Key genetic variants associated with variation of milk oligosaccharides from diverse human populations.

Human milk oligosaccharides (HMO), the third most abundant component of human milk, are thought to be important contributors to infant health. Studies have provided evidence that geography, stage of lactation, and Lewis and secretor blood groups are associated with HMO profile. However, little is known about how variation across the genome may influence HMO composition among women in various populations. In this study, we performed genome-wide association analyses of 395 women from 8 countries to identify genetic regions associated with 19 different HMO. Our data support FUT2 as the most significantly associated (P < 4.23-9 to P < 4.5-70) gene with seven HMO and provide evidence of balancing selection for FUT2. Although polymorphisms in FUT3 were also associated with variation in lacto-N-fucopentaose II and difucosyllacto-N-tetrose, we found little evidence of selection on FUT3. To our knowledge, this is the first report of the use of genome-wide association analyses on HMO.

Uterine influences on conceptus development in fertility-classified animals.

A major unresolved issue is how the uterus influences infertility and subfertility in cattle. Serial embryo transfer was previously used to classify heifers as high-fertile (HF), subfertile (SF), or infertile (IF). To assess pregnancy loss, two in vivo-produced embryos were transferred into HF, SF, and IF heifers on day 7, and pregnancy outcome was assessed on day 17. Pregnancy rate was substantially higher in HF (71%) and SF (90%) than IF (20%) heifers. Elongating conceptuses were about twofold longer in HF than SF heifers. Transcriptional profiling detected relatively few differences in the endometrium of nonpregnant HF, SF, and IF heifers. In contrast, there was a substantial difference in the transcriptome response of the endometrium to pregnancy between HF and SF heifers. Considerable deficiencies in pregnancy-dependent biological pathways associated with extracellular matrix structure and organization as well as cell adhesion were found in the endometrium of SF animals. Distinct gene expression differences were also observed in conceptuses from HF and SF animals, with many of the genes decreased in SF conceptuses known to be embryonic lethal in mice due to defects in embryo and/or placental development. Analyses of biological pathways, key players, and ligand-receptor interactions based on transcriptome data divulged substantial evidence for dysregulation of conceptus-endometrial interactions in SF animals. These results support the ideas that the uterus impacts conceptus survival and programs conceptus development, and ripple effects of dysregulated conceptus-endometrial interactions elicit loss of the postelongation conceptus in SF cattle during the implantation period of pregnancy.

QTL-mapping and genomic prediction for bovine respiratory disease in U.S. Holsteins using sequence imputation and feature selection.

BACKGROUND: National genetic evaluations for disease resistance do not exist, precluding the genetic improvement of cattle for these traits. We imputed BovineHD genotypes to whole genome sequence for 2703 Holsteins that were cases or controls for Bovine Respiratory Disease and sampled from either California or New Mexico to construct and compare genomic prediction models. The sequence variation reference dataset comprised variants called for 1578 animals from Run 5 of the 1000 Bull Genomes Project, including 450 Holsteins and 29 animals sequenced from this study population. Genotypes for 9,282,726 variants with minor allele frequencies ≥5% were imputed and used to obtain genomic predictions in GEMMA using a Bayesian Sparse Linear Mixed Model. RESULTS: Variation explained by markers increased from 13.6% using BovineHD data to 14.4% using imputed whole genome sequence data and the resolution of genomic regions detected as harbouring QTL substantially increased. Explained variation in the analysis of the combined California and New Mexico data was less than when data for each state were separately analysed and the estimated genetic correlation between risk of Bovine Respiratory Disease in California and New Mexico Holsteins was - 0.36. Consequently, genomic predictions trained using the data from one state did not accurately predict disease risk in the other state. To determine if a prediction model could be developed with utility in both states, we selected variants within genomic regions harbouring: 1) genes involved in the normal immune response to infection by pathogens responsible for Bovine Respiratory Disease detected by RNA-Seq analysis, and/or 2) QTL identified in the association analysis of the imputed sequence variants. The model based on QTL selected variants is biased but when trained in one state generated BRD risk predictions with positive accuracies in the other state. CONCLUSIONS: We demonstrate the utility of sequence-based and biology-driven model development for genomic selection. Disease phenotypes cannot be routinely recorded in most livestock species and the observed phenotypes may vary in their genomic architecture due to variation in the pathogen composition across environments. Elucidation of trait biology and genetic architecture may guide the development of prediction models with utility across breeds and environments.

