Search

Nitrogen (N) use efficiency (NUE) is an economically important trait for dairy cows. Recently, we proposed a new N efficiency index (NEI), that simultaneously considers both NUE and N pollution. This study aimed to validate the genomic prediction for NEI and its composition traits and investigate the relationship between SNP effects estimated directly from NEI and indirectly from its composition traits. The NEI composition included genomic estimated breeding value of N intake (NINT), milk true protein N (MTPN) and milk urea N yield. The edited data were 132,899 records on 52,064 cows distributed in 773 herds. The pedigree contained 122,368 animals. Genotypic data of 566,294 SNP was available for 4514 individuals. A total of 4413 cows (including 181 genotyped) and 56 bulls (including 32 genotyped) were selected as the validation populations. The linear regression method was used to validate the genomic prediction of NEI and its composition traits using best linear unbiased prediction (BLUP) and single‐step genomic BLUP (ssGBLUP). The mean theoretical accuracies of validation populations obtained from ssGBLUP were higher than those obtained from BLUP for both NEI and its composition traits, ranging from 0.57 (MTPN) to 0.72 (NINT). The highest mean prediction accuracies for NEI and its composition traits were observed for the genotyped cows estimated under ssGBLUP, ranging from 0.48 (MTPN) to 0.66 (NINT). Furthermore, the SNP effects estimated from NEI composition traits, multiplied by the relative weight were the same as those estimated directly from NEI. This study preliminary showed that genomic prediction can be used for NEI, however, we acknowledge the need for further validation of this result in a larger dataset. Moreover, the SNP effects of NEI can be indirectly calculated using the SNP effects estimated from its composition traits. This study provided a basis for adding genomic information to establish NEI as part of future routine genomic evaluation programs. [ABSTRACT FROM AUTHOR]

The functionality of circulating leukocytes in dairy cows is suppressed after calving, with negative energy balance as a risk factor. Leukocyte transcriptomic profiles were compared separately in 44 multiparous (MP) and 18 primiparous (PP) Holstein–Friesian cows receiving diets differing in concentrate proportion to test whether immune dysfunction could be mitigated by appropriate nutrition. After calving, cows were offered either (1) low concentrate (LC); (2) medium concentrate (MC) or (3) high concentrate (HC) diets with proportions of concentrate to grass silage of 30%:70%, 50%:50% and 70%:30%, respectively. Cow phenotype data collected included circulating metabolites, milk yield and health and fertility records. RNA sequencing of circulating leukocytes at 14 days in milk was performed. The HC diet improved energy balance in both age groups. There were more differentially expressed genes in PP than MP cows (460 vs. 173, HC vs. LC comparison) with few overlaps. The MP cows on the LC diet showed upregulation of the complement and coagulation cascade and innate immune defence mechanisms against pathogens and had a trend of more cases of mastitis and poorer fertility. In contrast, the PP cows on the HC diet showed greater immune responses based on both gene expression and phenotypic data and longer interval of calving to conception. The leukocytes of MP and PP cows therefore responded differentially to the diets between age, nutrient supply and immunity affecting their health and subsequent fertility. [ABSTRACT FROM AUTHOR]

New Mendelian genetic conditions, which adversely affect livestock, arise all the time. To manage them effectively, some methods need to be devised that are quick and accurate. Until recently, finding the causal genomic site of a new autosomal recessive genetic disease has required a two-stage approach using single-nucleotide polymorphism (SNP) chip genotyping to locate the region containing the new variant. This region is then explored using fine-mapping methods to locate the actual site of the new variant. This study explores bioinformatic methods that can be used to identify the causative variants of recessive genetic disorders with full penetrance with just nine whole genome-sequenced animals to simplify and expedite the process to a one-step procedure. Using whole genome sequencing of only three cases and six carriers, the site of a novel variant causing perinatal mortality in Irish moiled calves was located. Four methods were used to interrogate the variant call format (VCF) data file of these nine animals, they are genotype criteria (GCR), autozygosity-by-difference (ABD), variant prediction scoring, and registered SNP information. From more than nine million variants in the VCF file, only one site was identified by all four methods (Chr4: g.77173487A>T (ARS-UCD1.2 (GCF_002263795.1)). This site was a splice acceptor variant located in the glucokinase gene (GCK). It was verified on an independent sample of animals from the breed using genotyping by polymerase chain reaction at the candidate site and autozygosity-by-difference using SNP-chips. Both methods confirmed the candidate site. Investigation of the GCR method found that sites meeting the GCR were not evenly spread across the genome but concentrated in regions of long runs of homozygosity. Locating GCR sites was best performed using two carriers to every case, and the carriers should be distantly related to the cases, within the breed concerned. Fewer than 20 animals need to be sequenced when using the GCR and ABD methods together. The genomic site of novel autosomal recessive Mendelian genetic diseases can be located using fewer than 20 animals combined with two bioinformatic methods, autozygosity-by-difference, and genotype criteria. In many instances it may also be confirmed with variant prediction scoring. This should speed-up and simplify the management of new genetic diseases to a single-step process. [ABSTRACT FROM AUTHOR]

