【作者】 陈仕毅；

【导师】 赖松家； 李学伟；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 畜禽品种资源的保护与开发利用首先建立在清楚了解其遗传结构的基础上。采用线粒体基因组全序列和大规模数据集研究家养动物的遗传结构,群体演化历史及起源进化关系是最近发展出来的全新技术手段和分析方法,能有效解决以前各研究的不足。线粒体DNA作为细胞质效应的内在遗传基础,研究其SNP位点与性状间的关联性可以为标记辅助选择提供重要依据。本文通过线粒体基因组全序列和大规模控制区序列数据集研究普通牛种的母系结构、群体历史学特征和地理区域分布模式,分析线粒体DNA ATPase 8和ATPase 6基因SNP位点与荷斯坦奶牛泌乳性状间的关联性,得到以下结果:1.基于线粒体基因组全序列对普通牛种母系结构进行剖分,除已经报道的T1、T2、T4和T5单倍型组外,在传统的T3单倍型组内剖分出2个新的单倍型组,分别由第16119和第16122位点突变而来,命名为T16119和T16122单倍型组。所有现代普通牛（B.taurus）以较低的遗传分歧度被聚为一大支,其中T16119和T16122单倍型组与T4单倍型组具有相对平等的系统发生学地位。结果表明,采用线粒体基因组全序列可以对普通牛种母系结构进行更深入详细地剖分。2.本研究所测定的5个四川通江黄牛线粒体基因组全序列能稳定地被聚在T1、T2、T3、T4和T16119单倍型组中,与根据控制区序列分析得到的结果一致。这是首次在线粒体基因组全序列水平上证实了非洲起源的T1单倍型组存在于中国黄牛群体中。3.T16119是仅次于T3的优势单倍型组,其遗传网络关系为星状结构,中性检验的Fs值为-25.9805（P＜0.00001）。该单倍型组中约70%的个体来自中国地区,23%的个体来自东北亚地区,其最高频率出现在中国的西南地区,其次依次为黄河中上游地区、韩国地区和日本地区。结果表明,T16119单倍型组出现过群体扩张,可能发生在中国的西南地区,随后向北进一步扩散到东北亚地区。4.T16122单倍型组的遗传网络关系为星状结构,该单倍型组中81%的样本来自东北亚地区。中性检验的Fs值为-8.2383,P值为0.00500。结果表明,T16122单倍型组在东北亚地区发生过群体扩张。5个中国北方地区本地黄牛样本也出现在该单倍型组内,说明T16122单倍型组对中国北方地区的黄牛群体存在一定程度的影响。5.在T1单倍型组的遗传网络结构中,来自东亚地区与欧洲地区的样本分别被连接到只存在1个碱基差异的2个中心节点,表明起源于非洲地区的T1单倍型组为独立迁入到东亚地区和欧洲地区。6.T4单倍型组中97%的样本来自于中国和东北亚地区,分别出现37次和46次。结果表明,T4单倍型组不仅分布于东北亚地区,在中国地区也有广泛的分布,且出现的频率基本相当。7.在中国和东北亚地区的样本中检测到P和Q两种欧洲野牛单倍型类型,这说明欧洲野牛对现代家养普通牛的影响到达了除欧洲外的其它地区。8.不管采用何种地理区域分类标准,单独对普通牛和瘤牛（B.indicus）样本进行分析,约85～90%的遗传方差出现在品种内部,固定指数(F_ST)在0.15左右。当把普通牛和瘤牛样本综合在一起分析时,品种内的遗传方差组分降到79%,F_ST为0.21。因此认为,中国现代家养黄牛在地理区域间表现出来的遗传差异主要是由于瘤牛对各地区的影响程度不同造成的,而普通牛种内部在地理区域间不存在显著的遗传差异。9.作为一个实例研究（Case study）,在54个通江黄牛样本中,共检测到5种单倍型组（T1～T4,T16119）。因此推断,在样本含量足够大的情况下,可以在同一中国黄牛品种内检测到大部分的单倍型组;品种间在母系结构上的差异可能仅仅表现在各单倍型组以不同的频率出现,而不存在单倍型组特异性的品种。10.在线粒体基因组16338 bp中共检测到139个简约信息位点,其中11个分布在细胞色素b基因,变异位点数仅次于控制区,表明细胞色素b基因是线粒体DNA中的另一个高变区。11.在包括ATPase 8和ATPase 6两个基因的936 bp片段中共检测到18个多态位点,确定了6种单倍型（H1～H6）。在本研究所分析荷斯坦奶牛的4个泌乳性状中,不同单倍型对脂肪含量和体细胞数2个指标不存在显著影响（P＞0.05）,而蛋白含量和305天产奶量在不同单倍型间差异显著（P＜0.05）,其中单倍型H2具有最高的蛋白含量,单倍型H4具有最高的305天产奶量。结果表明,H2单倍型和H4单倍型可能是牛奶蛋白含量、305天产奶量的候选基因型,有待进一步验证。更多还原

【Abstract】 To develop effective conservation and utilization proposal of genetic resources in farm animal,we must first understand the genetic structure clearly.One recently developed and useful analysis method to study the genetic structure,population expansion history,and phylogenetic relationship of domestic animals is refered to the mitochondrial genomes sequence variation.The michondrial DNA（mtDNA） is the genetic basis for cytoplasmic inheritance effect,and to study the correlation of SNPs of mtDNA with important economic traits is significiant to perform marker-assisted selection.In this study,we seuqneced the mtDNA genome entire sequence of Chinese indigenious cattles,and studied the matrilineal components,population expansion history,and phylogeographical pattern of Bos taurus combined with available data from GenBank.In addition,we studied the SNPs distribution in the mtDNA ATPase 8 and ATPase 6 genes and the correlation with milk production traits in Holstein.The results are followed:1.After dissecting the matrilineal components of Bos taurus based on mitochondrial genomes sequence variation,we detected two new haplogroups as well as four already reported T1,T2,T4,and T5 haplogrous.The two new haplogroups separated by 16119^th and 16122^nd mutations,respectively,were defined by T16119 and T16122.All modern Bos taurus were clustered together with low genetic divergence,and in which T16119 and T16122 haplogroups presented comparable phylogenetic location with T4 haplogroup.The results suggested that the complete mitochondrial genome sequence variation could be used to dissect systematically matrilineal components of Bos taurus.2.The five new complete mtDNA sequences of Tonjiang cattle from Southwest China were perfectly clustered into T1,T2,T3,T4,and T16119 haplogrous, respectively,which is consistent with former reports studied by mtDNA D-loop sequence variation.This is the first report to verify the African haplogroup（T1） exist in the Chinese indigenious cattle population