO:0051539 (4 iron, 4 sulfur cluster binding); GO:0003994 (aconitate hydratase

activity); GO:0046872 (metal ion binding); GO:0032543 (mitochondrial transla-

tion); GO:0006099 (tricarboxylic acid cycle).

Conserved domains

TIGR01340: aconitate hydratase, mitochondrial. [Energy metabolism, TCA cycle];

cl00215. Aconitase swivel domain. Aconitase (aconitate hydratase) catalyses the

reversible isomerisation of citrate and isocitrate as part of the TCA cycle. cl00285.

Aconitase catalytic domain. Both cl00215 and cl00285 are present in enzymes

involved in biosynthesis of leucine.

PST130_12299 Accession gi|403173188| ref|XM_003332239.2|

Homolog Pgt hypothetical protein (PGTG_14583) UniProtKB/ TrEMBL ID E3KU93

Protein name Uncharacterised protein

Associated Function or Cellular location

CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 192

Associated with the cytosol, nucleus and membrane.

GO terms

GO:0003723 (RNA binding) GO:0043130 (binding ubiquitin, involved in pro-

teolytic degradation) GO:0031081 (nuclear pore distribution) GO:0016973 &

GO:0006606 (poly(A)+ mRNA export / protein import from nucleus into the

cytoplasm / vice versa) GO:0000972 & GO:0000973 (transcriptional & posttran-

scriptional tethering of RNA polymerase II gene DNA at nuclear periphery)

GO:2000728 (regulates mRNA export from nucleus in response to heat stress)

GO:0006405 (RNA export from nucleus to the cytoplasm).

Conserved domains

COG2319: WD40 repeat [General function prediction only] sd00039: WD40 re-

peats in seven bladed beta propellers. The WD40 repeat is found in a number

of eukaryotic proteins that cover a wide variety of functions including adap-

tor/regulatory modules in signal transduction, pre-mRNA processing, and cy-

toskeleton assembly; cl02567: WD40 Superfamily.

CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 193

B.3 Nonsynonymous polymorphisms in candidate genes

SA1 M S FL S N T I L K F A L L L S V A L V Y Q L S G I N A N S I V S P K P N Q T L N P G E K L
SA2 M S L S N T I L K F A L L L S V A L V Y Q L S G I N A N S I V S P K P N Q T L N P G E K L
45
SA3 M S L S N T I L K F A L L L S V A L V Y Q L S G I N A N S I V S P K P N Q T L N P G E K L
SA4 M S L S N T I L K F A L L L S V A L V Y Q L S G I N A N S I V S P K P N Q T L N P G E K L

A V V V K K N S T D S T D Q T L A F A V G L S V Y K D S L G R P F L R T V D V G K G E A T
A V V V K K N S T D S T D Q T L A F A V G L S V Y K D S L G R P F L R T V D V G K G E A T
46 90
A V V V K K N S T D S T D Q T L A F A V G L S V Y K D S L G R P F L R T V D V G K G E A T
A V V V K K N S T D S T D Q T L A F A V G L S V Y K D S L G R P F L R T V D V G K G E A T

W N S H E S T Y T F E V T V P P T S D F I D Q F S K P Y N F A V S E Y Y L K G P S N V P T
W N S H E S T Y T F E V T V P P T S D F I D Q F S K P Y N F A V S E Y Y L K G P S N V P T
91 135
W N S H E S T Y T F E V T V P P T S D F I D Q F S K P Y N F A V S E Y Y L K G P S N V P T
W N S H E S T Y T F E V T V P P T S D F I D Q F S K P Y N F A V S E Y Y L K G P S N V P T

L G L S E T P V T I K Q D *
L G L S E T P V T I K Q D *
136 149
L G L S E T P V T I K Q D *
L G L S E T P V T I K Q DN *

Figure B.1: Translated sequence alignment of gene PST130_02001. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The signal
peptide, predicted using SignalP (version 2; Emanuelsson et al., 2007) is
indicated by the black box. Alternative amino acids resulting from nonsyn-
onymous SNPs at biallelic sites are indicated in the below diagonal triangles.
Colours were assigned according to the “Clustal X Colour Scheme” used in
Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 194

SA1 M L F S V L A V FL M M V Q G R S V I G A G F Q C LP D P A R A Q A L C S R P P T A P Q D H T
SA2 M L F S V L A V L M M V Q G R S V I G A G F Q C LP D P A R A Q A L C S R P P T A P Q D H T
45
SA3 M L F S V L A V FL M M V Q G R S V I G A G F Q C LP D P A R A Q A L C S R P P T A P Q D H T
SA4 M L F S V L A V FL M M V Q G R S V I G A G F Q C LP D P A R A Q A L C S R P P T A P Q D H T

V T I V K P Y R I G D D Y F C P P R L D A E I P V C C K T D M Y M R Y M A S G W K T I L P
V T I V K P Y R I G D D Y F C P P R L D A E IT P V C C K T D M Y M R Y M A S G W K T I L P
46 90
V T I V K P Y R I G D D Y F C P P R L D A E IT P V C C K T D M Y M R Y M A S G W K T I L P
V T I V K P Y R I G D D Y F C P P R L D A E IT P V C C K T D M Y M R Y M A S G W K T I L P

N D T Y S A A C F P P V H L P D P P K V D L T D A L R Y Y P A G D G I N L H V D T K T G G
N D T Y S A A C F P P V H L P D P P K V D L T D A L R Y Y P A G D G I N L H V D T K T G G
91 135
N D T Y S A A C F P P V H L P D P P K V D L T D A L R Y Y P A G D G I N L H V D T K T G G
N D T Y S A A C F P P V H L P D P P K V D L T D A L R Y Y P A G D G I N L H V D T K T G G

S F N C P V K T C K S S Y G G I G C T H D D I P G L G K A N Q T C S H L F G A K G A T Q I
S F N C P V K T C K S S Y G G I G C T H D D I P G L G K A N Q T C S H L F G A K G A T Q I
136 180
S F N C P V K T C K S S Y G G I G C T H D D I P G L G K A N Q T C S H L F G A K G A T Q I
S F N C P V K T C K S S Y G G I G C T H D D I P G L G K A N Q T C S H L F G A K G A T Q I

C C T F T D A *
C C T F T D A *
181 188
C C T F T D A *
C C T F T D A *

Figure B.2: Translated sequence alignment of gene PST130_02118. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The signal
peptide, predicted using SignalP (version 2; Emanuelsson et al., 2007) is
indicated by the black box. Alternative amino acids resulting from nonsyn-
onymous SNPs at biallelic sites are indicated in the below diagonal triangles.
Colours were assigned according to the “Clustal X Colour Scheme” used in
Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 195

SA1 M L K L T H V I L A C V L V L E A Y A L H I DG S G H S K R D I Y S E P K D H Y G GS H D Y T
SA2 M L K L T H V I L A C V L V L E A Y A L H I G S G H S K R D I Y S E P K D H Y G S H D Y T
45
SA3 M L K L T H V I L A C V L V L E A Y A L H I G S G H S K R D I Y S E P K D H Y G S H D Y T
SA4 M L K L T H V I L A C V L V L E A Y A L H I DG S G H S K R D I Y S E P K D H Y G GS H D Y T

P Y K P E P Q K K P E P S K Y Y P E P P K K P E P F K Y Y P V P P K E P E P F K H Y P E P
S
S Y K P E P Q K K P E P S K Y Y P E P P K K P E P F K Y Y P V P P K E P E P F K H Y P E P
46 90
S Y K P E P Q K K P E P S K Y Y P E P P K K P E P F K Y Y P V P P K E P E P F K H Y P E P
P Y K P E P Q K K P E P S K Y Y P E P P K K P E P F K Y Y P V P P K E P E P F K H Y P E P
S

P K K P E P F K Y Y P EV P P K K P E P F K HY Y P E P P K K P E P F K Y Y P T P P K K P D P
P K K P E P F K Y Y P V P P K K P E P F K H Y P E P P K K P E P F K Y Y P T P P K K P D P
91 135
P K K P E P F K Y Y P EV P P K K P E P F K HY Y P E P P K K P E P FS K Y Y P T P P K K P D P
P K K P E P F K Y Y P EV P P K K P E P F K H Y P E P P K K P E P F K Y Y P T P P K K P D P

S K Y Y P E P P P K P D P S K Y F P T P P Q E K P E T P K Y Y P E P P K Y K P E E P K Y A
S K Y Y P E P P P K P D P S K Y F P T P P Q E K P E T P K Y Y P E P P K Y K P E E P K Y A
136 180
S K Y Y P E P P P K P D P S K Y F P T P P Q E K P E T P K Y Y P E P P K Y K P E E P K Y A
S K Y Y P E P P P K P D P S K Y F P T P P Q E K P E T P K Y Y P E P P K Y K P E E P K Y A

S P K Y D AP P Y E K T P D E E P K Y S A P S Y D Y N P P K K D G Y R H *
S P K Y D AP P Y E K T P D E E P K Y S A P S Y D Y N P P K K D G Y R H *
181 216
S P K Y D AP P Y E K T P D E E P K Y S A P S Y D Y N P P K K D G Y R H *
S P K Y D AP P Y E K T P D E E P K Y S A P S Y D Y N P P K K D G Y R H *

Figure B.3: Translated sequence alignment of gene PST130_02403. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The signal
peptide, predicted using SignalP (version 2; Emanuelsson et al., 2007) is
indicated by the black box. Alternative amino acids resulting from nonsyn-
onymous SNPs at biallelic sites are indicated in the below diagonal triangles.
Colours were assigned according to the “Clustal X Colour Scheme” used in
Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 196

SA1 M N I Q L F P I M I F L L G H P S L I F G R P T E G K A V T Q E F G K L H V D C P G T E H
SA2 M N V Q L F P I M I V L L G H P S L I F G R P T E G K A V T Q E F G K L H V D C P G T E H
45
SA3 M N I Q L F P I M I F L L G H P S L I F G R P T E G K A V T Q E F G K L H V D C P G T E H
SA4 M N I Q L F P I M I F L L G H P S L I F G R P T E G K A V T Q E F G K L H V D C P G T E H

V E H V K N P F A E E D K H A S V I S D N S K N I S G S R H S S S P E S I P E E E K P F L
V E H V K N P F A E E D K H A S V I S D N S K N I S G S R H S S S P E S I P E E E K P F L
46 90
V E H V K N P F A E E D K H A S V I S D N S K N I S G S R H S S S P E S I P E E E K P L L
V E H V K N P F A E E D K H A S V I S D N S K N I S G S R H S S S P E S I P E E E K P L L

D R S Q S D R G S S K P S G P A P D Q P K Q G E D G K G R K M A E L Y A R F K K S L S T W
D R S Q S D R G S S K P S G P A P D Q P K Q G E D G K G R K M A E L Y A R F K K S L S T W
91 135
D R S Q S D R G S S K P S G P A P D Q P K Q G E D G K G R K M A E L Y A R F K K S L S T W
D R S Q S D R G S S K P S G P A P D Q P K Q G E D G K G R K M A E L Y A R F K K S L S T W

Y G G H S A V A R F L R R M V N Y F H P R K M S K S K E A K E A K E A E D A K K V E D A K
Y G G H S A V A R F L R R L V N Y F H P R K M S K S K E A K E A K E A E D A K K V EK D A K
136 180
Y G G H S A V A R F L R R L V N Y F H P R K M S K S K E A K E A K E A E D A K K V E D A K
Y G G H S A V A R F L R R L V N Y F H P R K M S K S K E A K E A K E A EKDE A K EKAVEK D AV K

K V K D V K K V G D V K K A E E A T K A E D A E K A Q E A K K A Q E T T G A V R V E A S M
K V K D V K K V G D V K K A E E A T K A E D A E K A Q E A K K A Q E T T G A V R V E A S M
181 225
K V K D V K K V G D V K K A E E A T K A E D A E K A Q E A K K A Q E T T G A V R V E A S M
K AVEK D V K K V E D V K K A E E A T K A E D A E K A Q E A K K A Q E T T G A V R V E A S M

P E L S V T E E K A A T A V K P E S P S A T S P S T G T V P A S S N F V K P G L F A T D E
P E L S V T E E K A A T A V K P E S P S A T S P S T G T V P A S S N F V K P G L F A T D E
226 270
P E L S V T E E K A A T A A K P E S P S A T S P S T G T V P A S S N F V K P G L F A T D E
P E L S V T E E K A A T A A K P E S P S A T S P S A G T V P A S S N F V K P G L F A T D E

S Q P R P Q T I W I A *
S Q P R P Q T I W I A *
271 282
S Q P R P Q T I W I A *
S Q P R P Q T I W I A *

Figure B.4: Translated sequence alignment of gene PST130_05023. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The signal
peptide, predicted using SignalP (version 2; Emanuelsson et al., 2007) is
indicated by the black box. Alternative amino acids resulting from nonsyn-
onymous SNPs at biallelic sites are indicated in the below diagonal triangles.
Colours were assigned according to the “Clustal X Colour Scheme” used in
Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 197

SA1 M R G L Q I C K I V F G I L V S F H H S I A A D A P P S V G I P S S V S P C G A V P L E I
SA2 M R G L Q I C K I V F G I L V S F H H S I A A D A P P S V G I P S S V S P C G A V P L E I
45
SA3 M R G L Q I C K I V F G I L V S F H H S I A A D A P P S V G I P S S V S P C G A V P L E I
SA4 M R G L Q I C K I V F G I L V S F H H S I A A D A P P S V G I P S S V S P C G A V P L E I

T G G T P P Y S I A I N AT A D N P S G P P L H T F A D V K Q P S S L A W P S G M S T G M V
T G G T P P Y S I A I N A A D N P S G P P L H T F A D V K Q P S S L A W P S G M S T G M V
46 90
T G G T P P Y S I A I N AT A D N P S G P P L H T F A D V K Q P S S L A W P S G M S T G M V
T G G T P P Y S I A I N AT A D N P S G P P L H T F A D V K Q P S S L A W P S G M S T G M V

L T M E V K D S K G L T T T S G Q S T V I P S A D C P Q S P G A G A T K N T T D I A T T G
L T M E V K D S K G L T T T S G Q S T V I P S A D C P Q S P G A G A T K N T T D I A T T G
91 135
L T M E V K D S K G L T T T S G Q S T V I P S A D C P Q S P G A G A T K N T T D I A T T G
L T M E V K D S K G L T T T S G Q S T V I P S A D C P Q S P G A G A T K N T T D I A T T G

P PS G G D G AS A K N W T Q G M P A L S S DN K T A G G P T P P A S A N S T D P A H P A N A FV
P PS G G D G S A K N W T Q G M P A L S S N K T A G G P T P P A S A N S T D P A H P A N A V
136 180
P P G G D G AS A K N W T Q G M P A L S S DN K T A G G P T P P A S A N S T D P A H P A N A FV
P P G G D G AS A K N W T Q G M P A L S S DN K T A G G P T P P A S A N S T D P A H P A N A FV

S T T A N A T G A V R L D S A D S N N A S M P D S A N AV T A T A D Q H G V M N M T D S T P
S T T A N A T G A V R L D S A D S NS N A S M P D S A N A T A T A D Q H G V M N M T D S T P
181 225
S T T A N A T G A V R L D S A D S NS N A S M P D S A N AV T A T A D Q H G V M N M T D S T P
S T T A N A T G A V R L D S A D S NS N A S M P D S A N AV T A T A D Q H G V M N M T D S T P

M S P S T A R AT T N M P P S N K T V NSHN N D N S K S G N N T S S S EK P G K I G G V *
M S P S T A R A T N M P P S N K T V N H N D N S K S G N N T S S S EK P G K I G G V *
226 267
M S P S T A R AT T N M P P S N K T V NSHN N D N S K S G N N T S S S EK P G K I G G V *
M S P S T A R AT T N M P P S N K T V NSHN N D N S K S G N N T S S S EK P G K I G G V *

Figure B.5: Translated sequence alignment of gene PST130_05454. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The signal
peptide, predicted using SignalP (version 2; Emanuelsson et al., 2007) is
indicated by the black box. Alternative amino acids resulting from nonsyn-
onymous SNPs at biallelic sites are indicated in the below diagonal triangles.
Colours were assigned according to the “Clustal X Colour Scheme” used in
Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 198

SA1 M T R L I I I L G L V A R L L A P K V F G A G L P D E N L A K L P A D L H I I K A D E S G
SA2 M T R L I I I L G L V A R L L A P K V F G A G L P D E N L A K L P A D L H I I K A D E S G
45
SA3 M T R L I I I L G L V A R L L A P K V F G A G L P D E N L A K L P A D L H I I K A D E S G
SA4 M T R L I I I L G L V A R L L A P K V F G A G L P D E N L A K L P A D FL H I I K A D E S G

S P Y V D P V T N V K F R D I P N K L D K E I T I H D G K E P W I I E P R Q N V R L D Y D
S P Y V D P V T N V K F R D I P N K L D K E I T I H D G K E P W I I E P R Q N V R L D Y D
46 90
S P Y V D P V T N V K F R D I P N K L D K E I T I H D G K E P W I I E P R Q N V R L D Y D
S P Y V D P V T N V K F R D I PQ N K L D K E I T I H DN G KQ E P W I I E P R Q N V R L D Y D

P N Y P Y L L I T D N E R V L L N K D F Y N R H V T T T A I E R L K E E A A E R P P A S D
P N Y P Y L L I T D N E R V L L N K D F Y N R H V T T T A I E R L K E E A A E R P P A S D
91 135
P N HY P Y L L I T D N E R V FL L NT K D FS Y DN R H V T T T A I E R L K E E A A E R P P A S D
P N Y P Y L L I T D N E R V L L N K D F Y N R H V T T T A I E R L K E E A A E R P P A S D

P E G P T G T S N S Q H E E W Y E N L A P N P V L G T G R T A D K Q L P T D K G E S Q K E
P E G P T G T S N S Q H E E W Y E N L A P N P V L G T G R T A D K Q L P T D K G E S Q K E
136 180
P E G P T G T S N S Q H E E W Y E N L A P N P V L G T G R T A D K Q L P T D K G E S Q K E
P E G P T G T S N S Q H E E W Y E N L A P N P V L G T G R T A D K Q L P T D K G E S Q K E

Q F I E S S R D Q A E L P D S T T G S S G E K R P T D A P M E E I Q D G S N S R P V E P R
Q F I E S S R D Q A E L P D S T T G S S G E K R P T D A P M E E I Q D G S N S R P V E P R
181 225
Q F I E S S R D Q A E L P D S T T G S S G E K R P T D A P M E E I Q D G S N S R P V E P R
Q F I E S S R D Q A E L P D S T T G S S G E K R P T D A P M E E I Q D G S N S R P V E P R

V P D L P I R R D F L T G R L A G Q K K P K Q K K L R I R L P T E V P L L R E P D F S Q H
V P D L P I R R D F L T G R L A G Q K K P K Q K K L R I R L P T E V P L L R E P D F S Q H
226 270
V P D L P I R R D F L T G R L A G Q K K P K Q K K L R I R L P T E V P L L R E P D F S Q H
V P D L P I R R D F L T G R L A G Q K K P K Q K K L R I R L P T E V P L L R E P D F S Q H

F L Q L V N G Q K C T E A V K L L D P S T Q K D Y F K L V T Y I Y D A Q T G R W V H Q P N
F L Q L V N G Q K C T E A V K L L D P S T Q K D Y F K L V T Y I Y D A Q T G R W V H Q P N
271 315
F L Q L V N G Q K C T E A V K L L D P S T Q K D Y F K L V T Y I Y D A Q T G R W V H Q P N
F L Q L V N G Q K C T E A V K L L D P S T Q K D Y F K L V T Y I Y D A Q T G R W V H Q P N