Simple Summary: Escherichia coli is a bacterium which infects cow udders causing clinical mastitis, a potentially severe disease with welfare and economic consequences. During an infection, white blood cells (leukocytes) enter the udder to provide immune defence and assist tissue repair. We sequenced RNA derived from circulating leukocytes to investigate which genes are up- or down-regulated in dairy cows with naturally occurring cases of clinical mastitis in comparison with healthy control cows from the same farm. We also looked for genetic variations between infected and healthy cows. Blood samples were taken either EARLY (around 10 days) or LATE (after 4 weeks) during the recovery phase after diagnosis. Many genes (1090) with immune and inflammatory functions were up-regulated during the EARLY phase. By the LATE phase only 29 genes were up-regulated including six haemoglobin subunits, possibly important for the production of new red blood corpuscles. Twelve genetic variations which were associated with an increased or decreased expression of some important immune genes were identified between the infected and control cows. These results show that the initial inflammatory response to E. coli continued for at least 10 days despite the cows having received prompt veterinary treatment, but they had largely recovered within 4 weeks. Genetic differences between cows may predispose some animals to infection. The risk and severity of clinical infection with Escherichia coli as a causative pathogen for bovine mastitis is influenced by the hosts' phenotypic and genotypic variables. We used RNA-Seq analysis of circulating leukocytes to investigate global transcriptomic profiles and genetic variants from Holstein cows with naturally occurring cases of clinical mastitis, diagnosed using clinical symptoms and milk microbiology. Healthy lactation-matched cows served as controls (CONT, n = 6). Blood samples were collected at two time periods during the recovery phase post diagnosis: EARLY (10.3 ± 1.8 days, n = 6) and LATE (46.7 ± 11 days, n = 3). Differentially expressed genes (DEGs) between the groups were identified using CLC Genomics Workbench V21 and subjected to enrichment analysis. Variant calling was performed following GATKv3.8 best practice. The comparison of E. coli(+) EARLY and CONT cows found the up-regulation of 1090 DEGs, mainly with immune and inflammatory functions. The key signalling pathways involved NOD-like and interleukin-1 receptors and chemokines. Many up-regulated DEGs encoded antimicrobial peptides including cathelicidins, beta-defensins, S100 calcium binding proteins, haptoglobin and lactoferrin. Inflammation had largely resolved in the E. coli(+) LATE group, with only 29 up-regulated DEGs. Both EARLY and LATE cows had up-regulated DEGs encoding ATP binding cassette (ABC) transporters and haemoglobin subunits were also up-regulated in LATE cows. Twelve candidate genetic variants were identified in DEGs between the infected and CONT cows. Three were in contiguous genes WIPI1, ARSG and SLC16A6 on BTA19. Two others (RAC2 and ARHGAP26) encode a Rho-family GTPase and Rho GTPase-activating protein 26. These results show that the initial inflammatory response to E. coli continued for at least 10 days despite prompt treatment and provide preliminary evidence for genetic differences between cows that may predispose them to infection. [ABSTRACT FROM AUTHOR]

Intrauterine growth restriction (IUGR) is a leading cause of neonatal morbidity and mortality in humans and domestic animals. Developmental adaptations of skeletal muscle in IUGR lead to increased risk of premature muscle loss and metabolic disease in later life. Here, we identified β‐Klotho (KLB), a fibroblast growth factor 21 (FGF21) co‐receptor, as a novel regulator of muscle development in IUGR. Using the pig as a naturally‐occurring disease model, we performed transcriptome‐wide profiling of fetal muscle (day 90 of pregnancy) from IUGR and normal‐weight (NW) littermates. We found that, alongside large‐scale transcriptional changes comprising multiple developmental, tissue injury and metabolic gene pathways, KLB was increased in IUGR muscle. Moreover, FGF21 concentrations were increased in plasma in IUGR fetuses. Using cultures of fetal muscle progenitor cells (MPCs), we showed reduced myogenic capacity of IUGR compared to NW muscle in vitro, as evidenced by differences in fusion indices and myogenic transcript levels, as well as mechanistic target of rapamycin (mTOR) activity. Moreover, transfection of MPCs with KLB small interfering RNA promoted myogenesis and mTOR activation, whereas treatment with FGF21 had opposite and dose‐dependent effects in porcine and also in human fetal MPCs. In conclusion, our results identify KLB as a novel and potentially critical mediator of impaired muscle development in IUGR, through conserved mechanisms in pigs and humans. Our data shed new light onto the pathogenesis of IUGR, a significant cause of lifelong ill‐health in humans and animals. Key points: Intrauterine growth restriction (IUGR) is associated with large‐scale transcriptional changes in developmental, tissue injury and metabolic gene pathways in fetal skeletal muscle.Levels of the fibroblast growth factor 21 (FGF21) co‐receptor, β‐Klotho (KLB) are increased in IUGR fetal muscle, and FGF21 concentrations are increased in IUGR fetal plasma.KLB mediates a reduction in muscle development through inhibition of mechanistic target of rapamycin signalling.These effects of KLB on muscle cells are conserved in pig and human, suggesting a vital role of this protein in the regulation of muscle development and function in mammals. [ABSTRACT FROM AUTHOR]

There is very little information about how the genome is regulated in domestic pigs (Sus scrofa). This lack of knowledge hinders efforts to define and predict the effects of genetic variants in pig breeding programs. To address this knowledge gap, we need to identify regulatory sequences in the pig genome starting with regions of open chromatin. We used the “Improved Protocol for the Assay for Transposase- Accessible Chromatin (Omni-ATAC-Seq)” to identify putative regulatory regions in flash-frozen semitendinosus muscle from 24 male piglets. We collected samples from the smallest-, average-, and largest-sized male piglets from each litter through five developmental time points. Of the 4661 ATAC-Seq peaks identified that represent regions of open chromatin, >50% were within 1 kb of known transcription start sites. Differential read count analysis revealed 377 ATAC-Seq defined genomic regions where chromatin accessibility differed significantly across developmental time points. We found regions of open chromatin associated with downregulation of genes involved in muscle development that were present in small-sized fetal piglets but absent in large-sized fetal piglets at day 90 of gestation. The dataset that we have generated provides a resource for studies of genome regulation in pigs and contributes valuable functional annotation information to filter genetic variants for use in genomic selection in pig breeding programs. [ABSTRACT FROM AUTHOR]

Background The domestic sheep (Ovis aries) is an important agricultural species raised for meat, wool, and milk across the world. A high-quality reference genome for this species enhances the ability to discover genetic mechanisms influencing biological traits. Furthermore, a high-quality reference genome allows for precise functional annotation of gene regulatory elements. The rapid advances in genome assembly algorithms and emergence of sequencing technologies with increasingly long reads provide the opportunity for an improved de novo assembly of the sheep reference genome. Findings Short-read Illumina (55× coverage), long-read Pacific Biosciences (75× coverage), and Hi-C data from this ewe retrieved from public databases were combined with an additional 50× coverage of Oxford Nanopore data and assembled with canu v1.9. The assembled contigs were scaffolded using Hi-C data with Salsa v2.2, gaps filled with PBsuitev15.8.24, and polished with Nanopolish v0.12.5. After duplicate contig removal with PurgeDups v1.0.1, chromosomes were oriented and polished with 2 rounds of a pipeline that consisted of freebayes v1.3.1 to call variants, Merfin to validate them, and BCFtools to generate the consensus fasta. The ARS-UI_Ramb_v2.0 assembly is 2.63 Gb in length and has improved continuity (contig NG50 of 43.18 Mb), with a 19- and 38-fold decrease in the number of scaffolds compared with Oar_rambouillet_v1.0 and Oar_v4.0. ARS-UI_Ramb_v2.0 has greater per-base accuracy and fewer insertions and deletions identified from mapped RNA sequence than previous assemblies. Conclusions The ARS-UI_Ramb_v2.0 assembly is a substantial improvement in contiguity that will optimize the functional annotation of the sheep genome and facilitate improved mapping accuracy of genetic variant and expression data for traits in sheep. [ABSTRACT FROM AUTHOR]

