【作者】 李隐侠；

【导师】 徐银学；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 家犬是人类最亲密的伙伴和朋友,也是人类第一个驯化的家养动物,有关家犬的起源、进化和品种形成等问题一直是各国学者研究的热点之一,关于家犬的起源问题,目前已基本达成共识,即世界范围内的所有家犬都起源于灰狼。但是,关于家犬的起源地和具体起源时间研究结果并不一致,仍然存在较大的分歧。那么对目前世界上现存的古老犬种进行分析将是揭示家犬发源地和起源时间的有效方法。藏獒是目前世界上最古老、最凶猛的大型家犬品种之一,因此藏獒起源以及与其它家犬品种系统发育关系的研究对于揭示家犬的起源、起源时间和起源地点都具有重要意义。但是目前藏獒的研究工作比较滞后,关于藏獒的起源、分类地位,以及与其它大型家犬品种间关系的研究报道较少。为了揭示藏獒的起源以及与其它家犬品种间的系统发育关系,我们测定了藏獒线粒体基因组全序列,分析了藏獒线粒体基因组的基因组成、特征,以及与犬属物种(家犬、灰狼和郊狼等)和哺乳动物的差异；利用线粒体基因组12个编码蛋白基因、COI基因、DNA条形码、控制区和高变区I序列分析了藏獒的起源、分类地位以及与其它家犬品种间的系统发育关系,推测藏獒和家犬的起源时间；测定了藏獒群体控制区序列,利用控制区和538bp高变区I序列分析了藏獒群体的遗传多样性。具体结果如下：(1)藏獒线粒体基因组全序列测定与分析通过克隆测序和序列拼接首次获得了藏獒线粒体基因组全序列(GenBank序列号为HM048871),发现藏獒线粒体基因组全长16710bp,包括13个编码蛋白基因、22个tRNA基因、2个rRNA基因和1个非编码区(即D-loop区),基因组成、排列方式和位置均与哺乳动物其他物种基本一致。藏獒线粒体基因组序列中T、C、A、G等4种碱基含量分别为28.7%、25.6%、31.6%和14.1%,其中AT含量(60.3%)明显高于GC含量(39.7%)。藏獒线粒体基因组13个编码蛋白基因序列长度为11410bp,其中序列最长的基因为ND5 (1821bp),最短的为ATPase8(204bp);共发现4种起始密码子,大多数编码蛋白基因(8个)均以ATG为起始密码子,ND2、ND3和ND5基因以ATA为起始密码子,ND6基因以AAT为起始密码子,而ND4L基因以GTG为起始密码子；11个编码蛋白基因的终止密码子为TAA,其中4个基因为不完全终止密码子,而CytB基因和ND2基因的终止密码子分别为AGA和TAG。藏獒线粒体基因组D-loop区序列全长为1250bp,定位在tRNA-Pro与tRNA-Phe之间,包括2个保守序列模块即CSBⅠ和CSBⅡ,而在2个保守序列模块之间有一个短片段重复区,这个重复区是由一个包含10个核苷酸构成的重复单元5’-GTACACG T(G/A) C-3’经过30个重复形成的。藏獒线粒体22个tRNA基因序列长度在66-75bp之间,总长度为1521bp,与引物源序列(家犬,GenBank序列号：U96639) tRNA的同源性为为99.08%,共有4个变异位点。藏獒线粒体12s rRNA和16s rRNA基因全长分别为954bp、1580bp,与与引物源序列的同源性为99.96%,仅发现1个变异位点,即在16s rRNA基因nt372位点发生了A/G转换。(2)藏獒的起源及与其它家犬品种的系统发育分析采用NJ法、MP法和网络法,分别根据线粒体基因组H链12个编码蛋白基因、COI基因、DNA条形码(648bp)、控制区和控制区582bp高变区Ⅰ等5种序列,结合我们测定的和GenBank中收录的家犬、灰狼和郊狼的相应序列,以郊狼为外类群,构建了藏獒、家犬品种和灰狼的系统发育树和单倍型网络关系图,系统发育树和网络关系图均显示：藏獒、家犬品种与灰狼聚在一起,而外类群郊狼单独聚为一类,说明藏獒和其它家犬一样起源于灰狼；且藏獒与家犬品种间线粒体基因组5种序列的序列差异均小于藏獒与灰狼间的,而大于家犬品种间的,认为藏獒是家犬亚种中的一个品种,在动物分类地位上应该隶属于食肉目(Carnivora)、犬科(Canidae)、犬属(Canis)、狼种(Canis lupus)、家犬亚种(Canis lupus familiaris)。藏獒、家犬品种聚为4个分支,说明家犬至少有4个母系起源,与前人的研究结果基本一致；藏獒和部分的大型家犬品种如圣伯纳犬、英国老式牧羊犬等属于分支A,且藏獒与圣伯纳犬、英国老式牧羊犬等大型家犬品种间的5种序列的序列差异、遗传距离均最小,说明藏獒与圣伯纳犬、英国老式牧羊犬等大型家犬品种间的亲缘关系较近,从分子水平上证实了圣伯纳犬、英国老式牧羊犬等大型犬在品种形成过程中可能掺入了藏獒的血统。利用Savolainen等(2002)的方法,线粒体基因组H链12个编码蛋白基因、COI基因、控制区和控制区582bp高变区Ⅰ的碱基替换率估计藏獒、家犬不同分支与灰狼间的分化时间。结果发现：综合考虑4个分支,家犬与灰狼间的分化时间分别为16000 YBP、32000 YBP、65000 YBP和50000 YBP,我们估计的家犬起源时间界于目前研究的最早的家犬起源时间(100000 YBP)和最晚的家犬起源时间(15000 YBP)之间,综合各种因素,我们认为家犬的起源时间至少在16000YBP,与Savolainen等(2002)、Pang等(2009)估计的家犬起源时间(分别为15000YBP、16300YBP)一致,也符合根据化石推测的起源时间(26000 YBP)：藏獒的起源时间分别为18000 YBP、33000 YBP、68000 YBP和56000 YBP,均早于相应序列估计的家犬起源,且系统发育分析发现藏獒与一些古老的家犬品种(如英国老式牧羊犬等)聚为一类,序列差异和遗传距离均较小,与英国老式牧羊犬共享DNA条形码单倍型,说明藏獒是目前世界上最古老的家犬品种之一(3)藏獒群体遗传多样性分析通过克隆测序获得了40个藏獒个体线粒体基因组控制区全序列,对藏獒控制区的结构、控制区和538bp高变区Ⅰ序列的遗传多样性进行了分析,并与犬属7个群体(金毛寻回猎犬、拉布拉多猎犬、伊利里亚牧羊犬、土耳其牧羊犬、中国灰狼、欧洲灰狼、郊狼)进行了比较。结果显示：藏獒群体控制区全序列的长度从1240bp到1320bp不等,其中主要的长度类型为1270bp(32.5%,13/40),进一步分析发现藏獒控制区全序列的长度多态性主要是由重复单元数量的不同引起的；在40个藏獒的控制区全序列中共检测到75个变异位点,多态位点百分率为5.68%,其中单一多态位点14个,占多态位点总数的18.67%,简约信息多态位点61个,占多态位点总数的81.33%；核苷酸变异类型中只有转换、插入/缺失,没有颠换出现,表现出高度的转换偏爱性；藏獒控制区全序列的单倍型多样度(H)为0.990,核苷酸多样度(pi)为1.201,平均核苷酸差异数目(k)为13.082,均高于拉布拉多犬和金毛寻回猎犬群体,但与哺乳动物其他物种相比,核苷酸多样度偏低。在犬属8群体中,野生群体(中国灰狼、欧洲灰狼和郊狼)线粒体控制区538bp高变区Ⅰ序列的单倍型多样度(H)、核苷酸多样度和平均核苷酸差异数目均明显高于5个家犬品种；在5个家犬品种内,土耳其牧羊犬的多态位点最多为21个(3.90%)、其次是藏獒和伊利里亚牧羊犬,发现了19个多态位点(3.53%)；家犬品种289个个体共检测到36个单倍型,其中29个单倍型为相应品种特有,有7个共享单倍型；藏獒的单倍型多样性最丰富,单倍型多样度为0.854,而金毛寻回猎犬的单倍型多样性最贫乏,单倍型多样度为0.471；但是,藏獒的核苷酸多样度和平均核苷酸差异数目最小,分别为0.845和4.519,伊利里亚牧羊犬的核苷酸多样度最丰富(1.427)、平均核苷酸差异数目最大(7.661)。藏獒群体的单倍型多样度比较高而核苷酸多样度较低,表明现存的藏獒群体可能是由一个较小的有效种群迅速增长形成的。总体来看,藏獒群体的遗传多样性相对贫乏。更多还原

