Diversity assessment of a lesser known buffalo population from Central India and its comparative evaluation reveals presence of sufficient genetic variation and absence of selection.

Planned breeding and conservation strategies for a lesser-known population require an assessment of complete genetic diversity and population structure analysis in addition to its morphometric characteristics. In the present study, a comparative analysis of the genetic structure of a rare buffalo population, namely Chhattisgarhi, was extensively studied using a panel of FAO-recommended microsatellite markers along with well-established breeds namely Murrah, Nili-Ravi, Gojri, Kalahandi, and Nagpuri. Mode shift analysis indicated the absence of genetic bottleneck in the recent past. Assessment of genetic diversity indices across all loci indicated the presence of sufficient genetic variation within and between populations. Analysis of molecular variance between the six different buffalo populations attributed 19.05% of the variations to between-population differentiation. Cluster analyses using DAPC and Bayesian approach along with the phylogenetic tree based on UPGMA grouped six populations into three groups. The Chhattisgarhi population was revealed to be genetically closer to Nagpuri and Kalahandi populations. The study reveals the presence of sufficient genetic diversity within the Chhattisgarhi population and indicates the absence of a systematic selection program. We suggest improvement and conservation programs should be planned for this breed in the near future through short-term selection.

Discriminant analysis reveals differences in breed structure in river buffaloes of Maharashtra (India).

Buffaloes are a major milch species of India, and owning a true-to-breed buffalo herd is a reverie for livestock keepers, as it substantiates their incomes. Multivariate discriminate analysis is an efficient methodology to classify different farm animal populations based on canonical discriminant function. The present study was conducted on 392 adult buffaloes using 18 body biometric traits. Except for body height and body length, all the body characters revealed significant differences amongst the four buffalo populations. Unlike cattle, distance between horns and rump width were more discriminating factors, compared to body height, leg length, chest girth, and body length. We report the most suitable discriminate function which can discriminate with maximum between-group variation in four studied populations. Mahalanobis distances were calculated between populations and they ranged from 0.786 to 1.227. Purnathadi has revealed a distinct phenotypic structure, and these buffaloes have a distinct native breeding region comprising Akola, Amravati, and Buldhana districts of Maharashtra state. On the other hand, Elachpuri animals failed to depict physical and biometric discriminations from Nagpuri, Marathwadi, and Purnathadi buffalo populations, and Elachpuri can be considered as a strain of Nagpuri buffalo.

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.

Expression profile of different classes of proteases in milk derived somatic cells across different lactation stages of indigenous cows (Bos indicus) and riverine buffaloes (Bubalus bubalis).

Proteases play a significant role in milk and its products by affecting flavor, texture and longevity. The expression of endogenous proteases varies across different stages of lactation. The study was conducted to understand the transcriptional pattern of different classes of protease-pathways associated genes (CTSB, CTSD, CTSH, CTSL, CTSK, CTSS, CTSZ, PLAU, PLAT) and potential protease inhibitors (SERPIN E2 and SERPIN F2) in 40 milk somatic cells (MSC) samples isolated during early, peak, mid and late lactation stages of Sahiwal cows and Murrah buffaloes - the two most important dairy breeds of India. In Sahiwal cows, except CTSK and PLAU, the expression of other proteases class was not affected significantly (p > 0.05) across lactation stages. However, in Murrah buffaloes, the expression of different proteases increased as the lactation progressed. Most of the proteases showed lower expression during early and peak lactation stages while their expression tends to increase during mid to late lactation stages. The overall trend was somewhat similar in both the dairy species albeit the level of expression was higher in buffalo MSC as compared to cow MSC. The study has provided valuable information on expression kinetics of different proteases in milk somatic cells of two major dairy breeds of India.

Identification of novel polymorphism in mammary-derived growth inhibitor gene of water buffalo and its expression analysis in the mammary gland.

Mammary-derived growth inhibitor (MDGI), a member of the lipophilic family of fatty acid-binding proteins, plays an important role in the development, regulation, and differentiation of the mammary gland. The aim of the study was to identify polymorphism in the MDGI gene and its expression analysis in the mammary gland at various stages of lactation, in Indian buffalo. Nucleotide sequence analysis of MDGI gene in different breeds of riverine and swamp buffaloes revealed a total of 16 polymorphic sites and one Indel. Different transcription factor binding sites were predicted for buffalo MDGI gene promoter sequence, using online tools and in-silico analysis indicating that the SNPs in this region can impact the gene expression regulation. Phylogenetic analysis exhibited the MDGI of buffalo being closer to other ruminants like cattle, yak, sheep, and goats. Further, the expression analysis revealed that buffalo MDGI being highly expressed in well-developed mammary glands of lactating buffalo as compared to involution/non-lactating and before functional development to start the milk production stage in heifers. Stage-specific variation in expression levels signifies the important functional role of the MDGI gene in mammary gland development and milk production in buffalo, an important dairy species in Southeast Asia.

