【作者】 刘刚；

【导师】 胡德夫；

【作者基本信息】 北京林业大学 ， 野生动植物保护与利用， 2011， 硕士

【摘要】 动物血液和肌肉等组织业已广泛用于遗传学研究,然而,采集动物组织会伤及动物且难以获得充足的样本数量,目前研究者积极探索非损伤采样方法并运用遗传学研究。迄今非损伤取样方法在濒危物种保护遗传学研究中仍面临DNA量少、DNA降解、PCR抑制物、背景DNA干扰等诸多问题,制约了该方法及其相关研究深入开展。普氏野马重引入是我国一项濒危物保护生物学的重大实践,亟待开展非损伤途径的保护遗传学研究。鉴于此,本研究以三种粪便保存方法和2种粪便DNA提取方法为自变量,以PCR成功率为因变量,比较每种处理组合的PCR成功率,建立了一种适合普氏野马粪便DNA的保存方法、提取方法以及PCR扩增方法,并探讨该方法运用于普氏野马遗传多样性研究的可行性。本研究将近缘种家马的4个微卫星位点用于50匹普氏野马粪便DNA的扩增,并扩增了线粒体DNA的12SrRNA。主要研究结果如下：(1)三种保存方法的PCR成功率存在显著性差异(P<0.05),采用酒精保存效果较好。源自保存方法与提取方法的显著交互作用(P<0.05)表明,酒精保存和手工提取方法这种处理组合的PCR成功率最高(66.1%)。(2)粪便DNA保存时效性研究表明,采用酒精保存粪样的PCR在9个月时成功率(30%)显著降低(P=0.016),而采用干燥保存方法(P=0.031)和冷冻保存方法(P=0.001)的粪便DNA的PCR成功率,则在6个月的时候显著降低。(3)本研究建立的提取方法表明CTAB、马铃薯淀粉以及BSA对于去除粪便中PCR的抑制物具有重要的作用。(4)4个微卫星位点在50个普氏野马个体中共检测到16个等位基因,等位基因最低为3个,最高为5个,平均为4个。所有检测个体中,4个微卫星微点的平均期望杂合度(He)为0.703,平均观测杂合度(Ho)为0.575。更多还原

【Abstract】 Collection of tissue or blood samples often requires capturing the target animal,which inevitably has risks associated with injuries and difficulties of acquiring an adequate sample size. Non-invasive genetic sampling provides a new approach for research and management applications in wildlife conservation. However, for Przewalsi’s horse(Equus Przewalskii) less non-invasive genetic sampling research was reported. DNA recovered from non-invasive samples such as feces has the typically drawbacks of low quantity and poor quality, which limited its application to the field research. This paper aimed to optimize the method of fecal DNA preservation and extraction and evaluated the availability of non-invasive genetic sampling in Przewalsi’s horse.We evaluated and compared six combinations of three extraction and two preservation methods for fecal DNA, analyzed the effective storage time utilizing the 3 method, and constructed a technique system of fecal DNA preservation, extraction and PCR for Przewalsi’s horse. In order to evaluate the availability of non-invasive genetic sampling, mitochondrial 12SrRNA was amplified and 4 microsatellite locus from domestic horse(Equus Caballus) were used to amplify 50 Przewalsi’s horses.The main results are showed as follows:(1) Microsatellite amplification differed significantly between 3 storage types(P<0.05), with the ethanol the best. However, the interaction term was also significant(P<0.05), feflecting the fact that the optimal extraction method varied between storage types.(2) The highest amplification PCR success was obtained with samples stored in ethanol and extracted with the manual method(66.1%). Time dependent decay in amplification success was observed for samples stored in ethanol, drying, freezing, respectively at 9 months,6 months and 6 months.(3) CTAB、potato starch and BSA improved PCR by removing and inhibiting the co-purified PCR inhibitors in our manual stool DNA extraction method.(4) 4 microsatellite loci were used to conduct a general survey on the genetic of Przewalski’s horse on 50 samples. A total of 16 distinct alleles were observed. The number of alleles per locus ranged from 3 to 5 with an average of 4. The mean expected heterozygosity (He) and observed heterozygosity (Ho) of these microsatellites were 0.703 and 0.575, respectively.更多还原