【作者】 李慧芳；

【导师】 李碧春；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 为全面了解中国家鸭遗传资源的现状、遗传多样性及遗传关系,本研究通过原产地调查并采集了来自中国不同地区的24个地方鸭种,通过微卫星标记技术,检测了24个中国地方鸭种1440个个体在28个微卫星基因座的基因型。分析了等位基因频率、群体杂合度(H)、有效等位基因数、多态信息含量、群体间的Nei氏标准遗传距离(DS)和DA遗传距离,采用邻近结合法、主成分分析和群体遗传结构分析等方法,对24个中国地方鸭种的群体内的遗传变异进行了分析。研究结果如下:1.原产地调查显示,我国26个地方鸭品种中,文登黑鸭和中山麻鸭濒临灭绝,现存的24个地方鸭种中,建昌鸭和四川麻鸭处于危险状态,其他22个鸭品种均处于正常状态。2.进行了血样保存方法的比较,研究了乙醇保存家禽血样的效果,结果表明,利用75%的乙醇按4:1的体积比处理家禽血样,不仅操作简便快捷,常温保存较长的时间;而且,从该血样中可以提取高质量的DNA。3.检测了28个微卫星座位在24个地方鸭种中的等位基因数及多态信息含量。28个微卫星座位总共检测到236个等位基因,拥有的等位基因数量非常丰富(5-13),平均值为8.428;检测到134.4个有效等位基因,平均值为4.800。有效等位基因数小于实际观察等位基因数。除座位APL23和APL79的PIC为中度多态外,其它座位的PIC均具有较高的多态性。平均期望杂合度为0.7613,显示出丰富的遗传多样性和较高的选择潜力。各座位的有效等位基因数、平均多态信息含量和期望杂合度三者呈正相关的关系。从品种的角度看,贵州省的三穗鸭和兴义鸭在28个微卫星标记中的平均等位基因数最高,皆为4.25,而平均等位基因数最低出现在福建省的连城白鸭,平均等位基因数只有3.36。4.检测了24个地方鸭品种在同一微卫星座位上等位基因数目和频率的差异。结果显示:24个地方品种拥有27个特有等位基因以及数量相当的优势等位基因,28个微卫星基因座都有优势等位基因存在。24个品种中,三穗鸭的期望杂合度最高,0.6166;其次为微山麻鸭;杂合度最低的为金定鸭,0.5137。24个鸭群体的平均杂合度为0.569,遗传多样性较低,选择的潜力相对较小。5.群体间的遗传变异经F统计量分析,单个座位偏离Hardy-Weinberg平衡的群体数从0到8不等。对于整个群体而言存在着极显著的遗传分化,群体间遗传分化系数达到0.264(P<0.001),并且所有座位都显著地贡献于这一结果(P<0.001);杂合子缺失的水平很低。24个鸭群体之间的Nei氏标准遗传距离DS和DA遗传距离的结果一致。金定鸭和山麻鸭的遗传距离最近;四川麻鸭和汉中麻鸭的遗传距离最远。Nm值变异范围为从0.4620(四川麻鸭—汉中鸭)到1.3692(金定鸭—山麻鸭)。6.利用微卫星DNA标记分析24个中国鸭种遗传距离与地理距离的关联性,对于特定的群体而言,遗传距离与地理距离表现出相当程度的关联性,但24鸭个种间遗传距离和地理距离回归公式:FST/(1-FST) = 0.1976736 +0.0000578ln(d)以及Mantel’s检验的结果(P= 0.19192)并不能为两者间的显著联系提供足够的证据,表明在中国地方鸭品种的形成过程中,各自的地理分布可能并不是影响其群体遗传结构的决定因素。7.利用28对微卫星标记基于DA遗传距离对24个群体的亲缘关系进行分析,结合中国地方鸭种的历史起源、地理位置、生态条件、特定性状以及形态特征等,NJ聚类法将24个中国地方鸭种分为5大类:恩施麻鸭、荆江麻鸭、沔阳麻鸭、建昌鸭、四川麻鸭、靖西大麻鸭和广西小麻鸭聚为一类群;三穗鸭、兴义鸭、云南麻鸭、、连城白鸭、莆田黑鸭、金定鸭和山麻鸭聚为一类群;绍兴鸭和高邮鸭聚为一类;巢湖鸭、汉中麻鸭和大余鸭聚类一类;攸县麻鸭、临武鸭、北鸭、微山麻鸭和淮南麻鸭聚为一类。8.群体结构分析表明24个地方鸭品种1440个个体的平均基因组分数在所属推断类别中的平均基因组分数都大于0.800,表明我国24个鸭群体各具有本品种的特征特性。9.根据实验数据,提出对现有中国地方鸭种的保种措施:可采集组织和血样,贮存DNA信息,分析评价遗传结构的动态变化;在活体保种中,要采取保种场和保种区相结合的保种措施。更多还原

【Abstract】 China, as one of the largest duck producers in the world, is rich in duck genetic resources,and there are many indigenous breeds scattered throughout the country. These breeds vary in size, plumage color and other characteristics. In order to roundly find out the status of genetic resources, genetic diversity and genetic relationship of Chinese indigenous ducks, we had surveyed their numbers, conservation situation, utilization, performance, and collected 24 indigenous ducks from different original regions, examined the genotype of 1440 individuals based on 28 microsatellite loci. The genetic diversity was disclosed by calculating the allele frequency, heterozygosity(H),effective number of alleles, polymorphism information content (PIC), Nei’s standard genetic distance and DA genetic distance. Phylogenetic Relationship was analyzed by the neighbor-joining method (NJ), principal components analysis (PCA), genetic structure analysis, and so on. Our main results were summarized as follows:(1) The survey of Chinese indigenous ducks from original regions showed that Wendeng black duck and Zhongshan duck breeds were at the verge of extinction.Among the existing 24 indigenous duck breeds, Jianchang duck and Sichuan duck breeds were at risk, and 22 others were in normal state.(2) This research had studied the effect of saving poultry’blood with ethanol and explored the method of extracting DNA from these blood samples. Dealing with the blood sample with 75% ethanol at 4:1 on volume, it was simple and rapid to operate and the samples could be saved for a long time at the normal temperature. Others, by the Method of Phenol-Chloroform, we could extract higher quality of DNA which could be used to further molecule biology research from blood.(3) The allele number and polymorphism information content of the 24 indigenous ducks were examined with 28 microsatellite DNA markers. The results showed that 236 alleles were identified, the allele number was abundant, which was from 5 to 13, and the mean allele number was 8.428 per locus.134.4 effective allele numbers was identified, which was less than the observed value actually, the mean effective allele number was 4.800. Except that the PIC of locus of APL23 and APL79 were of medium level genetic diversity, those of other loci were high. The overall average expected heterozygosity was 0.7613, which showed great genetic diversity and high select potentiality. The effective allele numbers, average polymorphism information content and expected heterozygosity per locus were positive correlation. From the breeds, Xingyi ducks and Sansui ducks in 28 microsatellite markers were of the highest average number of alleles,which were 4.25.And the average minimum number of alleles in the Liancheng white ducks, which were only 3.36.