V P A *
V P A *
316 319
V P A *
V P A *

Figure B.6: Translated sequence alignment of gene PST130_05944. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The signal
peptide, predicted using SignalP (version 2; Emanuelsson et al., 2007) is
indicated by the black box. Alternative amino acids resulting from nonsyn-
onymous SNPs at biallelic sites are indicated in the below diagonal triangles.
Colours were assigned according to the “Clustal X Colour Scheme” used in
Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 199

SA1 M Q S S L I V S I L I V C S G V I A L P T S N Q A Q I E T R A E K T R S S D K Y A S S E Y
SA2 M Q S S L I V S I L I V C S G V I A L P T S N Q A Q I E T R A E K T R S S D K Y A S S E Y
45
SA3 M Q S S L I V S I L I V C S G V I A L P T S N Q A Q I E T R A E K T R S S D K Y A S S E Y
SA4 M Q S S L I V S I L I V C S G V I A L P T S N Q A Q I E T R A E K T R S S D K Y A S S E Y

N E S D T Y A S A P N S A P S V I P V G F P S I P L P Q V S G S S P Q S G S Y F G G K G G
N E S D T Y A S A P N S A P S V I P V G F P S I P L P Q V S G S S P Q S G S Y F G G K G G
46 90
N E S D T Y A S A P N S A P S V I P V G F P S I P L P Q V S G S S P Q S G S Y F G G K G G
N E S D T Y A S A P N S A P S V I P V G F P S I P L P Q V S G S S P Q S G S Y F G G K G G

R I S S A F P G F V G G F G G K I S G K A G G K M D A G M G G K I A A G G S G G L N A A G
R I S S A F P G F V G G F G G K I S G K A G G K M D A G M G G K I A A G G S G G L N A A G
91 135
R I S S A F P G F V G G F G G K I S G K A G G K M D A G M G G K I A A G G S G G L N A A G
R I S S A F P G F V G G F G G K I S G K A G G K M D A G M G G K I A A G G S G G L N A A G

S V G G Q V A G G V Q A G I G A A G S I A G Q AV A G G A Q
S V G G Q V A G G V Q A G I G A A G S I A G Q A A G G A Q
136 P A A 164
SV G G Q V A G G VQ A G I GA A G S I A G Q A A G G A Q
S V G G Q V A G G V Q A G I G A A G S I A G Q A A G G A Q

Figure B.7: Translated sequence alignment of gene PST130_06503. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The signal
peptide, predicted using SignalP (version 2; Emanuelsson et al., 2007) is
indicated by the black box. Alternative amino acids resulting from nonsyn-
onymous SNPs at biallelic sites are indicated in the below diagonal triangles.
Colours were assigned according to the “Clustal X Colour Scheme” used in
Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 200

SA1 M T K N A I S L S V F L L S C V P K S Q Q T F G F F S T V L S S N G G D P N A S Y Y A G G
SA2 M T K N A I S L S V F L L S C V P K S Q Q T F G F F S T V L S S N G G D P N A S Y Y A G G
45
SA3 M T K N A I S L S V F L L S C V P K S Q Q T F G F F S T V L S S N G G D P N A S Y Y A G G
SA4 M T K N A I S L S V F L L S C V P K S Q Q T F G F F S T V L S S N G G D P N A S Y Y A G G

K V R Q V L A A S Q P G A K G G G Q A D A G A V V P P V K C A C E N G G P P G P S G S S D
K V R Q V L A A S Q P G A K G G G Q A D A G A V V P P V K C A C E N G G P P G P S G S S D
46 90
K V R Q V L A A S Q P G A K G G G Q A D A G A V V P P V K C A C E N G G P P G P S G S S D
K V R Q V L A A S Q P G A K G G G Q A D A G A V V P P V K C A C E N G G P P G P S G S S D

K G T A P P N S A G G T T P P S I S S G G P T P P V T S G G P P P N G P P P I T S G A P P
K G T A P P N S A G G T T P P S I S S G G P T P P V T S G G P P P N G P P P I T S G A P P
91 135
K G T A P P N S A G G T T P P S I S S G G P T P P V T S G G P P P N G P P P I T S G A P P
K G T A P P N S A G G T T P P S I S S G G P T P P V T S G G P P P N G P P P I T S G A P P

P G S T P S G G P P S T P L G G T P P S G P S G D S S A K P S D S P T K G D G S G D K N S
P G S T P S G G P P S T P L G G T P P S G P S G D S S A K P S D S P T K G D G S G D K N S
136 180
P G S T P S G G P P S T P L G G T P P S G P S G D S S A K P S D S P T K G D G S G D K N S
P G S T P S G G P P S T P L G G T P P S G P S G D S S A K P S D S P T K G D G S G D K N S

P P P V T S G G P P P V T S G G A A T P S S P G N G S S G G K Q K P K D T P S K T T D K D
P P P V T S G G P P P V T S G G A A T P S S P G N G S S G G K Q K P K D T P S K T T D K D
181 225
P P P V T S G G P P P V T S G G A A T P S S P G N G S S G G K Q K P K D T P S K T T D K D
P P P V T S G G P P P V T S G G A A T P S S P G N G S S G G K Q K P K D T P S K T T D K D

L P P P V T S G G T S S P G S P G D G S S Q G K P K P K S G D S G D T P S V S S G G G T S
L P P P V T S G G T S S P G S P G D G S S Q G K P K P K S G D S G D T P S V S S G G G T S
226 270
L P P P V T S G G T S S P G S P G D G S S Q G K P K P K S G D S G D T P S V S S G G G T S
L P P P V T S G G T S S P G S P G D G S S Q G K P K P K S G D S G D T P S V S S G G G T S

D K P K D T P S K P G G S A D T P S V S S G G S T S D K P K D T P S K P G G S E D T P S V
D K P K D T P S K P G G S A D T P S V S S G G S T S D K P K D T P S K P G G S E D T P S V
271 315
D K P K D T P S K P G G S A D T P S V S S G G S T S D K P K D T P S K P G G S E D T P S V
D K P K D T P S K P G G S A D T P S V S S G G S T S D K P K D T P S K P G G S E D T P S V

S S G G S T A D G K P K P K D T T S K P G G S E D T
S S G G S PT A D G K P K P K D T T S K P G G S E D T
316 341
S S G G S T A D G K P K P K D T T S K P G G S E D T
S S G G S PTAS D G K PS K P K D T T S K P G G S E D T

Figure B.8: Translated sequence alignment of gene PST130_06558. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The signal
peptide, predicted using SignalP (version 2; Emanuelsson et al., 2007) is
indicated by the black box. Alternative amino acids resulting from nonsyn-
onymous SNPs at biallelic sites are indicated in the below diagonal triangles.
Colours were assigned according to the “Clustal X Colour Scheme” used in
Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 201

SA1 M I F H T R T F Q L F S L T A M L C S R V Q A K C E G V M I V S A D A P E I P D M S A K D
SA2 M I F H T R T F Q L F S L T A M L C S R V Q A K C E G V M I V S A D A P E I P D M S A K D
45
SA3 M I F H T R T F Q L F S L T A M L C S R V Q A K C E G V M I V S A D A P E I P D M S A K D
SA4 M I F H T R T F Q L F S L T A M L C S R V Q A K C E G V M I V S A D A P E I P D M S A K D

Q T Y H P E V G R I S Y S L D S A G T L E L T S T T P G F N C G P I T N F V S S N A T S K
Q T Y H P E V G R I S Y S L D S A G T L E L T S T T P G F N C G P I T N F V S S N A T S K
46 90
Q T Y H P E V G R I S Y S L D S A G T L E L T S T T P G F N C G P I T N F V S S N A T S K
Q T Y H P E V G R I S Y S L D S A G T L E L T S T T P G F N C G P I T N F V S S N A T S K

T P V K D P S A H K S S R D K K E S Q D P V Q S V G A Q L H C A R D P D T V G V D L M T P
T P V K D P S A H K S S R D K K E S Q D P V Q S V G A Q L H C A R D P D T V G V D L M T P
91 135
T P V K D P S A H K S S R D K K E S Q D P V Q S V G A Q L H C A R D P D T V G V D L M T P
T P V K D P S A H K S S R D K K E S Q D P V Q S V G A Q L H C A R D P D T V G V D L M T P

W Q T I T F Y G S L F F Q I E M K N N T C A K P A E L V L D Y S R C S Y N A T T N T G R Q
W Q T I T F Y G S L F F Q I E M K N N T C A K P A E L V L D Y S R C S Y N A T T N T G R Q
136 180
W Q T I T F Y G S L F F Q I E M K N N T C A K P A E L V L D Y S R C S Y N A T T N T G R Q
W Q T I T F Y G S L F F Q I E M K N N T C A K P A E L V L D Y S R C S Y N A T T N T G R Q

G S A I P C N W S T C *
G S A I P C N W S T C *
181 192
G S A I P C N W S T C *
G S A I P C N W S T C *

Figure B.9: Translated sequence alignment of gene PST130_07448. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The signal
peptide, predicted using SignalP (version 2; Emanuelsson et al., 2007) is
indicated by the black box. Alternative amino acids resulting from nonsyn-
onymous SNPs at biallelic sites are indicated in the below diagonal triangles.
Colours were assigned according to the “Clustal X Colour Scheme” used in
Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 202

SA1 M K S F G I I A T L L A L A S S I H A D A A V R P K T A A P A S D I I E L T L E N F D T V
SA2 M K S F G I I A T L L A L A S S I H A D A A V R P K T A A P A S D I I E L T L E N F D T V
45
SA3 M K S F G I I A T L L A L A S S I H A D A A V R P K T A A P A S D I I E L T L E N F D T V
SA4 M K S F G I I A T L L A L A S S I H A D A A V R P K T A A P A S D I I E L T L E N F D T V

V A T T P L I L V E F M V P W C H F C Q D L G P E Y K R S A K I L K E Q G I P S A K V D C
V A T T P L I L V E F M V P W C H F C Q D L G P E Y K R S A K I L K E Q G I P S A K V D C
46 90
V A T T P L I L V E F M V P W C H F C Q D L G P E Y K R S A K I L K E Q G I P S A K V D C
V A T T P L I L V E F M V P W C H F C Q D L G P E Y K R S A K I L K E Q G I P S A K V D C

T E Q D E L C A E H L L P S Y P T L K V F S N G R M A V Y K G P EK K A D S I V S Y I E N K
T E Q D E L C A E H L L P S Y P T L K V F S N G R M A V Y K G P EK K A D S I V S Y I E N K
91 135
T E Q D E L C A E H L L P S Y P T L K V F S N G R M A V Y K G P EK K A D S I V S Y I E N K
T E Q D E L C A E H L L P S Y P T L K V F S N G R M A V Y K G P EK K A D S I V S Y I E N K

E Y L G S N K A R I S S R R D S N T V *
E Y L G HS N K AV R I S S R R D S N T V *
136 155
E Y L G HS N K AV R I S S R R D S N T V *
E Y L G HS N K AV R I S S R R D S N T V *

Figure B.10: Translated sequence alignment of gene PST130_07513. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 203

SA1 M L P S R T I W L L F L A S S I P I L Q V L A G T D Q G L S P V R R Q T L E K R W G V C M
SA2 M L P S R T I W L L F L A S S I P I L Q V L A G T D Q G L S P V R R Q T L E K R W G V C M
45
SA3 M L P S R T I W L L F L A S S I P I L Q V L A G T D Q G L S P V R R Q T L E K R W G V C M
SA4 M L P S R T I W L L F L A S S I P I L Q V L A G T D Q G L S P V R R Q T L E K R W G V C M

V P N R R K G C V V W G S Q S C C R D C C S E Y L Q G I R P E S W R I Q C G C P P LR H AP P
V P N R R K G C V V W G S Q S C C R D C C S E Y L Q G I R P E S W R I Q C G C P P R H A P
46 90
V P N R R K G C V V W G S Q S C C R D C C S E Y L Q G I R P E S W R I Q C G C P P LR H AP P
V P N R R K G C V V W G S Q S C C R D C C S E Y L Q G I R P E S W R I Q C G C P P LR H AP P

H T V V V V Q Q A A P P P P P A P A P A P A P A Q G P T I V I N H P G A Q P A V A Y P Q P
H T V V V V Q Q A A P P P P P A P A P A P A P A Q G P T I V I N H P G A Q PT A V A Y P Q P
91 135
H T V V V V Q Q A A P P P P P A P A P A P A P A Q G P T I V IVNT H P G AG Q PT A V A Y P Q P
H T V V V V Q Q A A P P P P P A P A P A P A P A Q G P T I V I N H P G A Q P A V A Y P Q P

V V A Y P A Q P G V
V V A Y P A Q P G V
136 145
V V A Y P A Q P G V
V V A Y P A Q P G V

Figure B.11: Translated sequence alignment of gene PST130_07564. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 204

SA1 M T R I F F A L L S I L A I I N T I Y A R S S L N D F L R R A I K G G V S Y Y L S N M G A
SA2 M T R I F F A L L S I L A I I N T I Y A R S S L N D F L R R A I K G G V S Y Y L S N M G A
45
SA3 M T R I F F A L L S I L A I I N T I Y A R S S L N D F L R R A I K G G V S Y Y L S N M G A
SA4 M T R I F F A L L S I L A I I N T I Y A R S S L N D F L R R A I K G G V S Y Y L S N M G A

I S T D L M K D E D P K E E C V F Y V N S Y Q S T R E K N A A I A F A A M R N R Q L T A S
I S T D L M K D E D P K E E C V F Y V N S Y Q S T R E K N A A I A F A A M R N R Q L T A S
46 90
I S T D L M K D E D P K E E C V F Y V N S Y Q S T R E K N A A I A F A A M R N R Q L T A S
I S T D L M K D E D P K E E C V F Y V N S Y Q S T R E K N A A I A F A A M R N R Q L T A S

G G R P T A N T L Y D A F D L N L A F G D S G T L M R E A M A G G P A Y L R S Y F K V T S
G G R P T A N T L Y D A F D L N L A F G D S G T L M R E A M A G G P A Y L R S Y F K V T S
91 135
G G R P T A N T L Y D A F D L N L A F G D S G T L M R E A M A G G P A Y L R S Y F K V T S
G G R P T A N T L Y D A F D L N L A F G D S G T L M R E A M A G G P A Y L R S Y F K V T S

G A Y A Q R C R G T V W L I V K K G A E I Y H D A I W L T D E Y P Q L I R P G S G V T A I
G A Y A Q R C R G T V W L I V K K G A E I Y H D A I W L T D E Y P Q L I R P G S G V T A I
136 180
G A Y A Q R C R G T V W L I V K K G A E I Y H D A I W L T D E Y P Q L I R P G S G V T A I
G A Y A Q R C R G T V W L I V K K G A E I Y H D A I W L T D E Y P Q L I R P G S G V T A I

W E I D P A E I E A A I A L D N P N H D L H P T P Y
W E I D P A E I E A A I A L D N P N H D L H P T P Y
181 206
W E I D P A E I E A A I A L D N P N H D L H P T P Y
W E I D P A E I E A A I A L D N P N H D L H P T P Y

Figure B.12: Translated sequence alignment of gene PST130_08031. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 205

SA1 M S F S N T I L K F A L L F S V A L V Y Q L S G I N A N S I V S P K P N Q T L N P G T K L
SA2 M S F S N T I L K F A L L F S V A L V Y Q L S G I N A N S I V S P K P N Q T L N P G T K L
45
SA3 M S F S N T I L K F A L L F S V A L V Y Q L S G I N A N S I V S P K P N Q T L N P G T K L
SA4 M S F S N T I L K F A L L F S V A L V Y Q L S G I N A N S I V S P K P NT Q T L N P G ET K L

V V V V K K N S T D S T D Q T L A F A V G L S V Y K D S L G R P F L R T V E V G K G E A A
V V V V K K N S T D S T D Q T L A F A V G L S V Y K D S L G R P F L R T V E V G K G E A A
46 90
V V V V K K N S T D S T D Q T L A F A V G L S V Y K D S L G R P F L R T V E V G K G E A A
V V V V K K N S T D S T D Q T L A F AV V G L S V Y KRDE S L G R P F L R T V E V G K G E A A

W N S H E S T Y T F E V T L P P T S E F I D Q F T K
W N S H E S T Y T F E V T L P P T S E F I D Q F T K
91 116
W N S H E S T Y T F E V T L P P T S E F I D Q F T K
W N S H E S T Y T F E V T L P P T S E F I D Q F T K

Figure B.13: Translated sequence alignment of gene PST130_08984. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 206

SA1 M P R S I L H T S C L A L Y V I A A I H V A T R P T I C Y G A S L A K R A I E R E T D R T
SA2 M P R S I L H T S C L A L Y V I A A I H V A T R P T I C Y G A S L A K R A I E R E T D R T
45
SA3 M P R S I L H T S C L A L Y V I A A I H V A T R P T I C Y G A S L A K R A I E R E T D R T
SA4 M P R S I L H T S C L A L Y V I A A I H V A T R P T I C Y G A S L A K R A I E R E T D R T

L L R A T P S R K R V R L F G V D L S D E H N T R L E E A R V G R E K D D P Q S I P L S L
L L R A T P S R K R V R L F G V D L S D E H N T R L E E A R V G R E K D D P Q S I P L S L
46 90
L L R A T P S R K R V R L F G V D L S D E H N T R L E E A R V G R E K D D P Q S I P L S L
L L R A T P S R K R V R L F G V D L S D E H N T R L E E A R V G R E K D D P Q S I P L S L

K P E D T L G T I P L E A Y A A L V P E L F V C Q F G S K G T I P E L L E Y L R N P P F G
K P E D T L G T I P L E A Y A A L V P E L F V C Q F G S K G T I P E L L E Y L R N P P F G
91 135
K P E D T L G T I P L E A Y A A L V P E L F V C Q F G S K G T I P E L L E Y L R N P P F G
K P E D T L G T I P L E A Y A A L V P E L F V C Q F G S K G T I P E L L E Y L R N P P F G

F P G N A P W I Q R I D N T A T W L Q S K D I G V S N R F K P W D L L P R T Y K Q V E S D
F P G N A P W I Q R I D N T A T W L Q S K D I G V S N R F K P W D L L P R T Y K Q V E S D
136 180
F P G N A P W I Q R I D N T A T W L Q S K D I G V S N R F K P W D L L P R T Y K Q V E S D
F P G N A P W I Q R I D N T A T W L Q S K D I G V S N R F K P W D L L P R T Y K Q V E S D

F N M I K A R E V L K E M K N H D L E S E S Q E H L V Q N L L K D L M K V L E K K T L I LS
F N M I K A R E V L K E M K N H D L E S E S Q E H L V Q N L L K D L M K V L E K K T L I S
181 225
F N M I K A R E V L K E M K N H D L E S E S Q E H L V Q N L L K D L M K V L E K K T L I S
F N M I K A R E V L K E M K N H D L E S E S Q E H L V Q N L L K D L M K V L E K K T L I S

K D GR A G P S GR K Q F R F S G V G E H N E H N T G L K E A Q V Q R G K G H T Q S H T F S F
K D GR A G P S GR K Q F R F S G V G E H N E H N T G L K E A Q V Q R G K G H T Q S H T F S F
226 270
K D G A G P S R K Q F R F S G V G E H N E H N T G L K E A Q V Q R G K G H T Q S H T F S F
K D G A G P S R K Q F R F S G V G E H N E H N T G L K E A Q V Q R G K G H T Q S H T F S F

