Development And Cross-species Amplification Of Microsatellite Markers From Three Groupers

Microsatellite markers have been broadly used to assess genetic population structure, construct genetic linkage map and so on because of their high level of polymorphism and co-dominant inheritance. Groupers of the genus Epinephelus, family Serranidae, are one of the economically important marine cultured fish around the world. However, genetic information on groupers is still limited to date and there are not enough polymorphism microsatellite markers in groupers. More microsatellite markers are needed urgently in order to assess the genetic diversity of the groupers and provide theoretical and technical foundation for molecular-assisted breeding. Enriched genomic libraries were constructed for seven-band grouper {Epinephelus septemfasciatus), yellow grouper (Epinephelus awoara) and black seabass (Centropristis striata) using the FIASCO method, and the microsaellite sequences and their characteristics were analyzed in this study. Briefly, the whole genomic DNA was digested with Mse I and Mse I adaptors were ligated to the fragments. Bio-labelled probes were used to hybridize with the products of the restriction-ligation reaction. After the nonrepetitive DNA fragments were removed by washing, single-stranded DNA containing microsatellite DNA was collected. Then the single-stranded DNA was amplified using Mse I primers to obtain double-stranded targeted fragments. The double-stranded fragments were ligated into pMD18-T vector and transformed into Escherichia coli DH5a competent cells. The positive clones were sequenced by ABI Prism 3730 automated DNA sequencer. From these libraries, we isolated 66, 56, 39 microsatellite sequences, respectively. Using the software Primer 3, we design 54 pairs of primers for seven-band grouper, 36 for yellow grouper, and 28 for black seabass. Of these microsatellite markers, 12 were polymorphic for seven-band grouper and yellow grouper, respectively, 11 for black seabass by 6% denaturing polyacrylamide gel electrophoresis. The parameters of the polymorphic microsatellite loci including number and size range of alleles, observed and expected heterozygosities were also caculated in the study. The results indicate that FIASCO is a good method for enhancing the efficiency of development of microsatellite markers. Cross-species amplification were further investigated in the three species. High success rate of cross-amplification implied that these microsatellites would be also potentially useful for other related species. Therefore, these microsatellites would provide various of candidates for comparative genome analysis, marker-assisted selection and analysis of quantitative trait loci location of groupers.