Indian Buffalo Breeds Research Articles

Polymorphic Variants Analysis in Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-Alpha (PPARGC1A) Gene of Indian and Egyptian Buffaloes

Background-Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) is a candidate gene for milk fat in livestock. The present study was carried out to investigate polymorphism in PPARGC1A and study its potential association. Methods-Two hundred forty animals from Murrah and Bhadawari Indian buffalo breeds and Egyptian buffaloes were selected. The statistical analysis used Tukey’s honest significance test to investigate the association of different genotypes. PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) assays and sequencing technologies were used to confirm the SNPs. Results-Three Novel single nucleotide polymorphisms (SNP1- g.993A greater than T, SNP2- g.1237T greater than A and SNP3-g.1238G greater than C) and one insertion (g.1240_ greater than G bp) were identified in this gene. Due to nucleotide mutations, four non-synonymous mutations were observed amino acid sequences of buffalo breeds. Significant differences in protein and SNF output were discovered in Egyptian buffalo breeds. In both Murrah and Bhadawari buffaloes, fat, protein, and SNF yield were substantially correlated with detected genotypes while there was non-significant association of protein and SNF percent in buffalo breeds. The information provided will be valuable in studying the involvement of PPARGC1A in regulating fat synthesis and enhancing milk quality in buffaloes.

Performance of Illumina® BovineHD BeadChip in genotyping Indian riverine buffalo breeds

The current study tested the suitability of Illumina® BovineHD BeadChip in genotyping three riverine breeds of buffalo viz. Jaffarabadi, Murrah and Mehsana. Of the 777962 SNPs, 670955 SNPs were called in all animals and only 13150 SNPs were found to be polymorphic at a call rate of 90%, and had Minor Allele Frequency (MAF) greater than 0.05. The average Minor Allele Frequency (MAF) of polymorphic SNPs was 0.23 and average expected heterozygosity (HE) of 0.321. Principal Component Analysis (PCA) revealed 2 different clusters where Murrah and Mehsana breeds clustered together, while Jaffarabadi breed formed a distinct cluster. Result obtained in this study indicated that the SNPs available in Illumina BovineHD BeadChip may not be good enough for studying population structure and genetic analyses in Indian buffalo breeds. Hence it might be necessary to develop a custom SNP marker panel for Indian riverine buffaloes.

Identification of Y chromosome haplogroup and estimation of genetic diversity in Indian buffalo breeds using Y specific STR markers

The present investigation was to identify Y chromosome haplogroup using STR markers and to study genetic diversity in Indian breeds of buffalo. A total of 139 semen samples of five different Indian buffalo breeds were collected from different semen station of India for the study. DNA was extracted using standard phenol chloroform method. Based of Y specific microsatellite UMN1113 and UMN0304, the population was divided in 47 haplo groups. Subsequently, analysis of microsatellites revealed that overall haplogroup diversity in whole population of 139 buffalo bulls was (0.798±0.048), Murrah breed had maximum (0.899±0.024) and Surti had minimum (0.600±0.215) diversity. AMOVA showed that between breeds variation was 16.69% and differed significantly among the breeds (P<0.0001). Further, the pair wise genetic distance (FST value) between different breeds showed that Mehsana lies in between Surti and Murrah, which also signifies the fact that Mehsana breed originated as cross of Murrah and Surti, while Nili Ravi and Jaffarabadi were clustered together sharing Y specific signatures.

Comparative Signatures of Selection Analyses Identify Loci Under Positive Selection in the Murrah Buffalo of India.