K P E D T L D K T S L E A Y A A L V P D L Y R C R F G N K G T I P E L S K Y L D A R N P P
K P E D T L D K T S L E A Y A A L V P D L Y R C R F G N K G T I P E L S K Y L D A R N P P
271 315
K P E D T L D K T S L E A Y A A L V P D L Y R C R F G N K G T I P E L S K Y L D A R N P P
K P E D T L D K T S L E A Y A A L V P D L Y R C R F G N K G T I P E L S K Y L D A R N P P

P S L P K D E A V R K R I Y D T R A W L H S K D I E I N T S Y K H W S W G P S M Y R E V E
P S L P K D E A V R K R I Y D T R A W L H S K D I E I N T S Y K H W S W G P S M Y R E V E
316 360
P S L P K D E A V R K R I Y D T R A W L H S K D I E I N T S Y K H W S W G P S M Y R E V E
P S L P K D E A V R K R I Y D T R A W L H S K D I E I N T S Y K H W S W G P S M Y R E V E

S D F N T I S L E M Y L E L A P V V L G Y P H D W N Q D L R H F L G K K Y D L Q T K N Q G
S D F N T I S L E M Y L E L A P V V L G Y P H D W N Q D L R H F L G K K Y D L Q T K N Q G
361 405
S D F N T I S L E M Y L E L A P V V L G Y P H D W N Q D L R H F L G K K Y D L Q T K N Q G
S D F N T I S L E M Y L E L A P V V L G Y P H D W N Q D L R H F L G K K Y D L Q T K N Q G

A M A Q F L M N D L V K A F K E K M F K P R N P L *
A M A Q F L M N D L V K A F K E K M F K P R N P L *
406 431
A M A Q F L M N D L V K A F K E K M F K P R N P L *
A M A Q F L M N D L V K A F K E K M F K P R N P L *

Figure B.14: Translated sequence alignment of gene PST130_09018. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 207

SA1 M I S T N F L A C L T P I F L N G L L A L K V T S P T E N S Q W D L Q A T N T I T W T S V
SA2 M I S T N F L A C L T P I F L N G L L A L K V T S P T E N S Q W D L Q A T N T I T W T S V
45
SA3 M I S T N F L A C L T P I F L N G L L A L K V T S P T E N S Q W D L Q A T N T I T W T S V
SA4 M I S T N F L A C L T P I F L N G L L A L K V T S P T E N S Q W D L Q A T N T I T W T S V

A T D P K T F D I V L T N IN N P S C A P T G F T Q A I K Q N I A S S D G K F D I S G V S S
A T D P K T F D I V L T N IN N P S C A P T G F T Q A I K Q N I A S S D G K F D I S G V S S
46 90
A T D P K T F D I V L T N IN N P S C A P T G F T Q A I K Q N I A S S D G K F D I S G V S S
A T D P K T F D I V L T N IN N P S C A P T G F T Q A I K Q N I A S S D G K F D I S G V S S

M K A C S G Y Q I N L V A S S T P D N GS A H N A G I L A Q S A P F N V T Q T S G P S M S E
M K A C S G Y Q I N L V A S S T P D N GS A H N A G I L A Q S A P F N V T Q T S G P S M S E
91 135
M K A C S G Y Q I N L V A S S T P D N GS A H N A G I L A Q S A P F N V T Q T S G P S M S E
M K A C S G Y Q I N L V A S S T P D N GS A H N A G I L A Q S A P F N V T Q T S G P S M S E

S L P L A G A N S T A N T P A A S T P V A N T T S P T Q S T S S T G A P K Y N S G T A A P
S L P L A G A N S T A N T P A A S T P V A N T T S P T Q S T S S T G A P K Y N S G T A A P
136 180
S L P L A G A N S T A N T P A A S T P V A N T T S P T Q S T S S T G A P K Y N S G T A A P
S L P L A G A N S T A N T P A A S T P V A N T T S P T Q S T S S T G A P K Y N S G T A A P

G A K Y S F A P R I S G S F Q K V T A C A L L L V T F M L A *
G A K Y S F A P R I S G S FL Q K V T A C A L L L V T F M L A *
181 211
G A K Y S F A P R I S G S F Q K V T A C A L L FLIV T F M L A *
G A K Y S F A P R I S G S FL Q K V T A C A L L L V T F M L A *

Figure B.15: Translated sequence alignment of gene PST130_09275. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 208

SA1 M Q I Q Q L I T I L C L C F S Q A L A A S V E A FL K P K I Q S L V V D L T E R R V I P G E
SA2 M Q I Q Q L I T I L C L C F S Q A L A A S V E A FL K P K I Q S L V V D L T E R R V I P G E
45
SA3 M Q I Q Q L IT T I L C L C F S Q A L A A S V E A FL K P K I Q S L V V D L T E HR R V I P G E
SA4 M Q I Q Q L IT T I L C L C F S Q A L A A S V E A FL K P K I Q S L V V D L T E HR R V I P G E

R A S G T K Y D H A L R L D M D E P V A D P N Y T P A F Y R D Y I Q G M NY P L T Y V D K E
R A S G T K Y D H A L R L D M D E P V A D P N Y T P A F Y R D Y I Q G M NY P L T Y V D K E
46 90
R A S G T K Y D H A L R L D M D E P V A D P N Y T P A F Y R D Y I Q G M NY P L T Y V D K E
R A S G T K Y D H A L R L D M D E P V A D P N Y T P A F Y R D Y I Q G M NY P L T Y V D K E

S T N S F L D A R A A Y E E T L R D D F T G N Y R V Q R R R L R I C Q N A M Y S R L C D I
S T N S F L D A R A A Y E E T L R D D F T G N Y R V Q R R R L R I C Q N A M Y S R L C D I
91 135
S T N S F L D A R A A Y E E T L R D D F T G N Y R V Q R R R L R I C Q N A M Y S R L C D I
S T N S F L D A R A A Y E AE T L R DG D F T G N FY R V Q R R R L R I C Q N A M Y S R L C D I

V K K G D D D T V A H V L K T Y H E Y V K S L I N K H S N A F P Q I Q T S E R A P S K P Q
V K K G D D D T V A H V L K T Y H E Y V K S L I N K H S N A F P Q I Q T S E R A P S K P Q
136 180
V K K G D D D T V A H V L K T Y H E Y V K S L I N K H S N A F P Q I Q T S E R A P S K P Q
V K K G D D D T V A H V L K T Y H E Y V K S L I N K H S N A F P Q I Q T S E R A P PS K P Q

S A F V Y R T K E Q I N K E L L A T N Q A E T D V P K A R L I D G T S Q K T F E D F L F N
S A F V Y R T K E Q I N K E L L A T N Q A E T D V P K A R L I D G T S Q K T F E D F L F N
181 225
S A F V Y R T K E Q I N K E L L A T N Q A E T D V P K A R L I D G T S Q K T F E D F L F N
L A F V Y R TK EL A D K E L LA T N Q A E T D V P K A R L I D G T S Q K T F E D F L F N
S P Q Q I N K

H S Q K Q W Q L V H G S P S N T R P Q I F L E T G E R Y S *
H S Q K Q W Q L V H G S P S N T R P Q I F L E T G E R Y S *
226 255
H S Q K Q W Q L V H G S P S N T R P Q I F L E T G E R Y S *
H S Q K Q W Q L V H G S P S N T R P Q I F L E T G E R Y S *

Figure B.16: Translated sequence alignment of gene PST130_10286. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 209

SA1 M F G S S T I L L A C S L L S Y V L A A P A R L S N L P S L D G T L S N A P S P S W Q L T
SA2 M F G S S T I L L A C S L L S Y V L A A P A R L S N L P S L D G T L S N A P S P S W Q L T
45
SA3 M F G S S T I L L A C S L L S Y V LS A A P A GR L S N L PQ S L D G T L S N A P S P S W Q L T
SA4 M F G S S T I L L A C S L L S Y V L A A P A R L S N L P S L D G T L S N A P S P S W Q L T

I D N G Q I R N R R F M V E A S A P K V E P P M S K Q M A C F D S K V G K P S I E Q T E R
I D N G Q I R N R R F M V E A S A P K V E P P M S K Q M A C F D S K V G K P S I E Q ST E KR
46 90
I D N G Q I R N R R F M V E A S A P K V E P P M S K Q M A C F D S K V G K P S I E Q ST E KR
S
I D N G Q I R N R R F M V E A S A P K V E P P M S K Q M A C F D S K V G K P S I E Q TE R K

I E N Y L K H C K T G K A Y K V P A N G D I Y P M P K S D S T Y G Y I F G K V Q F Y D D C
I E N Y L K H C KN T G K A Y K V P AE N G D I Y P M P K S D S T Y G Y I F G K V Q F Y D D C
91 135
I E N Y L K H C KN T G K A Y K V P AE N G D I Y P M P K S D S T Y G Y I F G K V Q F Y D D C
I E N Y L K H C KN T G K A Y K V P A N G D I Y P M P K S D S T Y G Y I F G K V Q F Y D D C

D R L I H E T G C C Y G K P S D R E G Y N A M E S C C I V A G A C Y G C I C C T A F S A I
D R L I H E T G C C Y G K P S D R E G Y N A M E S C C I V A G A C Y G C I C C T A F S A I
136 180
D R L I H E T G C C Y G K P S D R E EGFYNTATGM E ST C C I GV A G A C CY G C I C C T A F S A I
D R L I H E T G C C Y G K P S D R E G Y N A M E S C C I V A G A C Y G C I C C T A F S A I

L N F K L T V D I K L V W S S N P *
L N F K L T V D I K L V W S S N P *
181 198
L N F K L T V D I K L V W S S N P *
L N F K L T V D I K L V W S S N P *

Figure B.17: Translated sequence alignment of gene PST130_12487. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 210

SA1 M R S F V A V A V T L A L L Q S T S A L P I F E K R A E T E G T G K G E S S S R S L G G C
SA2 M R S F V A V A V T L A L L Q S T S A L P I F E K R A E T E G T G K G E S S S R S L G G C
45
SA3 M R S F V A V A V T L A L L Q S T S A L P I F E K R A E T E G T G K G E S S S R S L G G C
SA4 M R S F V A V A V T L A L L Q S T S A L P I F E K R A E T E G T G K G E S S S R S L G G C

S N Q V G L L N I A L S T N T H C G Q N G P A S G S G G A G G L LV P G G G G L L P G G G I
S N Q V G L L N I A L S T N T H C G Q N G P A S G S G G A G G L L P G G G G L L P G G G I
46 90
S N Q V G L L N I A L S T N T H C G Q N G P A S G S G G A G G L L P G G G G P L P G G G I
S N Q V G L L N I A L S T N T H C G Q N G P A S G S G G A G G L L P G G G G L L P G G G I

D G L L P A G G L L P D G G I D G L L P A G G L L P G G G V D G L L P G G G I D G L L P G
D G L L P A G G L L P D G G I D G L L P A G G L L P G G G V D G L L P G G G I D G L L P G
91 135
D G L L P A G G L L P D G G I D G L L P A G G L L P G G G V D G L L P G G G I D G L L P G
D G L L P A G G L L P D G G I D G L L P A G G L L P G G G V D G L L P G G G I D G L L P G

G G A G G L L P A G G T G G F L P G G G G L L P G G G I D G L L P G G G I D G L L P A G G
G G A G G L L P A G G T G G F L P G G G G L L P G G G I D G L L P G G G I D G L L P A G G
136 180
G G A G G L L P A G G T G G F L P G G G G L L P G G G I D G L L P G G G I D G L L P A G G
G G A G G L L P A G G T G G F L P G G G G L L P G G G I D G L L P G G G I D G L L P A G G

I D
I D
181 182
I D
I D

Figure B.18: Translated sequence alignment of gene PST130_12491. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 211

SA1 M R S F G F L A T L F A L A S S I H A D A G L D P N D A P D D V I E L T S E N F D T V V T
SA2 M R S F G F L A T L F A L A S S I H A D A G L D P N D A P D D V I E L T S E N F D T V V T
45
SA3 M R S F G F L A T L F A L A S S I H A D A G L N P N D A P D D V I E L T S E N F D T V V T
SA4 M R S F G F L A T L F A L A S S I H A D A G L N P N D A P D D V I E L T S E N F D T V V T

P A P L I L V E F M A P W C G H C K A L M P E Y K R A A T L L K K G G I P V A K A D C T E
P A P L I L V E F M A P W C G H C K A L M P E Y K R A A T L L K K G G I P V A K A D C T E
46 90
P A P L I L V E F M A P W C G H C K A L M P E Y K R A A T L L K K G G I P V A K A D C T E
P A P L I L V E F M A P W C G H C K A L M P E Y K R A A T L L K K G G I P V A K A D C T E

Q S E L C A K Y E I Q G Y P T L K I F T N G V S S E Y K G P R K A D G I V S Y M E K R A H
Q S E L C A K Y E I Q G Y P T L K I F T N G V S S E Y K G P R K A D G I V S Y M E K R A H
91 135
Q S E L C A K Y E I Q G Y P T L K I F T N G V S S E Y K G P R K A D G I V CS Y M E K R A H
Q S E L C A K Y E I Q G Y P T L K I F T N G V S S E Y K G P R K A D G I V CS Y M E K R A H

P V V T I V T S D N H T D F T K S G N V V
P V V T I V T S D N H T D F T K S G N V V
136 156
P V V T I V T S D N H T D F T K S G N V V
P V V T I V T S D N H T D F T K S G N V V

Figure B.19: Translated sequence alignment of gene PST130_12956. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 212

SA1 M M T S S K A T L F Y V A L R T L F A S Q M V L A F P L G D V S P E M T S G I L S A G D T
SA2 M M T S S K A T L F Y V A L R T L F A S Q M V L A F P L G D V S P E M T S G I L S A G D T
45
SA3 M M T S S K A T L F Y V A L R T L F A S Q M V L A F P L G D V S P E M T S G I L S A G D T
SA4 M M T S S K A T L F Y V A L R T L F A S Q M V L A F P L G D V S P E M T S G I L S A G D T

A M T K P P R E Y F Q R V R Y G E Y G G H T D I A S N Q L P Q Y N K G E S D F S K L Y S T
A M T K P P R E Y F Q R V R Y G E Y G G H T D I A S N Q L P Q Y N K G E S D F S K L Y S T
46 90
A M T K P P R E Y F Q R V R Y G E Y G G H T D I A S N Q L P Q Y N K G E S D F S K L Y S T
A M T K P P R E Y F Q R V R Y G E Y G G H T D I A S N Q L P Q Y N K G E S D F S K L Y S T

I L L T L D L L G Q V A E V D S M E S A S R Q I R Q K I G K L K L I I P A A G R K G R E Y
I L L T L D L L G Q V A E V D S M E S A S R Q I R Q K I G K L K L I I P A A G R K G R E Y
91 135
I L L T L D L L G Q V A E V D S M E S A S R Q I R Q K I G K L K L I I P A A G R K G R E Y
I L L T L D L L G Q V A E V D S M E S A S R Q I R Q K I G K L K L I I P A A G R K G R E Y

S L H L A S Q FL E F I H N Q L S T E F Q W G L S H P N V E W A E L Y H G P A L V E A P P K
S L H L A S Q FL E F I H N Q L S T E F Q W G L S H P N V E W A E L Y H G P A L V E A P P K
136 180
S L H L A S Q FL E F I H N Q L S T E F Q W G L S H P N V E W A E L Y H G P A L V E A P P K
S L H L A S Q FL E F I H N Q L S T E F Q W G L S H P N V E W A E L Y H G P A L V E A P P K

V E P I K W D D L Y H GV P A L D K A S L E V Q P V R K S G I N P E V F Q D N Y N S L IT D W
V E P I K W D D L Y H GV P A L D K A S L E V Q P V R K S G I N P E V F Q D N Y N S L IT D W
181 225
V E P I K W D D L Y H GV P A L D K A S L E V Q P V R K S G IM N P E V F Q D N WY N S L IT D W
V E P I K W D D L Y H GV P A L D K A S L E V Q P V R K S G IM N P E V F Q D N WY N S L IT D W

L T K P E V DN G I T R K S P E F Y A A V A DE I I F L IL N N Y M I K Y K H T L P D F P K P L
L T K P E V DN G I T R K S P E F Y A A V A E I I F L L N N Y M I K Y K H T L P D F P K P L
226 270
L T K P E V DN G I T R K S P E F Y A A V A DE I I F L IL N N Y M I K Y K H T L P D F P K P L
L T K P E V DN G I T R K S P E F Y A A V A DE I I F L IL N N Y M I K Y K H T L P D F P K P L

R R F E P E E I A Y V I E N F A R S E K R L L E D I R L P F P P V D S E G W K T S A S I N
R R F E P E E I A Y V I E N F A R S E K R L L E D I R L P F P P V D S E G W K T S A S I N
271 315
R R F E P E E I A Y V I E N F A R S E K R L L E D I R L P F P P V D S E G W K T S A S I N
R R F E P E E I A Y V I E N F A R S E K R L L E D I R L P F P P V D S E G W K T S A S I N

F L I S S D I S K A F R G E I K A L D D E G Q E L V A K A F Q R G T A K L L E Q I R G K E
F L I S S D I S K A F R G E I K A L D D E G Q E L V A K A F Q R G T A K L L E Q I R G K E
316 360
F L I S S DE I FS K A F R G E I K A L D D E G Q E L V A K A F Q R G T A K L L E Q I R G K E
F L I S S DE I FS K A F R G E I K A L D D E G Q EK L V AV K A F Q R G T A K L L E Q I R G K E

I R GR S E Q A Y A Y L R R S A Q P K S P S R L G S P T H L T A E A LV *
I R GR S E Q A Y A Y L R R S A Q P K S P S R L G S P T H L T A E A LV *
361 395
I R GR S E Q A Y A Y L R R S A Q P K S P S R L G S P T H L T A E A LV *
I R GR S E Q A Y A Y L R R S A Q P K S P S R L G S P T H L T A E A LV *

Figure B.20: Translated sequence alignment of gene PST130_13969. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 213

SA1 M N N R F N I I I L L F I T S L D S L F A S Q H P H S T I N H L K T R D Q P N G I S K P C
SA2 M N N R F N I I I L L F I T S L D S L F A S Q H P H S T I N H L K T R D Q P N G I S K P C
45
SA3 M N N R F N I I I L L F I T S L D S L F A S Q H P H S T I N H L K T R D Q P N G I S K P C
SA4 M N N R F N I I I L L F I T S L D S L F A S Q H P H S T I N H L K T R D Q P N G I S K P C

Q T Y Y S A N T P H A V A H N C Q L D S S S Q N T T Q T C S V A F S Q T S E S A Y L C N T
Q T Y Y S A N T P H A V A H N C Q L D S S S Q N T T Q T C S V A F S Q T S E S A Y L C N T
46 90
Q T Y Y S A N T P H A V A H N C Q L D S S S Q N T T Q T C S V A F S Q T S E S A Y L C N T
Q T Y Y S A N T P H A V A H N C Q L D S S S Q N T T Q T C S V A F S Q T S E S A Y L C N T

P E G A Y T C T G P Q S G G V V C H N C V S T P N G V L P S N T T S N A K N Q A H S G S N
P E G A Y T C T G P Q S G G V V C H N C V S T P N G V L P S N T T S N A K N Q A H S G S N
91 135
P E G A Y T C T G P Q S G G V V C H N C V S T P N G V L P S N T T S N A K N Q A H S G S N
P E G A Y T C T G P Q S G G V V C H N C V S T P N G V L P S N T T S N A K N Q A H S G S N

