Maternal genetic diversity and phylogenetic analysis of Indian riverine and swamp buffaloes: insights from complete mitochondrial genomes.

This study explored the genetic diversity and evolutionary history of riverine and swamp buffaloes in India, utilizing complete mitochondrial genome sequences. Through comprehensive sampling across varied agro-climatic zones, including 91 riverine buffaloes from 12 breeds and 6 non-descript populations, along with 16 swamp buffaloes of the Luit breed, this study employed next-generation sequencing techniques to map the mitogenomic landscape of these subspecies. Sequence alignments were performed with the buffalo mitochondrial reference genome to identify mitochondrial DNA (mtDNA) variations and distinct maternal haplogroups among Indian buffaloes. The results uncovered the existence of 212 variable sites in riverine buffaloes, yielding 67 haplotypes with high haplotype diversity (0.991), and in swamp buffaloes, 194 variable sites resulting in 12 haplotypes, displaying haplotype diversity of 0.950. Phylogenetic analyses elucidated the genetic relationships between Indian buffaloes and the recognized global haplogroups, categorizing Indian swamp buffaloes predominantly into the SA haplogroup. Intriguingly, the haplogroup SB2b was observed for the first time in swamp buffaloes. Conversely, riverine buffaloes conformed to established sub-haplogroups RB1, RB2, and RB3, underscoring the notion of Northwestern India as a pivotal domestication site for riverine buffaloes. The study supports the hypothesis of independent domestication events for riverine and swamp buffaloes, highlighting the critical role of genetic analysis in unraveling the complex evolutionary pathways of domestic animals. This investigation contributes to the global understanding of buffalo mitogenome diversity, offering insights into this important livestock species' domestication and dispersal patterns.

Genetic differentiation of Indian dromedary and Bactrian camel populations based on mitochondrial ATP8 and ATP6 genes.

Camelids are acknowledged worldwide to endure hostile conditions prevalent in the hot as well cold deserts across the globe. Adaptations to climatic extremes have been associated with mitochondrial protein variants such as ATP8 and ATP6 in different species. The camel genetic resources of India are represented by 9 breeds of dromedary camels which inhabit hot arid and semi-arid zones of the country and a small population of Bactrian camels found in the cold desert of Ladakh. In this study, within and between breed genetic diversity in Indian dromedaries and their divergence from Bactrian camels was investigated based on ATP8/6 genes. Sequence analysis of a mitochondrial DNA fragment encompassing ATP8 and ATP6 genes identified 15 haplotypes in the dromedaries of India and 3 haplotypes in Bactrian camels. The values of haplotype diversity and nucleotide diversity were 0.647 and 0.00187 in the former and 0.679 and 0.00098, respectively in the latter. AMOVA analysis revealed 97.81% variance between the two species. Median-Joining network delineated three distinct mitochondrial haplogroups for Camelus dromedarius, Camelus ferus and Camelus bactrianus. Clear demarcation of the old world (Dromedary and Bactrian camels) and new world camelids (Alpaca, llama, guanaco and vicugna) was evident through the phylogenetic analysis.

Genetic characterization of donkeys of Braj region of India.

The grey type donkeys of Braj region of Uttar Pradesh in India were genetically characterized using ten heterologous microsatellite loci. At these loci the PCR product size ranged from 75-95 bp at locus HTG6 to 251-277 bp at locus COR18. The observed number of alleles varied from 4 (HTG15) to 10 (HTG7 and AHT4) with a mean of 7.50 ± 1.96. The observed heterozygosity ranged from 0.46 (HTG6 and NVHEQ54) to 0.79 (COR18) with a mean of 0.66 ± 0.12. The mean genetic diversity estimate (FIS) was 0.157. When these donkeys were compared on the basis of allelic frequency data at these loci to the brown type donkeys of Ladakh, Spiti and Rayalseema regions in the union territories/states of Ladakh, Himachal Pradesh and Andhra Pradesh, respectively, they clustered independently from these three donkey populations in a dendrogram based on Goldstein's average square distances indicating their genetic distinctness.