Identification of loci associated with conception rate in primiparous Holstein cows.

BACKGROUND: Subfertility is a major issue facing the dairy industry as the average US Holstein cow conception rate (CCR) is approximately 35%. The genetics underlying the physiological processes responsible for CCR, the proportion of cows able to conceive and maintain a pregnancy at each breeding, are not well characterized. The objectives of this study were to identify loci, positional candidate genes, and transcription factor binding sites (TFBS) associated with CCR and determine if there was a genetic correlation between CCR and milk production in primiparous Holstein cows. Cows were bred via artificial insemination (AI) at either observed estrus or timed AI and pregnancy status was determined at day 35 post-insemination. Additive, dominant, and recessive efficient mixed model association expedited (EMMAX) models were used in two genome-wide association analyses (GWAA). One GWAA focused on CCR at first service (CCR1) comparing cows that conceived and maintained pregnancy to day 35 after the first AI (n = 494) to those that were open after the first AI (n = 538). The second GWAA investigated loci associated with the number of times bred (TBRD) required for conception in cows that either conceived after the first AI (n = 494) or repeated services (n = 472). RESULTS: The CCR1 GWAA identified 123, 198, and 76 loci associated (P < 5 × 10- 08) in additive, dominant, and recessive models, respectively. The TBRD GWAA identified 66, 95, and 33 loci associated (P < 5 × 10- 08) in additive, dominant, and recessive models, respectively. Four of the top five loci were shared in CCR1 and TBRD for each GWAA model. Many of the associated loci harbored positional candidate genes and TFBS with putative functional relevance to fertility. Thirty-six of the loci were validated in previous GWAA studies across multiple breeds. None of the CCR1 or TBRD associated loci were associated with milk production, nor was their significance with phenotypic and genetic correlations to 305-day milk production. CONCLUSIONS: The identification and validation of loci, positional candidate genes, and TFBS associated with CCR1 and TBRD can be utilized to improve, and further characterize the processes involved in cattle fertility.

Validation of 46 loci associated with female fertility traits in cattle.

BACKGROUND: Subfertility is one challenge facing the dairy industry as the average Holstein heifer conception rate (HCR), the proportion of heifers that conceive and maintain a pregnancy per breeding, is estimated at 55-60%. Of the loci associated with HCR, few have been validated in an independent cattle population, limiting their usefulness for selection or furthering our understanding of the mechanisms involved in successful pregnancy. Therefore, the objectives here were to identify loci associated with HCR: 1) to the first artificial insemination (AI) service (HCR1), 2) to repeated AI services required for a heifer to conceive (TBRD) and 3) to validate loci previously associated with fertility. Breeding and health records from 3359 Holstein heifers were obtained after heifers were bred by AI at observed estrus, with pregnancy determined at day 35 via palpation. Heifer DNA was genotyped using the Illumina BovineHD BeadChip, and genome-wide association analyses (GWAA) were performed with additive, dominant and recessive models using the Efficient Mixed Model Association eXpedited (EMMAX) method with a relationship matrix for two phenotypes. The HCR1 GWAA compared heifers that were pregnant after the first AI service (n = 497) to heifers that were open following the first AI service (n = 405), which included those that never conceived. The TBRD GWAA compared only those heifers which did conceive, across variable numbers of AI service (n = 712). Comparison of loci previously associated with fertility, HCR1 or TBRD were considered the same locus for validation when in linkage disequilibrium (D' > 0.7). RESULTS: The HCR1 GWAA identified 116, 187 and 28 loci associated (P < 5 × 10- 8) in additive, dominant and recessive models, respectively. The TBRD GWAA identified 235, 362, and 69 QTL associated (P < 5 × 10- 8) with additive, dominant and recessive models, respectively. Loci previously associated with fertility were in linkage disequilibrium with 22 loci shared with HCR1 and TBRD, 5 HCR1 and 19 TBRD loci. CONCLUSIONS: Loci associated with HCR1 and TBRD that have been identified and validated can be used to improve HCR through genomic selection, and to better understand possible mechanisms associated with subfertility.

Genome-wide association study for feed efficiency and growth traits in U.S. beef cattle.

