ABSTRACT
Genetic diversity and species-diagnostic markers of 5 oysters in Thailand, Crassostrea belcheri (Sowerby, 1871), Crassostrea iredalei (Faustino, 1932), Saccostrea cucullata (Born, 1778), Saccostrea forskali (Gmelin, 1791), and Striostrea Parastriostrea) mytiloides (Lamarck, 1819), were investigated by randomly amplified polymorphic DNA (RAPD) analysis. In a total, 135, 127, and 108 genotypes were observed from primers OPA09, OPB01, and OPB08 (Operon Technologies Inc., kits A and B), and 131 and 122 genotypes from primers UBC210 and UBC220 (University of British Columbia), respectively. Two hundred fifty-four reproducible and polymorphic fragments (200-2500 bp in length) were generated across the 5 investigated species. The average number of bands per primer varied between 12.4 and 32.2. The percentage of polymorphic bands within Crassostrea (53.23%-77.67%) was lower than that within Saccostrea and Striostrea oysters (86.21%-99.36%). Nine, species-specific markers were found in C. belcheri, 4 in C. iredalei, and 2 in S. cucullata. The mean of a ratio between the number of genotypes generated by each primer and the number of investigated specimens of C. belcheri (0.58) was lower than that of the remaining species (0.90-1.00). Genetic distances between pairs of oyster samples were between 0.105 and 0.811. A neighbor-joining tree indicated distant relationships between Crassostrea and Saccostrea oysters, but closer relationships were observed between the latter and Striostrea mytiloides.
ABSTRACT
Randomly amplified polymorphic DNA (RAPD) analysis was used to identify species-specific markers of 5 oyster species in Thailand: Crassostrea belcheri, Crassostrea iredalei, Saccostrea cucullata, Saccostrea forskali, and Striostrea (Parastriostrea) mytiloides. Species-specific markers were found in C. belcheri, C. iredalei, and S. cucullata but not in S. forskali and S. mytiloides. Three C. belcheri-specific RAPD fragments were cloned and sequenced. A primer set was designed from each of the recombinant clones (pPACB1, pPACB2, and pPACB3). The polymerase chain reaction products showed expected sizes of 536, 600, and 500 bp, respectively, with the sensitivity of detection approximately 30 pg of C. belcheri total DNA template. The specificity of pPACB1 was examined against 135 individuals of indigenous oyster species in Thailand and against outgroup references S. commercialis (N = 12) and Perna viridis (N = 12). Results indicated the species-specific nature of primers developed from pPACB1. This primer set can be used for broodstock selection and determination of C. belcheri larvae to assist the selective breeding program for this commercially important species.
