【作者】 孙玉江；

【导师】 芒来；

【作者基本信息】 内蒙古农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 中国西南马是我国马种主要生态类型之一,经历了遗传变异和自然选择的长期进化过程,在西南地区逐渐分化形成了百色马、贵州马、文山马、乌蒙马、大理马、建昌马、腾冲马、中甸马等优良类群。受其特殊历史、自然、社会诸多条件的影响,西南马形成体形矮小、结构紧凑、抗逆性强、善走山路等特点,可以广泛用于科学研究、观光旅游、艺术表演、骑乘培训、驮负运输等,是中国乃至世界原始矮马资源的宝贵基因库。可见,中国西南马在遗传资源保护利用和现代马业发展中具有重要地位。但国内外关于西南马遗传多样性、起源进化、亲缘关系、资源分布、矮小机理等方面的研究还相对缺乏。本课题在对西南地区马进行初步调查的基础上,运用SAS、SPSS统计分析软件对西南马资源特征进行分析;通过PCR-SSCP技术、PCR产物纯化直接测序等方法,对七个类群的西南马及引入品种Shetland马共150个样品的mtDNA D-Loop区、ND3、ND4、ND4L基因和核内SHOX基因的多态性进行了检测,并进行了多态性与体尺指标相关分析,了解了西南马群体资源表型、分子特征。本文得出以下基本结论:(1)中国西南马在数量上已经成为我国五大类型马的第一大类群,影响其数量分布的主要因子是人均农机、人口密度、辖区比重等。非密度制约因子如年无霜期、降水量、日照时数和平均气温等自然因素均与中国西南马体高呈强相关,对不同类群马的形成可能起重要作用。(2)对中国西南马mtDNA D-Loop位点进行了研究,结果表明:中国西南马种内遗传多样性相对丰富,在类型间和类型内遗传分化不显著(P>0.05);中国西南马具有多个母系起源,且与蒙古国蒙古马亲缘关系较近,部分西南马起源于普氏野马或蒙古野马。(3)在ND4L、ND4基因上游和下游检测到多态:ND4L和ND4上游片段出现两种单倍型(定义为A和B),而且B单倍型出现频率均略高于A单倍型;ND4下游片段中,出现四种单倍型,其中一个为共享单倍型,三个为稀有单倍型。(4)西南马线粒体ND4基因和ND4L基因核苷酸组成的偏倚不严重,但在密码子第二和第三位点上,C的含量远远大于G含量,而且密码子的使用存在偏倚。(5)所研究的西南马(不包括建昌马)各类群内核苷酸多样性(Pi)较高,在0.00467至0.00737之间,其中大理马最低,乌蒙马最高。各类群间的遗传距离很小,而乌蒙马与其它西南马的遗传距离最大(0.006);类群间的核苷酸分化系数(Nst)在-0.35665至0.01694之间,乌蒙马与其它马种的核苷酸分化系数均较大,且同处云南地区的乌蒙马与大理马之间的Nst值最大,可见乌蒙马与大理马存在遗传分化。(6)本文对ND4下游片段的多态性与体尺性状进行了关联分析,结果表明基因型对体长影响显著(P<0.05):D基因型的体长较其余基因型大;对体高和管围的影响接近显著水平(P =0.0809和0.0753),对胸围影响不显著。(7)西南马种内遗传多样性低,类群间的遗传距离小,遗传相似度高。乌蒙马独立性最高(Pi=0.00737),可作为独立的管理单元;大理马核苷酸多样度最低(Pi=0.00467),分化最小(k=3.167),保持着类群特有的遗传特质,应该得到良好的保护。(8)本研究还首次克隆了西南马矮小性状基因SHOX的部分序列。其长度为520bp,与其他物种已知的SHOX基因序列比对,发现克隆所得的序列是马SHOX基因编码区的部分序列,包含了完整的第二外显子。(9)通过对马SHOX基因第二外显子序列与其它物种相应区域序列进行比较发现:不同物种间的第二外显子较保守,未出现不同片段的插入、缺失及短串联重复序列多态现象,且马的SHOX基因富含GC(7个类群马平均为67.85%)。(10)通过PCR-SSCP分析,检测到在SHOX基因P2位点268处有一个G→A的突变, 431处有一个T→G的突变,导致了等位基因B变为等位基因A。(11)不同类群马SHOX基因P2位点纯合度(Ho)、杂合度(He)、有效等位基因数(Ne)及多态信息含量(PIC)分析表明:多态信息含量(PIC)以文山马最高,为0.513,处于高度多态;以设特兰马为最低,仅为0.222。更多还原

