Wheat Tan Spot Pan-Genomics High Through Put Analysis (2019)

Pyrenophora has a number of necrotrophic fungal pathogens that cause major diseases in wheat and barley. This project has acquired a world-wide catalogue of pathogenic isolates for comparative and pan genomics analysis, and now explores host responses during disease. This project sets out to provide essential genomics-based resource support to better understand the host pathogen interactions that underlie fungal infection. This expansion of Ptr and wheat genome analyses directly facilitate research aimed at controlling fungal disease in wheat and barley.

Principal investigator

Paula Moolhuijzen paula.moolhuijzen@curtin.edu.au
Magnifying glass

Area of science

Agricultural And Veterinary Sciences, Biology

Systems used

Magnus, Zeus and Nimbus

Applications used

Canu, STAR, Exonerate, EvidenceModeller, KAKS Calculator
Partner Institution: Curtin University| Project Code: FL3

The Challenge

To ensure comprehensive comparative analyses, fully annotated genomes are produced to identify important regions of variation related to pathogen virulence and host defence. As the number and size of the genomes under investigation increases, a high through put environment is vital to process the data. For instance, currently in this project a typical pathogen pan-genomics analysis involves 351K individual computational tasks. A job this size on a 16-core machine would take more than 8 months.

The Solution

Our approach is to batch the hundreds of thousands computational tasks and run these in parallel on Pawsey’s HPC Magnus. To date, job runs on Magnus have been highly successful with results returning well within the 24 hours wall time. An array of diverse on-analyses is then completed on Pawsey Nimbus cloud resources, which include the development of a NextFlow pipeline for reproducible, parallel and scalable analysis of Nanopore sequence data.

The Outcome

Through Pawsey HPC project FL3 and Nimbus allocations we have expanded genomic resources for research into Pyrenophora tritici-repentis (tan spot wheat disease) and three new barley pathogens, Pyrenophora teres f. teres (net form net blotch disease), Pyrenophora teres f. maculata (spot form net blotch disease) and Blumeria graminis f. sp hordei (powdery mildew). Since 2018, we have increased the number of high-quality genomes for Pyrenophora from 20 to 32. In 2019, eight annotated pathogen genomes (PacBio sequenced) were publish in DDBJ/EMBL/GenBank under accessions (SAXQ00000000, WJSO00000000, WJSN00000000, WJSM00000000, WJSL00000000, WJSK00000000, OCTH02000000 and OCTF02000000). While another two PacBio sequenced genomes and a MinION Nanopore sequenced genome were deposited in DDBJ/EMBL/GenBank, and await publication. Furthermore, in 2019, analyses were expanded to transcriptomic (RNA-Seq) data of wheat during tan spot infection, barley during net blotch infection and barley during powdery mildew infection. Three draft manuscripts have been produced for the analysis of the three transcriptomic (RNA-Seq) data sets.
Another outcome for 2019, as part of Pawsey’s Perth Nextflow-hackathon, was the development of a workflow for the analysis of Nanopore sequence generated by an in-house MinION to run on Nimbus, https://github.com/ccdmb/PathoPore.

List of Publications

The following three peer-reviewed publications were based directly on the high-through-put analyses conducted in 2019 using the Pawsey’s HPC project FL3 and Nimbus allocations.

1: Rawlinson C, See PT, Moolhuijzen P, Li H, Moffat CS, Chooi YH, Oliver RP. The identification and deletion of the polyketide synthase-nonribosomal peptide
synthase gene responsible for the production of the phytotoxic triticone A/B in
the wheat fungal pathogen Pyrenophora tritici-repentis. Environ Microbiol. 2019 Dec;21(12):4875-4886. doi: 10.1111/1462-2920.14854. Epub 2019 Nov 21. PubMed PMID: 31698543; PubMed Central PMCID: PMC6915911.

2: Moolhuijzen P, See PT, Moffat CS. A new PacBio genome sequence of an Australian Pyrenophora tritici-repentis race 1 isolate. BMC Res Notes. 2019 Oct
4;12(1):642. doi: 10.1186/s13104-019-4681-6. PubMed PMID: 31585535; PubMed Central PMCID: PMC6778365.

3: Martin A, Moolhuijzen P, Tao Y, McIlroy J, Ellwood SR, Fowler RA, Platz GJ,
Kilian A, Snyman L. Genomic Regions Associated with Virulence in Pyrenophora teres f. teres Identified by Genome-Wide Association Analysis and Biparental Mapping. Phytopathology. 2020 Apr;110(4):881-891. doi: 10.1094/PHYTO-10-19-0372-R. Epub 2020 Mar 2. PubMed PMID: 31855502. Accepted in 2019.

Figure 1. (A) Dot plots showing the whole genome nucleotide alignments for V1 contigs (vertical axis) to M4 and Pt-1C-BFP scaffolded genomes (horizontal axes). *Pt-1C-BFP contigs that are not assigned to a Pt-1C-BFP chromosome.
Figure 2. Titicones Figure S9. Triticone biosynthetic gene cluster, Ttc, in Ptr compared to cpa gene cluster in Curvularia pallescens showing nucleotide percent identity between the clusters. Homologues genes are presented in turquoise, non-homologous in orange and 4 non cluster genes from Ptr in green.