India is home to a large and diverse buffalo population. The Murrah breed of North India is known for its milk production, and it has been used in breeding programs in several countries. Selection signature analysis yield valuable information about how the natural and artificial selective pressures have shaped the genomic landscape of modern-day livestock species. Genotype information was generated on six buffalo breeds of India, namely, Murrah, Bhadawari, Mehsana, Pandharpuri, Surti, and Toda using ddRAD sequencing protocol. Initially, the genotypes were used to carry out population diversity and structure analysis among the six breeds, followed by pair-wise comparisons of Murrah with the other five breeds through XP-EHH and F ST methodologies to identify regions under selection in Murrah. Admixture results showed significant levels of Murrah inheritance in all the breeds except Pandharpuri. The selection signature analysis revealed six regions in Murrah, which were identified in more than one pair-wise comparison through both XP-EHH and F ST analyses. The significant regions overlapped with QTLs for milk production, immunity, and body development traits. Genes present in these regions included SLC37A1, PDE9A, PPBP, CXCL6, RASSF6, AFM, AFP, ALB, ANKRD17, CNTNAP2, GPC5, MYLK3, and GPT2. These genes emerged as candidates for future polymorphism studies of adaptability and performance traits in buffaloes. The results also suggested ddRAD sequencing as a useful cost-effective alternative for whole-genome sequencing to carry out diversity analysis and discover selection signatures in Indian buffalo breeds.

Revealing inheritance of white markings in Nili Ravi buffalo

Among registered Indian buffalo breeds, Nili Ravi is well known for its production potential as well as unique typical five white markings, because of which it is also known as Panch-Kalyani. In this study an attempt was made to understand the inheritance pattern of white markings in 720 progenies sired by 21 bulls and belonging to 381 dams at an organized farm. Based on extent of white markings, animals were classified into typical, under and over white categories and significance of inheritance was tested by Chi- Square, Likelihood ratio and Mc-NemarBowker tests to confirm the results. The results showed distribution of inheritance pattern from sire to progeny, and dam to progeny was that the identical in white markings classes ratios and inheritance of skin/coat colour patterns could not be explained. Further, studies at genomic and epigenetic levels needed to reveal the genetic/methylation differentiation basis of white markings variation in Nili Ravi buffalo.

Semen quality and production potential of Indian buffalo breeds

The objective of the study was to evaluate non-genetic factors on semen traits. The overall means for the semen quality traits: ejaculate volume, sperm concentration, total sperm, mass activity, initial and post thaw motility, hypoosmotic swelling test, acrosome integrity of fresh and frozen test were 4.39±0.01 ml, 1.29±0.001 billion, 5.48±0.01 billion, 2.55±0.003, 73.41±0.0004%, 55.9±0.0002%, 59.01±0.001%, 84.98±0.002% and 71.13±0.002%, respectively. Breeds were found to differ in all the semen production and quality traits studied except acrosome integrity of fresh semen. Summer for semen production, and winter and monsoon for semen quality traits were comparatively better season. The quality traits improved with age and declined slightly in the later life.

Sequence-based structural analysis and evaluation of polymorphism in buffalo Nod-like receptor-1 gene.

In this study, we have sequence characterized and analyzed the polymorphism in buffalo NOD1 (nucleotide-binding oligomerization domain 1) gene as well as its expression analysis. Full-length sequence analysis of NOD1 revealed this gene in buffalo being conserved with respect to the domain structures, similar to other species. Alternate splice variants having exon3 skipping also identified for the first time in the gene expressed in buffalo-purified peripheral blood mononuclear cells (PBMCs). Phylogenetically ruminant species were found to be clustering together and buffalo displaying maximum similarity with cattle. Sequencing of NOD1 across 12 Indian buffalo breeds identified 23 polymorphic sites within coding region, among which 16 were synonymous and 7 changes found to be non-synonymous. Four SNPs (single nucleotide polymorphisms) of them were genotyped in 393 animals belonging to 12 riverine, swamp and hybrid (riverine × swamp) buffalo populations of diverse phenotypes and utilities, showing variable allelic frequencies. Principal component analysis revealed, riverine and swamp buffaloes being distinctly placed with the distribution of breeds within the group based on the geographical isolation. Further, quantitative real-time PCR detected NOD1 expression in multiple tissues with PBMCs and lungs showing highest expression among the tissues examined. Structural analysis based on the translated amino acid sequence of buffalo NOD1 identified four protein interaction motifs LxxLL important for ligand binding. Molecular interaction analysis of iE-DAP and NOD1-LRR and their complex stability and binding-free energy studies indicated variable binding energies in buffalo and cattle NOD1. Overall, the study reveals unique structural features in buffalo NOD1, important for species-specific ligand interaction.