BACKGROUND: Single nucleotide polymorphism (SNP) arrays for domestic cattle have catalyzed the identification of genetic markers associated with complex traits for inclusion in modern breeding and selection programs. Using actual and imputed Illumina 778K genotypes for 3887 U.S. beef cattle from 3 populations (Angus, Hereford, SimAngus), we performed genome-wide association analyses for feed efficiency and growth traits including average daily gain (ADG), dry matter intake (DMI), mid-test metabolic weight (MMWT), and residual feed intake (RFI), with marker-based heritability estimates produced for all traits and populations. RESULTS: Moderate and/or large-effect QTL were detected for all traits in all populations, as jointly defined by the estimated proportion of variance explained (PVE) by marker effects (PVE ≥ 1.0%) and a nominal P-value threshold (P ≤ 5e-05). Lead SNPs with PVE ≥ 2.0% were considered putative evidence of large-effect QTL (n = 52), whereas those with PVE ≥ 1.0% but < 2.0% were considered putative evidence for moderate-effect QTL (n = 35). Identical or proximal lead SNPs associated with ADG, DMI, MMWT, and RFI collectively supported the potential for either pleiotropic QTL, or independent but proximal causal mutations for multiple traits within and between the analyzed populations. Marker-based heritability estimates for all investigated traits ranged from 0.18 to 0.60 using 778K genotypes, or from 0.17 to 0.57 using 50K genotypes (reduced from Illumina 778K HD to Illumina Bovine SNP50). An investigation to determine if QTL detected by 778K analysis could also be detected using 50K genotypes produced variable results, suggesting that 50K analyses were generally insufficient for QTL detection in these populations, and that relevant breeding or selection programs should be based on higher density analyses (imputed or directly ascertained). CONCLUSIONS: Fourteen moderate to large-effect QTL regions which ranged from being physically proximal (lead SNPs ≤ 3Mb) to fully overlapping for RFI, DMI, ADG, and MMWT were detected within and between populations, and included evidence for pleiotropy, proximal but independent causal mutations, and multi-breed QTL. Bovine positional candidate genes for these traits were functionally conserved across vertebrate species.

A metagenomics and case-control study to identify viruses associated with bovine respiratory disease.

UNLABELLED: Bovine respiratory disease (BRD) is a common health problem for both dairy and beef cattle, resulting in significant economic loses. In order to identify viruses associated with BRD, we used a metagenomics approach to enrich and sequence viral nucleic acids in the nasal swabs of 50 young dairy cattle with symptoms of BRD. Following deep sequencing, de novo assembly, and translated protein sequence similarity searches, numerous known and previously uncharacterized viruses were identified. Bovine adenovirus 3, bovine adeno-associated virus, bovine influenza D virus, bovine parvovirus 2, bovine herpesvirus 6, bovine rhinitis A virus, and multiple genotypes of bovine rhinitis B virus were identified. The genomes of a previously uncharacterized astrovirus and picobirnaviruses were also partially or fully sequenced. Using real-time PCR, the rates of detection of the eight viruses that generated the most reads were compared for the nasal secretions of 50 animals with BRD versus 50 location-matched healthy control animals. Viruses were detected in 68% of BRD-affected animals versus 16% of healthy control animals. Thirty-eight percent of sick animals versus 8% of controls were infected with multiple respiratory viruses. Significantly associated with BRD were bovine adenovirus 3 (P < 0.0001), bovine rhinitis A virus (P = 0.005), and the recently described bovine influenza D virus (P = 0.006), which were detected either alone or in combination in 62% of animals with BRD. A metagenomics and real-time PCR detection approach in carefully matched cases and controls can provide a rapid means to identify viruses associated with a complex disease, paving the way for further confirmatory tests and ultimately to effective intervention strategies. IMPORTANCE: Bovine respiratory disease is the most economically important disease affecting the cattle industry, whose complex root causes include environmental, genetics, and infectious factors. Using an unbiased metagenomics approach, we characterized the viruses in respiratory secretions from BRD cases and identified known and previously uncharacterized viruses belonging to seven viral families. Using a case-control format with location-matched animals, we compared the rates of viral detection and identified 3 viruses associated with severe BRD signs. Combining a metagenomics and case-control format can provide candidate pathogens associated with complex infectious diseases and inform further studies aimed at reducing their impact.

Susceptibility loci revealed for bovine respiratory disease complex in pre-weaned holstein calves.

