Screening and suitability analysis of microsatellite markers in Cheilinus undulatus

Genomic DNA of Cheilinus undulatus was digested by restriction endonuclease Bsp143Ⅰand electrophored on agarose gel.The DNA fragments from 400 to 1000 bp were recovered and ligated to Bsp143Ⅰadaptor.Purified and adaptor ligated fragments were hybridized to biotin-labelled(CA) 15 probe and captured by Streptavidin-coated magnetic beads.Target fragments were eluted,and PCR amplified,then the purified PCR products were inserted to PMD18-T vector and transformed into Top 10 component cell.Positive clones in the enriched genomic DNA bank were screened out through PCR method and sequenced.Of 120 positive clones,88 sequences contained repetition that repeated no less than 5 times,none redundant sequences were found after the multiple sequence alignment analysis,and the 88 non-redundant microsatellite-contained sequences,of which about 73.33% contained positive sequence,and the largest repeat number of perfect type microsatellite was 26.Among the 88 non-redundants,28(or 31.82%) had enough flanking region(150 bp),which is enough to design microsatellite primer pairs.28 primer pairs were synthesized and tested with the compound DNA of 3 C.undulatus individuals.24 loci were produced with clear bands.A population of 39 individuals were tested with the 24 loci.20 loci revealed polymorphic,while 4 loci was monomorphic.The primer pairs amplified the loci with relatively high numbers of alleles ranging from 2 to 12 with an average of 3.5 per locus among 20 ploymorphic loci.The polymorphism information content(PIC),observed heterozygosity(H o) and expected heterozygosity(H e) were 0.0782,0.8513,and 0.5667,respectively.The 20 ploymorphic loci could be useful for the analysis of population structure from C.undulatus.