New insights into the spatial genetic structure of the Indian riverine buffalo populations

Several programmes for the genetic improvement and conservation Indian buffalo breeds have been implemented by various agencies since 1970. These programmes may have shaped the present genetic diversity and thereby the evolutionary potential of buffalo populations across India. In this study, we attempted to provide an insight into the contemporary genetic structure of buffaloes by simultaneously analyzing the genetic and geospatial data. A battery of 100 microsatellite markers was genotyped across 1055 buffalo samples representing different geographical regions of the country. Bayesian and multivariate approaches were used for analyses. Although the clustering results from the different approaches were not convergent, these methods were able to identify sub-structuring within the UP buffalo population. In our study BAPS was able to distinguish most of the buffalo breeds with better spatial distribution. The genetic structuring detected in our study is mainly characterised by isolation by distance and also reflects the effect of improvement programmes. Our analysis suggested genetic connectivity of Murrah and Nili-Ravi with several buffalo breeds, as they have been used for breed improvement. The information may be useful in identifying ecologically distinct or connected populations for future improvement and conservation programmes.

Establishment of a Somatic Cell Bank for Indian Buffalo Breeds and Assessing the Suitability of the Cryopreserved Cells for Somatic Cell Nuclear Transfer.

Biobanks of cryopreserved gametes and embryos of domestic animals have been utilized to spread desired genotypes and to conserve the animal germplasm of endangered breeds. In principle, somatic cells can be used for the same purposes, and for reviving of animals, the somatic cells must be suitable for animal cloning techniques, such as somatic cell nuclear transfer. In the present study, we derived and cryopreserved somatic cells from three breeds of riverine and swamp-like type buffaloes and established a somatic cell bank. In total, 350 cryovials of 14 different individual animals (25 cryovials per animal) were cryopreserved and informative data such as breed value, origin, and others were documented. Immunostaining of the established cells against vimentin and cytokeratin suggested a commitment to the fibroblast lineage. In addition, microsatellite analysis was performed and documented for unambiguous parentage verification of clones in the future. Subsequently, the cryopreserved cells were tested for their suitability as nuclear donors (n = 7) using handmade cloning, and the reconstructed embryos were cultured in vitro. The cleavage rates (95.99% ± 2.17% vs. 82.18% ± 2.50%) and blastocyst rates (37.73% ± 1.54% vs. 24.31% ± 1.78%) were higher (p < 0.05) for riverine buffalo cells than that of swamp-like buffalo cells, whereas the total cell numbers of blastocysts (258.16 ± 36.25 vs. 198.16 ± 36.25, respectively) were similar. In conclusion, we demonstrated the feasibility of biobanking of buffalo somatic cells, and that the cryopreserved cells can be used to produce cloned embryos. This study encourages the development of somatic cell biobanks of domestic livestock, including endangered breeds of buffalo, to preserve valuable genotypes for future revitalization by animal cloning techniques.

Evolution and diversity studies of innate immune genes in Indian buffalo (Bubalus bubalis) 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.

Microsatellite based polymorphism and genetic diversity among Indian buffalo breeds

Autosomal microsatellite markers allow estimation of expected heterozygosity and allele frequencies which may reveal the effects of genetic isolation, inbreeding, introgression and subdivision within livestock breeds. In the present study, 7 buffalo breeds (Bhadawari, Jaffrabadi, Murrah, Mehsana, Nagpuri, Pandharpuri and Surti) were studied using a panel of eight microsatellite markers selected based on polymorphic information content (PIC) for buffalo (Bubalus bubalis) species as per the guidelines of Food and Agricultural Organization. Frequency of breedspecific alleles was found lower in all the breeds studied; 4 out of 49 alleles showed a frequency of 8% while only one allele showed &gt;10% frequency. In the present study, highest genetic distance was observed between Murrah and Surti (0.896) indicating their isolated breeding over several generations.

Breed traceability of buffalo meat using microsatellite genotyping technique.