BACKGROUND: Bovine respiratory disease complex (BRDC) is an infectious disease of cattle that is caused by a combination of viral and/or bacterial pathogens. Selection for cattle with reduced susceptibility to respiratory disease would provide a permanent tool for reducing the prevalence of BRDC. The objective of this study was to identify BRDC susceptibility loci in pre-weaned Holstein calves as a prerequisite to using genetic improvement as a tool for decreasing the prevalence of BRDC. High density SNP genotyping with the Illumina BovineHD BeadChip was conducted on 1257 male and 757 female Holstein calves from California (CA), and 767 calves identified as female from New Mexico (NM). Of these, 1382 were classified as BRDC cases, and 1396 were classified as controls, with all phenotypes assigned using the McGuirk health scoring system. During the acquisition of blood for DNA isolation, two deep pharyngeal and one mid-nasal diagnostic swab were obtained from each calf for the identification of bacterial and viral pathogens. Genome-wide association analyses were conducted using four analytical approaches (EIGENSTRAT, EMMAX-GRM, GBLUP and FvR). The most strongly associated SNPs from each individual analysis were ranked and evaluated for concordance. The heritability of susceptibility to BRDC in pre-weaned Holstein calves was estimated. RESULTS: The four statistical approaches produced highly concordant results for 373 top ranked SNPs that defined 126 chromosomal regions for the CA population. Similarly, in NM, 370 SNPs defined 138 genomic regions that were identified by all four approaches. When the two populations were combined (i.e., CA + NM) and analyzed, 324 SNPs defined 116 genomic regions that were associated with BRDC across all analytical methods. Heritability estimates for BRDC were 21% for both CA and NM as individual populations, but declined to 13% when the populations were combined. CONCLUSIONS: Four analytical approaches utilizing both single and multi-marker association methods revealed common genomic regions associated with BRDC susceptibility that can be further characterized and used for genomic selection. Moderate heritability estimates were observed for BRDC susceptibility in pre-weaned Holstein calves, thereby supporting the application of genomic selection to reduce the prevalence of BRDC in U.S. Holsteins.

QTLs associated with dry matter intake, metabolic mid-test weight, growth and feed efficiency have little overlap across 4 beef cattle studies.

BACKGROUND: The identification of genetic markers associated with complex traits that are expensive to record such as feed intake or feed efficiency would allow these traits to be included in selection programs. To identify large-effect QTL, we performed a series of genome-wide association studies and functional analyses using 50 K and 770 K SNP genotypes scored in 5,133 animals from 4 independent beef cattle populations (Cycle VII, Angus, Hereford and Simmental×Angus) with phenotypes for average daily gain, dry matter intake, metabolic mid-test body weight and residual feed intake. RESULTS: A total of 5, 6, 11 and 10 significant QTL (defined as 1-Mb genome windows with Bonferroni-corrected P-value<0.05) were identified for average daily gain, dry matter intake, metabolic mid-test body weight and residual feed intake, respectively. The identified QTL were population-specific and had little overlap across the 4 populations. The pleiotropic or closely linked QTL on BTA 7 at 23 Mb identified in the Angus population harbours a promising candidate gene ACSL6 (acyl-CoA synthetase long-chain family member 6), and was the largest effect QTL associated with dry matter intake and mid-test body weight explaining 10.39% and 14.25% of the additive genetic variance, respectively. Pleiotropic or closely linked QTL associated with average daily gain and mid-test body weight were detected on BTA 6 at 38 Mb and BTA 7 at 93 Mb confirming previous reports. No QTL for residual feed intake explained more than 2.5% of the additive genetic variance in any population. Marker-based estimates of heritability ranged from 0.21 to 0.49 for residual feed intake across the 4 populations. CONCLUSIONS: This GWAS study, which is the largest performed for feed efficiency and its component traits in beef cattle to date, identified several large-effect QTL that cumulatively explained a significant percentage of additive genetic variance within each population. Differences in the QTL identified among the different populations may be due to differences in power to detect QTL, environmental variation, or differences in the genetic architecture of trait variation among breeds. These results enhance our understanding of the biology of growth, feed intake and utilisation in beef cattle.

Identifying Regions of the Genome Associated with Conception Rate to the First Service in Holstein Heifers Bred by Artificial Insemination and as Embryo Transfer Recipients.

