2个云南原始鸡种遗传多样性及其与红色原鸡的亲缘关系

引用本文

陆俊贤, 贾晓旭, 唐修君, 樊艳凤, 唐梦君, 高玉时, 苏一军. 2个云南原始鸡种遗传多样性及其与红色原鸡的亲缘关系[J]. 浙江大学学报(农业与生命科学版), 2016, 42(3): 385-390. DOI:10.3785/j.issn.1008-9209.2015.06.113 复制到剪切板

LU Junxian, JIA Xiaoxu, TANG Xiujun, FAN Yanfeng, TANG Mengjun, GAO Yushi, SU Yijun. Genetic diversity of two local Yunnan chicken breeds and their relationships with red jungle fowl.[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2016, 42(3): 385-390. DOI: 10.3785/j.issn.1008-9209.2015.06.113. 复制到剪切板

2个云南原始鸡种遗传多样性及其与红色原鸡的亲缘关系

[PDF全文]

陆俊贤, 贾晓旭, 唐修君, 樊艳凤, 唐梦君, 高玉时, 苏一军

江苏省家禽科学研究所,江苏扬州225125

基金项目: 国家自然科学基金(31372277;31501917)

通信作者: 高玉时(http://orcid.org/0000-0002-3170-5105), E-mail:gaoys100@sina.com

第一作者联系方式: 陆俊贤(http://orcid.org/0000-0002-1999-0310), E-mail:yzljx1972@sina.com

收稿日期(Received): 2015-06-11; 接受日期(Accepted): 2015-09-10; 网络出版日期(Published online): 2016-05-19

摘要: 对60只个体的线粒体基因组(mitochondrial genome,mtDNA)D-loop区全序列进行聚合酶链反应(polymerase chain reaction,PCR)扩增和测序,并结合GenBank中红色原鸡的D-loop区序列,分析其线粒体D-loop区多态性。结果发现,2个原始地方品种鸡mtDNA D-loop区全长1 231~1 232 bp,在60只个体中共发现19处单核苷酸多态位点和8种单倍型。系统发育分析发现,8种单倍型可以分为A、B、D和E 4个分支,其中A和B为主要分支。A和B分支各有3种单倍型,D和E分支分别只有1种单倍型。A和B分支与红色原鸡滇南亚种(Gallus gallus spadiceus)聚为一类,E分支与红色原鸡印度亚种(Gallus gallus murghi)聚为一类,D分支与4个红色原鸡(Gallus gallus)亚种聚为一类。表明2个品种有多个母系起源,红色原鸡滇南亚种在2个原始鸡种形成过程中贡献最大。研究为家鸡的起源和遗传分化提供基础材料,同时也为2个品种的选育改良、遗传资源保护以及进一步的开发利用提供重要的理论依据.

关键词: 云南家鸡红色原鸡线粒体DNA D-loop区

Genetic diversity of two local Yunnan chicken breeds and their relationships with red jungle fowl.

LU Junxian, JIA Xiaoxu, TANG Xiujun, FAN Yanfeng, TANG Mengjun, GAO Yushi, SU Yijun

Jiangsu of Poultry Science, Yangzhou 225125, Jiangsu, China

Summary: Chickens are an important animal genetic resource and the conservation of local breeds is an issue for preservation. With these highly diversified geographical environments, coupled with copious precipitation, adequate sunlight, and long frost-free periods, Yunnan Province has high biodiversity, including habitats for several ancestral species of domestic chicken and which has been suggested to be a center of domestication of chicken. Daweishan mini and Chahua are typical local chicken breeds in Yunnan Province. This study evaluated the genetic diversity and origins of the two breeds by analyzing their mtDNA sequence polymorphisms. Blood samples were collected from 30 of each breed. Their complete D-loop sequences were PCR amplified, sequenced and compared with the DNA data of five red jungle fowl (Gallus gallus) subspecies annotated in the GenBank. The results indicated that D-loop regions of the two local Yunnan chicken breeds were 1 231-1 232 bp, with a base C deficiency from 859 bp site in the 1 231 bp haplotype. Nineteen polymorphic sites were identified across the complete mtDNA D-loop region sequence. The eight haplotypes observed in the two local chicken breeds belonged to four previously published clades, i. e. Clades A, B, D and E. Of these clades, A and B were dominant. Clades A and B both contained three haplotypes, whereas Clades D and E both contained one haplotype. Clades A and B were clustered with G. g. spadiceus indicating that the two clades may originated from this subspecies. Clade E was clustered with G. g. murgh indicating that this clade may originated from this continental subspecies. The other haplotypes were contained in Clade D, which originated from multiple maternal origins. There were diversities in the middle of the mtDNA D-loop sequences of the two local breeds, which may result from multiple maternal origins. These data indicate that G. g. spadiceus contributed more to the evolution of the two local Yunnan breeds than the other four subspecies tested. The haplotypic diversity within the Daweishan mini chicken (0.685) and Chahua (0.476) was lower than those of breeds studied using the hypervariable I (HVI) segment of the D-loop in other chickens of Yunnan Province. Determining the genetic background of domestic chicken breeds local to Yunnan Province and their relationships with red Jungle fowl could improve the understanding of chicken domestication. In contrast to other chickens of Yunnan Province, the two chicken breeds studied here had lower diversity at mitochondrial levels. Due to their possible contribution to past and recent domestication, the two breeds deserve a conservation attention.