【Abstract】 Domestic dog is the closest friend of mankind and was probably the first animal to be domesticated. As the intimate relationship with people grew, the origin, domestication and breed formation of the dog had become a major topic of studies among the scholars in the world. With regard to the origin, there has been a confirmed conclusion that all the domestic dogs originated from the grey wolf. However, about the region and time of origin, there were greatly differing views. So the analysis of the most archaic extant domestic dog breeds in the world at present will be an effective method to reveal the region and time of domestic dog origin. Tibetan Mastiff is one of the most archaic and fierce large breed of domestic dogs, so the research about the origin of Tibetan Mastiff and its relationship between other large breed dogs will contribute to reveal the origin, origin time and origin regions. At present the study about Tibetan Mastiff is lagged, there have been few reports on the origin of the Tibetan Mastiff and its relationship with other domestic dogs. Therefore, we sequenced the complete mitochondrial genome of Tibetan Mastiff and analyzed its characteristics in comparison to those of other Canidae(domestic dogs, grey wolves and coyotes) and some mammals. Based on the mitochondrial genome (mtDNA) of 12 protein-coding genes, COI gene, DNA barcoding, D-loop and its hypervariable region I sequence, we analyzed the origin, taxonomic status of Tibetan Mastiff and its relationship between other domestic dogs, speculated the origin time of Tibetan Mastiff and domestic dog; we identified the structure characters of control region in Tibetan Mastiff, analyzed the genetic diversity based on the control region and hypervariable region 538bp sequence. The results followed as below:1. Sequencing and analyzing the complete sequence of the Tibetan Mastiff mitochondrial genomeThe complete sequence of the Tibetan Mastiff mitochondrial genome (mtDNA) was determined (GenBank No. HM048871) for the first time. The complete nucleotide sequence of the Tibetan Mastiff mtDNA was 16710 bp, and included 22 tRNA genes, 2S rRNA gene,13 protein-coding genes, and one non-coding region (D-loop region), the constitution, arrangements and location of genes is consistent with other mammalian mitochondrial genomes. The base composition of the Tibetan Mastiff mtDNA is 28.7%T,25.6%C,31.6% A, and 14.1% G. The AT content (60.3%) is significantly higher than the GC content (39.7%). The total length of the Tibetan Mastiff mtDNA that encodes the 13 protein-coding genes is 11410bp, in which the longest gene is ND5 (1821bp), the shortest gene is ATPase8 (204bp), The genome uses four initiation codons:eight genes use ATG; NADH2, NADH3, and NADH5 use ATA; NADH6 uses AAT; and NADH4 uses GTG. The stop codons of 11 genes are complete termination codons (TAA, TAG, or AGA) and four genes (COⅢ, NADH3, NADH4, and NADH6) use incomplete termination codons; The complete length of the D-loop region in the Tibetan Mastiff is 1250bp, and is located between the tRNA-Pro and tRNA-Phe genes; there were two conserved sequenced blocks (CSB), that is, CSB I and CSB II in the mitochondrial control region, and a short tandem repeat region between the two CSBs, which was composed of 30 