Heifer conception rate to the first service (HCR1) is defined as the number of heifers that become pregnant to the first breeding service compared to the heifers bred. This study aimed to identify loci associated and gene sets enriched for HCR1 for heifers that were bred by artificial insemination (AI, n = 2829) or were embryo transfer (ET, n = 2086) recipients, by completing a genome-wide association analysis and gene set enrichment analysis using SNP data (GSEA-SNP). Three unique loci, containing four positional candidate genes, were associated (p < 1 × 10-5) with HCR1 for ET recipients, while the GSEA-SNP identified four gene sets (NES ≥ 3) and sixty-two leading edge genes (LEGs) enriched for HCR1. While no loci were associated with HCR1 bred by AI, one gene set and twelve LEGs were enriched (NES ≥ 3) for HCR1 with the GSEA-SNP. This included one gene (PKD2) shared between HCR1 AI and ET services. Identifying loci associated or enriched for HCR1 provides an opportunity to use them as genomic selection tools to facilitate the selection of cattle with higher reproductive efficiency, and to better understand embryonic loss.

Identification by whole genome sequencing of genes associated with delayed postoperative hemorrhage in Scottish deerhounds.

BACKGROUND: Delayed postoperative hemorrhage (DEPOH) is an important health concern for Scottish deerhounds. HYPOTHESIS/OBJECTIVES: Identify genes associated with DEPOH in Scottish deerhounds. ANIMALS: Two hundred sixty-nine privately owned Scottish deerhounds. METHODS: Retrospective case-control study. DEPOH cases and controls were identified through an owner health survey. Genome-wide association analysis was performed using whole genome sequences from 8 cases and 17 controls. All cases and controls were genotyped for selected variants. RESULTS: Of 269 dogs, 10 met inclusion and exclusion criteria for DEPOH, while 62 controls had undergone similar surgical procedures without DEPOH. Genome-wide association analysis identified a single locus on chromosome 9 spanning 40 genes. One of these genes (SERPINF2 encoding alpha-2 antiplasmin) was directly linked to the pathophysiology of DEPOH. The entire cohort was genotyped for a missense SERPINF2 variant (c.605 C>T; p.A202V). Compared to dogs with the reference C/C genotype, the likelihood of DEPOH was significantly higher for dogs with the T/T genotype (odds ratio [OR] = 1235; 95% confidence interval [CI] = 23-6752; P = 0.0005) and with the C/T genotype (OR = 28; 95% CI = 1.4-542; P = 0.03). CONCLUSIONS AND CLINICAL IMPORTANCE: SERPINF2 is associated with DEPOH in Scottish deerhounds. Genetic testing might be able to identify dogs that are susceptible to DEPOH.

Genomic regions associated with pseudorabies virus infection status in naturally infected feral swine (Sus scrofa).

Pseudorabies virus (PRV)-the causative agent of Aujeszky's disease-was eliminated from commercial pig production herds in the United States (US) in 2004; however, PRV remains endemic among invasive feral swine (Sus scrofa). The circulation of PRV among abundant, widespread feral swine populations poses a sustained risk for disease spillover to production herds. Risk-based surveillance has been successfully implemented for PRV in feral swine populations in the US. However, understanding the role of host genetics in infection status may offer new insights into the epidemiology and disease dynamics of PRV that can be applied to management strategies. Genetic mechanisms underlying host susceptibility to PRV are relatively unknown; therefore, we sought to identify genomic regions associated with PRV infection status among naturally infected feral swine using genome-wide association studies (GWAS) and gene set enrichment analysis of single nucleotide polymorphism data (GSEA-SNP). Paired serological and genotypic data were collected from 6,081 feral swine distributed across the invaded range within the contiguous US. Three complementary study populations were developed for GWAS: 1) comprehensive population consisting of feral swine throughout the invaded range within the contiguous US; 2) population of feral swine under high, but temporally variable PRV infection pressure; and 3) population of feral swine under temporally stable, high PRV infection pressure. We identified one intronic SNP associated with PRV infection status within candidate gene AKAP6 on autosome 7. Various gene sets linked to metabolic pathways were enriched in the GSEA-SNP. Ultimately, improving disease surveillance efforts in feral swine will be critical to further understanding of the role host genetics play in PRV infection status, helping secure the health of commercial pork production.

Genome-wide association with footrot in hair and wool sheep.

