【作者】 杜文兴；

【导师】 王林云；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 我国地方鹅品种资源丰富,品种间体型外貌,生产性能及遗传特性差异较大。为全面了解中国家鹅遗传资源现状、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11个中国家鹅品种及7个江苏省内品种(类群),2个引进的欧洲家鹅品种和来自2个动物园的灰雁、鸿雁,利用双重抑制PCR技术获得的10对鹅微卫星引物对437个鹅个体基因组DNA进行分析。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UPGMA法、主成分分析(FCA)和群体遗传结构分析等方法,对11个中国家鹅品种及7个江苏省内地方品种的群体内的遗传变异进行了分析。基于线粒体DNA COI基因序列中的681bp片段对13个品种79个个体和GenBank中相关序列的分析进行了中国鹅品种分类地位探讨。其研究结果如下：1.经双重抑制PCR技术分离得到鹅的微卫星引物12对,其中仅一对不能完全扩增出微卫星片段,其余均能扩出,且退火温度变化范围较大,很容易进行PCR扩增反应。有一对引物扩增后在所有品种上都呈单态,其余10对引物均具多态性。从微卫星引物扩增出的片段中选择两个相对较小且清晰的等位基因片段A和B,A等位基因为11个(CA/TG)重复,B为14个重复。结果显示,在鹅的基因组中可能存在大量的(TG)n重复序列。2.从上述获得的微卫星引物中筛选出10对具有较好多态性的引物,对13个国内外的鹅种基因组DNA进行PCR扩增和基因型判断；由Fstat软件计算结果显示,13个品种中共检测到61个等位基因；每个座位的等位基因数为4～11个。61个等位基因中仅有7个在13个鹅品种中出现；有12个在11个中国鹅群体中出现,中国鹅中共有等位基因数占总等位基因数的19.56%。但有些特有的等位基因仅在某一群体中出现,如溆浦鹅、豁眼鹅、太湖鹅、狮头鹅分别在四个不同位点出现稀有的等位基因。除G08的143 bp位点在狮头鹅中出现较高的等位基因频率外(0.868),其余三个稀有等位基因均只有较低的频率。同时还发现,个别品种存在个别位点等位基因缺失现象,如伊犁鹅中在G01的143bp位点上出现等位基因缺失。3.10个微卫星在13个中外鹅品种中的平均群体杂合度(H)为0.545；除G04、G09和G12位点平均杂合度低于0.5外,其余7个位点的平均杂合度都超过0.5。我国11个地方鹅品种的群体杂合度较高,最高的长乐鹅(0.605),最低的东北籽鹅(0.488),两个外来品种莱茵鹅和朗德鹅的群体杂合度较低,分别为0.470和0.421；表明我国地方鹅品种的遗传变异相对较大；外来鹅种遗传变异相对较小、品种比较纯、选育程度较高。13个品种所有基因座的平均多态信息含量(PIC)为0.485,仍以中国鹅种较高,外来鹅种较低；表明我国地方鹅品种内的遗传基因丰富,外来鹅种遗传基因相对较纯。10个微卫星在13个品种的平均等位基因观察数为3.53、平均有效等位基因数为2.539；中国鹅种普遍高于外来鹅种。品种间的遗传变异分析表明：10个微卫星在13个鹅种的FST(群体分化系数即遗传分化系数)为21.39%(p<0.001),所有位点都出现极显著分化(p<0.001)。13个鹅品种的F(>)(总近交系数)值为23.7%,达到极显著水平(p<0.001)；其中G04、G06、G08、G09、G10和G12位点的Frr值偏离Hardy-Weinberg期望,达极显著水平(p<0.001)。13个鹅品种的FIs(鹅种间的近交系数)值为2.98%,达到显著水平(p<0.05)；其中G04、G08、G09、G10的FIs值偏离Hardy-Weinberg期望,达极显著水平(p<0.001)。Nm(基因流值)变化范围从G04的0.3863到G10的1.7854,平均值为0.8890。从FST和Nm值可知,FST值越大、Nm值越小,也即群体(品种间)分化程度越大、基因流值越小。通过分子方差分析得出我国新疆伊犁鹅与我国其它10个地方鹅品种间的FST值差异达到显著水平(P<0.05)。欧洲的莱茵鹅、朗德鹅与我国地方10个鹅品种(伊犁鹅除外)间的FST值差异达到极显著水平(P<0.01)。我国地方大型鹅品种与中、小型鹅之间的FST值差异达到显著水平(P<0.05)。从基因流值来看,皖西白鹅和雁鹅之间基因流值最大(Nm=9.554)；四川白鹅和雁鹅之间次之(Nm=7.947)；而朗德鹅和东北籽鹅之间基因流值最小(Nm=0.325)。莱茵鹅和朗德鹅两品种间遗传分化系数较小(FST=0.072);皖西白鹅和雁鹅间遗传分化程度最小(FST=0.026)。但总体来说,中国地方鹅品种间(除伊犁鹅外)群体遗传分化系数较小,说明中国家鹅的起源均受到了同一祖先的影响；中国鹅品种间的基因流值大于与外来鹅种间的基因流值,说明中国鹅种间相互迁移率更高。4.UPGMA和NJ法聚类均能把13个鹅群体在总体上分为两大类：中国鹅种(伊犁鹅除外)聚为一类,外来2个鹅种先聚后再与伊犁鹅聚为第二类。用Structure程序对13个鹅种的遗传结构进行分析,当K=2时,13个鹅种分成二类,其中伊犁鹅和欧洲鹅聚在一类；当K=3时,伊犁鹅仍和欧洲鹅聚在一类；中国其他鹅种中的浙东白鹅、长乐鹅、太湖鹅、皖西白鹅聚为一类,其除中国鹅聚为一类；当K=4、5时,浙东白鹅、长乐鹅、太湖鹅聚为一类,豁眼鹅、东北籽鹅、皖西白鹅聚为一类,四川鹅、雁鹅、溆浦鹅聚为一类,狮头鹅单独一类；当K=6时,伊犁鹅从欧洲鹅中分离出来,其除中国鹅种间聚类基本不变。此结果与UPGMA和NJ法聚类结果基本一致。5.选择11个品种79个个体的线粒体DNA COI基因序列中的681bp片段进行分析。结果表明：11个群体的Hd(单培型多样度)为0.6219±0.036；Pi(核苷酸多样度)为0.00120±0.00011；11个群体共有6种单倍型,且以H2和H3为主体单倍型,两者频率分别为51.9%和31.65%；而H4、H5和H6单倍型仅为一个个体的少数单倍型。采用Mega4软件进行多序列比对后发现,其中保守位点677个,可变位点4个,简约信息位点4个,单信息位点2个。群体内单倍型多样度(Hd)以莱茵鹅最高(0.8100±0.0169),其次为灰雁(0.7140±0.0151),伊犁鹅品种Hd为0。将中国地方家鹅品种间的Hd进行比较,皖西白鹅明显高于其它鹅品种。群体内核苷酸多样度(Dx/Dy)以菜茵鹅最高(0.00168),伊犁鹅最低为0。群体间核苷酸分歧度(Dxy)以鸿雁与中国鹅(伊犁鹅除外)间、灰雁与欧洲鹅及伊犁鹅间较小。群体间遗传分化指数(GST),以中国伊犁鹅与鸿雁的GsT遗传分化大于与灰雁的遗传分化程度；除伊犁鹅之外,中国地方鹅种群体间的GST相对较小,表明除伊犁鹅外的中国鹅间遗传关系更一致。6.