【作者】 包文斌；

【导师】 陈国宏；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 本试验使用多重PCR结合全自动电泳技术,利用29对国际通用的微卫星引物对仙居鸡、茶花鸡、鹿苑鸡、白耳鸡、藏鸡、固始鸡、大骨鸡、河南斗鸡、狼山鸡、泰和乌骨鸡、萧山鸡、北京油鸡、淮南麻黄鸡和皖南三黄鸡等14个中国家鸡品种以及红色原鸡Gallus gallus spadiceus亚种和Gallus gallus gallus亚种的570个个体进行扫描,讨论样本量、性别和微卫星座位数对微卫星分析中群体遗传多样性指标的影响以及近缘物种间微卫星引物的适用性,同时对中国家鸡和红色原鸡的256个个体mtDNA D-loop序列进行系统分析,利用微卫星DNA和mtDNA共同评估群体的遗传结构和遗传关系,分析群体间和群体内的遗传变异,探讨中国家鸡和红色原鸡的亲缘关系。主要研究结果如下:1.利用29对微卫星标记对16个群体内和群体间的遗传多样性进行分析,共检测到286个等位基因,平均值为9.86±6.36,所有群体的期望杂合度为0.6708±0.0251,PIC值为0.52,29个微卫星位点均具有较高的多态性。单个位点偏离Hardy-Weinberg平衡的群体数从0到7不等。群体间平均遗传分化为16.7%(P <0.001),所有的位点都极显著地贡献于这一结果(P <0.001);杂合子缺失的水平很高,为0.015(P <0.01);中国家鸡和红色原鸡16个群体间存在着极显著的遗传分化。群体间的Reynolds’遗传距离从0.036(萧山鸡-鹿苑鸡)到0.371(泰国红色原鸡-河南斗鸡)不等,而Nm值变异范围从0.583(泰国红色原鸡-河南斗鸡)到5.833(萧山鸡-鹿苑鸡)。2.利用29对微卫星标记对16个群体的亲缘关系进行分析,NJ系统发生树及Structure程序运行结果显示, 16个群体可以分成轻体型的鸡种(包括8个群体:泰国红色原鸡、中国红色原鸡、茶花鸡、藏鸡、仙居鸡、固始鸡、白耳鸡和泰和乌骨鸡)和重体型的鸡种(包括8个群体:皖南三黄鸡、淮南麻黄鸡、大骨鸡、河南斗鸡、狼山鸡、北京油鸡、鹿苑鸡和萧山鸡)两大类。藏鸡、皖南三黄鸡和淮南麻黄鸡遗传基础非常复杂,鹿苑鸡和萧山鸡彼此之间遗传基础十分类似。茶花鸡和藏鸡与中国红色原鸡存在着较近的亲缘关系,与泰国红色原鸡亲缘关系相对较远。中国家鸡和红色原鸡两个亚种的亲缘关系从近到远的排序是:进化型品种-原始型品种(茶花鸡与藏鸡)-中国红色原鸡(Gallus gallus spadiceus亚种)-泰国红色原鸡(Gallus gallus gallus亚种)。3.利用微卫星DNA和mtDNA遗传标记分析15个中国鸡种遗传距离与地理距离的关联性,对于特定的群体而言,微卫星DNA和mtDNA遗传距离与地理距离表现出相当程度的关联性,但15个鸡种间遗传距离和地理距离回归公式:FST/(1-FST) =–1.0283–0.0407ln(d)以及Mantel’s检验的结果(P=0.596)并不能为遗传距离与地理距离之间的显著联系提供足够的证据。线粒体D-loop序列的差异与群体间的地理分布也没有相关。中国地方鸡种的形成过程中,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。4.以实际测定的29个微卫星座位的基因频率为基础,分析样本量、性别和微卫星座位数对微卫星分析中群体遗传多样性指标的影响。结果表明:当样本量超过20后,期望杂合度值趋于稳定,样本量以20-25较为适宜,样本量与期望杂合度无显著相关,而与平均等位基因数呈正相关;微卫星位点多态性的高低直接影响到检测所需的样本量,在使用平均等位基因数分析群体遗传多样性时,应该充分考虑样本量对检测结果的影响;期望杂合度受样本量变动的影响较小,可作为度量群体遗传多样性的一个最适参数;在微卫星分析中性别对群体遗传多样性指标不表现出显著影响;随着微卫星数目的增多,遗传距离估测精确度精度也随着升高。5.对近缘物种间微卫星引物的适用性进行尝试,利用29对鸡微卫星标记对孔雀基因组DNA进行种间扩增,发现14对引物能扩增出特异性条带,每对引物扩增的平均等位基因数为1.71,有7对引物具有较丰富的多态性,其中MCW0080和MCW0098最为理想。蓝孔雀和绿孔雀群体间和群体内的遗传分析结果表明,绿孔雀和蓝孔雀两个群体的期望杂合度分别为0.7422和0.6943,群体间的遗传分化系数为0.078,Reynolds’遗传距离和基因流分别为0.0603和3.896,结果显示这两个孔雀群体的杂合度和遗传多样性水平都很低,且有相互混杂的趋势。6.对中国家鸡和红色原鸡的256个个体mtDNA D-loop序列进行系统分析,测定16个群体线粒体D-loop部分序列大小约为560bp,A、C、G、T这4种核苷酸的平均比例分别为25.00%、37.40%、4.40%和33.20%。A+T含量58.20%,G+C含量41.80%, A+T含量高于G+C含量;共发现44个变异位点,约占分析位点总数的7.86%,没有观测到插入/缺失,颠换和转换之比为0.13;共具有32种单倍型,9种为共享单倍型,其它23个单倍型均为各群体所特有;16个群体内单倍型多样度差异很大,从0到0.964,单倍型变异度总体为0.909±0.014,固始鸡的核苷酸多样度最低,淮南麻黄鸡和皖南三黄鸡的核苷酸多样度较高,红色原鸡两个亚种和14个中国地方鸡种整体的平均核苷酸差异数为7.276,核苷酸多样度为1.851%。16个群体表现出较高水平的遗传多态性。群体间核苷酸分歧度(Dxy)在0.747%～3.125%之间变化,核苷酸净遗传距离(Da)为-0.015%～2.633%,核苷酸分歧度(Dxy)和核苷酸净遗传距离差异均较大。红色原鸡2个亚种和14个中国地方鸡种间kimura双参数距离变异范围为0.007～0.031。mtDNA D-loop环序列群体间(Va)的方差组分占总变异的23.83%,Fst=0.38155,差异显著(P<0.05)。红色原鸡两个亚种和14个中国地方鸡种间表现出显著的遗传分化。7.对16个群体mtDNA D-loop单倍型进行分子系统树和网络关系分析,以日本鹌鹑(Coturnix japonica)为外群(Genbank登录号:D82924),16个鸡种mtDNA D-loop区32种单倍型的NJ、ME和UPGMA分子系统树均分为明显的4个类群,单倍型类群A中包含有泰国红色原鸡Gallus gallus gallus亚种的单倍型;单倍型类群B和单倍型类群C中包含有中国红色原鸡Gallus gallus spadiceus亚种的单倍型;单倍型类群D中同时含有这两个红色原鸡的单倍型。单倍型网络关系图中序列明显也聚为4个聚类簇,与单倍型系统发生树的结果完全一致。利用Kimura双参数模型构建D-loop区的分子系统树中,固始鸡、仙居鸡始终与泰国红色原鸡Gallus gallus gallus亚种聚在一起,茶花鸡、藏鸡、泰和乌骨鸡、河南斗鸡和白耳鸡也始终出现在一个类群。8.对中国红色原鸡Gallus gallus spadiceus亚种与泰国红色原鸡Gallus gallus gallus亚种微卫星DNA和mtDNA的多态性进行分析,中国红色原鸡Gallus gallus spadiceus亚种与泰国红色原鸡Gallus gallus gallus亚种的微卫星DNA遗传距离为0.167,Nm值为1.040。