Marker assisted evaluation of morphological and genetic attributes of sub-populations of Nili-Ravi buffalo: A vulnerable dairy type riverine breed of India

In the present study, we report the distribution of true to type and atypical Nili-Ravi buffalo, a vulnerable dairy type riverine breed of North India and its underlying genetic structure. Out of total investigated buffaloes 73.5% had bilateral wall eyes while 5.4% had unilateral wall eyes and 21.1% had no wall eyes. 41.15% of Nili-Ravi buffaloes maintained in the breeding farm were having typical true to the type characteristics (both eyes walled, white markings in forehead, muzzle/chin, all the four legs and tail) while only 28.5% of Nili-Ravi buffaloes were true to the type under field conditions. Genotypic data were generated in four groups of Nili-Ravi buffalo (FMTNR—Typical Nili-Ravi from farm; FMANR—Atypical Nili-Ravi from farm; FDTNR—Typical Nili-Ravi from field; FDANR—Atypical Nili-Ravi from field) at 16 microsatellite loci. Comparative genetic analysis of various groups of Nili-Ravi buffaloes with Murrah revealed significant between group differences with an estimated global F ST of 0.063. Pair-wise F ST values ranged from 0.003 (between FDTNR and FDANR) to 0.112 (between FMTNR and FDTNR). Phylogenetic analysis and multi-dimensional scaling revealed clustering of FDTNR and FDANR together while FMTNR and FMANR clustered separately with Murrah in between farm and field Nili-Ravi buffaloes. Based on the results, the paper also proposes three pronged strategy for conservation and sustainable genetic improvement of Nili-Ravi buffalo in India.     

Identification of X- and Y-chromosome bearing buffalo (Bubalus bubalis) sperm

The aim of this study was to identify the difference in DNA content characterizing the X- and Y-chromosome bearing sperm of buffalo. Sperm from six Murrah buffaloes and six Nili-Ravi buffaloes were collected and stained with Hoechst 33342 followed by flow cytometry analysis of the DNA content. Two symmetrical, separate but overlapping peaks presumed to be X- and Y-chromosome bearing sperm were detected. The difference in fluorescence intensity, which related to the DNA content, between the X- and Y-sperm was 3.59+/-0.11% for Murrah buffalo and 3.55+/-0.14% for Nili-Ravi buffalo, respectively. Significant differences were observed among males within each breed, but there were no differences between the averages of the two breeds. The results indicate that flow cytometric sorting of X- and Y-sperm of buffalo is feasible.     

Biometry of frozen-thawed sperm from eight breeds of Indian buffaloes (Bubalus bubalis)

Sperm morphometry, in combination with other objective traits, can be useful for developing a fertility index. The objective of the present study was to measure various biometric end points of frozen-thawed sperm from eight breeds of Indian buffaloes (Murrah, Surti, Tarai, Mehsana, Jaffrabadi, Bhadawari, Pandharpuri and Nili-Ravi). The sperm head of Pandharpuri buffaloes had the greatest length (10.21 microm), width (6.05 microm), area (52.31 microm(2)) and perimeter (31.86 microm). The ratio of sperm width to length was also greatest (0.61) in Pandharpuri as well as in two other breeds, viz. Nili-Ravi and Jaffrabadi. Murrah had the smallest sperm head width (4.75 microm), area (41.65 microm(2)) and perimeter (29.17 microm), but its sperm tail was longest (57.02 microm), along with that of Jaffrabadi buffaloes (56.96 microm). Based on mean values of sperm tail length, mid piece length and its width the eight buffalo breeds were categorized into three, four and five groups, respectively. Multivariate analysis and clustering put six breeds (Surti, Tarai, Mehsana, Jaffrabadi, Bhadawari and Nili-Ravi) in one cluster, whereas Murrah and Pandharpuri appeared as separate entities.     

