Detection and genotyping of SNPs tightly linked to two disease resistance loci, Rsv1 and Rsv3, of soybean

Allele‐specific polymerase chain reaction (AS‐PCR) for assaying single nucleotide polymorphisms (SNPs) would be more widely used with increased availability of AS primers sufficient to distinguish between SNP alleles. AS‐PCR could be a means unambiguously to detect the presence or absence of PCR products. Examples are given here of the detection and genotyping of SNPs in the genomic DNA fragments tightly linked to two soybean mosaic virus resistance genes, Rsv1 and Rsv3, with a modified AS‐PCR procedure in soybean. The modified AS‐PCR that introduces an additional base mismatch closest to the 3′‐end of the AS primers and uses publicly available microsatellite markers as positive controls directly determined SNP alleles from primary PCR of genomic DNAs. It was demonstrated that a set of AS primers designed from two adjacent SNP loci could simultaneously detect the two SNP loci. Using the modified procedure, many SNP loci in eight soybean parental lines and F₂ individuals of three mapping populations could be genotyped. The modified AS‐PCR procedure could greatly facilitate small‐to‐medium scale marker‐assisted selection programmes for agronomically important genes.