Phenotypic and genetic characterization of unexplored, potential cattle population of Madhya Pradesh.

Bawri or Garri, a non-descript cattle population managed under an extensive system in Madhya Pradesh state of India, was identified and characterized both genetically and phenotypically to check whether or not it can be recognised as a breed. The cattle have white and gray colour and are medium sized with 122.5 ± 7.5 cm and 109.45 ± 0.39 cm height at withers in male and female, respectively. Double-digest restriction site associated DNA (ddRAD) sequencing was employed to identify ascertainment bias free SNPs representing the entire genome cost effectively; resulting in calling 1,156,650 high quality SNPs. Observed homozygosity was 0.76, indicating Bawri as a quite unique population. However, the inbreeding coefficient was 0.025, indicating lack of selection. SNPs found here can be used in GWAS and genetic evaluation programs. Considering the uniqueness of Bawri cattle, it can be registered as a breed for its better genetic management.

Characterization of a unique Sikkimese yak population of India: a multivariate approach.

Native Sikkimese yak in Sikkim state of India is a pastoral treasure being raised through centuries-old transhumance practices and has evolved in response to natural and man-made selection. Currently, the population of Sikkimese yak is at risk with about five thousand total headcounts. Characterization is essential for taking appropriate decisions for conservation of any endangered population. In an attempt to phenotypically characterize the Sikkimese yaks, this study recorded phenotypic morphometric traits information, viz., body length (LG), height at withers (HT), heart girth (HG), paunch girth (PG), horn length (HL), horn circumference (HC), distance between horns (DbH), ear length (EL), face length (FL), face width (FW), and tail length with switch (TL), on 2154 yaks of both sexes. Multiple correlation estimation highlighted that HG and PG, DbH and FW, and EL and FW were highly correlated. Using principal component analysis, LG, HT, HG, PG, and HL were found to be the most important traits for phenotypic characterization of Sikkimese yak animals. Discriminant analysis based on different locations of Sikkim hinted at the existence of two separate clusters, however, broadly, phenotypic uniformity could be observed. Subsequent genetic characterization can offer greater insights and can pave the way for future breed registration and conservation of the population.

Genome-wide association study revealed suggestive QTLs for production and reproduction traits in Indian Murrah buffalo.

The present study was aimed to identify the genome-wide SNPs associated with production and reproduction traits in 96 Indian Murrah buffalo genotyped based on ddRAD approach using Genome-Wide Association Study (GWAS) along with phenotypes of contemporary animals using mixed linear model for production and reproduction traits. A total of 27,735 SNPs identified using ddRAD approach in 96 Indian Murrah buffaloes were used for GWAS. A total of 28 SNPs were found to be associated with production and reproductive traits. Among these, 14 SNPs were present in the intronic region of AK5, BACH2, DIRC2, ECPAS, MPZL1, MYO16, QRFPR, RASGRF1, SLC9A4, TANC1, and TRIM67 genes and one SNP in long non-coding region of LOC102414911. Out of these 28 SNPs, 9 SNPs were found to have pleiotropic effect over milk production traits and were present in chromosome number BBU 1, 2, 4, 6, 9, 10, 12, 19, and 20. SNPs in the intronic region of AK5, TRIM67 genes were found to be associated with milk production traits. Eleven and five SNPs in the intergenic region were associated with milk production and reproduction traits respectively. The above genomic information may be used for selection of Murrah animals for genetic improvement.

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.

Identification of novel allelic patterns and evolutionary lineage of BoLA MHC class II DQA locus in indicine and taurine cattle.

