【作者】 冀德君；

【导师】 常洪；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 牛亚科是一个巨大的分类学群体,动物学上分类有不同观点。属的划分影响我国牛亚科家畜遗传资源评价、保护和利用实践的问题之一。一种观点认为牛亚科家畜包括家牛属(Bos)、牦牛属(Poephagus)、野牛属(Bison)、准野牛属(Bibos)、水牛属(Bubalus)、非洲野水牛属(Syncerina)。牛属主要包括普通牛(Bos taurus)和瘤牛(Bos indicus),牦牛属仅包含牦牛(Poephagus grunniens)一个种,准野牛属(Bibos)主要包括大额牛(Bibos frontalis)、爪哇牛(Bibos javanicus)和印度野牛(Bibos gaurus)。野牛属(Bison)包括美洲野牛(Bison bison)和欧洲野牛(Bison bonasus)。水牛属包括亚洲水牛(Bubalus bulalus)、菲律宾水牛(Bubalus mindorensis)和印尼水牛(Bubalus depressicornis)等,亚洲水牛又包括沼泽型(Swamp)和河流型(River)两种。非洲野水牛属(Syncerus)包括克鲁斯水牛(Syncerus caffer)和非洲野水牛(Syncerus nanus)两个种。另一种观点认为牦牛、大额牛列为牛属(Bos)。众多专家学者对牛亚科内不同物种的起源及分化也存在各种观点。一般认为中国水牛品种完全属于沼泽型,有专家学者认为沼泽型水牛应划分为两个支系,另有专家学者认为沼泽型水牛和江河型水牛相互独立地分别在中国和印度次大陆进化和驯化,两者在驯化后分别传播至东南亚地区;一般认为雷琼牛可能起源于与印度瘤牛(Bos indicus),另有专家学者认为雷琼牛可能起源于与印度瘤牛不同的其他肩峰牛的祖先,同时混有一定的爪哇牛(Bos javanicus)血缘,并推断海南可能是世界的一个牛属的发源地之一;还有专家学者认为中国牦牛在母缘遗传上分为两大支系;等等。这些不同观点反映了国内学术界对牛亚科内家畜物种的起源及分化问题认识不够透彻,因此在牛亚科物种的遗传资源保护工作中缺乏足够理论指导,不利于遗传资源保护工作的及时有效的开展。为了解牛亚科家畜的遗传变异信息,进一步解决我国牛亚科家畜的宏观进化与分类问题,开展了本项研究工作。本研究以中心产区典型群内随机抽样方法,采集6个中国牛亚科物种的代表性群体,蒙古牛(MG, Bos taurus),雷琼牛(LQ, Bos indicus),巴音郭楞州牦牛(MN, Bos grunniens),独龙牛(DL, Bos frontalis),海子水牛(HZ, Swamp Bubalus bubalis)和尼里-拉菲水牛(NR, River Bubalus bubalis),的血液样本共计103份,提取各样本基因组DNA;以各样本基因组DNA为模板,采用PCR分段扩增和测序方法,测定该6个牛种群体的4个结构基因(MSTN、GH、ADH和Cyt b)的编码基因序列,采用相关软件比较分析了6个牛亚科物种的代表性群体的4个结构基因的碱基组成上的差异以及核苷酸序列上的差异,并分别以4个结构基因座上核苷酸序列差异为基础,进行群体间基因分化和基因流分析,并重建中国牛亚科种群的系统发生树,以探讨中国牛亚科种群的遗传分化和系统起源。结果显示:1.各基因碱基组成上,Cyt b基因的G含量远低于其它3个基因(低约8.3%-13.6%);GH基因的G+C含量约为60%,远远高于其它3个基因;6个牛种群体内核基因的核苷酸歧异度较小,表示多样性水平低;6个牛种群体内Cyt b基因分化水平不一,以独龙大额牛最高(0.03807),蒙古牛最低(0-0.00019),蒙古牛和雷琼牛群体各自具有比较单一的母缘血统。从基因多样性上看,综合6个牛种群体,以Cyt b基因多样性水平最高,其它3个核基因的多样性水平均较低。研究结果符合公认的关于核基因和线粒体基因进化速率的认识。2.水牛属和传统的牛属各物种群体间基因分化水平普遍较高,基因流水平很低,或无基因交流,例外的是,独龙大额牛和水牛群体间存在中等水平的基因分化和微弱的基因流,总体研究结果符合两者传统的属分类地位;蒙古牛群体未受其它母缘血统的混杂;独龙大额牛和雷琼牛群体间在Cyt b、GH和ADH基因上的基因流水平较高;独龙大额牛和蒙古牛群体间仅在Cyt b基因上有较高的基因流水平;独龙大额牛和巴音郭楞州牦牛在MSTN基因上有较高的基因流水平;牦牛和其它牛属物种群体间存在中等水平的基因分化和微弱的基因流;本研究中基因流对不同基因的影响力存在差异,其中MSTN基因可能对基因流的影响不敏感。3.牦牛在母缘遗传上分为野生牦牛与家养牦牛两大支;牦牛在母缘遗传上与美洲野牛的关系近于欧洲野牛;传统的牛属各物种与欧洲野牛亲缘关系近于水牛属或其它牛亚科属分类群体;单一的瘤牛母缘血统不支持雷琼牛群体中含有爪哇牛或者巴厘牛血缘的观点,支持雷琼牛与印度瘤牛亲缘关系较近的观点;独龙大额牛群体与普通牛或瘤牛群体在母系血缘上关系很近;海子水牛分为两支的结果支持了沼泽型水牛划分为两个支系的论点,也同时支持了沼泽型水牛独立驯化的观点;所有海子水牛与菲律宾水牛亲源关系近于印尼水牛的结果部分支持了沼泽型水牛通过两条路线传播的推测。4.依据进化速率适中的基因座位上分离位点在群体间的差异,可以采用PCR技术对不同牛种产品进行鉴别,且所采用的基因座位无特别要求。研究结果一方面获得了所检测群体的遗传多样性水平,为认识种间分化提供有价值的信息,为保护及其开发利用牛种资源提供了部分遗传学依据;另一方面探讨了所检6个牛种群体以及与近缘物种之间的遗传分化关系,为中国牛亚科家畜的起源和进化的宏观研究提供了客观的参考资料。更多还原

【Abstract】 Different taxonomic systems in Bovinae in China have long coexisted in explaining the divergence of Bovinae species. The key problem that affected the evaluation, protection and utilization of genetic resources in Bovinae livestock was the decision of each genus. One opinion prones to classify domestic animals in Bovinae into 6 genuses, Bos, Poephagus, Bison, Bibos, Bubalus and Syncerina. The genus Bos includes species normal cattle (Bos taurus) and zebu (Bos indicus); The genus Poephagus contains only yak (Poephagus grunniens); The genus Bibos includes species gayal(Bibos frontalis), banteng (Bibos javanicus) and India gayal(Bibos gaurus). The genus Bison includes species American bison (Bison bison) and