Although buffalo has emerged as a major meat producing animal in Asia, major research on breed traceability has so far been focused on cattle (beef). This research gap on buffalo breed traceability has impelled development and validation of buffalo breed traceability using a set of eight microsatellite (STR) markers in seven Indian buffalo breeds (Bhadawari, Jaffaarabadi, Murrah, Mehsana, Nagpuri, Pandharpuri and Surti). Probability of sharing same profile by two individuals at a specific locus was computed considering different STR numbers, allele pooling in breed and population. Match probabilities per breed were considered and six most polymorphic loci were genotyped. Out of eight microsatellite markers studied, markers CSSMO47, DRB3 and CSSM060 were found most polymorphic. Developed technique was validated with known and unknown, blood and meat samples; wherein, samples were genetically traced in 24 out of 25 samples tested. Results of this study showed potential applications of the methodology and encourage other researchers to address the problem of buffalo traceability so as to create a world-wide archive of breed specific genotypes. This work is the first report of breed traceability of buffalo meat utilizing microsatellite genotyping technique.

Genetic analysis of glycosylation sites, TM 8 and consensus transport motif of buffalo SLC11A1 gene

The present study aimed to explore polymorphisms at exon X-XI, encompassing two potential N-linked Glycosylation sites (GLY, GLY), complete transmembrane domain 8 (TM8) and one conserved ‘Consensus Transport Motif’ (CTM) of buffalo (Bubalus bubalis) SLC11A1 (formerly NRAMP1) gene by PCR-RFLP and automated DNA sequencing. PCR–RFLP genotyping of animals (n=150) from three Indian buffalo breeds viz., Murrah, Surti, and Mehsana by Alu I, Taq I, Hae III, Rsa I, Pst I and Mva I reveals monomorphic pattern. Sequence comparisons demonstrate no change at nucleotide level among the buffalo breeds. However, results of this study show three nucleotide changes in buffaloes as compared to that of B taurus (NM_174652): one transition (C?T at amino acid position 349) and two transversion (C?A at amino acid position 321 and C?G at amino acid position 356). Transversion C?A and C?G, resulting in deduced amino acid exchange Asp321 –Glu321 and Pro356 – Ala356, respectively has been identified. The non-synonymous single nucleotide variation (nsSNV) (C?A at amino acid position 321), observed in all the three buffalo breeds under study, leads to the loss of a potential N-glycosylation motif of SLC11A1 gene and therefore may alter the property of the protein. A comprehensive study of this N-glycosylation motif of SLC11A1 gene in different species followed by structural analysis of the protein is required to better understand the functional impact of nsSNV.

Characterization and association of the Pit-1 gene in the Indian buffalo

Pituitary-specific transcription factor (Pit-1) is responsible for pituitary development and hormone expression in mammals. In present study, a comparison of buffalo Pit-1 gene sequences with Bos taurus revealed no nucleotide changes however, 7 single nucleotide polymorphisms (SNPs), one of which was non-synonymous were found within Indian buffalo breeds (Bubalus bubalis). SNP c.160 T&gt;A alters the encoded amino acid from methionine to lysine. Association of all SNPs with milk production traits was assessed in Mehsana buffalo populations and the results demonstrated that SNP c.160 T&gt;A was significantly associated with the milk fat percentage (P=0.0358). Sequence analysis showed that the buffalo Pit-1 protein shares high homology with cattle (Bos taurus) (100%), goat (99%), sheep (99%), human (96%) and pig (98%). Polymorphisms detected in buffalo Pit-1 gene are expected to have functional significance and might be useful as genetic markers in association studies for milk fat and milk yield in buffalo.

H19 gene methylation study in Indian buffalo (Bubalus bubalis)

The methylation of DNA at cytosine residues within CpG dinucleotides is associated with transcriptional repression and is implicated in maintaining genomic stability and also the silencing of repetitive elements. These imprinted genes are unique as they are expressed exclusively from one parental allele. The present study was carried out to detect methylation status in H19 gene promoter CTCF III region in three Indian buffalo breeds (Jaffarabadi, Surti and Mehsani) by bisulfite sequencing. Methylation percent in Jaffarabadi, Surti and Mehsani buffaloes were found to be 50.19, 70.85 and 52.24, respectively, with mean incidence of methylation percent in H19 in all three breeds as 57.36. Apart from CpG methylation, unexpected nucleotide conversion (T>C, A>G, G>A) and deletion (A and G) after bisulfite sequencing were also observed. We observed no significant relationships in milk yield and milk fat per cent with methylation pattern in H19 gene in any of the three breeds.