Ovine footrot is an infectious disease with important contributions from Dichelobacter nodosus and Fusobacterium necrophorum. Footrot is characterized by separation of the hoof from underlying tissue, and this causes severe lameness that negatively impacts animal wellbeing, growth, and profitability. Large economic losses result from lost production as well as treatment costs, and improved genetic tools to address footrot are a valuable long-term goal. Prior genetic studies had examined European wool sheep, but hair sheep breeds such as Katahdin and Blackbelly have been reported to have increased resistance to footrot, as well as to intestinal parasites. Thus, footrot condition scores were collected from 251 U.S. sheep including Katahdin, Blackbelly, and European-influenced crossbred sheep with direct and imputed genotypes at OvineHD array (>500,000 single nucleotide polymorphism) density. Genome-wide association was performed with a mixed model accounting for farm and principal components derived from animal genotypes, as well as a random term for the genomic relationship matrix. We identified three genome-wide significant associations, including SNPs in or near GBP6 and TCHH. We also identified 33 additional associated SNPs with genome-wide suggestive evidence, including a cluster of 6 SNPs in a peak near the genome-wide significance threshold located near the glutamine transporter gene SLC38A1. These findings suggest genetic susceptibility to footrot may be influenced by genes involved in divergent biological processes such as immune responses, nutrient availability, and hoof growth and integrity. This is the first genome-wide study to investigate susceptibility to footrot by including hair sheep and also the first study of any kind to identify multiple genome-wide significant associations with ovine footrot. These results provide a foundation for developing genetic tests for marker-assisted selection to improve resistance to ovine footrot once additional steps like fine mapping and validation are complete.

Resolving the evolution of extant and extinct ruminants with high-throughput phylogenomics.

The Pecorans (higher ruminants) are believed to have rapidly speciated in the Mid-Eocene, resulting in five distinct extant families: Antilocapridae, Giraffidae, Moschidae, Cervidae, and Bovidae. Due to the rapid radiation, the Pecoran phylogeny has proven difficult to resolve, and 11 of the 15 possible rooted phylogenies describing ancestral relationships among the Antilocapridae, Giraffidae, Cervidae, and Bovidae have each been argued as representations of the true phylogeny. Here we demonstrate that a genome-wide single nucleotide polymorphism (SNP) genotyping platform designed for one species can be used to genotype ancient DNA from an extinct species and DNA from species diverged up to 29 million years ago and that the produced genotypes can be used to resolve the phylogeny for this rapidly radiated infraorder. We used a high-throughput assay with 54,693 SNP loci developed for Bos taurus taurus to rapidly genotype 678 individuals representing 61 Pecoran species. We produced a highly resolved phylogeny for this diverse group based upon 40,843 genome-wide SNP, which is five times as many informative characters as have previously been analyzed. We also establish a method to amplify and screen genomic information from extinct species, and place Bison priscus within the Bovidae. The quality of genotype calls and the placement of samples within a well-supported phylogeny may provide an important test for validating the fidelity and integrity of ancient samples. Finally, we constructed a phylogenomic network to accurately describe the relationships between 48 cattle breeds and facilitate inferences concerning the history of domestication and breed formation.

A high-density genome-wide association with absolute blood monocyte count in domestic sheep identifies novel loci.

Monocytes are a core component of the immune system that arise from bone marrow and differentiate into cells responsible for phagocytosis and antigen presentation. Their derivatives are often responsible for the initiation of the adaptive immune response. Monocytes and macrophages are central in both controlling and propagating infectious diseases such as infection by Coxiella burnetii and small ruminant lentivirus in sheep. Genotypes from 513 Rambouillet, Polypay, and Columbia sheep (Ovis aries) were generated using the Ovine SNP50 BeadChip. Of these sheep, 222 animals were subsequently genotyped with the Ovine Infinium® HD SNP BeadChip to increase SNP coverage. Data from the 222 HD genotyped sheep were combined with the data from an additional 258 unique sheep to form a 480-sheep reference panel; this panel was used to impute the low-density genotypes to the HD genotyping density. Then, a genome-wide association analysis was conducted to identify loci associated with absolute monocyte counts from blood. The analysis used a single-locus mixed linear model implementing EMMAX with age and ten principal components as fixed effects. Two genome-wide significant peaks (p < 5x10-7) were identified on chromosomes 9 and 1, and ten genome-wide suggestive peaks (p < 1x10-5) were identified on chromosomes 1, 2, 3, 4, 9, 10, 15, and 16. The identified loci were within or near genes including KCNK9, involved into cytokine production, LY6D, a member of a superfamily of genes, some of which subset monocyte lineages, and HMGN1, which encodes a chromatin regulator associated with myeloid cell differentiation. Further investigation of these loci is being conducted to understand their contributions to monocyte counts. Investigating the genetic basis of monocyte lineages and numbers may in turn provide information about pathogens of veterinary importance and elucidate fundamental immunology.