8个亚洲水牛群体的遗传结构分析

应用13个微卫星标记结合荧光–多重PCR技术, 对德昌水牛、兴隆水牛、富钟水牛、温州水牛、东流水牛、福安水牛和两个引进品种摩拉水牛、尼里－拉菲水牛进行遗传结构分析。结果表明: 8个水牛群体在13个微卫星座位中共检测到157个等位基因, 其中7个群体具有各自的特有等位基因, 其和为23; 8个群体的有效等位基因数在2.2908～4.2308之间, 杂合度在0.4951～0.7194之间, 多态信息含量在0.4495～0.6776之间; 有11个座位为高度多态座位, 是适合分析水牛遗传多样性的多态标记; 聚类分析表明富钟水牛和东流水牛先聚在一起, 再与兴隆水牛聚在一起, 然后与温州水牛和福安水牛聚在一起, 德昌水牛独自聚为一类; 两个引进品种聚为一类。     

Genetic diversity and differentiation of Chinese domestic buffalo based on 30 microsatellite markers

To determine genetic diversity and evolutionary relationships among Chinese buffalo populations, 18 indigenous swamp buffalo populations and two introduced river buffalo breeds were genotyped for 30 microsatellite loci. The mean number of alleles across the 30 loci was 8.13, and the expected heterozygosity ranged from 0.517 (Yanjin) to 0.609 (Dehong). Although there was abundant genetic variation, genetic differentiation between Chinese buffalo populations was low, with only 2.8% of the total genetic variance among populations. The genetic differentiation pattern and genetic relationships among Chinese buffalo populations were consistent with their geographic distribution. The Dehong population was discerned as a distinct indigenous population, but suffered genetic admixture with river buffalo. The remaining populations were divided into four major clusters, i.e. the Upper and Middle Reaches of Yangtze Valley cluster (Guizhou, Guizhoubai, Yanjin, Fuling, Enshi and Jianghan), the Lower Reaches of Yangtze Valley cluster (Haizi, Shanqu and Dongliu), the South China cluster (Fuan and Xinfeng) and the Southwest China cluster (Xinglong, Xilin, Diandongnan and Dechang).     

Genetic variation within and relationships among populations of Asian water buffalo (Bubalus bubalis)

Genetic variation at 53 protein-coding loci (25 polymorphic) was analysed for 17 water buffalo populations – 12 swamp, three Lankan and two of the Murrah breed (river type), to determine the magnitude of genetic differentiation and the genetic relationships among the populations. In accord with previous cytological studies, the Lankan buffalo clearly are river type. Significant deviations from Hardy–Weinberg equilibrium were shown for a number of locus–population combinations, with all populations but one showing significant heterogeneity in these deviations among loci. By contrast, heterogeneity among populations for each locus was much less, indicating locus-specific deviations, which suggest selection affecting allele frequencies at some loci. There was significant genetic differentiation among populations of both the swamp and river types. The differentiation among the swamp populations may reflect the geography of south-east Asia and the presumed spread of the swamp buffalo through this region. Phylogenies derived from pairwise genetic distance estimates show the clear separation of swamp and river types, but the topology of the swamp populations shows rather poor consistency with their geographic locations. For at least one population (Australia), it is clear that bottleneck effects have distorted the phylogenetic topology. Average genetic distances for both the swamp and river types, as compared with previous studies of livestock breeds, show that the genetic differentiation of each of these sets of populations is of the same order of magnitude as that among well-recognized and established breeds of other species.     

