Isolation,characterization and polymorphism of microsatell markers in triangle mussel Hyriopsis cumingii

Microsatellite enriched library of Hyriopsis cumingii was constructed according to the strong affinity between biotin and streptavidin.Total DNA was extracted using Wizard Genomic DNA Purification Kit(Promega),digested with restriction enzym Mse I and then ligated to the adapters.The treated DNA sample was then pooled and fragments were separated on a 1.2 % agarose gel prior to size selection.The product fragments(200-1 000 bp) were excised,purified and amplified with Mse I-N primers.The amplified DNA was hybridized with 5'-biotinylated(CA)13-3' repeat oligos.The hybrid mixture was incubated with magnetic beads coated with streptavidin.The selected DNAs were then amplified using primers designed complementary to the linkers.Cleaned products were ligated into a pMD18-T vector(TaKaRa) and transformed into DH5a competent cells.Recombinant clones were detected by PCR amplification with Mse I-N primers.A total of 100 clones with foreign inserts were sequenced.A total of 54 clones were screened for usable microsatellites.Of this clones,33 were deemed unique,of sufficient length(more than 8 repeats) and possessed adequate flanking regions for primer designment.Among 33 primer pairs designed,25 yielded scorable amplification products.Thirteen polymorphic microsatellite markers were separated on 10% denaturing polyacrylamide gel and visualized by silver staining,pBR322 marker was used as standard for scoring.These loci showed high levels of genetic polymorphism testing on 30 individuals sampled from Dongting Lake of Hunan Province,China.The observed number of alleles per locus ranged from 4 to 12.The ranges of observed and expected heterozygosity were 0.167 8 to 1.000 0 and 0.362 9 to 0.849 2,respectively and the average of polymorphic information content was 0.518 2.Two microsatellite loci were significantly deviated from Hardy-Weinberg equilibrium due to the presence of null alleles and no linkage disequilibrium was found.Construction microsatellite enriched library of triangle mussel according to the strong affinity between biotin and the streptavidin was successed.These microsatellite loci will be useful for assessment of genetic diversity and population structure in H.cumingii.