Assessment of genetic variability and structuring of riverine buffalo population (Bubalus bubalis) of Indo-Gangetic basin.

The buffalo population of Uttar Pradesh (UP) constitutes 26.1% of the total buffalo population of India, yet this population has not been classified into distinct breeds or subpopulations due to lack of systematic study. Genetic variation at 30 microsatellite loci was examined and statistical analysis was carried out to reveal genetic diversity, demographic parameters of these buffaloes and to investigate the existence of population substructures underlying geographical distribution. The mean number of alleles per locus was 13.26 and mean effective number of alleles was 3.74, whereas mean observed and expected heterozygosities were found to be 0.57 and 0.67 in UP buffaloes. Principal component analysis (PCA) based on allele frequency data revealed subclustering of UP buffalo population. Bayesian analysis result also revealed clear membership of individuals into five clusters indicating a genetic subdivision within the UP buffalo population. The buffaloes of Western and Central regions of UP were subtly separated while buffaloes of Tarai area and Bhadawari buffaloes revealed distinctive population structure. The buffaloes of Mau, Ballia and Ghazipur districts of Eastern region also had a distinctive genetic structure. The analysis of data on buffaloes of Indo-Gangetic plains revealed that population was in mutation drift equilibrium. The observed mean M ratio in the population was above the critical significance value (Mc) suggesting that it has not suffered any severe reduction in effective population size. The statistical tests revealed a historical constancy of size of buffalo in this geographical area. The high level of genetic variability indicates UP buffalo population is a vast reservoir of genetic diversity and this shall help in taking informed conservation decisions and sustainable utilization.

Characterization of hsp70 gene promoter for cis- acting elements in Indian zebu cattle of Hariana breed.

The promoter region of hsp70 gene was characterized for cis-acting elements in zebu cattle of Hariana breed. The basal regulatory domain of CAAT box identified as CAAT/enhancer binding protein (C/EBP) and CAAT binding transcription factor (CTF) binding sites, as well as GC box identified as sp1 binding site, were localized in at least two regions in the hsp70 gene promoter. A highly conserved heat shock element was found between position -108 to -95, which exactly matched at all eight positions with the consensus sequence. These cis-acting elements were found to be conserved between Holstein-Friesian and studied zebu breed.

Genetic variation and phylogenetic relationships of Indian buffaloes of uttar pradesh.

India possesses a total buffalo population of 105 million out of which 26.1% inhabit Uttar Pradesh. The buffalo of Uttar Pradesh are described as nondescript or local buffaloes. Currently, there is no report about the genetic diversity, phylogenetic relationship and matrilineal genetic structure of these buffaloes. To determine the origin and genetic diversity of UP buffaloes, we sequenced and analysed the mitochondrial DNA D-loop sequences in 259 samples from entire Uttar Pradesh. One hundred nine haplotypes were identified in UP buffaloes that were defined by 96 polymorphic sites. We implemented neutrality tests to assess signatures of recent historical demographic events like Tajima's D test and Fu's Fs test. The phylogenetic studies revealed that there was no geographic differentiation and UP buffaloes had a single maternal lineage while buffaloes of Eastern UP were distinctive from rest of the UP buffaloes.

Mitogenomic phylogeny reveals the predominance of the Nubian lineage of African wild ass in Indian donkeys.

