Multiple domestication of swamp buffalo in China and South East Asia

The domestication of swamp buffalo (Bubalus bubalis carabanesis) has been discussed for years and still remained unclear. To obtain a better understanding of where, when and how the swamp buffaloes were domesticated, the complete mitochondrial DNA D-loop sequences of 1,788 individuals from China, Vietnam, Laos, Thailand, Burma, Bangladesh and India were investigated. Our results revealed swamp buffalo with abundant genetic diversity. The lineage SA of swamp buffalo may be first domesticated in Lower Yangtze and then migrated following two different routes: one migrated along the Upper Yangtze to Southwest China (L1); the other migrated to Southeast China to Southeast Asia through Guangxi province (L2). During the migration process several later domestication events may be inspired in the Upper Yangtze (SB3), Southwest China and North of Southeast Asia (SB1 and SB2). In this study, we proposed a non-independent multiple domestication pattern in swamp buffalo.     

利用微卫星DNA标记分析槟榔江水牛群体遗传特征

槟榔江水牛是近年在我国云南西部发现的第一个本土河流型水牛.为了揭示其群体遗传多样性、群体遗传组成、其与河流型和沼泽型水牛的遗传关系及沼泽型水牛对该水牛的基因渗入等重要遗传背景信息,本研究采用30个微卫星DNA标记对141份槟榔江水牛样品和对照群体24份河流型水牛(摩拉水牛)样品、41份沼泽型水牛(滇东南水牛)样品进行了检测分析.结果,在槟榔江水牛群体中共检测到253个等位基因,其中,54个为3个群体所共享的等位基因,58个为只在槟榔江水牛与摩拉水牛间共享的等位基因,73个为只在槟榔江水牛与滇东南水牛间共享的等位基因,其余68个等位基因为该群体所独有.槟榔江水牛平均等位基因数、平均表观杂合度、平均期望杂合度和多态信息含量等参数均显著高于对照组群体,分别为8.433 3、0.640 5、0.600 9和0.594 7.该水牛群体近交系数FIS值为0.061 9.槟榔江水牛与摩拉水牛间的DA遗传距离为最小(0.114 4),在NJ树上聚为一支;而滇东南水牛与槟榔江水牛和摩拉水牛间的遗传距离较大(分别为0.382 9和0.555 3),其在NJ树上单独聚为一支.群体遗传结构分析显示,槟榔江水牛遗传组分为河流型与沼泽型2种类型水牛混合的模式(当K=2时),整个群体中有相当数量的个体存在沼泽型水牛的遗传渗入(群体中沼泽型水牛遗传组分为0.067 2).结果揭示,槟榔江水牛遗传资源独特,群体遗传多样性丰富,但该群体杂合子缺乏,且存在一定沼泽型水牛的基因渗入.     

我国首例本土河流型水牛——槟榔江水牛的种质特征

槟榔江水牛在云南西部槟榔江上游的腾冲县饲养已有500年以上的历史。该水牛经过长期闭锁繁育和风土驯化，已适应当地亚热带气候环境，是我国发掘的首例本土河流型水牛类群。具有典型的河流型水牛体型外貌特征，但体型相对较小。遗传学分析表明：槟榔江水牛体细胞核型染色体数多为2n=50；其群体内遗传变异水平较高，遗传多样性丰富；该水牛与河流型水牛表现出较近的遗传关系，与沼泽型水牛遗传关系较远；确认在群体遗传组成和血统来源上，该水牛由差异十分明显的河流型和沼泽型两个世系所构成，但河流型水牛世系在该水牛群体中占主体，揭示该水牛存在一定的沼泽型水牛基因渗入。生产性能测定结果显示：该水牛一个泌乳期泌乳量为（2452．2±553．8）kg，比河流型摩拉和尼里一拉菲水牛高8．4％～15．0％，比沼泽型本地乳用水牛高124．4％～140．3％；且其乳质佳，乳蛋白率和乳脂率分别为4．60％和6．82％。该水牛在2008年经国家畜禽遗传资源委员会颁布为我国遗传资源的牛种之一，发展前景看好，将有望培育成中国首个河流型乳用水牛新品种。     

