Isolation and Characterization of Microsatellite from Genome of Leuciscus waleckii Dybowski

Magnetic beads enriched method was used to isolate microsatellite DNA from Leuciscus waleckii Dybowski genome.L.waleckii genomic DNA was extracted and then digested with restriction enzyme Sau 3A I.Targeted segments of 400-900 base pairs were collected by centrifugation of sucrose density gradient and ligated to adaptors.Then the purified ligated DNA was hybridized with Biotin-labeled simple sequence repeats probes(CAG)8,(TGA)8,(AGAT)6 and(GATT)6.After capture of target fragments by magnetic beads and PCR amplification,the selected DNA s were cloned into the pMD18-T vector and transformed into competent Escherichia coli DH 5α,and finally a microsatellite library was obtained.One thousand,four hundred and sixteen positive colonies were obtain through twice screens by colony PCR.Sequencing of these positive colonies confirmed that 1 047 contained microsatellite loci(number of repeats ≥5).Among these sequences,737 repeat motifs(about 67.99%)were perfect,166 repeat motifs(about 15.31%)were imperfect,and 181 repeat motifs(about 16.70%)were compound.We design 105 pairs of primers from 160 microsatellite sequence and compose them.As a result,56 pairs were screened and used successfully to amplify special fragment,among which 23 pairs were polymorphism.Using 18 pairs of these microsatellite markers,we analyzed genetic structures of one L.waleckii population sampled from Dali Lake.The result showed that the alleles were ranged from 3-11,while the polymorphic information content(PIC)were 0.207 7-0.882 0.The statistic results indicated that 67%loci(12/18)were in high polymorphic level.