Field pathogenomics of wheat yellow (stripe) rust

Diane Saunders

Diane.Saunders@tgac.ac.uk

http://yellowrust.com/

Pathogen diversity at the field level applying gene sequence technology to

the surveillance of emerging and re-emerging plant pathogens

Puccinia striiformis f. sp. tritici

•  Despite the success of the UKCPVS and our understanding of the phenotypic diversity of wheat yellow rust, we know almost nothing about its genetic diversity in the UK

Field pathogenomics of wheat yellow rust

No. of samples

1 43Unknown UK location: 2

Mean incidence of YR on leaves

1998-2007

UKCPVS

212 samples for 2013

Field pathogenomics to complement traditional pathology

1. Receive sample from the field

2. Purify and multiply field isolates

3. Infect wheat lines and score infection type 4. Assess phenotypic diversity

4.0

3.0

2.0

1.00.0

Susceptible

Resistant

Phenotype

Results reported 6+ months later

Field pathogenomics to complement traditional pathology

2. Extract RNA

1. Receive sample from the field

3. Sequence genes with latest technology

Gene sequence data from the pathogen

Gene sequence data from the host (wheat)

4. Assess pathogen genotypic diversity

5. Determine wheat variety

Discussed in: Bayles, Saunders & Uauy (2013) NIAB-TAG Landmark Bulletin

Results reported within 1-2 months

ID Location Variety 13/65 Cambridge Claire RB1 Cambridge Oakley RB2 Cambridge Torch

13/520 Cambridge Warrior 13/182 Cambridge Crusoe T13/1 Dorset Triticale T13/2 Dorset Triticale T13/3 Dorset Triticale 13/27 Gloucestershire Oakley 13/26 Gloucestershire Haratio 13/28 Gloucestershire Solstice 13/20 Hampshire KWS Rowan 13/23 Lincolnshire KWS Santiago 13/42 Lincolnshire Audace 13/09 Lincolnshire Oakley 13/33 Lincolnshire Recital 13/38 Lincolnshire ReR138

13/123 Lincolnshire Ambition 13/40 Lincolnshire Fairplay 13/25 Lincolnshire KWS Dali 13/30 Lincolnshire ReR22 13/12 Lincolnshire RW41088 13/35 Lincolnshire ReR64 13/34 Lincolnshire ReR32 13/22 Lincolnshire Solstice 13/39 Lincolnshire Toisandor 13/37 Lincolnshire ReR11O 13/24 Lincolnshire KWS Kielder 13/36 Lincolnshire Allez-Y 13/32 Lincolnshire ReRO4

13/120 Lincolnshire Ambition 13/21 Norfolk Solstice CL1 Norfolk Triticale

13/29 Oxford Solstice 13/17 Suffolk Duxford 13/15 Suffolk Torch 13/19 Suffolk LGW56 (Panacea) 13/18 Suffolk KWS196 (KWS Dali) 13/14 Suffolk Oakley 13/71 Yorkshire Laurier

•  Gene sequence data from 40 samples

•  Samples selected based on location and wheat variety

Field pathogenomics data UK 2013

1

9  

Res

ista

nce

ratin

g

2012/13

Crusoe (8)

Torch (4)

Oakley (2)

How are these samples related to other archived isolates?

Warrior isolate different to older UK isolates

•  Based on full genome sequences

•  Lengthy and time-consuming process

SCPRID partners  

2013 field isolates: similar genetically to Warrior

Maximum likelihood 4275 genes

3rd codon position

Four distinct populations in 2013 field isolates

Bar chart: STRUCTURE, K=4

LincolnshireNorfolk

Gloucestershire

Suffolk

Cambridgeshire

Yorkshire

Oxfordshire

Hampshire

Dorset

Location

0001

13/123

13/19

13/520

13/26

13/15 13/1

8

RB1

RB2

13/65

13/32

13/7113/4013/29

13/30

13/1

2

13/3

513/3

9

13/36

13/34

13/14

13/24

13/20

13/25

13/37

13/28

13/2

2

13/42

13/23

13/33

13/182

13/21

13/09

13/38

T13/3

CL1

T13/2

T13/1

13/1

20

13/27

Cluster I

Cluster II

Cluster III

Cluster IV

100

100

100

100

100

100

57

51

Triticale

Tri'cale  

Correlation between genotypic and phenotypic data

SP, Spaldings Prolific; War, Warrior; Amb, Ambition; Tim, Timber; Ren, Rendezvous.