repeated units (5’-GTACACGT(G/A)C-3’).22 tRNA lengths range from 66 to 75bp, the total length of the Tibetan Mastiff mtDNA that encodes the tRNA genes is 1521bp, showing 99.08% homology with domestic dog (GenBank No. U96639) and 4 variable sites. The lengths of the 12s rRNA and 16s rRNA genes are 954bp and 1580bp respectively, showing 99.96% homology with domestic dog (GenBank No. U96639) and one variable site.2. Origin of Tibetan Mastiff and its phylogenetic analysis with other domestic dogsAccording to the methods of NJ, MP and network, based on the 5 sequences of 12 protein-coding genes in H chain, COI gene, DNA barcoding (648bp), control region and hypervariable region I (582bp) respectively, combining the sequences sequenced by us with the corresponding sequences of domestic dogs, grey wolves and coyotes gathered by GenBank, using coyotes as the outgroup, we constructed the phylogenetic trees and haplotypes network among Tibetan Mastiff, domestic dogs and grey wolves. Phylogenetic trees and network also showed that Tibetan Mastiff, domestic dogs and grey wolves clustered together, while the coyotes clustered alone, which suggesting that Tibetan Mastiff, as other domestic dogs, originated from grey wolves;the sequence divergence, based on the 5 types of sequences, between Tibetan Mastiff and domestic dogs were smaller than that of between Tibetan Mastiff and grey wolves but bigger than that of interspecies, so the Tibetan Mastiff could be considered as one species of domestic dogs, which belonged to the Carnivora, Canidae, Canis, Canis lupus, Canis lupus familiaris in the animal taxonomic status. Tibetan Mastiff and domestic dogs clustered into four clades, implying at least four maternal origins, which consistent with the before reports. Tibetan Mastiff and some large breed dogs such as Saint Bernard and the Old English Sheepdog belonged to clade A, the sequence divergence and genetic distance between the Tibetan Mastiff and these domestic dogs was smallest, indicating that Tibetan Mastiff appears to be closely related to large breed dogs such as the Saint Bernard and the Old English Sheepdog and confirming the point many of famous domestic dogs in the world such as Saint Bernard were possible of having blood lineage of Tibetan Mastiff at molecular level. According to the method of Savolainen et al (2002) based on the sequences of mitochondrial 12 protein-coding genes, COI gene, DNA barcoding, control region and hypervariable region I, we calculated the rate of base transition, speculated the divergence time between Tibetan Mastiff and grey wolves and between domestic dogs and grey wolves. The results showed that:considering the four clades synythetically, the divergence time between domestic dogs and grey wolves were 16000 YBP,32000 YBP,65000 YBP and 50000 YBP respectively, which was in the middle of the earliest originated time (100000 YBP) and the latest originated time (15000 YBP) of domestic dogs; we considered, synthetizing various factors,the originated time of domestic dogs was about at least 16000 YBP, which consistent with the results speculated by Savolainen et al (2002) and Pang et al (2009) (15000 YBP and 16300 YBP respectively) and fossil record (26000 YBP); the origin time of the Tibetan Mastiff was about 18000 YBP,33000 YBP,68000 YBP and 56000 YBP, which were