Identification and characterization of population-specific marker of Penaeus monodon in Thailand

Abstract

Randomly amplified polymorphic DNA (RAPD) analysis was used to identify a specific marker which could distinguish between the wild populations of Penaeus monodon from the Andaman Sea and the Gulf of Thailand. The eleven selected RAPD primers were screened. Two primers, OPB 08 and OPB15, showed a DNA band with size about 850 bp and 1,000 bp, respectively, which consistenly appeared only in the sample from the Gulf of Thailand. By increasing the number of samples in each group using primer OPB 08 and OPB 15, these bands also appeared inthe samples from the Andaman Sea. Therefore, the primer 428 which has been previously reported to provide a 950 bp specific to the population of the Andaman Sea was used in this study. The primer 428 appeared to identify a more variable region among the samples of Thai P. monodon from the Andaman Sea so a band with size about 950 bp which present in all of Satun Trang, but adsent in samples from Trad was used as a population-specific marker. The 950 bp fragment was cloned and transformed into E.coli: XL1-blue. Three different types of recombinant clones, A, B and C were obtained with the insert fragment size of 900, 950 bp (which upon digestion with Bam HI providing two bands with size about 650 and 350 bp) and 350 bp, respectively. To ensure that the insert fragment of the 3 clones were from the 950 bp RAPD marker, Southern blot hybridization was performed using the three insert fragments (900 bp, 650 bp and 350 bp) as DNA probes. A single specific band was appeared only in the samples form the Andaman Sea when using the 650 and 350 bp fragments while a positive band was found in all sample when using the 900 bp fragment. The insert fragment of the 3 clones were sequenced by using the ABI-PRISM automated sequencer and then aligned to other genes in the GenBank using BLAST program. Comparison of the 900 bp sequence showed similarity with the sequence of asparagine synthetase while the 650 and the 350 bp insert fragment were not sinificantly similar to any sequence in the GenBank. The specific primers were designed from the nucleotide sequences of the 350 bp fragment but could not distinguish the population of P. monodon from the Andaman Sea and the Gulf of Thailand. Therefore, a method which can be to distinguish the two populations was to amplified the genomic DNA using primer 428, followed by Southern blot hybridization with the 650 or 350 bp fragments. This method yielded a single band of 950 bp specific to the population of the Andaman Sea.