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 (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 focused on increasing the number of animals with whole-genome sequence, and beefing up the sequence coverage where known genes lie, to expand our knowledge of genomic variation existing in U.S. beef cattle herds. Specifically, 270 influential sires of our USMARC populations were sequenced, in addition to 94 bulls used by cooperating seedstock producers, to provide improved representation of the 7 predominant beef breeds used in the U.S. The sequence obtained this year provided a big step toward the ultimate goal of knowing or predicting the complete genome sequence of the USMARC GPE7 herd, which has records on many traits and represents a resource to associate genomic variation in a wide range of traits including the animal's ability to resist respiratory disease or to have high reproductive success, traits of high value in modern beef production systems. We have also been trying to facilitate technology transfer of our research to commercial herds through a cooperative research and development agreement (CRADA) to develop low-cost genotyping assays for specific markers shown to have predictive merit. Hampering our efforts in genomic studies, which are now largely based on mapping the sequence reads obtained for each animal to the public reference genome assembly, is the fact that the reference assembly is fraught with problems and inconsistencies. Fortunately, the latest DNA sequencing technology, in place at USMARC, is capable of generating a much improved assembly for a very small fraction of the cost (<$100,000) of the original assembly ($54 million). Based on sequence reads 10-25 times as long as the public assembly, this year we created assemblies of the goat and pig genomes that are approximately 30-40 times higher contiguity (a key measure of quality and utility) than the current public reference. We collected all the sequence data for the same cow used for the current bovine reference and are currently working on the assembly in collaboration with University of California at Davis, which will become the new public reference when complete. In addition, we collected sequence of RNA from ten tissues of the animal using a new technology called IsoSeq available at USMARC, which gives much higher quality data than short-read-based technology used in the past and will be useful to improve the total quality of the new reference assembly in collaboration with the University of Missouri at Columbia. The third foundation of the genomics program, phenotype collection, will be a primary focus of FY2016 endeavors; however, we have made substantial progress on this aspect of the program in FY2015. Specifically, we have accumulated over 5,000 nasal swab samples from calves, which are being used to characterize the microbial inhabitants of the respiratory tract to look for associations between cattle genome variation and the presence of respiratory pathogens. Only a low fraction of animals contract respiratory disease, so we must collect samples from a large number of animals at preconditioning and weaning timepoints, since our goal is to characterize changes in the microbial populations as animals progress from healthy to diseased. Approximately 500 calves exhibited symptoms of disease and have associated samples, providing a good start for the project. Another respiratory disease-related phenotype is the presence of lesion on the lung of animals, indicating a history of disease. We collected over 12,000 tissue samples from a commercial beef production line after examining the lungs for signs of lesions, and successfully used a commercially available genotyping assay to identify association of genome variation with the presence of historical infection. We characterized the bacterial composition in target environments in the ileum, jejunum and colon of cattle for feed efficiency and food safety studies. Specific species of bacteria that are associated with particular components of feed efficiency were identified, which will provide the basis for future studies attempting to monitor, predict, or influence feed efficiency. In addition, we identified and sequenced bacterial isolates from the bovine nasopharynx that have not previously had complete genome sequence, information that will be useful in gaining a complete understanding of the interplay of host and pathogen in the microbial milieu of the bovine respiratory system.
1. Identification of genetic variants linked to the presence of lung lesions, a proxy for respiratory disease. Development of genetic markers that could potentially identify animals at increased risk for experiencing respiratory disease would be an initial step toward reducing the impact of this issue for the beef cattle industry, which costs up to $1 billion dollars annually and is a primary reason for the use of antibiotics in beef production. Previous efforts have relied on the identification of animals with disease by observing them for symptoms, which has been shown to be too subjective to be very reliable. ARS researchers at Clay Center, Nebraska, tried a different approach by examining the lungs of animals postmortem, with lesions being probable indicators of previous infection. The project identified 85 sequence variants associated with the incidence of severe lung lesions, which can be used to help identify biomarkers for incorporation into national sire genetic evaluation and select cattle with reduced susceptibility to respiratory disease, and potentially reduce the use of antibiotics to control disease.
2. Improvement of livestock reference genomes. 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 improve the goat and pig reference genome assemblies. Both assemblies have 30-40 times better contiguity, a key measure of quality and utility, compared to the current public reference genomes for those two species. The improved reference genomes are supporting advances in genome analysis and identification of biomarkers, for example DNA variants associated with susceptibility to emerging swine disease.
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Heaton, M.P., Harhay, G.P., Smith, T.P.L., Bono, J.L., Chitko-McKown, C.G. 2015. Complete closed genome sequences of a Mannheimia haemolytica serotype A1 leukotoxin deletion mutant and its wild-type parent strain. Genome Announcements. 3(3):e00417-15. DOI: 10.1128/genomeA.00417-15.
Clawson, M.L., Redden, R., Schuller, G., Heaton, M.P., Workman, A.M., Chitko-McKown, C.G., Smith, T.P.L., Leymaster, K.A. 2015. Genetic subgroup of small ruminant lentiviruses that infects sheep homozygous for TMEM154 frameshift deletion mutation A4delta53. Veterinary Research. 46:22.
Cushman, R.A., Tait Jr, R.G., McNeel, A.K., Forbes, E.D., Amundson, O.L., Lents, C.A., Lindholm-Perry, A.K., Perry, G.A., Wood, J.A., Cupp, A.S., Smith, T.P., Freetly, H.C., Bennett, G.L. 2015. A polymorphism in myostatin influences puberty but not fertility in beef heifers, whereas µ-calpain affects first calf birth weight. Journal of Animal Science. 93(1):117-126.
Lindholm-Perry, A.K., Kuehn, L.A., Freetly, H.C., Snelling, W.M. 2015. Genetic markers that influence feed efficiency phenotypes also affect cattle temperament as measured by flight speed. Animal Genetics. 46(1):60-64.
Casas, E., Lippolis, J.D., Kuehn, L.A., Reinhardt, T.A. 2015. Seasonal variation in vitamin D status of beef cattle reared in the central United States. Domestic Animal Endocrinology. 52:71-74. DOI: 10.1016/j.domaniend.2015.03.003.