Andre Laroche, AAFC

1. Molecular characterization of Pst isolates
from Western Canada
André Laroche, Michele Frick, Yong Xu, Byron Puchalski, Brent Puchalski,
Harpinder Randhawa, Robert Graf and Denis Gaudet
Agriculture and Agri-Food Canada,
Lethbridge Research Centre,
Lethbridge, Alberta, CANADA T1J 4B1
#bgri2014

2. Presentation Outline
• Puccinia striiformis f.sp. tritici (Pst)
causal agent of stripe (yellow) rust
• Molecular characterization of old
and new isolates of Pst
• Conclusions

3. Puccinia striiformis f.sp. tritici
• Until year 2000, a lower optimum germination
temperature (12-13°C) which limited its impact around
major areas of the world
• Since year 2000, different isolates are growing under
higher temperature (optimally germinate 16-18°C) and
this is a major issues in many wheat growing regions
around the world.

4. Puccinia striiformis f.sp. tritici
4

5. Puccinia striiformis f.sp. tritici
• Genome size 117 MB – 3 assembled reference genome
• PST-78 (2000) reference assembly of 17,295 contigs
and 9,175 scaffolds (79.3 Mb – 72% of genome size) and
annotated transcriptome (Puccinia Group Sequencing Project, Broad
Institute of Harvard and MIT;
http://www.broadinstitute.org/annotation/genome/puccinia_group)
• PST-130 (2007) reference assembly of 29,178 contigs
(64.8 Mb – 59% of genome size) and 22,815 putative
coding sequences (Cantu et al 2011)

6. Puccinia striiformis f.sp. tritici
• PST-CY32 (1991) reference assembly of 12,833 contigs
and 4,283 scaffolds covering 130.7 Mb and annotated
transcriptome of 25,288 elements (Zheng W et al. 2013)

7. Puccinia striiformis f.sp. tritici
• PST-78 common with PST-130: 14,525 contigs
• PST-78 unique contigs: 2770
• PST-130 common with PST-78: 28,683 contigs
• PST-130 unique contigs:495

8. Puccinia striiformis f. sp. tritici
• Overview of gene content and identity in Pst
• Identify the genetic elements in Pst that
correspond to virulence and high temperature
germination
• Using next generation sequencing (NGS)
techniques isolate-specific sequences to further
facilitate their recognition
• First step is to group the isolates by molecular
phylogenetic analysis

9. Genomic DNA isolation & sequencing
• Samples from field infected leaves
• Single pustule purification 2 cycles and
amplification
• Ureidiniospores ground in Precellys 24
• QIAGEN fungal DNA isolation
• Illumina HiSeq 2000 (NRC-Saskatoon)
1 µg DNA 100 bp-pe 200 M reads
• Illumina MiSeq (Lethbridge) 50 ng DNA 250
bp-pe 17-20 reads

10. Assembly and mapping
• Velvet de novo assembly
• Map to PST-78 reference (Broad Institute) +
PST-130 using Bowtie or Geneious
• Mugsy genome alignment
• Sequence concatination
• RAxML bootstrapping to generate
phylogenetic trees
• SignalP

11. Stripe rust NGS library information
Isolate
name Id name Location Year
Isolate
type Sequencer Clean reads
108E42 SWS_9_90Vaux Vauxhall 1990 Old HiSeq 75,442,412
46E162 SRBI91 Bow Island 1991 Old MiSeq 4,749,091
42E0 SRBI93 Bow Island 1993 Old HiSeq 81,131,790
126E134 SRCReed2K Creston, BC 2000 ? HiSeq 71,986,246
238E150 SRLReed2K Lethbridge 2000 ? HiSeq 44,640,459
238E182 Reed2K Lethbridge 2000 ? MiSeq 17,800,348
New1 Beavers Magrath 2007 New MiSeq 23,893,285
New2 Foremost2011 Foremost 2010 New HiSeq 112,308,174
New4 Teal2011 Lethbridge 2011 New Hiseq 55,617,877
New9-4 LethSW1_2012 Lethbridge 2012 New MiSeq 12,538,011
New9-4 LethSW1_2012 Lethbridge 2012 New HiSeq 96,549,494
New9-A LethSW1_2012 Lethbridge 2012 New HiSeq 72,364,178
New10 LethSW3_2012 Lethbridge 2012 New HiSeq 53,745,698

12. Rust differential ratings
Isolate name ID name Yr5 Yr1 Yr10 Yr15 YrSP Yr8 Yr9 Yr23 YrCV Yr26 Yr6 Yr7
108E42 SWS_9_90Vaux + + +
46E162 SRBI91 + + + +
42E0 SRBI93 +
126E134 SRCReed2K + + + + +
238E150 SRLReed2K + + + + + +
238E182 Reed2K + + + + + + +
New1 Beavers 2007 nd nd nd nd nd nd nd nd nd nd nd nd
New2 Foremost2011 nd nd nd nd nd nd nd nd nd nd nd nd
New4 Teal2011 nd nd nd nd nd nd nd nd nd nd nd nd
New9-4 LethSW1_2012 +? + + + + + + + nd +
New10 LethSW3_2012 +? + + nd + + + nd +

13. Schematic of rRNA locus in P. striiformis
18S 28S 18S
5.8S 5S
ITS1 ITS2 IGS2IGS1
AY117126
1 13291 1758
Consensus
GQ457306
GU382673
HM057122
HM057130
HM057136
EU851142

