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United States Department of Agriculture

Agricultural Research Service

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Research Project: Whole Genome Sequencing of Magnaporthe Oryzae Isolates and Gene Expression Analysis of Infected Wheat

Location: Foreign Disease-Weed Science

Project Number: 1920-22000-041-08
Project Type: Specific Cooperative Agreement

Start Date: Sep 01, 2012
End Date: Sep 30, 2015

Objective:
The two objectives of the proposed research are (1) to develop molecular diagnostic assays to distinguish South American isolates of M. oryzae that infect wheat from native U.S. isolates of M. oryzae that are non-pathogenic on wheat and (2) to identify M. oryzae genes involved in pathogenesis and wheat genes involved in resistance. Specifically, we will (1) Perform whole genome sequencing of South American M. oryzae isolates infecting wheat and other related grasses; (2) perform whole genome sequencing on select isolates of M. oryzae collected in North America that do not infect wheat; (3) assemble and compare genomic sequences to identify unique regions; (4) develop and evaluate candidate genomic regions for diagnostic purposes; (5) obtain and evaluate gene expression data from M. oryzae infected wheat; and (6) genotype wheat varieties that have been tested for wheat blast resistance / susceptibility.

Approach:
Whole genome sequencing and post-sequencing comparisons will be used to rapidly identify unique loci between the M. oryzae pathotypes that infect wheat and the pathotypes that infect related grass species. This approach is made feasible by virtue of “next generation” sequencing platforms that enable fast and cost-effective whole genome sequencing projects. We have previously utilized this technology to sequence an isolate of M. oryzae obtained from a 2011 outbreak of wheat blast in Bolivia. As part of this agreement, we will collect and prepare isolates of M. oryzae from North and South America collected from wheat and other grasses. Although the virulent pathotype of M. grisea (i.e. "South American wheat blast") has not been found outside of South America, it is important to sequence isolates collected from both continents in order to design a robust detection assay that is not prone to signaling false-positives. Additionally, RNA from wheat plants infected with M. oryzae will be used for deep sequencing to ascertain the genes involved in pathogenicity/resistance, and we will perform genotyping by sequencing of wheat varieties that have been tested for wheat blast resistance / susceptibility. Preparation of the M. oryzae isolates and isolation of high-quality genomic DNA and RNA will be performed at the FDWSRU at Ft. Detrick in the necessary containment laboratory/greenhouse facilities, and the sequencing will be performed by collaborators at Kansas State University. Post-sequencing analysis will be performed jointly between ARS and KSU, and final testing of the molecular diagnostic assays will be performed by ARS staff at Ft. Detrick.

Last Modified: 9/29/2014
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