Genetic variation and relationships among eight Indian riverine buffalo breeds

Twenty-seven microsatellite loci were used to define genetic variation and relationships among eight Indian riverine buffalo breeds. The total number of alleles ranged from 166 in the Toda breed to 194 each in the Mehsana and the Murrah. Significant departures from the Hardy-Weinberg equilibrium were observed for 26 locus-breed combinations due to heterozygote deficiency. Breed differentiation was analysed by estimation of F(ST) index (values ranging from 0.75% to 6.00%) for various breed combinations. The neighbour-joining tree constructed from chord distances, multidimensional scaling (MDS) display of F(ST) values and Bayesian clustering approach consistently identified the Toda, Jaffarabadi, and Pandharpuri breeds as one lineage each, and the Bhadawari, Nagpuri, Surati, Mehsana and Murrah breeds as admixture. Analysis of molecular variance refuted the earlier classification of these breeds proposed on the basis of morphological and geographical parameters. The Toda buffaloes, reared by a tribe of the same name, represent an endangered breed from the Nilgiri hills in South India. Divergence time of the Toda buffaloes from the other main breeds, calculated from Nei's standard genetic distances based on genotyping data on seven breeds and 20 microsatellite loci, suggested separation of this breed approximately 1800-2700 years ago. The results of the present study will be useful for development of rational breeding and conservation strategies for Indian buffaloes.     

The role of MC1R gene in buffalo coat color

Melanocortin-1 receptor (MC1R) plays a major role in pigmentation in many species. To investigate if the MC1R gene is associated with coat color in water buffalo, the coding region of MC1R gene of 216 buffalo samples was sequenced, which included 49 black river buffalo (Murrah and Nili-Ravi), 136 swamp buffalo (Dehong, Diandongnan, Dechang, Guizhou, and Xilin) with white and gray body, and 31 hybrid offspring of river buffalo Nili-Ravi (or Murrah) and swamp buffalo. Among the three variation sites found, SNP684 was synonymous, while SNP310 and SNP384 were nonsynonymous, leading to p.S104G and p.I128M changes, respectively. Only Individuals carrying homozygote EBR/EBR were black. The genotype and phenotype analysis of the hybrid offspring of black river buffalo and gray swamp buffalo further revealed that the river buffalo type allele EBR or the allele carrying the amino acid p.104S was important for the full function of MC1R. The in silico functional analysis showed that the amino acid substitutions p.G104S and p.M128I had significant impact on the function of MC1R. Above results indicate that the allele EBR or the allele carrying the amino acid p.104S was associated with the black coat color in buffalo.     

Evolution and diversity studies of innate immune genes in Indian buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) breeds using next generation sequencing

Indian buffalo (Bubalus bubalis) breeds are acknowledged for their disease resistance power, hardiness and considerable milk production. River buffaloes are more disease resistance despite of tropical environment of India than cattle breeds of the world. The genetic polymorphisms of innate immune genes play a prominent role in disease susceptibility of livestock. Here in our study, we mainly focused on SNPs in ten selected candidate innate immune genes viz CHGA, CHGB, CHGC, Slc11a1, Slc11a2, DEFB1, BNBD4, BNBD5, LAP and TAP among three most economically important Indian buffalo breeds viz., Jaffrabadi, Mehsani and Murrah using amplicon sequencing on Ion torrent PGM sequencing platform. Polymorphism identification using by GATK tool of Unified Genotyper revealed 274, 245 and 224 high quality SNPs in Jaffrabadi, Mehsani and Murrah buffalo breeds, respectively as well as 168 common SNPs among these three breeds and uniquely detected SNPs were 31, 15 and 37 SNPs in Jaffrabadi, Mehsani and Murrah breeds respectively. Consequences of non-synonymous SNPs were analysed using several computational tools viz., I-Mutant, SIFT, PolyPhen and PROVEAN which identified three deleterious nsSNPs. This wealth of sequence information will be productive for conservation studies, selective breeding and designing future strategies for identifying disease associations.     

Analysis of mitochondrial D-loop region casts new light on domestic water buffalo (Bubalus bubalis) phylogeny

The phylogeny of water buffaloes (Bubalus bubalis) is still a matter of discussion, especially if the two types of domestic water buffalo (swamp and river) derived from different domestication events or if they are products of human selection. To obtain more insight, we analyzed the entire mitochondrial D-loop region of 80 water buffaloes of four different breeds, i.e., 19 swamp buffaloes (Carabao) and 61 river buffaloes (Murrah, Jafarabadi, and Mediterranean), sampled in Brazil and Italy. We detected 36 mitochondrial haplotypes with 128 polymorphic sites. Pooled with published data of South-East Asian and Australian water buffaloes and based on comprehensive median-joining network and population demography analyses we show evidence that both river and swamp buffaloes decent from one domestication event, probably in the Indian subcontinent. However, the today swamp buffaloes have an unravelled mitochondrial history, which can be explained by introgression of wild water buffalo mtDNA into domestic stocks. We are also discussing indications for an independent domestication of buffaloes in China.     

