Research Project: Identifying Genomic Solutions to Improve Efficiency of Swine Production

Location: Genetics and Animal Breeding

2021 Annual Report

Objectives
Objective 1: Utilize next-generation sequencing technologies to improve the contiguity of the swine genome assembly and better characterize genomic variation in pigs. Subobjective 1.A: Utilize segregation analysis to improve the porcine genome assembly. Subobjective 1.B: Develop more comprehensive gene models for the swine genome. Subobjective 1.C: Develop an electronic warehouse of genomic variants that can be utilized by the swine genomics research community. Objective 2: Develop genotyping products for commercial swine producers to increase the efficiency of swine production. Subobjective 2.A: Identify predictive genetic markers for traits associated with production efficiency in commercial swine populations. Subobjective 2.B: Develop strategies for inclusion of predictive markers in selection programs.

Approach
The principal goals of this project are to enhance our understanding of the biological processes important to swine production and provide the U.S. swine industry with genetic tools that will ensure that it remains the global leader in providing safe, nutritious, and economic pork products. The swine industry has been faced with significant challenges, many of which revolve around the production and performance of feeder pigs. The environment in which females are housed is continually evolving, and the increasing cost of feed has resulted in continuous shifts in the utilization of feed stuffs. Each new challenge requires an assessment of potential solutions. Genetic selection can be used to address many production issues. If DNA variants associated with changes in phenotype can be identified, then marker assisted selection can be implemented to expedite genetic progress. Predictive genetic markers need to be transferred to commercial entities that will rapidly evaluate and adopt them. The increasing improvements to the porcine genome, better annotations of genes from model organisms, and enhanced bioinformatics technologies provide researchers with the necessary tools to identify functional genetic variants. Objective 1 focuses on improving the porcine genome assembly and detecting polymorphisms from data generated by next-generation sequencing. Objective 2 will strive to effectively transfer the results of the research from Objective 1 to producers. Development of marker panels along with economical genotyping platforms will be essential. Our research will focus on the evaluation of genetic markers based on their predicted effects on gene products to discover causal genetic variants of phenotypic variation. This will lead to the development of marker panels and economical genotyping platforms for industry applications.

Progress Report
Research for Objective 1B ‘Develop more comprehensive gene models for the swine genome’ has been conducted. To identify genes and pathways involved in failure to reach puberty at an acceptable age or estrus expression in gilts, ribonucleic acid sequencing (RNA-Seq) and gene expression analysis of ovarian tissues from delayed puberty, behavioral anestrus and normal cycling control gilts was completed. Of 17,000 expressed genes in ovarian tissue, over 1,000 genes were differentially expressed in delayed puberty gilts compared to normal puberty gilts. The top biological pathways involved DNA replication and cell cycle. The contrast for behavioral anestrus and normal cycling gilts showed six and fifteen differentially expressed genes for ovarian and follicular tissues, respectfully. The pathways involved nuclear transport protein localization to the nucleus. In a second study to identify genes that affect litter size, RNA-seq libraries were constructed and sequenced from ovarian tissue from sows that were most extreme for differences in ovulation rate at harvest and litter size through four parities. These data will be analyzed for differential gene expression and biological pathways related to litter size and ovulation rate. We began a collaboration with the University of Edinburgh in conjunction with the Farm Animal Genotype-Tissue Expression atlas (FarmGTEx) and Pig Genotype-Tissue Expression atlas (PigGTEx) consortium. The goal is to provide an atlas of tissue-specific gene expression and regulation in livestock species. As part of this effort, we contributed RNA-Seq data and single nucleotide polymorphism (SNP) genotypes for these pigs, as our population will be used for validation of a transcript-wide association study (TWAS) that is being conducted using the PigGTEx data. We received gene expression matrices for multiple swine tissues (data collected from all public repositories as well as contributions to PigGTEx) that are being used in developing a new approach to identify differential gene expression. This new approach is based on prior knowledge of the distribution of gene expression of individual genes within a given tissue. And finally, a more user-friendly computational pipeline for identifying novel transcripts (same approach that was used with the milk transcripts in Fiscal Year (FY) 2020 was developed and is currently being used to identify novel transcripts from total RNA-Seq data of boar testicles. Research supporting Objective 1C ‘Develop an electronic warehouse of genomic variants that can be utilized by the swine genomics community’ was enhanced by our FarmGTEx collaboration as creation of this warehouse is part of their goals, too. This collaboration has reduced redundancy and will expedite release of the variants to the research community. Research for Objective 2B ‘Identify predictive genetic markers for traits associated with production efficiency in commercial swine populations’ has made considerable progress. Data collection has continued in acyclic gilts, feed efficiency in growing pigs and objective measures of lameness and structural soundness in sows. Data collection for prediction of lameness in 5-month-old gilts was completed, and analyses are underway. A genome-wide association study was completed for age at puberty in a large cohort of about 5,000 records using imputed genotypes for over 71,600 markers. Twenty-one genome-wide SNP associations were found on four chromosomes. Several genes in these quantitative trait loci (QTL) regions were differentially expressed in the ovaries of delayed puberty gilts and potentially functional DNA variants were identified from transcriptomic data of these gilts. These results confirmed associations from previous studies, as well as identified new candidate loci for age at puberty. Substantial progress to impute genome-level SNP genotypes from commercial array SNP platforms was made. A total of 14,103 animals have been genotyped with at least one commercial SNP array. Ancestors of these pigs were identified from pedigree records to create a 16,401 animal pedigree. Pedigree imputation using Findhap was performed to impute all genotyped animals to a common set of 71,634 array SNP. As the result of a newly established research collaboration with the University of Edinburgh, we have access to a large whole genome sequence (WGS) SNP reference panel with approximately 42 million WGS SNP. We filtered our ~27 million WGS SNP to remove SNP with minor allele frequency < 0.05, SNP that had inconsistent Mendelian inheritance patterns (derived using 12 sets of sequenced trios in the population) and overlapped with the PigGTEx panel, resulting in a final set of 11,890,826 WGS SNP. To optimize the imputation protocol for imputing the 71,634 array SNP to the 11,890,826 WGS SNP, a total of 8 haplotype phasing/imputation software pipelines are being evaluated for accuracy and computational efficiency. The set of SNP on chromosome 18 (SSC18), which includes 1,872 array SNP and 320,856 WGS SNP, are being used for evaluating the pipelines.

