Whole Genome Sequencing and Comparative Genomics of Fusarium Head Blight Fungi. Sean Walkowiak

Transcription

1 Whole Genome Sequencing and Comparative Genomics of Fusarium Head Blight Fungi Sean Walkowiak

2 Research Objective To better understand the genetics of toxin production and disease by FHB pathogens. Approach Sequencing and comparative genomics of different FHB fungi.

3 Comparing Isolates Isolate Species Disease on Wheat Toxin Host Origin Location PH-1 F. graminearum High 15-ADON Maize USA CS3005 F. graminearum High 15-ADON Barley Australia DAOM F. graminearum High 15-ADON Wheat USA DAOM F. graminearum High 3-ADON Wheat Canada DAOM F. graminearum High 15-ADON Maize Canada NRRL28336 F. graminearum Medium 3-ADON Wheat USA NRRL6101 F. asiaticum None 3-ADON Barley Japan NRRL28720 F. asiaticum Medium DON Maize Nepal NRRL28721 F. meridionale Low 4A-NIV, NIV Maize Nepal NRRL28723 F. meridionale Low NIV Maize Nepal

4 Comparing Isolates Fusarium Isolate #1 3-ADON VS Fusarium Isolate #2 15-ADON Infects Barley Well Grows quickly in vitro Warm climate Infects Wheat Makes spores Well Grows Filamentous growth more slowly Cool climate

5 Comparing Genomes Fusarium Isolate #1 VS Fusarium Isolate #2 ACCESORY GENOME CORE GENOME ACCESSORY GENOME

6 Comparing Genomes Isolate Species Disease on Wheat Toxin Host Origin Location PH-1 F. graminearum High 15-ADON Maize USA CS3005 F. graminearum High 15-ADON Barley Australia DAOM F. graminearum High 15-ADON Wheat USA DAOM F. graminearum High 3-ADON Wheat Canada DAOM F. graminearum High 15-ADON Maize Canada NRRL28336 F. graminearum Medium 3-ADON Wheat USA NRRL6101 F. asiaticum None 3-ADON Barley Japan NRRL28720 F. asiaticum Medium DON Maize Nepal NRRL28721 F. meridionale Low 4A-NIV, NIV Maize Nepal NRRL28723 F. meridionale Low NIV Maize Nepal

7 Comparing Genomes Isolate Genome Size (Mb) N50 Coverage (fold) NCBI Accession Genome Reference PH AACM Cuomo 2007 CS JATU Gardiner 2014 DAOM LAJZ0 Walkowiak 2015 DAOM LAKA Walkowiak 2015 DAOM LHUC Walkowiak (Accepted) NRRL LHUD Walkowiak (Accepted) NRRL LHTY Walkowiak (Accepted) NRRL LHTZ Walkowiak (Accepted) NRRL LHUA Walkowiak (Accepted) NRRL LHUB Walkowiak (Accepted)

8 1) >93% genome alignment Genome Alignment

9 Genome Alignment 1) >93% genome alignment 2) >700,000 variable positions sort isolates into species and populations

10 Genome Alignment 1) >93% genome alignment 2) >700,000 variable positions sort isolates into species and populations 15-ADON 3-ADON

11 Genome Alignment 1) >93% genome alignment 2) >700,000 variable positions sort isolates into species and populations Hybrid?

12 Genome Alignment 1) >93% genome alignment 2) >700,000 variable positions sort isolates into species and populations 3) Sequence variation is co-localized

13 Comparing Genes Fusarium Isolate #1 VS Fusarium Isolate #2 ACCESORY GENOME CORE GENOME ACCESSORY GENOME Walkowiak et al. BMC Genomics. Submitted

14 Comparing Genes 1) 1,827 Genes are absent in at least one genome, while >100 are unique each species

15 Comparing Genes 1) 1,827 Genes are absent in at least one genome, while >100 are unique each species 2) genes are unique and 1-34 are uniquely absent for each isolate Isolate Uniquely Present Genes Uniquely Absent Genes PH DAOM DAOM CS NRRL DAOM NRRL NRRL NRRL NRRL

16 Comparing Genes 1) 1,827 Genes are absent in at least one genome, while >100 are unique each species 2) genes are unique and 1-34 are uniquely absent for each isolate 3) Accessory genes are involved in toxin production i.e. apicidin polyketide synthase only in F. asiaticum

17 Comparing Genes 1) 1,827 Genes are absent in at least one genome, while >100 are unique each species 2) genes are unique and 1-34 are uniquely absent for each isolate 3) Accessory genes are involved in toxin production 4) Core genes are also variable (allelic, diversifying selection) 163 Genes (including Tri8)

18 Detailed Analyses of Accessory Genes Contig 207:90 kb (20 genes) Walkowiak, Mirmiran et al. Unpublished.

19 Detailed Analyses of Accessory Genes 1) Confirmation of the region s structure by overlapping PCR Overlapping PCR primers Results of PCR Walkowiak, Mirmiran et al. Unpublished.

