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.     

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.     

Y‐chromosomal variation confirms independent domestications of swamp and river buffalo

Y‐chromosomal variation in the water buffalo was analysed by sequencing of DBY, ZFY and SRY gene segments. A clear separation of the paternal lineages of the river and swamp types parallels the differences between their maternal lineages and nuclear DNA. Sequence divergence was found to be comparable to the divergence of taurine cattle and zebu, and this divergence predated domestication, confirming that river and swamp buffalo originated from different wild populations. Within a sample of 23 Thai swamp buffaloes, we identified four haplotypes with different geographical distributions, two of which were shared by Thai wild buffaloes.     

Independent maternal origin of Chinese swamp buffalo (Bubalus bubalis)

To obtain more knowledge on the origin and genetic diversity of the swamp buffalo (Bubalus bubalis) in China, the complete mitochondrial D-loop sequences of 119 samples representing seven native types were compared. Two mitochondrial DNA (mtDNA) lineages (lineages A and B) were determined for the Chinese swamp buffalo. Examination of the diversity patterns suggest that lineage A has undergone a population expansion event. Divergence of lineages A and B was estimated at 18,000 years ago. Combined analyses of mtDNA sequences from Chinese, Indian, Brazilian/Italian and Southeast Asian/Australian buffalo samples showed independent domestication events in the swamp buffalo from China and the river buffalo from the India subcontinent. The spread of swamp and river buffalo from China and India respectively to mainland Southeast Asia suggests that Southeast Asia is a hybrid zone for buffalo. Our data support the hypothesis of the evolution of domesticated swamp and river buffalo from ancestral swamp-like animals. These ancestral animals were extensively distributed across mainland Asia and most likely are represented today by the wild Asian buffalo (Bubalus arnee).     

Genomic diversity and selection sweeps identified in Indian swamp buffaloes reveals it's uniqueness with riverine buffaloes

The present investigation was focused to study genomic diversity of Indian swamp buffalo populations through reduced representation approach (ddRAD). The heterozygosity (F_ST) among the swamp buffaloes was 0.11 between Assam and Manipuri; 0.20 between swamp (Manipuri) and riverine buffaloes; 0.30 between swamp (Manipuri) and cattle. The average observed and expected heterozygosity in swamp buffalo populations was 0.254 and 0.221 respectively. The Inbreeding coefficient (F_IS) value was 0.02 among the swamp buffaloes. PCA and structure analysis revealed Manipuri swamp buffalo was genetically distinct and closely related to Nagaland swamp buffalo and least to Assam swamp buffalo. Identification of selective sweeps revealed 1087 regions to have undergone selection related to immune response, adaptation and nervous system. A total of 3451 SSRs were identified in the genome of swamp buffaloes. The study evidenced the genomic diversity in the swamp buffalo populations and its uniqueness in comparison with riverine buffalo and cattle.     

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.     

Analysis of genetic diversity of domestic water buffaloes and anoas based on variations in the mitochondrial gene for cytochrome b

There are two major groups of domestic water buffaloes in East and Southeast Asia: swamp buffaloes and river buffaloes. Genetic diversity among swamp and river buffaloes was studied by DNA sequence analysis of the mitochondrial gene for cytochrome b. The results showed that each of the two groups has mitochondrial DNA (mtDNA) with a specific cytochrome b haplotype. The pairwise nucleotide sequence divergence was calculated to be 2·67% between swamp and river buffaloes, suggesting that they might have diverged from the ancestral populations of Asiatic domestic water buffaloes, approximately 1 million years ago. In addition, the sequences of the same gene from three subspecies of anoa (lowland, mountain and quarles anoa) were determined and compared with that of a domestic water buffalo. The sequence divergence was 1·2% for mountain anoa vs quarles anoa, 3·6% for mountain anoa vs lowland anoa and 3·3% for quarles anoa vs lowland anoa. Moreover, the sequence divergence between water buffaloes and anoas was found to be approximately 3·33%. Our results provide molecular evidence to support the taxonomic classification, namely, that Asiatic buffaloes may be classified into four lineages, swamp buffalo, river buffalo, lowland anoa and mountain plus quarles anoa. However, the sequence divergence values among these four groups were lower than the sequence divergence values found in the genus and subgenus levels within the subfamily Bovinae. In particular, in contrast to some proposed taxonomic classifications, our results indicated that mtDNA in the water buffaloes and anoas did not diverge at the genus level.     