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。     

家养水牛催乳素基因外显子2多态性及其群体遗传特征

催乳素基因(prolactin gene,PRL)被视为与水牛产奶性状相关的重要候选基因,目前还未见有水牛PRL基因外显子2遗传特征的报道。本研究采用PCR-SSCP及PCR产物直接测序技术,检测了12个沼泽型水牛群体和3个河流型水牛群体共581个样本PRL基因外显子2的遗传变异。结果:水牛PRL基因外显子2由182个核苷酸组成,编码61个氨基酸,在进化上高度保守,测序结果显示沼泽型水牛群体中PRL基因外显子2有c.G192A替换,为同义替换,与泌乳性状之间没有显著相关性。在12个沼泽型水牛群体中均检测到该多态位点,PG基因频率在0.719～0.897之间,群体平均值为0.785±0.015,该基因为优势等位基因。Hardy-Weinberg平衡检测显示,10个沼泽型水牛群体处于平衡状态,而德宏水牛群体和福安水牛群体处于不平衡状态。在3个河流型水牛群体中,第192位碱基均为G,未检测到G>A替换。     

盱眙水牛遗传多样性及系统地位的研究

对江苏省盱眙县183头水牛外形特征及体尺指标进行了调查和测量,同时随机抽取48头母牛,用垂直平板聚丙烯酰胺凝胶电泳(PAGE)和水平淀粉凝胶电泳(SGE)对编码血液蛋白18个基因座的基因频率进行检测,同时引用相同方法所得东南亚21个水牛群体5个座位的研究资料,进行聚类分析,结果表明:成年公牛体高、体长、胸围和管围分别为129.16,147.69,198.66和23.12 cm,成年母牛分别为128.43,143.81,193.11和21.62 cm,估计体重公母牛分别为516.39和479.83 Kg。在所检测18个基因座中,有5个存在多型,多态位点百分比为27.78%。群体内遗传变异水平较低,平均杂合度和平均有效等位基因数分别为0.0793和1.1355。聚类分析表明:盱眙水牛与台湾和泰国水牛亲缘关系较近,孟加拉国为沼泽型水牛和河流型水牛的分界线,不同水牛群体的聚类结果基本与其地理分布吻合。     

The transfer of fresh and frozen embryos in an elite swamp buffalo herd

To investigate the development of fresh and frozen swamp buffalo embryos after transfer to synchronized recipients, 14 fresh embryos and 28 frozen embryos, collected from Thai swamp buffalo cows of an elite herd at the Surin Breeding Center, were transferred nonsurgically to 31 synchronized recipients buffalo cows. One fresh embryo was transferred to each of 14 recipients. Twenty eight frozen embryos were transferred to 17 recipients of which 7 cows received I embryo, 9 cows received 2 embryos and 1 cow received 3 embryos. Pregnancy was diagnosed by real time B-mode ultrasonography one month after transfer and confirmed by rectal palpation one and two months later. The pregnant cows were kept under observation until calving. The results of fresh embryo transfer showed that 5/14 (35.7%) were pregnant after 30 days, 4/14 (28.6%) remained pregnant until the 3rd month and 2/14 (14.3%) calved. With the frozen embryos, only one cow which received three embryos became pregnant and remained so for 3 months although the embryo did not survive to full term. The overall pregnancy rate using frozen embryos was 5.9% (1/17). The study demonstrated the possibility of performing embryo transfer in elite buffalo herds for genetic improvement, however the use of frozen embryos needed further investigation.     

To catch a buffalo: field immobilisation of Asian swamp buffalo using etorphine and xylazine

OBJECTIVE: To demonstrate the efficacy of a mixture of etorphine and xylazine to safely immobilise wild buffalo (Bubalus bubalis) in the field. METHODS: Body mass was estimated (to calculate mass-specific dosages) by deriving a predictive relationship between morphometric measurements (body length, height) and mass based on a dataset collected in Vietnam, because the study animals could not be weighed in the field. RESULTS: Mass-specific dosages varied between 0.02 and 0.03 mg/kg for etorphine and between 0.14 and 0.22 mg/kg for xyalazine; induction times varied between 10 and 33 min, mean recumbency time was 68 min, and the mean time to standing was 10 min (range: 10-17 min). CONCLUSIONS: The mixture of ethorphine and xylazine was effective for immobilisation of this species and appeared to have a relatively large safety margin, based on the mass-specific dosages used. The allometric relationships described here should prove useful for those working with wild swamp buffalo.     