Accomplishments
1. Genetic variation in swine linked to onset of puberty. A genome-wide association study was completed by ARS scientists in Clay Center, Nebraska, for age at puberty in a large group of 5,000 females (gilts) for over 71,600 markers. Twenty-one genome-wide markers were found on four chromosomes. Several genes in these quantitative trait loci (QTL) regions, were differentially expressed in the ovaries of delayed puberty gilts and potentially functional DNA variants were identified in these gilts. Results confirmed markers from previous studies, as well as identified new candidate markers and biological pathways for modifying age at puberty. Understanding puberty in swine is critical to select gilts at an early age that will improve lifetime productivity in commercial herds. Gilts that attain puberty earlier are more fertile and produce more piglets over their lifetime than later puberty gilts, improving pork production and efficiency as well as animal well-being and producer profitability. Each additional litter a sow produces results in $80-100 of profit, so methods to improve this important trait will greatly benefit producers.

Review Publications
Nonneman, D.J., Lents, C.A., Rempel, L.A., Rohrer, G.A. 2021. Potential functional variants in AHR signaling pathways are associated with age at puberty in swine. Animal Genetics. 52(3):284-291. Article 13051. https://doi.org/10.1111/age.13051.
Abbas, W., Keel, B.N., Kachman, S.D., Fernando, S.C., Wells, J.E., Hales, K.E., Lindholm-Perry, A.K. 2020. Rumen epithelial transcriptome and microbiome profiles of rumen epithelium and contents of beef cattle with and without liver abscesses. Journal of Animal Science. 98(12):1-13. https://doi.org/10.1093/jas/skaa359.
Snelling, W.M., Hoff, J.L., Li, J.H., Kuehn, L.A., Keel, B.N., Lindholm-Perry, A.K., Pickrell, J.K. 2020. Assessment of imputation from low-pass sequencing to predict merit of beef steers. Genes. 11(11). Article 1312. https://doi.org/10.3390/genes11111312.
King, D.A., Shackelford, S.D., Nonneman, D.J., Rohrer, G.A., Wheeler, T.L. 2020. Sire variation in the severity of the ham halo condition. Meat and Muscle Biology. 4(1). https://doi.org/10.22175/mmb.9743.
Lents, C.A., Supakorn, C., Dedecker, A.E., Phillips, C.E., Boyd, R.D., Vallet, J.L., Rohrer, G.A., Foxcroft, G.R., Flowers, W.L., Trottier, N.L., Salak-Johnson, J.L., Bartol, F.F., Stalder, K.J. 2020. Dietary lysine-to-energy ratios for managing growth and pubertal development in replacement gilts. Applied Animal Science. 36(5):701-714. https://doi.org/10.15232/aas.2020-02016.
Lents, C.A., Lindo, A.N., Hileman, S.M., Nonneman, D.J. 2020. Physiological and genomic insight into neuroendocrine regulation of puberty in gilts. Domestic Animal Endocrinology. 73. Article 106446. https://doi.org/10.1016/j.domaniend.2020.106446.
Keel, B.N., Oliver, W.T., Keele, J.W., Lindholm-Perry, A.K. 2020. Evaluation of transcript assembly in multiple porcine tissues suggests optimal sequencing depth for RNA-Seq using total RNA library. Animal Gene. 17-18. Article 200105. https://doi.org/10.1016/j.angen.2020.200105.
Leonard, S.M., Xin, H., Brown-Brandl, T.M., Ramirez, B.C., Johnson, A.K., Dutta, S., Rohrer, G.A. 2021. Effects of farrowing stall layout and number of heat lamps on sow and piglet behavior. Applied Animal Behaviour Science. 239. Article 105334. https://doi.org/10.1016/j.applanim.2021.105334.