【作者】 雷初朝；

【导师】 杨公社； 陈宏；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2002， 博士

【摘要】 从我国陕西省、山西省、湖南省、内蒙古自治区及河南省采集秦川牛(QC)、西镇牛(XZ)、晋南牛(JN)、岳阳牛(YY)、蒙古牛(MO)、南阳牛(NY)、郏县牛(JX)与中国黑白花奶牛(BW)共8个品种的样品，利用PCR技术，自行设计了一对特异性引物，用于扩增中国黄牛线粒体DNA D-loop区910bp全序列。用Clustal W(1.82)软件对8个黄牛品种22个个体的D-loop区全序列进行同源序列比对与深入分析，结果表明： 1．1 中国黄牛线粒体DNA D-loop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。碱基转换以T〈=〉C形式为主，占62.96％，A〈=〉G占37.04％。本文还发现一个特殊位点，即266位点，出现碱基突变C〈=〉G〈=〉A〈=〉T，在22个黄牛个体中，碱基G出现12次，占54.55％，碱基A6次，占27.27％，碱基C3次，占13.64％，碱基T1次，占4.55％。本文把266位点称为转换与颠换共存位点。 1．2 中国8个黄牛品种22个个体D-loop区全序列中，A+T平均含量为61.65％，G+C平均含量为38.35％。在黄牛D-loop 910bp序列中，共检测到核苷酸多态位点66个，约占核苷酸总数的7.25％。其中转换54个，约占核苷酸多态位点的81.82％；颠换4个，占6.06％；插入4个，占6.06％；缺失3个，占4.54％，转换与颠换共存位点1个，占1.52％。 1．3 以Anderson(1982)测定的欧洲牛mtDNA D-loop全序列(910bp)为标准，8个黄牛群体D-loop的平均核苷酸变异率分3个层次，西镇牛、蒙古牛、黑白花奶牛及秦川牛的核苷酸变异率最低，分别为0.37％、0.44％、0.52％和0.66％；南阳牛与郏县红牛的核苷酸变异率居中，分别为1.91％和2.02％；晋南牛与岳阳牛的核苷酸变异率最高，分别为4.47％和4.73％。 1．4 发现中国黄牛D-loop全序列中存在两个高变区，第一个主要的高变区，中国四个畜种(黄牛、水牛、耗牛、家驴)mONA遗传多样性研究与欧洲牛相同，位于241~61obP区域，长度为37ObP，约占核昔酸总变异位点的60.00%;第二个次要的高变区位于713一850bp区域，长度为138bp，约占20.00%。 1.5中国黄牛品种内D一looP区全序列变异率为0.55%~5.39%。其中蒙古牛D一loop区全序列变异率最低(0.55%)，南阳牛与郊县红牛变异率最高(大于5.0%)。中国黄牛品种间D一loop区全序列变异率为1.21%~6.59%。中国黄牛品种间D一100p区第一高变区的序列变异率为0.54%~9.46%，第二高变区的序列变异率为0.74%~8 .82%。 1.6对22条mtDNAD一1。。p的全序列进行同源序列比对，结果发现19种单倍型，其中南阳牛1(NYI)、秦川牛1(QCI)与秦川牛3(QC3)共享一种单倍型;晋南牛1(州l)与岳阳牛(YY)共享一种单倍型;其余17个个体分别单独享有一种单倍型，单倍型比例为86.36%，表明中国黄牛遗传多态性很丰富。 1.7通过对中国8个黄牛品种mtDNAD一loop区910饰全序列进行NJ法聚类分析，发现中国黄牛有三种单倍型组，分别代表着中国黄牛的三大母系起源。单倍型组I代表欧洲普通牛起源，单倍型组n代表印度瘤牛起源，单倍型组m代表一种不明身份的母系起源。首次从mtDNAD一loop母性遗传标记上证明中国黄牛是欧洲普通牛和印度瘤牛为主的混合起源，但受普通牛的影响更大一些。发现所研究的晋南牛1号、2号及岳阳牛起源于瘤牛，黑白花奶牛、秦川牛、蒙古牛与西镇牛起源于普通牛，晋南牛3号有另一种不明身份的母系起源。南阳牛与郊县牛品种内个体间分别含有普通牛与瘤牛的血缘。中国黄牛品种间与品种内有较大的分歧，表明中国黄牛品种间和个体之间具有丰富的遗传多态性。 从我国陕西省、甘肃省、云南省及新疆自治区采集关中驴(GZ)、凉州驴(LZ)、云南驴(YN)与新疆驴(XJ)共4个品种的血样，利用PCR技术，用一对驴的特异性引物(该引物为克罗地亚Ivankovic与斯洛文尼亚Dovc(2002)先生设计并惠赠的)扩增了中国家驴和关中马D一1。oP区部分序列，其长度分别为399bp和396bp。用Clustalw(l .82)软件对4个家驴品种21个个体和2匹关中马的D一foop区部分序列进行同源序列比对与分析，结果表明: 2.1以xu(1996)己发表的驴D一foop序列为对照，对家驴21个个体的mtDNAD一fo叩区399bp序列分析表明，中国4个家驴品种D一Icop序列核昔酸变异只有转换一种形式，没有发生D·lonp序列长度变异，说明中国家驴D一loop区序列核昔酸突变比较稳定。 2.2中国4个驴品种21个个体D一loop区部分序列中，A+T平均含量为59.06%，G+C含量为40.94%。共检测到核昔酸多态位点22个，只有转换一种类型，约占核 中国四个畜种(黄牛、水牛、耗牛、家驴)mtDNA遗传多样性研究且昔酸总数的5.51%。其中A<=> G10次，约占核昔酸多态位点的45.45%;C<=>T12次，占54.55%。 2.3关中马2个个体D一loop区396bp序列由2种单倍型组成，共检测到核营酸多态位点8个，全部为转换，约占核营酸总数的2.02%。其中A<=> G3次，约占核昔酸多态位点的37.50%;C<=>TS次，占62.50%。关中马n一loop区396bp序列中，A十T平均含量为57.95%，而G+C平均含量为42.05%。 2.4以xu(1996)己发表的欧洲驴D一loop作更多还原