Genetic differentiation of water buffalo (Bubalus bubalis) populations in China, Nepal and south-east Asia: inferences on the region of domestication of the swamp buffalo

Data from three published studies of genetic variation at 18 microsatellite loci in water buffalo populations in China (18 swamp type, two river type), Nepal (one wild, one domestic river, one hybrid) and south-east Asia (eight swamp, three river) were combined so as to gain a broader understanding of genetic relationships among the populations and their demographic history. Mean numbers of alleles and expected heterozygosities were significantly different among populations. Estimates of θ (a measure of population differentiation) were significant among the swamp populations for all loci and among the river populations for most loci. Differentiation among the Chinese swamp populations (which was due primarily to just one population) was much less than among the south-east Asian. The Nepal wild animals, phenotypically swamp type but genetically like river type, are significantly different from all the domestic river populations and presumably represent the ancestral Bubalus arnee (possibly with some river-type introgression). Relationships among the swamp populations (D(A) genetic distances, principal component analysis and structure analyses) show the south-east Asian populations separated into two groups by the Chinese populations. Given these relationships and the patterns of genetic variability, we postulate that the swamp buffalo was domesticated in the region of the far south of China, northern Thailand and Indochina. Following domestication, it spread south through peninsular Malaysia to Sumatra, Java and Sulawesi, and north through China, and then to Taiwan, the Philippines and Borneo.     

Phylogeography and Domestication of Chinese Swamp Buffalo

To further probe into whether swamp buffaloes were domesticated once or multiple times in China, this survey examined the mitochondrial DNA (mtDNA) Control Region (D-loop) diversity of 471 individuals representing 22 populations of 455 Chinese swamp buffaloes and 16 river buffaloes. Phylogenetic analysis revealed that Chinese swamp buffaloes could be divided into two distinct lineages, A and B, which were defined previously. Of the two lineages, lineage A was predominant across all populations. For predominant lineage A, Southwestern buffalo populations possess the highest genetic diversity among the three hypothesized domestication centers (Southeastern, Central, and Southwestern China), suggesting Southwestern China as the most likely location for the domestication of lineage A. However, a complex pattern of diversity is detected for the lineage B, preventing the unambiguous pinpointing of the exact place of domestication center and suggesting the presence of a long-term, strong gene flow among swamp buffalo populations caused by extensive migrations of buffaloes and frequent human movements along the Yangtze River throughout history. Our current study suggests that Southwestern China is the most likely domestication center for lineage A, and may have been a primary center of swamp buffalo domestication. More archaeological and genetic evidence is needed to show the process of domestication.     

Identification of novel single nucleotide polymorphisms in the DGAT1 gene of buffaloes by PCR-SSCP

Diacylglycerol O-acyltransferase 1 (DGAT1) is a microsomal enzyme that catalyzes the final step of triglyceride synthesis. The DGAT1 gene is a strong functional candidate for determining milk fat content in cattle. In this work, we used PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) and DNA sequencing to examine polymorphism in the region spanning exon 7 to exon 9 of the DGAT1 gene in Murrah and Pandharpuri buffaloes. Three alleles (A, B and C) and four novel single-nucleotide polymorphisms were identified in the buffalo DGAT1 gene. The frequencies of the alleles differed between the two buffalo breeds, with allele C being present in Murrah but not in Pandharpuri buffalo. The allele variation detected in this work may influence DGAT1 expression and function. The results described here could be useful in examining the association between the DGAT1 gene and milk traits in buffalo.     

