An efficient method for developing SNP markers based on EST data combined with high resolution melting (HRM) analysis

Abstract

BackgroundIn order to identify single nucleotide polymorphisms (SNPs) efficiently from a species with a large genome, SNPs were identified from an expressed sequence tag (EST) database combined with High Resolution Melting (HRM) analysis.FindingsA total of 574 sequence tagged sites (STSs) were generated from Cryptomeria japonica and HRM analysis was used to screen for polymorphisms in these STS markers. STSs were designed in two ways: 1) putative SNP sites were identified by comparing ESTs from specific contigs, then 226 primer pairs designed for the purpose to amplify these SNPs; 2) 348 primer pairs were randomly designed using reads from the 3' end of cDNA. HRM analysis revealed that 325 markers among eight individuals were polymorphic, and that STSs, including putative SNP sites, exhibited higher levels of polymorphism.ConclusionOur results indicate that the combination of SNP screening from an EST database combined with HRM analysis is a highly efficient way to develop SNP markers for expressed genes. This method will contribute to both genetic mapping and the identification of SNPs in non-model organisms.