【作者】 黄磊；

【导师】 王义权；

【作者基本信息】 南京师范大学 ， 动物分子生物学， 2004， 硕士

【摘要】 本文主要从微卫星DNA和mtVNTR两个方面对扬子鳄（Alligator sinensis）种群进行了研究。 1 微卫星分子标记技术及其在濒危动物遗传保护研究中的应用 微卫星DNA是广泛分布于真核生物基因组中的短串连重复序列，具有突变速率快，多态性高、共显性标记、孟氏遗传、选择中性等特点，是一种极具应用价值的遗传标记。本章简述微卫星分子标记的基本原理、研究方法、主要特点与技术发展，并介绍了微卫星分子标记在濒危动物遗传保护研究中的应用。 2 扬子鳄种群的微卫星DNA多态及其遗传多样性保护对策分析应用微卫星DNA分子标记对扬子鳄野生群、宣州F₁代及F₂代饲养群共39个个体进行研究，分析结果显示：扬子鳄种群在微卫星水平表现出很低的遗传多样性，平均等位基因数A=2.38、平均有效等位基因数N_e=1.60、平均观察杂合度H_o=0.374、平均期望杂合度H_e=0.350、平均多态信息含量PIC=0.327，3个群体间A、N_e、H_o、H_e、PIC及各微卫星座位等位基因频率分布无显著差异，但F₂代饲养群在Ami-μ-6和Ami-μ-222两个位点表现出极显著的遗传不平衡。扬子鳄种群遗传多样性水平极低主要是近几十年来种群数量大幅减少所致，现阶段应将全部现存的扬子鳄作为一个整体加以保护，在建立新的繁殖群体时，应考虑保存物种遗传多样性所必需的有效种群大小，在种群的遗传管理上应注重低频等位基因的筛查和保护。 3 扬子鳄mtDNA控制区多态与异质性分析 由于近几十年来严重的种群衰退，扬子鳄在已进行的多种分子标记研究中均表现出极低的多样性，但在对扬子鳄mtDNA控制区3’端研究中我们发现，串连重复序列区段表现出较丰富的片段长度多态，48个个体中共发现12种长度的单元型片段和21种扩增片段表型，其中12个个体稳定检测到2～4条扩增片段，呈现明显的异质性。mtVNTR表现中文摘要出较丰富的长度多态，可能是经历严重瓶颈效应后，现存扬子鳄饲养种群在不同层次的多样性恢复水平存在较大差异，同时与其特殊的重复序列组成可能也有很大关系，而在部分个体中检测到的异质性现象提示动物mtDNA的复制与遗传机制可能较为复杂，异质性现象的确切起因仍有待进一步研究。 4应用SSR与mtvNTR分子标记进行扬子鳄个体识别研究为有效避免扬子鳄种群的种质衰退，最大限度的保持现有的遗传多样性，有必要对现有种群进行个体识别，筛查稀有等位基因及其携带个体。我们应用SSR与mtVNTR两种分子标记对扬子鳄39个个体进行了个体识别研究，结果显示8个SSR座位的累计个体识别率与累计父权排除率分别达0.9968、0.7697;mtVNTR的个体识别率为0.9146，联合SSR与mtVNTR两种分子标记的累计个体识别率理论值达0.9997，并在实际分析中将所研究扬子鳄39个个体完全区分开，其区分能力较扬子鳄已用RAPD、AFLP及mtDNA序列分析等标一记强，并可对某些低频等位基因及其携带个体作有效筛查，对于日后进行扬子鳄的个体识别，及后续遗传谱系建立、核心种质确定等工作将具有一定实际意义。 5扬子鳄分子遗传学与遗传多样性研究现状扬子鳄作为中国特有的珍稀物种，其遗传资源的保护受到广泛关注和重视。本章对近年来有关扬子鳄在分子系统学、遗传多样性与种群遗传结构、线粒体基因组、个体识别以及性别决定方面的分子遗传学研究进展进行了综述。更多还原

【Abstract】 SSR polymorphism and mitochondria! DNA length variation and heteroplasmy in the population of Chinese alligator (Alligator sinensis) were studied in the present research.1 The technique of microsatellites DNA markers and its application in conservation genetics of endangered animals Microsatellites DNA are simple tandemly repeated sequence motifs consisting of repeat units of 1-5 bp in length. As genetic markers, they are widely dispersed in eukaryotic genomes. The advantages of microsatellites include high polymorphism, high abundance, codominance and selective neutrality. In present review, the primary principle, researching methods, main characters and technology development of microsatellite DNA makers were summarized, and its applications in the genetic conservation of endangered animal were illustrated.2 SSR polymorphism of Alligator sinensis and conservation strategy of genetic diversity In order to reveal the genetic structure of Chinese alligator population, total of 39 individuals including 7 wild individuals outside of the research center were sampled to construct wild, F1 and F2 groups according to their generations, and 10 micorsatellite loci selected from 25 primer pairs originally designed for Alligator mississippiensis were employed for investigating the genetic diversity of Alligator sinensis. The results indicated that contrasting with Alligator mississippiensis and some other endangered species, Chinese alligator had a extremely low genetic diversity level with ,4=2.38, Ne=1.60, H0=0.374, He = 0.350 and PIC=0.327. There were no significant differences of A, Ne, H0, He, PIC and each SSR locus alleles frequency distribution among 3 groups. However, Hardy-Weinberg equilibrium analysis revealed that F2 captive group showed a remarkable genetic disequilibrium at loci Ami-#,-6 and Ami-#-222. The reason accounting for the current genetic status ofXChinese alligator is dramatically shrink of the population in past decades. Due to the lack of significant difference between wild group and captive group, all survived Chinese alligator should be treated as one ESU in the next conservation practice. More attention regarding the effective population size and low frequency alleles should be emphasized in genetic management of captive alligators and establishing new separated propagation.3 Mitochondrial DNA Length Variation and Heteroplasmy in Captive Populations of Chinese Alligator Due to the severe population shrink and depression in past decades, Chinese alligator was in very poor genetic diversity status which was revealed by RAPD, AFLP, SSR and mtDNA sequencing. However, apparent variance of fragment length was found in tandemly repeated region of mtDNA control region 3’ end, 12 haplotype fragments and 21 genotypes were detected in 48 individual samples, and there were 12 individuals exhibited heteroplasmy that 2~4 fragments were detected in their amplification products respectively. Compared with other molecular makers, the high variance revealed by mtVNTR might be attributed to the apparent difference of the genetic diversity recovery at different lever despite the individual number recovered obviously in Chinese alligator captive populations which were set up sooner after the severe bottleneck, and also relate to its special composition in tandemly repeated sequences. The heteroplasmy detected in some individuals indicated that the mechanisms of mtDNA replicating and inheriting might be more complicated than we know, and it is necessary to carry out further research to determine the exact reason for length heteroplasmy of mtDNA detected in Chinese alligator.4 The individual identification of Chinese alligator with SSR and mtVNTR To prevent the genetic depression of Chinese alligator and keep its existing genetic diversity, it is necessary to carry out individual identification and screening for rare alleles and their holders. SSR and mtVNTR markers were applied to discriminate 39 Chinese alligator individuals, and the result showed th更多还原