European bison (Bison bonasus). The genus Bubalus includes species Asiatic water buffalo (Bubalus bulalus), tamaraw(Bubalus mindorensis)and the lowland anoa(Bubalus depressicornis), and sometimes the mountain anoa(Bubalus quarlesi), Asiatic water buffalo contains two types, swamp and river type. The genus Syncerina includes species Syncerus caffer and Syncerus nanus. Another opinion proposes that the genuses Poephagus and Bibos be combined into the Bos genus. Moreover, many scholars have different viewpoints on the origins and divergences of some Bovinae species. Chinese water buffalo breeds are generally regarded as swamp type, while some scholars suggested two subbranches in swamp-type water buffalo in China, and others demonstrated that swamp- and river-type water buffaloes independently evoluted and domesticated in China and Indian subcontinentant, and spread to southeast Asia;It is generally thought that Leiqiong cattle originates from Indian zebu (Bos indicus), while some scholars suggested another probable ancient humped cattle origin and a mixture of certain Banteng (Bos javanicus) blood, and deduced that Hainan region in China was probably one of the centers for early origination of Bos species; some scholars suggested that Chinese yak has two subbranches in maternal kinship; to name a few. These different opinions reflect a partial understanding of the issue on the origination and divergence in Bovinae, which leads to a lack of correct theoretical guide for protection of Bovinae genetic resources and an obstacle for effective execution of protection. To collect the information of genetic variations in domestical animals in Bovinae and explore the issue of their evolution and classification, we conducted this research. In the research, by applying simple random sampling in typical colony methods in the central area of habitat, blood samples of 6 typical bovinae species, Mongolia cattle (MG, Bos taurus), Leiqiong cattle (LQ, Bos indicus), Bayingolin yak (MN, Bos grunniens), Dulong gayal (DL, Bos frontalis) , Haizi water buffalo(HZ, Swamp Bubalus bubalis) , Nili-Ravi water buffalo (NR, River Bubalus bubalis), were collected. The genome DNA templates were then extracted from these samples by the regular method in literature. Using the methods of PCR amplification and sequencing, sequences of 4 structural genes, MSTN, GH, ADH and Cyt b, were detected in these 6 populations, and variations of base composition and nucleotides were analyzed by DnaSP and MEGA3.1 softwares, based on which, interpopulational gene flow was analyzed and phylogenic relations among Bovinae were reconstructed to explore the genetic divergence and phylogeny of Bovinae species. The results showed that:1. For the base composition of each gene, G content of Cyt b gene was far (8.3%-13.6%) lower than any of the other three genes; the G+C content of GH gene was about 60%, which was far higher than any of the other three genes; the nucleotide diversities of the three nuclear genes were relatively low among the 6 bovine populations, showing a low genetic diversity;The gene diversities of Cyt b gene were uneven among the 6 bovine populations, highest