Rapid isolation and characteristics analysis of microsatellites from Lutjanus russellii

The (CA)n DNA sequences in Lutjanus russelli were isolated through PCR arrays, and thecharacteristics of the microsatellite were analyzed. DNA was extracted from a sample of Lutjanus russelli, and digested with Hae+Dra. The fragments were ligated to pUCm-T vector and transferred into DH5alpha to construct a genomic library. The positive clones were isolated with universal M13 reverse and forward primers, M13 forward primer and the simple sequence repeats (SSRs) probes (CA)15, M13 reverse primer and (CA)15. After twice isolation, 121 positive clones, whose PCR products with M13 forward primer and probes (CA)15 or M13 reverse primer and (CA)15 were smaller than those with universal M13 reverse and forward primers probably contained microsatellites, were obtained. Then, 53 (CA)n(n> or =7) microsatellites were obtained by sequencing. The repeat length mainly distributed in 7-15(80.77%). Besides (CA)n repeats, other repeat motifs, such as An, (CAC)n and (AACA)n, were also obtained. Scorable and constant amplification of DNA fragments were observed with 48 pairs of SSR primers designed from the flank sequences. This research makes a positive contribution to explorating genomes of Lutjanus russelli, offers genetic tools to examine the genetic variations and constructs genetic linkage map