1a. Objectives (from AD-416):
Objective 1: Improve the draft bovine genome sequence and enhance annotation of genes (both protein-coding and noncoding), gene functions, and gene-gene interactions (functional networks). Objective 2: Identify inter-individual genome sequence variation in beef cattle and sheep, and explore the effect of this variation on a wide range of production traits. Objective 3: Assess variation in metagenomes associated with microenvironments within animals or their production settings, to identify potential novel strategies and techniques that manipulate microbial populations for improved production methods less reliant on antimicrobial use, while improving growth and production efficiencies in cattle and sheep.
1b. Approach (from AD-416):
Current challenges to the beef industry include pressure to reduce use of antibiotics, create healthier products, and a need to accommodate dietary changes imposed as corn is diverted to use as a fuel. The Project is designed to interact with and complement approved Projects in (1) the Nutrition Research Unit on feed efficiency and impacts of using distiller’s grain as a feedstuff, (2) the Animal Health Research and Meat Safety and Quality Research Units on reducing antibiotic use, and creating a healthier product, and (3) the Reproduction Research Unit to explore lifetime productivity of cows. This Project Plan is the primary vehicle for including genomics tools and approaches in these collaborating Projects, and the goals are to use genomics and related technologies to begin to address the current industry challenges. Our hypothesis is that substantial genetic variation exists among beef cattle that could be used to meet these challenges through selection. We expect that some desirable genetic effects may be exerted through interactions with the microbiome, and propose that enhanced knowledge of the bovine genome and microbial communities associated with the animals and their production environment can be utilized to target improvements in production, health, food safety, and product quality traits. The goals of the Project are to use molecular genetics and genomics techniques to identify inter-individual genome variation associated with the health, lifetime reproductive efficiency, feed efficiency, and food safety phenotypes recorded on the large research herd maintained in cooperation with the other approved Project Plan in the Genetics and Breeding Unit at USMARC. The Project will also develop knowledge of the microbial communities associated with beef production, and examine putative interactions between the bovine genome and microbiome variation. Since the current draft cattle genome assembly is inadequate to support our approaches, we will participate in international efforts to improve it. The Project will provide the industry with technology to support prediction of genetic merit for measures of animal health, fertility, and efficiency that are difficult to record outside a research setting. It will also provide basic knowledge to address the role(s) of microbial populations in beef production, while continuing commitment to support basic research and tools for investigation of genome biology of ruminants, historically a key role of USMARC in cattle genomics. We will expand this role to microbiomes associated with beef cattle production.
3. Progress Report:
The three foundations of the U.S. Meat Animal Research Center (UAMARC),Clay Center, Nebraska cattle genomics program are (1) a database of genotype/genome sequence of the Germplasm Evaluation Population (GPE) that is comprised of a multi-generation pedigree including representation of the top 18 beef breeds used in the U.S., (2) a high quality reference sequence to put the database in context, and (3) phenotype data on animals to use in determining genotype associations. These foundations will support identification of chromosomal regions, genes, and in some cases specific DNA differences associated with variation in important production traits. We have expanded the database of genotype/genome sequence of GPE as part of Objective 2 of the project plan, by genotyping of over 2,000 select animals in the GPE population for functional variants (i.e., changes in DNA sequence that are predicted to impact protein structure or function). This dataset allows us to predict the functional genotypes of other GPE animals by imputation. The imputation accuracy based on this group of animals exceeds 98% across most of the population. We have completed analysis of the previously-identified functional variants to determine genes affected, impacts of the variants on gene function, allele frequencies and correlations among variant genotypes in the GPE population, supporting Objective 1 as well as 2. This has positioned us to perform analysis for association between variants and quantitative traits such as birth or weaning weight, and other traits with recorded phenotypes. In addition to the variants previously discovered, we have performed whole genome sequencing (WGS) of over 100 animals to identify novel variants, to evaluate the possibility that rare variants that may not have been discovered in other populations could influence phenotypic traits in GPE, as has been observed in the case of human quantitative traits. A set of uncommon variants in the bovine EPAS1 gene was uncovered by WGS, and the potential relationship of these variants with the occurrence of pulmonary hypertension and diagnosed respiratory disease, a novel discovery of potentially high impact, is being studied by our collaborator in the Animal Health Project Plan 3040-32000-034-00D. All of the previously-described and novel variants found in GPE WGS data, were identified by mapping