没有表现出较近的遗传距离和较大的基因流动,群体的遗传分化系数为0.194(P<0.01),所有位点都极显著地贡献于这一结果(P<0.01)。红色原鸡两个亚种没有共享单倍型,它们各自具有不同的单倍型,群体间mtDNA D-loop Fst值差异显著(P=0.0360)。Gallus gallus spadiceus亚种和Gallus gallus gallus亚种群体具有不同的群体遗传结构,群体之间存在明显的遗传分化,本研究支持这两个亚种并非实际上是同一个亚种的观点。9.综合分析中国家鸡和红色原鸡微卫星DNA和mtDNA多态性和亲缘关系的结果,推测泰国红色原鸡Gallus gallus gallus亚种被驯化后,有部分群体演化形成了一些中国家鸡的群体如固始鸡和仙居鸡,而中国红色原鸡Gallus gallus spadiceus亚种被驯化后,也演化形成了一些中国家鸡的群体如茶花鸡和藏鸡等。泰国红色原鸡Gallus gallus gallus亚种中性检验的Tajima’s D值为-1.79995(P< 0.05),不符合中性突变。在泰国红色原鸡Gallus gallus gallus亚种群体一段时间内的群体扩张过程中,对一些在中国本地起源的家鸡群体中的一些亚群产生了影响,因此在一些中国地方鸡种同时具有这两种红色原鸡的遗传贡献。本研究认为中国家鸡起源于泰国或单纯起源于中国的观点都是不全面的,支持红色原鸡的驯化是多次、多地、长期的人类活动的结果这一观点。更多还原

【Abstract】 Combining the technique of multiplex-PCR and the fluorescent automated diction, genetic diversity and phylogenetic relationship among 570 individuals of 14 Chinese domestic chicken breeds(Xianju chicken, Chahua chicken, Luyuan chicken, Baier chicken, Tibetan chicken, Gushi chicken, Dagu Chicken, Henan Game, Langshan chicken, Taihe Silkies chicken, Xiaoshan chicken,Beijing Fatty chicken,Huainan Partridge and Wannan Three-yellow chicken) and two red jungle fowl subspecies(Gallus gallus spadiceus in China and Gallus gallus gallus in Thailand)were evaluated with 29 microsatellite loci. The effects of sample size, sex and number of microsatellite loci on various genetic diversity measures were estimated, and similar part of mtDNA D-loop of these 16 populations were sequenced and analyzed. Genetic variability within populations and genetic differentiation among populations were estimated, thereafter genetic diversity and phylogenetic relationship among red jungle fowls and Chinese domestic fowls were analyzed. Our main results were summarized as follows:1. The genetic variability within populations and genetic differentiation among populations were estimated, a total of 286 alleles were detected in 16 populations with 29 microsatellite markers, and the average number of observed alleles was 9.86±6.36. The overall expected heterozygosity of all populations and PIC of all loci were 0.6708±0.0251 and 0.52, respectively. All 29 microsatellite loci in this study showed high levels of polymorphism. The number of populations deviated from Hardy-Weinberg equilibrium per locus ranged from 0 to 7. In the whole population, the average of genetic differentiation among populations, measured as FST value, was 16.7% (P <0.001), and all loci were contributed significantly (P <0.001) to this differentiation. Significant genetic differentiation was observed among two subspecies of red jungle fowl and 14 Chinese domestic chicken breeds, and the deficit of heterozygote was observed very high (0.015) (P <0.01). Reynolds’distance values varied between 0.036 (Xiaoshan chicken-Luyuan chicken pair) and 0.371 (Gallus gallus gallus–Henan Game chicken pair). The Nm value was ranged from 0.583 (between Gallus gallus gallus and Henan Game chicken) to 5.833 (between Xiaoshan chicken and Luyuan chicken).2. The phylogenetic relationship among Chinese domestic fowls and red jungle fowls were analyzed, an un-rooted