Genetic characterization and diversity assessment in 'Bhangor' indigenous swamp buffalo population using heterologous microsatellite markers.

'Bhangor' newly identified swamp buffalo population from North East Indian, was characterized using microsatellite markers. Genomic DNA was isolated from blood samples of 76 unrelated animals, 15 microsatellite markers (CSSM33, BM1818, CSRM60, HEL13, ILSTS019, ILSTS025, ILSTS028, ILSTS029, ILSTS033, ILSTS036, ILSTS056, ILSTS058, ILSTS061, ILSTS089 and ETH003) were found to be highly polymorphic in the population of the selected markers. A total of 114 alleles were observed, which ranged from 3 in CSRM60 and ILSTS025 locus to 12 in ILSTS056 and ILSTS061. The mean effective number of alleles across all polymorphic loci was found to be 3.76. The overall mean expected heterozygosity and unbiased expected heterozygosity values were 0.67 and 0.68, ranging from 0.067 (ILSTS025) to 0.85 (ILSTS058) and 0.068 (ILSTS025) to 0.86 (ILSTS058), respectively. Within the population, the inbreeding estimates (FIS) ranged between -0.4352 and 0.804, with an average FIS of 0.114 ± 0.033. The outcome for infinite allele model (IAM), two-phase model (TPM) and test for mode shift revealed the absence of any recent bottleneck in the investigated buffalo population. The population was found to be in optimum diversity based on polymorphic microsatellite markers. With fast changing agro-climatic conditions; there is an urgent need to characterize the nondescript livestock populations.

Demographic pattern of A1/A2 beta casein variants indicates conservation of A2 type haplotype across native cattle breeds (Bos indicus) of India.

Genetic variations of the beta casein gene hold importance because of their probable association with human health. Comparative sequence analysis of ß-casein gene across Indian native, crossbred and exotic breeds in India revealed 15 SNPs and 4 INDELs corresponding to 14 haplotypes. The frequency of A2 type haplotype was maximum (0.941) across all Indian native breeds. Among the 15 variants reported for taurine breeds, only three (A1, A2 and B) were observed in analysed populations. Allelic profiling of A1/A2 ß-casein variants in ~ 4000 animals belonging to three cattle types and breeding bulls also revealed the predominance of A2 allele (0.95) in Indian cattle. The high proportion of A2 allele/haplotype indicates that Indian native cattle are the best suited to meet the demands for A2 milk globally. However, a higher percentage of heterozygous genotype (A1A2) in breeding bulls warrants the need to screen sire lines so as to drift the herd towards A2. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03232-0.

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.

Assessment of genetic diversity and bottleneck in Purnathadi buffaloes using short tandem repeat markers.

This study is the maiden attempt for genetic characterization of the Purnathadi, a phenotypically distinct buffalo population of the western Vidarbha region of Maharashtra and to explore genetic diversity using STR markers. A total of 48 unrelated Purnathadi buffaloes from the entire native tract were genotyped using a battery of 25 heterologous microsatellite markers. 5' end of forward primer of each microsatellite marker was labeled with one of the fluorescent dyes, viz., FAM (Blue), VIC (Green), NED (Yellow) or PET (Red) to assess the fragment length of genotyped PCR product with automated DNA sequencer (ABI 3100). 23 microsatellite loci (except ETH003 and ILSTS030) amplified successfully and adequately high allelic diversity (observed: 0.615 ± 0.043 and expected: 0.655 ± 0.037) was reported with 162 distinct microsatellite alleles. Sufficiently high Shannon index and PIC indicated the suitability of markers to evaluate genetic diversity in Purnathadi buffaloes. Within-population inbreeding estimates (FIS) for Purnathadi buffalo ranged between -0.171 and 0.495 with global FIS average of 5.9%. The outcome for IAM, TPM and test for mode shift revealed the absence of any recent bottleneck in Purnathadi buffalo. Pairwise FST (genetic differentiation) and gene flow between Purnathadi, Nagpuri and Marathwadi buffaloes were estimated using genotype data of 19 microsatellite markers. Lowest FST (0.031) was observed between Nagpuri and Purnathadi buffaloes with highest gene flow of 7.91% and highest FST (0.094) was between Purnathadi and Marathwadi populations. Present findings will definitely support in designing breeding plan for genetic improvement, as well as for developing conservation strategies of Purnathadi buffalo population. The comparative molecular study with other breeds of the regions is needed.