对雁亚科32条COI基因序列的统计分析发现,4种碱基(T、C、A、G)的平均含量分别为23.8%、34.6%、24.9%、16.7%,嘌呤平均含量与嘧啶平均含量相近,说明序列碱基不存在明显的偏向性。31条雁亚科mtDNACoI基因序列中共检测到24种单倍型,作为外群的鹊雁亚科(Anseranatinae)的鹊鹅(magpie goose)形成一个独立的单倍型,所有物种Hd为0.9819；其中中国家鹅、鸿雁与下载鸿雁序列共享同一单倍型,伊犁鹅、欧洲鹅与灰雁共享同一单倍型。7.基于10对鹅微卫星引物分析结果显示：鸿雁与莱茵鹅的遗传分化程度最高,为0.386；莱茵鹅与朗德鹅、伊犁鹅的遗传距离较近,分别为0.1484和0.2099,还与灰雁的距离相对较近,分别为0.2850和0.3031；中国家鹅(除伊犁鹅外)与鸿雁的遗传距离较近,为0.1768。鸿雁、中国家鹅与朗德鹅间的遗传距离最大,分别为0.855和0.854；相应地,它们之间的品种分化时间也最早,达到了777.2年和776.1年。中国家鹅(除伊犁鹅外)与上述集合中的所有群体的遗传距离均较远,伊犁鹅与欧洲鹅的分化时间较晚于与中国家鹅的分化时间。聚类分析结果发现：鸿雁与中国家鹅聚为一类,莱茵鹅与朗德鹅先聚,再分别与伊犁鹅、灰雁聚为一类。每个分枝上都获得较高的支持率。综合研究结果表明：中国家鹅(除伊犁鹅外)起源于鸿雁,欧洲鹅(包括伊犁鹅)起源于灰雁。8.基于10对鹅微卫星引物对江苏省鹅种遗传基因研究结果表明,江苏省内鹅品种资源可以分为四大类型。一类是太湖鹅：包括苏州种鹅场的太湖鹅、南农大种鹅场的太湖鹅、以及盐城建湖的太湖鹅。第二类是四季鹅：包括句容和溧水二地的四季鹅,以及含有浙东白鹅和皖西白鹅血缘的洪泽鹅。第三类是四川鹅：主要是引进饲养推广的四川白鹅,实际上常熟饲养的不是太湖鹅,而是引入的四川白鹅。第四类是扬州鹅：由于扬州鹅是通过引入四川白鹅等品种与太湖鹅杂交后选育的鹅种,所以遗传基因丰富,可以自成一体进行继续选育和推广。而豁眼鹅和狮头鹅至今仍游离于江苏养鹅业外。更多还原

【Abstract】 China have rich local goose breeds, and body conformation, performance and genetic traits were quite different among different varieties. To fully understand the genetic resources, genetic diversity and genetic relationships of geese in our country, the study collected 11 China local varieties which come from different locations of China,7 varieties of Jiangsu province,2 European domestic goose breeds, swan geese and gray geese. we obtained 10 pairs of microsatellite primers goose by using dual-suppression-PCR technique to analysis 437 Goose individual genomic DNA. Analysis of allele frequencies, heterozygosity (H), effective number of alleles, polymorphism information content, Nei’s standard genetic distance (DS) among populations, and the DA genetic distance, we analyzed 11 China local varieties and 7 local varieties in Jiangsu province by using UPGMA method, principal component analysis (FCA) and population genetic structure analysis method. Based on 79 individuals’ 681bp fragment of mitochondrial DNA COI gene sequences of 13 varieties and related sequences in GenBank to discussion taxonomic status of goose breeds in China. The results were as follows:1.12 pairs of microsatellite primers isolated from geese by dual-suppression PCR, of which only a pair of primer not completely amplified microsatellite fragments, the rest can do it, and the annealing temperature varied greatly, so it is easy for PCR amplification. Only G11 primer amplified fragments in all species were tested on a single state, the remaining 10 primers were polymorphic. we selected relatively small and clear alleles A and B from the amplified fragments by microsatellite primers, A allele was 11 (C A/TG)n, B was 14, The results suggest that there were a lot of (TG) n repeats in the genome of the geese.2.10 pairs of microsatellite primers with good polymorphism were screened from the obtained primers, which used to amplified genomic DNA of 13 breeds and to determined genotypes. The results calculated by Fstat software showed that 61 alleles were detected in 13 varieties; number of alleles ranged