Background: Since their domestication 10,500 years ago, goat populations with distinctive genetic backgrounds have adapted to a broad variety of environments and breeding conditions. The VarGoats project is an international 1000-genome resequencing program designed to understand the consequences of domestication and breeding on the genetic diversity of domestic goats and to elucidate how speciation and hybridization have modeled the genomes of a set of species representative of the genus Capra. Findings: A dataset comprising 652 sequenced goats and 507 public goat sequences, including 35 animals representing eight wild species, has been collected worldwide. We identified 74,274,427 single nucleotide polymorphisms (SNPs) and 13,607,850 insertion-deletions (InDels) by aligning these sequences to the latest version of the goat reference genome (ARS1). A Neighbor-joining tree based on Reynolds genetic distances showed that goats from Africa, Asia and Europe tend to group into independent clusters. Because goat breeds from Oceania and Caribbean (Creole) all derive from imported animals, they are distributed along the tree according to their ancestral geographic origin. Conclusions: We report on an unprecedented international effort to characterize the genome-wide diversity of domestic goats. This large range of sequenced individuals represents a unique opportunity to ascertain how the demographic and selection processes associated with post-domestication history have shaped the diversity of this species. Data generated for the project will also be extremely useful to identify deleterious mutations and polymorphisms with causal effects on complex traits, and thus will contribute to new knowledge that could be used in genomic prediction and genome-wide association studies. [ABSTRACT FROM AUTHOR]

Background: Previous studies have identified many immune pathways which are consistently altered in humans and model organisms as they age. Dairy cows are often culled at quite young ages due to an inability to cope adequately with metabolic and infectious diseases, resulting in reduced milk production and infertility. Improved longevity is therefore a desirable trait which would benefit both farmers and their cows. This study analysed the transcriptome derived from RNA-seq data of leukocytes obtained from Holstein cows in early lactation with respect to lactation number. Results: Samples were divided into three lactation groups for analysis: i) primiparous (PP, n = 53), ii) multiparous in lactations 2–3 (MP 2–3, n = 121), and iii) MP in lactations 4–7 (MP > 3, n = 55). Leukocyte expression was compared between PP vs MP > 3 cows with MP 2–3 as background using DESeq2 followed by weighted gene co-expression network analysis (WGCNA). Seven modules were significantly correlated (r ≥ 0.25) to the trait lactation number. Genes from the modules which were more highly expressed in either the PP or MP > 3 cows were pooled, and the gene lists subjected to David functional annotation cluster analysis. The top three clusters from modules more highly expressed in the PP cows all involved regulation of gene transcription, particularly zinc fingers. Another cluster included genes encoding enzymes in the mitochondrial beta-oxidation pathway. Top clusters up-regulated in MP > 3 cows included the terms Glycolysis/Gluconeogenesis, C-type lectin, and Immunity. Differentially expressed candidate genes for ageing previously identified in the human blood transcriptome up-regulated in PP cows were mainly associated with T-cell function (CCR7, CD27, IL7R, CAMK4, CD28), mitochondrial ribosomal proteins (MRPS27, MRPS9, MRPS31), and DNA replication and repair (WRN). Those up-regulated in MP > 3 cows encoded immune defence proteins (LYZ, CTSZ, SREBF1, GRN, ANXA5, ADARB1). Conclusions: Genes and pathways associated with lactation number in cows were identified for the first time to date, and we found that many were comparable to those known to be associated with ageing in humans and model organisms. We also detected changes in energy utilization and immune responses in leukocytes from older cows. [ABSTRACT FROM AUTHOR]

Bovine mastitis, an inflammatory disease of the mammary gland, is classified as subclinical or clinical. Circulating neutrophils are recruited to the udder to combat infection. We compared the transcriptomic profiles in circulating leukocytes between healthy cows and those with naturally occurring subclinical or clinical mastitis. Holstein Friesian dairy cows from six farms in EU countries were recruited. Based on milk somatic cell count and clinical records, cows were classified as healthy (n = 147), subclinically (n = 45) or clinically mastitic (n = 22). Circulating leukocyte RNA was sequenced with Illumina NextSeq single end reads (30 M). Differentially expressed genes (DEGs) between the groups were identified using CLC Genomics Workbench V21, followed by GO enrichment analysis. Both subclinical and clinical mastitis caused significant changes in the leukocyte transcriptome, with more intensive changes attributed to clinical mastitis. We detected 769 DEGs between clinical and healthy groups, 258 DEGs between subclinical and healthy groups and 193 DEGs between clinical and subclinical groups. Most DEGs were associated with cell killing and immune processes. Many upregulated DEGs in clinical mastitis encoded antimicrobial peptides (AZU1, BCL3, CAMP, CATHL1, CATHL2, CATHL4,CATHL5, CATHL6, CCL1, CXCL2, CXCL13, DEFB1, DEFB10, DEFB4A, DEFB7, LCN2, PGLYRP1, PRTN3, PTX3, S100A8, S100A9, S100A12, SLC11A1, TF and LTF) which were not upregulated in subclinical mastitis. The use of transcriptomic profiles has identified a much greater up-regulation of genes encoding antimicrobial peptides in circulating leukocytes of cows with naturally occurring clinical compared with subclinical mastitis. These could play a key role in combatting disease organisms. [ABSTRACT FROM AUTHOR]

Great progress has been made over recent years in the identification of selection signatures in the genomes of livestock species. This work has primarily been carried out in commercial breeds for which the dominant selection pressures are associated with artificial selection. As agriculture and food security are likely to be strongly affected by climate change, a better understanding of environment-imposed selection on agricultural species is warranted. Ethiopia is an ideal setting to investigate environmental adaptation in livestock due to its wide variation in geo-climatic characteristics and the extensive genetic and phenotypic variation of its livestock. Here, we identified over three million single nucleotide variants across 12 Ethiopian sheep populations and applied landscape genomics approaches to investigate the association between these variants and environmental variables. Our results suggest that environmental adaptation for precipitation-related variables is stronger than that related to altitude or temperature, consistent with large-scale meta-analyses of selection pressure across species. The set of genes showing association with environmental variables was enriched for genes highly expressed in human blood and nerve tissues. There was also evidence of enrichment for genes associated with high-altitude adaptation although no strong association was identified with hypoxia-inducible-factor (HIF) genes. One of the strongest altitude-related signals was for a collagen gene, consistent with previous studies of high-altitude adaptation. Several altitude-associated genes also showed evidence of adaptation with temperature, suggesting a relationship between responses to these environmental factors. These results provide a foundation to investigate further the effects of climatic variables on small ruminant populations. [ABSTRACT FROM AUTHOR]

