Comprehensive selection signature analyses in dairy cattle exploiting purebred and crossbred genomic data.

The main objective of the current research was to locate, annotate, and highlight specific areas of the bovine genome that are undergoing intense positive selection. Here, we are analyzing selection signatures in crossbred (Bos taurus X Bos indicus), taurine (Bos taurus), and indicine (Bos indicus) cattle breeds. Indicine cattle breeds found throughout India are known for their higher heat tolerance and disease resilience. More breeds and more methods can provide a better understanding of the selection signature. So, we have worked on nine distinct cattle breeds utilizing seven different summary statistics, which is a fairly extensive approach. In this study, we carried out a thorough genome-wide investigation of selection signatures using bovine 50K SNP data. We have included the genotyped data of two taurine, two crossbreds, and five indicine cattle breeds, for a total of 320 animals. During the 1950s, these indicine (cebuine) cattle breeds were exported with the aim of enhancing the resilience of taurine breeds in Western countries. For this study, we employed seven summary statistics, including intra-population, i.e., Tajima's D, CLR, iHS, and ROH and inter-population statistics, i.e., FST, XP-EHH, and Rsb. The NCBI database, PANTHER 17.0, and CattleQTL database were used for annotation after finding the important areas under selection. Some genes, including EPHA6, CTNNA2, NPFFR2, HS6ST3, NPR3, KCNIP4, LIPK, SDCBP, CYP7A1, NSMAF, UBXN2B, UGDH, UBE2K, and DAB1, were shown to be shared by three or more different approaches. Therefore, it gives evidence of the most intense selection in these areas. These genes are mostly linked to milk production and adaptability traits. This study also reveals selection regions that contain genes which are crucial to numerous biological functions, including those associated with milk production, coat color, glucose metabolism, oxidative stress response, immunity and circadian rhythms.

Genome-wide detection of copy number variations in Tharparkar cattle.

Copy number variations (CNVs) are major forms of genetic variation with an increasing importance in animal genomics. This study used the Illumina BovineSNP 50 K BeadChip to detect the genome-wide CNVs in the Tharparkar cattle. With the aid of PennCNV software, we noticed a total of 447 copy number variation regions (CNVRs) across the autosomal genome, occupying nearly 2.17% of the bovine genome. The average size of detected CNVRs was found to be 122.2 kb, the smallest CNVR being 50.02 kb in size, to the largest being 1,232.87 Kb. Enrichment analyses of the genes in these CNVRs gave significant associations with molecular adaptation-related Gene Ontology (GO) terms. Most CNVR genes were significantly enriched for specific biological functions; signaling pathways, sensory responses to stimuli, and various cellular processes. In addition, QTL analysis of CNVRs described them to be linked with economically essential traits in cattle. The findings here provide crucial information for constructing a more comprehensive CNVR map for the indigenous cattle genome.

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.

Uncovering genes underlying coat color variation in indigenous cattle breeds through genome-wide positive selection.

The identification of candidate genes related to pigmentation and under selective sweep provides insights into the genetic basis of pigmentation and the evolutionary forces that have shaped this variation. The selective sweep events in the genes responsible for normal coat color in Indian cattle groups are still unknown. To find coat color genes displaying signs of selective sweeps in the indigenous cattle, we compiled a list of candidate genes previously investigated for their association with coat color and pigmentation. After that, we performed a genome-wide scan of positive selection signatures using the BovineSNP50K Bead Chip in 187 individuals of seven indigenous breeds. We applied a wide range of methods to find evidence of selection, such as Tajima's D, CLR, iHS, varLD, ROH, and FST. We found a total of sixteen genes under selective sweep, that were involved in coat color and pigmentation physiology. These genes are CRIM1 in Gir, MC1R in Sahiwal, MYO5A, PMEL and POMC in Tharparkar, TYRP1, ERBB2, and ASIP in Red Sindhi, MITF, LOC789175, PAX3 and TYR in Ongole, and IRF2, SDR165 and, KIT in Nelore, ADAMTS19 in Hariana. These genes are related to melanin synthesis, the biology of melanocytes and melanosomes, and the migration and survival of melanocytes during development.

Biomarker discovery and authentication of cold-slaughtered chicken through classical analytical procedures and mass spectrometry based proteomic approaches.

