Isolation and Characterization of Microsatellite from Genome of Aspiorhynchus laticeps

Magnetic beads enriched method was used to isolate microsatellite DNA from Aspiorhynchus laticeps genome.Aspiorhynchus laticeps genomic DNA was extracted and then digested with restriction enzyme Sau3AI.Targeted segments of 250-750 bp 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(CA)8,(TGA)8.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 DH5α,and finally a microsatellite library was obtained.563 positive colonies were obtain through twice screens by colony PCR.Sequencing of these positive colonies confirmed that 53 contained microsatellite loci(number of repeats ≥5).In these sequences,36 repeat motifs(about 67.92%) were perfect,12 repeat motifs(about 22.64%) were imperfect,and 5 repeat motifs(about 9.43%) were compound.We design 24 pairs of primers from 50 microsatellite sequence and compose them.As a result,56 pairs were screened and used successfully to amplify special fragment,among which 24 pairs were polymorphism.Using 24 pairs of these microsatellite markers,we analyzed genetic structures of one Aspiorhynchus laticeps population.The result showed that the alleles were ranged from 3-6,while the polymorphic information content(PIC) were 0.625 3.Twelve of the loci were in high polymorphic level.