Construction of microsatellite library and sequences analysis of the swimming crab (Portunus trituberculatus)

Abstract:

Microsatellite sequences were isolated from swimming crab (Portunus trituberculatus) by using the magnetic bead hybridization enrichment method.Total genomic DNA was extracted and digested with restriction enzyme Sau3AI.Fragments in range of 200-1 000 bp were agarose-gel purified and ligated with special adaptors,and the combinations was hybridized with biotin-labelled microsatellite probe (CA) ₁₂ and (GA) ₁₂.By using the high binding affinity of biotin to streptavidin-coated magnetic beads,single-stranded DNA containing the selected microsatellite sequences were captured.The targeted DNA was used as the template to conduct PCR amplification using the adaptor as primers.The fragments were inserted into PMD18T vector,and thansformed into DH5α competent cell.Of the 60 positive clones for sequencing,42 microsatellite repeat were acquired (GenBank accession number: HQ283153-HQ283194). Except the CA / GT and GA /CT,the microsatellite sequences also had repeat motif of GAGT.The 42 microsatellite sequences could be categorized into 31 perfect type (73.8%), 9 imperfect type (21.4%), and 2 compound type (4.8%). The percentage of perfect type was much higher than the imperfect type in swimming crab,which was same as other eukaryote.39 microsatellite sequences (92.9%) had at least 10 times of repeats numbers.27 microsatellite containing sequences had 10-19 times of repeats numbers,and 12 microsatellite had more than 20 times of repeats numbers.The present study might provide a set of useful molecular markers for future studies on genetic diversity evaluation,population genetic structure identification and resources conservation of the swimming crab.