Mining single nucleotide polymorphisms from EST data of hard clam Meretrix meretrix

In this study, we describe the first set of single nucleotide polymorphism (SNP) genotyping assays for hard clam (Meretrix meretrix) that were discovered through expressed sequence tag (EST) database mining. A total of 3,129 ESTs were assembled and 228 putative SNPs were identified from 51 SNP-containing contigs, with an average of 0.64 SNPs per 100 bp. A/G (T/C) and A/C (T/G) types had a high frequency and accounted for 46.2% and 35.6%, respectively. Thirty-two SNP genotyping assays were designed for validation based on allele-specific polymerase chain reaction (PCR) and melting temperature (T-m)-shift analysis, and 15 (46.9%) of them showed polymorphisms with the minor allele frequency ranging from 0.075 to 0.475. Twelve (37.5%) assays failed due to amplification failure of one or both allele specific primers, and five (15.6%) assays lacked sufficient temperature discrimination for accurate genotype scoring. BLASTX gave significant hits for all 15 genotyped SNP-containing contigs but all were synonymous coding SNPs. These results indicate that EST data constitutes a valuable resource for SNP discovery and further maker development.