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

A 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.Our 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.Sugi, Cryptomeria japonica, is one of the most important commercial tree species in Japan. Recently, linkage maps have been constructed for this species, based on co-dominant markers including RFLP (Restriction Fragment Length Polymorphism), CAPS (Cleaved Amplified Polymorphic Sequences), and SSR (Simple Sequence Repeat) markers [1-3]. Although these linkage maps include hundreds of markers, more DNA markers are required to generate a denser linkage map. Furthermore, some markers do not segregate in all crosses, thus increasing the number of markers can greatly enhance the scope and resolution of QTL analyses using the progenies of controlled crosses.C. japonica has large genome (~10100 Mbp) [4], therefore full genomic sequences have not been obtained. On the other hand, because of the importance of this species to Japanese forestry, expressed sequence tags (ESTs) have been generated using several types of tissues from several individuals [5-7]. Redundant sequences found in EST databases can be a useful resource for mining SNPs or developing DNA markers, since mapping expressed genes to a linkage map makes the m