Molecular characterization of Pst isolates from Western Canada

Transcription

1 #bgri2014 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

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 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; 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 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 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, E134 SRCReed2K Creston, BC 2000? HiSeq 71,986, E150 SRLReed2K Lethbridge 2000? HiSeq 44,640, E182 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 E162 SRBI E0 SRBI E134 SRCReed2K E150 SRLReed2K E182 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 ITS1 ITS2 IGS1 18S 28S 18S 5.8S 5S IGS2 Consensus GQ GU HM HM HM EU AY

14 Phylogenetic tree based on rrna consensus UPGMA

15 Phylogenetic tree of 1700 MiSeq sequences

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

17 Isolate name Id name Location Year Isolate type Puccinia striiformis f.sp. tritici 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, E134 SRCReed2K Creston, BC 2000 New HiSeq 71,986, E50 SRLReed2K Lethbridge 2000 New HiSeq 44,640, E182 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 New isolates Puccinia striiformis f.sp. tritici Old isolates Genom. Transcr. Mito. Genom. Transcr. Mito. Total reads (M) Assembled R (M) Unassemb. R (M) paired (M) unpaired (M) Contigs 16,665 20, ,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 NA 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 restrictionmodification 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 Functional annotation of new isolates 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.

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 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 Yong Xu Byron Puchalski Brent Puchalski Eric Amundsen Arsh Singh Denis Gaudet Harpinder Randhawa Robert Graf NRC-Saskatoon Pierre Fobert, EFRT Project AAFC Peer Reviewed Projects Alberta Crop Industry Development Fund