Identification of seven Crassostrea oysters from the South China Sea using PCR-RFLP analysis

We have developed a PCR-RFLP assay of two mitochondrial [16S rDNA and cytochrome oxidase I (COI)] and one nuclear (ITS-1) gene fragment for the identification of seven Crassostrea oysters from the South China Sea. These include the morphologically distinct Crassostrea sikamea and three pairs of morphologically similar species: Crassostrea hongkongensis and Crassostrea ariakensis, Crassostrea gigas and Crassostrea angulata, Crassostrea iredalei and an unidentified Crassostrea sp. All seven species can be distinguished with both mitochondrial markers: DdeI/DraI or AluI/MseI digestion of 16S rDNA and NisI/AluI or NisI/DdeI digestion of COI. Five of the seven species can be distinguished using a single endonuclease digestion of the ITS-1 fragment. Based on genetic distance estimates and on morphology, the newly discovered Crassostrea sp. appears most closely related to C. iredalei. Despite the existence of intra-specific variation at low frequency in some restriction sites, the PCR-RFLP assay developed here can be reliably applied to identify all seven oyster species.