Isolation of microsatellite DNA in Epinephelus akaara

A partial genomic library of Epinephelus akaara was constructed and a PCR-based strategy was developed to screen the microsatellites in the library.A total of 2112 recombinant colonies were picked and regrown overnight in 96-well cell culture plates with 100 μL of LB medium plus ampicillin each well at 37℃.Twenty two plates were obtained and there were 20 mixing-pools in each plate.A PCR-based screening library method was used to screen these mixing-pools via the universal primer of M13 and repeat motif primers(CA)7C and(GA)7G,respectively.There were 111 microsatellites isolated after 61 positive clones were sequenced.The results indicated that microsatellite sequences characterized by(AC/GT)n and(GA/CT)n were abundant in genomic DNA of Epinephelus akaara.A portion of microsatellites repeat motifs and their flanking sequences were described.Among the 111 microsatellites,there were 54 perfect ones(48.7%),46 imperfect ones(41.4%),and 11 compound ones(9.9%).The 22 microsatellites sequences were submitted to the GenBank.It is supposed that these loci could be used for further genetic diversity and population structure analysis of Epinephelus akaara.