Background: Minimally invasive diagnostic imaging techniques to detect intestinal inflammation in dogs are lacking. Contrast‐enhanced ultrasound (CEUS) and endoscopic narrow band imaging‐like endoscopy (Storz Professional Image Enhancement System [SPIES]) might allow quantification of intestinal mucosal perfusion and microvessel density in chronic inflammatory enteropathy (CIE) of dogs. Hypothesis/Objectives: Markers of mucosal perfusion as determined by CEUS and SPIES endoscopy are potentially useful diagnostic markers to help characterize CIE and correlate with histological inflammation type and severity. Animals Thirty client‐owned dogs diagnosed with CIE at a referral hospital were prospectively enrolled. Materials and Methods: Data from CEUS, SPIES, and white light (WL) endoscopy were correlated with World Small Animal Veterinary Association (WSAVA) endoscopy and histology scores and vessel density as determined by immunohistochemistry for von Willebrand factor (vWF). Automated linear modeling was used to determine predictors of endoscopic and histologic severity. Results: Duodenal histology correlated with SPIES data (area percentage value, rho = 0.424, P =.04). White light endoscopy parameters correlated with SPIES parameters in the duodenum. Colonic histology correlated positively with CEUS, whereas colonic CEUS parameters correlated inversely with vWF expression. Several duodenal parameters combined predicted duodenal histology scores to a level of 73.4%. For the colon, 2 parameters contributed more than others to 73.1%. Conclusion and Clinical Importance: Minimally invasive CEUS and SPIES appear feasible to assess intestinal perfusion in CIE. Use of SPIES endoscopy may be promising for assessing small intestinal inflammation, whereas CEUS could be used to assess colonic perfusion and inflammation. Both techniques need to be investigated further for their clinical utility. [ABSTRACT FROM AUTHOR]

Bovine Tuberculosis (bTB) caused by Mycobacterium bovis is a livestock disease of global economic and public health importance. There are currently no effective vaccines available for livestock and so control relies on animal level surveillance and pasteurization of dairy products. A new alternative control approach is to exploit the genetic variability of the host; recent studies have demonstrated that breeding European taurine cattle, such as Holsteins for increased resistance to bTB is feasible. The utility of such an approach is still unknown for African cattle populations. This study aims to assess genetic variation in bTB resistance and the underlying genomic architecture of cattle in Cameroon. We conducted a cross-sectional study of 2,346 slaughter cattle in Cameroon. Retropharyngeal lymph node samples were collected and cultured on Lowenstein Jensen media and the BACTEC MGIT 960 system, and M. bovis was identified using the Hain® Genotype kits. A total of 153 cattle were positive for M. bovis and were archived along with a random selection of negative samples. In this study, we genotyped archived samples from 212 cattle. Their genomic diversity was characterized using PCA, hierarchical clustering and admixture analysis. We assessed genetic variation in bTB resistance using heritability analysis and compared quantitative trait loci. Previous research on this study population have shown that Fulani cattle are more susceptible to bTB than mixed breeds. However, here we show that these apparent phenotypic differences in breeds are not reflected by clear genomic differences. At the genetic level, both the Fulani and mixed cattle show similar patterns of admixture with evidence of both taurine and indicine ancestry. There was little European taurine introgression within the studied population. Hierarchical clustering showed clusters of cattle that differed in their susceptibility to bTB. Our findings allude to bTB resistance being polygenic in nature. This study highlights the potential for genetic control of bTB in Africa and the need for further research into the genetics of bTB resistance within African cattle populations. [ABSTRACT FROM AUTHOR]

The overall aim of the Ovine FAANG project is to provide a comprehensive annotation of the new highly contiguous sheep reference genome sequence (Oar rambouillet v1.0). Mapping of transcription start sites (TSS) is a key first step in understanding transcript regulation and diversity. Using 56 tissue samples collected from the reference ewe Benz2616, we have performed a global analysis of TSS and TSS-Enhancer clusters using Cap Analysis Gene Expression (CAGE) sequencing. CAGE measures RNA expression by 5′ cap-trapping and has been specifically designed to allow the characterization of TSS within promoters to single-nucleotide resolution. We have adapted an analysis pipeline that uses TagDust2 for clean-up and trimming, Bowtie2 for mapping, CAGEfightR for clustering, and the Integrative Genomics Viewer (IGV) for visualization. Mapping of CAGE tags indicated that the expression levels of CAGE tag clusters varied across tissues. Expression profiles across tissues were validated using corresponding polyA+ mRNA-Seq data from the same samples. After removal of CAGE tags with <10 read counts, 39.3% of TSS overlapped with 5′ ends of 31,113 transcripts that had been previously annotated by NCBI (out of a total of 56,308 from the NCBI annotation). For 25,195 of the transcripts, previously annotated by NCBI, no TSS meeting stringent criteria were identified. A further 14.7% of TSS mapped to within 50 bp of annotated promoter regions. Intersecting these predicted TSS regions with annotated promoter regions (±50 bp) revealed 46% of the predicted TSS were "novel" and previously un-annotated. Using whole-genome bisulfite sequencing data from the same tissues, we were able to determine that a proportion of these "novel" TSS were hypo-methylated (32.2%) indicating that they are likely to be reproducible rather than "noise". This global analysis of TSS in sheep will significantly enhance the annotation of gene models in the new ovine reference assembly. Our analyses provide one of the highest resolution annotations of transcript regulation and diversity in a livestock species to date. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis (bTB), caused by Mycobacterium bovis , is a chronic zoonotic disease where host genetics is thought to contribute to susceptibility or resistance. One of the genes implicated is the SLC11A1 gene, that encodes for the natural resistance-associated macrophage protein 1 (NRAMP1). The aim of this study was to identify SLC11A1 polymorphisms and to investigate any resulting functional differences in NRAMP1 expression that might be correlated with resistance/susceptibility to M. bovis infection. Sequencing of the SLC11A1 gene in cDNA isolated from Brown Swiss, Holstein Friesian, and Sahiwal cattle identified five single nucleotide polymorphisms (SNPs) in the coding region, but only one of these (SNP4, c.1066C>G, rs109453173) was present in all three cattle breeds and therefore warranted further investigation. Additionally, variations of 10, 11, and 12 GT repeats were identified in a microsatellite (MS1) in the SLC11A1 3′UTR. Measurement of NRAMP1 expression in bovine macrophages by ELISA showed no differences between cells generated from the different breeds. Furthermore, variations in the length of the MS1 microsatellite did not impact on NRAMP1 protein expression as analyzed by luciferase reporter assay. However, further analysis of the ELISA data identified that the presence of the alternative G allele at SNP4 was associated with increased expression of NRAMP1 in bovine macrophages. Since NRAMP1 has been shown to influence the survival of intracellular pathogens such as M. bovis through the sequestering of iron, it is possible that cattle expressing the alternative G allele might have an increased resistance to bTB through increased NRAMP1 expression in their macrophages. [ABSTRACT FROM AUTHOR]