Among different cattle types, Bos indicus are known for their ability to better resist the tropical microbial infections comparatively, wherein MHC molecules play a significant role. In this study allelic diversity at MHC locus, DQA of Bos indicus, Bos taurus and crossbred of taurine-indicus has been explored to understand the possible role of MHC region in differential immune response. Thirty nine different DQA alleles were identified, out of which 14 were novel, along with documentation of duplication of DQA alleles. Indicus cattle population presented diverse types of DQA alleles compared to crossbred and exotic. Translated amino acid sequence analysis indicated, codon 64 and 50 of peptide binding sites being highly polymorphic and most of the indicus cattle presented alanine and arginine amino acid at position 64 and 50. Within breed genetic variation found to be higher than between breeds. Because of their ability to bind and subsequently respond to a wide array of antigens, the newly identified DQA alleles with high diversity present in the form of duplicated haplotypes in different combinations in cattle populations provided significant insights into probable role of this MHC locus in better tropical disease combating ability and genetic fitness of indicus cattle.

Casein (CSN) gene variants and parity affect the milk protein traits in crossbred (Bos taurus x Bos indicus) cows in sub-tropical climate.

Genotypes at four casein (CSN) loci-A26181G of CSN1S1, C6227T of CSN1S2, A8101C of CSN2, and A13104C of CSN3-along with non-genetic factors were studied for their effects on various milk protein traits in 100 crossbred cows with major inheritance of Holstein Frisian (Bos taurus) and Tharparkar (Bos indicus). Results demonstrated the presence of all CSN genotypes with a predominance of heterozygotes. At CSN2 (A8101C; His67Pro) locus, the A2 allele, desirable for human health, was present in 62% as heterozygous and 29% in homozygous condition. Among non-genetic factors, parity of the cows had a significant influence on the milk protein traits in these crossbreds. The genotypes at CSN1S1, CSN2, and CSN3 loci were found to influence (p<0.05 to 0.01) the casein and whey protein yields and composition traits. The casein index and total milk yield were most influenced by the CSN1S2 locus. The AA (A1 milk) genotype of CSN2 had significantly higher yields and percentages of casein and whey proteins. Positive influence of CC genotype of CSNS3 on milk proteins of was observed similar to Bos taurus cows; however, such influence of AA genotype of CSN2 locus may be distinctive to the crossbred cows maintained in subtropical condition. Overall, the results revealed the diverse effects of CSN genotypes on milk proteins in crossbred cattle.

Reduced representation approach for identification of genome-wide SNPs and their annotation for economically important traits in Indian Tharparkar cattle.

The present study was carried out in Tharparkar cattle for identification of genome-wide SNPs and microsatellites, and then annotate the identified high-quality SNPs to milk production, fertility, carcass, adaptability and immune response of economically important traits. A total of 146,011 SNPs were identified with respect to Bos taurus reference genome which are indicus specific, out of which 10,519 SNPs were found to be novel. Similarly, a total of 87,047 SNPs were identified with respect to Bos indicus reference genome. After final annotation of SNPs identified with respect to Bos indicus reference genome, 2871 SNPs were found to be associated in 383 candidate genes having to do with milk production, fertility, carcass, immune response and adaptability traits. Following that, 2571 microsatellites were identified. The information mined from the data might be of importance for the future breed improvement programs, conservation efforts and for enhancing the SNPs density of the existing bovine SNP chips.

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.

Genomic divergence reveals unique populations among Indian Yaks.

The present study focused upon identification of genome-wide SNPs through the reduced representation approach and to study the genomic divergence of the Indian yak populations. A total of 80 samples belonging to Arunachali yak (N = 20), Himachali yak (N = 20), Ladakhi yak (N = 20) and Sikkimi yak (N = 20) of India were used in the study. The results of the study revealed a total of 579575 high quality SNPs along with 50319 INDELs in the Indian yaks. The observed heterozygosity was found to be high in Himachali yak, followed by Arunachali yak, Ladakhi yak and Sikkimi yaks. The Sikkimi yaks was found to be genetically distant, followed by Ladakhi yaks which was observed to have some few individuals from Arunachali and Himachali yaks. Arunachali and Himachali yaks are found to get clustered together and are genetically similar. The study provides evidence about the genomic diversity in the Indian yak populations and information generated in the present study may help to formulate a suitable breeding plan for endangered Indian yaks. Moreover, the unique yak populations identified in the study will further help to focus attention for future characterization and prioritization of the animals for conservation purposes through the ddRAD approach.

