【作者】 孙玉华；

【导师】 余其兴； 刘思阳；

【作者基本信息】 武汉大学 ， 遗传学， 2004， 博士

【摘要】 亚口鱼科(Catostomidae)，淡水鱼类，呈泛北极(holarctic)分布。全球范围内共14属，约80种。绝大多数种类分布于北美洲和中美洲，约有13属75种；仅有少数分布在亚洲，即分布于西伯利亚的Catostmus catostomus以及我国的Myxocyprinus asiaticus。中国胭脂鱼，Myxocyprinus asiaticus，是亚口鱼科在亚洲大陆分布的唯一单型属、种，主要生活于我国长江的干支流和附属湖泊以及闽江流域。由于人类活动的影响，中国胭脂鱼已被列为国家二级野生保护动物，濒危等级：易危。围绕这一珍稀物种，主要进行了三个方面的研究：一，应用限制性酶切片断长度多态(RFLP)、随机扩增长度多态性(RAPD)、微卫星标记(SSR)、DNA直接测序(DNA sequencing)等分子检测技术，分别在基因组DNA和线粒体DNA层次上分析了中国胭脂鱼长江中上游四个采样群体(宜宾，万州，宜昌，武汉)的遗传多样性，并且探讨了其形成机制。二，分别以核DNA和线粒体DNA的18S-ITS1-5.8S序列片段和细胞色素b(cytochrome b)基因片段作为分子标记，对亚口鱼科鱼类进行了分子系统学研究，并比较了中国胭脂鱼与亚口鱼科代表属种的分子变异模式。三，据化石证据、地球地质演化等最新进展，结合以上分子标记，进行了亚口鱼科鱼类的生物地理学研究。 在对中国胭脂鱼进行保护和人工繁殖工作中，调查其种群遗传结构是十分重要和基础性的工作。我们首次用分子生物学方法在基因组DNA和线粒体DNA层次上分析了中国胭脂鱼长江中上游四个采样群体的遗传结构及其遗传分化。在RAPD分析中，对约80个RAPD引物进行了筛选扩增，仅有3个引物的扩增带型具多态性。中国胭脂鱼宜昌江段群体内个体之间的遗传相似度平均为0.9274，武汉江段群体内个体之间的遗传相似度平均为0.931，群体之间遗传相似度为0.900。在线粒体ND-5／6基因(NADH dehydrogenase)的PCR-RFLP分析中，12个限制性内切酶酶切只检测出了2种单倍型(haplotype)，基因型间的核苷酸序列歧化距离为0.235％，核苷酸多样性为0.004。PCR-RFLP及RAPD分析共同揭示：长江中游宜昌和武汉江段群体的遗传多样性水平较低，且两江段群体之间出现了一定的遗传分化。对长江中上游四个群体70尾中国胭脂鱼线粒体DNA的控制区(D-Loop)序列进行了测序，序列长度约为920bp。分析表明，其平均碱基组成具有与其它鱼类D-Loop相似的碱基组成特点:平均AT含量明显高于GC含量(21.55C: 28.94T:犯.09A:17.42G)。控制区序列共观察到223(约为23.45%)多态位点，由此定义了39个单倍型。D一Loop序列间基于Jukes一Cantor距离测度得到的成对序列距离变异范围在0.000一0.167之间，平均为0.052。平均核普酸多样性是0.052士0.025，平均单倍型多样性为0.958士0.012。我们对长江中上游胭脂鱼四个群体进行了分子变异分析 (analysis or moleculary而ation)，揭示其遗传变异主要是来源于群体之间(占60.29%)，群体内的变异较少(占39.71%)。成对Fst值提示中国胭脂鱼群体之间存在一定的遗传分化，特别是宜宾江段群体与其它群体之间分化显著。我们还用微卫星标记初步调查了以上4个群体%尾中国胭脂鱼的群体遗传结构，筛选的3个微卫星位点分析的结果与D一Loop测序分析类似。综合以上研究，我们得出以下初步结论:由于小群体的遗传漂变，遗传结构易受各种随机事件的影响，中国胭脂鱼种群结构较为单一;不同江段群体具有适应当地水文特点的基因库，加之生境的片段化造成相互之间基因交流的机会较少，长江不同江段群体之间已存在一定的遗传分化。 我们用PCR方法得到了亚口鱼科17个代表属种线粒体DNA的细胞色素b基因和核DNA的185一ITSI一5.85部分序列，并进行了序列对比以分析其遗传变异模式。从细胞色素b基因成对序列变异来看，种间核昔酸变异的范围较大，变异率从最小的Thoburnia rkothoeca和Catostomus macroc人eilus之间(2.60%)到最大的Cyc妙tuselongates和人勿工ostoma anisurum之间(19.10%)。中国胭脂鱼与亚口鱼科其它属种之间核普酸的差异总体来说较大。其中与Ictiobus cyPrinellus之间核普酸差异最小 (共38个核营酸替代，其中转换32个，颠换6个)，与均夕entelium nigricans之IbJ核普酸差异最大(共55个核普酸替代，其中转换47个，颠换8个)。从6属7种亚口鱼的185一ITsl一5.85部分序列的变异模式来看，185一5.85序列较为保守，而ITsl长度变化较大，主要是存在较大片段的缺失。ITSI区长度变异范围很大，在从玖{ostoma robustom最短仅53bP，在Ictiobus呷rinellus最长达289bP。Ictiobus心尹rinellus和入为优o。岁rinus asiaticu、的ITSI区域明显比其它属种长些，分别为289饰和285 bp。以非加权对组算术平均法(UPGMA)及最大简约数法(MP)构建的分子系统发育树提示:中国胭脂鱼与CyclePtus etong以tus是并系关系。这与H翻s (2001)等基于125和165川NA基因研究的结果相同，而与Smith(1990)基于化石、生活史和形态等证据研究的结果不同(认为中国胭脂鱼与仰‘7ePtus elongatus是单系类群)。Tho石urnia rko功oeea和功夕entelium nigrieans是复系类群，关系较远。Thoburnia rkothoeca和Catostominae亚科中的Catostomin更多还原

