【作者】 刘保忠；

【导师】 相建海；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2003， 博士

【摘要】 群体遗传学和分子系统学是遗传学研究的重要内容。本文对扇贝群体遗传学研究中的主要分子方法同工酶和RAPD标记进行了研究，利用RAPD标记分析了海湾扇贝野生群体、养殖群体和地理亚种群体的遗传结构和遗传分化，探讨了人工养殖对群体遗传多样性的影响；利用数学统计方法研究了海湾扇贝壳性状与生产性能的相关关系，得出了海湾扇贝壳性状对活体重和软体重的回归方程，分析了3个不同养殖群体的生长和产量差异；实验克隆和分析了海湾扇贝、栉孔扇贝等8种扇贝的16S rDNA基因部分序列，结合已有的5个扇贝物种的序列，利用分子数据分析了扇贝科物种的分类地位和系统演化关系。 实验以海湾扇贝为材料，研究了样品不同保存条件下DNA的提取方法，结果显示，鲜活样品、冷冻样品以及70％乙醇固定样品，都可以方便地获得高质量的DNA，但甲醛固定的样品在本实验中没能获得满意的结果。以获得的样品DNA为模板进行RAPD扩增显示，上述3种方法获得的DNA扩增后的得到稳定的产物，并且样品间电泳检测的结果表现出很好的一致性，而甲醛固定样品的提取物在进行扩增后电泳检测无阳性结果。 RAPD和同工酶是群体遗传分析中常用标记，实验以野生栉孔扇贝群体为材料，筛选了10个可用于群体分析的RAPD标记，分析显示，多态位点比例为40.74％，群体平均杂合度为0.194；通过对同工酶的筛选，共得到13种酶的22个位点，其中7个为多态位点，多态位点比例和群体平均杂合度分别为31.82％和0.113。实验表明，同一扇贝群体采用RAPD标记分析得到的多态位点比例和平均杂合度均高于同工酶标记分析结果，但两种手段得到的结果所揭示的趋势表现出较好的一致性。 利用RAPD技术分析了海湾扇贝野生种群、两个不同养殖世代的养殖群体和一个海湾扇贝地理亚种墨西哥湾扇贝养殖群体的遗传结构和群体间的遗传距 刘保志海琦扇贝娜体遗传学和扁贝料分于系挽演化的研究博士学位论丈离。研究结果显示，各群体多态位点比例介于31.54%至35.28%之间，平均杂合度为0.1248至0.163，野生海湾扇贝群体的群体多态性水平最高。大连养殖群体的多态位点比例和平均杂合度均高于莱州养殖群体，但对两群体的壳高和体重分析表明两群体无显著差异。通过对群体遗传距离的比较，墨西哥湾扇贝和野生海湾扇贝群体间的遗传距离大于海湾扇贝野生群体和养殖群体间的遗传距离。实验结果发现，随着人工养殖世代的增加，群体遗传结构发生了改变。群体遗传多样性的降低会导致扇贝抗逆性的下降，而人工养殖过程中较高的养殖密度和恶化的水质条件，也是造成养殖扇贝较高死亡率的重要原因。 为了分析海湾扇贝壳性状同体重等经济性状的关系，实验测量了3个养殖群体共139个个体样品的壳长、壳高、壳宽、前耳宽、后耳宽等壳性状和活体重、壳重和软体重等经济性状，通过计算分析表明，海湾扇贝各性状的相关系数均达到极显著水平(尸<0.01)。利用壳性状作为自变量，活体重和软体重作为依变量进行多元回归分析，回归关系达到极显著水平(尸<0.01)，活体重对壳长、壳高的偏回归系数极显著(尸<0.01)，对壳宽的偏回归系数达到显著 (尸<0.05)，软体熏对壳长、高、宽的偏回归系数均极显著(尸<0.01)，前耳和后耳宽度对活体重和软体重的回归系数均不显著。多元回归分析建立的壳长(Xl)、壳高(溉)和壳宽(X3)估计活体亚(Y)和软体重(附)的回归方程为: 卜一4 1 .427+7.SOSXI+3.877X2+0.93lX3)不乞一25.771+4.574丫!十2.421X2+0.7llX3。 通过采用多参数方差分析和新复极差法对海湾扇贝3个养殖群体的壳长、壳高和活体重、软体重等经济性状进行了比较分析，结果显示，陵水桥群体各性状指标均显著高于其它两群体，棋盘磨群体和大李家群体则没有表现出显著差异，说明陵水桥群体的生产性能和预期产量最高。 165 rDNA基因序列是系统演化研究中有效的分子手段。实验对栉孔扇贝、华贵栉孔扇贝、褶纹肋扇贝和长肋日月贝等4种我国沿海分布的扇贝和海湾扇贝、墨西哥湾扇贝、虾夷扇贝和欧洲大扇贝等4种外来物种的线粒体DNA的165 rDNA基因部分序列进行了扩增，扩增产物转入T载体后进行了序列测定。各物种165 rDNA基因部分序列长度在548一642bp之间，物种间表现出较高的长度差异，通过序列比对，表现出较高的同源性。 利用得到的8种扇贝的165 rDNA基因部分序列和文献发表的5种扇贝的刘保忠海沟扁贝群体遗传学和扇贝科分子从统演化的研究博士学位论文165 rDNA基因部分序列进行了比较，采用邻接法，最大简约法和最大似然法构建了扇贝科的分子系统树。系统分析显示，又用NJ和ML方法构建的系统树表现出完全相同的拓扑结构，MP法的系统树除了一个物种的分枝位置出现差异，其它物种的排列循序跟上述两种方法得到的系统树相同。实验结果显示，海湾扇贝和墨西哥湾扇贝以及AqueiPecten opercularis和Chla州lys glabra所在属所属的亚科，很可能同栉孔扇贝亚科和扇贝亚科居于平等的分类阶元上。实验结果显示，165 rDNA序列所提供的分子数据，在扇贝科的系统演化分析中?更多还原