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 (FST) 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 (FIS) 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.

Differential expression of cytokines in PBMC of Bos indicus and Bos taurus × Bos indicus cattle due to Brucella abortus S19 antigen.

Brucellosis is the most dreadful disease among bovines, although breed differences have been observed in prevalence of disease, worldwide. In present study, antibody response and relative expression of proinflammatory cytokines was compared in Bos indicus (zebu) and Bos taurus × Bos indicus (crossbred) cattle vaccinated by live attenuated Brucella abortus S19 antigen. Six female calves (4-6 months age) of both groups were vaccinated with B.abortus S19 strain. Blood samples were collected before vaccination (0d) and 7th (7d), 14th (14d) and 28th (28d) days after vaccination. Indirect ELISA showed high (p < .05) anti-Brucella antibody level after vaccination; with no significant difference between the groups. During Real-time expression, IFNγ, TNFα, IL6 and IL10 genes initially showed down regulation followed by upregulation in both the groups; however, the trend was much prominent in crossbreds. The expressions of IFNγ, TNFα and IL6, proinflammatory molecules important for initial containment of the Brucella were significantly (p < .01) higher in crossbred. The study showed that the Sahiwal cattle were less responsive to B.abortus S19 antigen than crossbreds, indicating its lower sensitivity to the Brucella, comparatively. In contrary, higher expression of the proinflammatory molecules in crossbreds could be important for containment of the organism during initial stage of infection.

Development of PCR based assays for detection of lethal Holstein haplotype 1, 3 and 4 in Holstein Friesian cattle.

Holstein haplotype (HH) 1, 3 and 4 are lethal mutations, responsible for early embryonic losses in Holstein Friesian (HF) cattle, worldwide. Three PCR based assays - tetra Amplification Refractory Mutation System PCR, PCR primer induced restriction analysis and PCR-restriction fragment length polymorphism techniques for screening of HH1, 3 and 4, respectively were developed and validated. During screening, six among 60 HF bulls were found as carrier for either of three mutations. These PCR assays are highly accurate and reproducible and can be used for screening of the haplotypes in HF cattle.

Genome-wide discovery of SNPs in candidate genes related to production and fertility traits in Sahiwal cattle.

The present study was carried out to identify genome-wide genetic markers and variants in candidate genes for production and reproduction traits in Sahiwal cattle using a cost-effective reduced representation sequencing method. A total of 258,231 genome-wide SNPs were identified in Sahiwal cattle with reference to Bos indicus genome, of which 150,231 were novel SNPs. Among the high-confidence SNPs identified, 91.86% and 27.30% were genotyped in 50% and 100% of the samples. Mapping of the identified SNPs revealed 525 SNPs in candidate genes related to production traits while 333 SNPs were mapped to candidate genes related to reproduction traits. The SNPs identified in this study will facilitate further insights on tropical adaptation, domestication history and population structure of indigenous cattle. The variants in candidate genes identified in this study will serve as useful genetic tools, in the quest for phenotype modifying nucleotide change and help in designing appropriate genetic improvement programs.

Identification of novel polymorphism in buffalo stanniocalcin-1 gene and its expression analysis in mammary gland under different stages of lactation.

Stanniocalcin-1 (STC1) is secreted by the variety of tissues having a major role in the regulation of calcium ions in the involuting mammary gland. The present work aims to sequence and structural characterization as well as expression profiling of STC1 gene in buffalo. Polymorphism identified in the 3-untranslated region (UTR) was analysed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) genotyping in riverine and swamp buffaloes. Expression profiling of STC1 was performed in different lactation stages of mammary gland and peripheral blood mononuclear cells to study the impact of 3'-UTR polymorphism on its expression. Different polymorphic sites were detected in the entire coding and noncoding regions of riverine and swamp buffaloes, including two INDELs. An identified polymorphic nucleotide locus A324G, having target sites for two miRNAs, namely bta-miR-2382 and bta-miR-1343, reported in cattle, was genotyped by PCR-RFLP to reveal variable allelic distribution among swamp and riverine buffaloes. Gene expression profiling across buffalo mammary tissues representing different lactation stages showed maximum expression of the STC1 gene in the involuting mammary gland. Ruminants' specific genetic variation has been observed in STC1 and its implication in buffalo mammary gland involution as well as coregulation of gene expression through miRNA binding in the 3'-UTR is suggested.