S T N E H Q E H P W F EK D P I T E G C F W H F I R V I E N K L P *
S T N E H Q E H P W F K D P I T E G C F W H F I R V I E N K L P *
136 168
S T N E H Q E H P W F K D P I T E G C F W H F I R V I E N K L P *
S T N E H Q E H P RW F EK D P I IT E G C F W H F I R V I E N K L P *

Figure B.21: Translated sequence alignment of gene PST130_14091. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 214

SA1 M K I P A I I I L L G A V C S L T N A A P M V G D V V R A G E L D V R G T G L E G T P F A
SA2 M K I P A I I I L L G A V C S L T N A A P M V G D V V R A G E L D V R G T G L E G T P F A
45
SA3 M K I P A I I I L L G A V C S L T N A A P M V G D V V R A G E L D V R G T G L E G T P F A
SA4 M K I P A I I I L L G A V C S L T N A A P M V G D V V R A G E L D V R G T G L E G T P F AT

L A W L A Y M V L E R P G E L K N F M E G T E E G W K F S K F L P H V L G P H A L I G D I
L A W L A Y M V L E R P G E L K N F M E G T E E G W K F S K F L P H V L G P H A L I G D I
46 90
L A W L A Y M V L E R P G E L K N F M E G T E E G LW K F S K F L P H V L G P H A L I G D I
L A W L A Y M V L E R P G E L K N F M E G T E E G LW K F S K F L P H V L G P H A L I G D I

G L V T K A L EQ K T D P A L A E K A L A Y I K S I R S A A Y N D V L E A T R P A G G H V A
G L V T K A L E K T D P A L A E K A L A Y I K S I R S A A Y N D V L E A T R P A G G H V A
91 135
G L V T K A L EQ K T D P A L A E K A L A Y I K S I R S A A Y N D V L E A T R P A G G H V A
G L V T K A L EQ K T D P A L A E K A L A Y I K S I R S A A Y N D V L E A T R P A G G H V A

I A A T *
I A A T *
136 140
I A A T *
I A A T *

Figure B.22: Translated sequence alignment of gene PST130_14831. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 215

SA1 M M I L S L N L I L V L V A F F H S I P S ST I S T P A Y Y G R S S G D F R S P L M A H L G
SA2 M M I L S L N L I L V L V A F F H S I P S S I S T P A Y Y G R S S G D F R S P L M A H L G
45
SA3 M M I L S L N L I L V L V A F F H S I P S ST I S T P A Y Y G R S S G D F R S P L M A H L G
SA4 M M I L S L N L I L V L V A F F H S I P S ST I S T P A Y Y G R S S G D F R S P L M A H L G

D G L P L Q V S P D V I A A A L E R A Q R K A E A E A E V S A D G R M R I A T P T F R KT A
D G L P L Q V S P D V I A A A L E R A Q R K A E A E A E V S A D G R M R I A T P T F R K A
46 90
D G L P L Q V S P D V I A A A L E R A Q R K A E A E A E V S A D G R M R I A T P T F R K A
D G L P L Q V S P D V I A A A L E R A Q R K A E A E A E V S A D G R M R I A T P T F R KT A

G S D S K A R D A E W T S A R HN Q R K A E A A A A Y H A N G R S A K A A T A E K V H P E E
G S D S K A R D A E W T S A R N Q R K A E A A A A Y H A N G R S A K A A T A E K V H P E E
91 135
G S D S K A R D A E W T S A R N Q R K A E A A A A Y H A N G R S A KS A A T A E K V H P E E
G S D S K A R D A E W T S A R N Q R K A E A A A A Y H A N G R S A K A A T A E K V H P E E

F K V E P Y R S P SV M E L T S K L L G N T F V V L D D L S Y Q W K V E I R *
F K V E P Y R S P S M E L T S K L L G N T F V V L D D L S Y Q W K V E I R *
136 173
F K V E P Y R S P SV M E L T S K L L G N T F V V L D D L S Y Q W K V E I R *
F K V E P Y R S P S M E L T S K L L G N T F V V L D D L S Y Q W K V E I R *

Figure B.23: Translated sequence alignment of gene PST130_16778. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 216

SA1 M A F K S M T V A S L L V A F S F P S G L L A K D D D V K T C F T Y T G A N T T T A S C N
SA2 M A F K S M T V A S L L V A F S F P S G L L A K D D D V K T C F T Y T G A N T T T A S C N
45
SA3 M A F K S M T V A S L L V A F S F P S G L L A K D D D V K T C F T Y T G A N T T T A S C N
SA4 M A F K S M T V A S L L V A F S F P S G L L A K D D D V K T C F T Y T G A N T T T A S C N

D I P N M V C S G G C T G G L T A T K C T T S H E M N D Q R G P L T D E K C T I A Y G K S
D I P N M V C S G G C T G G L T A T K C T T S H E M N D Q R G P L T D E K C T I A Y G K S
46 90
D I P N M V C S G G C T G G L T A T K C T T S H E M N D Q R G P L T D E K C T I A Y G K S
D I P N M V C S G G C T G G L T A T K C T T S H E M N D Q R G P L T D E K C T I A Y G K S

S A T M A V C I A E H Q T Y T C Y G P V S G T A Q C K G C K N T Y I P P P N D Q Q N G G G
S A T M A V C I A E H Q T Y T C Y G P V S G T A Q C K G C K N T Y I P P P N D Q Q N G G G
91 135
S A T M A V C I A E H Q T Y T C Y G P V S G T A Q C K G C K N T Y I P P P N D Q Q N G G G
S A T M A V C I A E H Q T Y T C Y G P V S G T A Q C K G C K N T Y I P P P N D Q Q N G G G

G S G N G N G G K G S G G N G S G E S G N K P P G G S S S P T P G N S P A P G Q S P T P L
G S G N G N G G K G S G G N G S G E S G N K P P G G S S S P T P G N S P A P G PQ S P T P L
136 180
G S G N G N G G K G S G G N G S G E S G N K P P G G S S S P T P G N S P A P G PQ S P T P L
G S G N G N G G K G S G G N G S G E S G N K P P G G S S S P T P G N S P A P G Q S P T P L

I S P A P G S N G N S S T P P Q T P S G G S E A P P S S S G A T T D N S K K L N S S D S K
I S P A P G S N G N S S T P P Q T P S G G S E A P P S S S G A T T D N S K K L N S S D S K
181 225
I S P A P G S N G N S S T P P Q T P S G G S E A P P S S S G A T T D N S K K L N S S D S K
I S P A P G S N G N S S T P P Q T P S G G S E A P P S S S G A T T D N S K K L N S S D S K

P S A Y D I F L M S C S R S *
P S A Y D I F L M S C S R S *
226 240
P S A Y D I F L M S C S R S *
P S A Y D I F L M S C S R S *

Figure B.24: Translated sequence alignment of gene PST130_17605. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 217

SA1 M A F K S M T V A S L L V A F S F P S G L L A K D D D V K T C F T Y T G A N T T T A S C N
SA2 M A F K S M T V A S L L V A F S F P S G L L A K D D D V K T C F T Y T G A N T T T A S C N
45
SA3 M A F K S M T V A S L L V A F S F P S G L L A K D D D V K T C F T Y T G A N T T T A S C N
SA4 M A F K S M T V A S L L V A F S F P S G L L A K D D D V K T C F T Y T G A N T T T A S C N

D I P N M V C S G G C T G G L T A T K C T T S H E M N D Q R G P L T D E K C T I A Y G K S
D I P N M V C S G G C T G G L T A T K C T T S H E M N D Q R G P L T D E K C T I A Y G K S
46 90
D I P N M V C S G G C T G G L T A T K C T T S H E M N D Q R G P L T D E K C T I A Y G K S
D I P N M V C S G G C T G G L T A T K C T T S H E M N D Q R G P L T D E K C T I A Y G K S

S A T M A V C I A E H Q T Y T C Y G P V S G T A Q C K G C K N T Y I P P P N D Q Q N G G G
S A T M A V C I A E H Q T Y T C Y G P V S G T A Q C K G C K N T Y I P P P N D Q Q N G G G
91 135
S A T M A V C I A E H Q T Y T C Y G P V S G T A Q C K G C K N T Y I P P P N D Q Q N G G G
S A T M A V C I A E H Q T Y T C Y G P V S G T A Q C K G C K N T Y I P P P N D Q Q N G G G

G S G N G N G G K G S G G N G S G E S G N K P P G G S S S P T P G N S P A P G Q S P T P L
G S G N G N G G K G S G G N G S G E S G N K P P G G S S S P T P G N S P A P G PQ S P T P L
136 180
G S G N G N G G K G S G G N G S G E S G N K P P G G S S S P T P G N S P A P G PQ S P T P L
G S G N G N G G K G S G G N G S G E S G N K P P G G S S S P T P G N S P A P G Q S P T P L

I S P A P G S N G N S S T P P Q T P S G G S E A P P S S S G A T T D N S K K L N S S D S K
I S P A P G S N G N S S T P P Q T P S G G S E A P P S S S G A T T D N S K K L N S S D S K
181 225
I S P A P G S N G N S S T P P Q T P S G G S E A P P S S S G A T T D N S K K L N S S D S K
I S P A P G S N G N S S T P P Q T P S G G S E A P P S S S G A T T D N S K K L N S S D S K

P S A Y D I F L M S C S R S *
P S A Y D I F L M S C S R S *
226 240
P S A Y D I F L M S C S R S *
P S A Y D I F L M S C S R S *

Figure B.25: Translated sequence alignment of gene PST130_17605. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 218

SA1 ML S I N Y L L L V L S S V V L L A H S N D S L P P S S P R K S I N Y G P E L S T H S I K
SA2 ML S I N Y L L L V L S S V V L L A H S N D S L P P S S P R K S I N Y G P E L S T H S I K
45
SA3 ML S I N Y L L L V L S S V V L L A H S N D S L P P S S P R K S I N Y G P E L S T H S I K
SA4 ML S I N Y L L L V L S S V V L L A H S N D S L P P S S P R K S I N Y G P E L S T H S I K

T S V Y S N H N H N D QF QA S L T S F N A A S A S S L L P I K D T F H QT D S S S L K Q
T S V Y S N H N H N D QF QA S L T S F N A A S A S S L L P I K D T F H QT D S S S L K Q
46 90
T S V Y S N H N H N D QF QA S L T S F N A A S A S S L L P I K D T F H QT D S S S L K Q
T S V Y S N H N H N D QF QA S L T S F N A A S A S S L L P I K D T F H QT D S S S L K Q

F G I K I A T E F L H H L H P S DE S FL T F QL T S A H I S K H T K V L H A Y F V QT I P L
F G I K I A T E F L H H L H P S DE S FL T F QL T S A H I S K H T K V L H A Y F V QT I P L
91 135
F G I K I A T E F L H H L H P S DE S FL T F QL T S A H I S K H T K V L H A Y F V QT I P L
F G I K I A T E F L H H L H P S DE S FL T F QL T S A H I S K H T K V L H A Y F V QT I P L

G D L D HY H V K V H N A V A N L N L N L D P R S A N F G H V L S H S D S F H P I V E H P S
G D L D HY H V K V H N A V A N L N L N L D P R S A N F G H V L S H S D S F H P I V E H P S
136 180
G D L D HY H V K V H N A V A N L N L N L D P R S A N F G H V L S H S D S F H P I V E H P S
G D L D HY H V K V H N A V A N L N L N L D P R S A N F G H V L S H S D S F H P I V E H P S

S S E A V N F I N A F D G QQG D R C T H L K N K F D G V L QS L S T N N QL L N QQV M
S S E A V N F I N A F D G Q Q G D R C T H L K N K F D G V L QR S L S T N N QL L N QQV M
181 225
S S E A V N F I N A F D G Q Q G D R C T H L K N K F D G V L QR S L S T N N QL L N QQV M
S S E A V N F I N A F D G Q Q G D R C T H L K N K F D G V L QR S L S T N N QL L N QQV M

G L F S T K S S Q DH S A G D E K T L L T D F S E E E L R I I A E C E MS N P T K K A I R S
G L F S T K S S Q DH S A G D E K T L L T D F S E E E L R I I A E C E MS N P T K K A I R S
226 270
G L F S T K S S Q DH S A G D E K T L L T D F S E E E L R I I A E C E MS N P T K K A I R S
G L F S T K S S Q DH S A G D E K T L L T D F S E E E L R I I A E C E MS N P T K K A I R S

E I V D P R I A L V S F L T L A A D P E T E N H L R S R S L E D L V E S I D I V K K T P S
E I V D P R I A L V S F L T L A A D P E T E N H L R S R S L E D L V E S I D I V K K T PS S
271 315
E I V D P R I A L V S F L T L A A D P E T E N H L R S R S L E D L V E S I D I V K K T PS S
E I V D P R I A L V S F L T L A A D P E T E N H L R S R S L E D L V E S I D I V K K T PS S

S S S F Y A A G D S D G S A T K E S P T F E L F N V P G A L G A D S L D G S S S T K A T S
S S S F Y A A G D S D G S A T K E S P T F E L F N V P G A L G A D S L D G S S S T K A T S
316 360
S S S F Y A A G D S D G S A T K E S P T F E L F N V P G A L G AS D S L D G S S S T K A T S
S S S F Y A A G D S D G S A T K E S P T F E L F N V P G A L G AS D S L D G S S S T K A T S

A E L A WL S V D D G E R E L K MV WR F E Y R S N S N WY E A Y V D A S S P G L V P MV
A E L A WL S V D D G E R E L K MV WR F E Y R S N S N WY E A Y V D A S S P G L V P MV
361 405
A E L A WL S V D D G E R E L K MV WR F E Y R S N S N WY E A Y V D A S S P G L V P MV
A E L A WL S V D D G E R E L K MV WR F E Y R S N S N WY E A Y V D A S S P G L V P MV

I D W V N D F R P T S E L A D S Y S E H V A I Q T A I V E E F K R L PS T T P E
S S HP R HR N P
I D W V N D F R P T S E L A D S Y S E H V A I Q T A I V E E F K R L PS T T P E
S S H R H N P
406 450
I D W V N D F R P T S E L A D S Y S E H V A I Q T A I V E E F K R L P T T PS E S HP R HR N P
I D W V N D F R P T S E L A D S Y S E H V A I Q T A I V E E F K R L PS T T PS E S HP R HR N P

A Q S Q S E V D L P V L P E G A T D E K R T A T Y R V F P WS V N D P T L G K R Q I V V T
A Q S Q S E V D L P V L P E G A T D E K R T A T Y R V F P WS V N D P T L G K R Q I V V T
451 495
A Q S Q S E V D L P V L P E G A T D E K R T A T Y R V F P WS V N D P T L G K R Q I V V T
A Q S Q S E V D L P V L P E G A T D E K R T A T Y R V F P WS V N D P T L G K R Q I V V T
>>>

Figure B.26: See continuation on next page.

CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 219

<<<
P S N P T A S P L G WH T I P A T Q R N S E Q R D I S H MS T G WS R H V P R H G L R A T
P S N P T A S P L G WH T I P A T Q R N S E Q R D I S H MS T G WS R H V P R H G L R A T
496 540
P S N P T A S P L G WH T I P A T Q R N S E Q R D I S H MS T G WS R H V P R H G L R A T
P S N P T A S P L G WH T I P A T Q R N S E Q R D I S H MS T G WS R H V P R H G L R A T

D T R G N N V Y A Q E N WE G L D N WE A N H R P N G T D D L E F K F H L G WK H P D N P
D T R G N N V Y A Q E N WE G L D N WE A N H R P N G T D D L E F K F H L G WK H P D N P
541 585
D T R G N N V Y A Q E N WE G L D N WE A N H R P N G T D D L E F K F H L G WK H P D N P
D T R G N N V Y A Q E N WE G L D N WE A N H R P N G T D D L E F K F H L G WK H P D N P

S E T H V N P K R Y I D A A I S E L F F T C N E F H D L T Y L Y G F D E E S G N F QQH N
S E T H V N P K R Y I D A A I S E L F F T C N E F H D L T Y L Y G F D E E S G N F QQH N
586 630
S E T H V N P K R Y I D A A I S E L F F T C N E F H D L T Y L Y G F D E E S G N F QQH N
S E T H V N P K R Y I D A A I S E L F F T C N E F H D L T Y L Y G F D E E S G N F QQH N

F G H G G K G D D A V I A N A Q D G S G Y N N A N F A T P P D G R N G R MR MY V WN G A
F G H G G K G D D A V I A N A Q D G S G Y N N A N F A T P P D G R N G R MR MY V WN G A
631 675
F G H G G K G D D A V I A N A Q D G S G Y N N A N F A T P P D G R N G R MR MY V WN G A
F G H G G K G D D A V I A N A Q D G S G Y N N A N F A T P P D G R N G R MR MY V WN G A

E P WR D G D L E A G I V I H E Y S H G V S I R L T G G P A N S G C L G Y G E S G G MG E
E P WR D G D L E A G I V I H E Y S H G V S I R L T G G P A N S G C L G Y G E S G G MG E
676 720
E P WR D G D L E A G I V I H E Y S H G V S I R L T G G P A N S G C L G Y G E S G G MG E
E P WR D G D L E A G I V I H E Y S H G V S I R L T G G P A N S G C L G Y G E S G G MG E

G WG D F F A T L I R MH Q S K P V D F T MG E WA S G V K G G I R K Y K Y S L D N K V N
G WG D F F A T L I R MH Q S K P V D F T MG E WA S G V K G G I R K Y K Y S L D N K V N
721 765
G WG D F F A T L I R MH Q S K P V D F T MG E WA S G V K G G I R K Y K Y S L D N KIVN
G WG D F F A T L I R MH Q S K P V D F T MG E WA S G V K G G I R K Y K Y S L D N K V N

P E T Y Q T L D K P G Y WG V H A I G E V WA E ML F T V A E E L I A K H G F Q P S L F P
P E T Y Q T L D K P G Y WG V H A I G E V WA E ML F T V A E E L I A K H G F Q P S L F P
766 810
P E T Y Q T L D K P G Y WG V H A I G E V WA E ML F T V A E E L I A K H G F Q P S L F P
P E T Y Q T L D K P G Y WG V H A I G E V WA E ML F T V A E E L I A K H G F Q P S L F P

P S G E A D E E G F Y K V S K L S D K K V P K H G N T L I F Q L V L D G MK I Q R C R P G
P S G E A D E E G F Y K V S K L S D K K V P K H G N T L I F Q L V L D G MK I Q R C R P G
811 855
P S G E A D E E G F Y K V S K L S D K K V P K H G N T L I F Q L V L D G MK I Q R C R P G
P S G E A D E E G F Y K V S K L S D K K V P K H G N T L I F Q L V L D G MK I Q R C R P G

F F D A R D A I L E A D S I L T G G E N Q C E I WK G F S K R G L G P K A A I K G N T P W
F F D A R D A I L E A D S I L T G G E N Q C E I WK G F S K R G L G P K A A I K G N T P W
856 900
F F D A R D A I L E A D S I L T G G E N Q C E I WK G F S K R G L G P K A A I K G N T P W
F F D A R D A I L E A D S I L T G G E N Q C E I WK G F S K R G L G P K A A I K G N T P W

G G G I R T N D F S L P T G V P R V H Y Y K P R I E *
G G G I R T N D F S L P T G V P R V H Y Y K P R I E *
901 927
G G G I R T N D F S L P T G V P R V H Y Y K P R I E *
G G G I R T N D F S L P T G V P R V H Y Y K P R I E *

