【作者】 朱泽远；

【导师】 乐国伟；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2008， 博士

【摘要】 微卫星DNA是理想分子标记之一,广泛地应用于食品跟踪、群体遗传多样性等领域。中华绒螯蟹是绒螯蟹属中最具经济价值品种,为我国重要水产养殖和遗传保护对象。有关中华绒螯蟹遗传标记研究较薄弱,处于起步阶段,对于个体鉴定和跟踪还有较远距离。本研究首次采用磁珠富集法构建中华绒螯蟹微卫星文库,分离微卫星;在此基础上,采用荧光标记和毛细管电泳技术,分析微卫星特征并构建复合PCR高效扩增系统;应用所得标记,分析中华绒螯蟹的群体遗传多样性和个体识别;本研究还采用实时PCR技术首次报道中华绒螯蟹的基因组大小。1.中华绒螯蟹微卫星DNA文库的构建中华绒螯蟹微卫星的单富集文库主要构建过程有高分子量基因组DNA经Rsal消化、连接接头、PCR扩增、变性、Dynabeads^?磁珠杂交和捕获、转化、克隆PCR、测序及序列分析等过程。为提高效率,对磁珠一次杂交后捕获的回收片段再次富集,构建双富集文库。测序结果表明,磁珠富集法比传统文库筛选法的微卫星分离效率高一个数量级,双富集文库中78.1%的克隆含有侧翼序列的微卫星。2.中华绒螯蟹微卫星的特征分析基于20个微卫星位点设计PCR引物,其中有14个位点在退火温度55℃、Mg²⁺的浓度为1.5mM能特异性扩增。采用FAM标记10个位点引物,HEX标记4个位点引物,PCR扩增经Acroprep^TM过滤板吸附法快速提取的32个中华绒螯蟹样品基因组DNA。以分子长度标准ROX500为内标,PCR扩增产物经ABI3730XL毛细管电泳检测,Genemapper 3.5分析,结果表明毛细管电泳能精确检测微卫星的多态性。14个位点中ES10和ES26存在基因重复,ES38为单态位点,其它11个多态位点可以用于中华绒螯蟹的群体多样性的研究。3.荧光复合PCR的构建与优化将9个微卫星位点分成两组,5个位点用FAM（蓝色）标记,4个位点用HEX（绿色）标记;两种荧光类型分组优化,用琼脂糖胶电泳检测。随后,荧光标记的复合PCR扩增8个中华绒螯蟹样品的9个微卫星位点,采用毛细管电泳检测,调整各微卫星位点引物比例使所有位点均匀扩增。最后,逐一检测复合PCR基本参数对复合PCR产物的影响,优化PCR条件。结果表明荧光复合PCR的最优参数为dNTP 200μM、36个循环、Ta 55℃45s、退火时间为60s,摸板DNA20-500ng均可以稳定扩增。4.微卫星评估中华绒螯蟹的群体遗传多样性对无锡、苏州和上海的三个中华绒螯蟹群体共计102个样品,采用过滤板Acroprep^TM吸附基因组DNA,使用已经特征分析的11个微卫星标记对基因组DNA进行PCR扩增,PCR产物经毛细管电泳检测。位点ES37可能存在无效等位基因,其余10个位点分析统计结果为:平均每个位点有高达22.9个等位基因,PIC和E值分别高达0.826和7.700,这一结果清楚表明中华绒螯蟹群体具有较高的遗传多样性。欧氏遗传距离和遗传相似性平均值分别为0.439和0.825;瓶颈分析表明有必要对中华绒螯蟹采取科学的品种保护计划。5.中华绒螯蟹的个体来源识别初步分析应用GeneClass 2.0软件,比较频率法、贝叶斯法、DAS、标准内氏、最小内氏、内氏DA、Cavalli-Sforza和Goldstein等8种个体来源识别不同算法,结果表明贝叶斯法识别率最高,达93.14%;单一微卫星位点的个体识别分析表明,等位基因数最多的ES35标记个体识别能力最高,达87.25%;选择多态性高的微卫星位点,有助于减少微卫星位点的使用数量,且不降低个体正确识别率。6.实时PCR测定中华绒螯蟹基因组大小以已经测序的水稻样品（Nipponbare）为对照,建立一种实时PCR方法定量检测中华绒螯蟹基因组大小。整个过程约120 min,PCR效率为97.8%。标准曲线为Y=-3.768X+44.568,标准曲线的相关系数（R²）为0.992,结果表明中华绒螯蟹的基因组大小（c值）为1.72±0.25pg。研究不仅首次报道了中华绒螯蟹的基因组大小,还表明基于SYBR GreenI的定量PCR方法可为基因组大小的定量检测提供了一种特异、快速和简洁的方法。更多还原

【Abstract】 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.更多还原