【Abstract】 The family Catostomidae has 14 genera and about 80 species all over the world, mainly in North America. The Chinese sucker, Myxocyprinus asiaticus, is an endemic freshwater fish in China and the only representative of family Catostomidae in Asia. The fish is naturally distributed mainly in the Yangtze River, especially in the upper reaches. The wild fish source was greatly damaged due to over-harvesting and other human activities, to date the Chinese sucker has been an endangered species and has been listed in the second class of preserved animals in China. About this precious fish species, we have performed research work on 3 aspects. Firstly, on genome DNA and mitochondrial DNA levels, we detected the genetic diversity and population differentiation among 4 Chinese sucker geographical populations (Yibin, Wanzhou, Yichang, and Wuhan) from the Yangtze River based on RFLP, RAPD, SSR and DNA sequencing analyses. Secondly, we sequenced the 18S-ITS1-5.8S partial sequences from nuclear DNA and cytochrome b partial sequences from mitochondrial DNA respectively. By using these two markers, we performed molecular systematics research on Catostomidae fishes and compared the molecular variation model between Chinese sucker and other representative Catostomids. Thirdly, we studied the biogeography of Catostomidae fishes based on the molecular, fossil, and biogeographical evidences, especially about the biogeographical history of the Chinese sucker.It is necessary to learn the genetic background of the Chinese sucker in the Yangtze River in order to preserve and restore this endangered fish. We are the first research group to do this work using molecular methods. The RAPD and PCR-RFLP methods were used to detect genetic structure of Chinese sucker populations from the middle reaches of Yangtze River: Yichang section and Wuhan section. We screened about 80 RAPD primers, and only 3 primer produced polymorphism. The genetic similarity within the Yichang and the Wuhan population is 0.9274, 0.9313 respectively. The geneticsimilarity between them is about 0.9000. In PCR-RFLP analysis, 4uL aliquots of the PCR products of mitochondrial NADH dehydrogenase (ND-5/6) genes were digested by 12 restriction enzymes. Of 12 restriction enzymes types, 11 are monomorphic except Ncil enzyme. Only two kinds of haplotypes were found. The nucleotide diversity was 0.004, and the pair-wise sequence divergence was about 0.235%, which suggested that the genetic diversity of Chinese sucker was low. Seen from the electrophorisis patterns in the PCR-RFLP and RAPD analysis, we found that the genetic differentiation was formed between the two populations to some extent. Genetic diversity and population structure of the Chinese sucker was also assessed by sequencing the mtCR (mitochondrial control region) in 4 natural populations from the Yibin, the Wanzhou, the Yichang, and the Wuhan section of the Yangtze River. We obtained the mtCR sequences of about 920 base pairs. The average base composition was similar to other fishes that the average A and T contents were much higher than the average G and C contents (21.55C: 28.94T: 32.09A: 17.42G) . A total of 223 nucleotide positions were polymorphism, and these defined 39 haplotypes. Of the 39 haplotypes, 37(90%) are private and multi-sharing of haplotypes was seldom observed among populations. The average haplotype diversity (0.958) and the average nucleotide diversity (0.052) indicated higher level of genetic diversity of Chinese sucker globally. Analysis of molecular variation (AMOVA) of data revealed significant partitioning of variance (P<0.001) that most variation was from among populations (60.29%), and less from within populations (39.71%). The dendrogram or phylogenetic trees by the NJ (neighbor joining) and MP (maximum parsimorny) methods showed mosaic composition of the 39 haplotypes, suggesting that the populations were not significantly divergent. Nevertheless, we still recognized two divergence phylogenetic clusters: haplotype 34, 35, 36, and 39 consisted of the cluster B and the更多还原