Development And Application Of Microsatellite Markers In Spotted Halibut (Verasper Variegatus)

Spotted halibut (Verasper variegatus), as a cold-temperate macrobenthos, is high-level edible fish with delicious taste. In this study, new microsatellite markers for spotted halibut were developed and the genetic diversity of a wild population was evaluated. In addition, the univeral applicability of microsatellite primers developed from V. variegates in other flatfish species were discussed.The construction of satellite-enriched genomic library was proved to be one of the efficient approaches for isolating microsatellites in this study. The genomic DNA of was digested with AluI and size-selected DNA fragments (400–1400 bp) were ligated to an adaptor. Microsatellite-containing sequences were captured by using a combination of (AC)n and (AG)n probes that were attached to a nylon membrane. The microsatellite enrichment library constructed in this study consisted of approximately 2000 clones, of that about 600 were positive clones. Of all the clones sequenced, 429 clones were found to contain microsatellites from the genomic library. Of them, 294 pairs of primers were designed from the sequenced fragments and 93 primer pairs provided clear and polymorphic amplification products. To estimate genetic variation in spotted halibut, we analyzed 21 microsatellite loci in 30 individuals of one wild population. The number of alleles per locus ranged from 4 to 14. The values of observed heterozygosities (HO) varied from 0.1371 to 1 while the expected heterozygosities (HE) varied from 0.0487 to 0.9117, respectively. Five loci showed significant departure from the Hardy-Weinberg equilibrium,(HWE, p<0.005) , but nonsignificant linkage disequilibrium between pairs of loci was observed (p<0.05) . The above research results show that these DNA markers are therefore potentially useful for studies of the population structure of the species.Moreover, the universal applicability of 21 microsatellite markers of spotted halibut were tested in two affinis species, olive flounder (Paralichthys olivaceus), and stone flounder (Kareius bicoloratus L.). 9 loci were found to be applicable in K. bicoloratus L., and 9 loci were applicable in P. olivaceus. The universal rates were 61.9% and 47.6% respectively. Likewise, 231 pairs of primers optimized successfully from spotted halibut were examined in half smooth lounge sole Cynoglossus semilaevis Günther, of which 56 pairs of primers were proved to be applicable. The universal rates were 13.4%. These results indicated that the universal rate of spotted halibut microsatellite markers in Pleuronectiodei (P. olivaceus and K. bicoloratus L.) was higher than in Soleoidei (C. semilaevis). It revealed that the conservation of microsatellite markers in these species made it possible for the phylogenetic analysis of the affinis species. The development of microsatellite markers provided powerful molecular tool for population genetic studies as well as genetic breeding in spotted halibut (V. variegatus).