1a.Objectives (from AD-416)
1. Use virus induced gene silencing (VIGS) to turn off candidate resistance genes in the Brown Stem Rot resistant genotypes. Test silenced plants for susceptibility to Brown Stem Rot.
2. Use Solexa transcript sequencing to determine which resistance genes in the Rbs3 locus are expressed preferentially in the resistant parent and correlate with Brown Stem Rot resistance.
3. Use the Williams 82 genome sequence to fine map Rbs3 in a population segregating for Brown Stem Rot resistance (BSR101 x PI437654, developed by Lewers et al. 1999). Develop SSR markers to be used for marker-assisted selection of BSR resistance.
1b.Approach (from AD-416)
Our approach will identify candidate Brown Stem Rot resistance genes for Rbs3. We will leverage the Williams 82 genome sequences and use it to identify candidate genes from the resistant parents. We have compared sequence from the Williams 82 soybean genome to markers that have been used to map Rbs3. The region corresponding to Rbs3 has been bioinformatically screened for the presence of genes with similiarity to known resistance genes. The sequences of these R-genes has been used to develop Virus Induced Gene Silencing Constructs. These constructs will be tested on Brown Stem Rot resistant genotypes to determine if we can turn off resistance to Brown Step Rot. This information will identify a cluster of genes responsible for resistance. To identify the actual genes, we will use solexa transcript sequences to compare Brown Stem Rot resistant and susceptible genotypes before and after infection with Brown Stem Rot. Significantly differentially expressed genes in the Rbs3 cluster will be candidates for Rbs3. In addition, we will use fine mapping to further define the candidate genes.
The Brown Stem Rot (BSR) resistance gene Rbs3 was previously mapped to chromosome 16. We have compared the markers used to map the gene with the soybean genome sequence. Within this region we identified two different classes of receptor-like proteins (RLPs) with homology to known resistance genes. We have developed virus induced gene silencing (VIGS) constructs to test the function of the RLP candidate genes. While VIGS can be used to turn off all the genes within a cluster, it cannot target individual genes. Therefore, using the genome sequence, we developed 24 simple sequence markers to further define the location of the Rbs3 resistance gene. Of these, 17 amplified differences between the resistant and susceptible parent could be used for fine mapping. These markers were then screened against a population segregating for BSR resistance. We are currently incorporating the mapping and disease resistance data to narrow the location of Rbs3. At the same time, our collaborator is using VIGS to silence the expression of the RLP candidate genes. Progress on this project was monitored through telephone calls, data exchange and written reports.