Karyotypic evolution of ribosomal sites in buffalo subspecies and their crossbreed

Domestic buffaloes are divided into two group based on cytogenetic characteristics and habitats: the “river buffaloes” with 2n = 50 and the “swamp buffaloes”, 2n = 48. Nevertheless, their hybrids are viable, fertile and identified by a 2n = 49. In order to have a better characterization of these different cytotypes of buffaloes, and considering that NOR-bearing chromosomes are involved in the rearrangements responsible for the karyotypic differences, we applied silver staining (Ag-NOR) and performed fluorescent in situ hybridization (FISH) experiments using 18S rDNA as probe. Metaphases were obtained through blood lymphocyte culture of 21 individuals, including river, swamp and hybrid cytotypes. Ag-NOR staining revealed active NORs on six chromosome pairs (3p, 4p, 6, 21, 23, 24) in the river buffaloes, whereas the swamp buffaloes presented only five NOR-bearing pairs (4p, 6, 20, 22, 23). The F1 cross-breed had 11 chromosomes with active NORs, indicating expression of both parental chromosomes. FISH analysis confirmed the numerical divergence identified with Ag-NOR. This result is explained by the loss of the NOR located on chromosome 4p in the river buffalo, which is involved in the tandem fusion with chromosome 9 in this subspecies. A comparison with the ancestral cattle karyotype suggests that the NOR found on the 3p of the river buffalo may have originated from a duplication of ribosomal genes, resulting in the formation of new NOR sites in this subspecies.     

Mitochondrial DNA analyses of Indian water buffalo support a distinct genetic origin of river and swamp buffalo

Water buffalo (Bubalus bubalis) is broadly classified into river and swamp categories, but it remains disputed whether these two types were independently domesticated, or if they are the result of a single domestication event. In this study, we sequenced the mitochondrial D-loop region and cytochrome b gene of 217 and 80 buffalo respectively from eight breeds/locations in northern, north-western, central and southern India and compared our results with published Mediterranean and swamp buffalo sequences. Using these data, river and swamp buffalo were distinguished into two distinct clades. Based upon the existing knowledge of cytogenetic, ecological and phenotypic parameters, molecular data and present-day distribution of the river and swamp buffalo, we suggest that these two types were domesticated independently, and that classification of the river and swamp buffalo as two related subspecies is more appropriate.     

Genetic variants of ribosomal DNA and mitochondrial DNA between swamp and river buffaloes

To clarify the genetic relationship between Swamp and River buffaloes, the restriction fragment length polymorphisms (RFLPs) of nuclear genomic ribosomal DNA (rDNA) and cytoplasmic mitochondrial DNA (mtDNA) were analysed. Blood or liver samples from 73 Swamp and three River buffaloes were collected in East and South-east Asian countries. DNA samples from cattle, goats and sheep were used for comparisons. The analysis of rDNA allowed water buffaloes, cattle, goats and sheep to be characterized by four distinct repeat-types. However, swamp and river buffaloes showed the same repeat-type. Divergence of water buffalo and cattle is considered to have occurred approximately four to six million years ago. The RFLPs for mtDNA divided water buffaloes into three haplotypes, swamp-1, swamp-2 and river types. Swamp-1 accounted for 91% of all swamp buffaloes while swamp-2 was observed only in water buffaloes from Thailand (9%). All river buffaloes were of the same haplotype. No differences were observed between swamp and river buffaloes at the rDNA level. In contrast, a few distinct differences between them were found at the mtDNA level. Therefore, mtDNA polymorphisms provide an adequate means for classifying water buffaloes into either swamp or river buffaloes.     

Genetic diversity analysis of an indigenous Chinese buffalo breed and hybrids based on microsatellite data

Chinese native buffaloes have faced the threat of extinction, along with an increase in crossbreeding with domesticated river buffaloes; consequently, conservation of local buffalo genetic resources has become a priority. A Chinese native breed, Jianghan, is often crossed intentionally and unintentionally with imported breeds from India and Pakistan, Murrah, and Nili-Ravi. A total of 128 buffaloes of the breeds Jianghan, Murrah, and Nili-Ravi and their presumed hybrid offspring were genotyped for 10 microsatellite markers. Heterozygosity and Wright's F-statistics were calculated to determine the genetic variation in those populations. The observed average heterozygosities ranged from 0.836 (Murrah) to 0.986 (Jianghan), higher than the expected heterozygosities and all the inbreeding values within the populations were negative. The genetic distances between the presumed hybrid buffaloes and the two imported river type dairy buffalo breeds (Murrah and Nili-Ravi) were lower than with the native Jianghan, indicating strong contributions of the imported breeds to this presumed hybrid buffalo population. This information will be useful for the development of rational breeding for the dairy buffalo industry and for conservation strategies for the Jianghan buffalo.     

