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 USMARC cattle genomics program are (1) a database of genotype/genome sequence of the population, (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. For the first foundation, this year we greatly increased the number of animals with whole-genome sequence, and used this data to identify variants predicted to affect protein sequence or gene expression. These type of variants have a high likelihood of representing underlying phenotypic effects relative to variants lying outside of known gene elements. This effort identified previously unknown variants with putative effect on brisket disease, a pulmonary hypertension pathology that has been known to affect certain breeds when living at high altitude, and now has become more common even at lower altitudes. The data we generated contributed to development of a public database of variation, supporting the identification of variation in any gene annotated in the current cattle genome draft assembly. This year, coverage of minor breeds was expanded in concert with their inclusion in the Germplasm Evaluation (GPE) project, bringing us closer to the goal of being able to impute genome sequence to all the animals in the multi-generational, deeply phenotyped GPE population. In addition, associations between single nucleotide polymorphisms (SNP) from high-density genotyping arrays and sequence variant genotypes were examined to gain insight into imputation and improve accuracy of methods to impute sequence from these genotypes (Objective 2a). Inaccuracies in the current reference assembly limit the ability to accurately impute sequence, in addition to limiting the identification of gene-related variation because gene annotation is incorrect/lacking in mis-assembled regions. This is the second foundation of the genomics program, a high quality reference assembly. We created a new assembly of the cattle genome with >50-fold higher contiguity (lack of gaps in the sequence) and accuracy than the current public reference using long-read sequencing technology combined with other new assembly technologies. This was possible as result of experience gained while leading a long-read assembly of the genome of domestic goat. The new cattle assembly is currently undergoing quality checks and annotation prior to public release (Objective 1). The third foundation of the genomics program is phenotype collection. Two major phenotypes targeted this year were microbial profile from the nasal passage of cattle, and overall incidence of respiratory disease. We collected thousands (>6,000 now collected) of nasal swabs from animals at different times of their life, with the aim of characterizing differences between apparently healthy animals and those that eventually develop respiratory disease. Characterization of the microbes present by a technique called 16S profiling was conducted on most of the samples from both respiratory disease cases and matched controls, to begin identification of any correlations between early life microbial populations and eventual disease, and any associations of cattle genome variation with either disease or microbial profile. In addition, we collected approximately 4,000 sample pairs of animals treated for respiratory disease and matched controls with no apparent disease to look for association of markers with incidence of disease. As part of a broader effort to look at all aspects of respiratory disease, we also collected complete genome sequence of >200 species of bacteria isolated from the nasopharynx of cattle that did not have genome sequence in the public database. Another phenotype collected was presence of liver abscesses post-harvest, and we reported SNP markers associated with susceptibility to these abscesses, since we anticipate that reduced use of antibiotics will increase the incidence of disease linked to this phenotype (Objective 2a, 3a and 3b). Objectives for sheep genomic research were added in FY2015. This year we participated in construction of a long-read sheep genome assembly by selecting, housing, and sampling the animal whose genome will become the new reference. The assembly is nearing completion and should be released prior to the end of calendar year 2016. We initiated several projects to generate genome assemblies of internal parasites affecting sheep, but inadequacies of methods for DNA extraction of these species have impeded progress. Long read sequencing technology has very exacting requirements for DNA quality. Our attempts to use commercial DNA extraction technologies have not yet met these high standards. Research to improve DNA quality continues (Objective 1 and 3a).
1. Identification of markers associated with susceptibility to liver abscesses. Liver abscesses occur in approximately 13% of cattle being fed high energy density rations, as this diet makes them more susceptible to infection from ruminant bacteria that cause subacute acidosis. The acidosis leads to reduced carcass weight and quality, and economic impact of $20 to $80 per affected animal. ARS researchers at Clay Center, Nebraska, predict the prevalence of abscesses will dramatically increase as the use of antibiotics is decreased, bringing urgency to the need for alternate management practices to prevent them. They identified 35 genetic markers associated with abscess susceptibility, that may be useful in genetic selection programs to reduce the incidence of disease.
2. Identification of variation beyond single-base changes in the cattle genome. Most of the efforts to identify genetic variation among cattle contributing to differences in productivity measures, has targeted simple single-base changes along the chromosomes called single nucleotide polymorphisms (SNP). However, it has been known for some time that larger variations including deletions or duplications of larger sections of chromosomes, or insertions of other sequence, exist among individuals and are at least as likely as SNP to contribute to variation in performance. ARS researchers at Clay Center, Nebraska, examined the occurrence of these larger variations, called copy number variants or CNV, in the sequence of 154 bulls representing the most numerous breeds in production in the U.S. About 7% of the genome, including 12% of protein coding genes, were found to harbor CNV, including genes involved in development and immunity. The researchers predict that these variants could have significant impact on production characteristics, and present the results as a resource to open investigation on the effect they have on beef production and disease susceptibility traits.