consensus tree was constructed using the Neighbour-Joining method and the clustering results are generally in accordance with the results obtained from STRUCTURE. The tree topology revealed two main clusters, although the relationship between breeds was not always supported by high bootstrap values. The heavy-body sized chicken breeds, Huainan Partridge, Wannan Three-yellow chicken, Dagu chicken, Henan Game, Langshan chicken, Beijing Fatty chicken, Luyuan chicken and Xiaoshan chicken formed in one cluster; and the light-body sized chicken breeds, including Red Jungle Fowl in China, Red Jungle Fowl in Thailand, Chahua chicken, Tibetan chicken, Xianju chicken, Gushi chicken, Baier chicken and Taihe Silkies chicken formed in the second main cluster. The results suggested that the Tibetan, Huainan Partridge and Wannan Three-yellow chickens have mixed genetic structures, while the genetic bases of Luyuan chicken and Xiaoshan chicken are nearly same. Chahua chicken and Tibetan chicken had closer genetic relationship with Gallus gallus spadiceus but appeared rather farer phylogenetic relationship with Gallus gallus gallus. The evolutional dendrogram was as follows: evolutional breeds ? primitive breeds (Chahua chicken and Tibetan) ? red jungle fowl in China (Gallus gallus spadiceus ) ? red jungle fowl in Thailand (Gallus gallus gallus )3. The geographical elements may own to the close relationship for particular population pairs, however, the equation FST/ (1-FST) =–1.0283–0.0407ln (d) and the result from Mantel’s test (P=0.596) 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 mtDNA D-loop 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.4. Based on the analysis of genetic diversity in 4 Chinese indigenous chicken breeds at 29 microsatellite loci, the effects of sample size and sex on various genetic diversity measures were estimated, the accuracy of pair wise genetic distance among 4 Chinese native chickens were analyzed at 5, 10, 15, 20 and 25 microsatellite loci levels. The results indicated that the expected heterozygosity was fairly stable when sample size was over 20, the mean number of allele over loci was significantly affected by sample size, while there is no significant correlation between sample size and expected heterozygosity, the suitable sample size in microsatellite analysis ranged from 20 to 25. The sample size needed in the study was affected by the polymorphism of microsatellite loci. When the mean number of allele over loci has been chosen to detect the population diversity, the effect of sample size variation should be assessed deliberately, while the expected heterozygosity was not sensitive to sample size variation and can be used as a reliable parameter to estimate the genetic diversity. The genetic diversity measures were not significantly difference between sexes, and the accuracy of genetic distance estimation increased along with the numbers of the loci increased.5. The applicability of microsatellite primers from chicken to peafowl population was analyzed, the results showed that the 14 of the 29 pairs of microsatellite primers