Diane Saunders, The Genome Analysis Centre

1. Field pathogenomics of
wheat yellow (stripe) rust
Diane Saunders
Diane.Saunders@tgac.ac.uk
http://yellowrust.com/

2. 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

3. • 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 43
Unknown UK location: 2
Mean incidence of
YR on leaves
1998-2007
UKCPVS
212 samples for 2013

4. 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
1.0
0.0
Susceptible
Resistant
Phenotype
Results reported 6+ months later

5. 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

6. 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
Resistancerating
2012/13
Crusoe (8)
Torch (4)
Oakley (2)

7. How are these samples related to other
archived isolates?

8. Warrior isolate different to older UK isolates
• Based on full
genome sequences
• Lengthy and time-
consuming process
SCPRID partners

9. 2013 field isolates: similar genetically to Warrior
Maximum likelihood
4275 genes
3rd codon position

10. Four distinct populations in 2013 field isolates
Bar chart:
STRUCTURE, K=4
Lincolnshire
Norfolk
Gloucestershire
Suffolk
Cambridgeshire
Yorkshire
Oxfordshire
Hampshire
Dorset
Location
0001
13/123
13/19
13/520
13/26
13/15
13/18
RB1
RB2
13/65
13/32
13/71
13/40
13/29
13/30
13/12
13/35
13/39
13/36
13/34
13/14
13/24
13/20
13/25
13/37
13/28
13/22
13/42
13/23
13/33
13/182
13/21
13/09
13/38
T13/3
CL1
T13/2
T13/1
13/120
13/27
Cluster I
Cluster II
Cluster III
Cluster IV
100
100
100
100
100
100
57
51
Triticale

11. 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
13/182
13/24
13/36
Yr7
SP
War
Amb
Tim
Ren
3.1
3.0
3.5
4.0
3.5
1.2
3.5
3.1
3.3
4.0
3.2
1.9
2.0
1.0
2.1
0.0
0.0
3.0
3.2
0.0
1.9
0.0
0.0
3.3
3.0
3.0
1.8
0.6
0.6
2.2
3.2
3.5
2.1
0.1
2.2
3.2
Cluster I Cluster III Cluster IV
13/120
2.8
3.1
3.0
3.5
3.1
0.0
13/123
3.0
3.0
3.0
3.2
4.0
1.4
13/29
3.2
4.0
2.1
0.4
0.3
2.8
4.0
3.0
2.0
1.0
0.0
Susceptible
Resistant
Phenotype
0001
13/1
23
13/19
13/520
13/26
13/15
13/18
RB1
RB2
13/65
13/32
13/71
13/40
13/29
13/30
13/12
13/35
13/39
13/36
13/34
13/14
13/24
13/20
13/25
13/37
13/2
8
13/22
13/42
13/23
13/33
13/182
13/2
1
13/09
13/38T13/3
CL1
T13/2
T13/1
13/120
13/27
Cluster I
Cluster II
Cluster III
Cluster IV
100
100
100
100
100
100
57
51
13/26
13/65
13/09
13/1
Yr7
SP
War
Amb
Tim
Ren
3.1
3.0
3.5
4.0
3.5
1.2
3.5
3.1
3.3
4.0
3.2
1.9
2.0
1.0
2.1
0.0
0.0
3.0
3.2
0.0
1.9
0.0
0.0
3.3
Cluster I Cluster III
13/120
2.8
3.1
3.0
3.5
3.1
0.0
13/123
3.0
3.0
3.0
3.2
4.0
1.4

12. 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
13/182
13/24
13/36
Yr7
SP
War
Amb
Tim
Ren
3.1
3.0
3.5
4.0
3.5
1.2
3.5
3.1
3.3
4.0
3.2
1.9
2.0
1.0
2.1
0.0
0.0
3.0
3.2
0.0
1.9
0.0
0.0
3.3
3.0
3.0
1.8
0.6
0.6
2.2
3.2
3.5
2.1
0.1
2.2
3.2
Cluster I Cluster III Cluster IV
13/120
2.8
3.1
3.0
3.5
3.1
0.0
13/123
3.0
3.0
3.0
3.2
4.0
1.4
13/29
3.2
4.0
2.1
0.4
0.3
2.8
4.0
3.0
2.0
1.0
0.0
Susceptible
Resistant
Phenotype
0001
13/1
23
13/19
13/520
13/26
13/15
13/18
RB1
RB2
13/65
13/32
13/71
13/40
13/29
13/30
13/12
13/35
13/39
13/36
13/34
13/14
13/24
13/20
13/25
13/37
13/2
8
13/22
13/42
13/23
13/33
13/182
13/2
1
13/09
13/38T13/3
CL1
T13/2
T13/1
13/120
13/27
Cluster I
Cluster II
Cluster III
Cluster IV
100
100
100
100
100
100
57
51

13. How many genes are specifically
differentially expressed for a particular
population cluster?

14. 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

15. 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)

16. Could these genes encode polymorphic
effector candidates?
Secreted candidate effector protein

17. Can we differentiate the wheat variety
in the transcriptome data?

18. 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

19. 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

20. 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)

21. 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