to the existing reference assembly and using the annotation of that genome. The inaccuracies in the public reference assembly limited our ability to identify gene-related variation because gene annotation is incorrect/lacking in mis-assembled regions. Objective 1 of the project was thus the creation of a high quality, annotated reference assembly, which we succeeded in producing in FY2017. The new assembly of the cattle genome has >100-fold higher contiguity (lack of gaps in the sequence) and accuracy than the current public reference, and was created by combining a suite of new genome technologies. The basis of this approach was developed in our collaboration on the genome of domestic goat, in a high-profile publication in the April 2017 edition of the journal Nature Genetics, which featured the work on the cover of the journal and a two-page “news and views” story in the issue. For the improved cattle genome, we preformed extensive studies of gene expression in more than 20 tissues to support annotation of genes, and produced the highest quality combination of genome assembly and annotation yet achieved for a de novo assembly of any livestock animal. More work remains in annotation and to compare genomes of different breeds and subspecies of cattle, which is proposed for the replacement Project Plan under review that would commence in mid-FY2018. We also provided support for a similar approach to improve the sheep genome reference, being performed in collaboration with Baylor College of Medicine and anticipated public release of the assembly in August 2017. In addition, we collaborated with an international group to produce two high-quality reference genomes of pigs, publicly released. For all three species, the genome assemblies created have been accepted by the global research communities as the definitive reference genomes for each species, and will have high impact on livestock genome research. The third foundation of the genomics program is phenotype collection. We collected data on over 4,000 USMARC and 3,300 commercial collaborator animals for female reproductive traits in pursuit of Objective 2a. We routinely collect phenotypes for “classical” traits, such as growth rate, age at puberty, calf birth weight, as well as novel traits such as feed intake/feed efficiency, heifer fertility, lifetime female productivity, disease resistance, and carcass quality. These traits have been recorded on another generation of GPE animals in FY2017, as well as in animals from collaborators in the Midwest and Southeast, supporting detection of association with segregating functional variants. In addition, two novel phenotypes targeted for this project were microbial profile from the nasal passage of cattle, and overall incidence of respiratory disease. We collected thousands (>9,000 now collected) of nasal swabs from approximately 4,900 calves at two time points of early growth, with the aim of identifying differences in the nasal microbiomes between apparently healthy animals and those that eventually develop respiratory disease. Approximately 600 calves have been also sampled after displaying signs of respiratory disease after the second early growth sampling, along with 1,200 matching healthy cohorts as controls. New this year was a collaboration with our new virologist in the animal health group, which identified bovine corona virus and mycoplasma as putative agents of disease, providing finer-grained phenotypes for association analysis. In a collaboration with Colorado State University using feedlot records, a marker in the Usher gene of cattle was found to be associated with appearance of respiratory disease. This gene plays a role in coordinated cilia movement (i.e. clearance) in airways and lungs, and is significant because a known pathway of disease in children involves primary ciliary dyskinesia. As part of a broader effort to look at all aspects of respiratory disease, we also collected complete genome sequence of >400 species of bacteria isolated from the nasopharynx of cattle that did not have genome sequence in the public database. This work was related to Objectives 2a, 3a, and 3b. We progressed in efforts to generate genome assemblies of internal parasites affecting sheep, but did not achieve the goal of a high-quality genome assembly due to technical and bioinformatics challenges. These challenges are being addressed through international collaborative efforts. This work is related to Objective 3a.
1. Production and public release of an improved reference genome assembly for cattle. All efforts to use genomics to study cattle rely upon the reference genome to accurately represent all the genes and regulatory sequences, in their correct order and orientation. The reference genome for cattle has been the Hereford breed assembly produced in 2007 (published in 2009) that was created using what has become outdated technology, and as a result has many inaccuracies and deficiencies. ARS researchers at Clay Center, Nebraska, in collaboration with researchers at University of California, Davis; University of Missouri, Columbia; University of Maryland; the National Human Genome Research Institute; and ARS researchers in Beltsville, Maryland, released an improved reference assembly of the same animal using a combination of modern technologies available at Clay Center, Nebraska. This assembly is over 100 times more continuous (a key measure of accuracy and quality) than the existing reference. Significantly, genes related to immune functions, which are notoriously difficult to assemble using older technology, are now accurately represented in the reference. The Clay Center, Nebraska-led assembly is now the accepted reference for genomic studies in cattle.
