Genetic diversity and population structure of the blue swimming crab Portunus pelagicus in Thailand

Abstract

Genetic diversity and population differentiation of the blue swimming crab (Portunus pelagicus) in Thai waters was examined by SSCP analysis. Thirty eight primers pairs were initially tested against genomic DNA of P. pelagicus (N = 3). The positive amplification product of cytochrome oxidase subunit I (COI), M122/135RAP and P[subscript +3]4M[subscript +3]1_454 was cloned and sequenced. A pair of primer was designed from each sequence (called PP-COI[subscript 270]-F/R, PP-SCARRAP[subscript 318]-F/R and PP-SCARAFLP[subscript 300]-F/R, respectively) and tested against P.pelagicus from Chanthaburi (N = 29), Prachuap Kriri Khan (N = 40), Suratthani (N = 35), Ranong (N = 35) and Krabi (N = 35). SSCP analysis was carried out. A total of 9, 19 and 7 SSCP fragments were found across overall investigated individuals and generated 8, 56 and 21 SSCP genotypes, respectively. Large numbers of SSCP genotypes found in Thai P. pelagicus suggested high genetic diversity in this species. The average genetic identity between pairs of geographic samples was 0.8871 - 0.9902. Genetic distance between pairs of geographic samples was 0.0099 - 0.1198. Generally, larger genetic distance was observed between samples from different coastal regions (0.0346 - 0.1198) than that between geographic locations within coastal regions (0.0099 – 0.0198 and 0.0135 for Gulf of Thailand and Andaman samples, respectively). Significant geographic heterogeneity was observed across overall samples (P < 0.01 for FST based statistics, θ and P < 0.0001 for exact test) suggested that the gene pool of P. pelagicus in Thai waters is not panmictic but genetically fragmented at the microgeographic level. The estimated gene flow level of Thai P. pelagicus was 0.39 – 5.37 individuals per generation. A UPGMA dendrogram constructed from the average unbiased genetic distance between pairs of geographic samples allocated 5 geographic samples to 2 evolutionary lineages; Chanthaburi, Suratthani and Prachuap Kriri Khan (Gulf of Thailand, A) and Ranong and Krabi (Andaman Sea, B). In addition, species-diagnostic markers for authentication of P. pelagicus were successfully developed. Initially, cytochrome oxidase subunit I (COI, 706 bp) and 12S ribosomal (r) DNA (406 bp) gene segments of P. pelagicus were amplified using universal primers. These gene segments were cloned and sequenced. Gene-specific primers were designed (PP-COI[subscript 270]-F/R for COI and PP-12S[subscript 312]-F/R for 12S rDNA) and tested for the species specificity against genomic DNA of various species. The expected product of PP-COI[subscript 270] (270 bp) was specifically found in all individuals of P. pelagicus (N = 174) but not in the mud crabs (Scylla oceanica, N = 18, S. serrata, N = 7, S. tranquebarica, N = 9), the swimming crab, Charybdis crucifera (N = 20) and the three spot swimming crab, P. sanguinolentus (N = 10). The expected product of PP-12S[subscript 312] (312 bp) was 100% successfully amplified in P. pelagicus (N = 174) and all non-target species. The PCR product of all investigated species was further analyzed by SSCP. Five SSCP genotypes were found in P. pelagicus and the most common genotype was observed in 91.4% (159/174 individuals) of investigated specimens. Non-overlapping SSCP genotypes were found in other crab species. These molecular markers can be directly applied to authenticate the products of P. pelagicus from Thailand. Temperature-stress response transcripts (TSRT) in haemocytes of P. pelagicus were identified by cDNA-AFLP. A total of 110 primer combination were screened using cDNA of haemocytes of normal crab (N = 4) and those at 0, 3, 6, 12 and 24 hours (N = 4 for each group) after temperature stressed at 33ºC for 3 hours. Seven TSRT fragments were cloned and sequenced. Expression levels of three TSRTs (PP-TSRT[subscript 152], PP-TSRT[subscript 200] and PP-TSRT[subscript 339]) were examined by semiquantitative RT-PCR. PP-TSRT[subscript 152] and PP-TSRT[subscript 339] were significantly down-regulated at 12 and 24 hpt and 12 hpt, respectively (P < 0.05).