The aim of this study was to identify genomic regions associated with 305‐day milk yield and lactation curve parameters on primiparous (n = 9,910) and multiparous (n = 11,158) Holstein cows. The SNP solutions were estimated using a weighted single‐step genomic BLUP approach and imputed high‐density panel (777k) genotypes. The proportion of genetic variance explained by windows of 50 consecutive SNP (with an average of 165 Kb) was calculated, and regions that accounted for more than 0.50% of the variance were used to search for candidate genes. Estimated heritabilities were 0.37, 0.34, 0.17, 0.12, 0.30 and 0.19, respectively, for 305‐day milk yield, peak yield, peak time, ramp, scale and decay for primiparous cows. Genetic correlations of 305‐day milk yield with peak yield, peak time, ramp, scale and decay in primiparous cows were 0.99, 0.63, 0.20, 0.97 and −0.52, respectively. The results identified three windows on BTA14 associated with 305‐day milk yield and the parameters of lactation curve in primi‐ and multiparous cows. Previously proposed candidate genes for milk yield supported by this work include GRINA, CYHR1, FOXH1, TONSL, PPP1R16A, ARHGAP39, MAF1, OPLAH and MROH1, whereas newly identified candidate genes are MIR2308, ZNF7, ZNF34, SLURP1, MAFA and KIFC2 (BTA14). The protein lipidation biological process term, which plays a key role in controlling protein localization and function, was identified as the most important term enriched by the identified genes. [ABSTRACT FROM AUTHOR]

Goats (Capra hircus) are an economically important livestock species providing meat and milk across the globe. They are of particular importance in tropical agri-systems contributing to sustainable agriculture, alleviation of poverty, social cohesion, and utilisation of marginal grazing. There are excellent genetic and genomic resources available for goats, including a highly contiguous reference genome (ARS1). However, gene expression information is limited in comparison to other ruminants. To support functional annotation of the genome and comparative transcriptomics, we created a mini-atlas of gene expression for the domestic goat. RNA-Seq analysis of 17 transcriptionally rich tissues and 3 cell-types detected the majority (90%) of predicted protein-coding transcripts and assigned informative gene names to more than 1000 previously unannotated protein-coding genes in the current reference genome for goat (ARS1). Using network-based cluster analysis, we grouped genes according to their expression patterns and assigned those groups of coexpressed genes to specific cell populations or pathways. We describe clusters of genes expressed in the gastro-intestinal tract and provide the expression profiles across tissues of a subset of genes associated with functional traits. Comparative analysis of the goat atlas with the larger sheep gene expression atlas dataset revealed transcriptional similarities between macrophage associated signatures in the sheep and goats sampled in this study. The goat transcriptomic resource complements the large gene expression dataset we have generated for sheep and contributes to the available genomic resources for interpretation of the relationship between genotype and phenotype in small ruminants. [ABSTRACT FROM AUTHOR]

Pervasive allelic variation at both gene and single nucleotide level (SNV) between individuals is commonly associated with complex traits in humans and animals. Allele-specific expression (ASE) analysis, using RNA-Seq, can provide a detailed annotation of allelic imbalance and infer the existence of cis-acting transcriptional regulation. However, variant detection in RNA-Seq data is compromised by biased mapping of reads to the reference DNA sequence. In this manuscript, we describe an unbiased standardized computational pipeline for allele-specific expression analysis using RNA-Seq data, which we have adapted and developed using tools available under open license. The analysis pipeline we present is designed to minimize reference bias while providing accurate profiling of allele-specific expression across tissues and cell types. Using this methodology, we were able to profile pervasive allelic imbalance across tissues and cell types, at both the gene and SNV level, in Texel×Scottish Blackface sheep, using the sheep gene expression atlas data set. ASE profiles were pervasive in each sheep and across all tissue types investigated. However, ASE profiles shared across tissues were limited, and instead, they tended to be highly tissue-specific. These tissue-specific ASE profiles may underlie the expression of economically important traits and could be utilized as weighted SNVs, for example, to improve the accuracy of genomic selection in breeding programs for sheep. An additional benefit of the pipeline is that it does not require parental genotypes and can therefore be applied to other RNA-Seq data sets for livestock, including those available on the Functional Annotation of Animal Genomes (FAANG) data portal. This study is the first global characterization of moderate to extreme ASE in tissues and cell types from sheep. We have applied a robust methodology for ASE profiling to provide both a novel analysis of the multi-dimensional sheep gene expression atlas data set and a foundation for identifying the regulatory and expressed elements of the genome that are driving complex traits in livestock. [ABSTRACT FROM AUTHOR]

