中国地方猪种线粒体基因组遗传多样性及其系统发育分析

为从分子水平上探究中国地方猪种遗传多样性和分类地位,本试验采用生物信息学方法比较了6个类型共22个中国地方猪种的线粒体基因组全序列,分析了其多态性,并构建了6个类型猪种线粒体D-loop区单倍型的网络中介图以及基于线粒体D-loop序列、Cytb基因、完整编码区序列的系统进化树。结果表明,6个类型22个猪种中共检测到了144个多态位点,22种单倍型,说明地方猪种具有丰富的遗传多样性;地方猪线粒体基因组序列中核苷酸变异以转换为主,且Ti/Tv大于转换/颠换比临界值（2.0）,变异位点均符合中性突变。6个类型猪种间遗传距离均较小,且有共享单倍型。系统进化树结果表明,6种类型地方猪种主要聚为两个支系。表明线粒体D-loop序列及Cytb基因均可作为研究种内系统发育、起源进化的分子标记。     

The phylogenetic status of typical Chinese native pigs: analyzed by Asian and European pig mitochondrial genome sequences

China is one of the most diverse countries, which have developed 88 indigenous pig breeds. Several studies showed that pigs were independently domesticated in multiple regions of the world. The purpose of this study was to investigate the origin and evolution of Chinese pigs using complete mitochondrial genomic sequences (mtDNA) from Asian and European domestic pigs and wild boars. Thirty primer pairs were designed to determine the mtDNA sequences of Xiang pig, Large White, Lantang, Jinhua and Pietrain. The phylogenetic status of Chinese native pigs was investigated by comparing the mtDNA sequences of complete coding regions and D-loop regions respectively amongst Asian breeds, European breeds and wild boars. The analyzed results by two cluster methods contributed to the same conclusion that all pigs were classified into two major groups, European clade and Asian clade. It revealed that Chinese pigs were only recently diverged from each other and distinctly different from European pigs. Berkshire was clustered with Asian pigs and Chinese pigs were involved in the development of Berkshire breeding. The Malaysian wild boar had distant genetic relationship with European and Asian pigs. Jinhua and Lanyu pigs had more nucleotide diversity with Chinese pigs although they all belonged to the Asian major clade. Chinese domestic pigs were clustered with wild boars in Yangtze River region and South China.     

陆川猪mtDNA D-loop序列遗传多样性分析

本研究测定了陆川猪17个个体mtDNA D-loop高变区序列,共检测到5个单倍型,单倍型多样度和核苷酸多样度分别为0.640和0.0074,表明陆川猪有较高的遗传多样性。结合已报道的中国地方猪种的mtDNA D-loop序列进行分析,结果发现,陆川猪和属于华中型的宁乡猪、大花白猪有很近的遗传关系,但与传统上同属华南型的香猪和滇南小耳猪的遗传关系较远,这与传统的分类方法不一致。     

The Genetic Diversity and Phylogenetic Relationships of Five Guangxi Pig Breeds and Three Exotic Pig Breeds by Analysis of mtDNA D-loop Sequences

In order to compare the genetic diversity and phylogenetic relationships between Guangxi pig breeds and three exotic pig breeds,one pair of primers was used to amplify 85 mtDNA D-loop sequences of five Guangxi pig breeds (Bama Xiang pig,Dongshan pig,Guizhong spotted pig,Luchuan pig,Huanjiang Xiang pig),and we downloaded 45 mtDNA D-loop sequences of three exotic pig breeds (Duroc,Yorkshire,Landrace) from GenBank.Then the haplotype,genetic diversity and phylogenetics analysis were carried out.The results showed that 38 haplotypes were identified in 130 sequences,and the mean distances among Guangxi pig breeds were smaller than that among Duroc,Landrace and Guangxi pig breeds.The NJ phylogenetic tree classified all samples into two clusters.One contained all of Guangxi pig breeds and partial Yorkshire,the other was composed wholly of exotic pig breeds,which mean there were two distinct maternal origins.In the cluster of Guangxi pig breeds,Luchuan pig were clustered,and the others were rather dispersive.It indicated that Luchuan pig had simple maternal bloods,and the other breeds had complex maternal bloods,and they had gene flow with each other.The maternal pig resources in Guangxi were not affected by the introduction of exotic pig breeds,and they were still suitable for use as a female parent in their development and utilization.     