Analysis of Genetic Diversity of Indian Buffalo Breeds by DNA Markers

Buffalo is an important species contributing significantly to Indian economy. There is urgent need to study genetic diversity between and within breeds using appropriate set of microsatellite markers which can be further supplemented by using Genome sequencing data, phonemics and high density arrays. Animal resources are valuable assets of a country. Present buffalo breeds have gene pools of wide representation and valuable combination of genes. Genetic variability also provides the opportunity for tracing the history of populations, species, and their ancestors through DNA based markers. For the overall breed improvement and to meet future challenges there is an urgent need to characterize buffalo breeds. It is our responsibility to conserve, preserve and maintain the animal genetic diversity.

Single nucleotide polymorphism (SNP) identification and sequence analysis of 5′ flanking region of lactoferrin gene in Indian buffaloes (Bubalus bubalis)

Lactoferrin, an iron (Fe 3+) binding glycoprotein, found in a variety of body secretions of mammals, is a potential candidate gene in dairy cattle breeding for increasing resistance against infections especially in the mammary gland. The present study was undertaken with the objective of examining the 5′ flanking region of bubaline ( Bubalus bubalis) lactoferrin gene to gain insight into the polymorphism existing in this region across different buffalo breeds. The 5′ flanking region of lactoferrin gene was analyzed by single strand conformation polymorphism (SSCP) in five different Indian buffalo breeds, viz. Murrah, Jaffarabadi, Marathwada, Toda and Pandharpuri. A total of 10 distinct SSCP patterns were observed which further revealed 13 polymorphisms with 8 transitions, 4 transversions and one deletion mutation upon sequence analysis. A total of 1056 bp 5′flanking region of bubaline lactoferrin gene was sequenced which was found to have 96% homology with Bos taurus, 91% with Capra hircus, 95%, Bos indicus, 92% with Ovis aries, 57% with Sus scrofa and 28% with Homo sapiens. From the polymorphisms identified in the 1056 bp 5′ flanking region, polymorphism at nucleotide position − 209 was found to lie within a potential SP1 transcription factor binding site, while SNP at − 19 was found in the vicinity of the TATA box. Two SNPs identified at − 57 and − 61 positions were found to be present in the vicinity of putative estrogen response element. Another SNP at − 636 position was found to be present in the vicinity of putative recognition sequence for IRF-1 which serves as a transcriptional activator for interferon genes and interferon inducible genes. The SNPs identified in the lactoferrin promoter region may serve as potential candidate genetic marker(s) in buffaloes for disease resistant traits.

Assessment of Genetic Variability in Two North Indian Buffalo Breeds Using Random Amplified Polymorphic DNA (RAPD) Markers

Murrah and NiliRavi are the important North Indian buffalo breeds occupying the prominent position of being the highest milk producers. These breeds are more or less similar at morphological as well as physiological levels. The technique of RAPD-PCR was applied in the present study to identify a battery of suitable random primers to detect genetic polymorphism, elucidation of the genetic structure and rapid assessment of the differences in the genetic composition of these two breeds. A total of 50 random primers were screened in 24 animals each of Murrah and NiliRavi buffaloes to generate RAPD patterns. Of these, 26 (52%) primers amplified the buffalo genome generating 263 reproducible bands. The number of polymorphic bands for the 26 chosen RAPD primers varied from 3 (OPG 06 and B4) to 26 (OPJ 04) with an average of 10.1 bands per primer and size range of 0.2 to 3.2 kb. DNA was also pooled and analyzed to search for population specific markers. Two breed specific RAPD alleles were observed in each of Murrah (OPA02 and OPG16) and NiliRavi (OPG09) DNA pools. RAPD profiles revealed that 11 (4.2%) bands were common to all the 48 individuals of Murrah and NiliRavi buffaloes. Pair-wise band sharing calculated among the individual animals indicated considerable homogeneity of individuals within the breeds. Within breed, band sharing values were relatively greater than those of interbreed values. The low genetic distance (Nei's) value (0.109) estimated in this study is in accordance with the origin and geographical distribution of these breeds. The RAPD analysis indicated high level of genetic similarity between these two important North Indian buffalo breeds.