Genetic diversity of the blue swimming crab portunus pelagicus in Thailand analyzed by AFLP and RAPD

Abstract

Genetic diversity and population differentiation of the blue swimming crab (Portunus pelagicus) was analyzed by RAPD and AFLP analyses. A total of 112 RAPD fragments were generated from analysis of 109 individuals of P. pelagicus with OPA02, OPA14, OPB10, UBC122 and UBC158. The percentage of polymorphic bands in each sample was 72.73% - 85.05% The mean genetic distance between samples across overall primers was 0.1151-0.2440. Results from geographic heterogeneity analysis using the exact test and F[subscript ST]-based statistics ([theta]) indicated statistically significant differences between all pairwise comparisions (P<0.001) indicating microgeographic differentiation of investigated samples. Fifteen RAPD fragments were regarded as candidate species-specific fragments. Eight of these were cloned and sequenced. Five pairs of primers were designed. Three RAPD-derived SCAR makers (pPP122-510, pPP158-1200 and pPP158-1500) generated the expected product (152, 397 and 262 bp) in95%, 100% and 100% in the target species without any false positive in the non-target species. The sensitivity of detection was approximately 400 pg, 200 pg and 12.21 pg, respectively. Stability of P. pelagicus-specific markers was tested against genomic DNA of frozen, saline-preserved and boiled P. pelagicus meat using either a standard phenol/chloroform or a chelex-based methods. The positive amplification product was consistently found in all specimens regardless sources of genomic DNA and extraction methods. High genetic diversity was also observed when analyzed 72 individuals of P. pelagicus with 4 primer combinations (P[subscript+3]-2/M[subscript+3]-1,P[subscript+3]-4/M[subscript+3]-1, P[subscript+3]-4/M[subscript+3]-2 and P[subscript+3]-7/M[subscript+3]-1). A total of 227 AFLP fragment were generated and the percentage of polymorphic band in each samples was 66.19%-94.38%. The mean genetic distance between paired sample was 0.0929 -0.247I. Like results from RAPD analysis, geographic heterogeneity analysis of AFLP data using the exact test and F[subscript ST]-based statistics ([theta]) indicated statistically significant differences between all pairwise comparisions (P < 0.001). This also indicated a fine scale genetic differentiation in this species. Thirteen candidate species-specific AFLP fragments were found. Two of these were cloned and sequenced. A primer pair was designed from nucleotide sequence of each fragment. One AFLP-derived SCAR marker (P4M1-300) gave the positive amplification product in 30% of investigated P. pelagicus individuals while the other (pP4M2-420) consistently generated the expected product in 97.0% of the target species. Nevertheless, the positive amplification product of pP4M2-420 was cross amplified with genomic DNA of C. crucifera (N=6) but not mud crabs (N=30). SSCP analysis was successfully applied to further authenticate the species origin of P. pelagicus. The sensitivity of detection of pP4M2-420 was approximately 400 pg of the target DNA template. High stability of the developed markers was also observed in frozen, saline-preserved and boiled P. pelagicus specimens.