不同保存方法对川金丝猴粪便DNA提取效果的影响

Effect of Different Preservation Methods on the Fecal DNA Extracted Quality of Sichuan Snub-nosed Monkey

目的:川金丝猴(Rhinopithecus roxellana)是我国特有珍稀物种,其粪便作为一种非损伤性样品,为珍稀濒危动物的种群数量调查、遗传多样性评价、亲缘关系、系统进化等研究带来了很大便利,本研究试图建立高效、简便的粪便样品保存方法。方法:在现有珍稀濒危动物粪便样品保存方法的基础上,分别使用干燥法、冷冻法和干燥-冷冻法保存川金丝猴的粪便样品,比较了不同保存方法的DNA提取效果,以及对mtDNA控制区片段的PCR扩增成功率和微卫星基因的PCR扩增效率。结果:干燥法、冷冻法和干燥-冷冻法三种不同保存方法保存粪便1周时间后,提取的粪便DNA样本扩增mtDNA片段的成功率均为92%,微卫星基因的扩增成功率分别为79%、78%、80%;保存2个月后,mtDNA片段扩增成功率分别为80%、76%和80%,微卫星基因扩增成功率分别为65%、61%、67%;保存6个月后,mtDNA片段扩增成功率分别为56%、52%和64%,微卫星基因扩增成功率分别40%、34%、46%。因此,随着保存时间的增长,三种方法的保存效率都将明显降低,但干燥-冷冻法得到的DNA样本扩增成功率相对较高。结论:粪便样品能够为川金丝猴的遗传多样性评价等相关研究提供有效信息,干燥-冷冻法保存能够更为有效的保证DNA的提取和基因扩增效率。

Objective： Sichuan snub-nosed monkey （Rhinopithecus roxellana）, a endemic rare species in China and to be list to a national first class protected animal species, has characteristic of natural alert and arboreal life. Fecal, as a non-invasive sample, gives great convenience to researches on population survey, genetic diversity, genetic relationship, phylogeny. However, appropriate sampling and preservation methods are the premise to get useful DNA for the researches of endangered animals with non-invasive fecal sample. This study attempts to establish a high efficient, convenient method. Methods： Combined the reported preservation methods of the endangered animal fecal samples with the actual fact, three methods included drying, freezing and drying-freezing are used. Taking blood samples and hair samples as a reference, it is to analyze the effects of different fecal samples preservation methods on the efficiency of DNA extraction and the success rate of mtDNA and microsatellite DNA loci gene amplification, which included drying, freezing and drying-freezing in 1 week, 2 months and 6 months storage time respectively. Results： The amplification success rate of mtDNA gene is 92% with the three preservation methods and microsatellite loci amplification success rate of drying, freezing and drying-freezing was 79%, 78%, 80% respectively after a week. For the mtDNA gene amplification success rate with drying method, freezing method and drying-freezing method, it is 80%, 76%, 80% respectively after two months, and 56%, 52% and 64% respectively after six months. For microsatellite loci success rate with three methods, it is 65%, 61% and 67% respectively after two months, 40%, 34% and 46% respectively after six months. It showed that DNA amplification success decreases with the increasing of the storage time. Compared with two other methods, the samples with drying-freezing method can get better DNA extracted quality and high amplification success rate of mtDNA gene and microsatellite loci. Conclusion： The fecal of Sichuan snub-nosed monkey can be the research sample for providing effective information on some genetics study such as the evaluation of genetic diversity. Compared with other two methods, thedrying-freezing method for fecal preservation with long-way transportation is a better way to get quality DNA extracted and high efficiency of gene amplification.