2011 Annual Report
The objective of this proposal is to utilize recent advances in massively parallel sequencing technology to study the regulation of genes during the S. sclerotiorum and P. sativum host-pathogen interaction. To identify pea resistance genes and Sclerotinia pathogenicity genes we will compare gene expression in individuals from a pea recombinant inbred line that is segregating for partial resistance to S. sclerotiorum. The identification of genes involved in partial resistance may lead to the development of markers for marker assisted breeding for resistance to S. sclerotiorum. The expression profiling studies will also provide valuable information on the genetics of the interaction between host and pathogen which may lead to further insights and solutions for developing resistant material not only in pea but in other crops affected by Sclerotinia spp. Additional objectives of this proposal are to provide characterization of genes for improved annotation of the Sclerotinia genome project and gene function discovery for both pea and S. sclerotiorum.
A 35Mb expressed sequence tag (EST) data set was developed from S.sclerotiorum infected pea tissue with massively parallel sequencing on a 454 Roche platform; 145,049 reads, each averaging >200 nucleotides. Data were trimmed, based on sequence quality and adaptor sequence removal, and 128,720 reads were assembled into 10,158 contiguous sequences (contigs); 51 contigs were discarded due to high similarity to viruses and viroids. To separate S. sclerotiorumcontigs from Pisumsativum reads we utilized a tBlastx search algorithm against a fungal genome library (Sclerotinia sclerotiorum, Botrytis cinerea, Chaetomiumglobosum, Fusariumgraminearum, Magnaporthegrisea, Neurosporacrassa, and Verticillium dahlia) and a legume genome library (Glycine max, Lotus japonicus and Medicagotruncatula). This method was first developed and tested with a set of pea and Sclerotinia ESTs downloaded from public databases (NCBI and BROAD). On the test data set the tBlastx parsing method demonstrated that 90% of ESTs could be assigned with a very low (0.1%) false allocation rate. The tBlastxparsing method was applied to our 10,107 EST contigs; 53.8% of contigs were assigned to pea, 27.4% to S. sclerotiorum, 5.4% of reads were ambiguous with hits to both genome libraries, and 13.4% were unassigned. The EST contig assignment method was validated by selecting 50 pea and 50 S. sclerotiorumassigned ESTs at random, PCR primers were designed to these ESTs and PCR was run against cDNA from pea infected with S. sclerotiorum, pea only and S. sclerotiorum only. All 50 pea EST primers amplified the same size amplicons from pea infected with S. sclerotiorum and pea cDNA, indicating correct assignment of reads. Likewise, the S. sclerotiorum EST primer sets amplified 47 out of 50 predicted amplicons. Two S. sclerotiorum ESTs only amplified correct sized PCR products from the pea-Sclerotinia cDNA sample, perhaps indicating that this transcript is only expressed during the interaction with pea. One Sclerotinia primer set failed to amplify from any template.We are currently completing analysis of the 454 EST data set for publication, including gene ontology analysis to further characterize the EST data set. Simple sequence repeat (SSR) markers identified in the pea ESTs are also being screened for polymorphism among pea parental lines as additional markers for mapping studies. Once this study is published the EST data sets will be made publicly available for the Sclerotinia and pea community.