几个地方绵羊品种线粒体DNA(mtDNA)细胞色素b基因多态性研究

用12种限制性内切酶分析了6个地方绵羊品种线粒体DNA（mtDNA）细胞色素b（cytb）基因的RFLP。结果表明，在71只绵羊个体中，检出的14种限制性态型可归结成4种单倍型，其间的差异主要来源于几个限制性位点的点突变；4种单倍型间和6个绵羊群体间的平均遗传距离（D）分别为0．412％和0．041％，遗传多态程度（π）为0．028％，说明遗传多样性较为贫乏；单倍型聚类结果和群体聚类结果提示，我国地方绵羊品种的起源可能是单一的。     

深县猪mtDNA CytB基因的遗传多样性与母系起源研究

为探讨深县猪群体内的遗传多样性及母系起源分化,采用PCR直接测序法测定了40头深县猪的mtDNA CytB基因长1 140 bp片段的全序列,并结合GenBank已公布的15个猪种mtDNA CytB基因全序列,采用邻接法构建深县猪、部分其他地方猪种和引进猪种的系统发育树。结果显示,在1 140 bp长的序列中A、T、G、C的含量分别为25.93%、31.72%、28.90%及13.45%,其中A+T的含量(57.65%)高于G+C的含量(42.35%);共发现2个变异位点,无插入和缺失突变,全部为转换位点,其中简约信息位点2个,未发现单一信息位点。40条序列共定义了3种单倍型,单倍型多样性(HD)为0.312,核苷酸多样性(Pi)为0.00026,平均核苷酸差异数(K)为0.327。在深县猪mtDNA CytB基因全序列NJ进化树中,三种单倍型聚为同一分支,母系来源单一,与莱芜猪、大蒲莲猪等山东品种遗传距离较近。结果表明,深县猪群体内遗传多样性比较贫乏,与山东地区的华北型黑猪种群有更近的亲缘关系。     

五指山猪线粒体控制区序列分析及遗传多样性研究

为了研究五指山猪遗传多样性和系统地位,本研究采用PCR产物直接测序法得到55条五指山猪线粒体控制区(mtDNA D-Loop)全序列,并结合GenBank中发表的1条五指山猪mtDNA D-Loop全序列进行结构分析、遗传多样性研究及系统发育分析。结果发现,56条序列中出现21次保守区变异,即"TTATAAAACAC"序列重复,共定义13个单倍型,发现14个变异位点,单倍型多样度(Hd)和核苷酸多样度(Pi)分别为0.838和0.00334,表明五指山猪遗传多样性较丰富。五指山猪线粒体控制区序列构建的系统发育树和Network网络分析呈现两大分支,推测有两个母系起源,且与GenBank中其他23个猪种的聚类结果表明,五指山猪与长江流域以南地区的猪种有着较近的亲缘关系。     