【Abstract】 Population genetics and molecular phylogeny are the important contents for research of mollusc genetics. Allozyme and RAPD markers were developed and applied in scallop genetic structure analysis in this dissertation. The population genetic variation and growth difference of bay scallop, Argopecten irradians, were studied by RAPDs and quantitative traits. 16S rDNA genes partial fragments of 8 species of scallop were sequenced and the molecular phylogeny of Pectinidae was analyzed based on the 16S rDNA genes.In order to search for the suitable method for the tissue preservation and DNA isolation of marine animals, the adductor muscle of bay scallop (Argopecten irradians) were treated with different ways, such as frozen at -20℃, fixed with 70% ethanol or 10% formaldehyde for more than 10 days. DNA templates about 20 kb were extracted successfully from the fresh, frozen and 70% ethanol fixed tissues. RAPD analysis indicated that PCR products had no obvious distinction among them in the same individual. No usable DNA was gotten from the fixed tissue in formaldehyde. The result suggests that the 70% ethanol is appropriate for tissue preservation andDNA extraction for RAPD analysis.To compare genetic markers for the use of population genetics analysis, allozyme electrophoresis and RAPD (Random Amplified Polymorphic DNA) were used to detect the genetic structure of wild Zhikong scallop Chlamys farreri population. A total of 40 individuals from the same species were investigated using 10 arbitrarily selected primers (10 base). 40.74% of 54 RAPD fragments were polymorphic and revealed an average of heterozygosity with 0.194. 13 enzymes (IDH, GPI, PGM, PEP-B, PEP-D, ACP, AK, PK, GOT, SOD, EST) were selected and 22 loci were used for the population genetic analysis, among them 7 loci (Gpi, Pgm, Pep-Bl, Pep-D, Got-2, Est-2, Est-3) were polymorphic which attributed 31.82% of polymorphism.The average of heterozygosity was 0.113 and most of the studied loci showed a heterozygote deficiency. RAPDs revealed more genetic variation than isozyme electrophoresis, but the two kinds of markers reflected the consistent trend about the genetic structure of the population.Bay scallop, Argopecten irradians, was introduced to China from America in 1982, it has become one of mair) cultured species in the north of China. After culturing for more than decadal generations, its genetic structure might has changed because of the inbreeding. The RAPD markers were used to investigate the population genetic structure and their variation in four populations of A. irradians. Three populations of A. irradians irradians, one wild population was from Massachusetts of America, two cultured populations from Laizhou reared 12th generation and Dalian reared 6th generation respectively, the other cultured population, subspecies A. irradians concentricus, from Qingdao. The value of population polymorphism fluctuated from 31.54% to 35.28%, and the average of heterozygosity varied from 0.1248 to 0.1631, the wild population had the highest level of polymorphisms as well as the average heterozygosity. Dalian population revealed the higher level of polymorphisms and heterozygosity than that in Laizhou population, but there was no significant difference in body weight and shell height between them by statistical method, this result indicated that there had no distinct association between heterozygosity and growth of bay scallop. There was greater genetic distance between the population of wild A. i. irradians and A, i. concentricus (D=0.0093) than that between the wild and two cultured populations of A. i. irradians (D=0.0028, 0.0034). Our data provided some evidences that some genetic variation occurred between the different generations. The lost of genetic diversity usually caused decrease the repellency of scallop. However, the authors think the high cultured density and the deteriorating water environment are still the main reasons that caused high mortality in cultured scallop.A total of 139 individuals from three cultured bay scallop, A. irr更多还原