Targeted resistance gene mapping in soybean using modified AFLPs

The soybean [Glycine max (Merr.) L.] linkage group F contains a vital region of clustered genes for resistance to numerous pathogens including the soybean mosaic virus resistance gene, Rsv1. In order to develop new genetic markers that map to this gene cluster, we employed a targeted approach that utilizes the speed and high-throughput of AFLP, but modified it to incorporate sequence information from the highly conserved nucleotide binding site (NBS) region of cloned disease resistance genes. By using a labeled degenerate primer corresponding to the p-loop portion of the NBS region of resistance genes, such as N, L6, and Rps2, we were able to quickly amplify numerous polymorphic bands between parents of a population segregating for resistance to Rsv1. Of these polymorphic bands, bulk segregant analysis revealed four markers that were closely linked to Rsv1. These markers were cloned and used as probes for RFLP analysis. The four clones mapped to within a 6-cM region surrounding Rsv1, the closest being 0.4 cM away from the gene. Sequence analysis showed that all four clones contain the p-loop sequence corresponding to the degenerate primer and that one of the four clones contains an open reading frame sequence which when translated is related to the NBS region of other cloned disease resistance genes. The rapid identification of four markers closely linked to Rsv1 in soybean demonstrates the utility of this method for generating markers tightly linked to important plant disease resistance genes.