In Silico Analysis of HSP70 Gene Family in Bovine Genome.

Heat shock proteins (HSPs), members of molecular chaperones families fulfill essential roles under normal conditions and provide protection and adaptation during and after stress. Among different HSPs, HSP70 kDa family of proteins is most abundant and well-studied in human and mouse but has not yet been characterized in bovines. In silico analysis was performed to characterize members of HSP70 gene family in bovine genome and a total of 17 genes of bovine HSP70 gene family were identified. The members of HSP70 family were distributed over 12 chromosomes with gene size ranging from 1911 (HSPA2) to 54,017 bp (HSPA4). Five genes were intronless, while rest of 12 genes were multiexonic. Phylogenetic analysis of HSP70 gene family distinguished them into eight major evolutionary groups wherein members of group 1 were most divergent and quite dissimilar than from rest of the HSP70 sequences. Domain structure of all bovine HSP70 genes was conserved and three signature patterns HSP70_1, HSP70_2, and HSP70_3 were identified. HSPA8, HSP9, and HSPA1A showed comparatively higher expression in majority of tissues. Like humans, bovine HSP70 family was characterized by remarkable evolutionary diversity. The analysis also suggested resemblance of bovine HSP70 family to that of human compared to mouse. Overall, the study indicates the presence of diversity for structure, function, localization, and expression in the bovine HSP70 family chaperons which could form the basis to understand thermotolerance/adaptive changes in the bovines.

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 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.

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.

Milk-derived mammary epithelial cells as non-invasive source to define stage-specific abundance of milk protein and fat synthesis transcripts in native Sahiwal cows and Murrah buffaloes.

The molecular physiology of milk production of two important dairy species; Sahiwal cows (Bos indicus) and Murrah buffaloes (Bubalus bubalis) are not fully understood due to constraints in obtaining mammary tissue samples because of sacred and ethical reasons. The present study suggests the use of milk-derived mammary epithelial cells (MECs) as a non-invasive method to understand molecular aspects of lactation biology in dairy animals. A total of 76 MECs were collected from five different lactation periods viz. colostrum (0-2), early (5-20), peak (30-50), mid (90-140) and late lactation (> 215 days) stages from Sahiwal cows and Murrah buffaloes to study the transcription kinetics of milk protein, fat synthesis, and their regulatory genes. Significant changes were observed in milk composition of both dairy species with lactation stages. High mRNA abundance of all milk protein and fat synthesis genes was observed in MECs of Murrah buffaloes as compared to Sahiwal cows. The mRNA abundance of caseins (CSN1S1, CSN1S2, CSN2, and CSN3) and whey protein (LALBA, LF) were higher in early lactation stage. Similarly, the expression of milk fat synthesis genes (SCD, BTN1A1, ACACA, GPAM, FAPB3, FASN) was also high in early lactation stage. The relative abundance of 4 regulatory genes (JAK2, STAT5, SREBF1 and EIF4BP41) remained high during early lactation indicating their regulatory roles in lactogenesis process. Overall, results suggested a significant effect of lactation stages on milk composition and transcription abundance of milk protein and fat synthesis genes. The present study establishes the fact that milk-derived MECs could be utilized as a valuable source to understand mammary gland functioning of native cows and buffaloes.

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.

Comparative modeling and mutual docking of structurally uncharacterized heat shock protein 70 and heat shock factor-1 proteins in water buffalo.

