【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， 2014， 博士

【摘要】 普氏野马(Equus przewalskii)是欧亚大陆草原和荒漠地带的代表性物种,野生种群于上世纪60年代灭绝,现存种群是19世纪末少数野外捕捉并圈养个体的后嗣。普氏野马圈养种群经历了严重的瓶颈效应和近交。迄今有关该物种的遗传多样性研究仅限于部分圈养种群。自2001年以来,中国先后在新疆和甘肃实施了普氏野马重引入,这是濒危物种保护的重大科学实践。然而,中国普氏野马的遗传多样性本底并不清楚,缺乏圈养和放归种群的遗传多样性研究及监测,由此严重制约了该物种重引入的后续推进。鉴于此,本研究以2个圈养种群(新疆野马繁育中心,略为WHBC;甘肃濒危物种研究中心,略为GESRC)和1个放归种群(卡拉麦里自然保护区,略为KNR)为研究样本,于2011-2013年利用线粒体DNA控制区(mtDNA)、微卫星、谱系和计算机模拟技术,分析和评价了普氏野马的种群遗传多样性。本研究得到的主要结果为：1. mtDNA控制区扩增的总体成功率达到96.2%(93.8%-100%),表明粪便DNA应用于马科动物遗传多样性研究具有较高的可行性。2.分析了105匹野马的粪便DNA,获得了普氏野马的2个mtDNA控制区单倍型(PR1, PR2),2个单倍型的核苷酸差异为1.63%,2个单倍型的频率在3个研究种群中分布不均匀。单倍型PRl在三个种群中均为优势单倍型,达到76.2%(60-91.9%),但放归的KNR种群的PR2仅占8.1%。甘肃圈养种群(GESRC)和放归种群(KNR)存在显著遗传分化(P=0.00267)。线粒体单倍型系的个体相似性概率(PI)高于母系分支,表明单倍型系是衡量母系遗传多样性的良好指标。3.分析了126匹普氏野马的粪便DNA,在10个微卫星座位上共检测到45个等位基因。WHBC、GESRC和KNR种群的等位基因丰富度分别为3.77,3.39,3.33,平均值为3.49。KNR的等位基因丰富度均显著低于WHBC (P=0.007)和GESRC(P=0.047)。WHBC和GESRC的平均期望杂合度分别为0.485(介于0.447到0.521)和0.460(介于0.392到0.532),均高于KNR的0.439(介于0.320到0.537)。亲缘关系系数(IR)与基于谱系的近交系数(fp)呈正相关(r=0.50,P=0.005),且fp和基于微卫星的近交系数(fm)也为正相关(r=0.34,P=0.014)。4.根据微卫星计算的FST,WHBC和KNR种群间的遗传分化最高(FsT为0.115,P<0.05),而两个圈养种群WHBC和GESRC的遗传分化程度最低(FsT为0.063,P<0.05)。应用STRUCTURE软件对群体基于Bayesian或然率数学模型的类群划分,在所分析的8个假设类群中,根据Ln P(D)最大值为-2359.02时,K=3,可判断126匹普氏野马可划分为3个遗传结构有差异的类群。5.采用计算机模拟程序,预测了普氏野马种群未来的遗传多样性水平。Bottlesim预测表明若要在未来100年内保持普氏野马种群90%的遗传多样性,种群数量的阈值须不低于100；AlleleRetain预测表明若要达到保留90%稀有等位基因的目标,当设定初始放归数量(startN)为20,则需要每隔5年引入20个新个体.6.依据本论文的研究结果,提出普氏野马圈养和放归种群的保护建议：首先,须做好谱系登录工作,确保完整性和准确性,及时上传谱系数据到国际野马谱系登录系统。定期结合谱系和分子标记数据评价圈养种群的遗传多样性,以此为基础调整繁殖群的结构,避免圈养种群的遗传多样性丧失。其次,选择圈养个体作为放归的建群者时,应选择亲缘关系较远且遗传变异高的个体组建放归群。既要保留原圈养种群的遗传多样性,又不能破坏其遗传结构。同时需确保放归群体的遗传代表性,减少遗传漂变的影响,避免种群分化。再者,在选择放归地点时,应考虑选择承载量大的放归地,以满足放归最少的建群者,节约放归成本。放归初期,应加强监测,确保种群数量的稳定和增长利于稀有等位基因的保留。同时还需定期监测种群近交情况,加强世界范围内各种群的交流,并且严格避免同域家马和野马杂家现象的发生。更多还原

【Abstract】 The Przewalski’s horse (Equus przewalskii) is a flagship species for conservation that once inhabited the Eurasian steppes, but was extirpated in the wild in the mid1960s. The present-day Przewalski’s horse population originated from individuals captured at the turn of the19th century. The captive population went through a severe bottleneck and suffered from inbreeding. Studies of genetic diversity within Przewalski’s horses have been sparse and limited to the captive population. Starting in2001, reintroduction programs were initiated in Xinjiang and Gansu provinces and proved to be one important practice in worldwide conservation biology. However, knowledge on genetic diversity in China’s horse populations is limited, but would help improving the genetic management and assess the success of the reintroduction.This aim of this study was to evaluate the genetic diversity in two captive (Wild Horse Breeding Center, WHBC; Gansu Endangered Species Research Center, GESRC) and one released population (Kalameili Nature Reserve, KNR) of Przewalski horse populations in China using mtDNA, microsatellite, pedigree data and computer simulations during2011-2013. Main results are as listed below:1. The mean PCR success rate of mtDNA control region was96.2%(93.8%-100%) in fecal DNA of Przewalski’s horse, which indicated a high feasibility of using non-invasive sampling approach in the genetic studies on the equids.2. Two mtDNA