Key words: Yunnan chicken red jungle fowl mitochondrial DNA D-loop region

红色原鸡根据地理分布和外貌可以分为滇南亚种(Gallus gallus sadiceus,GGS),指名亚种(Gallus gallus gallus,GGG),印度亚种(Gallus gallus murghi,GGM),海南亚种(Gallus gallus jabouillei,GGJ),滇南亚种(Gallus gallus spadiceus,GGS)5个亚种。对于家鸡母系起源问题,早期FUMIHITO，等^[1]提出家鸡母系单一起源于泰国及其周边地区的红色原鸡指名亚种(G. g. gallus)。KANGINAKUDRU，等^[2]研究结果表明,红色原鸡滇南亚种和印度亚种在家鸡进化中也同样做出了贡献。现在大多数学者支持家鸡由红色原鸡多个母系在中国西南部、南亚和东南亚经过多次独立驯化而来的观点^[3-5]。

中国西南部主要指云南及周边地区,云南特殊的地理位置和复杂的自然地理环境,形成了立体气候和多样化的生境条件,孕育了极为丰富的生物多样性资源,再加上山脉隔离、众多少数民族与外界极少交流以及他们特有的经济文化活动,形成了形形色色的优良地方鸡品种,至今还存在一些与现存家禽祖先为同类的禽类.茶花鸡和大围山微型鸡就是云南省2个典型的原始鸡种.红色原鸡和家鸡能够繁殖后代,因此认为是同一物种.

线粒体D-loop区具有高度变异性的非编码区,其变异速率约为其他区段的5~10倍,具有高度多态性,已被广泛用于家禽的群体遗传多样性、起源与进化、系统发育关系等方面的研究^[6-7].本研究通过PCR扩增茶花鸡和大围山微型鸡线粒体D-loop区的全长,并结合NCBI上红色原鸡(Gallus gallus)的D-loop区序列,一方面研究2个原始鸡种的遗传多样性,另一方面探讨红色原鸡5个亚种在中国家鸡驯化中的贡献.

1 材料与方法 1.1 试验动物及材料

试验样本均采自国家级地方鸡种基因库(江苏),是国内最大,世界上保存资源最多的家禽活体基因库.从大围山微型鸡和茶花鸡群体中各随机采取30只(10♂,20♀),翅静脉采血,肝素钠抗凝,采用常规的酚-氯仿法提取血液基因组DNA.

选取NCBI数据库中,指名亚种Gallus gallus gallus(AB007725、NC007236)、印尼亚种Gallus gallus bankiva(NC007237)、海南亚种Gallus gallus jabouillei(GU261674、GU261696)、印度亚种Gallus gallus murghi(HE793430、GU261707~GU261709)、滇南亚种Gallus gallus spadiceus(GU261690、GU261692、GU261693、GU261695、GU261702~GU261704、GU261706、GU261716、NC007235)等红色原鸡19条序列,与2个原始鸡种进行系统发育分析.

1.2 试验方法 1.2.1 引物合成

根据红色原鸡mtDNA的已知序列(NC_007235)作为参考序列,设计引物(Primer Premier 5.0软件)扩增2个原始鸡种mtDNA D-loop区.上下游引物分别为:5′-AAACACCCAAACTCACT AAC-3′/5′-CACTGGGATGCGGATACTTGC-3′,送上海生工生物工程有限公司合成。

1.2.2 PCR扩增和测序

聚合酶链反应(polymerase chain reaction,PCR)扩增体系(50 μL):聚合酶链反应扩增试剂盒(2×PCR Master Mix购自南京博尔迪公司)25 μL,10 μmol/L引物各1.5 μL,模板2 μL,灭菌水20 μL。反应程序:95 ℃ 5 min，95 ℃ 30 s，53 ℃ 60 s，72 ℃ 60 s,30个循环;72 ℃ 4 min。琼脂糖(15 g/L)凝胶电泳检测PCR产物,用试剂盒(Promega公司)回收纯化,送华大基因上海公司,然后用测序仪(ABI 3730)进行双向测序.