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.

Genomewide identification and annotation of SNPs in Bubalus bubalis.

The present study was carried out to identify and annotate the genome wide SNPs in Murrah buffalo genome. A total of 21.2 million raw reads from 4 pooled female Murrah buffalo samples were obtained using restriction enzyme digestion followed by sequencing with Illumina Hiseq 2000. After quality filtration, the reads were aligned to Murrah buffalo genome (ICAR-NBAGR) and Water buffalo genome (UMD_CASPUR_WB_2.0) which resulted in 99.37% and 99.67% of the reads aligning, respectively. A total of 130,688 high quality SNPs along with 35,110 indels were identified versus the Murrah bufffalo genome. Similarly 219,856 high quality SNPs along with 15,201 indels were identified versus the Water buffalo genome. We report 483 SNPs in 66 genes affecting Milk Production, 436 SNPs in 38 genes affecting fertility and 559 SNPs in 72 genes affecting other major traits. The average genome coverage was 13.4% and 14.8% versus the Murrah and Water buffalo genomes, respectively.

Identification of genetic variation in NOD-like receptor 2 gene and influence of polymorphism on gene structure and function in buffalo (Bubalus bubalis).

Nucleotide-binding oligomerization domain (NOD)-like receptor 2 is one of the important mediators of innate as well as adaptive immune response to microbial infections. In this study, NOD-like receptor-2 was characterized by determining the full gene sequence and analyzing genetic diversity in Indian buffaloes. Sequence analysis of buffalo NOD2 revealed 3042 nucleotides long ORF, encoding 1013 amino acids from 12 exons. Domain structure analysis indicated existence of 8 leucine-rich repeat (LRR) domains in buffalo, cattle, sheep and mouse, along with central NACHT/NOD domain and two N-terminal CARD domains. Comparative sequence analysis among different buffalo breeds identified 46 polymorphic sites in NOD2 gene. Among coding region SNPs, 10 were non-synonymous, 7 synonymous and 3 were present in 5'UTR. Genotyping of two nsSNPs, revealed significant differences in the allele frequencies, distinguishing swamp and riverine buffaloes, having different utilities. Association analysis with mastitis in dairy buffaloes indicated significant variation in allelic frequencies at G1135A locus, between mastitis affected and non-affected animals. Further, NOD2 gene expression was quantified in different riverine buffalo tissues, using real-time PCR and lymph node displayed highest expression, compared to others organs included in the study. Overall, the study revealed buffalo NOD2 gene attributes, important to understand species specific immune response in ruminants.

High genetic diversity and distribution of Bubu-DQA alleles in swamp buffaloes (Bubalus bubalis carabanesis): identification of new Bubu-DQA loci and haplotypes.

In this study, genetic diversity analysis of MHC class II-DQA locus helped in identification of 25 new Bubu-DQA nucleotide sequences in swamp buffaloes (Bubalus bubalis carabanesis, Bubu). Phylogenetic analysis revealed the distribution of the buffalo DQA sequences in two major clusters of DQA1 and DQA2 genes, sharing common lineages with corresponding cattle alleles, possibly due to trans-species evolution. However, a highly divergent sequence, Bubu-DQA*2501, homologous to cattle (BoLA) DQA3 allele, was identified, indicating the existence of an additional locus; putative DQA3 in buffalo. PCR-RFLP analysis revealed extensive duplication of DQA locus in swamp buffaloes, sharing DQA1, DQA2, and DQA3 alleles in different combinations in duplicated haplotypes. Higher dN than dS values and Wu-Kabat variability at peptide-binding regions in Bubu-DQA indicated high polymorphism with balancing selection. Levels of genetic diversity within DQA sequences and duplication in a small population of swamp buffalo indicate the genetic richness of the species, important for fitness.