in Dulong gayal population(0.03807), lowest in Mongolia and Leiqiong cattle populations (0-0.00019), indicating both Mongolia and Leiqiong cattle had relatively a single maternal kinship. For the gene diversities of the 4 genes, Cyt b gene had a highest diversity, the gene diversities of the three nuclear genes were relatively low. These findings were identical with the general knowledge of the evolutionary rate of nuclear and mitochondrial genes.2. The gene diversities between the Bubalus populations and the traditional Bos populations were commonly high, with a low or no gene flow. An exception was that a moderate gene diversity and a faint gene flow were observed. The overall results were in agreement with the traditional taxonomic positions of Bubalus and Bos; Mongolia cattle had not been affected by other maternal kinship; Only in Cyt b gene, a high gene flow level was observed between Dulong gayal and Leiqiong cattle population; a high gene flow level was observed in MSTN gene between Dulong gayal and Bayingolin yak population; a moderate gene diversity and a faint gene flow were observed between the traditional Bos populations and Bayingolin yak population; the gene flow had different effect on these 4 genes, MSTN gene probably insensitive to the pressure of gene flow.3. the yak population was maternally grouped into two subbranches, wild yak and domestic yak; yak was more closely related with American bison than European bison, while the traditional Bos populations were more closely related with European bison than the Bubalus populations or other bovinae populations; the single maternal kinship of the zebu population would not support the supposition of Leiqiong cattle population containing blood of Banteng cattle or Bali cattle, but supported the close relationship between Leiqiong cattle and Indian zebu; Dulong gayal population had maternally close relationships with normal cattle and zebu cattle; the results of two subbranches in Haizi water buffalo population supported the supposition of dividing swamp-type water buffalo into two subbranches, and meanwhile, supported the supposition of its independent domestication; the closer relationship between Haizi water buffalo and tamaraw(Bubalus mindorensis)than the lowland anoa(Bubalus depressicornis)supported partially the supposition of swamp-type water buffalo spreading through two different routes to Southeast Asia.4. on the basis of interpopulational differences at separating sites of genes with moderate evolutionary rate, PCR technique could be used to identify products from diffent bovinae populations, with no special requirements for target gene loci. The overall findings aquired the genetic diversity of these target populations, providing important information for a further understanding of interspecies divergence and partial genetic evidence for the protection and utilization of genetic resources in Bovinae livestock; meanwhile, the study explored the genetic divergence of these 6 target Bovinae populations and species with near relationships, providing objective genetic data for overall researches on the origination and evolution of Bovinae livestocks.更多还原