【作者】 张红平；

【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， 2003， 博士

【摘要】 测定了饲养在中国的16个家养山羊品种(包括4个引进品种)96个个体的线粒体DNA(mtDNA)控制区(D-环)全序列，结果表明控制区全序列长度为1,212bp或1,213bp，共检测到90个变异(突变)位点，占分析位点总数的7.43％，其中简约信息位点69个，单一多态位点20个，所有这些突变均为两核苷酸间的变异。在1,075位有一个核苷酸的插入／缺失。所有的核苷酸替换均为转换，没有颠换发生。 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，且所有序列Tajima D中性检验结果不显著(P＞0.10或0．05＜P＜0.10)，说明序列均为选择性中性，表明中国山羊在过去没有出现群体扩张。无论中国山羊，还是引进品种均表现出了较高的核苷酸多样度(Pi)和单倍型多样性(H)。整个家山羊群体的平均核苷酸差异数为19.475，核苷酸多样度为(1.608±0.09)％，这些差异共定义了54种单倍型，单倍型多样性为0.977。其中，中国家养山羊11个品种有41种单倍型，引进的4个品种有16种单倍型。中国山羊的单倍型多样性(0.973)比引进品种(0.913)高，但引进品种的核苷酸多样性(1.731％)、单倍型间的平均距离(0.0187)、单倍型间的平均核苷酸差异数(20.96)高于中国山羊品种(分别为1.531％、0.0151和18.554)，但差异均不显著(P＞0.05)。表明中国家养山羊的遗传多样性较为丰富。用Alrequin2.0软件进行了分子方差分析(AMOVA)，品种间的方差组分只占总变异的24.22％，经检验不显著(P＞0.05)。表明家山羊品种(系)间还没有出现显著的遗传分化，品种间表现出弱的遗传结构。 将本研究测定的96条mtDNA的D环全序列与文献报道的16条家山羊序列、2种野山羊(角(羊骨)羊和捻角山羊)的D环序列结合分析发现，文献报道的单倍型仅有3种在中国山羊中存在。各家养山羊品种间平均核苷酸差异数为19.475(7.50～30.42)，平均核苷酸差异率为1.607％(0.619％～2.512％)。品种间的Kimura双参数距离变异较大(0.0008～0.0315)。许多品种都具有独特的单倍型类型，只有10种单倍型在品种间有共享。各单倍型在品种间和品种内的分布和频率也不平衡。 用PAUP4.0软件构建了56条单倍型和2条野山羊D环全序列的MP和ML系统发育无根树，在Kimura双参数模型下，用MEGA软件构建了ME树，并进行了Bootstrap检验。结果三种一致树各分支的统计置信度(凡值)都在50%以上，三种树的拓扑结构是相似的，家山羊的所有单倍型分成了3个有明显区别的簇一一1、n、m，表明中国家养山羊有多个母系来源。但中国家养山羊有哪些母系来源，野生种间是先杂交、再家养，还是先有独立的起源、后来再有野生种的基因渗入等需要进一步研究。 细胞色素b(cytb)基因在线粒体基因组中进化速度适中，较短的1个DNA片段就能包含从种下水平到属水平乃至纲水平的系统发育信息，而且在一定的进化尺度内cytb基因不受饱和效应的严重影响，所提供的系统发育信息和遗传分化水平适于分析种间和属间差异。实验测定了位于线粒体DNA上的唯一的蛋白质编码基因—细胞色素b全序列，结果表明细胞色素b基因序列全长为1，14obp，编码379个氨基酸和一个终止密码子。16个家养品种的42条序列中，共有17个多态位点，其中单一多态位点7个，简约信息位点10个，在215、241和302三个位置产生了氨基酸的变化。在第一位密码子和第三位密码子位置富含碱基A，而第二位富含T。第三密码子的碱基组成存在较大偏倚，第3位G的含量最低，只有3.6%一6.3%。与7个野山羊的11条序列比较，家养山羊同各野生山羊的序列差异变异范围很大，平均核营酸差异数为16.512一89.905，序列差异百分比为 1.448%一7.886%。转换发生的数目显著高于颠换，无论是转换还是颠换，在密码子的第三位碱基发生的概率均远高于第一和第二位密码子。氨基酸的密码子使用频率极不均衡。 用绵羊作为外群，16个家养品种的42条细胞色素b序列以及7个野生种的11条序列构建了系统发育MP树，结果表明除角滑羊外，家山羊、野生山羊分别聚在不同的枝上，但在家山羊群体内部出现了遗传分化，本实验中测定的所有家山羊个体聚为3大类。野生山羊种，如角招，羊，同家山羊的分化不明显。更多还原

