【作者】 赖松家；

【导师】 李学伟；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2004， 博士

【摘要】 本研究采用PCR产物，Sanger双脱氧链终止法自动测序技术，测定了我国饲养的14个黄牛品种(其中两个引进品种)84个个体和5个牦牛品种(类群)33个个体的线粒体DNA控制区(D-loop)全序列，分析结果表明： 我国饲养的黄牛D—loop全序列长度为910bp、911bp和912bp，检测到83个多态位点，占分析位点总数的9.10％，其中单一多态位点19个，简约信息位点64个；核苷酸位点突变类型有五种，即转换、颠换、插入／缺失及转换与颠换共存。确定了45种单倍型，其中单倍型H10和H6为中国黄牛的主体单倍型，中国本地黄牛品种38种单倍型，其中32种单倍型为相应品种所特有。各单倍型在品种间和品种内的分布和频率均不平衡，所测定的14个黄牛品种单倍型平均多样度为0.9593±0.0126，各单倍型间的平均遗传距离为0.016(0.000—0.055)，说明我国黄牛D-loop单倍型类型丰富。黄牛品种内各序列平均核苷酸差异数19.675，核苷酸多样度2.164％，黄牛品种间核苷酸分歧度(Dxy)从0.292％—4.571％；品种间双参数距离范围较大(0.000—0.053)。结果表明我国黄牛具有非常丰富的遗传多样性。群体核苷酸不配对分布曲线是一条不规则的多峰波浪型曲线，所有序列Tajima’s D中性检验结果不显著(p＞0.01)，表明中国黄牛在过去没有出现群体扩张。分子方差分析表明，品种间的方差组分占总变异的26.5％，品种内的方差组分为73.50％，经检验差异极显著(p＜0.01)，进一步分析各品种间的Fst P值，大部分品种间差异不显著，部分品种间差异显著或极显著。表明我国黄牛品种间出现了显著的遗传分化。 本研究在国际上首次测定了牦牛线粒体DNA控制区(D-loop)全序列，并采用了克隆测序，反向测序进行验证，表明结果是准确可靠的。牦牛线粒体DNA控制区全序列长度为891 bp至895 bp。T、C、A、G四种核苷酸的含量分别为28.5％、25.3％、32.5％、13.7％。检测到47个变异位点，约占分析位点总数的5.23％，其中简约信息位点36个，单一多态位点11个；核甘酸变异类型有转换、颠换、插入／缺失四种。确定了24种单倍型，单倍型H6和H4为中国牦牛的主体单倍型，各单倍型在品种间分布不平衡，所测定的5个牦牛品种(类群)单倍型平均多样度为0.9697±0.0180，各单倍型间的平均遗传距离0.014(0.000—0.037)，说明我国牦牛D—loop单倍型类型丰富。牦牛的品种内各序列平均核苷酸差异数10.936，核苷酸多样度1.231%;耗牛品种间核昔酸分歧度(Dxy)从0.760%一2.155%，品种间双参数距离范围为0.003一0.029。结果表明我国耗牛遗传多样性丰富。 我国耗牛控制区全序列核营酸数量比我国黄牛少巧一21 bp，核昔酸变异率耗牛 (5.23%)较黄牛(9.10%)低，核营酸多样度黄牛(2.164%)较耗牛(1.231%)高约一倍。表明我国黄牛遗传多样性较耗牛丰富。 试验测定了我国饲养的黄牛13个品种82个个体、耗牛5个品种(类群)31个个体和水牛4个地方类群18个个体的线粒体DNA细胞色素b部分(420 bp)序列，分析结果表明: 黄牛中发现11个多态位点，单一信息位点6个，简约信息位点5个，确认了13种单倍型，单倍型核营酸平均差异数为2.331，核营酸多样度为0.555%，品种间核普酸分歧度(Dxy)从0.048%一1 .23%，双参数距离从0.000一0.012;在10、17、78、81四个位置检测到氨基酸序列变异，黄牛各品种间在cys、Leu、Ala、ne、Thu、.Val、Tyr七种氨基酸组成含量有差异。耗牛中发现9个变异位点，单一信息位点4个，简约信息位点5个，确定了6种单倍型，单倍型核营酸平均差异数1，798，核营酸多样度为0.428%，在69、118两个位置检测到氨基酸变异，耗牛各品种间在Ile，Thr两种氨基酸组成含量存在差异。水牛中发现5个多态位点，单一信息位点2个，简约信息位点3个，确定了5种单倍型，单倍型核普酸平均差异数为1.065，核昔酸多样度为0.254%，没有发现氨基酸变异。结果表明，中国黄牛遗传多样性较耗牛丰富，中国水牛遗传多样性贫乏;线粒体DNAD一loop区遗传多样性较Cyth基因遗传多样性丰富。 Cytb基因核昔酸的变异类型在黄牛中为转换和颠换，在水牛和耗牛中只有转换，三种牛均以转换为主，没有缺失/插入;核昔酸的替换三种牛均发生在密码子的第三位，三种牛均以同义替换为主，黄牛各品种仅在安西黄牛中检测到非同义替换，耗牛和水牛中没有检测到非同义替换，同义替换的速率(ds)高于非同义替换的速率(ka)。 黄牛、耗牛和水牛3种牛的Cytb序列中，不同位点碱基含量不同，3种牛均为密码子的第一位点和第三位点富含碱基A，第二位点富含碱基T。同一碱基在不同的密码子位点和不同的牛种中含量不同，黄牛中T、A在第2位点最高，G在第一位点最高，C在第三位点最高;耗牛和水牛中T在第二位点最高，G在第一位点最高，C、A在第三位点最高。可见，在不同位点和不同牛种间密码子的碱基组成存在较大偏倚。比较黄牛、水牛、耗牛密码子的使用频率，不同的密码子在同一牛种使用频率不同，同一密码子在不同的牛种中使用不同;同义密码子在牛种内和牛种间使用频率不平衡，结果表明，密码子使用频率存在偏倚性。 采用黄牛和耗牛mtDNA控制区全序列单倍型，黄牛、耗牛和水牛m更多还原