To contribute to the knowledge of maternal genetic diversity in domestic donkeys, this study investigated the mitochondrial DNA variations and analyzed the genetic structure in Indian donkeys based on 31 mitogenome sequences representing four breeds/populations (Agra, Halari, Kachchhi and Spiti). A total of 27 haplotypes with a haplotype diversity value of 0.989 were evident in the donkey genetic resources of India. The genetic differentiation between the investigated populations was evaluated using population pairwise FST values, which showed maximum differentiation between Kachchhi and Halari donkeys. The Neighbor-Joining (NJ) tree based on the whole mitogenome sequence and the Median-Joining (MJ) network for partial D-loop fragment showed clear demarcation of Indian donkeys into Nubian and Somali clades, substantiating African maternal origin of Indian domestic donkeys. The topology of the MJ network excluded the Asian wild asses as the possible progenitors of Indian donkeys. Halari and Agra donkeys showed conformity exclusively to the Nubian lineage of the African wild asses. However, representation of both the Nubian and Somali lineages was observed in Kachchhi and Spiti donkeys. Comprehensive analysis carried out by retrieving D-loop sequences from different countries representing Asia, Africa, Europe and South America revealed existence of shared haplotypes across geographically isolated regions of the globe. This observation is indicative of utility of donkeys as pack animals across inter-continental trading routes during development of human civilizations. Our results represent a valuable contribution to maternal genetic diversity of Indian donkeys and provide insights into the worldwide spread of the species following initial domestication in Africa.

Mitochondrial DNA diversity divulges high levels of haplotype diversity and lack of genetic structure in the Indian camels.

Camels represent an important genetic resource of the desert ecosystems of India, with the dromedary and Bactrian camels inhabiting the hot and cold deserts, respectively. This study is the first attempt to investigate mitochondrial DNA based genetic diversity in the Indian camel populations and explores their relationship in the context of global genetic diversity of all the three large camel species (Camelus ferus, Camelus bactrianus and Camelus dromedaries). A mitochondrial DNA fragment encompassing part of cytochrome b gene, tRNAThr, tRNAPro and the beginning of the control region was amplified and analyzed in 72 dromedary and 8 Bactrian camels of India. Sequence analysis revealed that the haplotype and nucleotide diversity (Hd: 0.937 and π: 0.00431) in the Indian dromedaries was higher than the indices reported so far for the dromedary or Bactrian camels across the globe. The corresponding values in the Indian Bactrian camels were 1.000 and 0.00393, respectively. Signals of population expansion were evident in the dromedaries of India on the basis of mismatch analysis and Fu's Fs values. The analysis of molecular variance attributed most of the genetic variance (92.15%) between the dromedary, wild Bactrian and domestic Bactrian camels indicating separate maternal origins. The existence of three mitochondrial lineages in the old world camels (C. bactrianus: Lineage A; C. ferus: Lineage B and C. dromedarius: Lineage C) was also substantiated by the topology of the Median-Joining network.

Sequence of GDF 8 (myostatin) gene in Bubalus bubalis.

The sequence of myostatin gene (growth differentiation factor 8 [GDF 8]) in Indian riverine buffalo (Bubalus bubalis) is reported. The genomic DNA as well as mRNA were sequenced. The sequence is conserved across all the livestock species. Five nonsynonymous changes as compared to cattle were found in this study and were also confirmed by mRNA sequence. Two intronic single nucleotide polymorphisms (SNPs) were identified in buffalo.

Genetic diversity and differentiation of dromedarian camel of India.

Estimation of genetic variability and relationship among different livestock breeds is important for management of genetic resources for their sustainable utilization and conservation. This is more important when the livestock species, like camel, have shown a sharp decline in head count during the last decade. In the present study we estimated genetic variability and relationship among four camel breeds of India using 23 microsatellite loci. A total of 252 alleles were observed across all the four populations with mean number of alleles per locus as 8.04, 7.30, 6.39, and 7.43 for Bikaneri, Jaisalmeri, Kutchi, and Mewari breeds, respectively. The mean observed heterozygosity of the four breeds were 0.58, 0.57, 0.56, and 0.60 for Bikaneri, Jaisalmeri, Kutchi, and Mewari breeds, respectively and were lower than expected heterozygosity values. The mean estimates of F statistics were 0.227+/-0.044 (F(IT)), 0.157+/-0.038 (F(IS)), and 0.082+/-0.019 (F(ST)). The values were significantly different from zero for all the three measures and point towards the existence of population structure and moderate differentiation in four camel breeds. The exact test also indicated significant population differentiation (P < 0.001). The analysis of molecular variance revealed 12% of the variation attributed to among populations and 88% within populations. Sixty-nine percent of the individuals could be correctly assigned using "leave one out" procedure. All the individuals of Mewari and 42 out of 44 Jaisalmeri were correctly assigned. The existence of strong population structure in Jaisalmeri and Mewari camel was further substantiated by Nei's standard genetic distance as well as interindividual allele sharing distance. Thus these two breeds owing to selection for specific traits are distinct from other camel breeds.

