【作者】 亐开兴；

【导师】 昝林森； 张亚平；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 线粒体的生理功能是产热维持体温和为机体的活动提供ATP。线粒体突变对供能相关的选择压力相当敏感,适宜于检测物种对外界环境的适应性,因此,研究线粒体基因可以揭示物种对缺氧、低温的高海拔环境的适应性进化。牛伴随着人类的迁移与农耕文明,在人类的历史文明中扮演着重要的经济、文化及宗教角色,其中家养牛受到了人类的驯化,而牦牛经受着低氧、寒冷等多重高原考验,它们为研究牛的驯化及对极端环境的适应性进化遗传机制提供了很好的生物学模型。本研究在探讨牛亚科物种的系统发育关系和群体扩张的基础上,既了解其起源和进化历史,还对家养牛和牦牛的线粒体DNAATPase复合体基因开展研究,探讨牛亚科物种在驯化后及对不同地域的适应性进化机制。此外,我们还开展了云南大额牛的遗传多样性研究以及大额牛(Bos frontalis,冻精)与婆罗门牛(Bos indicus)的种间杂交试验,从细胞遗传学与分子生物学的角度为大额牛与婆罗门牛种间杂交的可行性提供科学依据,为今后大额牛的保护、杂交利用提供遗传学信息。1、本研究通过对牛亚科物种mtDNA ATPase复合体基因(ATP8+ATP6)882bp的合并,并以牦牛(家牦牛+野牦牛)、家养牛(普通牛+瘤牛)为两个数据集同时融入下文的分析中,得到以下结论：1.1基于牛亚科物种mtDNA ATPase复合体基因构建的系统发育树将牛亚科分为5个明显的支系(以海口水牛为外群),即美洲野牛Bison.瘤牛Zebu.普通牛Taurus.牦牛Yak和亚洲野牛/大额牛(Gaur/Mithun)。美洲野牛Bison与牦牛Yak、普通牛Taurus与瘤牛：Zebu互为两组姊妹群,与亚洲野牛关系较远。所构建的牛亚科系统发育关系与以前的结果相吻合。此外,所有的拓扑结构表明大额牛中有一支聚为单系,与别的牛种分开。在云南大额牛ATPase复合体基因单倍型中,20.6%属于普通牛支系,20.6%属于瘤牛支系,其余58.8%属于亚洲野牛/大额牛支系,因此云南大额牛具有其独特的遗传基础,与印度大额牛关系最亲近。牛亚科物种的单倍型多样度与核苷酸多态度较低,分别在0.348～0.724之间与0.058%～0.259%之间。1.2Network中介网络图和碱基错配分布图显示,牛亚科物种中,牦牛(Tajima’s D=-1.838,P<0.05:Fu and Li’s D*=-5.489,P<0.02)、家牦牛(Tajima’s D=-1.744,0.10>P>0.05;Fu and Li’s D*=-4.487,P<0.02)和普通牛Taurus(Tajima’s D=-2.198, P<0.01;Fu and Li’s D*=-3.133,P<0.05)者经历了显著的群体扩张；尽管瘤牛Zebu的Tajima’s D检验未达到显著偏离中性,且为一条不太光滑的碱基错配分布图,Fu and Li’s D*检验却表明Zebu显著偏离了中性进化,有群体扩张迹象(Fu and Li’s D*=-3.691, P<0.02;Tajima’s D=-1.314,P>0.10)。而美洲野牛Bison(Tajima’s D=-0.118,P>0.10)与亚洲野牛/大额牛(Gaur/Mithun,Tajima’s D=1.977,P>0.10)无群体扩张,与目前二者分布地域狭窄,群体较小相符合。1.3基于系统发育树的比较分析,mtDNA ATPase复合体基因在牛亚科物种中的Ka/Ks<<1,表明该基因经受了强烈的纯净化选择。相比之下,MK检验两两比较群体内与群体间的非同义突变与同义突变之比值的差异水平,结果显示牦牛与其它牛种之间呈显著差异(P<0.05或P<0.01),表明ATPase复合体基因可能在牦牛经历了适应性进化过程,以提供它们适应低氧高海拔环境所需的能量供应。此外,MK检验结果还显示,家养牛与其它牛种间也呈显著差异(P<0.05或P<0.01),推测可能与家养牛在驯化后发生了显著的体型变化及基础代谢速率的调整有关。中性指数NI值高于1,表明mtDNA ATPase复合体基因在牛亚科上积累了过多的非同义突变和氨基酸多态性。对应的ATP6基因的分析结果与ATPase复合体基因出入不大,但ATP8基因由于序列太短,信息较少,功能保守而与ATPase复合体基因的结果差异较大。同样的分析表明,ND6基因在牛亚科物种经历了强烈的纯净化选择。2、通过同期发情处理的婆罗门牛经人工授精(大额牛冻精)成功获得种间杂交大婆F1代,通过传统的染色进行细胞遗传学的核型观察及大额牛第1号染色体为整条探针进行荧光原位杂交,以及微卫星DNA标记扫描揭示大额牛的遗传多样性和种间杂交F1代的亲子鉴定,得到以下结果：2.1大额牛的核型2n=58(N=3,3♀),而外形一致的大额牛中也有核型异常的2n=59个体(N=2,1♂1♀)。2.2大额牛与婆罗门牛种间杂交的大婆F1代核型为2n=59,为双亲的中间类型,染色体间发生了rob(2；28)罗伯逊易位。大额牛与婆罗门牛种间杂交F1代的母牛无论在放牧条件下本交或是进行人工授精都能育,对15月龄和24月龄的F1代公牛进行电刺激采精,观察不到精子的生成,推测F1代公牛没有生育能力。杂交后代雌性能育,雄性不育。2.3通过用牛微卫星引物分子标记对云南大额牛群体扫描,发现云南大额牛的期望杂合度(Hexp)和多态信息含量(PIC)低于其它肉牛品种,分别为0.6332和0.5965。经Bottleneck检验,云南大额牛并未经历瓶颈效应,而是受到了来自家养牛的基因渐渗。这些结果也表明牛微卫星引物对大额牛的实用性,利于对其遗传背景的揭示和系统发育关系的构建。2.4对大婆F1代进行的亲子鉴定表明,已知一亲本基因型、已知一亲本基因型与性别两种情形的联合非父排除率(PEc)分别为99.9858%与99.9999%,证实大额牛(BOS frontalis)与婆罗门牛(Bos indicus)种间杂交的可行性,从分子生物学的角度证实了大额牛与家养牛种间杂交的可行性。鉴于此结果,我们也建议对Bos属的大额牛遗传资源进行圈养或保护区划定进行保护,以避免这一珍稀牛种再受来自家养牛的遗传侵蚀。更多还原

【Abstract】 The major functions of mitochondrion are to generate heat to maintain body temperature and ATP for action in organisms. The mitochondrial DNA (mtDNA) mutations were considered as extremely sensitive to energy-related selective pressure and available to test the adaptation of species to the environment via mtDNA genes. Cattle have played important roles in economic, cultural and religious values accompanying the migration and farming in human history. Domestic cattle were domesticated by human, while yak have always undergone hypoxia and cold temperature besides domestication in