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。     

河流型水牛尼里-拉菲和摩拉水牛群体遗传结构分析

为了对摩拉水牛、尼里-拉菲水牛进行合理有效的保护,研究其遗传多样性,掌握其遗传背景,试验利用15个微卫星位点对96个个体(尼里-拉菲、摩拉水牛各48头)进行遗传多样性检测。结果表明:在尼里-拉菲水牛15个微卫星位点上共检测到92个等位基因,平均等位基因为6. 133 3,平均有效等位基因为2. 942 3,平均多态信息含量(PIC)为0. 565 5,平均期望杂合度值为0. 618 4;在摩拉水牛15个微卫星位点上共检测到76个等位基因,平均等位基因为5. 133 3,平均有效等位基因为2. 545 4,平均PIC为0. 493 7,平均期望杂合度值为0. 541 3。说明摩拉水牛、尼里-拉菲水牛群体的遗传多样性比较丰富,尼里-拉菲水牛群体的遗传多样性高于摩拉水牛群体。     

我国中东部地区地方水牛品种的遗传多样性分析

采用中心产区典型群随机抽样方法,检测了峡江水牛、海子水牛、西林水牛、恩施山地水牛、江汉水牛及一个对照群体尼里-拉菲水牛位点的遗传多态性并作分析,来探讨群体的遗传分化关系.研究表明:(1)五个群体及一个对照群体在11个微卫星座位中共检测到174个等位基因,其中有28个等位基因为品种所特有.(2)根据主成份分析结果聚类,先是江汉水牛与恩施水牛聚为一类,然后是与峡江水牛聚为一类,接着与海子水牛聚为一类,再后与西林水牛聚为一类,最后与尼里-拉菲水牛聚类.群体间亲缘关系的远近与其所处地理位置远近表现出了紧密相关.     

我国中东部地区地方水牛品种的遗传多样性分析

采用中心产区典型群随机抽样方法,检测了峡江水牛、海子水牛、西林水牛、恩施山地水牛、江汉水牛及一个对照群体尼里—拉菲水牛位点的遗传多态性并作分析,来探讨群体的遗传分化关系。研究表明:(1)五个群体及一个对照群体在11个微卫星座位中共检测到174个等位基因,其中有28个等位基因为品种所特有。(2)根据主成份分析结果聚类,先是江汉水牛与恩施水牛聚为一类,然后是与峡江水牛聚为一类,接着与海子水牛聚为一类,再后与西林水牛聚为一类,最后与尼里—拉菲水牛聚类。群体间亲缘关系的远近与其所处地理位置远近表现出了紧密相关。     

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.     

摩拉水牛和尼里-拉菲水牛PRKAA2基因SNPs检测及遗传多样性分析

为研究水牛蛋白激酶AMP活化的催化亚基α2(protein kinase AMP-activated catalytic subunit alpha 2,PRKAA2)基因多态性,本试验以摩拉水牛和尼里-拉菲水牛基因组DNA为模板,扩增PRKAA2基因外显子4及内含子3部分序列,通过常规测序法检测其SNP并进行遗传多样性分析。结果发现,PRKAA2基因外显子4内存在1个SNP位点(c.462GA),PRKAA2基因内含子3部分序列存在3个SNPs位点(IVS3.557TC、IVS3.560CT和IVS3.565GA)。经遗传多样性分析表明,在c.462GA位点的野生纯合型和杂合型比突变纯合型更有优势,IVS3.557TC和IVS3.560CT位点的突变纯合型为非优势基因型,IVS3.565GA位点杂合型为优势基因型。IVS3.565GA位点在摩拉水牛群体中处于Hardy-Weinberg非平衡状态;c.462GA位点在尼里-拉菲水牛群体中处于Hardy-Weinberg非平衡状态。4个SNPs位点在摩拉水牛群体中均为中度多态;c.462GA、IVS3.557TC位点在尼里-拉菲水牛群体中为低度多态,IVS3.560CT、IVS3.565GA位点为中度多态。IVS3.557TC位点在两个水牛群体中杂合度较低。说明摩拉水牛IVS3.565GA位点和尼里-拉菲水牛c.462GA位点的基因型频率和基因频率遗传状态不平衡,尼里-拉菲水牛群体中IVS3.557TC位点遗传变异小,选择潜力不高。4个多态位点可以构建5种单倍型,其中T-C-G-G是摩拉水牛群体和尼里-拉菲水牛群体的优势单倍型。综上,本研究检测的摩拉水牛和尼里-拉菲水牛PRKAA2基因上4个SNPs位点可为水牛标记辅助选择育种提供参考。     