1.2.3 数据处理和分析

将测序得到的序列用DNAStar 5.02软件的SeqMan程序拼接,剪掉引物及多余的序列,与NCBI数据库中红色原鸡参考序列进行比对.用DnaSP 5.10软件统计单倍型数、多态位点数、核苷酸平均差异数(K)、单倍型多样性(Hd)和核苷酸多样性(Pi)^[8].用Network 4.6构建中介网络图(median-joining)分析单倍型间的进化关系(www.fluxus-technology.com).用MEGA 4.0进行碱基组成分析和转换与颠换数目的统计,并采用邻接法(neighbor-jointing,NJ)构建系统发育树.发育树选用Kimura-2模型,重复1 000次^[9].

2 结果与分析 2.1 2个原始品种mtDNA D-loop区全长序列多态性

设计的引物得到了较好的扩增,与目的片段大小一致(图 1),测序得到60个样本的扩增序列,经过软件的校对和编辑有效序列全长为1 231~1 232 bp.茶花鸡30只个体中没有发现1 231 bp单倍型,全部为1 232 bp.大围山微型鸡群体中发现1 231 bp单倍型(859 bp C碱基缺失).碱基含量统计显示,2个品种群体的A、T、C、G碱基差异不大,A、T、C和G 4 种碱基含量分别为26.7%、33.5%、26.5%和13.3%,其中G+C含量(39.8%)明显低于A+T含量(60.2%),家鸡D-loop区为非编码区,富集T/A碱基,作为转录的起始序列,能被特异分子(RNA聚合酶等)识别.

M:DL2000标志物;(1~2):PCR产物. M: DL2000; (1-2): PCR products. 图1 PCR扩增结果 Fig. 1 PCR amplification results

大围山微型鸡mtDNA D-loop区核苷酸多样性(Pi)为0.004 43±0.000 14,单倍型多样性(Hd)为0.685±0.079,核苷酸平均差异数(K)为5.448。茶花鸡mtDNA D-loop区核苷酸多样性(Pi)为0.003 01±0.000 81,单倍型多样性(Hd)为0.476±0.091,核苷酸平均差异数(K)为3.703。这2个原始鸡种mtDNA D-loop变异区在167~1 215 bp,共发现19处多态位点,其中单一信息位点4个,简约信息位点15个,其中18处为转换,T-C转换14处,A-G转换4处,1处颠换位于391 bp(C-A).

2.2 2个原始品种单倍型分析

根据2个群体D-loop区序列中19个多态位点,用DnaSP 5.10软件进行单倍型统计分析,界定了8个单倍型(Hap1~8,图 2)。其中Hap5为2个群体共享单倍型,Hap3、Hap4为茶花鸡单独拥有,其余为大围山微型鸡单独拥有。参照LIU，等^[4]划分标准可以分为A、B、D、E 4个分支。

DWS:大围山微型鸡;CH:茶花;N:数目;Clade:分支. DWS: Daweishan mini; CH: Chahua; N: number； Clade: clade. 图2 基于mtDNA D-loop区的多态位点划分的单倍型 Fig. 2 Identified haplotypes with the mitochondrial D-loop sequence polymorphisms

在茶花鸡群体中,有3种单倍型(Hap3~5),所有单倍型公母均有分布,有A和B 2个分支,A分支有21个个体,1种单倍型,占70%(21/30);B分支有9个个体,2种单倍型,占30%(9/30).

在大围山微型鸡群体中,有6种单倍型(Hap1、Hap2和Hap5~8),Hap2均为公鸡,Hap7和Hap8均为母鸡,其余公母均有。有A、B、D和E 4个分支.A分支有7个个体,2种单倍型,占23.33%(7/30);B分支有19个个体,2种单倍型,占63.33%(19/30);D分支有3个个体,1种单倍型,占10%(3/30).

2.3 2个原始品种与红色原鸡系统发育分析

基于本研究测得的60条序列和红色原鸡19条序列,用DnaSP 5.10软件分析,共计得到27种单倍型,通过MEGA 4.0软件构建了2个原始品种与红色原鸡的系统发育树(图 3).红色原鸡滇南亚种GGS1和Hap4 D-loop区序列相同,红色原鸡滇南亚种GGS7与Hap1~3聚为一类;滇南亚种GGS1、GGS4与Hap4~6聚为一类;红色原鸡海南亚种GGJ1~2与Hap1~6聚为一大类.Hap7与红色原鸡滇南亚种GGS2、GGS8、指名亚种GGG1和GGG2、印度亚种GGM4、印尼亚种GGB聚为一类,红色原鸡印度亚种GGM1~3与Hap8聚为一类.