from 4-11 in each locus.12 alleles exist in all 11 local goose populations of China, the number of alleles accout for 19.56% in the total number of alleles; only seven alleles appeared in 11 chinese local goose populations and two exotic species. However, some specific alleles only occured in certain groups, such as in Xupu goose, Shitou goose, taihu goose, Huoyan goose. The results show that in addition to 143 bp fragment of G08 appeared in Huoyan goose has high allele frequency (0.868), while the rest rare alleles only appeared lower frequencies. Also we found that some breeds lack some alleles, such as the 143 bp fragment of G01 weren’t detected in Yili goose.3. The average heterozygosity of 10 microsatellites in 13 breeds was 0.545, in addition to average heterozygosity of G04, G09 and G12 are lower than 0.5, the remaining seven were more than 0.5. The heterozygosity of Changle geese was highest (0.605), and Zi Geese was lowest (0.488) in 11 local goose breeds of China. The heterozygosity of two exotic species were lower, so the heterozygosity of Rhine and Landes goose were 0.470 and 0.421, respectively, indicated that China local goose breeds have large genetic variation. Local breeds of China have a high genetic diversity, showed a high heterozygosity, the heterozygosity of Rhin and Landes goose are lower than our local varieties, indicated that the two exotic species were lower. The average of all loci polymorphic information content (PIC) were 0.485 in 13 goose breeds. Local breeds of China have a high PIC, two exotic species were lower PIC. The average of observed number alleles in 10 microsatellite loci were 3.53; the number of effective alleles were 2.539, that of Local breeds of China were high than the number of the two exotic species. Effective alleles of Sichuan white goose was 2.8270. FST (population differentiation factor) in the all groups, the average genetic differentiation was 21.39%(p<0.001), all sites were significant contribution to this result (p<0.001). FIT (total inbreeding coefficient) of 13 goose breeds was 23.73%, reached a significant level (p<0.001); and FIT value of G04, G06, G08, G09, G10 and G12 loci deviated from Hardy-Weinberg expectations, reached extremely remarkable level. Nm (gene flow value) range of 0.3863 of G04 to 1.7854 of G10, the average Nm was 0.8890. From the known Fst and Nm, Fst was larger, Nm was smaller, the degree of species differentiation was greater, so Nm was smaller. FST values of Yili geese and other 11 geese species were remarkable (P<0.05) by analysis of molecular variance. FST between two exotic species and 10 different varieties of China reached a extremely remarkable level (P<0.01). FST between large breeds and small breeds in China local Goose varieties were remarkable (P<0.05). From the results of Nm, Nm between Wanxi White goose and wild goose was maximum (9.554); Nm between Sichuan white goose and wild geese was followed (7.947); Nm