Isolation and Characterization of Microsatellite Markers for Sea Cucumber (Apostichopus japonicus) by Screening the SSR-Enriched Library

We isolated and characterized 33 novel microsatellite markers of sea cucumber(Apostichopus japonicus) using the method of enrichment-colony hybridization.A total of 900 clones derived from the SSR-enriched library were screened and 415 clones gave the positive response(46.1%).One hundred positive clones were randomly selected for sequencing and the results showed that all of the clones contained microsatellites.Fifty-seven primer pairs were designed using the software Primer Premier 5.0,of which 33 pairs can be amplified scorable PCR products.The polymorphisms of these loci were assessed using 48 individuals sampled from the natural habitat.The results showed that all 33 loci showed polymorphism.A total of 222 alleles were identified with an average of 6.7 alleles over all loci.The values of observed and expected heterozygosities ranged from 0.050 0 to 1.000 0 and from 0.203 2 to 0.915 9,respectively.The results indicated that the method of SSR-enriched library hybridization was of high efficiency,sensitiveness,stability and low cost for isolation of a large amount of microsatellite markers in the target species.These microsatellite markers can be used in a wide range of genetic studies and breeding programs.