DWS:大围山微型鸡;CH:茶花;GGS:滇南亚种;GGJ:海南亚种;GGG:指名亚种;GGB:印尼亚种;GGM:印度亚种. DWS: Daweishan mini; CH: Chahua; GGS: G. g. spadiceus; GGJ: G. g. jabouillei; GGG: G. g. gallus; GGB: Gallus g. bankiva; GGM: G. g. murghi. 图3 基于mtDNA D-loop区序列采用NJ法构建的系统发育树 Fig. 3 Neighbor-joining tree based on mtDNA D-loop sequences

将所检测到的27种单倍型,通过Network软件构建了Median Joining网络聚类图(图 4)。由图 4可见,网络聚类图结果和系统发育树结果一致,Hap1~3与红色原鸡滇南亚种GGS7构成了一个大的单倍型群A,Hap4~6与红色原鸡滇南亚种GGS1、GGS4构成了一个大的单倍型群B,这2个单倍型群占了2个群体的绝大部分。

Hap1~8为2个原始鸡种的单倍型;GGB:印尼亚种;GGS:滇南亚种;GGJ:海南亚种;GGG:指名亚种;GGM:印度亚种. HAP1-8 is haplotype of two local Yunnan chicken breeds; GGB: Gallus g. bankiva; GGS: G. g. spadiceus; GGJ: G. g. jabouillei; GGG: G. g. gallus; GGM: G. g. murghi. 图4 基于单倍型构建的网络结构图 Fig. 4 Median network profile of the mtDNA D-loop haplotypes observed in the present study

3 讨论 3.1 2个原始品种群体变异程度和序列结构

前人有关鸡D-loop区的研究一般集中在高变区,特别是高变Ⅰ区(hypervariable region Ⅰ,HVS-Ⅰ)^{[4, 10-12]},用于研究的DNA片段约为500 bp。而家鸡和红色原鸡线粒体D-loop区全长1 231~1 232 bp,除了本研究发现的859 bp处存在C碱基缺失。792、1 214和1 215 bp处存在碱基转换之外,在686、889、904和1 174 bp等处也存在转换或者颠换^[5]。如果只用线粒体D-loop HVS-Ⅰ区进行研究则无法发现这些信息。

物种进化和适应环境的基础是遗传多样性.遗传多样性的下降不但会使种群遗传结构发生改变,还会导致群体内对环境变化的适应能力及抗病力下降.群体单倍型多样度和核苷酸多样度值越大,遗传多样性越丰富.大围山微型鸡D-loop区核苷酸多样(Pi)为0.004 43,单倍型多样性(Hd)为0.685.茶花鸡D-loop区核苷酸多样(Pi)为0.003 01,单倍型多样性单倍型多样性(Hd)为0.476.与同样用D-loop区研究云南其他品种瓢鸡(Pi=0.015 03、Hd=0.883)^[13]、西畴乌骨鸡(Pi=0.014 52、Hd=0.884)等^[14]得出的结果相比偏低.

3.2 2个原始品种群体和红色原鸡亲缘关系

大围山微型鸡分布在云南大围山地区,地理环境较为封闭,处于半野生半家养状态,体型较小,成年公鸡平均857 g,母鸡平均682 g。茶花鸡产区为原始森林,林中的红色原鸡,在谷物收获季节觅食过程中,有的公原鸡与母家鸡交配,因此红色原鸡与茶花鸡的亲缘关系较近,也属半野生型家鸡^[15]。2个品种都是红色原鸡在热带、亚热带生态条件下,经过当地少数民族长期驯养培育成为家鸡的一个地方品种,是中国不可多得的原始鸡种。

3.3 2个原始品种群体和红色原鸡亲缘关系

2个原始品种,茶花鸡A分支为优势分支,个体数占整个群体70%(21/30),大围山微型鸡B分支为优势分支,个体数占整个群体63.33%(19/30).说明2个品种在母系来源上各有偏重.茶花鸡和大围山微型鸡A和B分支个体数占2个群体总数93.33%(56/60),而在这2个分支中,红色原鸡滇南亚种GGS7(GU261695)与A分支聚为一类,红色原鸡滇南亚种GGS4(GU261704)、GGS1(NC_007235)与B分支为一类;红色原鸡滇南亚种GGS1(NC007235)和Hap4序列相同,可能红色原鸡滇南亚种在这2个原始品种鸡形成过程中贡献最大.A和B分支与红色原鸡海南亚种聚为一类.推测海南亚种在我国家鸡母系起源也做了一定贡献.