1. This study evaluated the suitability of routine analytical procedures and used mass spectrometry-based proteomic approaches to distinguish meat from dead chicken/ cold-slaughtered birds (CS), electrically stunned and slaughtered birds, as per standard protocols (ES), and birds slaughtered according to halal guidelines (HS).2. Meat from CS birds had lower (P < 0.05) pH, water-holding capacity and higher (P < 0.05) lipid oxidation, haem iron content, residual blood and total viable counts relative to ES and HS meat indicating poor quality.3. The results demonstrated the presence of unique protein bands on SDS-PAGE only in CS meat that can be used for routine screening.4. Protein analysis using MALDI-TOF mass spectrometry identified haemoglobin subunit alpha-A and alpha-D; Adenylate kinase isoenzyme 1 as reliable and stable marker proteins for authentication of dead chicken meat under raw and cooked conditions and halal slaughtered chicken, respectively.5. The methods used may be employed by the food safety and regulatory agencies for regular screening of meat quality and to authenticate CS or HS chicken.

Characterisation and comparison of immune response mechanisms in an indigenous and a commercial pig breed after classical swine fever vaccination.

The live attenuated classical swine fever (CSF) vaccine has been successfully used to prevent and control CSF outbreaks for 6 decades. However, the immune response mechanisms against the vaccine remain poorly understood. Moreover, very few reports exist regarding the breed differences in the response to CSF vaccine. In this study, we generated the peripheral blood mononuclear cell transcriptomes of indigenous Ghurrah and commercial Landrace pig breeds, before and 7 days after CSF vaccination. Subsequently, between and within-breed differential gene expression analyses were carried out. Results revealed large differences in pre-vaccination peripheral blood mononuclear cell transcriptome profiles of the two breeds, which were homogenised 7 days after vaccination. Before vaccination, gene set enrichment analysis showed that pathways related to antigen sensing and innate immune response were enriched in Ghurrah, while pathways related to adaptive immunity were enriched in Landrace. Ghurrah exhibited greater immunomodulation compared to Landrace following the vaccination. In Ghurrah, cell-cycle processes and T-cell response pathways were upregulated after vaccination. However, no pathways were upregulated in Landrace after vaccination. Pathways related to inflammation were downregulated in both the breeds after vaccination. Key regulators of inflammation such as IL1A, IL1B, NFKBIA and TNF genes were strongly downregulated in both the breeds after vaccination. Overall, our results have elucidated the mechanisms of host immune response against CSF vaccination in two distinct breeds and revealed common key genes instrumental in the global immune response to the vaccine.

Temporal dysregulation of genes in lamb testis cell during sheeppox virus infection.

The present study was aimed to elucidate the host-virus interactions using RNA-Seq analysis at 1 h and 8 h of post-infection of sheeppox virus (SPPV) in lamb testis cell. The differentially expressed genes (DEGs) and the underlying mechanisms linked to the host immune responses were obtained. The protein-protein interaction (PPI) network analysis and ingenuity pathway analysis (IPA) illustrated the interaction between the DEGs and their involvement in cell signalling responses. Highly connected hubs viz. AURKA, CHEK1, CCNB2, CDC6 and MAPK14 were identified through PPI network analysis. IPA analysis showed that IL-6- and ERK5-mediated signalling pathways were highly enriched at both time points. The TP53 gene was identified to be the leading upstream regulator that directly responded to SPPV infection, resulting in downregulation at both time points. The study provides an overview of how the lamb testis genes and their underlying mechanisms link to growth and immune response during SPPV infection.

Genome-wide assessment of genetic diversity, linkage disequilibrium and haplotype block structure in Tharparkar cattle breed of India.

