Genetic Structure Of Wild Haliotis Ovina And Comparative Analysis Of MtDNA Cytb Gene Sequence Between H.ovina And H.asinina

Haliotis ovina, which is of great nutrition and medicinal value, has been tremendouslydestroyed due to overexploitation for its high commercial value and its worse habitatenvironment.9high polymorphism microsatellite loci have been developed in our study usingmagnetic bead hybridization method. And the genetic diversity and differentiation of H.ovinapopulation have been analyzed by the nine microsatellite loci. MtDNA Cytb gene fragments ofthree H.ovina species and one H.asinina species were amplified by PCR, and the PCR productswere sequenced and analyzed.The aim of our study is to provide scientific basis for thesustainable utilization and protection the resource of H.ovina as well as its selective breeding.The used of magnetic bead hybridization method to enrich the H. ovina microsatellite loci.H. ovina genomic DNA was extracted and digested with restriction enzyme MboI. Fragments of300-900bp were isolated and then was hybridized with a biotin-labeled microsatellite probe(GT)15,(CT)15. The hybrid mixture was incubated with magnetic beads coated with streptavidin.After washing to remove the non-SRS fragments, the eluted single-stranded DNA contains theselected microsatellite DNA. The selected DNAs are then cloned into the pMD19-T vector.Positive clones in the enriched genomic DNA bank were screened out through PCR method andsequenced. We selected150sequences length≥500bp to design40pairs of primers. The test ofpolymorphism in12pair primers indicated that number of alleles per locus ranged from2to5,polymorphism information content ranged from0.153to0.637, the observed and expectedheterozygosities ranged from0.0000to0.7857and from0.0734to0.6019, respectively. Thestudy also indicated that the polymorphism information content of9primer pairs were high andthese primer pairs can be used in the population analysis.The genetic diversity and differentiation of3wild H. ovina populations in China weredetected by using9microsatellite primers. The mean number of alleles per locus (A) ranged from2.7778to3.5556, with an average of3.2593. And the mean effective number of alleles per locus(Ae) ranged from1.8662to2.0512, with an average of1.9322. The mean number of thePolymorphism Information Content per locus (PIC) from0.4596to0.5434, with an average of0.4978. The observed heterozygosities (Ho) ranged from0.3650to0.4259, with an average of0.3964. the expected heterozygosities (He) ranged from0.4274to0.4779, with an average of0.4480. In general, the genetic differentiation among the three populations was small, the geneticdistance among the three populations ranged from0.0174to0.0256. The FSTvalues (FST) among three populations was0.0178. Based on the FSTvalues and the genetic distance, the3wildpopulations clustered into two major groups, the Yingzhou and Yalongwan populations belong toone group, and the Anyou population belong to another group.MtDNA Cytb gene fragments of three H.ovina populations and one H.asinina species wereamplified by PCR, and the PCR products were purified and sequenced. The results showed thatall the MtDNA Cytb gene fragments were758bp for nucleotide sequence with similar basecomposition and the content of A+T were more than that of G+C. The genetic distance amongthe three H.ovina populations ranged from0.007to0.019, the genetic distance among H.ovinaand H.asinina was0.113. H.ovina and H.asinina clustered into two major groups. H.ovinabelong to one group. H.asinina clustered into another group.