Isolation Of Microsatellite Markers And Population Genetic Structure Analysis Between Sexes And Ages In Hyriopsis

Hyriopsis cumingii is a principal freshwater pearl mussel and be rich in the fivelargest freshwater lakes in China. However, due to overexploitation and environmentpollution, resource of H.cumingii declined significantly, natural germplasm resourceprotection is the foundation of breeding of H.cumingii.20polymorphic microsatelliteswere isolated using repeat-enrichment method with streptavidin magnetic beads, thegenetic structure between different sexes and ages of H.cumingii from PoyanghuLake(PY) and Taihu Lake(TH) were evaluated with10polymorphic microsatellites,analysed the variety regulation of these nature populations of H.cumingii, PY stock andTH stock were collected from Duchang in Jiangxi Province and Taihu village inZhejiang Province, respectively. And the effect between DNA extracted from freshtissues and tissues preserved in ethanol for different time and the effect of in vivosampling on growth of juveniles were compared. The aim is to provide theoretical basisfor germplasm resource protection, stock accessment and utilization rational ofH.cumingii. The main contents are as follows:1. Development and Genetic Diversity Analysis of Microsatellite in Hyriopsis cumingiiMicrosatellite-enhanced genomic Library of the H.cumingii was constructed usingrepeat-enrichment method with biotin-labeled oligos (CA)10and streptavidin magneticbeads. To amplify24individuals sampled from Dongting Lake,32pair primers couldamplify expected PCR products were selected and labeled by fluorophore.20microsatellite loci were found to be polymorphic.Among20polymorphic loci, thenumber of alleles per locus varied from5to20, the number of effective alleles per locusranged from2.3213to13.2603. The observed and expected heterozygosity varied from 0.3182to1.0000and from0.5825to0.9461, respectively; polymorphism informationcontent (PIC) ranged from0.5472to0.9199.14of20markers conformed toHardy–Weinberg equilibrium(P＞0.05). These microsatellite loci would be useful forstudies of population genetics, stock management and other relevant research inH.cumingii.2. Genetic Diversity Analyse between Ages of Hyriopsis cumingii297samples of PY stock with the age ranged from3to8and217samples of THstock with the age ranged from7to15were collected,10polymorphic microsatelliteprimers were used to analyse genetic diversity of different age stocks above-mentioned,respectively. Results indicated no change between the average expected heterozygosityand average Nei’s genetic diversity of different age stocks, indicated the difference ofgenetic diversity of different age stocks in Poyanghu Lake and Taihu Lake is notobvious. The comparison results of the average number of alleles, averagepolymorphism information content showed7years stock and8years stock are lowerthan other stocks in Poyanghu Lake,9years stock is lower than10years stock,13years stock is lower than12years stock and15years stock in Taihu Lake, but theaverage expected heterozygosity is relatively high. The result of individual geneheterozygosity showed the proportion of individual gene heterozygosity at high levelof7years stock and8years stock in Poyanghu Lake and9years stock and10yearsstock in Taihu Lake are relatively high. Different age groups were divided accordingto environmental change great, the genetic parameter of different age groups showedthe genetic diversity of PY1is lower than PY2, TH1showed the highest diversity,followed by TH3, TH2showed the lowest genetic diversity. The difference of10microsatellite loci showed alleles missing after PY6Y except loci HcuCA0007andloci HcuCA0104in Poyanghu Lake, new alleles were detected begin with TH9Yexcept loci HcuCA0100in Taihu Lake.3. Genetic Diversity Analyse between Sexes of Hyriopsis cumingii 131female samples and166male samples of PY stock,118female samples and99male samples of TH stock were collected,10polymorphic microsatellite primers wereused to analyse genetic diversity of male and female wild stocks of H.cumingiiabove-mentioned, respectively. Results indicated the average number of alleles for16.6and17.8, average expected heterozygosity for0.811and0.813, average Nei’s geneticdiversity for0.8079and0.8106of male and female stock in Poyanghu Lake,respectively. The average number of alleles for14.2and17.6, average expectedheterozygosity for0.7633and0.7747, average Nei’s genetic diversity for0.7601and0.7708of male and female stock in Taihu Lake, respectively. The average expectedheterozygosity and average Nei’s genetic diversity of male stock are higher than femalestock, result showed the genetic diversity of male is higher than female stock.4. Quality Comparison of DNA Extracted from Fresh Tissues and Tissues Preserved inEthanol for Different Time in Hyriopsis cumingiiThe genomic DNA from different tissues of H. cumingii, which were four freshtissues (gill, foot, mantle, adductor muscle), four tissues fixed in ethanol for7days andmantle tissues preserved in ethanol for1year,3years,4years and5years, wasextracted by phenol/chloroform method respectively. The DNA quantity and the effectof PCR ampilification among them were compared. The result showed that foot, mantle,adductor muscle were appropriate for DNA extraction, but not gill. Genomic DNA waswell extracted from tissues treated with ethanol compared to fresh tissues, but noobvious difference in SSR-PCR product among them were observed. It showed that thequality of DNA extracted from tissues treated with ethanol could be improved.Compared with mantle samples fixed in ethanol for7days, DNA extracted from mantlesamples preserved for more than one year all presented degradation in more or lessdegrees. Mantle samples preserved for more than one year were not appropriate for highquality DNA extraction. If DNA amount was not increased in PCR reaction, DNAextracted from mantle samples preserved in ethanol for4years and5years affected PCR ampilification largely in normal reaction system.5. Growth Comparison of In Vivo Sampling of juveniles in Hyriopsis cumingiiCompared and analysed the effect of after minor mantle tissues peeled off wereused for DNA extraction on growth of juveniles, the result showed genomic DNA waswell extracted, juveniles could come to growth steady state, the trunk length and bodyweight were growth, indicated juveniles with the trunk length ranged from1.8to2.1cmand the body weight ranged from284to391mg were appropriate for in vivo sampling,and minor tissues peeled off have little effect on growth of juveniles, genomic DNAextracted could satisfy Molecular Biology study.