Identifying Loci Associated With Bovine Corona Virus Infection and Bovine Respiratory Disease in Dairy and Feedlot Cattle.

Bovine coronavirus (BCoV) is associated with respiratory and enteric infections in both dairy and beef cattle worldwide. It is also one of a complex of pathogens associated with bovine respiratory disease (BRD), which affects millions of cattle annually. The objectives of this study were to identify loci and heritability estimates associated with BCoV infection and BRD in dairy calves and feedlot cattle. Dairy calves from California (n = 1,938) and New Mexico (n = 647) and feedlot cattle from Colorado (n = 915) and Washington (n = 934) were tested for the presence of BCoV when classified as BRD cases or controls following the McGuirk scoring system. Two comparisons associated with BCoV were investigated: (1) cattle positive for BCoV (BCoV+) were compared to cattle negative for BCoV (BCoV-) and (2) cattle positive for BCoV and affected with BRD (BCoV+BRD+) were compared to cattle negative for BCoV and BRD (BCoV-BRD-). The Illumina BovineHD BeadChip was used for genotyping, and genome-wide association analyses (GWAA) were performed using EMMAX (efficient mixed-model association eXpedited). The GWAA for BCoV+ identified 51 loci (p < 1 × 10-5; 24 feedlot, 16 dairy, 11 combined) associated with infection with BCoV. Three loci were associated with BCoV+ across populations. Heritability estimates for BCoV+ were 0.01 for dairy, 0.11 for feedlot cattle, and 0.03 for the combined population. For BCoV+BRD+, 80 loci (p < 1 × 10-5; 26 feedlot, 25 dairy, 29 combined) were associated including 14 loci across populations. Heritability estimates for BCoV+BRD+ were 0.003 for dairy, 0.44 for feedlot cattle, and 0.07 for the combined population. Several positional candidate genes associated with BCoV and BRD in this study have been associated with other coronaviruses and respiratory infections in humans and mice. These results suggest that selection may reduce susceptibility to BCoV infection and BRD in cattle.

A DNA Regulatory Element Haplotype at Zinc Finger Genes Is Associated with Host Resilience to Small Ruminant Lentivirus in Two Sheep Populations.

Small ruminant lentivirus (SRLV) causes Maedi-Visna or Ovine Progressive Pneumonia in sheep and creates insidious livestock production losses. This retrovirus is closely related to human immunodeficiency virus and currently has no vaccines or cure. Genetic marker assisted selection for sheep disease resiliency presents an attractive management solution. Previously, we identified a region containing a cluster of zinc finger genes that had association with ovine SRLV proviral concentration. Trait-association analysis validated a small insertion/deletion variant near ZNF389 (rs397514112) in multiple sheep breeds. In the current study, 543 sheep from two distinct populations were genotyped at 34 additional variants for fine mapping of the regulatory elements within this locus. Variants were selected based on ChIP-seq annotation data from sheep alveolar macrophages that defined active cis-regulatory elements predicted to influence zinc finger gene expression. We present a haplotype block of variants within regulatory elements that have improved associations and larger effect sizes (up to 4.7-fold genotypic difference in proviral concentration) than the previously validated ZNF389 deletion marker. Hypotheses for the underlying causal mutation or mutations are presented based on changes to in silico transcription factor binding sites. These variants offer alternative markers for selective breeding and are targets for future functional mutation assays.

Functional Variants Surrounding Endothelin 2 Are Associated With Mycobacterium avium Subspecies paratuberculosis Infection.

