Construction on enriched microsatellite library and characterization of microsatellite markers from swimming crab

Thirty microsatellite markers of swimming crab Portunus trituberculatus were isolated and characterized by screening SSR-enriched library.Genomic DNA were digested by HaeⅢ,then DNA fragments containing microsatellites were captured by a piece of nylon membrane(Hybond N+) bounded with the probe combinations of(AC)15 and(AG)15.After transformation to Escherichia coli strain DH5α,the clones were regularly re-arrayed in a new agar plate with the density of about 300 clones per plate and were screened with(AC)15 and(AG)15 probes labeled by DIG luminescent Detection Kit system(Roche).One hundred and fifty clones were randomly selected for sequencing.One hundred and five primer pairs were designed using the software Primer Premier 5.0,of which 56 pairs could amplify distinct PCR products.Polymorphism of these distinct loci was assessed using 30 swimming crab individuals,and the results showed that the 30 loci were all polymorphic.The values of Ho,He and PIC varied from 0.222 2 to 1.000 0,from 0.436 7 to 0.909 9 and from 0.350 to 0.892,respectively.These microsatellite markers would be useful for the studies of genome mapping,QTL,parentage determination and population genetics for this species.