based on the mitochondrial genome entire sequence.3.The occurrence frequency of T16119 haplogroup was only inferior to T3 haplogroup.The network profile of T16119 haplogroup was the star-like shape,and the Fs value of neutrality test was-25.98045（P＜0.00001）.In T16119 haplogroup, about 70%samples came from China and 23%from Northeast Asia.The highest frequency of T16119 haplogroup occurred in Southwest China,and followed by the middle and upstream region of the Yellow River,Korea region,and Japan region.The results suggested that T16119 haplogroup might have undergone population expansion in the past,which would occur in Southwest China.Subsequently,T16119 haplogroup spread north to Northeast Asia.4.The network profile of T16122 haplogroup was the star-like shape,in which more than 80%samples was from Northeast Asia.The Fs value of neutrality test was -8.2383（P=0.00500）.The results suggested that T16122 haplogroup might have undergone population,and perhaps occurred in Northeast Asia.Five samples from North China region was also detected in T16122 haplogroup,which suggested that the influence of T16122 haplogroup presented in North China region.5.Although network profile of T1 haplogroup almost showed the star-like shape,all the samples could be clustered into two potential central nodes with one mutation distance,which were further occurred by East Asian and European samples, respectively.Then we proposed that T1 haplogroup was introduced into East Asia and Europe regions with independent events,and the one nucleotide mutation already existed before initial diffusion.6.97%samples in T4 haplgroup came from China（37 individuals） and Northeast Asia（46 individuals）.The results suggested that the distribution of T4 haplgroup was not only in Northeast Asia but also in China with comparable frequency.7.Two extinct anrochsen mtDNA haplogroups（P and Q） were detected in China and Northeast Asia cattle population with much low frequency.The results suggested the influence of Aurochs on modern cattle population would be not restricted only in Europe region.8.Whichever geographical classifications were adopted,about 85-90%of the genetic variation was distributed within breeds when the Bos taurus and Bos indicus type samples were separately studied.The population pairwise F_ST value was about 0.15.The genetic variation within breeds and F_ST value were changed to 79%and 0.21 in all Bos taurus and Bos indicus samples,respectively.Then we proposed that the genetic difference of Chinese cattle among ecologically geographical areas was resulted from the different influence degree of Bos indicus on these geograpchical populations.For Bos taurus type alone,there was no significiant genetic difference among ecologically geographical areas.9.As one case study,total five haplogroups（T1-T4,T16119） were detected in Tongjiang cattle with 54 samples.We proposed that there was no haplogroup-specific breed for Chinese indigenious cattle.The difference of matrilineal components of Chinese cattle populations would be only referred to different occurrence frequency of haplogroups.10.Total 139 parsimony informative sites were detected in complete mtDNA sequence.Among them,92 sites were distributed in coding region,and the Cyt b gene was the secondary mutational hot spots fragment.11.Total 18 polymorphism sites were detected in the 936 bp fragment of ATPase 6 and ATPase 8 genes,which further determined six haplogroups（H1-H6）.The least squares analyses revealed that the haplotypes had no significant influence on fat content and somatic cell count（SCC） traits.In contrast,the protein content and 305 days milk production differed significantly among haplotypes（P＜0.05）.Haplotype H2 had the highest protein content and haplotype H4 ocurred by the highest 305 days milk production.The results suggested that H2 and H4 haplotypes could be used as candidate gentypes for protein content and 305 days milk production traits,which should be further confirmed.更多还原