建昌马mtDNA D-loop区遗传多样性及系统进化分析

试验旨在以线粒体DNA(mitochondrial DNA,mtDNA)为切入点,研究建昌马的母系遗传多样性与系统进化。从建昌马(n=39)血液中提取基因组DNA,用PCR方法扩增mtDNA D-loop区并直接测序,分析其高变区247 bp序列信息,统计mtDNA D-loop区的单倍型及变异位点,计算单倍型多样性(haplotype diversity,Hd)、核苷酸多样性(nucleotide diversity,Pi)和平均核苷酸变异数(average number of nucleotide differences,K)。构建包括建昌马在内的19个品种马的NJ系统进化树,计算各品种间的遗传距离。结果显示,试验获得了清晰的PCR扩增产物,并通过直接测序方法获得了约1200 bp的序列。39匹建昌马mtDNA D-loop区247 bp序列(其中1个样品缺失1 bp)的AT碱基含量为61.45%,属AT碱基对富集区,检测到33个多态性位点,共显示26种单倍型,其中4种为共享单倍型,且Hap7和Hap1为优势单倍型,单倍型多样性为0.947,核苷酸多样性为0.02399,平均核苷酸变异数为5.901,显示丰富的母系遗传多样性;NJ系统进化树显示,建昌马分布在A、C、D、E、F、G共6个支系中,约50%的样品分布在A支系,显示出复杂的母系起源;建昌马与关中马的遗传距离最小(0.021),其次是三河马、文山马、韩国车巨马(0.024),与韩国济州岛马遗传距离最大(0.032)。本研究结果表明,建昌马的mtDNA D-loop高变区遗传多样性丰富,具有多个母系起源,且A支系占有明显优势,与关中马、文山马可能有共同的母系起源。     

Complete Mitochondrial DNA D-Loop Sequence and Genetic Diversity of Wuzhishan Pig

In order to research the genetic diversity and phylogenies of Wuzhishan pig, 56 complete mtDNA D-Loop were analyzed.55 sequences were amplified by polymerase chain reaction (PCR) and 1 sequence was downloaded from GenBank.The results showed that repetitive sequence "TTATAAAACAC" appeared in the conservative regions of 21 mtDNA D-Loop sequences.13 haplotypes and 14 variable sites were observed in 56 sequences.Haplotype diversity and nucleotide diversity (Hd =0.838 and Pi=0.00334) indicated that Wuzhishan pig had high genetic diversity.Two branches in the phylogenetic tree and Network analysis diagram indicated that there were two maternal origins in Wuzhishan pig.The phylogenetic tree of Wuzhishan pig and other 23 species showed that Wuzhishan pig had close genetic relationship with the pig breeds in the south region of the Yangtze river.     

江淮水牛线粒体D-loop区和Cytb基因遗传多态性及系统发育分析

为研究安徽江淮水牛群体的分子种质特性,本试验测定了江淮水牛2个亚群共82个个体的线粒体D-loop区和细胞色素b基因完整序列,分析了序列遗传多态性及系统进化关系,并结合GenBank中已发表的141条中国水牛D-loop序列,进行了联合分析。结果在D-loop区内共发现核苷酸多态位点91个,组成104个单倍型,其中32个是在江淮水牛中新发现的单倍型。总体mtDNA D-loop区核苷酸多样性为(0.015 43±0.001 42),单倍型多样性为(0.948±0.009)。其中,江淮水牛的mtDNA D-loop区核苷酸多样性为(0.014 89±0.002 32),单倍型多样性为(0.955±0.013),表明其群体遗传多样性丰富,群体变异性水平与中国其他水牛群体接近。根据线粒体D-loop区单倍型构建系统树和进化网络,显示江淮水牛存在沼泽型水牛线粒体支系A和B,表明其具有2个线粒体母系来源,其中B支又分为b1和b2两个亚支;针对细胞色素b基因的进化分析也支持这一结论。这些研究结果为今后开展江淮水牛遗传资源的保护和利用提供了客观依据。     

几个中国地方猪群体遗传结构和产仔数性状选择信号分析

旨在对几个中国地方猪品种进行群体遗传结构分析,并筛选与中国地方猪产仔数相关的基因组选择信号及候选基因.本研究下载了6个中国地方品种猪共计102头个体的Illumina PorcineSNP60芯片数据,构建了包括19头迪庆藏猪、16头明光小耳猪、16头五指山猪在内的低产仔数组和包括11头姜曲海猪、20头蓝塘猪、20头梅...     