Knowledge about genetic diversity is very essential for the management and sustainable utilization of livestock genetic resources. In this study, we presented a comprehensive genome-wide analysis of genetic diversity, ROH, inbreeding, linkage disequilibrium, effective population size and haplotype block structure in Tharparkar cattle of India. A total of 24 Tharparkar animals used in this study were genotyped with Illumina BovineSNP50 array. After quality control, 22,825 biallelic SNPs were retained, which were in HWE, MAF > 0.05 and genotyping rate >90%. The overall mean observed (HO) and expected heterozygosity (HE) were 0.339 ± 0.156 and 0.325 ± 0.129, respectively. The average minor allele frequency was 0.234 with a standard deviation of ± 0.131. We identified a total of 1832 ROH segments and the highest autosomal coverage of 13.87% was observed on chromosome 23. The genomic inbreeding coefficients estimates by FROH, FHOM, FGRM and FUNI were 0.0589, 0.0215, 0.0532 and 0.0160 respectively. The overall mean linkage disequilibrium (LD) for a total of 133,532 pairwise SNPs measured by D' and r2 was 0.6452 and 0.1339, respectively. In addition, we observed a gradual decline in effective population size over the past generations.

Genomic scans for selection signatures revealed candidate genes for adaptation and production traits in a variety of cattle breeds.

Domestication and selection are the major driving forces responsible for the determinative genetic variability in livestock. These selection patterns create unique genetic signatures within the genome. BovineSNP50 chip data from 236 animals (seven indicine and five taurine cattle breeds) were analyzed in the present study. We implemented three complementary approaches viz. iHS (Integrated haplotype score), ROH (Runs of homozygosity), and FST, to detect selection signatures. A total of 179, 56, and 231 regions revealed 518, 277, and 267 candidate genes identified by iHS, ROH, and FST methods, respectively. We found several candidate genes (e.g., NCR3, ARID5A, HIST1H2BN, DEFB4, DEFB7, HSPA1L, HSPA1B, and DNAJB4) related to production traits and the adaptation of indigenous breeds to local environmental constraints such as heat stress and disease susceptibility. However, further studies are warranted to refine the findings using a larger sample size, whole-genome sequencing, and/or high density genotyping.

Comparative analysis of five different methods to design a breed-specific SNP panel for cattle.

Single nucleotide polymorphisms (SNPs) have now replaced microsatellite markers in several species for various genetic investigations like parentage assignment, genetic breed composition, assessment for individuality and, most popularly, as a useful tool in genomic selection. However, such a resource, which can offer to assist breed identification in a cost-effective manner is still not explored in cattle breeding programs. In our study, we have tried to describe methods for reducing the number of SNPs to develop a breed-specific panel. We have used SNP data from Dryad open public access repository. Starting from a global dataset of 178 animals belonging to 10 different breeds, we selected five panels each comprising of similar number of SNPs using different methods i.e., Delta, Pairwise Wright's FST, informativeness for assignment, frequent item feature selection (FIFS) and minor allele frequency-linkage disequilibrium (MAF-LD) based method. MAF-LD based method has been recently developed by us for construction of breed-specific SNP panels. The STRUCTURE software analysis of MAF-LD based method showed appropriate clustering in comparison to other panels. Later, the panel of 591 breed-specific SNPs was called to their respective breeds using Venny 2.1.0 and UGent web tools software. Breed-specific SNPs were later annotated by using various Bioinformatics softwares.

Revelation of genomic breed composition in a crossbred cattle of India with the help of Bovine50K BeadChip.

The aim of the present study was to assess the population structure and admixture levels in the Vrindavani composite population in India by using Bovine50KSNP BeadChip data. Genotypic data were generated for randomly selected animals (n = 72) of Vrindavani population and the data for parental breeds i.e., Hariana (n = 10), Holstein-Friesian (n = 63), Jersey (n = 28) and Brown Swiss (n = 22) were retrieved from a public repository. The indices of population structure were calculated using PLINK software and R-program. The merged dataset was analysed for assessing admixture levels and population stratification using three different approaches i.e., principal component analysis (PCA), multi-dimensional scaling (MDS) approach and the model-based approach in STRUCTURE software. The average minor allele frequency (MAF) value for Vrindavani population was estimated to be 0.235. Vrindavani population was found to possess an average ancestry of 39.5, 22.9, 26.9, and 10.7% inheritance levels from Holstein Friesian, Jersey, Hariana and Brown Swiss cattle breeds, respectively.

Ancestry informative markers derived from discriminant analysis of principal components provide important insights into the composition of crossbred cattle.