摩拉水牛和尼里-拉菲水牛PRKAA2基因SNPs检测及遗传多样性分析

为研究水牛蛋白激酶AMP活化的催化亚基α2（protein kinase AMP-activated catalytic subunit alpha 2,PRKAA2）基因多态性,本试验以摩拉水牛和尼里-拉菲水牛基因组DNA为模板,扩增PRKAA2基因外显子4及内含子3部分序列,通过常规测序法检测其SNP并进行遗传多样性分析。结果发现,PRKAA2基因外显子4内存在1个SNP位点（c.462 G>A）,PRKAA2基因内含子3部分序列存在3个SNPs位点（IVS3.557 T>C、IVS3.560 C>T和IVS3.565 G>A）。经遗传多样性分析表明,在c.462 G>A位点的野生纯合型和杂合型比突变纯合型更有优势,IVS3.557 T>C和IVS3.560 C>T位点的突变纯合型为非优势基因型,IVS3.565 G>A位点杂合型为优势基因型。IVS3.565 G>A位点在摩拉水牛群体中处于Hardy-Weinberg非平衡状态;c.462 G>A位点在尼里-拉菲水牛群体中处于Hardy-Weinberg非平衡状态。4个SNPs位点在摩拉水牛群体中均为中度多态;c.462 G>A、IVS3.557 T>C位点在尼里-拉菲水牛群体中为低度多态,IVS3.560 C>T、IVS3.565 G>A位点为中度多态。IVS3.557 T>C位点在两个水牛群体中杂合度较低。说明摩拉水牛IVS3.565 G>A位点和尼里-拉菲水牛c.462 G>A位点的基因型频率和基因频率遗传状态不平衡,尼里-拉菲水牛群体中IVS3.557 T>C位点遗传变异小,选择潜力不高。4个多态位点可以构建5种单倍型,其中T-C-G-G是摩拉水牛群体和尼里-拉菲水牛群体的优势单倍型。综上,本研究检测的摩拉水牛和尼里-拉菲水牛PRKAA2基因上4个SNPs位点可为水牛标记辅助选择育种提供参考。     

西林水牛和富钟水牛mtDNA D-loop序列的遗传多样性研究

该研究旨在探究西林水牛和富钟水牛mtDNA的遗传多样性和母系起源。采用PCR扩增、测序及生物信息学方法,测定了60头西林水牛和富钟水牛的mtDNA D-loop序列。结合GenBank数据库已公布的25条西林水牛和富钟水牛的mtDNA D-loop序列联合进行多态性分析,共检测到60个多态位点,定义了38种mtDNA单倍型。西林水牛和富钟水牛的单倍型多样度分别为0.909±0.028和0.913±0.035,核苷酸多样度分别为0.019±0.004和0.013±0.005,这一结果表明西林水牛和富钟水牛具有丰富的遗传多样性。NJ系统发育树显示,西林水牛和富钟水牛均属于沼泽型水牛,且有A支系和B支系两个母系起源。     

德宏水牛与尼里拉菲水牛及其杂交水牛的细胞遗传学研究

为了弄清河川型水牛与沼泽型水牛杂交后代的核型,以及与其育性和亲本核型的关系,用外周血液淋巴细胞培养法对尼里拉菲水牛、德宏水牛及尼里拉菲水牛×德宏水牛F1代公牛进行淋巴细胞培养,制作染色体标本,并进行G-带染色;利用BEION染色体核型分析系统对染色体标本分别进行染色体核型分析,确定其染色体核型。根据染色体核型分析结果,并结合其育性,分析染色体核型与其育性的关系。结果显示,尼里拉菲水牛核型为2n=50,XY;德宏水牛核型为2n=48,-4,-4,-9,-9,+t(4;9),+t(4;9),XY;尼里拉菲水牛×德宏水牛F1代公牛核型为:2n=49,-4,-9,+t(4;9),XY。     

尼里·拉菲水牛与德宏水牛杂交效果观察

20世纪90年代以后,随着农机具的推广运用,作为役用型的德宏水牛存栏数逐年下降.为提高该品种牛的乳肉役兼用性能,1997年借助中欧德宏水牛开发项目,我们开始引进印度的摩拉水牛与本地水牛(德宏水牛)进行杂交试验,因其后代产奶量达不到理想要求,2000年又开始引进尼里·拉菲水牛与本地水牛杂交,并及时测定尼×德杂交一代水牛不同生长发育时期的有关数据.通过十多年的试验研究,取得了较为理想的改良效果,现将尼×德杂交结果报告如下.     