Figure B.27: Translated sequence alignment of gene PST130_07579. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 220

SA1 MY A L G Y R Q I V R L A S C C L L A T Q V V G V A T Q V V S V E P S I S E A K A T WK S
SA2 MY A L G Y R Q I V R L A S C C L L A T Q V V G V A T Q V V S V E P S I S E A K A T WK S
45
SA3 MY A L G Y R Q I V R L A S C C L L A T Q V V G V A T Q V V S V E P S I S E A K A T WK S
SA4 MY A L G Y R Q I V R L A S C C L L A T Q V V G V A T Q V V S V E P S I S E A K A T WK S

R F N A L F S A S T N P H D V E H D M S R S DG A S I G A Q E M D Q F T Y K P WHY E AT V S K
R F N A L F S A S T N P H D V E H D MS R S G A S I G A Q E MD Q F T Y K P WH E A V S K
46 90
R F N A L F S A S T N P H D V E H D MS R S G A S I G A Q E MD Q F T Y K P WH E A V S K
R F N A L F S A S T N P H D V E H D MS R S G A S I G A Q E MD Q F T Y K P WH E A V S K

K MD R K A I P L F L R E P N P Y V K P G P D SIT E S D L N L I S E G F D E W V E A TV I T
K MD R K A I P L F L R E P N P Y V K P G P D S I E S D L N L I S E G F D E WV E A V I T
91 135
K MD R K A I P L F L R E P N P Y V K P G P D SIT E S D L N L I S E G F D E W V E A TV I T
K MD R K A I P L F L R E P N P Y V K P G P D SIT E S D L N L I S E G F D E W V E A TV I T

K S L S E S P E E T E K F E E Q C K I L K P I L V F L N AG G E S DG S L K Y S E E N P E Q PS
K S L S E S P E E T E K F E E Q C K I L K P I L V F L N AG G E S DG S L K Y S E E N P E Q PS
136 180
K S L S E S P E E T E K F E E Q C K I L K P I L V F L N AG G E S DG S L K Y S E E N P E Q PS
K S L S E S P E E T E K F E E Q C K I L K P I L V F L N AG G E S DG S L K Y S E E N P E Q PS

K I V N S D D L S R NS L I S L W K S I G S P E I N E H E AP T L D S D L DI R A N H F L K Q K
K I V N S D D L S R NS L I S L WK S I G S P E I N E H E P T L D S D L D I A N H F L K QK
181 225
K I V N S D D L S R NS L I S L W K S I G S P E I N E H E AP T L D S D L DI R A N H F L K Q K
K I V N S D D L S R NS L I S L W K S I G S P E I N E H E AP T L D S D L DI R A N H F L K Q K

T F R T M D Y I Y N Y N I M S H E A L KN K V L S S D DN I L E I T G S N L F V A Y S H DN D L S
T F R T MD Y I Y N Y N I MS H E A L K K V L S S D D I L E I T G S N L F V A Y S H DN D L S
226 270
T F R T M D Y I Y N Y N I M S H E A L KN K V L S S D DN I L E I T G S N L F V A Y S H N D L
T F R T MD Y I Y N Y N I MS H E A L K K V L S S D D I L E I T G S N L F V A Y S H N D L

D F N H Y P I E Y N F F R R N D Q H E S K S F F Q V L D A K Q R R K V MY F Y A K S R Y T
D F N H Y P I E Y N F F R R N D Q H E S K S F F Q V L D A K Q R R K V MY F Y A K S R Y T
271 315
D F N H Y P I E Y N F F R R N D PQHV E S K S F F Q V L D A K Q R R K V M Y F Y A K S R Y T
D F N H Y P I E Y N F F R R N D Q H E S K S F F Q V L D A K Q R R K V MY F Y A K S R Y T

K Q K E D H L L R L R S K E S K D E D E I T E E R Y L KR L K A F S T D S I F K D N E F L I D S
K QK E D H L L R L R S K E S K D E D E I T E E R Y L K L K A S T D S I F K D N E L I D S
316 360
K QK E D H L L R L R S K E S K D E D E I T E E R Y L K L K A S T D S I F K D N E L I D S
K QK E D H L L R L R S K E S K D E D E I T E E R Y L K L K A S T D S I F K D N E L I D S >>>

Figure B.28: See continuation on next page.

CHAPTER B: EVOLUTION OF SOUTH AFRICAN PST 221

<<< L E A Y L E H A Q S H N S Q T K N A N P Y K S K E K L K E L F V T L L A L WD D K Y S P I
L E A Y L E H A Q S H N S Q T K N A N P Y K S K E K L K E L F V T L L A L WD D K Y S P I
361 405
L E A Y L E H A Q S H N S Q T K N A N P Y K S K E K L K E L F V T L L A L WD D K Y S P I
L E A Y L E H A Q S H N S Q T K N A N P Y K S K E K L K E L F V T L L A L WD D K Y S P I

R E D Y V D F L S S L C N F I E E S Y G I D I I IVE N Q P K GR K E F MI K Y K L V S S Y M
R E D Y V D F L S S L CS N F I E E S Y G I D I I I V E N Q P K GR K E F M I K Y KT L IVS S Y M
406 450
R E D Y V D F L S S L CS N F I E E S Y G I D I I I V E N Q P K GR K E F M I K Y KT L IVS S Y M
R E D Y V D F L S S L C N F I E E S Y G I D I I IVE N Q P K GR K E F M I K Y KT L IVS S Y M

K Y L E E L D K F R E Y L L N H P S D P N V P F S H F F K E S T Q Q K ML A L D E L T V I
K Y L E E L D KT F I R DE Y L L N H PS S D P N V P F S H F F EK E S T Q Q K M L A L D E L RT V I
451 495
K Y L E E L D KT F I R DE Y L L N H PS PS D P N I V P FS S H F F EK E G M Q Q K ML A L D E LR V I
S T T
K Y L E E L D KT F I R E Y L L N H P S D P N V P F S H F F K E S T Q Q K ML A L D E L T V I

E N Y S D H MQ R K I S K L K G H N L Y S S D L KIT Q A E Q T R L D V Q E L I S R A L WV
E N Y S D H M Q R K I S K L K G H N L Y NS S D L KIT Q A E Q T R L D V Q E L I S R A L WV
496 540
E N Y S D H I MQ R K I MS K L K G H N L Y S S D L KIT Q A E Q T R L D V Q E L I S R A L WV
E N Y S D H I MQ R K I MS KN L K G H N L Y NS S D L KIT Q A E Q T R L D V Q E L I S R A L WV

R FY L R L L *
R FY L R L L *
541 547
R F L R L L *
R FY L R L L *

Figure B.29: Translated sequence alignment of gene PST130_15131. This gene has been
identified to encode a putative effector protein (Cantu et al., 2013). The
signal peptide, predicted using SignalP (version 2; Emanuelsson et al.,
2007) is indicated by the black box. Alternative amino acids resulting from
nonsynonymous SNPs at biallelic sites are indicated in the below diagonal
triangles. Colours were assigned according to the “Clustal X Colour Scheme”
used in Jalview (Waterhouse et al., 2009), categorising amino acid profiles.
Appendix C

Gene Expression Analysis of

Candidate Effectors Identified in
South African Pst Isolates

222
CHAPTER C: GENE EXPRESSION ANALYSIS 223

C.1 Candidate gene inspection

PST130_02001
mRNA

SA1 1 AUGUCUUUCUCAAACACR AUCCUCAAGUUY GCCCUACUCUUGUCUGUGGCCCUAGUGUACCAAUUAUCUGGCAUCAAUGC 80

SA4 AUGUCUCUCUCAAACACGAUCCUCAAGUUY GCCCUACUCUUGUCUGUGGCCCUAGUGUACCAAUUAUCUGGCAUCAAUGC

SA1 81 C A A C U C G A U C G U C U C G C C U A A G C C C A A C C A A A C U C U C A A U C C A G G A G A G A A G C U A G C C G U G G U C G U C A A G A A A A A U U C C A 160
SA4 CAACUCGAUCGUCUCGCCUAAGCCCAACCAAACUCUCAAUCCAGGAGAGAAGCUAGCCGUGGUCGUCAAGAAAAAUUCCA

SA1 161 C C G A U U C G A C A G A U C A A A C A C U C G C U U U C G C C G U U G G A U U G U C G G U K U A U A A A G A C A G U U U A G G A A G A C C U U U U C U U C G U 240

SA4 CCGAUUCGACAGAUCAAACACUCGCUUUCGCCGUUGGAUUGUCGGUGUAUAAAGACAGUUUAGGAAGACCUUUUCUUCGU

SA1 241 A C U G U C G A C G U U G G A A A A G G G G A A G C U A C A U G G A A C U C G C A U G A G U C U A C U U A U A C C U U U G A A G U C A C U G U A C C C C C C A C 320

SA4 ACUGUCGACGUUGGAAAAGGGGAAGCUACAUGGAACUCGCAUGAGUCUACUUAUACCUUUGAAGUCACUGUACCCCCCAC

SA1 321 C A G C G A U U U C A U U G A C C A G U U C U C G A A G C C A U A U A A C U U U G C U G U C U C U G A G U A U U A C U U A A A A G G G C C C U C C A A C G U G C 400

SA4 CAGCGAUUUCAUUGACCAGUUCUCGAAGCCAUAUAACUUUGCUGUCUCUGAGUAUUACUUAAAAGGGCCCUCCAACGUGC

SA1 CY ACUUUAGGCUUAUCUGAR ACACCCGUGACGAUCAAACAGR ACUGA 480

SA4 401 C Y A C U U U A G G C U U A U C U G A R A C A C C C G U G A C G A U C A A A C A G R A C U G A

Translated peptide
SA1 1 M S F S N T I L K F A L L L S V A L V Y Q L S G I N A N S I V S P K P NQ T L N P G E K L A V V V K K N S T D S T DQ T L A F A V G L S V Y K D S L G R P F L R
SA4 80
M S L S N T I L K F A L L L S V A L V Y Q L S G I N A N S I V S P K P NQ T L N P G E K L A V V V K K N S T D S T DQ T L A F A V G L S V Y K D S L G R P F L R

SA1 81 T V D V G K G E A T WN S H E S T Y T F E V T V P P T S D F I DQ F S K P Y N F A V S E Y Y L K G P S N V P T L G L S E T P V T I K Q X *
160
SA4 T V D V G K G E A T WN S H E S T Y T F E V T V P P T S D F I DQ F S K P Y N F A V S E Y Y L K G P S N V P T L G L S E T P V T I K Q X *

Depth Maximum Depth Exon boundaries

Forward Primer
SA1 24x Nonsynonymous SNP
SA4 47x Amino acid change Reverse Primer

Figure C.1: Nonsynonymous polymorphisms and primer design of the candidate effector
gene PST130_02001 in SA1 and SA4.
CHAPTER C: GENE EXPRESSION ANALYSIS 224

PST130_02403
mRNA

SA1 1 A U G U U G A A G U U G A C A C A C G U C A U C U U G G C U U G C G U G C U A G U U C UMG A G G C MU A U G C G C U C C A C A U A G R U U C A G G A C A C U C 80
SA4 AUGUUGAAGUUGACACACGUCAUCUUGGCUUGCGUGCUAGUUCUAGAGGCAUAUGCGCUCCACAUAGR UUCAGGACACUC

SA1 81 A A A G C G C G A U A U C U A U U C C G A G C C C A A G G A U C A C U A C G G U R G C C A U G A U U A U A C G Y C C U A U A A G C C C G A G C C G C A A A A G A 160
SA4 AAAGCGCGAUAUCUAUUCCGAGCCCAAGGAUCACUACGGUR GCCAUGAUUAUACGY CCUAUAAGCCCGAGCCGCAR AAGA

SA1 161 A G C C C G A G C C G U C U A A G U A Y U A U C C U G A A C C G C C G A A G A A G C C C G A G C C G U U C A A G U A C U A U C C U G W G C C G C C G A A G A A G 240

SA4 AGCCCGAGCCGUCUAAGUAY UAUCCUGAACCGCCGAAGAAGCCCGAGCCGUUCAAGUACUAUCCUGUGCCGCCGAAGR AG

SA1 241 C C C G A G C C G U U C A A Y R A C U A U C C U G A A C C G C C G A A G A A G C C C G A G C C G U U C A A G U A C U A U C C U G W R C C G C C G A A G A A G C C 320

SA4 C C C G A G C C G U U C A A Y R A C U A U C C U G A A C C G C C G A A G A A G C C C G A G C C G U U C A A G U A C U A U C C U GWG C C G C C G A A G A A G C C

SA1 321 C G A G C C G U U C A A A A A C U A U C C U G A G C C G C C G A A G A A R C C C G A G C C G U U C A A G U A C U A U C C U A C G C C G C C G A A A A A G C C A G 400

SA4 CGAGCCGUUCAAACACUAUCCUGAGCCGCCGAAGAAACCCGAGCCGUUCAAGUACUAUCCUACGCCGCCGAAAAAGCCAG

SA1 401 A C C C G U C U A A A U A U U A U C C U G A G C C G C C G C C G A A G C C C G A C C C G U C C A A G U A C U U U C C U A C C C C G C C G C A A G A G A A G C C M 480

SA4 A C C C G U C WA A A U A U U A U C C U G A G C C G C C G C C G A A G C C C G A C C C G U C C A A G U A C UWU C C U A C C C C G C C G C A A G A G A A G C C M

SA1 G A A A C G C C C A A G U A U U A U C C C G A G C C G C C C A A G U A U A A G C C C G A G G A A C C C A A A U A U G C U A G U C C A A A A U A U G A U S C G C C 560
SA4 481 GAAACGCCCAAGUAUUAUCCCGAGCCGCCCAAGUAUAAGCCCGAGGAACCCAAAUAUGCUAGUCCAAAAUAUGAU SCGCC

SA1 C U A C G A G A A G A C C C C U G A U G A A G A G C C A A A A U A C U C G G C C C C A A G C U A C G A U U A C A A U C C A C C A A A G A A A G A C G G C U A C C 641
SA4 561 CUACGAGAAGACCCCUGAUGAAGAGCCAAAAUACUCGGCCCCAAGCUACGAUUACAAUCCACCAAAGAAAGACGGCUACC

SA1 641 GUCAUUGA 648

SA4 GUCAUUGA

Translated peptide
SA1 1
ML K L T HV I L AC V L V L E AY A L H I X S GH S K R D I Y S E P K DHY GX HDY T X Y K P E PQ K K P E P S K Y Y P E P P K K P E P F K Y Y P X P P K K
80
SA4 ML K L T HV I L AC V L V L E AY A L H I X S GH S K R D I Y S E P K DHY GX HDY T X Y K P E PQ K K P E P S K Y Y P E P P K K P E P F K Y Y P V P P K X

SA1 81 P E P F NX Y P E P P K K P E P F K Y Y P X P P K K P E P F K NY P E P P K K P E P F K Y Y P T P P K K P DP S K Y Y P E P P P K P DP S K Y F P T P PQ E K P
160
SA4 P E P F NX Y P E P P K K P E P F K Y Y P X P P K K P E P F K HY P E P P K K P E P F K Y Y P T P P K K P DP S K Y Y P E P P P K P DP S K Y X P T P PQ E K P

SA1 E T P K Y Y P E P P K Y K P E E P K Y A S P K Y DX P Y E K T P D E E P K Y S AP S Y DY NP P K K DGY R H *
SA4 161 E T P K Y Y P E P P K Y K P E E P K Y A S P K Y DX P Y E K T P D E E P K Y S AP S Y DY NP P K K DGY R H *
216

Depth Maximum Depth Exon boundaries

Forward Primer
SA1 23x Nonsynonymous SNP
SA4 36x Amino acid change Reverse Primer

Figure C.2: Nonsynonymous polymorphisms and primer design of the candidate effector
gene PST130_02403 in SA1 and SA4.
CHAPTER C: GENE EXPRESSION ANALYSIS 225

PST130_05023
mRNA

SA1 1 AUGAAUAUUCAAUUAUUCCCAAUCAUGAUCUUCUUGUUAGGCCACCCAAGCCUAAUAUUCGGGAGGCCGACGGAAGGAAA 80
SA4 AUGAAUAUUCAAUUAUUCCCAAUCAUGAUCUUCUUGUUAGGCCACCCAAGCCUAAUAUUCGGGAGGCCGACGGAAGGAAA

SA1 81 AGCUGUUACCCAAGAAUUCGGGAAGCUACACGUAGAUUGUCCUGGCACGGAACAUGUUGAACAUGUUAAAAAUCCGUUCG 160

SA4 AGCUGUUACCCAAGAAUUCGGGAAGCUACACGUAGAUUGUCCUGGCACGGAACAUGUUGAACAUGUUAAAAAUCCGUUCG

SA1 161 CCGAAGAAGACAAACACGCAUCUGUGAUCUCGGACAACAGCAAAAACAUUUCCGGCUCACGUCACUCCAGCUCACCAGAA 240

SA4 CCGAAGAAGACAAACACGCAUCUGUGAUCUCGGACAACAGCAAAAACAUUUCCGGCUCACGUCACUCCAGCUCACCAGAA

SA1 241 UCUAUACCAGAAGAAGAGAAACCACUCCUCGAUCGUUCACAAUCCGACCGCGGCUCUUCAAAGCCGUCAGGACCAGCUCC 320

SA4 UCUAUACCAGAAGAAGAGAAACCACUCCUCGAUCGUUCACAAUCCGACCGCGGCUCUUCAAAGCCGUCAGGACCAGCUCC

SA1 321 CGACCAACCAAAACAAGGAGAAGACGGAAAGGGAAGAAAAAUGGCCGAACUUUAUGCCAGGUUCAAAAAAUCUCUGUCAA 400

SA4 CGACCAACCAAAACAAGGAGAAGACGGAAAGGGAAGAAAAAUGGCCGAACUUUAUGCCAGGUUCAAAAAAUCUCUGUCAA

SA1 401 CUUGGUACGGUGGACAUUCGGCUGUGGCCAGGUUUUUGCGCCGCUUGGUUAAUUACUUUCACCCAAGAAAGAUGAGUAAG 480

SA4 CUUGGUACGGUGGACAUUCGGCUGUGGCCAGGUUUUUGCGCCGCUUGGUUAAUUACUUUCACCCAAGAAAGAUGAGUAAG

SA1 481 AGCAAGGAAGCCAAGGAAGCCAAGGAAGCCGAAGACGCCAAGAAAGY CR AAGACGY CAAGAAAGY CR AAGACGUCAAGAA 560

SA4 AGCAAGGAAGCCAAGGAAGCCAAGGAAGCCAAAGAAGCCAAGGAAGY CR AAGACGY CAAGAAAGY CR AAGACGUCAAGAA

SA1 561 AGCCGAAGACGUCAAGAAAGCCGAAGAAGCCACGAAAGCUGAAGACGCCGAGAAAGCCCAAGAGGCCAAGAAAGCCCAAG 640

SA4 AGCCGAAGACGUCAAGAAAGCCGAAGAAGCCACGAAAGCUGAAGACGCCGAGAAAGCCCAAGAGGCCAAGAAAGCCCAAG

SA1 641 AGACCACAGGCGCAGUGAGGGUCGAAGCAUCGAUGCCCGAAUUGUCGGUGACCGAAGAGAAGGCUGCCACGGCGGCGAAA 720

SA4 AGACCACAGGCGCAGUGAGGGUCGAAGCAUCGAUGCCCGAAUUGUCGGUGACCGAAGAGAAGGCUGCCACGGCGGCGAAA

SA1 721 CCUGAAAGCCCAUCUGCCACAUCCCCGUCCK CUGGUACUGUGCCGGCGUCAAGUAACUUCGACAAGCCUGGGCUCUUUGC 800

SA4 C C U G A A A G C C C A U C U G C C A C A U C C C C G U C C G C U G G U A C U G U G C C G G C G U C A A G U A A C U U C GMC A A G C C U G G G C U C U U U G C

