Classification of oysters genera crassostrea, saccostrea and striostrea in Thailand utilizing RAPD markers

Abstract

Randomly amplified polymorphic DNA (RAPD) analysis was used to determine levels of genetic diversity and to identify species-specific markers of five oyster species in Thailand; Crassostrea belcheri (Sowerby, 1871), C.iredalei (Faustino, 1932), Saccostrea cucullata (Born, 1778), S.forskali (Gmelin, 1791) and Striostrea (Parastriostrea) mytiloides (Lamarck, 1819). Five of 103 screened decanucleotide primers (OPA09, OPB01, OPB08, UBC210 and UBC220), were selected for the genetic analysis of oysters in Thailand. Two hundred and fifty-four reproducible and polymorphic fragments (200-2500 bp in length) were generated across investigated species. A total of 193, 192, 174, 192 and 181 genotypes was observed from each respective primer. The percentages of polymorphic bands were 52.23%, 74.67%, 97.69%, 99.40% and 98.50% for C.belcheri, C.iredalei, S.cucullata, S.forskali and S.mytiloides, respectively. Genetic differences between samples from different species were higher than those within the same species. A neighbor-joining tree indicated distant relationships between Crassostrea and Saccostrea oysters but closer relationships were observed within each genus. Ten, five and two species-specific markers were found in C.belcheri, C.iredalei, and S.cucullata, respectively. Three C.belcheri-specific RAPD fragments were cloned and sequenced. A pair of primer set was designed from each recombinant clones (pPACB1, pPACB2 and pPACB3). The PCR products showed expected product sizes of 536 bp, 600 bp and 506 bp, respectively. The sensitivity of detection using pPACB-1F/R, pPACB-2F/R and pPACB-3F/R primers reached approximately 30 pg of C.belcheri total DNA template. Specificity of pPACB1F/R primers was examined against 203 individuals of indigenous oyster species in Thailand, an ingroup (S.commercialis) and an outgroup (Perna viridis) references. Results indicated species-specific nature of pPACB1F/R primers to C.belcheri in Thailand.