20 Detailed Analyses of Accessory Genes 1) Confirmation of the region s structure by overlapping PCR 2) Most genes are more common in 3-ADON producers, and genes may have multiple origins Percent Conservation Fusarium - BLASTn F. graminearum (3-ADON) - PCR n = 27 F. graminearum (15-ADON) - PCR n = 27 0 Gene Accession Walkowiak, Mirmiran et al. Unpublished.

21 Detailed Analyses of Accessory Genes 1) Confirmation of the region s structure by overlapping PCR 2) Most genes are more common in 3-ADON producers, and genes may have multiple origins Percent Conservation Fusarium - BLASTn F. graminearum (3-ADON) - PCR n = 27 F. graminearum (15-ADON) - PCR n = 27 0 Gene Accession Walkowiak, Mirmiran et al. Unpublished.

22 Detailed Analyses of Accessory Genes 1) Confirmation of the region s structure by overlapping PCR 2) Most genes are more common in 3-ADON producers, and genes may have multiple origins Percent Conservation Fusarium - BLASTn F. graminearum (3-ADON) - PCR n = 27 F. graminearum (15-ADON) - PCR n = 27 0 Gene Accession Walkowiak, Mirmiran et al. Unpublished.

23 Detailed Analyses of Accessory Genes 1) Confirmation of the region s structure by overlapping PCR 2) Most genes are more common in 3-ADON producers, and genes may have multiple origins 2 Percent Conservation Fusarium - BLASTn F. graminearum (3-ADON) - PCR n = 27 F. graminearum (15-ADON) - PCR n = 27 Gene Accession RQ 1 0 FG1 DAOM (3-ADON) FG2 DAOM (15-ADON) Walkowiak, Mirmiran et al. Unpublished.

24 Detailed Analyses of Accessory Genes 1) Confirmation of the region s structure by overlapping PCR 2) Most genes are more common in 3-ADON producers, and genes may have multiple origins 3) Genes are expressed during early infection Normalized Number of cdna Copies FG1 DAOM (3-ADON) FG2 DAOM (15-ADON) 52.2 ± g3564 g3563 g3562 g3561 g3560 g3559 g3558 g3557 g3556 g3555 g3554 g3553 g3552 g3551 g3550 g3549 g3548 g3547 g3546 g3545 Gene Walkowiak, Mirmiran et al. Unpublished.

25 Detailed Analyses of Accessory Genes 1) Confirmation of the region s structure by overlapping PCR 2) Most genes are more common in 3-ADON producers, and genes may have multiple origins 3) Genes are expressed during early infection 4) Predicted gene functions include proteases and DNA/RNA interaction - Gene functions: 8 genes: DNA/RNA binding (replication, transcription and gene regulation, epigenetics, maintenance) 2 genes: proteolysis 10 genes: mostly unknown function Walkowiak, Mirmiran et al. Unpublished.

26 Take Home Message Comparing genomes of Fusarium Head Blight pathogens can help us understand/classify: a) fungal species (i.e. hybrid) b) genetic populations within species (i.e. 3-ADON/15-ADON) c) accessory genes involved in toxin production and disease (i.e. apicidin) d) allelic variation and diversification of core genes (i.e. Tri8) e) the origin, distribution, and biological role of genetic variation (i.e. 20 genes) Looking to the future: f) gene candidates for functional studies (i.e. mutants)

27 Acknowledgements Dr. Rajagopal Subramaniam and Team (especially Armand Mirmiran) (Agriculture and Agri-Food Canada, Carleton University, University of Toronto) Dr. Linda Harris and Team (Agriculture and Agri-Food Canada) Dr. Owen Rowland (Carleton University) Dr. Nicolas Rodrigue (Carleton University) Dr. Curtis Pozniak (University of Saskatchewan, Crop Development Center)