SA1 801 UAUCGACGACUUCCAGCCACGUCUACAGACCAUCUGGAUUGCGUGA 846

SA4 UAUCGACGACUUCCAGCCACGUCUACAGACCAUCUGGAUUGCGUGA

Translated peptide
SA1 1 MN I Q L F P I M I F L L GHP S L I F GR P T E GK A V T Q E F G K L HV DC P G T E HV E HV K NP F A E E DK HA S V I S DN S K N I S G S R H S S S P E 80
SA4 MN I Q L F P I M I F L L GHP S L I F GR P T E GK A V T Q E F G K L HV DC P G T E HV E HV K NP F A E E DK HA S V I S DN S K N I S G S R H S S S P E

SA1 81 S I P E E E K P L L D R S Q S D R G S S K P S G P A P DQ P K Q G E D G K G R K M A E L Y A R F K K S L S T WY G G H S A V A R F L R R L V N Y F H P R K M S K 160
SA4 S I P E E E K P L L D R S Q S D R G S S K P S G P A P DQ P K Q G E D G K G R K M A E L Y A R F K K S L S T WY G G H S A V A R F L R R L V N Y F H P R K M S K

SA1 161 S K E A K E A K E A E DA K K X X DX K K X X DV K K A E DV K K A E E A T K A E DA E K AQ E A K K AQ E T T G A V R V E A SMP E L S V T E E K A A T A A K 240

SA4 S K E A K E A K E A K E A K E X X DX K K X X DV K K A E DV K K A E E A T K A E DA E K AQ E A K K AQ E T T G A V R V E A SMP E L S V T E E K A A T A A K

SA1 241 P E S P S A T S P S X GT V P A S S N F DK P G L F A I DD FQ P R LQ T I W I A * 282

SA4 P E S P S A T S P S A GT V P A S S N F X K P G L F A I DD FQ P R LQ T I W I A *

Depth Maximum Depth Exon boundaries

Forward Primer
SA1 23x Nonsynonymous SNP
SA4 24x Amino acid change Reverse Primer

Figure C.3: Nonsynonymous polymorphisms and primer design of the candidate effector
gene PST130_05023 in SA1 and SA4.
CHAPTER C: GENE EXPRESSION ANALYSIS 226

PST130_06503
mRNA

SA1 1 AUGCAAUCCAGCUUAAUUGUCAGCAUCCUCAUCGUGUGCAGCGGUGUCAUUGCUUUACCUACUUCCAACCAAGCACAAAU 80
SA4 AUGCAAUCCAGCUUAAUUGUCAGCAUCCUCAUCGUGUGCAGCGGUGUCAUUGCUUUACCUACUUCCAACCAAGCACAAAU

SA1 81 C G A A A C U C G G G C C G A G A A G A C C C G U U C C A G C G A C A A A U A C G C C U C U U C C G A A U A C A A U G A A U C C G A C A C A U A C G C A U C G G 160
SA4 CGAAACUCGGGCCGAGAAGACCCGUUCCAGCGACAAAUACGCCUCUUCCGAAUACAAUGAAUCCGACACAUACGCAUCGG

SA1 161 C U C C U A A C U C C G C U C C A U C C G U G A U U C C U G U U G G C U U C C C U U C C A U U C C U C U U C C C C A A G U C U C U G G A U C G U C U C C C C A A 240

SA4 CUCCUAACUCCGCUCCAUCCGUGAUUCCUGUUGGCUUCCCUUCCAUUCCUCUUCCCCAAGUCUCUGGAUCGUCUCCCCAA

SA1 241 U C U G G A U C U U A C U U C G G C G G A A A G G G A G G C C G C A U U U C U U C U G C A U U C C C C G G A U U C G U U G G A G G A U U U G G C G G A A A A A U 320

SA4 UCUGGAUCUUACUUCGGCGGAAAGGGAGGCCGCAUUUCUUCUGCAUUCCCCGGAUUCGUUGGAGGAUUUGGCGGAAAAAU

SA1 321 C A G C G G G A A G G C C G G C G G U A A A A U G G A U G C G G G A A U G G G U G G A A A G A U C G C C G C U G G G G G U U C A G G G G G C C U C A A U G C C G 400

SA4 CAGCGGGAAGGCCGGCGGUAAAAUGGAUGCGGGAAUGGGUGGAAAGAUCGCCGCUGGGGGUUCAGGGGGCCUCAAUGCCG

SA1 401 C A G G A Y C A G U C G G C G G U C A G G U C G C G G G U G G U G Y C C A R G Y Y G G A A U C G S Y G C C G C A G G A U C A R U U G C Y G G U C A G G Y C G C W 480

SA4 CAGGAY CAGUCGGCGGUCAGGUCGCGGGUGGUGUCCAGGCUGGAAUCGGUGCCGCAGGAUCAAUUGCCGGUCAGGCCGCU

SA1 481 G G U G G U G C Y C A R 492

SA4 GGUGGUGCUCAG

Translated peptide
SA1
1 MQ
S S L I V S I L I V C S G V I A L P T S NQ A Q I E T R A E K T R S S D K Y A S S E Y N E S D T Y A S A P N S A P S V I P V G F P S I P L P Q V S G S S P Q
SA4 MQ S S L I V S I L I V C S G V I A L P T S NQ A Q I E T R A E K T R S S D K Y A S S E Y N E S D T Y A S A P N S A P S V I P V G F P S I P L P Q V S G S S P Q
80

SA1 S G S Y F G G K G G R I S S A F P G F V G G F G G K I S G K A G G K MD A GMG G K I A A G G S G G L N A A G X V G GQ V A G G X Q X G I X A A G S X A GQ V A
SA4 81 S G S Y F G G K G G R I S S A F P G F V G G F G G K I S G K A G G K MD A GMG G K I A A G G S G G L N A A G X V G GQ V A G G V Q A G I G A A G S I A GQ A A
160

SA1 G G AQ
SA4 161 G G A Q 164

Depth Maximum Depth Exon boundaries

Forward Primer
SA1 21x Nonsynonymous SNP
SA4 40x Amino acid change Reverse Primer

Figure C.4: Nonsynonymous polymorphisms and primer design of the candidate effector
gene PST130_06503 in SA1 and SA4.
CHAPTER C: GENE EXPRESSION ANALYSIS 227

PST130_07513
mRNA

SA1 1 AUGAAGUCGUUCGGGAUUAUCGCAACUCUACUUGCUCUAGCUUCUUCUAUCCAUGCCGACGCGGCCGUCAGACCCAAAAC 80
SA4 AUGAAGUCGUUCGGGAUUAUCGCAACUCUACUUGCUCUAGCUUCUUCUAUCCAUGCCGACGCGGCCGUCAGACCCAAAAC

SA1 81 U G C C G C K C C U G C A A G C G A U A U C A U C G A A U U G A C A U U A G A A A A C U U U G A C A C Y G U C G U C G C C A C U A C G C C U U U G A U C U U G G 160
SA4 UGCCGCK CCUGCAAGCGAUAUCAUCGAAUUGACAUUAGAAAACUUUGACACY GUCGUCGCCACUACGCCUUUGAUCUUGG

SA1 161 U C G A A U U U A U G G U A C C A U G G U G C C A C U U U U G U C A A G A C C U G G G W C C C G A G U A C A A A C G U U C G G C G A A A A U C U U G A A A G A G 240

SA4 U C G A A U U U A U G G U A C C A U G G U G C C A C U U U U G U C A A G A C C U G G GWC C C G A G U A C A A A C G U U C G G C G A A A A U C U U G A A A G A G

SA1 241 CAAGGCAUUCCAUCGGCCAAR GUUGACUGUACCGAGCAGGACGAAUUAUGUGCCGAGCAUUUACUUCCAAGUUACCCAAC

320
SA4 CAAGGCAUUCCAUCGGCCAAR GUUGACUGUACCGAGCAGGACGAAUUAUGUGCCGAGCAUUUACUUCCAAGUUACCCAAC

SA1 321 U C U C A A G G U G U U U U C A A A U G G A A G G A U G G C C G U A U A C AAAGGUCCUR AGAAGGCCGAUAGCAUCGUUUCCUACAUAGAGA

400
SA4 UCUCAAGGUGUUUUCAAAUGGAAGGAUGGCCGUAUAC AAAGGUCCUR AGAAGGCCGAUAGCAUCGUUUCCUACAUAGAGA

SA1 401 A U A A G G A A U A U C U A G G C U U C A A C A A G G Y C C G A A U U U C A U C A A G A C G A G A C A G U A A C A C C G U C U A A 465

SA4 A U A A G G A A U A U C U A G G C C MC A A C A A G G Y C C G A A U U U C A U C A A G A C G A G A C A G U A A C A C C G U C U A A

Translated peptide
SA1 M K S F G I I A T L L A L A S S I H A D A A V R P K T A A P A S D I I E L T L E N F D T V V A T T P L I L V E FM V P W C H F C Q D L G P E Y K R S A K I L K E
SA4 1 MK 80
S F G I I A T L L A L A S S I H A D A A V R P K T A A P A S D I I E L T L E N F D T V V A T T P L I L V E FM V P W C H F C Q D L G P E Y K R S A K I L K E

SA1 Q G I P S A K V DC T EQ D E L C A E H L L P S Y P T L K V F S NGR MA V Y K GP X K A D S I V S Y I E NK E Y L G F NK X R I S S R R D S NT V *
SA4 81 Q G I P S A K V DC T EQ D E L C A E H L L P S Y P T L K V F S NGR MA V Y K GP X K A D S I V S Y I E NK E Y L GX NK X R I S S R R D S NT V *
155

Depth Maximum Depth Exon boundaries

Forward Primer
SA1 22x Nonsynonymous SNP
SA4 41x Amino acid change Reverse Primer

Figure C.5: Nonsynonymous polymorphisms and primer design of the candidate effector
gene PST130_07513 in SA1 and SA4.
CHAPTER C: GENE EXPRESSION ANALYSIS 228

PST130_09275
mRNA

SA1 1 AUGAUUUCAACUAACUUCCUCGCGUGCCUCACUCCUAUCUUUCUCAAUGGACUUUUGGCCUUGAAAGUCACUAGUCCCAC
80
SA4 AUGAUUUCAACUAACUUCCUCGCGUGCCUCACUCCUAUCUUUCUCAAUGGACUUUUGGCCUUGAAAGUCACUAGUCCCAC

SA1 81 CGAGAAUUCCCAGUGGGAUUUACAGGCUACGAACACCAUAACAUGGACCAGUGUAGCGACUGACCCAAAAACCUUCGACA
160
SA4 CGAGAAUUCCCAGUGGGAUUUACAGGCUACGAACACCAUAACAUGGACCAGUGUAGCGACUGACCCAAAAACCUUCGACA

SA1 U A G U C C U C A C C A A C AWC A A C C C C U C A U G C G C U C C Y A C U G G C U U C A C C C A A G C G A U U A A A C A A A A C A U U G C C U C C U C C G A U
SA4 161 240
U A G U C C U C A C C A A C AWC A A C C C C U C A U G C G C U C C Y A C U G G C U U C A C C C A A G C G A U U A A A C A A A A C A U U G C C U C C U C C G A U

SA1 241 GGCAAGUUUGAUAUCAGUGGUGUUUCCUCAAUGAAGGCAUGCAGUGGCUACCAGAUCAAUCUUGUAGCCUCAAGUACCCC 320

SA4 GGCAAGUUUGAUAUCAGUGGUGUUUCCUCAAUGAAGGCAUGCAGUGGCUACCAGAUCAAUCUUGUAGCCUCAAGUACCCC

SA1 321 SGAUAAUR GUGCCCAUAACGCAGGCAUCUUGGCACAAUCGGCCCCAUUCAACGUGACCCAAACAUCCGGUCCAUCCAUGU 400

SA4 SGAUAAUR GUGCCCAUAACGCAGGCAUCUUGGCACAAUCGGCCCCAUUCAACGUGACCCAAACAUCCGGUCCAUCCAUGU

SA1 401 CGGAGUCGUUACCACUCGCUGGAGCGAACUCAACCGCUAAUACCCCUGCUGCAAGUACUCCUGUCGCUAACACGACCUCC 480

SA4 CGGAGUCGUUACCACUCGCUGGAGCGAACUCAACCGCUAAUACCCCUGCUGCAAGUACUCCUGUCGCUAACACGACCUCC

SA1 CCGACCCAAUCCACAUCCUCCACUGGUGCACCAAAAUAUAACUCGGGUACGGCUGCUCCUGGCGCCAAGUACUCUUUCGC 560

SA4 481 CCGACCCAAUCCACAUCCUCCACUGGUGCACCAAAAUAUAACUCGGGUACGGCUGCUCCUGGCGCCAAGUACUCUUUY GC

SA1 561 UCCCAGAAUUUCUGGCUCUUUCCAGAAGGUCACCGCUUGUGCUCUUCUACUUGUAACUUUCAUGUUGGCCUAG 633

SA4 UCCCAGAAUUUCUGGCUCUY UCCAGAAGGUCACCGCUUGUGCUCUUCUAY UUR UAACUUUCAUGUUGGCCUAG

Translated peptide
SA1 M I S T N F L A C L T P I F L N G L L A L K V T S P T E N S Q WD L Q A T N T I T WT S V A T D P K T F D I V L T N X N P S C A P T G F T Q A I K Q N I A S S D
SA4 1 80
M I S T N F L A C L T P I F L N G L L A L K V T S P T E N S Q WD L Q A T N T I T WT S V A T D P K T F D I V L T N X N P S C A P T G F T Q A I K Q N I A S S D

SA1 G K F D I S G V S SMK A C S G Y Q I N L V A S S T P DNX A H NA G I L AQ S A P F NV T Q T S G P SM S E S L P L A G A N S T A NT P A A S T P V A NT T S

SA4 81 160
G K F D I S G V S SMK A C S G Y Q I N L V A S S T P DNX A H NA G I L AQ S A P F NV T Q T S G P SM S E S L P L A G A N S T A NT P A A S T P V A NT T S

SA1 P T Q S T S S T GA P K Y N S GT A A P GA K Y S F A P R I S G S FQ K V T A C A L L X X T FM L A *
SA4 161 P T Q S T S S T GA P K Y N S GT A A P GA K Y S F A P R I S G S LQ K V T A C A L L X X T FM L A *
211

Depth Maximum Depth Exon boundaries

Forward Primer
SA1 23x Nonsynonymous SNP
SA4 24x Amino acid change Reverse Primer

Figure C.6: Nonsynonymous polymorphisms and primer design of the candidate effector
gene PST130_09725 in SA1 and SA4.
CHAPTER C: GENE EXPRESSION ANALYSIS 229

PST130_12487
mRNA

SA1 1
AUGUUCGGGUCCUCAACAAUAUUACUAGCAUGCUCUUUACUGAGCUACGUUUUGGCUGCCCCCGCGAGAUUAUCAAA CCU
80
SA4 AUGUUCGGGUCCUCAACAAUAUUACUAGCAUGCUCUUUACUGAGCUACGUUUUGGCUGCCCCCGCGAGAUUAUCAAA CCU

SA1 ACCAUCAUUAGACGGCACAUUGUCGAAUGCCCCAUCACCUUCGUGGCAACUGACUAUUGACAAUGGUCAAAUCAGGAACC
SA4 81 160
ACCAUCAUUAGACGGCACAUUGUCGAAUGCCCCAUCACCUUCGUGGCAACUGACUAUUGACAAUGGUCAAAUCAGGAACC

SA1 GUAGGUUUAUGGUGGAAGCAAGUGCACCAAAGGUGGAACCACCCAUGUCCAAACAGAUGGCCUGUUUUGACAGUAAGGUU
SA4 161 240
GUAGGUUUAUGGUGGAAGCAAGUGCACCAAAGGUGGAACCACCCAUGUCCAAACAGAUGGCCUGUUUUGACAGUAAGGUU

SA1 GGGAAACCUAGCAUUGAACAAACCGAGCGGAUCGAGAACUACCUAAAGCAUUGUAAAACUGGAAAGGCUUAUAAGGUUCC
SA4 241 G G G A A A C C U A G C A U U G A A C A A A S C G A GMR G A U C G A G A A C U A C C U A A A G C A U U G U A AMA C U G G A A A G G C U U A U A A G G U U C C
320

SA1 UGCAAACGGAGACAUCUACCCUAUGCCCAAAUCCGAUUCGACUUACGGGUACAUCUUCGGAAAGGUUCAGUUCUACGACG
SA4 321 UGCAAACGGAGACAUCUACCCUAUGCCCAAAUCCGAUUCGACUUACGGGUACAUCUUCGGAAAGGUUCAGUUCUACGACG
400

SA1 401 ACUGCGAUAGAUUGAUACACGAAACCGGCUGCUGCUAUGGAAAACCAAGUGACAGAGAGGGUUACAAUGCCAUGGAAUCC 480

SA4 ACUGCGAUAGAUUGAUACACGAAACCGGCUGCUGCUAUGGAAAACCAAGUGACAGAGAGGGUUACAAUGCCAUGGAAUCC

SA1 481 UGUUGUAUCGUUGCAGGCGCUUGCUAUGGUUGCAUCUGUUGCACUGCCUUUUCCGCCAUUCUCAAUUUCAAGUUAACAGU

560
SA4 UGUUGUAUCGUUGCAGGCGCUUGCUAUGGUUGCAUCUGUUGCACUGCCUUUUCCGCCAUUCUCAAUUUCAAGUUAACAGU

SA1 561 UGACAUCAAACUUGUCUGGUCAUCAAACCCUUGA 594

SA4 UGACAUCAAACUUGUCUGGUCAUCAAAY CCUUGA

Translated peptide
SA1 M F G S S T I L L A C S L L S Y V L A A P A R L S N L P S L D G T L S N A P S P S WQ L T I D N G Q I R N R R F M V E A S A P K V E P P M S K Q M A C F D S K V 80
SA4 1 M F G S S T I L L A C S L L S Y V L A A P A R L S N L P S L D G T L S N A P S P S WQ L T I D N G Q I R N R R F M V E A S A P K V E P P M S K Q M A C F D S K V

SA1 G K P S I EQ T E R I E NY L K H C K T G K A Y K V P A NG D I Y P MP K S D S T Y G Y I F G K VQ F Y DDC DR L I H E T G C C Y G K P S DR E G Y NAM E S

SA4 81 160
G K P S I EQ X E X I E NY L K H C X T G K A Y K V P A NG D I Y P MP K S D S T Y G Y I F G K VQ F Y DDC DR L I H E T G C C Y G K P S DR E G Y NAM E S

SA1
SA4 161
C C I V AGAC Y GC I C C T A F SA I L N F K L T V D I K L VWS SNP * 198
C C I V AGAC Y GC I C C T A F SA I L N F K L T V D I K L VWS SXP *

Depth Maximum Depth Exon boundaries

Forward Primer
SA1 22x Nonsynonymous SNP
SA4 28x Amino acid change Reverse Primer

Figure C.7: Nonsynonymous polymorphisms and primer design of the candidate effector
gene PST130_12487 in SA1 and SA4.
CHAPTER C: GENE EXPRESSION ANALYSIS 230