Qinghai-Tibetan Plateau. So, they had become the ideal biological models for testing domestication and adaptative evolution to the extreme inviroment. Herein, mtDNA ATPase synthesis genes were amplified to analyze the phylogeny, population expansion and adaptive evolution after domestication and in different zones in Bovini species. In addition, we investigated the genetic diversity of Yunnan mithun population and performed the interspecies crossing between mithun (Bos frontalis, frozen semen) and zebu(Bos indicus) to provide cytogenetic and molecular evidences that allowed for the potential of interspecies crossing. It will be beneficially to provide the genetic information to promote the protection and hybridization utility in mithun. The results were as follows:1Data set (including ATPase8and ATPase6genes,882bp) was concatenated as ATPase synthesis gene, and two combined data sets from Yak (Domestic yak+Wild yak) and Domestic cattle (Taurus+Zebu) were integrated into the analyzing in the context:1.1ATPase synthesis gene was combined to construct phylogenetic trees of Bovini species with five distinct clades, Bison, Yak, Zebu, Taurus and Gaur. The results were consistent with the previous phylogeny of Bovini, which clearly showed sister-groups relationships to Yak-Bison, Taurus-Zebu, with far relationship to Gaur, when Haikou buffalo used as outgroup. All tree topologies indicated that one of mithun formed its own monophyletic clade, distinct from other species of the Bovini; in addition, Yunnan mithun possessed its own58.8%genetic components except20.6%taurine and20.6%i ndicine lineages, closest to Indian mithun based on the haplotypes of ATPase synthesis gene. Haplotype diversity and nucleotide diversity in Bovini were lower, ranging from0.348-0.724and0.058-0.259%, respectively.1.2Star-burst patterns observed for the clades and differences in the shape of each mismatch distribution prompted an analysis of population demography. The presence of population expansions were supported for clades of Yak (Tajima’s D=-1.838, P<0.05; Fu and Li’s D*=-5.489, P<0.02), Domestic yak (Tajima’s D=-1.744,0.10> P>0.05; Fu and Li’s D*=-4.487, P<0.02), and Taurus (Tajima’s D=-2.198, P<0.01; Fu and Li’s D*=-3.133, P<0.05). The Zebu clade has an ambiguous signature that showed significant deviation from neutrality for the Fu and Li’s D*test (Fu and Li’s D*=-3.691, P<0.02; Tajima’s D=-1.314, P>0.10), although not for the Tajima’s D test and an uncompleted smoothly unimodal of mismatch distribution. The absences of population expansions were rejected for clades of Bison (Tajima’s D=-0.118, P>0.10) and Gaur (Tajima’s D=1.977, P>0.10), of which the scenarios were better to fit the narrow distributions and small population sizes of bison and mithun.1.3To detect variations in selective pressure along the different branches of a phylogenetic tree, all the comparisons of Ka/Ks for ATPase synthesis gene from the clades revealed Ka/Ks values were far less than1.0, indicating ATPase complex evolved globally under strong purifying selection in Bovini species. By comparing the relative rates of nonsynonymous and synonymous substitutions in ATPase synthesis gene from inter-and intra-clades, we inferred that Yak (domestics cattle