haplotypes were obtained (PR1, PR2) with a nucleotide difference of1.63%based on105Przewalski’s horses, but quite unequal in regards of haplotype frequency among populations. The PR1was dominant haplotype in three populations, averaging76.2%(60～1.9%), but PR2only accounted for8.1%in KNR population. The analysis of differentiation coefficient test revealed thatGESRC and KNR differentiated significantly (P=0.00267). Probability of Identity (PI) based on haplotypic information was higher than that from dam lines, indicating haplotypic information could be used to evaluate materinal diversity.3. A total of45alleles were identified for the10microsatellite loci in the126fecal DNA samples. Allelic richness (AR) in WHBC, GESRC and KNR was3.77,3.39,3.33, with mean number of3.49. AR in KNR population was lower than that of its founding WHBC population (the Wilcoxon’s signed rank test, P=0.01). The mean expected heterozygosity (HE) was0.49(ranging from0.45to0.52) and0.46(ranging from0.39to0.53) in the WHBC and GESRC respectively, as compared to0.44(ranging from0.32to0.54) in the KNR. When testing the relationship between fp and IR, fm, there was a positive relationship between fp and IR (r=0.50, R2=0.25, P=0.005) and fp was positively correlated with fm (r=0.34, R2=0.12, P=0.014).4. Based on FST, the highest genetic differentiation was estimated between populations in WHBC and KNR (FST=0.115, p<0.05), and the lowest between WHBC and GESRC (FST=0.063, p<0.05). The STRUCTURE Bayesian clustering yielded the most support for three, albeit admixed, genetic subpopulations within8simulated populations of126individuals (Ln P(D)=-2359.02, K=3).5. By means of simulation, a population size of100is required to retain90%of the genetic diversity in the reintroduced population (KNR) over the next100years; The number of immigrants needed to retain90%of rare alleles increased to20every5years, when the starting population was20.6. According to the results above, implications for conservation of Przewalski’s horse are listed below:(1) Managers should make complete and accurate studbook documentation, and timely upload local pedigree information to the International Studbook System of Przewalski’s horse. Molecular and pedigree approaches should be applied. Based on genetic analysis, the structure of the herd could be adjusted to avoid loss of genetic diversity.(2) When founding a released population, it is necessary to select the most distantly related individuals but with appropriate genetic structure. Another important issue to note is to maintain the genetic diversity of original captive population, and meanwhile, to ensure the genetic representation of the released population to minimize the genetic drift and avoid population subdividation.(3) Release sites with a larger carrying capacity should be selected in order to immigrate less individuals to retain rare alleles and save reintroduction cost. Upon release, temporal monitoring is crucial to ensure population maintainance and growth, which are beneficial to retain rare allels. Future reintroduction efforts should determine levels and directions of inbreeding of reintroduction projections and utilize multiple source populations, meanwhile avoiding hybridization with domestic horses.更多还原