AIM: In this study, a wide range of in silico investigation of Bubalus bubalis (BB) heat shock protein 70 (HSP70) and heat shock factor-1 (HSF1) has been performed, ranging from sequence evaluation among species to homology modeling along with their docking studies to decipher the interacting residues of both molecules. MATERIALS AND METHODS: Protein sequences of BB HSP70 and HSF1 were retrieved from NCBI database in FASTA format. Primary and secondary structure prediction were computed using Expasy ProtParam server and Phyre2 server, respectively. TMHMM server was used to identify the transmembrane regions in HSP70. Multiple sequence alignment and comparative analysis of the protein was carried out using MAFFT and visualization was created using ESPript 3.0. Phylogenetic analysis was accomplished by COBALT. Interactions of HSP70 with other proteins were studied using STRING database. Modeller 9.18, RaptorX, Swiss-Modeller, Phyre2, and I-TASSER were utilized to design the three-dimensional structure of these proteins followed by refinement; energy minimization was accomplished using ModRefiner and SPDBV program. Stereochemical quality along with the accuracy of the predicted models and their visualization was observed by PROCHECK program of PDBsum and UCSF Chimera, respectively. ClusPro 2.0 server was accessed for the docking of the receptor protein with the ligand. RESULTS: The lower value of Grand Average of Hydropathy indicates the more hydrophilic nature of HSP70 protein. Value of the instability index (II) classified the protein as stable. No transmembrane region was reported for HSP70 by TMHMM server. Phylogenetic analysis based on multiple sequence alignments (MSAs) by COBALT indicated more evolutionarily closeness of Bos indicus (BI) with Bos taurus as compared to BI and BB. STRING database clearly indicates the HSF1 as one of the interacting molecules among 10 interacting partners with HSP 70. The best hit of 3D model of HSP70 protein and HSF1 was retrieved from I-TASSER and Phyre2, respectively. Interacting residues and type of bonding between both the molecules which were docked by ClusPro 2.0 were decoded by PIC server. Hydrophobic interactions, protein-protein main-chain-side-chain hydrogen bonds, and protein-protein side-chain-side-chain hydrogen bonds were delineated in this study. CONCLUSION: This is the first-ever study on in silico interaction of HSP70 and HSF1 proteins in BB. Several bioinformatics web tools were utilized to study secondary structure along with comparative modeling, physicochemical properties, and protein-protein interaction. The various interacting amino acid residues of both proteins have been indicated in this study.

PCR-SSCP analysis of MDGI gene and its association with milk production traits in river buffalo (Bubalus bubalis).

In this study, we investigated the genetic variation within 3'UTR of Mammary-Derived Growth Inhibitor (MDGI) gene of buffalo using PCR-SSCP and sequencing; and also analyzed association of polymorphism with the milk production traits. The study revealed two conformational patterns, 'A' and 'B' among 234 Mehsana buffaloes maintained with their records in the field and at farm. The frequency of SSCP variant 'A' was found to be invariably high in the buffalo population under study. Further, association analysis of SSCP variants with various milk production and milk quality traits indicated no significant effect on any of the traits investigated. Sequencing of SSCP variant 'A' showed homozygous G/G and A/A and 'B' had heterozygous G/C and A/G at positions +124 and +140 respectively, in the 3'UTR of buffalo MDGI. The preliminary results showed the substantial variations in the distribution of SSCP variants' frequencies within Mehsana buffaloes, however these variants had non-significant association with milk yield, fat yield and fat percentage in Mehsana buffaloes.

Expression Analysis of Genes Associated with Prolificacy in FecB Carrier and Noncarrier Indian Sheep.

The effect of FecB mutation on the gene expression in FecB carrier and noncarrier estrous synchronized ewes, has been analyzed. For this study the whole ovarian tissues and Graafian follicles were collected from estrus synchronized FecB carrier Garole, and non-carrier Deccani Indian sheep, showing remarkable differences in the numbers of preovulatory follicles among two groups. Eleven potential candidate genes (BMP15, GDF9, BMP4, BMP7, BMPR1B, BMPR1A, SMAD9, LHCGR, FSHR, IGF1R, and STAT5) were selected for their expression analysis by SybrGreen based real-time PCR, across ovaries and Graafian follicles of different fecundity groups, for having better insights into the effect of FecB genotypes on follicular development. Variable expression was observed for almost all the genes included in the present study among high and low fecundity groups that was most significant for the BMP7, BMP4, LHCGR, and FSHR transcripts in the ovarian follicles of high and low fecundity ewes, indicating their importance in governing the fecundity in FecB carrier, Indian Garole sheep. BMP4 expression among the genes studied was significantly higher in FecB carrier Garole sheep. This study confirms the changes in mRNA expression of the genes implicated in follicular development in FecB carrier and noncarrier Indian sheep breeds.

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.