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.     

Genetic relationship and diversity analysis of Indian water buffalo (Bubalus bubalis)

The water buffalo (Bubalus bubalis) is an important dairy animal on the Indian subcontinent and in Southeast Asian countries. The diversity and differentiation among 12 populations or breeds of buffalo were studied. Data were generated and analyzed from 527 animals belonging to 10 recognized breeds and 2 additional populations of Indian buffalo by using 22 microsatellite loci. Relationships among buffalo breeds and populations were estimated based on genetic distances. The Bayesian analysis grouped 12 populations into 8 distinctive clusters. Geographically close breeds clustered together, except for the Jaffarabadi and Murrah, which were not in geographic contiguity. The Mantel test revealed nonsignificant correlations between genetic and geographic distances. This supports the hypothesis that buffaloes have been domesticated at different places for specific purposes. The phylogenetic relationship based on microsatellite loci supported the breed classification based on body size. The Toda breed, which is considered to be endangered, had genotypes similar to those of the surrounding buffalo populations.     

德昌水牛与杂交水牛乳生化组成的分析

对德昌水牛、广西水牛×尼里拉菲水牛F1代乳生化组成进行了分析。结果表明:德昌水牛及F1代乳中蛋白质、乳脂含量和pH较高;SDS-PAGE分析显示德昌水牛及F1代乳蛋白中乳清蛋白比例高,而酪蛋白相对含量低于普通牛乳;德昌水牛乳中的氨基酸含量显著高于杂交水牛和普通牛。     

Riverine status and genetic structure of Chilika buffalo of eastern India as inferred from cytogenetic and molecular marker-based analysis

The present study aimed at assessing the status of the Chilika buffalo population of eastern India employing cytogenetic and molecular markers. The Chilika buffaloes investigated cytogenetically possess a somatic chromosome count of 50, identical to that of typical riverine buffaloes. Various diversity estimates, viz. observed number of alleles (4.68), effective number of alleles (2.79), and observed (0.487) and expected (0.602) heterozygosity across 25 heterologous microsatellite markers indicated the presence of a moderate level of genetic diversity in Chilika buffaloes, comparable with three other prominent Indian riverine buffalo breeds (Murrah, Nagpuri and Toda) included in this study. Across the four buffalo populations, mean estimates of F -statistics from Jackknifing over loci were significantly different from zero (p < 0.05), with F _IT (total inbreeding estimate) = 0.315 ± 0.038, F _IS (within-population inbreeding estimate) = 0.178 ± 0.038, and F _ST (population differentiation) = 0.166 ± 0.025. Inter-breed analysis reflected Chilika buffaloes to be genetically close to Nagpuri followed by Murrah and Toda buffaloes. Factorial correspondence analysis (FCA) revealed low breed-specific clustering of Chilika and Nagpuri buffaloes. Additionally, the neighbour-joining tree structure of mitochondrial DNA D-loop haplotypes indicated clear grouping of the Chilika haplotypes with the riverine buffalo. Thus the cytogenetic, microsatellite and mitochondrial data analysed in the present study classify Chilika buffalo of eastern India to be of the riverine type and not swamp-type buffalo.     

Evaluation of genetic variability and mutation drift equilibrium of Banni buffalo using multi locus microsatellite markers

The present study was conducted to evaluate genetic diversity of Banni buffalo and its relationship/differentiation with Murrah using genotypic data on 24 heterologus bovine specific microsatellite marker loci. A total of 138 alleles were observed with a mean of 5.75 alleles/locus across two populations. The mean observed and expected heterozygosities were found to be 0.441 and 0.572 respectively in Banni buffaloes while it was 0.464 and 0.610 respectively in Murrah buffaloes. The average heterozygosity deficit was significantly positive with substantially higher values observed in Banni (22.3%) and Murrah (24%) buffalo populations. Banni buffalo population, when evaluated for mutation drift equilibrium revealed significant heterozygosity excess under IAM while no such excess was observed under SMM and TPM. The qualitative graphical test revealed a normal L-shaped distribution of allele frequencies indicating the absence of genetic bottleneck in Banni buffaloes. The mean estimates of F-statistics over all the loci were 0.376 for F_IT, 0.187 for F_ST and 0.232 for F_IS respectively. Analysis of molecular variance (AMOVA) revealed 18.95% of the total variation being explained by between breed differences while 14.36% of the variation explained differences between individuals within each breed. Genotype assignment test revealed distinct clustering of Banni and Murrah buffaloes. Genetic distance was estimated using three different methods, the results of which revealed considerable genetic differentiation between these two buffalo populations. The divergence time between Banni and Murrah buffaloes was estimated to be around 7286 years. The results of the present study may be helpful in decision making for conservation programs as Banni buffalo population is on decline.     