3. Public release of an improved goat genome assembly. Genome studies in livestock species have made significant advances in weeding out deleterious mutations, and in improving production traits that are difficult or expensive to measure. However, genome studies have great reliance on the reference genome sequence assemblies that were made at great expense in the preceding decade. ARS researchers at Clay Center, Nebraska, and Beltsville, Maryland, with university and federal contractor collaborators, have used the latest long-read sequencing technology available at Clay Center to create and release an improved reference genome assembly of the domestic goat. The assembly is >250 times better in contiguity (a key measure of quality and utility), compared to the previous public reference genome. The improved reference genome is supporting advances in genome analysis and identification of biomarkers.
5. Significant Activities that Support Special Target Populations:
Keele, J.W., Kuehn, L.A., McDaneld, T.G., Tait Jr, R.G., Jones, S., Keel, B.N., Snelling, W.M. 2016. Genomewide association study of liver abscess in beef cattle. Journal of Animal Science. 94(2):490-499.
Myer, P.R., Wells, J.E., Smith, T.P.L., Kuehn, L.A., Freetly, H.C. 2015. Cecum microbial communities from steers differing in feed efficiency. Journal of Animal Science. 93(11):5327-5340. doi: 10.2527/jas2015-9415
Myer, P.R., Wells, J., Smith, T.P., Kuehn, L.A., Freetly, H.C. 2015. Microbial community profiles of the colon from steers differing in feed efficiency. SpringerPlus. 4:454.
Dickey, A.M., Loy, J.D., Bono, J.L., Smith, T.P.L., Apley, M.D., Lubbers, B.V., DeDonder, K.D., Capik, S.F., Larson, R.L., White, B.J., Blom, J., Chitko-McKown, C.G., Clawson, M.L. 2016. Large genomic differences between Moraxella bovoculi isolates acquired from the eyes of cattle with conjunctivitis versus the deep nasopharynx of asymptomatic cattle. Veterinary Research. 47:31.
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 and closed genome sequences of 10 Salmonella enterica subsp. enterica serovar Anatum isolated from human and bovine sources. Genome Announcements. Vol. 4(3): e00447-16. doi: 10.1128/genomeA.00447-16.
Tait Jr, R.G., Cushman, R.A., McNeel, A.K., Casas, E., Smith, T.P., Freetly, H.C., Bennett, G.L. 2016. Estimates of epistatic and pleiotropic effects of casein alpha s1 (CSN1S1) and thyroglobulin (TG) genetic markers on beef heifer performance traits enhanced by selection. Journal of Animal Science. 94(3):920-926. doi:10.2527/jas2015-9860.
Harhay, D.M., Bono, J.L., Smith, T.P., Fields, P.I., Dinsmore, B.A., Santovenia, M., Kelley, C.M., Wang, R., Harhay, G.P. 2016. Complete closed genome sequences of Salmonella enterica subsp. enterica serotypes Anatum, Montevideo, Typhimurium and Newport, isolated from beef, cattle, and humans. Genome Announcements. 4(1):e01683-15. doi:10.1128/genomeA.01683-15.
Kern, R.J., Lindholm-Perry, A.K., Freetly, H.C., Snelling, W.M., Kern, J.W., Keele, J.W., Miles, J.R., Foote, A.P., Oliver, W.T., Kuehn, L.A., Ludden, P.A. 2016. Transcriptome differences in the rumen of beef steers with variation in feed intake and gain. Gene. 586:12-26.
Kern, R.J., Lindholm-Perry, A.K., Freetly, H.C., Kuehn, L.A., Rule, D.C., Ludden, P.A. 2016. Rumen papillae morphology of beef steers relative to gain and feed intake and the association of volatile fatty acids with kallikrein gene expression. Livestock Science. 187:24-30.
Lindholm-Perry, A.K., Kern, R.J., Kuehn, L.A., Snelling, W.M., Miles, J.R., Oliver, W.T., Freetly, H.C. 2015. Differences in transcript abundance of genes on BTA15 located within a region associated with gain in beef steers. Gene. 572(1):42-48. doi:10.1016/j.gene.2015.06.076.
Lindholm-Perry, A.K., Butler, A.R., Kern, R.J., Hill, R., Kuehn, L.A., Wells, J., Oliver, W.T., Hales, K.E., Foote, A.P., Freetly, H.C. 2016. Differential gene expression in the duodenum, jejunum and ileum among crossbred beef steers with divergent gain and feed intake phenotypes. Animal Genetics. 47(4):408-427. doi: 10.1111/age.12440.