Development of robust allele specific PCR based SNP markers for barley

Numerous methods have been developed for genotyping SNPs, however many of these depend on expensive equipment for analysis. In addition, as a result of the diversity of assay methods, markers developed in one laboratory are often not transferable to laboratories dedicated to a different method of analysis. An alternative cheap, simple, transferable and robust method of SNP genotyping is based on a three primer nested allele specific PCR. With this method, SNP genotyping can be carried out with a thermocycler and agarose gel electrophoresis. We have developed markers for 40 SNP sites in 28 barley genes based on the nested allele specific PCR method. Primers flanking 50 chosen SNP sites were designed for putative intervarietal SNPs identified from publicly available EST sequences. Two approaches were undertaken for the development of allele specific primers. In the first approach allele specific primers were designed to both alleles in both orientations, with a second set of primers designed with 3rd base back mismatches. We obtained a 95% success rate for the development of allele specific markers (for one or both alleles) with this approach. Markers for both alleles at a SNP site were developed in 84% of cases. In the second approach, designed to decrease costs of primer synthesis, only the best one or two allele specific primers were chosen for synthesis. With this approach we obtained a 77% success rate. This method of allele specific PCR shows promise for the widespread development of cheap transferable SNP based markers for plant genomics.