Genetic analysis of river,swamp and hybrid buffaloes of north‐east India throw new light on phylogeography of water buffalo (Bubalus bubalis)

This study analysed buffaloes from north‐east India and compared their nuclear and mitochondrial DNA variations with buffaloes of mainland India, China, Mediterranean and South‐East Asia. Microsatellite genotypes of 338 buffaloes including 210 from six north‐east Indian buffalo populations and three mainland Indian breeds were analysed to evaluate their genetic structure and evolutionary relationships. Phylogenetic analysis and multidimensional scaling plot of pairwise F_ST revealed the clustering of all swamp‐type buffaloes of north‐east India with Lower Assamese (significantly hybrid type) buffaloes in one plane and all the mainland river buffaloes in another plane while the upper Assamese buffaloes being distinct from both these clusters. Analysis of mtDNA D‐loop region of 530‐bp length was performed on 345 sequences belonging to 23 buffalo populations from various geographical regions to establish the phylogeography of Indian water buffalo. The swamp buffaloes of north‐east India clustered with both the lineages of Chinese swamp buffalo. Multidimensional scaling display of pairwise F_ST derived from mitochondrial DNA data showed clustering of upper Assamese, Chilika and Mediterranean buffaloes distinctly from all the other Indian buffalo populations. Median‐joining network analysis further confirmed the distinctness and ancestral nature of these buffaloes. The study revealed north‐east region of India forming part of the wider hybrid zone of water buffalo that may probably extend from north‐east India to South‐East Asia.     

槟榔江水牛保种及选育利用策略

槟榔江水牛是近年在云南西部发现的我国第一个本土河流型水牛群体,具有较高的产奶性能,是发展我国水牛奶业宝贵的遗传资源。本文基于近几年对槟榔江水牛遗传资源调查和种质特性及遗传背景分析的结果,就其遗传资源的保护和选育利用方略进行了探讨。     

水牛κ-酪蛋白基因（CSN3）外显子4序列多态性研究

 分别设计位于CSN3 5个外显子旁侧引物,采用DNA测序法对沼泽型和河流型水牛CSN3编码区结构进行了分析,并对10个水牛群体共106个样本CSN3第4外显子序列进行了变异检测。结果表明,两类水牛CSN3编码区由848个核苷酸组成,包括ORF序列573 bp、5′-UTR序列69 bp和3′-UTR序列206 bp。在水牛CSN3第4外显子中共检测到4个SNP,其中c.445G>A,c.467C>T和c.516A>C为异义替换,导致相应的κ-CN成熟肽中氨基酸发生p.Val128Ile、p.Thr135Ile和p.Glu151Asp改变,c.467C>T和c.516A>C替换可能对κ-CN功能产生了影响。群体遗传分析表明,在河流型水牛中,等位基因c.445G、c.467C、c.471C和c.516A均为优势等位基因,而在沼泽型水牛中,仅c.445G、c.467C和c.471C为优势等位基因,河流型和沼泽型水牛的遗传差异主要体现在SNP516位点的群体遗传组成上。     

云南水牛mtDNA D-loop遗传多样性研究

[目的] 探究云南3个水牛品种(德宏水牛、滇东南水牛、盐津水牛)的mtDNA D-loop区的遗传多样性与母系起源。[方法]采用PCR扩增、测序及生物信息学方法。[结果] 本研究共分析了215条云南水牛mtDNA D-loop全序列,检测到107个多态位点,定义了86种单倍型。结果表明,云南3个水牛品种的mtDNA遗传多样性非常丰富,其中,滇东南水牛的遗传多样性最高(Hd: 0.946±0.017,Pi: 0.0162±0.0018),盐津水牛的遗传多样性最低(Hd: 0.805±0.063,Pi: 0.0141±0.0031)。系统发育分析结果表明,在3个云南水牛品种中检测到2个主要支系(A和B支系及其亚支系)及一个稀有支系C,其中A支系为主要支系(79.07%),且经历了强烈的群体扩张。[结论] 云南3个水牛品种具有丰富的mtDNA遗传多样性,主要有A与B两个母系起源。     

德宏水牛的保种选育及利用

[目的]根据德宏水牛的品种特性及发展现状,做好德宏水牛的保种选育及合理的开发利用,保持德宏水牛原有的特性。[方法]采用常规保种方法：制定德宏水牛保种选育方案,划定保种区,建立保种核心群,采用各家系等量留种和防止近交的一系列措施。[结果]德宏水牛具有体格高大,结构匀称,产肉性能好,耐湿热,耐粗饲,繁殖力强,适应性、抗病力强等特点。[结论]德宏水牛是云南省著名的沼泽型水牛中的优良地方品种,屠宰率、净肉率高,具有较高的遗传变异和丰富的生物遗传多样性,保种选育工作亟待重视和加强。     