【Abstract】 Chinese Southwest horse distributes throughout the Southwestern region of China, and is one of the most important composition of Chinese horses owing to their large number and abundant breed types. During undergoing genetic variation and natural selection of a long-term evolutionary course, the different typed populations have been gradually differentiated, such as Baise horse, Guizhou horse, Wenshan horse, Wumeng horse, Dali horse, Jianchang horse, Tengchong horse and Zhongdian horse, and so on. Chinese Southwest horse populations are one of the original and natural pony breeds in the resource pool of China and even the world. Chinese southwest horse is valuable genetic resources of human being because of some good qualities including dwarf somatotype, compact structure, strong stress resistance and being good at walking rugged mountain road. So can be used as materials or tools of scientific research, sightseeing, artistic performance, ridding training, and transportation. In view of this, Chinese southwest horse type populations own important statuses for protective utilization of genetic resource. The profundity of research work was few about Chinese southwest horse’s genetic diversity, origin evolution, genetic relationship, resources distribution, and the mechanism of dwarfishness. Based on a preliminary investigation for southwest region of China, including seven local breeds and a foreign variety, Shetland, totaled 150 samples, we detected the polymorphisms of D-Loop region, ND3, ND4, ND4L on mitochondrial DNA and SHOX on nuclear DNA by PCR-SSCP, sequencing, and other methods, and analyzed the resource characteristics of South-west horse by using SAS, SPSS statistical analysis software. And we conducted a correlation analysis between polymorphisms and body measurements, further understanding the colony resource’s phenotypic and molecular characteristics of Chinese southwest horse populations, the basic conclusions were followed:(1) Considering the amount of Chinese southwest horse as it stands, it has become the largest group in the five main groups in China. The main factors of influencing the abundance distribution were: agricultural machinery per capita, population density, administrative district ratio, and so on. The density-independent factors were taken into account and annual frost-free period, precipitation, sunshine hours, the average temperature have powerful effect for Chinese southwest horses and they might play a leading role in the formation of different Chinese southwest horse types. (2) The mtDNA D-Loop of Chinese southwest horses were detected and sequenced. The results showed that there is relatively abundant genetic diversity in Chinese southwest horse populations and the he genetic differentiation of Chinese southwest horse within the types and among the types is not significant (P>0.05). And more Chinese southwest horse came of multiple female ancestors, close genetic relationship with Mongolian horse of Mongolia, and several South-west horse populations originated from Przewalskii horse, namely Mongolian wild horse.(3) Polymorphisms in ND4L, upstream and downstream of ND4 gene were detected: ND4L and upstream of the ND4 fragment both exist two haplotypes (defined as A and B), and the frequency of B haplotype were slightly higher than the A haplotype; in the downstream of ND4 fragments, there are four haplotypes, and one is shared haplotype, the others are rare ones.(4) The bias of nucleotide composition of ND4 and ND4L genes in South-west horse is not critical, but on the sites of the second and third codons, the content of C is far greater than the content of G, and found bias exist in the usage of the codons.(5) Nucleotide diversity (Pi) of six groups(excluding Jianchang horse) within Chinese southwest horse alternates from 0.00467 to 0.00737, and the results showed abundant polymorphism appeared inter-groups. Genetic distance is lower among all types, and the genetic distance between Wumeng horse and the others are the greatest (0.006). The Index of Nucleotide Differentiation (Nst) among six groups varies between -0.35665 to 0.01694 and Nst of Wumeng horse and other groups are all relatively big. The Nst between Wumeng and Dali horse is the largest one, despite in the same district, therefore genetic differentiation exists between Wumeng horse and Dali horse.(6) The correlation analysis between the polymorphisms of ND4 downstream fragment and body size showed that the genotype had significant impact on body length (P <0.05): the effect of genotype D is the largest one among all the genotype effects; the impacts of genotype on height and cannon circumference were close to a significant level (P = 0.0809 and 0.0753), the chest circumference was not significantly affected.(7) Chinese southwest horse showed low genetic diversity, close genetic distance, and high genetic similarity within groups. The independence of Wu Meng horse is the highest (Pi=0.00737) one, so can be tackled as an independent management unit; nucleotide diversity of Dali horse is minimum (Pi=0.00467), and the coefficient of division is smallest (k= 3.167), maintained as a unique group of genetic specialty, so it should be well protected. (8 In the paper, we cloned a partial sequence of the SHOX gene about dwarfism trait of pony for the first time. The length of the sequence is 520bp. After aligning, showed that the sequence we acquired is partial CDS of SHOX gene, including the complete the second exon.(9) By aligning with other species on the homologous sequence of the second exon of SHOX gene, we discovered that the second exon is more conservative among different species, no Indels(insertion and deletion) and STRs(short tandem repeat) were found in the region, and the SHOX gene of horse is GC-rich (the average value of seven groups is 67.85%).(10) Detecting the variations in the SHOX gene via PCR-SSCP, we found in the P2 locus of 268 existed a G→A transition, and at 431 was a T→G transversion, resulting in the B allele into allele A.(11) The analysis of SHOX gene P2 locus about the gene heterozygosity(He), the gene homogeneity index(Ho), the number of effective allele(Ne), and polymorphic information content (PIC) showed that: the value of PIC of Yunnan Wenshan horse is significant than the others, and it is 0.513, in a high polymorphic degree; Sheteland horses were the lowest, only is 0.222.更多还原