earlier than the originated time of domestic dogs speculated by corresponding sequences, phylogenetic analysis showed that Tibetan Mastiff clustered with some archaic domestic dog (such as Old English Sheepdog), there was small sequence divergence and genetic distance between Tibetan Mastiff and these archaic domestic dogs, Tibetan Mastiff shared the haplotype of DNA barcoding with Old English Sheepdog, which suggesting that Tibetan Mastiff was one of the most archaic breed of domestic dog.3. Genetic diversity of Tibetan Mastiff populationWe sequenced the complete sequences of mitochondrial D-loop of 40 Tibetan Mastiff, analyzed the structure of control region and genetic diversity of control region and its 538bp hypervariable region I sequences and compared the genetic diversity with other 7 population of Canids (Golden Retriever, Labrador Retriever, Illyrian sheepdog, Kangal, Chinese wolf, grey wolf and coyote). The results showed that:the length of D-loop in Tibetan Mastiff populations ranges from 1240bp to 1320bp, of which 1270bp was the primary length types, there were 13 (32.5%) individuals owning 1270-bp length. Further analysis found that the length diversity mainly due to the different number of repeated units (5’-GTACACGT(G/A)C-3’). In 40 Tibetan Mastiff individuals,75 sites were polymorphic, taking up 5.68% of total sits, of which 14 (18.67% variable sites) was singleton polymorphic sites and 61 (81.33% variable sites) was parsimony informative polymorphic sites. There were two types of mutation of nucleotide such as transition, insertion/deletion, there was no transversion, showing strong transition bias. The haplotype diversity, nucleotide diversity and average number of nucleotide differences of Tibetan Mastiff popultion was 0.990,1.201 and 13.082 respectively, which were higher than that of Golden Retriever population and Labrador Retriever population, but compared with other mammals nucleotide diversity was lower. In 8 populations of Canids, the haplotype diversity, nucleotide diversity and average number of nucleotide differences of 538bp control region I sequence in wild populations (Chinese wolf, European wolf and coyote) were obviously higher than that of domestic dog breeds; in 5 breeds of domestic dog, the most variable sites (21 variable sites) happened in kangal (3.90%), following the Tiebtan Mastiff and Illyrian sheepdog with also 19 variable sites (3.53%); 289 individuals of 5 domestic dogs breeds were defined 36 haplotypes in which 29 haplotypes were the characteristic of their corresponding breeds and 7 haplotypes were the sharing haplotypes among breeds; haplotype diversity of Golden Retriever was smallest (0.471) and haplotype diversity of Tibetan Mastiff was highest (0.854), but the nucleotide diversity and average number of nucleotide differences in Tibetan Mastiff was smallest (0.845 and 4.519 respectively), the nucleotide diversity and average number of nucleotide differences in Illyrian sheepdog was highest (1.427 and 7.661 respectively). In Tibetan Mastiff population, the high haplotype diversity and low nucleotide diversity suggested the extant Tibetan Mastiff population probably formed and rapidly increased from a small and effective population. From these, we concluded that the genetic diversity of Tibetan Mastiff was relative spareness.更多还原