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.     

Strong and stable geographic differentiation of swamp buffalo maternal and paternal lineages indicates domestication in the China/Indochina border region

The swamp type of the Asian water buffalo is assumed to have been domesticated by about 4000 years BP, following the introduction of rice cultivation. Previous localizations of the domestication site were based on mitochondrial DNA (mtDNA) variation within China, accounting only for the maternal lineage. We carried out a comprehensive sampling of China, Taiwan, Vietnam, Laos, Thailand, Nepal and Bangladesh and sequenced the mtDNA Cytochrome b gene and control region and the Y‐chromosomal ZFY, SRY and DBY sequences. Swamp buffalo has a higher diversity of both maternal and paternal lineages than river buffalo, with also a remarkable contrast between a weak phylogeographic structure of river buffalo and a strong geographic differentiation of swamp buffalo. The highest diversity of the swamp buffalo maternal lineages was found in south China and north Indochina on both banks of the Mekong River, while the highest diversity in paternal lineages was in the China/Indochina border region. We propose that domestication in this region was later followed by introgressive capture of wild cows west of the Mekong. Migration to the north followed the Yangtze valley as well as a more eastern route, but also involved translocations of both cows and bulls over large distances with a minor influence of river buffaloes in recent decades. Bayesian analyses of various migration models also supported domestication in the China/Indochina border region. Coalescence analysis yielded consistent estimates for the expansion of the major swamp buffalo haplogroups with a credibility interval of 900 to 3900 years BP. The spatial differentiation of mtDNA and Y‐chromosomal haplotype distributions indicates a lack of gene flow between established populations that is unprecedented in livestock.     

Genetic diversity of Asian water buffalo (Bubalus bubalis): mitochondrial DNA D-loop and cytochrome b sequence variation

Swamp and river buffalo mitochondrial DNA (mtDNA) was sequenced for 303 bp of the cytochrome b gene for 54 animals from 14 populations, and for 158 bp of the D-loop region for 80 animals from 11 populations. Only one cytochrome b haplotype was found in river buffalo. Of the four haplotypes identified in swamp buffalo, one found in all populations is apparently ancestral both to the other swamp haplotypes and to the river haplotype. The phylogenetic relationships among the 33 D-loop haplotypes, with a cluster of 11 found in swamp buffalo only, also support the evolution of domesticated swamp and river buffalo from an ancestral swamp-like animal, most likely represented today by the wild Asian buffalo ( Bubalus arnee ). The time of divergence of the swamp and river types, estimated from the D-loop data, is 28 000 to 87 000 years ago. We hypothesise that the species originated in mainland south-east Asia, and that it spread north to China and west to the Indian subcontinent, where the rive type evolved and was domesticated. Following domestication in China, the domesticated swamp buffalo spread through two separate routes, through Taiwan and the Philippines to the eastern islands of Borneo and Sulawesi, and south through mainland southeast Asia and then to the western islands of Indonesia.     

Identification of crossbred buffalo genotypes and their chromosome segregation patterns

Chromosome analysis on different breed types of water buffaloes (Bubalus bubalis) was undertaken to identify their karyotypes and to determine the pattern of chromosome segregation in crossbred water buffaloes. Altogether, 75 purebred and 198 crossbred buffaloes including 118 from Malaysia and 80 from the Philippines, were analyzed in this study. The diploid chromosome number of the swamp buffalo from both countries was 48 and that of the river buffalo was 50, while all F1 hybrids exhibited 49 chromosomes. The F2 hybrids consisted of three different karyotype categories (2n = 48, 2n = 49, and 2n = 50), whereas the backcrosses included two different karyotype categories each, with 2n = 48 and 2n = 49 in the three quarters swamp types and 2n = 49 and 2n = 50 in the three quarters river types. Chi-square tests on pooled data from Malaysia and the Philippines indicated that the distribution of different karyotype categories of F2 animals did not deviate significantly from the 1:2:1 ratio expected if only balanced gametes with 24 and 25 chromosomes were produced by the F1 hybrids. In the three quarters swamp and three quarters river types, the respective karyotypic categories were in ratios approximating 1:1. The distribution of chromosome categories among the F2 hybrids and backcrosses suggests that only genetically balanced gametes of the F1 hybrids are capable of producing viable F2 and backcross generations.     

