Pacific oyster Crassostrea gigas and Portuguese oyster C. angulata are morphologically
related species that are sympatrically distributed at nearshore blackish areas in
the subtropical zone of East Asia. Although these two species have thus far been known as inter-fertilizable under controlled conditions, molecular-based studies on mitochondrial DNA analysis verified obvious genetic differences between them. Huvet et al. (2004) reported a novel microsatellite DNA marker enabling to discriminate allelic sequence variants for C. gigas and C. angulata. In this study, we developed a rapid, reliable, and secure PCR-RFLP analysis of the biallelic microsatellite marker to diagnose hybrid oysters between C. gigas and C. angulata in wild stocks. An array of wild oyster specimens of C. gigas haplotypes and C. angulata haplotypes collected from Japan, Taiwan, and Korea were subjected to PCR amplification of the microsatellite marker and subsequent RFLP assay using FastDigest® Bsp1407I
restriction enzyme, followed by microchip electrophoretic diagnosis of both PCR and RFLP products. It took about 2 hours for the whole process of our analysis, and the frequencies of biallelic variants observed in C. gigas and C. angulata haplotype stocks were at the equivalent level of those of the previous report. In addition, a high frequency of the allelic variants for C. angulata in C. gigas haplotype stock in Tokyo Bay suggests the occurrence of natural hybrid oysters between C. gigas and C. angulata.