Nucleotide polymorphisms in the bovine lymphotoxin A gene and their distribution among Bos indicus zebu cattle breeds.

The present study was undertaken to characterize the genetic variation present in lymphoxin A gene (LTA gene) encoding for the lymphotoxin A protein also known as tumor necrosis factor beta, a cytokine produced by lymphocytes, known to be cytotoxic for a wide range of tumor cells both in vitro and in vivo, and, which is essential for normal immunological development; in 40 animals of 5 diverse Bos indicus Indian zebu cattle breeds. These breeds survive under the harsh and tough tropical climatic conditions of various parts of the Indian subcontinent. The LTA gene in the present study was observed to contain 33 SNPs and 3 small insertion/deletion polymorphisms. Four SNPs occurred in the coding regions of the gene viz. g.1327A>G and g.1400C>T in exon 2 and g.1840C>T and g.1942C>T in exon 3, of which the SNP g.1327A>G in exon 2 resulted in a non-synonymous amino acid change G38D. This amino acid change was however predicted not be affecting the protein function in any manner. The gene contained putative transcription factor binding sites for the c-Re1 and for Pax-4 transcription factors. A putative promoter region was also predicted on the reverse DNA strand from position 894 to 644. Several repeat elements and microsatellite repeats were detected to be occurring across the 3.2kb LTA gene sequence. The study showed the occurrence of 40 genotypes and 48 most probable haplotypes. The genotypes at the observed SNP positions in the LTA gene were in near Hardy-Weinberg equilibrium. A negative Tajima's D value that was not significant statistically at P>0.10 indicated that the neutral mutation hypothesis could not be excluded. The genetic variations observed in the LTA gene in the present study have not been reported earlier and these could possibly be used as molecular markers for further studies involving association of the gene variability with disease resistance/tolerance traits.

Genetic diversity analysis of buffalo fatty acid synthase (FASN) gene and its differential expression among bovines.

Fatty Acid Synthase (FASN) gene seems to be structurally and functionally different in bovines in view of their distinctive fatty acid synthesis process. Structural variation and differential expression of FASN gene is reported in buffalo (Bubalus bubalis), a bovine species close to cattle, in this study. Amino acid sequence and phylogenetic analysis of functionally important thioesterase (TE) domain of FASN revealed its conserved nature across mammals. Amino acid residues at TE domain, responsible for substrate binding and processing, were found to be invariant in all the mammalian species. A total of seven polymorphic nucleotide sites, including two in coding region of TE domain were identified across the 10 buffalo populations of riverine and swamp types. G and C alleles were found almost fixed at g18996 and g19056 loci, respectively in riverine buffaloes. Principal component analysis of three SNPs (g18433, g18996 and g19056) revealed distinct classification of riverine and swamp buffalo populations. Reverse Transcription-PCR amplification of mRNA corresponding to exon 8-10 region of buffalo FASN helped in identification of two transcript variants; one transcript of 565 nucleotides and another alternate transcript of 207 nucleotides, seems to have arisen through alternative splicing. Both the transcripts were found to be expressed in most of the vital tissues of buffalo with the highest expression in mammary gland. Semi-quantitative and real-time expression analysis across 13 different buffalo tissues revealed its highest expression in lactating mammary gland. When compared, expression of FASN was also found to be higher in liver, adipose and skeletal muscle of buffalo tissues, than cattle. However, the FASN expression was highest in adipose among the three tissues in both the species. Results indicate structural and functional distinctiveness of bovine FASN. Presence of alternate splicing in buffalo FASN also seems to be a unique phenomenon to the bovines, probably associated with mRNA based regulation of the biological functions of FASN in these species.