The cost of SNP genotyping to screen different breeds and to estimate the exact proportion of ancestry level is quite high, which can be compensated through deriving a small panel of ancestry informative markers (AIMs). Hence, we carried out the present study to provide an insight into ancestry level inferred from a panel of informative markers in the crossbred Vrindavani population developed at ICAR-IVRI, India. We have performed a new method i.e., discriminant analysis of principal components (DAPC) for the first time on the dataset of Vrindavani cattle. To confirm our method, we had performed DAPC on two other well-known crossbred cattle, i.e., Frieswal and Beefmaster. Three sets of panels (500, 1000 and 2000 markers) were tested for clustering of individuals. Among all the panels, we found the panel (1000 markers) with DAPC based contribution method was of the smallest size and comparatively of the highest accuracy.

Identification of genes affecting milk fat and fatty acid composition in Vrindavani crossbred cattle using 50 K SNP-Chip.

The aim of this study was to identify candidate genes associated with milk fat per cent and fatty acid (FA) composition in Vrindavani cattle using the Illumina 50 K single-nucleotide polymorphism (SNP) array. After quality control, a total of 41,427 informative and high-quality SNPs were used for a genome-wide association study (GWAS) for milk fat percentage and 16 different types of fatty acids. Lactation stage, parity, test day milk yield, and proportion of exotic inheritance were included as fixed effects in the GWAS model. A total of 67 genome-wide significant (P < 1.20 × 10-06) SNPs and 176 suggestive significant (P < 2.41 × 10-05) SNPs were identified. Out of these, 15 SNPs were associated with more than one trait. The strongest associations were found on BTA14 for milk fat percentage and on BTA2 and BTA16 for polyunsaturated fatty acids. Several significant SNPs were identified close to or within the genes ELOVL6, FABP4, PMP2, PLIN1, MFGE8, GHRL2, and LDLRAD3 which are known to be associated with fat percentage and FA composition in dairy cattle breeds. This study is a step forward to better characterize the molecular mechanisms of phenotypic variation in milk fatty acids in a taurine-indicine composite cattle breed reared in tropical environments.

Selection and validation of differentially expressed metabolic and immune genes in weaned Ghurrah versus crossbred piglets.

In the present investigation, differentially expressed genes (DEGs) were studied using RNA sequencing (RNA-seq) technique in porcine peripheral blood mononuclear cells (PBMC) of weaned Ghurrah and crossbred piglets at 3-month age. Transcriptomic analysis was done using three different packages, namely, EBSeq, DESeq2, and edgeR, to identify the DEGs between Ghurrah and crossbred piglets. Total 7717 DEGs were commonly identified by all three packages, out of which 4151 genes found to be up-regulated, and 3566 genes were down-regulated. Functional annotation of these DEGs indicated metabolism as the most commonly enriched category followed by the immune response. Genes related to metabolism and growth were up-regulated in crossbred piglets as compared with Ghurrah piglets, whereas immunity-related genes were up-regulated in Ghurrah piglets elucidating the disease resistance nature of this indigenous breed over crossbred counterparts. Further, eight DEGs, namely, LRP-1, ADCY4, ERRFI1, LDHD, ARG1, OASL, MGARP, and S100A8, were validated by qRT-PCR in a separate set of biological samples and found to be in concordance with RNA-seq results. Finding in the present study provides insight into genes and their molecular mechanisms governing difference in growth performance between Ghurrah and crossbred pigs.

Genome-wide integrated analysis of miRNA and mRNA expression profiles to identify differentially expressed miR-22-5p and miR-27b-5p in response to classical swine fever vaccine virus.

The present study was conducted to identify the differentially expressed miRNAs (DE miRNAs) in the peripheral blood mononuclear cells of crossbred pigs in response to CSF vaccination on 7 and 21 days of post vaccination as compared to unvaccinated control (0 dpv). Simultaneously, set of miRNA was predicted using mRNA seq data at same time point. The proportion of CD4-CD8+ and CD4+CD8+ increased after vaccination, and the mean percentage inhibition was 86.89% at 21 dpv. It was observed that 22 miRNAs were commonly expressed on both the time points. Out of predicted DE miRNAs, it was found that 40 and 35 DE miRNAs were common, obtained from miRNA seq analysis and predicted using mRNA seq data on 7 dpv versus 0 dpv and 21 dpv versus 0 dpv respectively. Two DE miRNAs, ssc-miR-22-5p and ssc-miR-27b-5p, were selected based on their log2 fold change and functions of their target genes in immune process/pathway of viral infections. The validations of DE miRNAs using qRT-PCR were in concordance with miRNA seq analysis. Two set of target genes, CD40 and SWAP70 (target gene of ssc-miR-22-5p) and TLR4 and Lyn (target gene of ssc-miR-27b-5p), were validated and were in concordance with results of RNA seq analysis at a particular time point (except TLR4). The first report of genome-wide identification of differentially expressed miRNA in response to live attenuated vaccine virus of classical swine fever revealed miR-22-5p and miR-27b-5p were differentially expressed at 7 dpv and 21 dpv.

