Development and characteristics of tri-and tetra-nucleotide microsatellites for silver carp(Hypophthalmichtys molitrix)

It was a study to isolate the tri-and tetra-nucleotide repeat microsatellite from sliver Carp(Hypophthalmichtys molitrix) with magnetic beads enriched method.Sliver carp(Hypophthalmichtys molitrix) genomic DNA was extracted from blood and digested with restriction Sau 3A I.The segments of 400-900 bp were collected by centrifugation of sucrose density gradient and then a whole genome PCR library was created.The genomic DNA was hybridized with biotin-labeled microsatellite probes(TGA)8,(CAG)8,(AGAT)8,and the single-stranded DNA containing the selected microsatellite DNA was obtained.Those selected DNAs were amplified with PCR,cloned into pMD 18-T vector,transformed into the competent Escherichia coli DH5α,and the microsatellite library was finally obtained.The second screen was performed with radiolabeled probes.1758 positive clones were obtained and 348 microsatellite clones contained 248 microsatellite sequences.There were 167(59.64%) perfect motifs,28(10%) imperfect motifs,85(30.36%) compound motifs.60 pairs of primers were designed with the software Primer 3.0 and detected with the wild population of sliver Carp(Hypophthalmichtys molitrix) from the Pearl River.The results showed that 22 pairs of primers could be used to amplify special fragments and a genetic tool to evaluate germplasm resources of sliver Carp(Hypophthalmichtys molitrix).