Aim:The purpose of this study was to investigate the effect of normal synovial fluid (SF) on exposed endogenous tendon-derived cells (TDCs) and engrafted mesenchymal stem cells (MSCs) within the tendon extracellular matrix.Methods:Explants from equine superficial digital flexor (extra-synovial) and deep digital flexor tendons (DDFTs) from the compressed, intra-synovial and the tensile, extra-synovial regions were cultured in allogeneic or autologous SF-media. Human hamstring explants were cultured in allogeneic SF. Explant viability was assessed by staining. Proliferation of equine monolayer MSCs and TDCs in SF-media and co-culture with DDFT explants was determined by alamarblue®. Non-viable Native Tendon matrices (NNTs) were re-populated with MSCs or TDCs and cultured in SF-media. Immunohistochemical staining of tendon sections for the apoptotic proteins caspase-3, −8, and −9 was performed.Results:Contact with autologous or allogeneic SF resulted in rapid death of resident tenocytes in equine and human tendon. SF did not affect the viability of equine epitenon cells, or of MSCs and TDCs in the monolayer or indirect explant co-culture. MSCs and TDCs, engrafted into NNTs, died when cultured in SF. Caspase-3, −8, and −9 expression was the greatest in SDFT explants exposed to allogeneic SF.Conclusions:The efficacy of cells administered intra-synovially for tendon lesion repair is likely to be limited, since once incorporated into the matrix, cells become vlnerable to the adverse effects of SF. These observations could account for the poor success rate of intra-synovial tendon healing following damage to the epitenon and contact with SF, common with most soft tissue intra-synovial pathologies. [ABSTRACT FROM AUTHOR]

Characterizing the locations of genetic regulatory elements is critical for understanding the regulatory mechanisms of complex phenotypic traits related to production traits and health in livestock species. The Ovine Functional Annotation of Animal Genomes (FAANG) Project aims to characterize transcriptional regulatory elements across the sheep genome to facilitate a better understanding of the biological mechanisms influencing phenotypic traits in sheep. Assays including sequencing of messenger RNA (mRNA-seq), cap analysis of gene expression (CAGE), chromatin immunoprecipitation of histones (ChIP-seq), assay for transposase-accessible chromatin (ATAC-seq), whole genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) were performed on tissues collected from the Rambouillet ewe used to assemble the reference genome ARS-UI_ Ramb_v2.0. Histone modifications were used to define nine chromatin states for tissues across the genome depicting promoters and enhancers (active, poised, and repressed) using ChromHMM. Chromatin states were overlayed with RNA-seq, ATAC-seq and DNA methylation. These data suggest that active promoter and enhancer states reside in open chromatin regions with a greater transcriptional activity and hypomethylated regions than other states. Further, poised and repressed enhancers did not primarily reside in open chromatin and had less transcriptional activity and more hypermethylated sites compared with active states. Collectively these data define transcriptional regulatory regions throughout the ovine genome which provides a valuable resource to better understand regulatory regions in the genome and how these influence economically important traits in sheep and other livestock species. [ABSTRACT FROM AUTHOR]

Phosphodiesterase (PDE) inhibitors have been utilized for in vitro maturation (IVM) of oocytes to manipulate the meiotic resumption and progression. Premature chromatin condensation and DNA replication of the oocytes, immediately after the decrease in the cAMP level, are the difficulties in canine IVM. Caffeine, a nonselective competitive PDE inhibitor, due to its structural similarity to adenosine molecule maintains the cAMP level by occupying PDE enzymes such as PDE-3A inside the oocyte and PDE-4 and PDE-5 in the cumulus cells. In this study, the effects of 12-hour caffeine pretreatment in a biphasic IVM protocol were assessed on maturation rates of canine oocytes. Sixty hours of culture after a 12-hour of 10 mM caffeine pretreatment resulted in 16.9% ± 2.4 of the oocytes reaching metaphase II stage (MII) and 25.9% ± 5.2 degeneration rate compared with the control group with 2.2% ± 2.2 MII and 37.6% ± 4.3 degeneration rates (P < 0.05). Caffeine pretreatment induced higher mitogen-activated protein kinases (MAPK1 and MAPK3) phosphorylation and maturation-promoting factor activity at 12 hours and activated MAPK1 and maturation-promoting factor at 48 hours after culture in cumulus-oocyte complexes (COCs) compared with the control group (P < 0.05). Fresh canine COCs were also analyzed before IVM using brilliant cresyl blue (BCB) staining. Oocytes showed difference in meiotic resumption (MI-MII) (BCB+ = 16.11% ± 5.5, BCB− = 9.86% ± 5.0; P < 0.05) after 60 hours of culture following 12-hour caffeine pretreatment. The BCB+ canine oocytes had higher MII rate than the BCB− group under caffeine pretreatment (10.2% ± 2.9 vs. 1.1% ± 1.1, respectively; P < 0.05). Results indicated that 12-hour caffeine pretreatment of canine COCs improves the MII maturation rates at 72 hours and BCB+ oocytes have higher competency in vitro for nuclear maturation. [ABSTRACT FROM AUTHOR]

Canine oocytes require an extended period of culture (72 h) in vitro for nuclear maturation to the metaphase II stage, which also results in high degeneration. Canine cumulus oocyte complexes were isolated by slicing from ovaries collected after ovariohysterectomy and cultured in serum-free synthetic oviductal fluid incubated at low (5%) or high (20%) oxygen levels. Changes in oocyte nuclear maturation rates, H2O2 levels within the oocytes and mRNAs of reactive oxygen species inhibitory genes superoxide dismutase 1 and 2 (SOD 1 and 2), glutathione reductase (GSR), glutathione peroxidase (GPXI), and catalase (CAT) were quantified. Higher meiotic resumption from germinal vesicle breakdown up to MII was observed in low 02 (41.8±13.1%) compared to high O2 (15.8±8.2%) (P= 0.014) after 52 h of culture (n= 112). Extension of the culture period up to 84 h at low O2 (n= 457 oocytes) produced the highest meiotic resumption at 72 h (64.1 ± 6.0%; P= 0.008), compared with 52 h. Oocytes (n= 110) cultured in high O2 contained higher levels of peroxidase measured using the 2′,7′-dichlorodihydrofluorescein diacetate fluorescence assay after 72 h of culture compared with low O2 (P=0.004). High O2-cultured oocytes also showed higher amounts of SOD1, 50D2, GSR, GPX1, and CAT mRNA. Vitamin E in high oxygen level was able to decrease degeneration (P= 0.008) but had no improving effect on percentage of oocytes in MII. These results for the first time showed that low oxygen gas composition improves nuclear maturation rates and alleviates the oxidative stress for canine oocytes during in vitro maturation. [ABSTRACT FROM AUTHOR]