江汉水牛与摩拉水牛顶间骨形态比较研究

对摩拉水牛头骨形态特征进行研究,并对2例(1,4号)摩拉水牛头骨的顶间骨进行研究,与2例(2,3号)江汉水牛头骨的顶间骨进行形态特征比较,结果表明摩拉水牛顶间骨发达,是由完整的对骨组成.该研究进一步论证了摩拉水牛头骨的顶间骨形态结构与江汉水牛头骨的顶间骨之间存在很明显差异,摩拉水牛顶间骨比江汉水牛的顶间骨发达.     

Investigation of transferability of BovineSNP50 BeadChip from cattle to water buffalo for genome wide association study

Cattle and water buffalo belong to the same subfamily Bovinae and share chromosome banding and gene order homology. In this study, we used genome-wide Illumina BovineSNP50 BeadChip to analyze 91 DNA samples from three breeds of water buffalo (Nili-Ravi, Murrah and their crossbred with local GuangXi buffalos in China), to demonstrate the genetic divergence between cattle and water buffalo through a large single nucleotide polymorphism (SNP) transferability study at the whole genome level, and performed association analysis of functional traits in water buffalo as well. A total of 40,766 (75.5 %) bovine SNPs were found in the water buffalo genome, but 49,936 (92.5 %) were with only one allele, and finally 935 were identified to be polymorphic and useful for association analysis in water buffalo. Therefore, the genome sequences of water buffalo and cattle shared a high level of homology but the polymorphic status of the bovine SNPs varied between these two species. The different patterns of mutations between species may associate with their phenotypic divergence due to genome evolution. Among 935 bovine SNPs, we identified a total of 9 and 7 SNPs significantly associated to fertility and milk production traits in water buffalo, respectively. However, more works in larger sample size are needed in future to verify these candidate SNPs for water buffalo.     

中国地方黄牛的遗传多样性*

中国黄牛起源复杂,我国地方黄牛群体不同品种在毛色、形态外貌、细胞遗传学、血液蛋白座位分析均表现出多样性。计算我国黄牛群体6个毛色座位平均杂合度和6个血液蛋白座位平均杂合度分别为0.3144和0.4873,表明我国地方黄牛群体的遗传多样性非常丰富。计算我国黄牛群体的6个毛色座位和6个血液蛋白座位的基因分化系数分别为0.3404和0.095,表明我国黄牛群体毛色差异中有34.04％是由品种间的差异造成的。血液蛋白的多态性有9.5％是由品种间的差异造成的。我国黄牛群体的遗传多样性主要来自品种内的遗传多样性。保存我国黄牛品种资源多样性不仅要从整个中国黄牛群体上考虑,而且要针对每个品种（或类群）进行保种。     

Asian water buffalo: domestication,history and genetics

The domestic Asian water buffalo (Bubalus bubalis) is found on all five continents, with a global population of some 202 million. The livelihoods of more people depend on this species than on any other domestic animal. The two distinct types (river and swamp) descended from different wild Asian water buffalo (Bubalus arnee) populations that diverged some 900 kyr BP and then evolved in separate geographical regions. After domestication in the western region of the Indian subcontinent (ca. 6300 years BP), the river buffalo spread west as far as Egypt, the Balkans and Italy. Conversely, after domestication in the China/Indochina border region ca. 3000–7000 years BP, swamp buffaloes dispersed through south-east Asia and China as far as the Yangtze River valley. Molecular and morphological evidence indicates that swamp buffalo populations have strong geographic genetic differentiation and a lack of gene flow, but strong phenotypic uniformity. In contrast, river buffalo populations show a weaker phylogeographic structure, but higher phenotypic diversity (i.e. many breeds). The recent availability of a high-quality reference genome and of a medium-density marker panel for genotyping has triggered a number of genome-wide investigations on diversity, evolutionary history, production traits and functional elements. The growing molecular knowledge combined with breeding programmes should pave the way to improvements in production, environmental adaptation and disease resistance in water buffalo populations worldwide.     

