SNP Mining in Crassostrea gigas EST Data: Transferability to Four Other Crassostrea Species, Phylogenetic Inferences and Outlier SNPs under Selection

Oysters, with high levels of phenotypic plasticity and wide geographic distribution, are a challenging group for taxonomists and phylogenetics. Our study is intended to generate new EST-SNP markers and to evaluate their potential for cross-species utilization in phylogenetic study of the genus Crassostrea. In the study, 57 novel SNPs were developed from an EST database of C. gigas by the HRM (high-resolution melting) method. Transferability of 377 SNPs developed for C. gigas was examined on four other Crassostrea species: C. sikamea, C. angulata, C. hongkongensis and C. ariakensis. Among the 377 primer pairs tested, 311 (82.5%) primers showed amplification in C. sikamea, 353 (93.6%) in C. angulata, 254 (67.4%) in C. hongkongensis and 253 (67.1%) in C. ariakensis. A total of 214 SNPs were found to be transferable to all four species. Phylogenetic analyses showed that C. hongkongensis was a sister species of C. ariakensis and that this clade was sister to the clade containing C. sikamea, C. angulata and C. gigas. Within this clade, C. gigas and C. angulata had the closest relationship, with C. sikamea being the sister group. In addition, we detected eight SNPs as potentially being under selection by two outlier tests (fdist and hierarchical methods). The SNPs studied here should be useful for genetic diversity, comparative mapping and phylogenetic studies across species in Crassostrea and the candidate outlier SNPs are worth exploring in more detail regarding association genetics and functional studies.