水牛黑色素皮质素1受体基因克隆、生物信息学分析及表达模式研究

本试验旨在对水牛黑色素皮质素1受体(MC1R)基因进行克隆、生物信息学分析及表达模式研究。参考牛MC1R基因(GenBank登录号:JN123363.1)序列设计引物,以本地沼泽水牛、白沼泽水牛、摩拉水牛和黄牛基因组DNA为模板,应用PCR方法扩增克隆MC1R基因片段并进行测序分析。运用QRT-PCR方法检测摩拉水牛、沼泽水牛、白沼泽水牛和黄牛皮肤组织中MC1R基因的表达模式,并通过Western blotting方法检测沼泽水牛和白沼泽水牛MC1R基因的蛋白表达差异。结果表明,应用PCR方法成功克隆了水牛MC1R基因,其编码区全长954 bp,共编码317个氨基酸。测序分析后发现沼泽水牛、白沼泽水牛、摩拉水牛和黄牛MC1R基因的核苷酸序列和氨基酸序列相似性很高。沼泽水牛与白沼泽水牛在476、618、881、930和931 bp位点上分别发生T→C、G→C、G→A、G→A和A→G突变,导致了沼泽水牛和白沼泽水牛第159位氨基酸由丝氨酸变成苯丙氨酸,第310位氨基酸由谷氨酸变成丙氨酸,第294位氨基酸由天冬氨酸变成丙氨酸,发生了非同义突变。QRT-PCR结果发现,MC1R基因在摩拉水牛、沼泽水牛和黄牛皮肤组织中的相对表达量均显著高于白沼泽水牛(P<0.05);Western blotting分析结果显示,沼泽水牛皮肤组织中MC1R蛋白的表达量高于白沼泽水牛。综上所述,白沼泽水牛MC1R基因的编码区发生氨基酸位点突变,且相对表达量和蛋白表达量均低于沼泽水牛,推测此为白沼泽水牛体内合成的黑色素缺失而导致毛色白化的主因。     

沼泽型水牛微卫星DNA的遗传多样性分析

为探究中国沼泽型水牛品种或类群的遗传多样性,本研究采用了沼泽型水牛30个微卫星标记结合LabChip芯片检测法对德昌水牛、德宏水牛、温州水牛、贵州水牛、西林水牛、富钟水牛及两个引进品种摩拉水牛、尼里－拉菲水牛等40个样本进行了检测和遗传多样性分析。结果表明,在30个微卫星DNA标记上共发现332个等位基因,平均基因多样性和PIC值分别为0.7808和0.7554。聚类分析结果显示,德宏水牛和德昌水牛先聚为一类,随后与富钟水牛、贵州水牛、温州水牛、西林水牛聚为一类;摩拉水牛与尼里－拉菲水牛聚为一类。本研究证实了所选的30个沼泽型水牛微卫星DNA标记可作为有效的遗传标记用于水牛品种间的遗传多样性分析,同时也丰富了当前的水牛微卫星标记资源。     

Genetic Diversity Analysis of Microsatellite DNA Markers in Swamp Buffalo

This study was aimed to investigate the genetic diversity of swamp buffalo breeds in China.The analysis of genetic diversity was performed in 40 buffalo individuals from 8 buffalo breeds（Dechang buffalo,Dehong buffalo,Wenzhou buffalo,Guizhou buffalo,Xilin buffalo,Fuzhong buffalo,Murrah buffalo and Nili-Ravi buffalo）by 30 microsatellite loci and LabChip chip test method.The results showed that 332 alleles at 30 microsatellite loci were found in 8 buffalo breeds,the average values of gene diversity and PIC were 0.7808 and 0.7554,respectively.Cluster analysis indicated that Dechang buffalo and Dehong buffalo firstly clustered together,followed by Fuzhong buffalo,Guizhou buffalo,Wenzhou buffalo and Xilin buffalo.Moreover,Murrah buffalo and Nili-Ravi buffalo clustered together.Our findings revealed that the 30 microsatellite loci could be used as an effective genetic marker for the analysis of genetic diversity among the buffalo breeds,which enriched the current SSR marker resources in buffalo.     