Bovine paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (MAP), continues to impact the dairy industry through increased morbidity, mortality, and lost production. Although genome-wide association analyses (GWAAs) have identified loci associated with susceptibility to MAP, limited progress has been made in identifying mutations that cause disease susceptibility. A 235-kb region on Bos taurus chromosome 3 (BTA3), containing a 70-kb haplotype block surrounding endothelin 2 (EDN2), has previously been associated with the risk of MAP infection. EDN2 is highly expressed in the gut and is involved in intracellular calcium signaling and a wide array of biological processes. The objective of this study was to identify putative causal mutations for disease susceptibility in the region surrounding EDN2 in Holstein and Jersey cattle. Using sequence data from 10 Holstein and 10 Jersey cattle, common variants within the 70-kb region containing EDN2 were identified. A custom SNP genotyping array fine-mapped the region using 221 Holstein and 51 Jersey cattle and identified 17 putative causal variants (P < 0.01) located in the 5' region of EDN2 and a SNP in the 3' UTR (P = 0.00009) associated with MAP infection. MicroRNA interference assays, mRNA stability assays, and electrophoretic mobility shift assays were performed to determine if allelic changes at each SNP resulted in differences in EDN2 stability or expression. Two SNPs [rs109651404 (G/A) and rs110287192 (G/T)] located within the promoter region of EDN2 displayed differential binding affinity for transcription factors in binding sequences harboring the alternate SNP alleles. The luciferase reporter assay revealed that the transcriptional activity of the EDN2 promoter was increased (P < 0.05) with the A allele for rs109651404 and the G allele for rs110287192. These results suggest that the variants rs109651404 and rs110287192 are mutations that alter transcription and thus may alter susceptibility to MAP infection in Holstein and Jersey cattle.

GSEA-SNP identifies genes associated with Johne's disease in cattle.

SNP-based gene-set enrichment analysis from single nucleotide polymorphisms, or GSEA-SNP, is a tool to identify candidate genes based on enrichment analysis of sets of genes rather than single SNP associations. The objective of this study was to identify modest-effect genes associated with Mycobacterium avium subsp. paratuberculosis (Map) tissue infection or fecal shedding using GSEA-SNP applied to KEGG pathways or Gene Ontology (GO) gene sets. The Illumina Bovine SNP50 BeadChip was used to genotype 209 Holstein cows for the GSEA-SNP analyses. For each of 13,744 annotated genes genome-wide located within 50 kb of a Bovine SNP50 SNP, the single SNP with the highest Cochran-Armitage Max statistic was used as a proxy statistic for that gene's strength of affiliation with Map. Gene-set enrichment was tested using a weighted Kolmogorov-Smirnov-like running sum statistic with data permutation to adjust for multiple testing. For tissue infection and fecal shedding, no gene sets in KEGG pathways or in GO sets for molecular function or cellular component were enriched for signal. The GO biological process gene set for positive regulation of cell motion (GO:0051272, q = 0.039, 5/11 genes contributing to the core enrichment) was enriched for Map tissue infection, while no GO biological process gene sets were enriched for fecal shedding. GSEA-SNP complements traditional SNP association approaches to identify genes of modest effects as well as genes with larger effects as demonstrated by the identification of one locus that we previously found to be associated with Map tissue infection using a SNP-by-SNP genome-wide association study.

The EDN2 rs110287192 gene polymorphism is associated with paratuberculosis susceptibility in multibreed cattle population.

Paratuberculosis (pTB), also known as Johne's disease (JD), is a contagious, chronic, and granulomatous inflammatory disease of the intestines of ruminants which is caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection, resulting in billions of dollars in economic losses worldwide. Since, currently, no effective cure is available for MAP infection, it is important to explore the genetic variants that affect the host MAP susceptibility. The aim of this study was to analyze a potential association between EDN2 synonymous gene mutations (rs110287192, rs109651404 and rs136707411), that modifies susceptibility to pTB. EDN2 rs110287192, rs109651404 and rs136707411 mutations were genotyped in 68 infected and 753 healthy animals from East Anatolian Red crossbred, Anatolian Black crossbred and Holstein breed cattle by using Custom TaqMan SNP Genotyping Assays. For pTB status, serum antibody levels S/P ≥ 1.0 were assessed in carriers of the different EDN2 genotypes. EDN2 rs110287192 mutation showed a significant association with bovine pTB (adj. p < 0.05). For rs110287192 locus, the odd ratios for GG and TG genotypes versus TT genotypes were 1.73; (95% CI = 0.34-8.59) and 0.53 (95% CI = 0.12-2.37) respectively, which indicated that proportion of TG heterozygotes were significantly higher in control animals as compared to pTB animals. On the other hand, while rs136707411 mutation showed a suggestive association with pTB status in the examined cattle population (nominal p < 0.05); no association was detected between rs109651404 genotypes and pTB status. Selecting animals against rs110287192-GG genotype may decrease the risk of pTB in cattle of the Bos taurus taurus subspecies.