2012 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.
Achievements to date both directly advance our ability to improve pea genotypes for resistance to white mold (i.e. development of additional markers for marker assisted breeding) and set the ground work for the identification of novel resistance genes and improved understanding of this host-pathogen interaction.
First, a 454 sequence data set derived from the pea-S. sclerotiorum interaction was analyzed and annotated. We found 597 pea ESTs which are putatively involved in plant defense and response to biotic or abiotic stress,and 155 S. sclerotiorum ESTs involved in pathogenicity or virulence. To assist in the development and refinement of pea linkage maps, 37 EST-derived SSR markersfrom the 454 sequence data were developed and screened for 23 pea individual cultivars including parents of four Pisumsativum recombinant inbred line (RIL) mapping populations developed for improved white mold resistance. Eleven SSR markers produced polymorphism in at least one population.
To further investigate the interaction and quantify gene expression over time global gene expression profiling of partially resistant and susceptible pea lines infected with S. sclerotiorum was conducted using an Illumina GA2 sequencing platform with a 75 bp paired end sequencing protocol. Pea stem samples from a susceptible pea cultivar ‘Lifter’ and a partially resistant cultivar ‘PI240515’either inoculated with S. sclerotiorum or mock–inoculated were collected at 12, 24 and 36 hour after inoculation. Additionally, RNA was extracted from S. sclerotiorum growing on the culture medium used for inoculation. After RNA extraction, cDNA library preparation and barcoding, a total of 30 cDNA samples were sequenced in 7 lanes of one Illumina Genome Analyzer flowcell, which produced more than 300 million paired-end reads. After first-round de novo assembly, those reads resulted in 60,656 Lifter contigs, 67,893 PI240515 contigs and 18,178 S. sclerotiorum contigs obtained from mock-inoculated samples with contigs larger than 200 nucleotides; 71,228 contigs and 81,533 contigs were assembled from the Lifter and PI240515 samples infected with S. sclerotiorum. A second round of de novo assemble was executed for the gene expression profiling study between two cultivars. All Lifter-Sclerotinia related reads were assembled into 61,056 contigs larger than 300 nucleotides and all PI240515-Sclerotinia reads into 65,047 contigs. Based on the tBLASTx method we developed previously and using the first-round contigs from mock control as reference, the second-round Lifter-Sclerotinia contigs were sorted into 44,998 pea ESTs, 14,436 S. sclerotiorum ESTs, 360 ambiguous ESTs and 1,262 ESTs cannot be assigned. The second-round PI240515-Sclerotinia contigs were sorted into 48,174 pea, 14,513 S. sclerotiorum, 383 ambiguous and 1,977 unassigned.
By comparing the contigs from the 2 pea cultivars, 541 simple sequence repeat (SSR) markers from pea were identified. These SSR markers have been provided for the construction of pea linkage maps. All pea ESTs from Lifter and the PI line were annotated based on Arabidopsis, Medicago and Soybean annotation databases, respectively. A total of 20,526 annotated pea ESTs (average length 1,454 nucleotides) with 95% similarity in the two cultivars have been selected for comparison of gene expression profiling. 10,596 S. sclerotiorum ESTs (average length 1,767 nucleotides) were also selected and will be used to determine the expression profiling of the fungus during this host-pathogen interaction.
Through gene expression profiling study, we hope to identify genes and pathways involved in resistance to facilitate the breeding of pea lines with a higher level of resistance to white mold. We also plan to identify S. sclerotiorum genes with putative roles in infection, growth, and pathogenicity and to help us better understand the mechanism of this pathogen.
In summary, during 2011, an expressed sequence tag (EST) resource of pea and S. sclerotiorum was characterized and a publication prepared for submission. From this EST resource 37 simple sequence repeat (SSR) markers for pea breeding were developed and a manuscript describing this was submitted for publication. A time course gene expression profiling study of S. sclerotiorum on pea was conducted with RNA seq, which produced approximately 300 million paired end reads and this RNA-seq data set was mined for an additional 541 SSR markers for use in the development of linkage maps for S. sclerotiorum resistance breeding.