13/26

13/65

13/09

13/18

2

13/24

13/36

Yr7SPWarAmbTimRen

3.1

3.0

3.5

4.03.51.2

3.53.1

3.3

4.03.21.9

2.0

1.0

2.1

0.00.03.0

3.20.0

1.9

0.00.03.3

3.0

3.0

1.8

0.60.62.2

3.23.5

2.1

0.12.23.2

Cluster I Cluster III Cluster IV

13/12

0

2.83.1

3.0

3.53.10.0

13/12

3

3.03.0

3.0

3.24.01.4

13/29

3.24.0

2.1

0.40.32.8

4.0

3.0

2.0

1.00.0

Susceptible

Resistant

Phenotype

0001

13/123

13/19

13/520

13/26

13/15 13/1

8

RB1

RB2

13/65

13/32

13/7113/4013/29

13/30

13/1

2

13/3

513/3

9

13/36

13/34

13/14

13/24

13/20

13/25

13/37

13/28

13/2

2

13/42

13/23

13/33

13/182

13/21

13/09

13/38T1

3/3

CL1

T13/2

T13/113

/120

13/27

Cluster I

Cluster II

Cluster III

Cluster IV

100

100

100

100

100

100

57

51

13/26

13/65

13/09

13/18

2

13/24

13/36

Yr7SPWarAmbTimRen

3.1

3.0

3.5

4.03.51.2

3.53.1

3.3

4.03.21.9

2.0

1.0

2.1

0.00.03.0

3.20.0

1.9

0.00.03.3

3.0

3.0

1.8

0.60.62.2

3.23.5

2.1

0.12.23.2

Cluster I Cluster III Cluster IV

13/12

0

2.83.1

3.0

3.53.10.0

13/12

3

3.03.0

3.0

3.24.01.4

13/29

3.24.0

2.1

0.40.32.8

Tri'cale  

Correlation between genotypic and phenotypic data

SP, Spaldings Prolific; War, Warrior; Amb, Ambition; Tim, Timber; Ren, Rendezvous.

13/26

13/65

13/09

13/18

2

13/24

13/36

Yr7SPWarAmbTimRen

3.1

3.0

3.5

4.03.51.2

3.53.1

3.3

4.03.21.9

2.0

1.0

2.1

0.00.03.0

3.20.0

1.9

0.00.03.3

3.0

3.0

1.8

0.60.62.2

3.23.5

2.1

0.12.23.2

Cluster I Cluster III Cluster IV

13/12

0

2.83.1

3.0

3.53.10.0

13/12

3

3.03.0

3.0

3.24.01.4

13/29

3.24.0

2.1

0.40.32.8

4.0

3.0

2.0

1.00.0

Susceptible

Resistant

Phenotype

0001

13/123

13/19

13/520

13/26

13/15 13/1

8

RB1

RB2

13/65

13/32

13/7113/4013/29

13/30

13/1

2

13/3

513/3

9

13/36

13/34

13/14

13/24

13/20

13/25

13/37

13/28

13/2

2

13/42

13/23

13/33

13/182

13/21

13/09

13/38T1

3/3

CL1

T13/2

T13/113

/120

13/27

Cluster I

Cluster II

Cluster III

Cluster IV

100

100

100

100

100

100

57

51

How many genes are specifically differentially expressed for a particular

population cluster?

How many genes are differentially expressed? No. of genes specifically differentially expressed in all pair-wise

comparisons

What is the function of the genes that are specifically differentially expressed?

FDR = 0.05; p-value 0.05

What are the functions of the proteins they encode?

•  Small proportion of proteins annotated

•  All predicted secreted proteins are non-annotated

•  Several secreted proteins are in our list of top effector candidates

(Cantu et al. Saunders & Uauy BMC Genomics, 2013)  

Could these genes encode polymorphic effector candidates?

Secreted candidate effector protein

Can we differentiate the wheat variety in the transcriptome data?

Can RNAseq data differentiate wheat varieties?

Match to Variety 1 AAGTGCCTCGGATCGAT

C 100% match

CT Partial match

T No match

Match transcriptome SNPs to variety of interest

Warrior sample: 52-65 SNPs

Sample from Warrior

Accelerating the response to changes in population dynamics

•  Rapid progress from sample submission to comprehensive genotyping

•  Ability to detect important new pathotypes that may emerge

•  Generation of a valuable countrywide record of field isolates ➔ New models for population dynamics

Rapid response to emerging/re-emerging pathogens is vital to keep pace with fast evolving

pathogens in a changing environment

Tools and data available via yellowrust.com

http://yellowrust.com/

•  Raw reads relating to genome sequences of 18 PST isolates •  Phenotypic data from field trials of the Watkins landraces

New tools to access published data from five PST isolates

Unpublished data available to download (SCPRID project)

Acknowledgments

TSL: Sophien Kamoun Kentaro Yoshida Dan MacLean Graham Etherington JIC: Cristobal Uauy Clare Lewis Albor Dobon

TGAC: Mark McMullan UEA: Cock Van Oosterhout NIAB: Tina Barsby Rosemary Bayles Jane Thomas Amelia Hubbard

SCPRID partners