Cloning,Bioinformatics and Expression Pattern Analysis of Buffalo MC1R Gene

The aim of this study was to clone and analyze the expression pattern of buffalo MC1R gene.A pair of specific primers was designed according bovine MC1R sequence (GenBank accession No.:JN123363.1),with genome DNA of swamp buffalo,White swamp buffalo,Murrah buffalo and Yellow cattle as template,MC1R was amplified by PCR.Then relative expression level of MC1R gene of swamp buffalo,White swamp buffalo,Murrah buffalo and Yellow cattle was analyzed using QRT-PCR,and protein expression was detected by Western blotting method.The results showed that the 954 bp coding region of buffalo MC1R gene was successfully cloned and sequenced,which code for 317 amino acids.The MC1R gene nucleotide sequences and amino acid sequences of swamp buffalo,White swamp buffalo,Murrah buffalo and Yellow cattle were highly conserved.Five polymorphic sites were found between White swamp buffalo and swamp buffalo of MC1R gene,including 476 T→C,618 G→C,881 G→A,930 G→A and 931 A→G,which caused three nonsynonymous mutation sites of Phe159Ser,Glu310Ala and Asp294Ala.The QRT-PCR result showed that relative expressions of MC1R gene of Murrah buffalo,swamp buffalo and Yellow cattle were significant higher than that of White swamp buffalo (P<0.05).The Western blotting results revealed that MC1R protein expression level in swamp buffalo was higher than that of White swamp buffalo.In conclusion,there were amino acids mutation in White swamp buffalo MC1R gene,and MC1R gene relative expression of White swamp buffalo was lower than that of other buffalo,which was the main reason of lacking of melanin production in White swamp buffalo.     

广西的尼里—拉菲水牛和摩拉水牛mtDNA D-loop区遗传多样性研究

[目的]探究引入广西南宁的尼里-拉菲水牛和摩拉水牛的mtDNA D-loop区遗传多样性与母系起源。[方法]采用PCR扩增、测序及生物信息学方法。[结果]对从广西南宁采集的52个江河型水牛（尼里-拉菲水牛25个,摩拉水牛27个）mtDNA D-loop序列与GenBank下载的20条尼里-拉菲水牛和23条摩拉水牛序列进行联合分析,共检测到112个变异位点,定义42个单倍型,发现摩拉水牛（Hd: 0.934±0.027）与尼里-拉菲水牛的遗传多样性（Hd: 0.929±0.017）都很丰富。NJ系统发育树显示尼里-拉菲水牛和摩拉水牛含有江河型和沼泽型水牛mtDNA支系,表明尼里-拉菲水牛和摩拉水牛引入中国后均与沼泽型水牛进行了杂交,在外貌上很难区分。[结论]引入广西南宁的尼里-拉菲水牛和摩拉水牛与沼泽型水牛存在广泛的血缘混杂现象。     

福安水牛的分子遗传多样性研究

福安水牛是福建省地方优良品种之一,为了进一步阐明其群体遗传结构和遗传多样性,采用8对微卫星引物对福建福安、闽清、古田及南靖等4个福安水牛群体,共计130头样本进行检测分析。结果表明:福安水牛4个群体8个微卫星座位共有78个等位基因,其中4个群体共享的等位基因为50个,非共享等位基因28个。各等位基因频率介于0.015 2~0.527 8之间;共检测到基因型6~29种;4个群体的平均多态信息含量、杂合度、有效等位基因数分别为0.817 2±0.023 0、0.820 4±0.029 4和5.85±0.84;根据奈氏标准遗传距离进行UPGMA聚类分析,结果表明:遗传距离最远的是闽清群体与南靖群体(0.351 8),遗传距离最近的是福安群体与闽清群体(0.183 2)。     

水牛FASN基因外显子37多态性及其生物信息学分析

[目的]脂肪酸合成酶(Fatty acid synthase,FASN)是影响哺乳动物脂肪酸合成的关键酶,但有关水牛FASN基因的群体遗传组成特征还不清楚。[方法]本研究采用PCR产物直接测序法和PCR-SSCP方法对88头河流型和122头沼泽型水牛、54头牦牛和40头大额牛FASN基因外显子37进行群体变异检测,并结合已发表的牛科物种序列进行生物信息学分析。[结果]结果表明,仅在水牛中发现2个SNP位点,为c.6363CT和c.6372CT,均为同义替换。河流型和沼泽型水牛共享这2个SNP位点,但它们在两类水牛中的群体遗传组成不同。确定水牛特有的核苷酸位点3个,即c.6183A,c.6255T和c.6394A,其中c.6394A导致水牛FASN蛋白第2132位氨基酸与其它牛科物种不同,在水牛中为M而其它牛科物种中为V;山羊特有的位点2个,为c.6189A和c.6267T。普通牛、山羊和绵羊中具有异义替换SNP位点,分别为1个、3个和7个。普通牛的异义替换SNP位点c.6365GA对蛋白质功能影响不显著(subPSEC-3);山羊的这些异义替换SNP位点c.6296TC、c.6301CT和c.6341TC对其蛋白质功能影响均有显著影响(subPSEC-3);绵羊7个异义替换SNP位点中的2个,即c.6286AC和c.6406AG对FASN功能有显著影响(subPSEC-3)。[结论]这些核苷酸差异引起的氨基酸差异可能引起不同牛科物种间FASN功能的差异。