Studies On The Characterization Of Microsatellite DNA And Genetic Diversity From Chinese Mitten Crab, Eriocheir Sinensis

Microsatellite DNA,one of the most popular molecular markers,has been used in many fields such as food traceability,genetic diversity.Chinese mitten crab(Eriocheir sinensis),the highest commercial value among the genus Eriocheir,has become the important species for captive culture and conservation genetics.It is difficult to assign individuals and trace the original source population,because the markers from E.sinensis are rare.In the present study, the first microsatellite library was constructed by using Dynabeads^?.With fluorescent labeled oligo and capillary electrophoresis,we characterized the loci and constructed one high throughput multiplex PCR system.With the characterized markers,genetic diversity and assignment of individuals have been analysed.This study was also the first report of the genome size in E.sinensis assessed through real time PCR.1.Construction of the mierosatelite enriched library for E.sinensisThe main procedure of construction of primary enriched microsatellite library from E. sinensis were as follow:digestion of the genomic DNA,ligation of adaptors,PCR amplification,fragment denaturing,hybridization and capturing with Dynabeads(?), transformation to competent cell,colony PCR,sequencing and the nucleotides analysis. To improve the efficiency,the secondary enriched microsatellite library was established by using the fragments which had been enriched twice.The result showed that the efficiency from Dynabeads(?) enriched library is much higher than that from classic library screening. The percentage of positive clones with flanking regions in the secondary enriched library was 78.1%.2.Characterization of microsatellite loci from E.sinensisPrimers were designed on 20 microsatellite loci,among which 14 can be amplified to get specific bands at Ta 55℃and Mg²⁺ 1.5mM.Using 10 FAM labeled oligos and 4 HEX labeled oligos,the 32 individuals' genomic DNA isolated through AcroPrep ^TM 96-well Filter Plate were PCR amplified.With ROX500 as a size standard,PCR product was analyzed using capillary electrophoresis and Genemapper 3.5.The results showed that capillary electrophoresis is precise for genotyping microsatellite.Among 14 microsatellite loci, besides gene duplication loci ES10 and ES26 and unpolimorphism locus ES38,11 loci could be useful in studying genetic diversity of E.sinensis.3.Construction and optimization of fluorescently labeled multiplex-PCRAccording to the expected length,nine microsatellite loci were divided into two groups, five labeled with FAM in one group and four labeled with HEX in another group.Different fluorescent groups were optimized separately using agar gel electrophoresis.Subsequently, eight Chinese mitten crabs were amplified by one set of nine fluorescently labeled primers.The multiplex-PCR products were detected with capillary electrophoresis.To get uniform signals, the ratio of primers was modified.Finally,the fundamental parameters were optimized one by one.This study showed the optimal parameters of this multiplex PCR were dNTP 200μM,36 cycles,Ta at 55℃for 45s and ripe for 60s.4.Microsatellite DNA based assessment of genetic diversity of E.sinensisThe genomic DNA of 102 E.sinensis samples from Wuxi,Suzhou and Shanghai was isolated with AcroPrep ^TM 96-well Filter Plate.The samples were genotyped by capillary electrophoresis with characterized loci.The results showed that the excepted ES37 might be null alleles,the average allele number for the other loci was 22.9 per locus and polymorphism information content and effective number of alleles are 0.826 and 7.700,respectively.Genetic distance(Nei,1978) and genetic identity were 0.439 and 0.825,respectively.The bottleneck analysis showed that scientific conservation program was necessary.5.Primary analysis of individual assignment for E.sinensisAfter comparison of algorithm,such as frequency method,Bayesian method,DAS,Nei standard,Nei minimum,Nei DA,Cavalli-Sforza and Goldstein,we found that Bayesian method with 93.14%efficiency is the most efficient in assigning individuals by using GeneClass2.0 software.For single locus,ES35 with maximum allele number showed the highest power of assigning individuals.Selection of the highly polymorphism microsatellite loci would reduce the loci used without decreasing the percentage of correct identification.6.Estimation of genome size from E.sinensis with real-time PCRUsing sequenced rice(Nipponbare) as a control,real-time PCR was established to check the genome size of E.sinensis..The whole procedure took about 120 min.The result showed the PCR efficiency was 97.8%,the standard curve was Y=-3.768X+44.568(R²=0.992) and the genome size of E.sinensis(c-value) was 1.72±0.25 pg.This study not only firstly reported the genome size of E.sinensis,but also indicated that SYBR Green I-based quatitive Real-time PCR was a specific,fast and simple method for estimation of genome size.