from chicken could amplify peafowl DNA and produce special allele patterns, with 1.71 mean alleles per locus. Seven pairs of primer had higher polymorphism, among them MCW0080 and MCW0098 primers were perfect markers for peafowl. Based on the analysis of genetic diversity within and between green peafowl and the blue peafowl population, our results demonstrated the expected heterozygosity of two peafowl populations were 0.2482 and 0.2744, respectively. Inbreeding index (FST), Reynolds’ genetic distance and gene flow between two populations were 0.078, 0.0603 and 3.896, respectively. These results indicated that the heterozygosity and the genetic diversity of two peafowl populations were very low, it was a mix-up tendency between two peafowl populations.6. Part of mtDNA D-loop among 256 individuals of 14 Chinese domestic chicken breeds and two red jungle fowl subspecies were sequenced and analyzed. The result showed that the length of D-loop in this study was about 560 bp.Content of nucleotide A, C,G, T were 25.00%, 37.40%, 4.40% and 33.20%, respectively. The percentage of A+T was 58.2% and G+C was 41.8%, showed high A+T of mtDNA D-loop in 16 chicken populations. There were 44 polymorphic sites represent 7.86% of total analyzed sites.Only transition and transversion but no insertion/deletion were found in this region, the ratio of transition and transversion in this study was 0.13.32 haplotypes,among them 14 haplotypes were shared among some chicken populations, 23 haplotypes were unique for one population. The distribution of all haplotypes among the populations was disequilibrium and the diversity of haplotypes was ranged from 0 to 0.964.The average diversity of haplotypes was 0.909±0.014, indicated that there was existed rather abundant mitochondrial genetic diversity in 16 chicken populations.The Gushi chicken showed the lowest diversity of haplotypes while the Huainan Partridge and Wannan Three-yellow chickens showed the highest one. The average number of nucleotide divergence (K) and average nucleotide diversity (Pi) were 7.276 and 1.851%, respectively. Inter-population Nucleotide Divergence (Dxy) in 16 chicken populations was ranged from 0.747%～3.125%, wheras Inter-population Net Nucleotide Divergence (Da) in 16 chicken populations was ranged from -0.015% to 2.633%. The results indicated that the genetic diversity of 16 chicken populations was very abundant.Kimura 2-parameter distance among these populations ranged from 0.007 to 0.031. Analysis of molecular variance showed that 23.83% of genetic variation was present within populations. FST value was 0.38155, which indicated the genetic variation was significant within populations (P < 0.05). There were significant divergence among 14 Chinese domestic chicken breeds and two subspecies of red jungle fowl.7. The NJ, ME and UPGMA phylogenetic dendograms of 32 haplotypes in 16 chicken populations and as Coturnix japonica an outgroup from GenBank (Accession No. D82924) were constructed. As a whole, the results of three kinds of trees are same and these 32 haplotypes were placed into four lineages. Lineage