(4) The allele frequency differences of the 24 indigenous duck breeds in the same micro-satellite loci were examined. The results showed that,there were 27 private alleles and a large number of dominate alleles at the 28 microsatellite loci of 24 indigenous ducks. The expected heterozygosity of Sansui duck was the highest, which was 0.6166; the second was Weishan duck, and the least was Jingding Duck, which was 0.5137. The overall average heterozygosity of the 24 indigenous ducks was 0.569, which relatively reflected the lower genetic diversity and narrow potentiality of selection.(5) Genetic differences was analyzed by the F statistic analysis, the number of the populations deviated from HWE per locus ranged from 0 to 8. The genetic differentiation among populations had reached to 0.264 (P <0.001), which was very significant for the whole population, all loci were contributed significantly (P <0.001) to this differentiation. And the deficit of heterozygote was observed at a low level. The concrete values of two genetic distances of DA and DS among the 24 indigenous ducks were consistent. The Nm value ranged from 0.4620 (Sichuan duck-Hanzhong duck) to 1.3692 (Jingding duck-Shanma duck). The genetic distance between Jingding duck and Shanma duck was the nearest, and that of Sichuan duck and Hanzhong duck was the most distant.(6) The geographical elements may own to the close relationship for particular population pairs, however, the equation FST/ (1-FST) = 0.1976736 +0.0000578ln (d) and the result from Mantel’s test (P= 0.19192) did not provide enough support for a significant correlation between the genetic and geographical pair wise distances. It was no significant correlation between the genetic diversity of genetic distance and the distributing of these populations. The results concluded that the geographical distributing maybe not the determinant influence on the genetic structure of Chinese chicken populations during the course of their developed history.(7) Combining with the origin of indigenous duck breeds, geography, entironment, special traits, morphologic characteristic, and so on, the phylogenetic relationship based on DA among Chinese indigenous ducks were analyzed, an un-rooted consensus tree was constructed, and the 24 indigenous ducks and divided them into 5 groups. The first group, Ehshi sheldrake, Jingjiang sheldrake and Mianyang sheldrake clustered first, the three populations were all sampled in Hubei province, then clustered with Jianchang duck and Sichuang sheldrake, both in Sichuan province. At last, we clustered a larger colony with Jingxi big sheldrake and Guangxi small sheldrake both from Guangxi province. The second group, Sansui duck and Xingyi duck in Guizhou province, Yunnan sheldrake in Yunnan province and four excellence ducks Liancheng white duck, Putian black duck, Jinding duck and Shan sheldrake in Fujian province clustered a larger colony. The third group, Shaoxing duck in Zhejiang province and Gaoyou duck in Jiangsu province clustered despite a long genetic distance between them. The fourth group, Chaohu duck in Anhui province and Hanzhong sheldrake in Shangxi province clustered first, then clustered with Dayu duck in Jiangxi province. The fifth group, Youxian sheldrake in Hunan province and Linwu duck clustered first, then a larger colony with Beijing duck, Weishan sheldrake in Shandong province and Huainan sheldrake in Henan province clustered at last.(8) For the analysis of population structure from STRUCTURE, the average gene score of the 1440 individuals of the 24 local ducks was more than 0.800 in their concluded sorts respectively. And this reflected the 24 populations all held their own special characteristics respectively.(9) Based on the experiment data and the actuality of Chinese indigenous duck breeds, it is suggested that we should collect the sample of tissue and blood to reserve DNA, which can be used to analyze dynamic change of the genetic structure. During the conservation in vivo, it is necessary to take the measures of combinations of conservation farm with conservation area.更多还原