2. Production and public release of improved reference genome assemblies for pigs. All efforts to use genomics to study pigs rely upon the reference genome to accurately represent all the genes and regulatory sequences, in their correct order and orientation. The reference genome for pigs has been the Duroc breed assembly produced in 2010 (published in 2012) that was created using what has become outdated technology, and as a result has many inaccuracies and deficiencies. ARS researchers at Clay Center, Nebraska worked in a collaboration led by the Roslin Institute in Scotland, which also included collaborators at two U.S. universities and three genome industry partners, to release two improved reference assemblies using a combination of modern technologies available at Clay Center, Nebraska and at a genome industry partner. The primary assembly used the same animal as the original reference, and is over 200 times more continuous (a key measure of accuracy and quality) than the existing reference. The second assembly was of a crossbred pig from Clay Center, Nebraska and is also over 100 times as continuous as the original reference. The new re-assembly of the original pig is now the accepted reference for genomic studies in swine, and the alternate crossbred pig assembly is being used to investigate genome structure and function of commercial pig populations.
3. Development of animal-friendly microbial profiling techniques for classifying respiratory disease in cattle. Study of respiratory disease in cattle, the most costly infectious disease in beef production, has been complicated by the fact that symptoms may be caused by a spectrum of viral and bacterial pathogens. A promising approach to surmounting this difficulty is the use of microbial profiles created from DNA, extracted out of swabs of the respiratory tract, by targeted sequencing of one gene (called the 16S rRNA gene) that has sequence specific to individual species of bacteria. However, initial work developing this approach was based on deep nasopharyngeal swabs, which is relatively uncomfortable for the animals. ARS researchers at Clay Center, Nebraska compared 16S rRNA gene profiling of deep nasopharyngeal swabs, with less invasive nasal swabs (6 inch-long swabs versus the 8-9 inch nasopharyngeal swabs), to determine that the nasal swab data could be used as a surrogate for the more invasive approach, in classifying the organisms associated with respiratory disease. This less invasive approach can now be used on large numbers (over 9600 animals total to be sampled) in studies to identify cattle genetic variation that may be more resistant to development of respiratory disease.
Wilson, W.C., Ruder, M.G., Jasperson, D.C., Smith, T.P., Naraghi Arani, P., Lenhoff, R., Stallknecht, D.E., Valdivia-Granda, W.A., Sheron, D. 2016. Molecular evolution of epizootic hemorrhagic disease viruses in North America based on historical isolates. Virus Genes. 52:495-508.
Nguyen, S.V., Harhay, D.M., Bono, J.L., Smith, T.P.L., Fields, P.I., Dinsmore, B.A., Santovenia, M., Kelley, C.M., Wang, R., Bosilevac, J.M., Harhay, G.P. 2016. Complete, closed genome sequences of 10 Salmonella enterica subsp. enterica serovar Typhimurium strains isolated from human and bovine sources. Genome Announcements. 4(6):e01212-16. doi:10.1128/genomeA.01212-16.
Myer, P.R., Kim, M.S., Freetly, H.C., Smith, T.P. 2016. Metagenomic and near full-length 16S rRNA sequence data in support of the phylogenetic analysis of the rumen bacterial community in steers. Data in Brief. 8:1048-1053. doi: 10.1016/j.dib.2016.07.027.
Clemmons, B.A., Reese, S.T., Dantas, F.G., Franco, G.A., Smith, T.P.L., Adeyosoye, O.I., Pohler, K.G., Myer, P.R. 2017. Vaginal and uterine bacterial communities in postpartum lactating cows. Frontiers in Microbiology. 8:1047. doi:10.3389/fmicb.2017.01047.
Zarek, C.M., Lindholm-Perry, A.K., Kuehn, L.A., Freetly, H.C. 2017. Differential expression of genes related to gain and intake in the liver of beef cattle. BMC Research Notes. 10:1. doi:10.1186/s13104-016-2345-3.
Keel, B.N., Lindholm-Perry, A.K., Snelling, W.M. 2016. Evolutionary and functional features of copy number variation in the cattle genome. Frontiers in Genetics. 7:207. doi:10.3389/fgene.2016.00207.
Snelling, W.M., Kuehn, L.A., Keel, B.N., Thallman, R.M., Bennett, G.L. 2017. Linkage disequilibrium among commonly genotyped SNP and variants detected from bull sequence. Animal Genetics. 48:516-522. https://doi.org/doi: 10.1111/age.12579.
Myer, P.R., Freetly, H.C., Wells, J.E., Smith, T.P., Kuehn, L.A. 2017. Analysis of the gut bacterial communities in beef cattle and their association with feed intake, growth, and efficiency. Journal of Animal Science. 95(7):3215-3224. doi: 10.2527/jas2016.1059.
Liu, H., Smith, T.P.L., Nonneman, D.J., Dekkers, J.C.M., Tuggle, C.K. 2017. A high-quality annotated transcriptome of swine peripheral blood. BMC Genomics. 18:479. doi:10.1186/s12864-017-3863-7.