【Abstract】 In the study, complete sequence of mitochondrial DNA(mtDNA) control region (CR, D-loop) was sequenced in 96 individuals from 16 domestic goat breeds (strains) raised in China (include 4 foreign breeds). The complete sequence of mtDNA CR is 1,212 or l,213bp(base pair). 90 sites were polymorphic (7.43% in 1,212) with 69 parsimony informative polymorphic sites, 20 singleton polymorphic sites and one insertion/deletion in 1075 sites. All polymorphic sites were two variants with transitition .The curve of nucleotide mismatch distributions in domestic goats population took on unimodal and wavilness. Tajima’s test of selective neutrality was not significant (P > 0.10 or 0.05 < P < 0.10) .It revealed that all sequence of domestic goats were selective neutrality and that goat population did not undergo expansions. High nucleotide diversity (Pi=0.977) and haplotype diversity(H=1.608%)were manifested either in Chinese goat or in foreign goat populations. All sequence polymorphic sites defined 54 haplotypes with 16 haplotypes within 4 foreign breeds , 41 within 11 Chinese breeds. Chinese indigenous goats showed higher haplotype diversity(0.973) than foreign breeds(0.913), while nucleotide diversity, pairwise distance and average number of nucleotide differences among haplotypes were less than foreign breeds. These results suggested richer genetic diversity within Chinese goat populations. The hierarchical components of mtDNA variation computed under the analysis of molecular variance(AMOVA) framework showed that smaller but not significant percentage existed among breeds(24.22%;P>0.05) .The AMOVA results revealed that there were no significant divergence and far weaker genetic structure among breeds.Only 3 haplotypes existed within Chinese goat populations by analyzing 96 sequences in the study, combined 16 domestic goats and 2 wild goats(Copra aegagrus and Capra falconeri) reported in documents. The average number of nucleotide differences , average number of nucleotide subs, per site between breeds were 19.475(7.50~30.42) and 1.607%(0.619%~2.512%), respectively. The Kimura 2-parameter distance between breeds varied from 0.0008 to 0.0315. Many breeds had its unique haplotypes and only 10 haplotypes were shared among some breeds. The distributions and frequencies were unequilibrium either among breeds or within breeds.Maximum parsimony tree and. maximum-likehood tree were constructed by using PAUP4.0 software , including 56 haplotypes and 2 wild capra sequences. While minumumevolution tree was established by MEGA software. All trees tested by 1000 times bootstrap replications. Three phylogenetic trees had same branch structure and higher confidence probabilities .All domestic haplotypes were divided three high obvious divergent clusters (I , II and III ) that showed multiple maternal origins of Chinese domestic goat population. More and deeper research should be continued if we want to get specific answer to some questions, for example, which wild capra maternal origins contributed to Chinese domestic goats ? Whether wild Capra crossed before domestication or independent domestication and subsequent introgression?The rate of cytochrome b (cyt &) evolution is relatively moderate. The cytochrome b genes that the only cytochrome coded by mitochondrial DNA, even small fragment sequence, contains phylogenetically informative and unlikely to be severely compromised by saturation effects. So it is thought to be variable enough for population level questions, and conserved enough for clarifying deeper phylogenetic relationships. In the study , cyt b gene were sequenced in 42 individuals from 16 domestic goat breeds(strains) in China. The complete sequence was l,140bp that coded 379 amino acid and one termination. 17 sites were polymorphic with 7 singleton polymorphic sites ,10 parsimony informative which caused 3 amino acid change at 215,242 and 302 sites. Rich adenine(A) presented in the 1st and 3rd codons, while the 2nd had rich thymine(T). The base composition showed more bias at 3rd codon that guanine (G) cont更多还原