In 2019, a novel coronavirus, SARS-CoV-2/nCoV-19, emerged in Wuhan, China, and has been responsible for the current COVID-19 pandemic. The evolutionary origins of the virus remain elusive and understanding its complex mutational signatures could guide vaccine design and development. As part of the international "CoronaHack" in April 2020, we employed a collection of contemporary methodologies to compare the genomic sequences of coronaviruses isolated from human (SARS-CoV-2; n = 163), bat (bat-CoV; n = 215) and pangolin (pangolin-CoV; n = 7) available in public repositories. We have also noted the pangolin-CoV isolate MP789 to bare stronger resemblance to SARS-CoV-2 than other pangolin-CoV. Following de novo gene annotation prediction, analyses of gene–gene similarity network, codon usage bias and variant discovery were undertaken. Strong host-associated divergences were noted in ORF3a, ORF6, ORF7a, ORF8 and S, and in codon usage bias profiles. Last, we have characterised several high impact variants (in-frame insertion/deletion or stop gain) in bat-CoV and pangolin-CoV populations, some of which are found in the same amino acid position and may be highlighting loci of potential functional relevance. [ABSTRACT FROM AUTHOR]

Simple Summary: Reproductive performance is an important factor, which determines productive life and drives culling decisions in dairy herds. There are strong motives for including reproductive performance in genetic selection programs of dairy cows; however, low heritability estimates reported for reproductive performance measures limit the genetic selection efficiency. More effective genetic selection could be achieved using genomic information. The aim of this study was to identify genomic region(s) associated with the length of the calving interval in Holstein cows. The accuracies of genomic estimated breeding values (GEBVs) with single-step genomic BLUP (ssGBLUP) and the pedigree-based BLUP were compared as well. The results showed that the accuracies of GEBVs using the single-step genomic BLUP were much higher than those estimated using the pedigree-based BLUP. We identified three genomic regions (BTA3, BTA6, and BTA7) associated with the length of the calving interval in Holstein dairy cows. The aim of the present study was to identify genomic region(s) associated with the length of the calving interval in primiparous (n = 6866) and multiparous (n = 5071) Holstein cows. The single nucleotide polymorphism (SNP) solutions were estimated using a weighted single-step genomic best linear unbiased prediction (WssGBLUP) approach and imputed high-density panel (777 k) genotypes. The effects of markers and the genomic estimated breeding values (GEBV) of the animals were obtained by five iterations of WssGBLUP. The results showed that the accuracies of GEBVs with WssGBLUP improved by +5.4 to +5.7, (primiparous cows) and +9.4 to +9.7 (multiparous cows) percent points over accuracies from the pedigree-based BLUP. The most accurate genomic evaluation was provided at the second iteration of WssGBLUP, which was used to identify associated genomic regions using a windows-based GWAS procedure. The proportion of additive genetic variance explained by windows of 50 consecutive SNPs (with an average of 165 Kb) was calculated and the region(s) that accounted for equal to or more than 0.20% of the total additive genetic variance were used to search for candidate genes. Three windows of 50 consecutive SNPs (BTA3, BTA6, and BTA7) were identified to be associated with the length of the calving interval in primi- and multiparous cows, while the window with the highest percentage of explained genetic variance was located on BTA3 position 49.42 to 49.52 Mb. There were five genes including ARHGAP29, SEC24D, METTL14, SLC36A2, and SLC36A3 inside the windows associated with the length of the calving interval. The biological process terms including alanine transport, L-alanine transport, proline transport, and glycine transport were identified as the most important terms enriched by the genes inside the identified windows. [ABSTRACT FROM AUTHOR]

Our aim was to examine size-specific effects of Hyaluronan (HA) on preimplantation embryo development. We investigated the effects of Hyalovet (HA, 500–750 kDa; the size produced by HA synthase-3, which is abundant in the oviduct), or HA treated with Hyaluronidase-2 (Hyal2; also expressed in the oviduct that breaks down HA into 20 kDa fragments). In experiment 1 ( in vivo ), oviducts of synchronized and superovulated ewes (n = 20) were surgically exposed on Day 2 post-mating, ligated, and infused with either Hyalovet, Hyalovet + Hyal2, Hyal2, or PBS (control). Ewes were killed 5 days later for recovery of embryos and oviductal epithelial cells (OEC). Blastocyst rates were significantly higher in Hyal2 and Hyalovet + Hyal2 oviducts. Hyaluronidase-2 infusion resulted in higher blastocyst cell numbers and hatching rates. This was associated with increased HSP70 expression in OEC. In contrast, Hyalovet resulted in the lowest development to blastocyst stage and lowest hatching rates, and decreased IGF2 and IGFBP2 expression in OEC. IGF1 and IL1α expression were not affected. In experiment 2, to rule out indirect effects of oviductal factors, ovine embryos were produced and cultured with the same treatments in vitro from Day 2 to 8. Hyaluronidase-2, but not Hyalovet, enhanced blastocyst formation and reduced inner cell mass apoptosis. Hyalovet inhibited hatching. In conclusion, the presence of large-size HA (500–750 kDa) in the vicinity of developing embryos appears to disturb the oviductal environment and embryo development in vivo and in vitro . In contrast, we show evidence that breakdown of HA into smaller fragments is required to maximize embryo development and blastocyst quality. [ABSTRACT FROM AUTHOR]

One of the most significant physiological challenges to neonatal and juvenile ruminants is the development and establishment of the rumen. Using a subset of RNA-Seq data from our high-resolution atlas of gene expression in sheep (Ovis aries) we have provided the first comprehensive characterization of transcription of the entire gastrointestinal (GI) tract during the transition from pre-ruminant to ruminant. The dataset comprises 164 tissue samples from sheep at four different time points (birth, one week, 8 weeks and adult). Using network cluster analysis we illustrate how the complexity of the GI tract is reflected in tissueand developmental stage-specific differences in gene expression. The most significant transcriptional differences between neonatal and adult sheep were observed in the rumen complex. Comparative analysis of gene expression in three GI tract tissues from age-matched sheep and goats revealed species-specific differences in genes involved in immunity and metabolism. This study improves our understanding of the transcriptomic mechanisms involved in the transition from pre-ruminant to ruminant by identifying key genes involved in immunity, microbe recognition and metabolism. The results form a basis for future studies linking gene expression with microbial colonization of the developing GI tract and provide a foundation to improve ruminant efficiency and productivity through identifying potential targets for novel therapeutics and gene editing. [ABSTRACT FROM AUTHOR]

We report the generation of an organism-wide catalog of 976,813 cis -acting regulatory elements for the bovine detected by the assay for transposase accessible chromatin using sequencing (ATAC-seq). We regroup these regulatory elements in 16 components by nonnegative matrix factorization. Correlation between the genome-wide density of peaks and transcription start sites, correlation between peak accessibility and expression of neighboring genes, and enrichment in transcription factor binding motifs support their regulatory potential. Using a previously established catalog of 12,736,643 variants, we show that the proportion of single-nucleotide polymorphisms mapping to ATAC-seq peaks is higher than expected and that this is owing to an approximately 1.3-fold higher mutation rate within peaks. Their site frequency spectrum indicates that variants in ATAC-seq peaks are subject to purifying selection. We generate eQTL data sets for liver and blood and show that variants that drive eQTL fall into liver- and blood-specific ATAC-seq peaks more often than expected by chance. We combine ATAC-seq and eQTL data to estimate that the proportion of regulatory variants mapping to ATAC-seq peaks is approximately one in three and that the proportion of variants mapping to ATAC-seq peaks that are regulatory is approximately one in 25. We discuss the implication of these findings on the utility of ATAC-seq information to improve the accuracy of genomic selection., (© 2023 Yuan et al.; Published by Cold Spring Harbor Laboratory Press.)