Interpopulation and intrapopulation maternal lineage genetics of the Lanyu pig (Sus scrofa) by analysis of mitochondrial cytochrome b and control region sequences

The Lanyu pig is an indigenous breed from the Lanyu Islet, which is southeast of Taiwan. Two herds of Lanyu pigs were introduced from the Lanyu Islet into Taiwan in 1975 and 1980. The current population of conserved Lanyu pigs consists of only 44 animals with unknown genetic lineage. The Lanyu pig possesses a distinct maternal genetic lineage remote from Asian and European pigs. The present study aimed to understand the phylogenetic relationship among conserved Lanyu, Asian, and European type pigs based on the cytochrome b coding gene, to ascertain the maternal lineage and genetic diversity within the conserved Lanyu pigs, and to address whether genetic introgression from exotic or Formosan wild pigs had occurred in the conserved Lanyu pigs. Entire mitochondrial genomes of both types of Lanyu pig comprised 2 ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes. Only 2 haplotypes of the mitochondrial DNA (mtDNA) control region and cytochrome b were identified in the conserved Lanyu pig herds. When maximum likelihood trees were constructed, the Type I Lanyu mitochondrial genes formed a unique clade with a large pairwise distance of both cytochrome b and the control region from Asian and European type breeds, Formosan wild pigs, and exotic breeds. Significant loss of genetic diversity of mtDNA within the conserved Lanyu pigs was demonstrated by low haplotype and nucleotide diversities, supported by Fu and Li's D* neutrality test (1.44055; P < 0.05). The mtDNA control region sequences of extant pigs in the Lanyu Islet, however, showed high haplotype and nucleotide diversity, and clustered with exotic pigs. These results indicate no maternal lineage mtD-NA gene introgression from Formosan wild pigs and introduced exotic pigs to conserved Type I Lanyu pigs, and a severe loss of heterozygosity of mtDNA in conserved Lanyu pigs. The remaining extant pigs on the Lanyu Islet have been introgressed with exotic breeds. Strategies for future conservation of native Lanyu pigs are now even more urgent and important.     

4种鼢鼠线粒体DNA D-loop区全序列分析

采用PCR测序技术对采自甘肃境内的4种鼢鼠22个个体的线粒体DAND-loop区全序列进行了测定。结果表明：鼢鼠线粒体DNAD-loop序列长度为893、894、895、896或899bp,核苷酸位点突变类型有5种,即转换、颠换、插入、缺失及转换与颠换共存。碱基转换以T〈=〉C形式为主。其中T、C、A、G4种核苷酸的平均比例分别为34．1％、24．3％、30．1％、11．5％,A＋T含量（64．2％）明显高于G＋C含量（35．8％）。这4种鼢鼠22条线粒体DNAD-loop区发现20种单倍型,单倍型比例为81．82％,说明中国鼢鼠mtDNA遗传多态性很丰富。从系统发育树分析,4种鼢鼠明显聚为两类,推测鼢鼠可能有两个起源。     

阿坝藏族羌族自治州若尔盖地区藏猪mtDNA D-Loop的遗传多样性分析

旨在通过获得和对比若尔盖地区藏猪mtDNA D-Loop高变区的部分序列,为该地区藏猪遗传资源的保护与利用提供参考。本试验收集了若尔盖地区9个乡镇共80头藏猪耳组织,以甘南州藏猪mtDNA D-Loop基因序列为模板设计引物,采用PCR扩增测序技术获得了80条核苷酸序列,并采用生物信息学的数据处理方法进行分析。结果表明,若尔盖地区藏猪mtDNA D-Loop高变区（435 bp）的A+T含量（56.46%）明显高于G+C含量（43.54%）,存在碱基偏倚性现象;在80条长度为435 bp的序列中,共检测到14个变异位点,鉴定了17个单倍型,单倍型多样度（Hd）、核苷酸多样度（Pi）和平均核苷酸差异数（k）分别为0.881、0.004 66和2.028,其中Hd、Pi和k在降扎乡藏猪群体中最高,Pi和k在占哇乡藏猪中最低;共享单倍型8个,特有单倍型9个,且若尔盖地区不同藏猪群体间特有单倍型数差异较大,其中,降扎乡藏猪的特有单倍型数量最多,占单倍型总数的17.65%（3/17）;Hap_1和Hap_15单倍型是4个乡镇（降扎乡、益哇乡、热尔乡和冻列乡）群体的共享单倍型,表明这4个乡镇藏猪群体存在两个共同的母系祖先单倍型。若尔盖地区藏猪的平均遗传距离为0.004 66,其中降扎乡和冻列乡藏猪间的遗传距离最大为0.006 90,包座乡和益哇乡藏猪间的遗传距离最小为0.002 16;构建的NJ分子系统进化树将若尔盖地区藏猪分为2支,而加入中国地方家猪、野猪和引种猪后,若尔盖地区藏猪在进化树中比较分散,说明该地区藏猪母源血统遗传复杂,彼此之间基因交流多。本研究进一步证实了若尔盖地区藏猪比西藏林芝、山南、日喀则、甘孜州、阿坝州藏猪的遗传多样性程度高,受到人工选择强度低,应强化对若尔盖地区藏猪遗传资源的保护与利用。     