and yak) had a higher excess of nonsynonymous mutations in the ATPase synthesis gene than those in other clades, indicating adaptive changes related to tolerate low temperature and hypoxia in yak than other Bovini leaning on the ATPase complex generated the energy of basal metabolism in high-altitude environment by MK test. Similarly, through MK test, domestic cattle had a higher excess of nonsynonymous mutations than those in other Bovini clades, probably indicating domestic cattle adjusted its basal metabolism and decreased in body size. Neutrality indices (NI) were higher than1.0in Bovini species showing an excess of nonsynonymous mutations and amino acid polymorphism. The counterparts from ATP6gene in the corresponding statistics were similar to those of ATPase synthesis gene, while they differentiated from ATP8gene because of short fragment, less informative and conservational functions. ND6gene was under strong purifying selection in Bovini species based on the same analyses.2Through estrus synchronization, Brahman cows were inseminated artificially with mithun’s frozen semen to gain the F1generations of interspecies crossing. Metaphase chromosome spreads were analyzed by conventional staining and fluorescence in situ hybridization with the whole chromosome1of mithun as a specific probe. And microsatellite DN A markers were scanned to reveal the genetic diversity of mithun and parentage testing of F1generation of interspecies crossing. The results were as follows:2.1Mithun (Bos frontalis) had a karyotype2n=58(N=3,3♀), but abnormal karyotype had2n=59(N=2,16(?)1♀) with a normal mithun phenotype.2.2The F1karyotypes of mithun×Brahman cow had2n=59, intermediate between their parents, consistent heterozygous carriers with a centric fusion involving rob(2;28). The female hybrids normally reproduced with whatever mated, both parents indicating their fertility when mating naturally or artificial insemination. Meanwhile, the F1sires did not produce sperm at the age of15and24months as assessed by electroejaculation to confirm that the F1bull is sterile. The female hybrids are fertile, and the male are sterile.2.3The expected heterozygosity (He) and polymorphism information content (PIC) of Yunnan mithun population were lowest amongst the beef breeds revealed by microsatellite markers,0.6332and0.5965, respectively. Yunnan mithun population hadn’t yet undergone severe bottleneck effect while suffering moderate gene introgression from local cattle (Bos taurus or Bos indicus) by Bottleneck, showing the availability of bovine microsatellite markers to facilitate the genetic background and phylogeny of mithun.2.4According to parentage testing from Cervus, two PEc of combined non-exclusion probability (first parent), combined non-exclusion probability (second parent) were99.9858%and99.9999%, respectively. These displayed the potential of interspecies crossing between mithun (Bos frontalis) and Brahman (Bos indicus) based upon molecular biological insights. As a rare genetic resource of genus Bos, it is suggested that mithun population will be under captive breeding, or to establish Mithun Preserve to protect and avoid subsequent genetic erosion from domestic cattle due to its crossing facilitation.更多还原