Genome-wide SNP based species identification of Chinemys reevesii, Ocadia sinensis and their hybrids

Freshwater turtles have a long history as medicine and food. Due to the increasing demand, many non-cultured turtles are overfished and endangered. It is difficult to distinguish the commercially cultured Chinemys reevesii, Ocadia sinensis and their hybrid offspring by conventional method, which brings harm to variety protection and market supervision. In order to identify these turtles, we generated genomic resources of the C. reevesii double digest restriction-site associated DNA (ddRADseq) and O. sinensis re-sequencing libraries. A total of 88,087 SNP loci with high-quality were found in the genomes, and 8,617 pairs of SNP primers were carefully designed, from which 20 SNP markers were randomly selected for experimental verification, among which four markers were confirmed to distinguish C. reevesii, O. sinensis and their hybrid offspring. In order to improve the practical efficiency of the species identification, a method combining multiplex ligation-dependent probe amplification (MLPA) with real-time quantitative polymerase chain reaction (RT-PCR) was established. According to the melting curve, these turtle species can be visually and rapidly distinguished in less than 3 h with the template concentration as low as 0.01 ng/μL. This study laid a foundation for the protection of wild resources and the regulation of turtle market, and also laid a molecular foundation for genetic research of turtles, such as genetic improvement and molecular marker-assisted selection.