Genetic variations and sequences analysis of MTATP6 and MTATP8 genes among different Chinese pig breeds

To investigate the genetic variations of mtATP6 and mtATP8 genes among different Chinese pig breeds, two fragments of 425 and 743 bp containing the whole coding region of mtATP8 and mtATP6 genes were amplified with 805 individuals from 23 Chinese local pig breeds, three types of Chinese wild boars and three European pig breeds. Sequence comparison identified a total number of 17 substitutions including six variable sites in mtATP8 and eleven substitutions in mtATP6 gene. The restriction enzyme Fok I revealed four polymorphic sites (nt8086, 8176, 8514 and 7784), and four RFLP haplotype patterns (A, B, C and D) were identified in mtATP6 and mtATP8 genes among all tested samples. Our data showed AC combined haplotype originated from Asia and BD was regarded as European origin. The average frequency of Asian mtDNA haplotypes was 38.3% across the investigated European breeds but varied within each breeds (13.3～76.7%). Phylogenetic analyses were performed also considering some published sequences in the databases; the sequences were divided into three distinct groups, denoted A, E1, and E2. The Asian AC haplotype existed among the European domestic pigs was fully consistent with the results of previous molecular studies and well-documented history. This study will help us to better understand the genetic variations of mitochondrial genes among different Chinese pig breeds.     

Genetic diversity of mtDNA D-loop sequences in four native Chinese chicken breeds

1. To explore the genetic diversity of Chinese indigenous chicken breeds, a 585 bp fragment of the mitochondrial DNA (mtDNA) region was sequenced in 102 birds from the Xichuan black-bone chicken, Yunyang black-bone chicken and Lushi chicken. In addition, 30 mtDNA D-loop sequences of Silkie fowls were downloaded from NCBI. The mtDNA D-loop sequence polymorphism and maternal origin of 4 chicken breeds were analysed in this study.

2. The results showed that a total of 33 mutation sites and 28 haplotypes were detected in the 4 chicken breeds. The haplotype diversity and nucleotide diversity of these 4 native breeds were 0.916 ± 0.014 and 0.012 ± 0.002, respectively. Three clusters were formed in 4 Chinese native chickens and 12 reference breeds. Both the Xichuan black-bone chicken and Yunyang black-bone chicken were grouped into one cluster. Four haplogroups (A, B, C and E) emerged in the median-joining network in these breeds.

3. It was concluded that these 4 Chinese chicken breeds had high genetic diversity. The phylogenetic tree and median network profiles showed that Chinese native chickens and its neighbouring countries had at least two maternal origins, one from Yunnan, China and another from Southeast Asia or its surrounding area.     