Evaluation of the genetic variability of 13 microsatellite markers in native Indian pigs.

We analysed polymorphism of 13 microsatellites in two Indian domesticated pig types (North Indian and Northeast Indian). Heterozygosity, polymorphism information content, and probability of identity of two random individuals were calculated for all microsatellites in both types. The number of alleles observed at a locus varied between five and 12. The evaluated microsatellites exhibited a very high heterozygosity and polymorphism information content. The probability of identity of two random individuals from different populations taking into account all the 13 microsatellites was as low as 3.51 x 10(-19). On the basis of these results, we propose that these microsatellite markers may be used with reliability for studying the genetic diversity and for identification of individuals in Indian pig types.

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.

Characterization of Osteopontin gene of Bubalus bubalis.

Osteopontin, a glycoprotein, is expressed in several tissues including the mammary gland. The gene has been reported to be associated with milk and its constituents in various livestock species. This gene was sequenced in buffalo and it coded for the protein of 280 amino acids with the conserved GRGDS domain. The sequence was confirmed from the cDNA sequence derived from the mammary gland of buffalo. The earlier-reported 9T/10T variation in the upstream region of the gene was investigated for its effect on milk in buffalo and it was found to be non-significant.

Genetic analysis of Ankamali pigs of India using microsatellite markers and their comparison with other domesticated Indian pig types.

Ankamali pigs, the domesticated native pigs of Kerala province of India were genetically characterized using 23 FAO recommended microsatellite markers and were compared with other native Indian pig types and Large White pigs. Twenty-six blood samples were collected from genetically unrelated animals from their breeding tract and DNA was isolated by standard procedure of phenol/chloroform. The genomic DNA was amplified by polymerase chain reaction (PCR) at these 23 microsatellite loci, which were also used earlier to characterize Desi (North Indian) and Gahuri (North-East Indian) pigs, the other two native domesticated pig types of India. The PCR products were resolved by denaturing urea-polyacrylamide gel electrophoresis and alleles were visualized after silver nitrate staining. The data were analysed for allele size range, number of alleles, allelic frequencies, heterozygosity and polymorphism information content for each locus. The allele size range varied between 92-108 bp at locus S0026 and 280-296 bp at locus IGF-1. The total number of alleles varied between five (S0178 and S0386) and 11 (S0355). The mean observed and expected heterozygosities were found to be 0.74 +/- 0.09 and 0.83 +/- 0.03 respectively. In the neighbour-joining dendrogram based on D(A) genetic distances developed after 1000 bootstraps, the Ankamali pigs did not show genetic closeness either with other native Indian pig types or exotic Large White pigs with high bootstrap values indicating genetic distinctness of Ankamali pigs.

Analysis of 22 heterologous microsatellite markers for genetic variability in Indian goats.

The genetic variability of 22 heterologous microsatellite markers was analyzed in two Indian goat breeds, namely Bengal and Chegu. The heterozygosity, polymorphism information content (PIC), and probability of identity of two individuals were calculated for all microsatellite loci in both the breeds. The observed number of alleles varied between 4 and 13 at the studied microsatellite loci. The evaluated microsatellite loci exhibited high mean heterozygosity of 0.69 +/- 0.11 and 0.66 +/- 0.07 in Bengal and Chegu goats, respectively. The mean PIC values of the studied loci in these breeds were 0.79 +/- 0.08 and 0.78 +/- 0.05, respectively. The probability of identity of two random individuals from different breeds, taking into account, all the 22 microsatellite loci was as low as 5.523 x 10(-40). On the basis of these results, we propose that these microsatellite markers may be used with reliability for studying genetic diversity and for identification of individuals in Indian goat breeds.