Isolation and characterization of microsatellite markers for Zhikong scallop by screening SSR-enriched library

In this study,40 microsatellite markers were isolated and characterized for Zhikong scallop(Chlamys farreri) using the method of screening SSR-enriched library.DNA fragments containing microsatellites were captured by a piece of nylon membrane(Hybond N+) bound with the probe combinations of(AG)15 and(AC)15.The nylon membrane was washed three times in 2×SSC,1% SDS at 62℃,two times for 15 min with the final wash for 30 min.The captured DNA was eluted in 0.1×TE buffer and used to construct an SSR enrichment library.After transformation,the clones were regularly re-arrayed in a new agar plate with the density of about 300 clones per plate and screened with(AG)15 and(AC)15 probes labelled by the ECL system(Amersham).A total of 1200 clones derived from the enrichment library were screened and 532 clones gave the positive response.One hundred clones were randomly selected for sequencing and the results showed that all of the clones contain at least one microsatellite.Sixty-five primer pairs were designed using the software Primer Premier 5.0,of which 40 pairs can be amplified scorable PCR products.The polymorphisms of these scorable loci were assessed using 48 Chlamys farreri individuals,and the results showed that 37 loci were polymorphic.The number of alleles per polymorphic locus ranged from 2 to 14 with an average of 7.0 alleles per locus,and the values of Ho,He and PIC varied from 0.1000 to 1.0000,0.1197 to 0.9831 and 0.1172 to 0.9782,respectively.The results indicated that the method of screening SSR-enriched library is efficient and suitable to isolate a large amount of microsatellite markers for the target species of interest.