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.     

Phylogeography and domestication of Indian river buffalo

Background  

The water buffalo- Bu balus bubalis holds tremendous potential in livestock sector in many Asian countries, particularly India. The origin, domestication and genetic structure of the Indian river buffalo are poorly understood. Therefore, to understand the relationship among the maternal lineages of Indian river buffalo breeds and their domestication process, we analysed mitochondrial D-loop region of 217 animals representing eight breeds from eight different locations in India along with published sequences of Mediterranean buffalo.     

Genomic differentiation between swamp and river buffalo using a cattle high-density single nucleotide polymorphisms panel

Buffalo (Bubalus bubalis) is an important livestock species in many tropical and subtropical regions. In recent decades, the interest in buffalo’s milk have expanded and intensive buffalo farms start to emerge. However, breeding programs and population genetics information for this species is scarce or inexistent. The present study aims to test the suitability of the commercial high-density single nucleotide polymorphisms (SNP) genotyping panel, the Illumina BovineHD BeadChip, to estimate population genetics parameters, pedigree control and identification of common variants in major production candidate genes. From a total of 777 962 SNPs included in the panel, 20 479 were polymorphic in water buffalo at a call rate of 86% and an average expected heterozygosity (H_E) of 0.306. From these, 357 were mapped within or around the flanking regions of several major candidate genes. A principal components analysis identified three different clusters, each representing pure swamp buffalo type, pure river buffalo type and admixed river buffalo. The hybrids between swamp and river buffalo were clearly identified as an intermediary cluster. The suitability of these SNPs data set for parentage and identity testing demonstrated that the combination of just 30 to 50 SNPs were enough to attain high probabilities of parentage exclusion (0.9999) in both types and identity (2.3×10⁻⁵ and 2.0×10⁻⁷) for river and swamp buffalo, respectively. Our analysis confirms the suitability of the BovineHD BeadChip to assess population structure, hybridization and identity of the water buffalo populations.     

Phylogenetic relationship among all living species of the genusBubalus based on DNA sequences of the cytochromeb gene

The cytochromeb genes of all living species ofBubalus, including the river type and the swamp type of domestic buffaloes (Bubalus bubalis), were sequenced to clarify their phylogenetic relationships. These sequences were compared together with the African buffalo (Syncerus caffer) and banteng (Bos javanicus) sequences as an outgroup. Phylogenetic trees ofBubalus species based on the DNA sequences of the cytochromeb gene demonstrated that the tamaraw (Bubalus mindorensis), endemic to the Philippines, could be classified into the subgenusBubalus, not the subgenusAnoa. The divergence time between the lowland anoa (B. depressicornis) and the mountain anoa (B. quarlesi) was estimated at approximately 2.0 million years (Myr), which is almost the same as the coalescence time for theBubalus sequences. This large genetic distance supports the idea that the lowland anoa and the mountain anoa are different species. An unexpectedly large genetic distance between the river and the swamp type of domestic buffaloes suggests a divergence time of about 1.7 Myr, while the swamp type was noticed to have the closest relationship with the tamaraw (1.5 Myr). This result implies that the two types of domestic buffaloes have differentiated at the full species level.     

Diversity analysis at MHC class II DQA locus in buffalo (Bubalus bubalis) indicates extensive duplication and trans-species evolution

Variation at MHC Class II-DQA locus in riverine and swamp buffaloes (Bubu) has been explored in this study. Through sequencing of buffalo DQA, 48 nucleotide variants identified from 17 individuals, reporting 42 novel alleles, including one pseudogene. Individual animal displayed two to seven variants, suggesting the presence of more than two Bubu-DQA loci, as an evidence of extensive duplication. dN values were found to be higher than dS values at peptide binding sites, separately for riverine and swamp buffaloes, indicating locus being under positive selection. Evolutionary analysis revealed numerous trans-species polymorphism with alleles from water buffalo assigned to at least three different loci (Bubu-DQA1, DQA2, DQA3). Alleles of both the sub-species intermixed within the cluster, showing convergent evolution of MHC alleles in bovines. The results thus suggest that both riverine and swamp buffaloes share con-current arrangement of DQA region, comparable to cattle in terms of copy number and population polymorphism.