PST130_12491
mRNA

SA1 1 AUGCGUUCCUUCGUAGCCGUCGCCGUCACCCUUGCUCUCCUCCAGAGCACUUCCGCCUUACCAAUUUUCGAGAAGCGUGC 80
SA4 AUGCGUUCCUUCGUAGCCGUCGCCGUCACCCUUGCUCUCCUCCAGAGCACUUCCGCCUUACCAAUUUUCGAGAAGCGUGC

SA1 81 CGAGACUGAAGGCACCGGAAAAGGUGAAUCAAGCUCCCGCUCCUUAGGUGGCUGCAGCAACCAAGUUGGCCUUCUCAACA 160

SA4 CGAGACUGAAGGCACCGGAAAAGGUGAAUCAAGCUCCCGCUCCUUAGGUGGCUGCAGCAACCAAGUUGGCCUUCUCAACA

SA1 161 UUGCCCUCUCGACCAACACUCACUGUGGACAAAAUGGUCCAGCCAGUGGCAGCGGUGGUGCCGGUGGCCUCK UACCUGGC 240

SA4 UUGCCCUCUCGACCAACACUCACUGUGGACAAAAUGGUCCAGCCAGUGGCAGCGGUGGUGCCGGUGGCCUCUUACCUGGC

SA1 241 GGGGGUGGUCY CUUACCUGGCGGUGGUAUCGAUGGUCU SUUACCUGCCGGUGGCCUCUUACCUGACGGUGGUAUCGAUGG 320

SA4 GGGGGUGGUCCCUUACCUGGCGGUGGUAUCGAUGGUCUGUUACCUGCCGGUGGCCUCUUACCUGACGGUGGUAUCGAUGG

SA1 UCUCUUACCUGCCGGUGGUCUCUUACCUGGCGGGGGUGUGGAUGGUCUCUUACCUGGCGGUGGUAUCGAUGGUCUCUUGC
SA4 321 400
UCUCUUACCUGCCGGUGGUCUCUUACCUGGCGGGGGUGUGGAUGGUCUCUUACCUGGCGGUGGUAUCGAUGGUCUCUUGC

SA1 CUGGCGGUGGCGCCGGCGGCCUCUUACCUGCCGGUGGUACCGGUGGCUUCUUACCUGGCGGGGGUGGUCUCY UACCUGGC

SA4 401 480
CUGGCGGUGGCR CCGGCGGCCUCUUACCUGCCGGUGGUACCGGUGGCUUCUUACCUGGCGGGGGUGGUCUCCUACCUGGC

SA1 GGUGGUAUCGAUGGUCUCUUGCCUGGCGGUGGUAUCGAUGGUCUCUUV CCUG SCGGUGGUAUCGAU

SA4 481 GGUGGUAUCGAUGGUCUCUUGCCUGGCGGUGGUAUCGAUGGUCUCUUGCCUGGCGGUGGUAUCGAU
546

Translated peptide
SA1 1 M R S F V A V A V T L A L L Q S T S A L P I F E K R A E T E G T G K G E S S S R S L G G C S NQ V G L L N I A L S T N T H C GQ N G P A S G S G G A G G L V P G 80
SA4 M R S F V A V A V T L A L L Q S T S A L P I F E K R A E T E G T G K G E S S S R S L G G C S NQ V G L L N I A L S T N T H C GQ N G P A S G S G G A G G L L P G

SA1 81 GGGP L P GGG I DG L L P AGG L L P DGG I DG L L P AGG L L P GGGV DG L L P GGG I DG L L P GGGAGG L L P AGGT GG F L P GGGG L L P G 160
SA4 GGGP L P GGG I DG L L P AGG L L P DGG I DG L L P AGG L L P GGGV DG L L P GGG I DG L L P GGGAGG L L P AGGT GG F L P GGGG L L P G

SA1 161 GG I DG L L P GGG I DG L L P GGG I D 182

SA4 GG I DG L L P GGG I DG L L P GGG I D

Depth Maximum Depth Exon boundaries

Forward Primer
SA1 21x Nonsynonymous SNP
SA4 32x Amino acid change Reverse Primer

Figure C.8: Nonsynonymous polymorphisms and primer design of the candidate effector
gene PST130_12491 in SA1 and SA4.
CHAPTER C: GENE EXPRESSION ANALYSIS 231

PST130_12956
mRNA

SA1 1 AUGAGGUCGUUUGGUUUUUUGGCAACGCUGUUUGCCCUAGCUUCUUCUAUCCAUGCCGACGCAGGACUCAACCCCAAUGA 80
SA4 AUGAGGUCGUUUGGUUUUUUGGCAACGCUGUUUGCCCUAGCUUCUUCUAUCCAUGCCGACGCAGGACUCAACCCCAAUGA

SA1 81 CGCUCCAGAUGACGUCAUCGAAUUGACAUCAGAGAACUUCGACACCGUCGUCACCCCUGCGCCUUUGAUCUUGGUCGAAU 160

SA4 CGCUCCAGAUGACGUCAUCGAAUUGACAUCAGAGAACUUCGACACCGUCGUCACCCCUGCGCCUUUGAUCUUGGUCGAAU

SA1 161 UCAUGGCACCAUGGUGUGGUCAUUGUAAAGCCCUCAUGCCCGAGUAUAAACGUGCGGCGACACUUUUGAAAAAGGGAGGU 240

SA4 UCAUGGCACCAUGGUGUGGUCAUUGUAAAGCCCUCAUGCCCGAGUAUAAACGUGCGGCGACACUUUUGAAAAAGGGAGGU

SA1 241 AUCCCAGUGGCCAAAGCUGACUGUACCGAGCAGAGUGAAUUAUGCGCUAAGUAUGAAAUY CAAGGUUACCCAACUCUCAA

320
SA4 AUCCCAGUGGCCAAAGCUGACUGUACCGAGCAGAGUGAAUUAUGCGCUAAGUAUGAAAUY CAAGGUUACCCAACUCUCAA

SA1 321 GAUCUUCACGAAUGGUGUGUCAUCCGAAUACAAAGGUCCUCGAAAGGCUGAUGGCAUCGUCUCCUACAUGGAGAAACGGG

400
SA4 GAUCUUCACGAAUGGUGUGUCAUCCGAAUACAAAGGUCCUCGAAAGGCUGAUGGCAUCGUCUGCUACAUGGAGAAACGGG

SA1 401 CACACCCUGUCGUCACUAUCGUCACAUCGGACAACCACACCGACUUCACCAAAUCUGGUAACGUGGUG

468
SA4 CACACCCUGUCGUCACUAUCGUCACAUCGGACAACCACACCGACUUCACCAAAUCUGGUAACGUGGUG

Translated peptide
SA1 MR S F G F L A T L F A L A S S I H A D A G L N P N D A P D D V I E L T S E N F D T V V T P A P L I L V E F MA P WC G H C K A L MP E Y K R A A T L L K K G G 80
SA4 1 MR S F G F L A T L F A L A S S I H A D A G L N P N D A P D D V I E L T S E N F D T V V T P A P L I L V E F MA P WC G H C K A L MP E Y K R A A T L L K K G G

SA1 81 I P V A K A DC T EQ S E L C A K Y E I Q GY P T L K I F T NGV S S E Y K GP R K A DG I V S YM E K R A HP V V T I V T S DNHT D F T K S GNV V 156

SA4 I P V A K A DC T EQ S E L C A K Y E I Q GY P T L K I F T NGV S S E Y K GP R K A DG I V C YM E K R A HP V V T I V T S DNHT D F T K S GNV V

Depth Maximum Depth Exon boundaries

Forward Primer
SA1 23x Nonsynonymous SNP
SA4 24x Amino acid change Reverse Primer

Figure C.9: Nonsynonymous polymorphisms and primer design of the candidate effector
gene PST130_12956 in SA1 and SA4.
CHAPTER C: GENE EXPRESSION ANALYSIS 232

C.2 Additional figures of statistical analyses

 60 3000

40

Residuals
2000 2000
Sample

Sample
20

1000 1000
0

0 -20 0
233

-2 0 2 -2 -1 0 1 2 0 200 400 600

Theoretical Theoretical Fitted Values

(i) Normal probability plot of residuals af- (ii) Normal probability plot of the random (iii) Assessment of equal variances after
ter the model was fitted to the relative intercepts after the model was fitted to the model was fitted to the relative
gene expression values. the relative gene expression values. gene expression values.

Figure C.10: Graphical tests for normality and equal variances of the residuals and random intercepts. The relative gene expression dataset
was evaluated applying the assumptions that linear mixed models are based on. Normal probability plots of the random
intercept dataset (i) and the residuals (ii) showed deviation from normality. The fan like pattern observed in the plot to assess
equal variances (iii) revealed that variances were not equal, as is required for using a linear model. This indicated that the
relative gene expression dataset was not a good fit for a linear mixed model, as it violated the assumptions of the model type.
 SA1 SA1 SA1 SA1 SA1 SA1 SA1 SA1 SA1
PST130_02001 PST130_02403 PST130_05023 PST130_06503 PST130_07513 PST130_09275 PST130_12487 PST130_12491 PST130_12956
80 0.3

300 200
4 500
8 80 60 60 0.2
150
200
40
100 250 0.1
4 2 30
100 40

50 20
0.0
0 0 0
0
0 0 0 0

-100 -0.1
-50
-2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2
Sample
234

SA4 SA4 SA4 SA4 SA4 SA4 SA4 SA4 SA4

PST130_02001 PST130_02403 PST130_05023 PST130_06503 PST130_07513 PST130_09275 PST130_12487 PST130_12491 PST130_12956

15 40
300 3 300
300
100
10 2 2000 30 0.2
200 200 200
1 20
5 50
100 1000
100 100
0 0.0
10
0
0 0
-1 0 0
0 0
-5
-0.2
-2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2
Theoretical

Figure C.11: Gene and isolate specific tests for equal variances after the model was fitted to the relative gene expression values.
 PST130_02001 PST130_02403 PST130_05023 PST130_06503 PST130_07513 PST130_09275 PST130_12487 PST130_12491 PST130_12956
3000

2000

SA1
1000

0
Residuals

3000
235

2000

SA4
1000

0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 600
Fitted Values

Figure C.12: Gene and isolate specific tests for equal variances after the model was fitted to the relative gene expression values.
 3 3
1.0

2 2

1 1
Sample

0.5

Sample
Sample
0 0

0.0
-1 -1

-2 -2
-0.5
236

-2 0 2 -2 -1 0 1 2 -2 0 2
Theoretical Theoretical Theoretical
(i) Normal probability plot of residuals af- (ii) Normal probability plot of the random (iii) Assessment of equal variances after
ter the model was fitted to the log10 intercepts after the model was fitted the model was fitted to the log10 trans-
transformed relative gene expression to the log10 transformed relative gene formed relative gene expression val-
values. expression values. ues.

Figure C.13: Graphical tests for normality and equal variances of the residuals and random intercepts following a log10 transformation.
The relative gene expression dataset was log10 transformed and revaluated for the assumptions that linear mixed models are
based on. (i) Normal probability plot of the residuals and (ii) random intercepts of the log10 transformed relative expression
values. A much closer relation was observed between the data and the curve indicating normality (in red), compared to the
untransformed data (Figure C.12), (iii) residuals randomly scattered around the horizontal axis were as expected in a normally
distributed dataset.
 SA1 SA1 SA1 SA1 SA1 SA1 SA1 SA1 SA1
PST130_02001 PST130_02403 PST130_05023 PST130_06503 PST130_07513 PST130_09275 PST130_12487 PST130_12491 PST130_12956
1.5 1.0 1
1
1.0 1
2 1 2
1.0 0.5
0.5 0
0 0.5 1 1 0
0.0
0
0.0
0.0
-0.5 -1
0 0 -1
-1 -0.5
-0.5
-1
-1.0
-1.0 -1 -1
-1.0 -2 -2
-1.5
-2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2
Sample

SA4 SA4 SA4 SA4 SA4 SA4 SA4 SA4 SA4

237

PST130_02001 PST130_02403 PST130_05023 PST130_06503 PST130_07513 PST130_09275 PST130_12487 PST130_12491 PST130_12956

3
1 1.0 1 1.0
2
1 1
2
0.5 0.5
1 0
0 1 0
0.0 0.0
0 0
0
-0.5 0 -0.5
-1 -1 -1
-1 -1 -1
-1.0 -1 -1.0

-1.5 -2 -1.5
-2
-2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2
Theoretical

Figure C.14: Gene and isolate specific normal probability plots of the residuals after the model was fitted to the log10 transformed relative
gene expression values.
 PST130_02001 PST130_02403 PST130_05023 PST130_06503 PST130_07513 PST130_09275 PST130_12487 PST130_12491 PST130_12956

SA1
0

-1

-2
Residuals
238

SA4
0

-1

-2

-2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2
Fitted Values

Figure C.15: Gene and isolate specific tests for equal variances after the model was fitted to the log10 transformed relative gene expression
values.
CHAPTER C: GENE EXPRESSION ANALYSIS 239

C.3 Variability in RT-qPCR

RT-qPCR is a sensitive, multi-step method where technical variation can easily

be introduced, yielding variable data. Ling et al. (2012) recommended the use

of three biological replicates with two to three RT-PCR repeats. It is a lengthy

process, where every step needs to be skillfully performed with high precision,

using calibrated instruments and keeping consumables constant. High quality

template DNA is needed for biologically useful results, as limited template

material can reduce the sensitivity of qPCR (Derveaux et al., 2010).

Relative quantification of target gene expression complicates the process

because only one reference gene can be used in the Pfaffl gene expression quan-

tification model (Pfaffl, 2001). The use of a single reference gene is not advised,

as more accurate results, with higher statistical significance are obtained when

multiple reference genes are used. This requires the use of more complicated

quantification models with built-in calibration schemes, efficiency calculators

and methods to determine confidence measures, all adding to the total cost of

qPCR experiments (Derveaux et al., 2010).

The technical sensitivity of the RT-qPCR assay would ideally require every

step in this five-step process to be replicated, for example, replicated RNA ex-

tractions of the same tissue sample and replicate cDNA synthesis. This is often

impractical, both in terms of time and cost. In this study, the amount of sample

tissue that could be harvested per time point was an additional limitation. To

include two more reference genes in the study would have increased the num-

ber of PCR reactions by three times, significantly increasing required time and

resources. Even if this was not a limiting factor, raw sample material would not

have yielded enough RNA to be used in all reactions, and a whole different exper-

imental approach would have been needed. The experimental setup performed

was the most ambitious approach that could be accommodated.

In the rest of this section best practices to keep technical noise to a minimum
CHAPTER C: GENE EXPRESSION ANALYSIS 240

will be discussed and suggestions of how to further improve this aspect in future

studies will be made.

C.3.1 Variation in the application of treatments to biological replicates

To keep technical variation to a minimum, special care must be taken to apply

inoculum evenly to all plants. In this study, variation could have been intro-

duced by the specific location of trays in the glasshouse, as some plants could

have received more sunlight than others. In future, a mock inoculation can be

considered as an alternative negative control.

Run-to-run variation is not considered as true biological variation, but should

not be regarded as negligible. The sample “maximisation assay setup”, described

in Section 6.2.6, was applied with the aim to avoid this variation. Due to all

the possible introduced variation, solid conclusions rely on the inclusion of

multiple, independent biological replicates (Derveaux et al., 2010). However, due

to physical capacity limitations, different biological replicates were assessed in

different PCR runs. Assays would be spread out over even more runs if mock

inoculation samples were added. Derveaux et al. (2010), offers a word of caution

about inter-run variation, proposing inclusion of an identical sample across all

plates as an inter-run calibrator. The limitation in the present study was that

the positive control sample was not identical between all plates due to quantity

constraints.

C.3.2 Variation introduced by the RNA extraction process

One must take the utmost care during the RNA extraction process as this is the

most vulnerable part of the RT-qPCR experiment, due to the unstable nature of

RNA. Fleige and Pfaffl (2006) also emphasise that it is important to use intact

RNA for RT-qPCR and states that the Bioanalyzer 2100 measurement is a stable

and reliable method for the quantification and quality assessment of RNA (Fleige

and Pfaffl, 2006). Bustin and Nolan (2004) argue that RNA purification is the
CHAPTER C: GENE EXPRESSION ANALYSIS 241

critical determinant of reproducibility and biological relevance of the subsequent

result.

Approximately half of the inoculated leaf sample for each time point was

used in the first attempt of RNA extraction. The quantity of sampling material

is therefore a limiting factor for RNA extraction replicates. With the current

method, a maximum of two RNA extraction replicates would be possible, but that

would leave no material to repeat an extraction that was unsuccessful. Sample

processing, storing and transportation were controlled and kept consistent to

minimise variability.

C.3.3 Variation introduced by the reverse transcription process

In this study, random hexamers were used as primers for the RT process. Using

this nonspecific random oligonucleotides allowed the assessment of multiple

targets in each sample and is known to yield the highest quantity and least bias

cDNA. The alternative to prime RT reactions with, are thymine oligonucleotides

(oligo-dTs). The advantages of this type of primers are that it is more specific

to mRNA. RNA needs to be intact and of very high quality for this method as

it will not prime RNAs without an adenine tail consisting of multiple adenine

nucleotides (polyA tail). Judging the RNA integrity number (RIN) range, the

methodology of random hexamers rendered it more suitable for the samples

used in this study. An alternative priming method could be tested in future

experiments, where a mixture of random hexamers and oligo-dTs is used, as

suggested by Taylor and Mrkusich (2014).

Some may argue that the one-step method of RT-qPCR reduces technical noise

to some extent. The two-step method of RT-qPCR was chosen in this work as

expression of multiple genes were assessed. Interest in more than one part of the

transcriptome also meant that qPCR experiments were done over a considerable

time frame of about three months and therefore storing a more stable form of

nucleic acid was beneficial.

CHAPTER C: GENE EXPRESSION ANALYSIS 242

C.3.4 Variation introduced by RT-qPCR

Two objectives drive experimental layout (Derveaux et al., 2010). Firstly, in the

“gene maximisation method”, the expression profiles of different genes in a sam-

ple are compared. Multiple genes are assessed together on the same sample DNA

per plate and samples are spread across different plates. In the present study, the

alternative layout was used. Nine biological replicates were assessed for each

gene and time point, allowing one biological replicate per 96-well PCR plate.

This type of layout is also known as the “sample maximisation method” where

samples that will be compared are preferably run together. The standardised

values of the two treatments were compared with one another in each plate at

each time point. In this way, gene expression changes can be investigated over

the course of the infection process, to assess differences in the expression pattern

between the two treatments. Because the number of wells per run is a limitation,

the nine genes were assessed in different PCR runs. To reduce technical variabil-

ity, three repeats of each sample were evaluated (Ling et al., 2012). Although

isolates could be compared across time points for each gene assay within a run,

inter-run variability could not be accounted for, as the nine biological replicates

were assessed on nine different plates and the internal control had to be taken

from different samples due to too little quantities. Standardisation as explained

by Willems et al. (2008) could have been considered if a third treatment or mock

treatment was present.

C.3.5 Variation introduced by primers

Primer design

Primers that were specific to target sequences were designed with the help of

online tools. Primers were designed with suitable characteristics regarding GC

content, with low probability to form secondary structures and with the desired
CHAPTER C: GENE EXPRESSION ANALYSIS 243

annealing temperature, using gene sequences.

Empirical proof of primer dimer absence can be found by assessment of the

melt curves. Primers-dimers are usually shorter than target amplicons and would

therefore form a peak at low melting temperatures which are clearly visible on

the melt curve (Kubista et al., 2006). In such a case the melt curve will have two

peaks, one for the secondary structures and one for the amplicons.