【Abstract】 The samples were collected from 8 Chinese yellow cattle breeds, including Qinchuan cattle (QC), Xizhen cattle (XZ), Jinnan cattle (JN), Yueyang cattle (YY), Mongolian cattle (MG), Nanyang cattle (NY), Jiaxian cattle (JX) and China Holstein cattle (BW) from Shaanxi, Shanxi, Hunan, Mongolian and Henan Province or Autonomous region in China. In order to amplify the complete control region (D-loop) sequence of mtDNA in Chinese cattle by PCR technique, the specific primers was designed by ourself in reference to Loftus (1991) Si Wu (2000). The complete mitochondrial D-loop sequence, 910bp in length, in 22 individuals from 8 cattle breeds were aligned by Clustal W (1.82) software and analyzed, the results showed that:1.1 Comparisons of these D-loop sequences revealed five types of mutation of nucleotide sites in Chinese cattle: transition, transversion, insertion, deletion and coexistence of transition and transversion. The nucleotide transitions existed mainly in T<=>C with 62.96%, A<=>G with 37.04%. A special polymorphic site of 266 position with C<=>G<=>A<=>T was revealed, of the 22 individual sites, 12 represented base G with 54.55%, 6 represented base A with 27.27%, 3 represented base C with 13.64%, 1 represented base T with 4.55%, the 266 position was named as coexistence site of transition and transversion.1.2 In the complete mitochondrial D-loop sequence, 910bp in length of 22 individuals from 8 Chinese cattle breeds, A% + T% was about 61.65%, G% +C% was about 38.35% and 66 polymorphic nucleotide sites were observed, with the percentage of 7.25% of 910bp. Of the 66variable positions, 54 represented transition with 81.82%, 4 represented transversion with 6.06%, 4 represented insertion with 6.06%, 3 representeddeletion with 4.54% and 1 represented coexistence site of transition and transversion with 1.52%.1.3 Complete mtDNA D-loop of Bos Taurus (Anderson, 1982) was as a control, 3 groups were divided according to the average percentage of mtDNA D-loop nucleotide variation in 8 Chinese cattle breeds. The first group with the lowest percentage of D-loop nucleotide variation, including Xizhen cattle (XZ) , Mongolian cattle (MG), China Holstein cattle (BW) and Qinchuan cattle (QC) with 0.37%, 0.44%, 0.52%, 0.66% respectively; the second group with the percentage of D-loop nucleotide variation, including Nanyang cattle (NY) and Jiaxian cattle (JX) with 1.91%, 2.02% respectively and the third group with the highest percentage of D-loop nucleotide variation, including Jinnan cattle (JN), Yueyang cattle (YY) with 4.47% , 4.73% respectively.1.4 An examination of the distribution of nucleotide mutations in the bovine whole D-loop revealed two major hypervariable regions in China cattle, the first major hypervariable regions was the same as that of the European Bos Taurus (Loftus, 1994), 370bp in length between the 241-610bp, containing almost 60.00% of the overall number of nucleotide mutations, and the second, less variable region, 138bp in length between the 713-850bp, containing almost 20.00% of mutations.1.5 The average sequence divergence estimated from total D-loop within breeds varied from 0.55%~5.39% in Chinese cattle. The average levels of divergence were seen within 8 cattle breeds: the lowest (0.55%) and the highest (above 5.0%) among Mongolian cattle, Nanyang cattle and Jiaxian cattle, respectively. The average sequence divergence estimated from total D-loop among breeds varied from 1.21%~6.59%. The average sequence divergence estimated from total D-loop from the 370bp and 138bp hypervariable region among breeds varied from 0.54%~9.46% and 0.74%~8.82%, respectively.1.6 Comparisons of these 22 complete D-loop sequences revealed 19 mitochondrial haplotypes in Chinese cattle, one haplotype sequence was shared by NY1, QC1 and QC3, another by JN1 and YY1. The haplotype rate is 86.36%, demonstrating that Chinese cattle existed abundant mitochondrial genetic diversity.1.7 Phylogenetic trees were constructed using the neighbor-joining algorithm (NJ) in complete mt更多还原