Rapid isolation of microsatellites from genome of silver crucian carp by magnetic beads

Nine microsatellite markers of silver crucian carp Carassius auratus gibelio were constructed by combination of biotin capture method with radioactive labeling hybridization.Genomic DNA was extracted by normal methods and digested with restriction enzyme MboI.The fragments of 400-900 bp were isolated by sucrose-density-gradient centrifugation and ligated to Brown adaptors and hybridized with biotin-labeled simple sequence repeats(SSR) probes(CA)16.The target fragments were ligated into plasmid cloning pMD18-T vector and transformed into competent Escherichia coli after collection by magnetic beads and PCR amplification,DH5α using the CaCl2 method.A microsatellite genomic library containing 1800 colonies was constructed in which 235 positive colonies were dected by radioactive γ-32P-ATP labeling hybridization.Sequencing of 235 positive colonies confirmed that there were 224 colonies containing microsatellite loci in which 36 were compound repeat motifs(18.5%),117 were perfect(52.8%) and 71 were imperfect(28.7%) respectively.163 pairs of PCR primers were designed with the software primer 5.0 and 30 pairs were synthesized and screened.Nine loci can give bands,6 had a degree of polymorphisms,and 3 were monomorphic.