在大围山微型鸡群体存在E分支仅有1个个体,与红色原鸡印度亚种聚为一类,可能是大围山微型鸡少数个体与印度红色原鸡或印度地方品种发生了杂交.大围山微型鸡D分支3个个体与红色原鸡滇南亚种,指名亚种,印度亚种,印尼亚种等4个亚种聚为一类,多个红色原鸡亚种在这一支系的形成过程中都做出了贡献.

参考文献

[1]	FUMIHITO A, MIYAKE T, SUMI S, et al. One subspecies of the red jungle fowl (Gallus gallus gallus) suffices as the matriarchic ancestor of all domestic breeds. Proceedings of the National Academy of Sciences, 1994,91 : 12505–12509.
[2]	KANGINAKUDRU S, METTA M, JAKATI R D, et al. Genetic evidence from Indian red jungle fowl corroborates multiple domestication of modern day chicken. BMC Evolutionary Biology, 2008,8 : 174.
[3]	OKA T, INO Y, NOMURA K, et al. Analysis of mtDNA sequences shows Japanese native chickens have multiple origins. Animal Genetics, 2007,38 : 287–293.
[4]	LIU Y P, WU G S, YAO Y G, et al. Multiple maternal origins of chickens: out of the Asian jungles. Molecular Phylogenetics and Evolution, 2006,38 (1): 12–19.
[5]	MIAO Y W, PENG M S, WU G S, et al. Chicken domestication: an updated perspective based on mitochondrial genomes. Heredity, 2013,110 : 277–282.
[6]	LI H F, ZHU W Q, CHEN K W, et al. Two maternal origins of Chinese domestic goose. Poultry Science, 2011,90 : 2705–2710.
[7]	包文斌, 束婧婷, 王存波, 等.中国家鸡和红色原鸡mtDNA控制区遗传多态性及系统进化分析.畜牧兽医学报,2008,39 (11):1449–1459. BAO W B, SHU J T, WANG C B, et al. Investigation on genetic diversity and systematic evolution in chinese domestic fowls and red jungle fowls by analyzing the mtDNA control region. Acta Veterinaria et Zootechnica Sinica, 2008,39 (11): 1449–1459. (in Chinese with English abstract)
[8]	ROZAS J, SANCHEZELBARRIO J C, MESSEGUER X, et al. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics, 2003,19 (18): 2496–2497.
[9]	TAMUIA K, DUDLEY J, NEI M, et al. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 2007,24 (8): 1596–1599.
[10]	LIU Z G, LEI C Z, LUO J, et al. Genetic variability of mtDNA sequences in Chinese native chicken breeds. Asian Australasian Journal of Animal Sciences, 2004,17 (7): 903–909.
[11]	MUCHADEYI F C, EDING H, SIMIANER H, et al. Mitochondrial DNA D-loop sequences suggest a Southeast Asian and Indian origin of Zimbabwean village chickens. Anim Genet, 2008,39 : 615–622.
[12]	NIU D, FU Y, LUO J, et al. The origin and genetic diversity of Chinese native chicken breed. Biochemical Genetics, 2002,40 (5): 163–174.
[13]	贡潘偏抽, 刘丽仙, 李大林, 等.基于线粒体DNA控制区(mtDNA D-loop)序列分析瓢鸡的遗传多样性.云南农业大学学报,2011,26 (2):211–214. GONG P P C, LIU L X, LI D L, et al. The investigation of genetic diversity of piao chicken based on mitochondrial DNA D-loop region sequence. Journal of Yunnan Agricultural University, 2011,26 (2): 211–214. (in Chinese with English abstract)
[14]	黄道平, 李大林, 袁峰, 等.西畴乌骨鸡mtDNA D-loop区遗传多样性分析.云南农业大学学报,2010,25 (3):373–376. HUANG D P, LI D L, YUAN F, et al. Genetic diversity of mitochondrial DNA D-loop region of Xichou black-bone Chicken. Journal of Yunnan Agricultural University, 2010,25 (3): 373–376. (in Chinese with English abstract)
[15]	国家畜禽遗传资源委员会.中国畜禽遗传资源志.北京:中国农业出版社,2012. China National Commission of Anima Genetic Resources. Animal Genetic Resources in China. Beijing: China Agriculture Press, 2012. (in Chinese)