四川地方乌骨鸡种群遗传变异的微卫星DNA分析

利用10个微卫星标记，对四川不同地区5个乌骨鸡种群的等位基因频率、各群体的遗传杂合度、群体间的遗传距离等进行了分析，并根据遗传距离对这几个乌骨鸡种群进行了聚类分析。结果表明，各乌骨鸡群体的遗传多样性较为丰富，并具有较高的选择潜力。各乌骨鸡种群间有一定的遗传距离，UPGMA聚类分析表明，四川山地乌骨鸡黑羽系与川南山地乌骨鸡被聚为一类，草科鸡与旧院黑鸡被聚为一类。     

摩拉水牛及德宏水牛瘤胃产甲烷菌多样性比较

本试验旨在比较分析摩拉水牛及德宏水牛瘤胃液中产甲烷菌的多样性。选取健康雌性摩拉水牛及德宏水牛各3头,采用口腔导管法收集瘤胃液,酚-氯仿-异戊醇抽提法提取瘤胃液总DNA,用产甲烷菌特异性引物Met86F/Met1340R扩增产甲烷菌16S rDNA,构建16S rDNA基因克隆文库。摩拉水牛及德宏水牛各获得96个16S rDNA基因序列,均归类于Methanobacteriales目,其中德宏水牛有82个序列（18个OTUs）与已知菌的16S rDNA序列相似性≥ 97%,占总序列的85.4%,有14个序列（9个OTUs）与已知菌16S rDNA序列相似性为89%~97%;摩拉水牛有94个序列（13个OTUs）与已知菌16S rDNA序列相似性≥ 97%,占总序列的97.9%,仅有2个序列（1个OTUs）与已知菌16S rDNA序列相似性为94%。系统进化树分析表明,所有序列分别聚集于两大分支上,其中德宏水牛有13个OTUs代表序列和摩拉水牛7个OTUs代表序列聚集在进化树顶端的同一分支上,且在系统发育距离上与Methanobacteriales目中任何已知相似序列都相隔较远。德宏水牛瘤胃中的SGMT簇序列和RO簇序列所占总序列比列分别为83.3%、9.4%,摩拉水牛瘤胃中的SGMT簇序列和RO簇序列所占总序列的比列分别为51.0%、9.4%。由此可见,摩拉水牛及德宏水牛瘤胃产甲烷菌序列以Methanobacteriales目为主,其中德宏水牛拥有更多未知的产甲烷菌序列;德宏水牛瘤胃中的SGMT簇产甲烷菌序列比例要高于摩拉水牛。     

PCR-SSCP analysis of leptin gene and its association with milk production traits in river buffalo (Bubalus bubalis)

Leptin gene has been found to be associated with various economic traits including milk production and fat quality in dairy animals. In the present study, we investigated genetic variations in intron 1 region of leptin gene in riverine buffaloes (Bubalus bubalis) using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing methods and associated them with milk traits. The study revealed three SSCP variants A, B and C among a total of 301 buffaloes from nine breeds. The frequency of variant C was found invariably high among all the breeds except in Marathwada buffalo. Variant A was found to be absent in Chilika, Nili-Ravi, Nagpuri and Pandharpuri breeds and also had the lowest frequencies in Mehsana, Jaffarabadi, Murrah and Toda breeds. Sequencing of SSCP variants revealed a total of five polymorphic sites, with three haplotypes. Statistical analysis revealed significantly high fat percentage at 150?days in SSCP variant B in Mehsana buffaloes. However, the associations of SSCP variants of leptin gene with total milk yield, 305?days milk yield and total fat yield were found to be non-significant. The present study is the first report on association analysis of leptin gene polymorphisms with milk production and milk quality traits in river buffalo.