Construction Of Microsatellite-Enriched Library And Genetic Diversity In Four Populations Of Scapharca Broughtonii

Genomic microsatellite library is constructed on Scapharca broughtonii using themethod of magnetic-bead hybridization enrichment to screen the microsatellitemolecular markers in this paper.48microsatellite markers are isolated and all of theseare polymorphic loci after the PCR optimized reaction.20microsatellite loci thatseparate out from the Scapharca broughtonii are selected for analyzing the geneticdiversity, genetic differentiation and genetic distance of the4populations（RiZhao,PengLai,HuangDao, Korea）. The main results were showed as follows:1. The method of magnetic-bead hybridization enrichment was used to screen themicrosatellite molecular markers of Scapharca broughtonii. Genomic DNA of S.broughtonii was digested with restriction enzyme Msp Ⅰ. The fragments of400bp-1200bp were recycled and purified. Then DNA fragments which containingmicrosatellite sequences were screened with biotinylated oligonucleotide probecombinations of （AC）₁₅and （AG）₁₅.The fragments were connected to pMD18-T vectorand transformed into E.coli TOP10strain,and then genomic DNA contained themicrosatellite-enriched1ibrary was constructed．295positive clones randomly pickedfrom890colonies were isolated for sequencing by PCR technique. Among the208microsatellite sequences,147（70.7%） were identified to be perfect microsatellites,35（16.8%） imperfect and26（12.5%） compound. Most of the repeat motif based onAC,AG and ranged from5to31.One hundred and forty primer pairs were designedusing a software, of which48polymorphic markers were screened out.Genetic diversity and polymorphism of these distinct loci were assessed using30S.broughtonii individuals. The numbers of alleles of48microsatellite loci was375,ranged from2to14;average alleles are7.8in every locus. The values of Ho, He andPIC ranged from0.1034to0.9655, from0.1831to0.9208and from0.1638to0.8946,respectively. The result showed that most of the selected microsatellite markers hadhigh polymorphism. 2. Genetic diversity of4populations（RZ,PL,HD, Korea） of S. broughtonii wasassessed by20microsatellite markers. Results showed that the numbers of alleles was196, alleles obtained from different primer ranged from5to17,average alleles are9.8inevery locus and the numbers of average effective alleles was7.3540.The polymorphism information content（PIC） ranged from0.6720to0.9224, theaverage PIC is0.8357（>0.5） which indicates that high level of genetic diversity wasshowed in these locations.The average observed heterozygosity value （Ho） and the expected heterozygosityvalue （He） ranged from0.7039to0.8265and0.8333to0.8524, respectively. Theobserved heterozygosities of the4populations obtained from20microsatellite loci weredifferent, the KH-42and KH-43loci showed the highest observedheterozygosity（0.8974） while the KH-26locus showed the lowest observedheterozygosity（0.5741）.The test of P value shows that only a little of loci deviatedsignificantly or very significantly from the Hardy-Weinberg equilibrium.The genetic differentiation index Fstof4populations ranged from0.0132to0.0314, revealing low level genetic differentiation in each group. Fstof the HD-HGpopulations（0.0314） showed largest differentiation, while the differentiation of RZ-HDpopulations was minimum （0.0132）. Genetic identity ranges from0.7821to0.8820andthe genetic distance ranges from0.1255to0.2458. UPGMA cluster analysis based ongenetic distance indicated that RiZhao and HuangDao populations were in one clusterfirst, and they were genetically closest among the4populations, and then PengLaipopulation clustered with it, while further clustered with the Korean population, whichshowed that Korean population was farthest relative with Chinese populations.These results and the information of population structure obtained in this workindicated that these microsatellite loci should be useful for the studies of moleculargenetic breeding, resources conservation，population genetics, genome mapping, QTLand other relevant research in S. broughtonii.In the mean time,this work also should behelpful to the achievement of marker-assisted selection in S. broughtonii.