【作者】 晏鹏；

【导师】 吴孝兵；

【作者基本信息】 安徽师范大学 ， 动物学， 2003， 硕士

【摘要】 扬子鳄(Alligator sinensis)是中国特有的珍稀物种，也是全球23种鳄鱼中最濒危的一种。尽管人工繁殖取得了成功，但扬子鳄仍未完全摆脱物种灭绝的危险，其保护现状主要呈现以下两特点：一方面饲养种群数量虽有增加，但出现近交衰退现象，种质资源有退化趋势；另一方面则是野生扬子鳄种群处于灭绝的边缘，总数已不足150条。当前应尽快将人工饲养的扬子鳄种源引入自然环境中，扩大野生种群间基因交流，以最终恢复和重建扬子鳄的自然种群。 扩增片段长度多态性(AFLP)是一种基于选择性扩增完全酶切消化后的基因组DNA片段的新型分子标记技术，该技术具有不需要预知基因组的序列特征，较高的多态分辨力等诸多优点。本文利用AFLP技术对来自安徽省扬子鳄繁殖研究中心的43个饲养个体的遗传多样性进行了检测，以期为野放扬子鳄个体的筛选提供更为翔实的遗传背景信息。研究中4对引物共获得了117个AFLP扩增条带，同时发现了六个个体的带谱极为特异，在统计中忽略这6个个体时，117个AFLP扩增位点中19个位点为单态性，98个多态位点，多态位点率为84％，当加入这6个个体时，则所有117个条带都表现为多态性。在忽略这6个个体时，37条扬子鳄群体内遗传相似系数GS在0.13～0.97之间，平均种群遗传相似性为0.7503±0.0064，而当考虑这六个个体时群体内的遗传相似系数明显下降为0.06～0.97，平均种群遗传相似性为0.6523±0.0079。利用遗传距离构建聚类树，也发现这6个个体的遗传背景差异较大。依据聚类分析和性别比例的搭配，筛选出了27条遗传背景差异较大的个体用于野外放归，部分个体已于2003年4月成功放归自然。 在保护遗传学研究中，微卫星DNA是另一种高效的分子遗传标记。文中对微卫星的结构及其多态性检测技术的原理进行了介绍，并通过对扬子鳄微卫星位点PCR引物筛选的初步研究，发现来自于密西西比鳄的12对微卫星引物中的7对引物可以在扬子鳄基因组DNA的扩增中得到扩增产物，但位点都表现为单态性。微卫星位点在近缘物种间的可用性上还有待进一步研究。研究同时表明利用经典分子生物学方法克隆、测序、设计引物，是目前获得扬子鳄特异性微卫星位点较为可行的方案。 通过设计一对位点特异性的鉴定引物Alli-M和Alli-R，仅需一步PCR反应，根据琼脂糖凝胶电泳检测扩增片断的有无，即可实现对扬子鳄肉的快速、准确鉴定。更重要的是，利用这种方法即使是熟肉样品也可被准确地鉴定。盲检以及对送检不明鳄爪的实例鉴定都证明了这对鉴定引物的准确性和实用性。尤其是在对公安部门所送检不明鳄爪的鉴定实例中，对样品12S基因的测序鉴定结果与利用所设计的特异性引物的鉴定结果完全吻合，这更证明了这对特异性鉴定引物的真实可靠性。更多还原

【Abstract】 Chinese alligator (Alligator sinensis) is an endemic species in China, and also has been recognized as the most endangered crocodile species among the 23 species of crocodiles in the world. Despite of the success in the artificial breeding and rising of Chinese alligator, the rare species is still in edge of extinction. Two points are showed clearly in the status quo of conservation for Chinese alligator. On the one hand, with the steadily increasing of the captive-bred population, the appearance of the inbreeding depression indicates the decline of the situation of germplasm resource. On the other hand, the wild population of Chinese alligator is on the verge of extinction. The current wild population is estimated at about 150 individuals. Currently, it is urgent to reintroduce the captive-bred alligators in nature environment, and enhance the gene exchange among the wild populations and thereby, reestablish the wild population ultimately.As a novel DNA molecular marker technique, the AFLP (Amplified Fragment Length Polymorphism) analysis is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The AFLP technique has some advantages, such as a high level of polymorphism detection, the applicability for any organism without any prior knowledge about the genomic makeup, and so on. In this article, by the AFLP analysis, the genetic variation of 43 Chinese alligator individuals from ARCCAR was detected. It is expected that the research can offer the full and accurate information of genetic background for selection of introduced alligators. 117 AFLP markers were obtained with four pairs of primers in the research. Of 6 individuals, the DNA fingerprintings are significantly different from others. When the 6 specific individuals were moved, it was observed that there were 19 monomorphic loci and 89 polymorphic loci with 84% of the proportion of polymorphic loci. Moreover, the genetic distance (GS) for 37 individuals ranged from 0.13 to 0.97, and the value of average GS was 0.7503±0.0064. However, when the 6 individuals were included, all the 117 loci were polymorphic. At the same time, the range of GS for all individuals was obtained from 0.06 to 0.97, and the value of average GS was 0.6523±0.0079. Analysis of clustering also showed that the 6 individuals were not in cluster with other alligators. According to the dendrograms and sexuality of 43 individuals of Chinese alligator, 27 alligators with much difference of genetic background were selected to reintroduce the wild environment Some individuals have been released successfully the wild habitat.Microsatellite DNA is another powerful molecular genetic marker in research ofconservation genetics. In this paper, the structure of microsatellite and the principle of the detection technique of microsatellite polymorphism are illustrated. In the primary study of the application of the microsatellite primers on Chinese alligator, 12 pairs of microsatellite primers for American alligator (Alligator mississippiensis) were tested on Chinese alligators. 7 pairs of primers were amplified successfully, but these loci were lower polymprphic. So the usability of microsatellite loci on related taxa needs a further research. To acquire the specific microsatellite loci of Chinese alligator, it is feasible to clone, sequence and design primers by the classical method of molecular biology.A pair of Allele-Specific PCR primers (Alli-M and Alli-R) was designed. According to the agarose gel electrophoresis of PCR products, Chinese alligator meat can be identified rapidly and accurately with a single PCR-reaction step. Moreover, even cooked meat could be successfully identified with this method. The accuracy and practicability of the specific primers were confirmed by a blind test and a case of identification for the pending claw sample. In the case of identification, the result of sequencing of 12S rRNA gene of the claw sample was further supported for the result from the identification with the primers of Alli-M and Alli更多还原