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

Presentation Outline

• Puccinia striiformis f.sp. tritici (Pst)

causal agent of stripe (yellow) rust

• Molecular characterization of old

and new isolates of Pst

• Conclusions

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.

Puccinia striiformis f.sp. tritici

4

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)

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)

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

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

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

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

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

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 +

Schematic of rRNA locus in P. striiformis

18S 28S 18S 5.8S 5S

ITS1 ITS2 IGS2 IGS1

AY117126 1 1329 1 1758

Consensus

GQ457306 GU382673 HM057122 HM057130 HM057136 EU851142

Phylogenetic tree based on rRNA consensus

UPGMA

Phylogenetic tree of 1700 MiSeq sequences

Gene annotation and Gene make up

Puccinia striiformis f.sp. tritici

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

Puccinia striiformis f.sp. tritici New 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

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

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

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

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

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

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.

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