1a.Objectives (from AD-416)
1. Sequence the Rps2, Rps3 and Rps8 loci from the resistant parents and use expression data to identify expressed candidate resistance genes.
2. Use virus induced gene silencing (VIGS) to assay the function of candidate resistance genes in response to Phytopthora sojae infection.
1b.Approach (from AD-416)
Our approach will identify candidate Phytophthora sojae resistance genes for Rps2, Rps3 and Rps8. We will leverage the Williams82 and PI 96983 genome sequences and use it to identify candidate genes from the resistant parents. Our approach will be to compare sequence from the Williams82 soybean genome and sequence from PI96983, which does not have an Rps gene in these regions, to sequence from lines with Rps2, Rps3 and Rps8. Real-time PCR will be used to monitor expression of candidate genes in resistant and susceptible parents following P. sojae infection. The function of expressed candidate resistance genes will be assayed using virus induced gene silencing to "turn off" resistance.
The Phytophthora sojae (Phytophthora root and stem rot) resistance Rps2 was previously mapped to soybean chromosome 16. Sequencing of this region in the susceptible genotype Williams82 identified a cluster of genes with homology to the nucleotide-binding site, leucine rich repeat family of resistance genes. In order to facilitate cloning of Rps2, we have developed a clone library from the resistant parent L76-1988. Markers used to map Rps2 have been used to screen the library for clones corresponding to the region. Three clones have been identified and are currently being sequenced. Once candidate genes have been identified, the next step will be to silence the expression of the genes using virus induced gene silencing (VIGS). However, the normal inoculation protocols used for VIGS are not compatible with inoculation protocols for P. sojae. Therefore, we are evaluating the vascular puncture inoculation method for introducing VIGS vectors. Progress on this project was monitored through telephone calls, data exchange and written reports.