A double-stranded probe coupled with isothermal amplification for qualitative and quantitative detection of avian reovirus.

We applied a probe-based real-time loop-mediated isothermal amplification (Cy5-RTqLAMP) technique targeting the avian reovirus (ARV) S3 gene to develop a rapid, sensitive, and specific method for virus detection and quantification. This test specifically detected the presence of ARV, but not other viruses or bacteria present in clinical or artificially spiked samples, including Newcastle disease virus, infectious bursal disease virus, fowl adenovirus, Marek's disease virus, Escherichia coli, and Salmonella spp. This test can detect ARV in less than one hour with an analytical sensitivity of 10 viral gene copies and 1 fg of total cDNA. The Cy5-RTqLAMP does not yield false positive results and is 100 times more sensitive than conventional PCR. This test was shown to be able to detect the presence of ARV in clinical samples. A similar strategy may be used for detection of other important human and animal viral pathogens.

EvaGreen-based Multiplex Real-time PCR Assay for Rapid Differentiation of Wild-Type and Glycoprotein E-Deleted Bovine Herpesvirus-1 Strains.

Bovine herpesvirus-1 (BoHV-1) is an important viral pathogen causing significant economic losses to the cattle industry. Glycoprotein E-deleted marker vaccines form the basis for BoHV-1 control programs widely, wherein detection and differentiation of wild-type and gE-deleted vaccine strains is of crucial importance for proper disease management. In the present study, we report an EvaGreen-based multiplex real-time polymerase chain reaction (EGRT-PCR) assay for rapid differentiation of wild-type and glycoprotein E-deleted strains of BoHV-1. The EGRT-PCR assay could simultaneously detect two viral genes (glycoprotein B and E) and an internal positive control gene (bovine growth hormone- bGH), in a single-tube reaction. The analytical sensitivity of the EGRT-PCR assay was as little as 10 copies of the BoHV-1 DNA per reaction. The modified real-time PCR assay could successfully differentiate wild-type and gE-deleted BoHV-1 strains based on gene specific melting temperatures (Tm) peaks. Our results have shown that the EGRT-PCR developed in this study might prove to be a promising tool in disease management by enabling rapid differentiation of wild-type and gE-deleted strains of BoHV-1.

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.

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.

Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay.

Several pathogens including Brucella spp. are shed in semen of infected bulls and can be transmitted to cows through contaminated semen during artificial insemination. The present study reports omp2a and bcsp31 gene based loop-mediated isothermal amplification (LAMP) assays for detection of Brucella genomic DNA in semen from infected bulls. The positive results could be interpreted visually by change in colour of reaction mixture containing hydroxyl naphthol blue (HNB) dye from violet to sky blue. LAMP assays based on omp2a and bcsp31 could detect as little as 10 and 100 fg of B. abortus S19 genomic DNA, respectively. Sensitivity of omp2a and bcsp31 LAMP assays for direct detection of organisms in bovine semen was 2.28 × 10(1) CFU and 2.28 × 10(2) CFU of B. abortus S19 in spiked bovine semen, respectively. The omp2a LAMP assay was found equally sensitive to TaqMan probe based real-time PCR and 100 times more sensitive than conventional PCR in identifying Brucella in spiked semen. The diagnostic applicability of the omp2a LAMP assay was evaluated with seventy-nine bovine semen samples and results were re-evaluated through TaqMan probe based real-time PCR and conventional PCR. Taken together, the omp2a LAMP assay is easy to perform, rapid and sensitive in diagnosis of Brucella spp. in bovine semen.