between Landes and Zhedong Goose was minimum (0.325). FST between Rhine and Landes geese was smaller (FST=0.072), showed that two populations have weak genetic differentiation. In local varieties, the degree of genetic differentiation between wild geese and Wanxi White was smallest (FST=0.026). But overall, gene differentiation coefficient among populations and gene flow were at a low level in various varieties of Chinese local goose, indicated that the origin of China local goose were affected by the same ancestor.4. In general,13 goose populations can be divided into two categories by UPGMA and NJ method:first, wild geese, Sichuan White geese, Wanxi White geese, Xupu geese, Shitou geese, Taihu geese, Huoyan geese, Zi Geese, Zhedong geese and Changle geese clustered together, second, Rhin and Landes geese firstly clustered together, and then clustered with Yili geese. Genetic differences can be distinguish between different varieties in FactorⅠ, corresponding to differences of origin between China goose and foreign breeds; FactorⅡcorresponds to the difference size of geese. The results analysed by Structure program by the of 13 groups consistent with the results of genetic distance reflects the local breeds genetic structure and relationship, also consistent with 13 local breeds’ geographical distribution, formation history and gene flow. we determined the real kind of contemplating the class based on theΔK method,13 varieties can obviously divided into two groups; and 11 China local varieties can be divide three groups.5.681bp fragment of mitochondrial DNA COI gene were analysed in 79 individual of the 11 varieties. The results are as follows:11 groups Hd (Haplotype diversity)was 0.6219±0.036; Pi(Nucleotide diversity) was 0.00120±0.00011 and have 6 haplotypes. H2 and H3 haplotypes were main haplotype,and frequency were 51.9% and 31.65%, respectively, and H4, H5 and H6 haplotype existed in only someone individual. Rhin haplotype was most(4), Yili geese was least, only one.677 conserved sites,4 variable sites,4 parsimony informative sites, and 2 single-informative sites were found by Mega4 software for multiple sequence alignment. which can be seen base content was no serious bias. Haplotype diversity in Rhin group was highest level (0.8100±0.0169), followed by Grey Goose Geese (0.7140±0.0151), and Yili haplotype diversity was 0. Nucleotide diversity in Grey Goose and Rhine geese were significantly higher than that in Landes and Yili geese; In China local domestic goose breeds, Huoyan geese and Wanxi White geese was significantly higher than other domestic goose breeds and swan goose. The net nucleotide divergence (Da) between grey goose and other two geese (Yili goose and Zhedong White goose) groups were highest, Da between swan goose and other two breeds (Yili goose and Zhedong). The degree of genetic differentiation between Yili goose and the swan goose larger than Yili goose and gray goose. The