Mitochondrial sequence-based evolutionary analysis of riverine–swamp hybrid buffaloes of India indicates novel maternal differentiation and domestication patterns

In this study, mitochondrial D-loop sequence data on riverine, swamp and hybrid buffaloes from India have been generated and compared with other reported Indian riverine, Chinese and Bangladeshi swamp buffalo populations. Sequence analysis revealed the presence of 132 haplotypes, with a haplotype diversity of 0.9611 ± 0.0045 and a nucleotide diversity of 0.04801 ± 0.00126. For the first time, the existence of riverine–swamp hybrids among the Indian Chilika buffalo population has been recorded, having 49 chromosomes, which was also confirmed by mitochondrial haplotype sharing between Chilika and Indian swamp as well as Chinese swamp buffalo populations in the network analysis. Phylogenetic analysis documents the sharing of reported pre-domestication haplogroups ‘SA1’, ‘SA2’, ‘SA3’ and ‘SB1’ between the Chilika and swamp buffalo populations of India, China and Bangladesh, an indication of the migration of swamp buffaloes towards Bangladesh and adjoining lower parts of India and north towards Chinese domestication sites. The results have also been supplemented by multidimension scaling, grouping Indian and Chinese swamp buffaloes more closely together with Bangladeshi buffaloes, but into a separate quadrant, whereas Chilika grouped away from other riverine as well as swamp buffaloes. These findings thus confirm the previous reports that the northeast region of India, close to the Indo-China border, is the point of evolution of swamp buffaloes with multiple sites of domestication.     

Single nucleotide polymorphism analysis on melanocortin receptor 1 (MC1R) of Chinese native pig

Thecoatcolorisanimportantcharacteristicofapigbreed,andcanbeclassifiedintomanytypes.Thecolorvariationsareeitherduetothedistributionofmelanocytesintheskinortotheabilityofmelano-cytestoproducemelaningranules.Thesynthesisofmelaninoriginatedfromtheformationofneuralcrest-derivedcells,whichhavetwomigrationroutes[1,2].Themelanocytesaretheramificationswhiletheneuralcrest-derivedcellsmigrateviadorsalroute[3].Andtheyprovidethefactoryformelaninsynthesis.Al-thoughtherearemanykindsofpigmentsinvertebralanim…     

Evaluating the potential for reintroducing the endangered wild water buffalo (Bubalus arnee) in Kanha National Park,central India

Reintroducing megafauna to their historic range is an effective strategy to halt their extinctions and restore ecosystems. Wild water buffalo (Bubalus arnee) is an endangered megaherbivore that is lost from 95% of its range. About 90% of its global population (less than 4000) resides within India, in two isolated populations: northeast and central India. The central Indian population is on the verge of extinction and warrants urgent conservation interventions. We assess the potential and provide a strategy for reintroducing buffaloes in Kanha National Park, India. Habitat suitability using the global occurrence of buffalo revealed low-lying grasslands with least human pressure found in Kanha (390 km²) to be suitable. Within this suitable range, we evaluated vegetation composition, forage biomass, and potential carrying capacity. Multidimensional ordination classified these suitable sites into moist and dry grassland clusters. Moist grasslands were found to better suit reintroduction due to lower grazing pressure, higher productivity, and availability of perennial water sources. Distinct matriarchal clades of swamp and river buffalo were observed. Within the river buffalo clade, Indian wild buffaloes formed a distinct cluster with close proximity between northeast and central Indian samples, suggesting northeast buffaloes could be sourced for a founding population in Kanha. Following IUCN guidelines, we discuss the reintroduction strategy that could sustain approximately 200 buffaloes in the area and subsequently expand to other suitable habitats in central India. If implemented as proposed, recovery of this lost ecosystem engineer will help to restore grasslands, and swamp habitats, as well as contribute to its global conservation efforts.