中国家驴mtDNA D-loop 遗传多样性与起源研究

对我国12个家驴品种126个个体（包括引用26个个体）的mtDNA D-loop区399 bp进行分析,共检测到36种单倍型37个多态位点,其单倍型多样度为0.466 7-0.977 8,核苷酸多样度为0.001 2-0.028 5,表明我国家驴的遗传多态性丰富。与3条努比亚野驴、3条索马里野驴和6条亚洲野驴的序列构建NJ系统发育树,首次证明我国家驴的母系起源为非洲野驴中的索马里驴和努比亚驴,亚洲野驴不是中国家驴的祖先。本文还讨论了我国家驴可能的迁徙路线。     

4个引进山羊品种mtDNA控制区序列变异和系统发生关系研究

本研究测定了四川4个引进山羊品种24个个体的线粒体控制区全序列,并从GenBank获得山羊属2个野山羊种的2每控制区序列。利用MEGA2．0软件构建分子系统发育无根树。序列分析表明:山羊控制区线粒体控制全序列长度为1 212bp或1 213 bp,A+T含量占59．9％,其中64个核苷酸位点存在变异(约占5．28％),核苷酸多样度为 1．731％,这些差异共定义了16种单倍型,单倍型多样性为0．913±0．048。安哥拉山羊、波尔山羊都有自己独特的单倍型,与其他品种间都没有共享类型。使用NJ法构建了系统发育树,结果表明:2个野山羊种中,角(?)羊与家养山羊的关系相对较近,4个家养山羊品种有2个母系来源,在4个家养山羊品种内部,安哥拉山羊的分化要比其他3个品种早。     

中国四个地方驴品种mtDNA D-Loop部分序列分析与系统进化研究

对中国4个家驴品种95个个体的mtDNAD—loop部分序列进行测序。用clustalX软件进行同源序列比对。Dnasp4-10软件用于遗传多样性分析，计算单倍型多样度、核苷酸多样度、平均核苷酸差异。MEGA3．1软件采用邻接法构建系统进化树并进行系统发生分析。以欧洲驴线粒体基因组为对照（Genbank登录号为X97337），中国家驴4个品种95个个体385bp序列共检测到核苷酸多态位点33个，其中31个位点为转换，1个位点为颠换，1个位点有插入现象。95个序列由31个单倍型组成，单倍型比例为32．6％。德州驴单倍型比例最高为100％，凉州驴单倍型比例最低为42．42％。31个单倍型与引用Genbank已登录的13条序列构建系统发育树，发现31个单倍型聚为两支，说明中国家驴可能有两个母系起源。以欧洲野驴线粒体D—loop为对照（登录号为AF403063、AF403063、AF403065），揭示本研究涉及的中国家驴品种与非洲野驴的进化亲缘关系较亚洲野驴近，并且从分子水平证明中国家驴可能起源于非洲野驴中的索马里野驴分支。     

Phylogenetic analysis of Myanmar indigenous chickens using mitochondrial D-loop sequence reveals their characteristics as a genetic resource

Myanmar indigenous chickens play important roles in food, entertainment, and farm business for the people of Myanmar. In this study, complete mitochondrial D-loop sequences (1232 bp) were analyzed using 176 chickens, including three indigenous breeds, two fighting cock populations, and three indigenous populations to elucidate genetic diversity and accomplish a phylogenetic analysis of Myanmar indigenous chickens. The average haplotype and nucleotide diversities were 0.948 ± 0.009 and 0.00814 ± 0.00024, respectively, exhibiting high genetic diversity of Myanmar indigenous chickens. Sixty-four haplotypes were classified as seven haplogroups, with the majority being haplogroup F. The breeds and populations except Inbinwa had multiple maternal haplogroups, suggesting that they experienced no recent purifying selection and bottleneck events. All breeds and populations examined shared haplogroup F. When 232 sequences belonging to haplogroup F (79 from Myanmar and 153 deposited sequences from other Asian countries/region) were analyzed together, the highest genetic diversity was observed in Myanmar indigenous chickens. Furthermore, Myanmar indigenous chickens and red junglefowls were observed in the center of the star-like median-joining network of 37 F-haplotypes, suggesting that Myanmar is one of the origins of haplogroup F. These findings revealed the unique genetic characteristic of Myanmar indigenous chickens as important genetic resources.