【作者】 凌英会；

【导师】 马月辉；

【作者基本信息】 中国农业科学院 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 中国拥有丰富的马种资源,长期以来与我国农业生产息息相关,但对中国马种遗传多样性和起源进化的研究还不够系统和深入。本文采用基因组微卫星标记、线粒体D-loop高变区序列和Y染色体DNA三类DNA分子标记分别从全基因组、母系及父系三个方面对中国地方马群体的遗传多样性和起源驯化进行系统的分析,从而为马种遗传资源的保护和开发利用提供科学依据。1.应用世界粮农组织(FAO)和国际动物遗传学会(ISAG)推荐的27对微卫星引物,对26个中国地方马群体和2个引进品种,共1273份样品进行了遗传多态性检测。通过统计各种遗传多样性参数、分析群体遗传分化程度和系统聚类,研究了中国地方马群体遗传多样性和群体间遗传关系。结果显示:中国地方马群体具有较高的等位基因数、多态信息含量和遗传杂合度等遗传参数,表明中国地方马群体具有丰富的遗传多样性。其中,平均观察杂合度(Ho)为0.743,各群体的平均PIC都在0.69以上。中国地方马群体遗传多样性参数明显高于纯血马。中国地方马群体遗传多样性存在着一定的群体间差异。部分马群体具有较多的特有等位基因(NPA)。但总体遗传分化程度较低,群体间的遗传变异仅占2.4%。系统发育树将中国地方马种分成五个主要类群。这五大类群基本上与中国马种的五大地理区域分布相一致,包括长江流域及以南类群;青藏高原类群;西北类群;东北类群和内蒙古类群。主成分分析和Structure分析进一步支持了系统聚类结果。2.通过测序所获得659条家马和28条普氏野马mtDNA D-loop区序列。统计显示,共发现82个变异位点,定义出178个家马单倍型。在普氏野马序列中仅发现了2种单倍型。中国地方马群体的单倍型多样度和核苷酸多样度都表明中国家马群体mtDNA具有较丰富的遗传多样性。178个家马单倍型定义了9个支系(A-I),新定义H和I两个支系。中国地方马群体的单倍型在A、D和F三大支系中占有明显的优势。NETWORK网络关系分析支持中国家马群体存在9个主要进化分枝(A-I)。中国家马9个支系分布存在着地理偏倚性。F支系个体在长江以北地区含量比较高,在长江以南地区比较低。G支系在长江以南的大部分地方马群体中所占比例比其它地区地方马群体的比例明显要高。中国家马具有丰富的遗传多样性和广泛的支系分布表明,中国很有可能是家马驯化中心之一,并支持中国家马具有多个母系起源。联合GenBank中家马序列(增加334条序列,共1021条序列)分析发现,来自欧洲、中东、亚洲各个区域家马群体mtDNA,支系分布存在比较明显的差异性。远东地区马mtDNA类型与F支系具有显著的相关性,新定义的I支系马在亚洲地区分布具有明显的优势,这两个支系很有可能与中国马种最早的驯化事件相关。3.六个Y-DNA微卫星标记,在家马、普氏野马及家驴雄性样本中得到很好的扩增,形成明显的微卫星峰图,并在家马、普氏野马及家驴之间显示出等位基因差异。总体上,家马的Y-DNA遗传变异非常有限。本研究在家马的Y染色体非重组区首次发现的遗传变异信息——在Eca.YA16位点上,发现了一种新的单倍型(单倍型B),通过两种测序方法获得变异序列并证实其可靠性。统计发现单倍型B个体主要分布在中国的西南及长江流域以南区域。更多还原

【Abstract】 There are numerous domestic horse breeds in China, but there has been no extensive study on genetic diversity, population demographic history and origin of Chinese horses.In this study, three kinds of molecular genetic markers including autosomal microsatellites, mitochondrial DNA and Y–DNA, were used to analyze the genetic diversity, and origin of Chinese domestic horse, and results of this study further provide scientific basis for its conservation and utilization.1. Genetic variation at 27 microsatellite loci was investigated for 1,273 individuals from 26 Chinese indigenous horse populations and two introduced horse populations. Genetic variation parameter statistics, population differentiation analysis and clustering analysis were carried out to determine the genetic diversity and evolutionary relationships among Chinese horse populations. The number of alleles, PIC and heterozygosity in this reseach showed that there was abundant genetic variation in Chinese indigenous horses. The mean Observed heterozygosity (Ho) was 0.743, and the mean Polymorphism information content (PIC) was above 0.69 in each Chinese horse population. Genetic diversity of Chinese horses was higher than Thoroughbred horse based on most genetic diversity estimators. However, the genetic differentiation in Chinese native horses was low and only 2.4% of the total genetic variance existed among populations.Dendrogram analysis showed that the genetic differentiation and genetic relationships between Chinese horse populations were almost consistent with their geographic distribution, and five groups were recognized: Yangtze River group, Qinghai-Tibet Plateau group, Northwest group, Northeast group and Inner Mongolia group. Cluster analysis was performed by the Multivariate Statistical Packsge (MVSP) and Structure analysis.2. 659 domestic horse and 28 Przewalskii’horse D-loop sequences were sequenced. Statistics showed that there were 82 variation sites and 178 haplotypes in domestic horses. There were only two haplotypes in Przewalskii’horses. Haplotype diversity (Hd) and Nucleotide diversity (Pi) showed abundant genetic variation in Chinese domestic horses. Phylogenetic analysis showed nine haplogroups (A-I) in Chinese domestic horses based on 178 haplotypes of domestic horse. The two haplogroups of new definition were Haplogroup H and Haplogroup I. There was high sample proportion in A, D and F haplogroups of Chinese domestic horses. NETWORK analysis supported the results of phylogenetic analysis. There was geographical bias of the distribution of the nine haplogroups in Chinese domestic horses. The samples of F haplogroup in north of the Yangtze River were more than the samples in the south of the Yangtze River. The samples of G haplogroup in south of the Yangtze River were more than the samples in other area. Abundant genetic diversity and wide haplogroup distribution of mtDNA showed that China is likely to be one of the horse domestication centers, and supported there were wide maternal origin of the domestic horses in China.Added 334 sequences from GenBank, a total of 1021 sequences from Europe, the Middle East, and Asian were analysed. Results of analysis showed that there was different distribution of haplogroups in different area. This concordance of haplogroup F with the Far Eastern population origin was highly significant. Samples of Haplogroup I in Asian were more than other aera. These results indicated the F and I haplogroups maybe correlated with domestication of Chinese horses.3. Genetic variations of the six equine Y-DNA microsatellites were assessed in the male domestic horses, male Przewalski horses and male domestic donkeys. And there were obvious allele differences between domestic horse, Przewalski’s horse and donkey. Though the six Y-specific microsatellites displayed limited genetic variation in the domestic horse breeds, this was the first time polymorphism was found in non-recombinant horse Y-DNA. Moreover, Chinese domestic horse breeds carrying two alleles were only in the southern area of the Yangtze River, mainly in the southwest of China. Horse breeds in these regions have distinct appearances from those in the North of China.更多还原