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 Phytopthora 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 same region in L76-1988. This region, which spans ~370 kb, has now been completely sequenced. Computational analyses have identified 25 candidate resistance genes, sharing greater than 90 percent nucleotide identity. To help narrow the number of candidate resistance genes, we are using Reverse-Transcriptase PCR (RT-PCR) to measure gene expression in P. sojae infected and mock-infected tissue from resistant and susceptible soybean genotypes. To control for differences in amplification efficiency, we have designed a single set of conserved primers that will amplify all of the candidate resistance genes. Sequencing of the RT-PCR product identifies base changes that will allow us to distinguish individual genes. By comparing amplification of genomic DNA to cDNA, we can calculate expression levels for the individual genes in the cluster.