A included the haplotypes of Gallus gallus gallus, Lineage B and C included the haplotypes of Gallus gallus spadiceus and Lineage D included the haplotypes of both Gallus gallus gallus and Gallus gallus spadiceus. The median-joining networks of the 16 chicken populations in the control region also showed the same results with phylogenetic trees and all 32 haplotypes appeared into four clusters. NJ, ME and UPGMA dendograms based on Kimura 2-parameter distance of mtDNA D-loop sequences in 16 chicken populations were also constructed, three kinds of dendograms all showed that Gushi chicken, Xianju chicken and Gallus gallus gallus were always in the same cluster, while other breeds such as Chahua chicken, Tibetan chicken, Taihe Silkies chicken, Henan Game and Baier chicken were confined into the same cluster.8. Genetic diversity of red jungle fowl in China(Gallus gallus spadiceus) and red jungle fowl in Thailand (Gallus gallus gallus)was evaluated with 29 microsatellite loci and mtDNA D-loop, Reynolds’ genetic distance and gene flow between two populations were 0.157 and 1.040, respectively, there was no closer relationship and large gene flow between these two subspecies. Genetic differentiation index (FST) of these two populations was 0.194 (P <0.01) and all loci contributed significantly (P <0.01) to this differentiation. There was no shared haplotypes between these two subspecies, the pair wise genetic differentiation index (Fst) of mtDNA D-loop was also significant (P=0.0360), all results indicated that there were different genetic structure and significant genetic differentiation between red jungle fowl in China and red jungle fowl in Thailand, and this provided enough support to identify these two red jungle fowl subspecies as the different subspecies. 9. Analysis of genetic diversity and phylogenetic relationships among Chinese domestic fowls and red jungle fowls estimated with 29 microsatellite loci and mtDNA D-loop sequences indicated that some Chinese domestic fowls such as Chushi chickens and Xianju chickens derived from some subpopulations of Gallus gallus gallus after their domestication, other Chinese domestic fowls such as Chahua chickens and Tibetan chickens derived from some subpopulations of Gallus gallus spadiceus. Tajima’s D values were -1.79995 (P< 0.05)for Gallus gallus gallus, which showed significant difference from neutrality. During the course of population expansion after the domestication, Gallus gallus gallus maybe have some effect on some subpopulations of some Chinese domestic breeds originated from Gallus gallus spadiceus in China, so the genetic contribution of these two subspecies of red jungle fowl can be detected in some Chinese chicken populations. The results in this study concluded that the Chinese domestic fowls don’t just derive from red jungle fowl in Thailand or just from red jungle fowl in China and gave support the viewpoint that the red jungle fowls were domesticated several times independently in different place and the domestication of red jungle fowls was the outcome of human activities during the long period.更多还原