The overall goal of this project is to obtain a complete, publicly available genomic sequence of Pyrenophora tritici-repentis (PTR), the causal agent of tan spot of wheat. The objectives of the project include: <OL> <LI>Creation of a high quality genome assembly for P. tritici-repentis with 7X sequence and 56X physical coverage. <LI>Generation of an optical genetic map and integration with the genome assembly. <LI> Perform automated annotation to identify genes, protein domains and repeat features. <LI> Generate Expressed Sequence Tags (ESTs) from two pathogenic races and from one nonpathogenic race of PTR. </OL>These ESTs will help to improve the annotation of the genome and could provide valuable resources for the investigation of pathogenicity. Also, this project will support training and outreach activities in several ways including: <OL> <LI> Development and implementation of a unit in genomics and molecular biology of agriculturally related microorganisms at Heritage University, a nondenominational private, accredited institution of higher education offering higher education to multicultural populations that have been educationally isolated. <LI> Development of an eleven-week internship program at Oregon State University (OSU) for select Heritage University students. <LI> Participation in a Genomics workshop for K-12 and community college teachers. <LI> Expand community interest and education in fungal genomics through development of a Seminar Series on microbial "omics" and selection of a fungal genomicist for the Special Knutson Lectureship at OSU.
Non-Technical Summary: Tan spot of wheat caused by the fungus Pyrenophora tritici-repentis (PTR) is a serious, economically significant disease, both in the U.S. and worldwide, on one of the world's most important food crops, wheat. The genus Pyrenophora includes a number of significant pathogens that reflect the type of lifestyle and pathogenic adaptations of a large number of related, economically and historically significant fungi. The information created through this project will help clarify the underlying molecular mechanisms of pathogenicity and disease susceptibility. PTR serves as a unique model for resolving these mechanisms, which are currently not well understood. Equally important, the information created will serve as a basis for research and discovery in controlling the significant disease caused by this plant pathogen. Experimentally, PTR is unique in displaying extensive race structure that involves the production of numerous, race-specific toxins which themselves are unique because they are predominantly single proteins. The sequence generated from this project will also provide a genome-wide view of this economically and scientifically important plant pathogen. For purposes of comparative genomics, there is a broad, extended community with interest in the genomic analyses of Pyrenophora that will benefit from immediate use of these data. This research is a partnership between the Pyrenophora scientific community and the Broad Institute. Information gained from this Project will be made readily available to the scientific community at large. <P> Approach: Due to the nature of the experimental approach, this project addresses the issue of Genomics and Future Food and Fiber Production. Also, because the effort will help to resolve fundamental issues related to plant disease susceptibility, it addresses issues related to Agricultural and Food Security and Natural Resources and Environmental Quality. The information about the genome of P. tritici-repentis (PTR) is viewed as highly valuable to an extended community of investigators. The pathogenic, race 1 isolate has been chosen for sequencing. Sequence and assembly will be conducted at the Broad Institute with their standardized sequencing approaches. The Broad will generate three types of whole-genome shotgun libraries using randomly sheared total genomic DNA, provided by the Ciuffetti lab. Two plasmid libraries with insert sizes of 4 and 10kb will be generated to provide the majority of sequence coverage. The Broad assembly program, Arachne, will be used to assemble the PTR genome sequence. An optical map will be generated at OpGen using high molecular weight genomic DNA prepeared in the Ciuffetti lab. The integration of the optical map and the assembly will anchor the sequence scaffolds to the chromosomes and will provide a comprehensive landscape of PTR genome structure. The automated annotation of the high quality PTR race 1 assembly will be performed using Calhoun, a whole genome analysis system developed at the Broad. To improve the annotation of the genome and to provide valuable resources to the Pyrenophora community for the investigation of pathogenicity, 4 normalized cDNA libraries (5000 clones from each library in both directions) from two pathogenic races and from the nonpathogenic race will be sequenced.