Discovery and validation of genic single nucleotide polymorphisms in the Pacific oyster Crassostrea gigas

The economic and ecological importance of the oyster necessitates further research on the molecular mechanisms, which both regulate the commercially important traits of the oyster and help it to survive in the variable marine environment. Single nucleotide polymorphisms (SNPs) have been widely used to assess genetic variation and identify genes underlying target traits. In addition, high-resolution melting (HRM) analysis is a potentially powerful method for validating candidate SNPs. In this study, we adopted a rapid and efficient pipeline for the screening and validation of SNPs in the genic region of Crassostrea gigas based on transcriptome sequencing and HRM analysis. Transcriptomes of three wild oyster populations were sequenced using Illumina sequencing technology. In total, 50-60million short reads, corresponding to 4.5-5.4Gbp, from each population were aligned to the oyster genome, and 5.8x10(5) SNPs were putatively identified, resulting in a predicted SNP every 47 nucleotides on average. The putative SNPs were unevenly distributed in the genome and high-density (2%), nonsynonymous coding SNPs were enriched in genes related to apoptosis and responses to biotic stimuli. Subsequently, 1,671 loci were detected by HRM analysis, accounting for 64.7% of the total selected candidate primers, and finally, 1,301 polymorphic SNP markers were developed based on HRM analysis. All of the validated SNPs were distributed into 897 genes and located in 672 scaffolds, and 275 of these genes were stress inducible under unfavourable salinity, temperature, and exposure to air and heavy metals. The validated SNPs in this study provide valuable molecular markers for genetic mapping and characterization of important traits in oysters.