【Abstract】 The mitochondrial DNA control region complete sequence of 84 individuals from 14 yellow cattle breeds or strains (including two foreign breeds) and 33 individuals from 5 Chinese yak breeds were individually determined. The results showed:The complete sequence of mtDNA D-loop of Chinese yellow cattle was 910 bp, 911bp and912 bp. 83 sites were polymorphic (9.10% in 910) with 19 singleton polymorphic sites and 64 parsimony informative polymorphic sites. There were five types of mutation of nucleotide. They were transition, transversion, insertion/deletion and the coexistence of transition and transversion. This research defined 45 haplotypes, in which haplotype H10 and H6 are major haplotypes of Chinese yellow cattle. Chinese native yellow cattle breeds include 38 haplotypes in which 32 haplotypes were the characteristic of their corresponding breeds. The distribution and frequency of all the haplotypes either within the breeds or between the breeds were unbalance. The average haplotypes diversity of the 14 yellow cattle breeds was 0.9593 ?.0126. The average genetic distance of haplotypes was 0.016(0.000-0.055). That indicated the abundance of D-loop haplotypes in Chinese yellow cattle. Average nucleotide differences and the nucleotide diversity in yellow cattle breeds were 19.675 and 2.164% respectively. Nucleotide divergence and Kimura 2-parameter distance between yellow cattle breeds were 0.292%~4.57% and 0.000~0.053 respectively. It indicated that genetic diversity of Chinese yellow cattle was very abundant. The curve of nucleotide mismatch distributions in yellow cattle population took on unimodal and wavilness. Tajima’s test of selective neutrality was not significant (P>0.10). It revealed that yellow cattle population did not undergo expansions. The AMOVA results revealed that variance component among the breeds took 26.5% while within the breeds it took 73.5%. The results revealed that there were very significant differences(P>0.01). Further research on the Fst P value among the breeds revealed that there were not significant differences among most breeds while significant or very significant differences among a few breeds. It indicated that there was very significant divergence among Chinese yellow cattle breeds.This research had first time in the world determined yak’s complete sequence ofmitochondrial DNA control region. It also adopted clone sequencing and reverse sequencing to testify the accuracy and credibility of the results. The length of yak’s mitochondrial DNA complete sequence of control region was 891bp~895bp. Content of nucleotide T, C, A, G were 28.5%, 25.3%, 32.5% and 13.7% respectively. There were 47 polymorphic sites (5.23% in total analysed sites) with 11 singleton polymorphic sites and 36 parsimony informative polymorphic sites. They were transition, transversion and insertion/deletion. This research defined 24 haplotypes , in which haplotype H4 and H6 were major haplotypes of Chinese yak. The distribution of all the haplotypes among the breeds was disequilibration. The average diversity and genetic distance of haplotypes were 0.9687 ?0.0180 and 0.014(0.000-0.037) respectively, which indicated the abundance of haplotypes of yak’s D-loop. The average nucleotide difference and the nucleotide diversity in Chinese yak breeds were 10.936 and 1.231% respectively. Nucleotide divergence and Kimura 2-parameter distance between yak breeds were 0.760-2.155%, and 0.003-0.029 respectively. It indicated that the genetic diversity of Chinese yak was very abundant.The nucleotide number of Chinese yak’s control region complete sequence was 5-12bp less than that of Chinese yellow cattle. Yak’s nucleotide variation rate (5.23%) was lower than yellow cattle’s (9.10%). Yellow cattle’s nucleotide diversity (2.164%) was higher than yak’s (1.231%). It revealed that the genetic diversity was more abundant in Chinese yellow cattle than in Chinese yak.The partial sequence (420bp) of mitochondrial DNA cytochrome b (Cytb) had determined among 82 individuals from 13 yellow cattle breeds or strains (includi更多还原