Identification of molecular genetic markers for taxonomy of oysters genera Crassostrea, Saccostrea and Striostrea in Thailand

Abstract

Genetic diversity of 5 oyster species of genera Crassostrea and Saccostrea; Crassostrea belcheri (Sowerby, 1871), C. iredalei (Faustino, 1932), Saccostrea cucullata (Born, 1778), S. forskali (Chemnitz, 1785) and Striostrea (Parastriostrea) mytiloides (Lamarck, 1819), were analyzed by PCR-RFLP of 16S (Acs I, Alu I, Dde I, Dra I, Rsa I and Taq I) and 18S (Hinf I) rDNAs and COI (Acs I, Dde I and Mbo I). A total of 54 composite haplotypes were observed. No overlapping haplotypes were found between different oyster species. Species-diagnostic composite haplotypes were specifically found in each commercially cultured oyster species (C. belcheri, C. iredalei and S. cucullata). Genetic distances between pairs of composite haplotypes within a particular species were lower than those between species. A neighbor-joining tree constructed from nucleotide divergence between pairs of species indicated large genetic differentiation between Crassostrea and Saccostrea(including S. mytiloides) but closer relationships were observed within each genus. Four unidentified oyster groups (Crassostrea sp., Saccostrea sp. group 1, 2 and 3) were also genetically analyzed. Results from restriction analysis indicated that Crassostrea sp. and Saccostrea sp. group 2 showed unique species-diagnostic markers for both single and composite haplotypes whereas Saccostrea sp. group 3 showed composite haplotypes commonly found in S. forskali and S. mytiloides. The Saccostrea sp. group 1 showed composite haplotypes DFGABABAHP which was also found in S. forskali. The remaining haplotypes of this species were not found in other species but were available at low frequencies. The amplified COI from a representative individual of C. belcheri showing AAAAAAAAAA haplotype, that of C. iredalei showing BBBBAAAABC haplotype and that of S. cucullata showing CDCCBBBBCD haplotype, were cloned and sequenced. The nucleotide sequences indicated that these cloned DNA fragment were COI as they exhibited significant similarity with COI sequences of other Crassostrea, Saccostrea and Ostrea oysters previously deposited in the GenBank (p<0.0001). This further confirmed that the amplified COI fragments from different oyster species used in this study were homologous. Two reverse primers (R301 and R353) were designed and used with the primer HCO2198. Only a single fragment showing greater amplification specificity was observed. Although both primer set provided a specific amplification fragment for C. belcheri and C. iredalei, HCO2198+R353 gave more consistent results than did HCO2198+R301. Digestion of the HCO2198+R353 product could differentiate C. belcheri and C. iredalei unambigously.