degree of genetic differentiation were small in China local geese breeds except Yili goose, Indicated that most of variation existed within local varieties.6.32 COI gene sequences of subfamily of geese were analysed showed that average content of bases (T, C, A, G) were 23.8%,34.6%,24.9%,16.7%, the average content of purine close to pyrimidine content, indicated that there was no significant base bias.24 haplotypes were detected in 31 mtDNA CoI sequences, the magpie geese as outgroup subfamilies (Anseranatinae) form a separate haplotype, all breeds’ Hd were 0.9819; domestic goose, swan geese and swan geese sequence was downloaded share the same haplotype, Yili geese, Landes geese and gray geese share the same haplotype.7. The results showed that COI gene sequences were analysed by 10 pairs of goose microsatellite primers as follows:the degree of genetic differentiation between swan geese and Rhine geese was highest (0.386). the genetic distance vaule between Rhin geese and Yili geese was 0.1484, between Landes geese and Yili geese was 0.2099, between Rhin geese and gray geese was 0.2850, between Landes geese and gray geese was 0.3031. The genetic distance between China local goose breeds and the collections of all the breeds are far, but closer to swan geese (0.1768). the genetic distance between Landes geese and swan geese was largest (0.855), the genetic distance between Landes geese and China local goose breeds was 0.854. Accordingly, the earliest time of species differentiation between them were also reached 777.2 years and 776.1 years. differentiation The divergence time between Yili goose and Europe geese later than between Yili goose goose and China local geese. The results of cluster analysis showed that:swan goose cluster with Chinese geese, Rhin and Landes firstly cluster together, and then clustered together with Yili geese and gray geese. On each branch was obtained a higher support rate.8. According to the results of geese varieties of jiangsu province were analyzed based on 10 pair of goose microsatellite seats, The geese varieties of jiangsu province can be divided into four major categories, one kind is taihu goose:including taihu goose in goose field of suzhou, taihu lake goose of Nanjing agricultural university, and currently Yancheng taihu goose; The second is four seasons goose in Lishui and Jurong, and Hongze geese which contain blood of eastern zhejiang white geese and Wanxi white geese; Three is sichuan white geese were introduced to breed and promotion, actually Changshu breeding is not taihu lake goose, but sichuan white geese, they Outward promotion sichuan white geese; Four is Yangzhou geese, which was breed by Hybrid breeding Sichuan white goose through the introduction and taihu goose, so the goose genetic was rich, can self-contained to continue breeding and promotion. And variable eyes goose and annals of goose still free in jiangsu province raising goose outside.更多还原