Understanding the genomic control of tissue-specific gene expression and regulation can help to inform the application of genomic technologies in farm animal breeding programs. The fine mapping of promoters [transcription start sites (TSS)] and enhancers (divergent amplifying segments of the genome local to TSS) in different populations of cattle across a wide diversity of tissues provides information to locate and understand the genomic drivers of breed- and tissue-specific characteristics. To this aim, we used Cap Analysis Gene Expression (CAGE) sequencing, of 24 different tissues from 3 populations of cattle, to define TSS and their coexpressed short-range enhancers (<1 kb) in the ARS-UCD1.2&#95;Btau5.0.1Y reference genome (1000bulls run9) and analyzed tissue and population specificity of expressed promoters. We identified 51,295 TSS and 2,328 TSS-Enhancer regions shared across the 3 populations (dairy, beef-dairy cross, and Canadian Kinsella composite cattle from 2 individuals, 1 of each sex, per population). Cross-species comparative analysis of CAGE data from 7 other species, including sheep, revealed a set of TSS and TSS-Enhancers that were specific to cattle. The CAGE data set will be combined with other transcriptomic information for the same tissues to create a new high-resolution map of transcript diversity across tissues and populations in cattle for the BovReg project. Here we provide the CAGE data set and annotation tracks for TSS and TSS-Enhancers in the cattle genome. This new annotation information will improve our understanding of the drivers of gene expression and regulation in cattle and help to inform the application of genomic technologies in breeding programs., Competing Interests: Conflicts of interest No commercial or academic conflict of interest were declared by any of the authors for this manuscript., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

The functionality of circulating leukocytes in dairy cows is suppressed after calving, with negative energy balance as a risk factor. Leukocyte transcriptomic profiles were compared separately in 44 multiparous (MP) and 18 primiparous (PP) Holstein-Friesian cows receiving diets differing in concentrate proportion to test whether immune dysfunction could be mitigated by appropriate nutrition. After calving, cows were offered either (1) low concentrate (LC); (2) medium concentrate (MC) or (3) high concentrate (HC) diets with proportions of concentrate to grass silage of 30%:70%, 50%:50% and 70%:30%, respectively. Cow phenotype data collected included circulating metabolites, milk yield and health and fertility records. RNA sequencing of circulating leukocytes at 14 days in milk was performed. The HC diet improved energy balance in both age groups. There were more differentially expressed genes in PP than MP cows (460 vs. 173, HC vs. LC comparison) with few overlaps. The MP cows on the LC diet showed upregulation of the complement and coagulation cascade and innate immune defence mechanisms against pathogens and had a trend of more cases of mastitis and poorer fertility. In contrast, the PP cows on the HC diet showed greater immune responses based on both gene expression and phenotypic data and longer interval of calving to conception. The leukocytes of MP and PP cows therefore responded differentially to the diets between age, nutrient supply and immunity affecting their health and subsequent fertility.

By their paternal transmission, Y-chromosomal haplotypes are sensitive markers of population history and male-mediated introgression. Previous studies identified biallelic single-nucleotide variants in the SRY, ZFY and DDX3Y genes, which in domestic goats identified four major Y-chromosomal haplotypes, Y1A, Y1B, Y2A and Y2B, with a marked geographical partitioning. Here, we extracted goat Y-chromosomal variants from whole-genome sequences of 386 domestic goats (75 breeds) and seven wild goat species, which were generated by the VarGoats goat genome project. Phylogenetic analyses indicated domestic haplogroups corresponding to Y1B, Y2A and Y2B, respectively, whereas Y1A is split into Y1AA and Y1AB. All five haplogroups were detected in 26 ancient DNA samples from southeast Europe or Asia. Haplotypes from present-day bezoars are not shared with domestic goats and are attached to deep nodes of the trees and networks. Haplogroup distributions for 186 domestic breeds indicate ancient paternal population bottlenecks and expansions during migrations into northern Europe, eastern and southern Asia, and Africa south of the Sahara. In addition, sharing of haplogroups indicates male-mediated introgressions, most notably an early gene flow from Asian goats into Madagascar and the crossbreeding that in the 19th century resulted in the popular Boer and Anglo-Nubian breeds. More recent introgressions are those from European goats into the native Korean goat population and from Boer goat into Uganda, Kenya, Tanzania, Malawi and Zimbabwe. This study illustrates the power of the Y-chromosomal variants for reconstructing the history of domestic species with a wide geographical range., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

In 2019, a novel coronavirus, SARS-CoV-2/nCoV-19, emerged in Wuhan, China, and has been responsible for the current COVID-19 pandemic. The evolutionary origins of the virus remain elusive and understanding its complex mutational signatures could guide vaccine design and development. As part of the international "CoronaHack" in April 2020, we employed a collection of contemporary methodologies to compare the genomic sequences of coronaviruses isolated from human (SARS-CoV-2; n = 163), bat (bat-CoV; n = 215) and pangolin (pangolin-CoV; n = 7) available in public repositories. We have also noted the pangolin-CoV isolate MP789 to bare stronger resemblance to SARS-CoV-2 than other pangolin-CoV. Following de novo gene annotation prediction, analyses of gene-gene similarity network, codon usage bias and variant discovery were undertaken. Strong host-associated divergences were noted in ORF3a, ORF6, ORF7a, ORF8 and S, and in codon usage bias profiles. Last, we have characterised several high impact variants (in-frame insertion/deletion or stop gain) in bat-CoV and pangolin-CoV populations, some of which are found in the same amino acid position and may be highlighting loci of potential functional relevance.