14. Phylogenetic tree based on rRNA consensus
UPGMA

15. Phylogenetic tree of 1700 MiSeq sequences

16. Gene annotation and Gene make up
Puccinia striiformis f.sp. tritici

17. Puccinia striiformis f.sp. tritici
Isolate
name Id name Location Year
Isolate
type Sequence Clean reads
108E42 SWS_9_90Vaux Vauxhall 1990 Old HiSeq 75,442,412
46E162 SRBI91 Bow Island 1991 Old MiSeq 4,749,091
42E0 SRBI93 Bow Island 1993 Old HiSeq 81,131,790
126E134 SRCReed2K Creston, BC 2000 New HiSeq 71,986,246
238E50 SRLReed2K Lethbridge 2000 New HiSeq 44,640,459
238E182 Reed2K Lethbridge 2000 ? MiSeq 17,800,348
New1 Beavers Magrath 2007 New MiSeq 23,893,285
New2 Foremost2011 Foremost 2010 New HiSeq 112,308,174
New4 Teal2011 Lethbridge 2011 New Hiseq 55,617,877
New9-4 LethSW1_2012 Lethbridge 2012 New MiSeq 12,538,011
New9-4 LethSW1_2012 Lethbridge 2012 New HiSeq 96,549,494
New9-A LethSW1_2012 Lethbridge 2012 New HiSeq 72,364,178
New10 LethSW3_2012 Lethbridge 2012 New HiSeq 53,745,698

18. Puccinia striiformis f.sp. triticiNew isolates Old isolates
Genom. Transcr. Mito. Genom. Transcr. Mito.
Total reads (M) 168 180 87 161 161 63
Assembled R (M) 80 50 2.7 98 61 1.1
Unassemb. R (M) 88 130 85 63 100 62
paired (M) 62 83 61 23 40 22
unpaired (M) 26 47 25 40 60 40
Contigs 16,665 20,180 1 16,774 20,201 1
>90% PW idty 14,865 17,605 NA 14,315 17,553 NA
>95% PW idty 9,800 13,484 NA 12,873 12,400 NA
<75% PW idty 178 149 NA 146 132 NA

19. Functional annotation of old isolates
234 unique enriched included GO terms under the
biological process category related to:
Phosphorylation: activation of MAPKK activity,
regulation of MAPK cascade, regulation of MAP kinase
activity, regulation of peptidyl-serine phosphorylation,
stress-activated MAPK cascade
cell signaling: cell-cell signaling, cell
communication, cellular developmental process, cell
differentiation, checkpoint, G2/M transition DNA
damage checkpoint, regulation of multi-organism
process

20. Functional annotation of old isolates
transport: lipid transport, regulation of
cholesterol & sterol transport
DNA modifications: DNA restriction-
modification system, restriction
endodeoxyribonuclease activity
117 terms were enriched with high homology (>95%
pairwise identity & >500 bp) to the reference PST-78
strain
14 enriched terms (43 contigs) with low homology
(<70% pairwise identity & >500 bp) were also
identified. These enriched terms were different than
those identified in the unique category

21. Functional annotation of new isolates
88 unique enriched included GO terms under the
biological process category for:
transport: calcium ion transmembrane
transport, divalent metal ion transport, establishment
of RNA localization, macromolecule localization,
mRNA export from nucleus, nucleic acid transport
nuclear import, nucleo-cytoplasmic transport, nucleic
acid transport nuclear import, protein targeting, RNA
export from nucleus, RNA transport, protein import
into nucleus, protein localization to nucleus and
organelle

22. Functional annotation of new isolates
response to exogenous molecules: defense
response, insecticide metabolic process, response to
bacterium, responses to DDT, insecticide,
organophosphorus, to other organism and to toxin,
toxin catabolic and metabolic processes, xenobiotic
catabolic and metabolic processes
RNA metabolism: regulation of mRNA
processing, regulation mRNA and RNA splicing
modification of cell wall: anatomical structure
development, cell wall macromolecule catabolic
process, glycosaminoglycan and peptidoglycan
catabolic and metabolic processes, histolysis, tissue
and system development

23. 137 terms enriched with high homology (>95%
pairwise identity & >500 bp) to the reference PST-78
strain were found
74 enriched terms with low homology (<70% pairwise
identity & >500 bp) were also identified.
These enriched terms were different than those
identified in the unique category.
Functional annotation of new isolates

24. Conclusions
• Illumina HiSeq mapping data has allowed us to
separate our various rust isolates into
phylogenetic groups
• Minimum depth of MiSeq sequence data is
required to compare Pst isolates to HiSeq data
• Pst rust continues to evolve rapidly as we see
difference from 2011 to 2012.
• Unique and enriched sequences were identified in
both old and new isolates. This suggests that a
genome reorganization has occurred between
these strains since genes involved in multiple
different biological processes were identified.

25. Acknowledgements
Associates and collaborators at LRC:
Michele Frick Denis Gaudet
Yong Xu Harpinder Randhawa
Byron Puchalski Robert Graf
Brent Puchalski
Eric Amundsen
Arsh Singh
NRC-Saskatoon
Pierre Fobert, EFRT Project
AAFC Peer Reviewed Projects
Alberta Crop Industry Development Fund