Designing primers for long gene sequences that include introns can be more

complex. The possibility of alternative splicing in short genes of fungal pathogens

has been indicated before (Grützmann et al., 2014), which sets the stage for future

investigations on alternative splicing in Pst effector coding genes. Effectors are

often short peptides (Saunders et al., 2012), which are consequently not rich in

intronic regions. However, when genes have alternatively spliced isoforms, target

identification can be tricky, complicating the primer design in turn (Derveaux

et al., 2010). Some of the candidates assessed in this work have been shown to be

alternatively spliced from RNA-Seq datasets.

By annealing primers at various points along the mRNA, information about

the expression of specific exons or the full transcript length can be gathered. To

avoid amplification of gDNA, primers can further be designed to span exon-exon

boundaries (Thellin et al., 1999). In future, more attention could be given to

incorporate this step in the primer design protocol to improve primer specificity.

This would not be possible in all cases however, as for example where short exons

exist with high sequence variability between compared entities, as other criteria

such as the absence of SNPs in primer sequences and identical amplicons need

to be met. This will furthermore highly increase the cost and time needed per

gene assay. End point analysis by gel electrophoresis remains a good indication

that a PCR product of the intended size is obtained (Wittwer et al., 1997). Gel

purification and sequencing of the product can be performed for a more specific

confirmation. In this study, after optimisation, one primer pair was used for each

gene assayed.
CHAPTER C: GENE EXPRESSION ANALYSIS 244

Efficiency

Primer efficiencies were determined by implementing dilution series assays.

Care should be taken that the starting concentration of the series is concentrated

enough to allow six or seven serial dilutions that still contain sufficient template

to yield an accurate result. Quantifications at low template concentrations were

either not successful in the programmed 40 cycle PCRs, or less reproducible.

C.3.6 Choice of reference genes

The Pst β-tubulin was used as reference gene. It has been used widely across many

species, but there are controversial reports in the literature about the stability of

many of the genes traditionally considered to have stable expression profiles and

specifically using them as reference genes in qPCR (Murphy and Polak, 2002; Jain

et al., 2006; Schmidt and Delaney, 2010). If the reference gene has not been tested

before in the same experimental conditions, it is recommended that more than

one reference gene should be included when the relative quantification method

is used (Thellin et al., 1999). Thellin et al. (1999) suggest using rRNA 18S and

28S as internal standards. The use of three to five reference genes is proposed for

accurate normalisation (Derveaux et al., 2010). It is advised that more reference

genes should be used to evaluate relative gene expression in future studies.

RT-qPCR remains to be a process full of grey areas, but it has become a prime

method in various biological research fields (Schmittgen and Livak, 2008). Al-

though the variability in data quality and reporting has been addressed by setting

up the MIQE guidelines (Bustin et al., 2009), vague reporting of methodology

and statistical analysis continues to mislead newcomers to the field.

The sensitivity of RT-qPCR makes it a powerful tool, but necessitates that

every step is performed with great accuracy to keep variability, which is inevitably

introduced into every part of the multi-step process, to a minimum. Technical

CHAPTER C: GENE EXPRESSION ANALYSIS 245

repeats aim to identify outliers that are not caused by true biological variation,

but rather due to accumulation of technical inconsistency. Even when no obvious

outliers can be identified, the measured CT values are always a combination of

true biological variance and technical noise introduced during the process (Ling

et al., 2012).

C.3.7 Results of efficiency corrected relative gene expression

Sufficient amounts of biological replicates are needed to make sound conclusions.

The trouble with RT-qPCR is that inter-plate variability can jeopardies conclu-

sions. Appendix C, Figure C.16, displays the relative expression data that was

standardised to the reference gene by the efficiency corrected method (Schmittgen

and Livak, 2008) and log10 transformed. From this data, it is difficult to conclude

a true biological result. Patterns seen across plates can indicate an experimental

error. For example, high relative expressions were seen in all genes in SA1, plate

four at time point 9. Similar behaviour was observed at time points 3 and 5,

although to a lesser extent.

Appendix C, Figure C.17, indicates relative gene expression profiles as de-

termined by the efficiency corrected relative quantification method. It is clear

from Figures C.16 and C.17 that the Pfaffl method is not suitable for data with so

much variability. To achieve success in RT-qPCR assays in future, it is advised

to use multiple reference genes and deploy the developed software available, as

reviewed (Ruijter et al., 2013). Improvement of both throughput and accuracy

can be achieved using a 384-well PCR platform.

CHAPTER C: GENE EXPRESSION ANALYSIS 246

PST130_02001 PST130_02403 PST130_05023

3

-1
Log(10) of the Relative Expression of SA1 to SA4

-2
PST130_06503 PST130_07513 PST130_09275 plate
3 1
2
2
3
1 4
5
0
6
-1 7
8
-2
9
PST130_12487 PST130_12491 PST130_12956
3

-1

-2
0 1 2 3 5 9 12 0 1 2 3 5 9 12 0 1 2 3 5 9 12
Days Post Inoculation

Figure C.16: High inter-run variability in relative expression patterns is due to the sum
of the effects of inter-assay variability and the variability between different
biological replicates. It is difficult to distinguish between the two sources
of variability. This highlights the need for a calibration method when
experiments include more than one qPCR that need to be compared.
CHAPTER C: GENE EXPRESSION ANALYSIS 247

PST130_02001 PST130_02403 PST130_05023

0.75

0.50

0.25

0.00

-0.25
Log(10) of the Relative Expression of SA1 to SA4

-0.50
PST130_06503 PST130_07513 PST130_09275
0.75

0.50

0.25

0.00

-0.25

-0.50
PST130_12487 PST130_12491 PST130_12956
0.75

0.50

0.25

0.00

-0.25

-0.50
0 1 2 3 5 9 12 0 1 2 3 5 9 12 0 1 2 3 5 9 12
Days Post Inoculation

Figure C.17: The Pfaffl method of relative gene expression shows the relative gene ex-
pression of SA1 to SA4. A positive value indicates a higher expression in
SA1, while a negative value indicates a higher expression in SA4. This
method does not correct for inter-run variability and risks making false
conclusions.
Appendix D

Analysis of the Current Stripe Rust

Threat in South Africa

248
CHAPTER D: CURRENT PST THREAT IN SOUTH AFRICA 249

13/SAZP1 14/SADL1 14/SADL2 14/SADL3 14/SADL4

250 1200 600
750 200 200 900
150 400
500 600
100 100 200
250 50 300
0 0 0 0 0
14/SADL5 14/SADL6 14/SATT1 14/SATT2 14/SATT3
1500 800 1000
600
200 600 750
1000
400
400 500
100 200 500
200 250
0 0 0 0 0
14/SATT4 14/SATT5 14/SAZP2 14/SAZP3 15/SAZP1
600
750 600 300
400 400
500 400 200
250 200 200 200
100
0 0 0 0 0
count

15/SAZP10 15/SAZP11 15/SAZP12 15/SAZP2 15/SAZP3

1200 1250 1000 300
900 1000 300
750 200
750
600 500 200
500 100
300 250 250 100
0 0 0 0 0
15/SAZP4 15/SAZP5 15/SAZP6 15/SAZP7 15/SAZP8
800 800 400
90 750
600 600 300
60 500
400 400 200
30 250 200 200 100
0 0 0 0 0
0.00
0.25
0.50
0.75
1.00

0.00
0.25
0.50
0.75
1.00

0.00
0.25
0.50
0.75
1.00

0.00
0.25
0.50
0.75
1.00

15/SAZP9

1000

500

0
0.00
0.25
0.50
0.75
1.00

frequency

Figure D.1: Read frequency graphs from heterokaryotic SNP sites for the South African
field isolates (analysed in Chapter 7) that were collected between 2013 and
2015. See Table 7.2 for further identification purposes.
CHAPTER D: CURRENT PST THREAT IN SOUTH AFRICA 250

14/ET2 14/ET3 14/ET4 14/ET5 14/K10

250 600 1000 500
90 200 400
400 750
60 150 300
500
100 200
30 200
50 250 100
0 0 0 0 0
14/K11 14/K12 14/K13 14/K14 14/K15
1000 1000
750 600 900
750 750
500 500 400 600
500
250 250 250 200 300

0 0 0 0 0
count

14/K16 14/K2 14/K4 14/K5 14/K6

800 600
1500 750 750
600
400
1000 500 500
400
500 250 250 200
200
0 0 0 0 0
0.00
0.25
0.50
0.75
1.00

0.00
0.25
0.50
0.75
1.00
14/K7 14/K8 14/K9
600 800
600
400 600
400 400
200 200
200
0 0 0
0.00
0.25
0.50
0.75
1.00

0.00
0.25
0.50
0.75
1.00

0.00
0.25
0.50
0.75
1.00

frequency

Figure D.2: Read frequency graphs from heterokaryotic SNP sites for the East African
field isolates (analysed in Chapter 7) that were collected in 2014. See Table 7.2
for further identification purposes.
 14/K
14/
14

K2
6
14
/K1
14
/ET 4

0
/E
14

5
T
/E 5
14 14

T3
/K

78.6SS1
88.45SS
/K

88.5SS1

03 4SS3
14 1 1
K1 2
/

88.4 40
11/1 1
14

§
08/2
/K 1

11 011 -02 5C
J0 085F

1
5

J0 /7
/10

m
14

J 02 5
/1 44 2
/K7 ET08

28 B
J 20
14
/K9 82 d-2
14/ /1 3 Ql -1 T2
K13 13 3/3 1 Qld -1 14/E ER181a
/11
14/K 1 3/2 R
16 1 AT -3
14/K
4 ATR
14/K1
4 ER179b/11
14/K8
KE74217
KE89069 13/38
0.0002 14/40
ET87094
13/25
ET03b/10 13/29
SA1 13/71
251

SA3 11/1
3
SA2
4 13/
SA 2 27
ZP
SA 3 13/
14/ ATT 1
/S TT2 13 23
14 /19
/S
A T5 11
14 SAT T1 13 08
/
/ T
14 4/S ADL
14 /SA 11 /15
15 AZ P7

/S AD 6
1 /S

14
AZ

AT L5
/0
2
/S P1

14

8*
/S ZP9
15 5/S

T4
14 SAD
/SA ZP5
1

14
8

/
A
/S

S
14/ ADL3
ZP
15/S AZP6

/
AD 4
A

14/
0

13/S
15/SA 11
1

SA
14/SA
ZP4
P3

L1
15/SAZP1
AZP

S
15

AZP

L
S

DL
15

15/SAZ

AZP
T13/1
15/

T13/2
T13/3

CL1

2
ZP3
15/S

Figure D.3: Circular relative distance maximum likelihood phylogenetic tree. The relative distance maximum likelihood phylogenetic tree
describes the relative relationship between isolates described in Figure 7.3 where branch lengths are ignored and only topology
was considered. The group East Africa (B) isolates absent from Figure 7.4 is displayed in this dendrogram. The key in Figure 7.3
also applies here.
CHAPTER D: CURRENT PST THREAT IN SOUTH AFRICA 252

Table D.1: Differential testing of South African Pst isolates previously defined as patho-
type 6E16A- on an extended set of wheat seedling testers

!"#$%&'(!) 674(89:497;< 4=>67?@4

*+'%&(,%-.'&/ 0'"."&%12' 0'34 0'35 0'34 0'35
!"#$%&'()&*+,- !"#$%#&' ./ ./ ./ ./
!0-"1123&45/6$78 !"#&(%#)* ./ ./ ./ ./
9$-$ !"#+,%#-." ./ ./ ./% ./%
:5;+$-5%&(< !"#/0%#$0 ./% ./% ./% ./%
=25%23&>$;12% !"#)%#1 <? <? <? <?
@22 !"#2 A A A A
B85%2323&CDD !"#+ . E E .
=25%23&:FF )%#)*%#3455 ./% ./% ./% ./%
GH!)!"&6 !"#&6 E E E E
GH!)!"/) !"#/)#7!"849 .CI/ E ./ E
B$+7,5- !"#: E E E <
J$-K&4237-2L !"#/0%#$0 ./ ./ ./ ./
=25%23&M26$ !"#)%#1%#)* ./% ./% ./% ./%
N25/82-312-O&A( !"#2%#)* ./% ./% ./% ./%
=P1-5K&AD !"#$; . . . .
B;2+2%0 !"#)%#<%#)*!"#$% . . . .
QR&!72;0, !"#* E E . .
>,;P,%3$%, !"#) A A A A
GH$/20&! !"3/&/$%0-$; A A <?? A
!"+)DS&GH! !"#+ . . . .
!"*)DS&GH! !"#* . E . .
!"1)DS&GH! !"#1 A A A A
!"2)DS&GH! !"#2 A A A A
!":)DS&GH! !"#: E E E <?
!"<)DS&GH! !"#< E E ./ E
!"+,)DS&GH! !"#+, E E E E
!"+*)DS&GH! !"#+* . E . .
!"+2)DS&GH! !"#+2 <I/ <I/ <I/ <I/
!")$)DS&GH! !"#)$ ./% ./% .%/ ./%
!")1)<S&GH! !"#)1 ./% E ./% E
!")2)DS&GH! !"#)2 .CI/% .CI/% ./% .CI/%
GH$/20&N& !"#= ./ ./ .C/% .C/
!"#&> !")2 C/% C/% .C/% (/
!2;65-6 !")2 .CI/% .CI/% .C/% .C/%
G+1505$% ?@>@.A@ ./% ./% ./% ./%
G7,/82 !"2%#!"+2%#BCDE" ./% ./% ./% ./%
T-5O,K52- !"+2%#BCDE" . . . .
B,K2%L, !"1%#!"2%#BCDE" ./% ./% ./% ./%
B,-302%3&: !"/)%#BCDE" . . . .
B;,5-2 !"+1 ./ ./ ./ ./
B-"3$2 ?@>@.A@ . . . .
42;785 ?@>@.A@ ./% ./% ./% ./%
>-,%5/8 ?@>@.A@ ./% ./% . .
>U!&!02-;5%O ?@>@.A@ . . . .
9$%02-2P ?@>@.A@ ./ ./ ./ ./
9$3,5/ ?@>@.A@ . . . .
N2%K2LH$"3 !"+2%#BCDE" . . . .
N2H2;,05$% ?@>@.A@ . . . .
N$15O"3 ?@>@.A@ ./ ./ ./ ./
!$;305/2 ?@>@.A@ . . . .
Q-5K2%0 !"+2 << <</ <</ </
Q,;$% !"/) . . . .
:"6, !"3/&/$%0-$;&VW>X ./% ./% ./% ./%
U,--5$- ?@>@.A@ . . . .
N27Y&-27;5/,02&Z&3"3/Y&3"3/270,1;2&Z&.&&[;2/6&Z&I&&H2-P&3+,;;&7"30";2&Z&CEA&35L2&$[&7"30";2&Z&/&&/8;$-$353&Z&%&%2/-$353
CHAPTER D: CURRENT PST THREAT IN SOUTH AFRICA 253

Table D.2: Differential testing of South African Pst isolates previously defined as patho-
type 6E22A+ on an extended set of wheat seedling testers

!"#$%&'(!) 678 49:67;<8

*+'%&(,%-.'&/ 0'"."&%12' 0'34 0'35 0'34 0'35
!"#$%&'()&*+,- !"#$%#&' ./ ./ ./ ./
!0-"1123&45/6$78 !"#&(%#)* 9:/ 9;; +5<2=>&./&?&( @
A$-$ !"#+,%#-." ./% ./ ./ ./
B5C+$-5%&(9 !"#/0%#$0 .D:/ .D:/ .D:/ .D:/
E25%23&F$C12% !"#)%#1 G G G G
H22 !"#2 G G G G
I85%2323&DJJ !"#+ . @ . @
E25%23&BKK )%#)*%#3455 .D;/% .D/% 9/ 9/
LM!)!"&6 !"#&6 @ @ @ @
LM!)!"/) !"#/)#7!"849 @ @ .D/&N35%OC2P @
I$+7,5- !"#: @ @ @ 9
Q$-=&4237-2R !"#/0%#$0 .D:/ .D:/ ./ ./D:/
E25%23&S26$ !"#)%#1%#)* 9@/ 9@/ 9@/ 9;/
T25/82-312-O&G( !"#2%#)* 9;;/ 9;;/ 9;;/ 9;;/
EU1-5=&GJ !"#$; . . . .
IC2+2%0 !"#)%#<%#)*!"#$% . . . .
VW&!72C0, !"#* . @ . @
F,CU,%3$%, !"#) G G G G
LM$/20&! !"3/&/$%0-$C G G G G
!"+)JX&LM! !"#+ . . . .
!"*)JX&LM! !"#* . @ @ @
!"1)JX&LM! !"#1 G G G G
!"2)JX&LM! !"#2 G G G G
!":)JX&LM! !"#: 9 @ @ @
!"<)JX&LM! !"#< . @ @ ./
!"+,)JX&LM! !"#+, @ @ @ @
!"+*)JX&LM! !"#+* . @ . @
!"+2)JX&LM! !"#+2 9:/ 9:/ 9;; 9;;
!")$)JX&LM! !"#)$ ./ ./ ./ ./
!")1)9X&LM! !"#)1 @ @ ./&N35%OC2P @
!")2)JX&LM! !"#)2 .D:/% .D:/% .D:/% @
LM$/20&T& !"#= G G +5<2=>&./&?&4 +5<2=>&./&?&4
!"#&> !")2 .D(/% .D(/% (;/ (;/
!2C65-6 !")2 (/% (/% 9:/% 9:/%
L+1505$% ?@>@.A@ ./% ./% ..D:D:/% ..D:/%
L7,/82 !"2%#!"+2%#BCDE" ./% ./% ./% ./%
Y-5O,=52- !"+2%#BCDE" . . . .
I,=2%R, !"1%#!"2%#BCDE" 9/ 9/ 9;/ 9;/
I,-302%3&B !"/)%#BCDE" ./% ./% ./% ./%
IC,5-2 !"+1 .D:/% ./% ./% ./%
I-"3$2 ?@>@.A@ . . . .
42C785 ?@>@.A@ ./% ./% ./% ./%
F-,%5/8 ?@>@.A@ ./% ./% ./% .D:/%
FZ!&!02-C5%O ?@>@.A@ ./% ./% ./% ./%
A$%02-2U ?@>@.A@ ./% ./% .D:/ .D:/
A$3,5/ ?@>@.A@ ./% ./% ./% ./%
T2%=2RM$"3 !"+2%#BCDE" ./% ./% ./% ./%
T2M2C,05$% ?@>@.A@ ./% ./% ./% ./%
T$15O"3 ?@>@.A@ ./% ./% ./% ./%
!$C305/2 ?@>@.A@ . . ./ ./
V-5=2%0 !"+2 9;; 9/ G 9;;/
V,C$% !"/) ./% ./% ./% ./%
B"6, !"3/&/$%0-$C&N[FP 9:/ 9:/ 9;; 9;;
Z,--5$- ?@>@.A@ . . . .
T27\&-27C5/,02&?&3"3/\&3"3/270,1C2&?&.&&]C2/6&?&:&&M2-U&3+,CC&7"30"C2&?&D@G&35R2&$]&7"30"C2&?&/&&/8C$-$353&?&%&%2/-$353
+5<2=\&+$-2&08,%&$%2&5%]2/05$%&0U72&,+$%O&5%=5M5=",C&7C,%03&5%&5%$/"C,05$%&-27C5/,02&?&35%OC2\&$%CU&,&35%OC2&7C,%0&3/$-2=
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