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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Genomics and Improvement Laboratory » Research » Research Project #442481
Research Project: Accelerating Genetic Improvement of Ruminants Through Enhanced Genome Assembly, Annotation, and Selection

Location: Animal Genomics and Improvement Laboratory

2025 Annual Report


Objectives
Objective 1: Develop biological resources and computational tools to enhance the representation and annotation characterization of dairy breed-specific bovine and other genomes. Sub-objective 1.A: Improve dairy breed-specific bovine and other genome assemblies for pangenome representation. Sub-objective 1.B: SNP and CNV mapping in cattle and other ruminants. Sub-objective 1.C: Evaluate digestive tract function and identify gastrointestinal microorganism effects on nutrient digestibility, milk production capacity, nutrient use efficiency, and health in dairy cattle. Objective 2: Apply novel tools to utilize genotypic and phenotypic data to enhance genetic improvement in ruminant production systems. Sub-objective 2.A: Continue work on community-based breeding programs and develop imputation pipeline and data in goats. Sub-objective 2.B: Characterize and localize within and across breed measures of dominance as observed in inbreeding depression and heterosis. Objective 3: Characterize functional genetic and epigenetic variations for improved fertility, growth, health, production, reproduction, and environmental sustainability of ruminants. Sub-objective 3.A: Epigenome-wide Association Study based on DNA methylation. Sub-objective 3.B: FarmGTEx for goats.


Approach
Completion of our objectives is expected, in the short term, to improve genome-wide selection in the U.S. dairy industry as well as facilitate new genome-enhanced breeding strategies to bring economic and genetic stability to various ruminant value chains. Ultimately, longer term objectives to identify and understand how causative genetic variation affects livestock biology will require a combination of genome sequencing and comparative genomics, quantitative genetics, epigenomics and metagenomics, all of which are components of this project plan and areas of expertise in our group. Efforts to characterize genome activity and structural conservation/variation are an extension of our current research program in applied genomics. This project plan completely leverages the resources derived from the Bovine Genomes, HapMap, 1000 Bull Genomes, FAANG, Bovien Pangenome, and FarmGTEx projects, and genotypic data derived from the Council on Dairy Cattle Breeding (CDCB) genome-enhanced genetic evaluations for North American dairy cattle.


Progress Report
This is the third report for the new NP101 Project 8042-31000-112-000D which started July 24, 2022, entitled “Accelerating Genetic Improvement of Ruminants Through Enhanced Genome Assembly, Annotation, and Selection”. For Objective 1.A, the Bovine Pangenome Consortium was launched to describe the full extent of genetic variation in cattle through the creation of genome assemblies for bovine species of economic and biodiversity importance. The Consortium has generated and collected 157 genome assemblies representing 67 cattle breeds originating from 27 countries. Pangenome construction is underway. Pangenome efforts are also nearly complete in sheep and approaching the construction phase for goats. The NIFA-funded developing the Ovine pangenome project has assembled and incorporated 14 breeds into a pangenome: Awassi, Damara, Dorper, Friesian, Katahdin, Merino, Native Churro, Polypay, Romanov, Romney, Shire, St. Croix, Suffolk, and Wiltshire. The goat pangenome is still in development, but has 11 breeds assembled thus far: Alpine, Appenzeller, Boer, Kiko, Saanen, La Mancha, Nigerian Dwarf, Nubian, Spanish, Toggenburg, and Valais Blackneck. The T2T Ruminant project is producing complete, gapless, assemblies across the ruminant clade. The project has released completed genome assemblies for bighorn and giraffe. It has also released the first complete assemblies of the cattle and sheep Y chromosomes. The project has nearly complete assemblies for representatives of all six ruminant families (Antilocapridae, Bovidae, Cervidae, Giraffidae, Moschidae, and Tragulidae). Livestock and closely related assemblies are also nearly complete for cattle (1960’s Holstein, Ayrshire, Charolais, Gyr, Piedmontese, Simmental, Tuli, and Wagyu) and close relatives of cattle (bison, gaur, river buffalo, and yak), sheep (Friesian, Native Churro, and Polypay) and close relatives of sheep (bighorn and muskox), goat (Boer, Kiko, Saanen, and Spanish) and close relatives of goat (chamois and ibex), as well as the newly emerging livestock species elk, red deer and reindeer. Gapless chromosome X and Y assemblies are now completed for cattle, bison, water buffalo, sheep, goats, and red deer. For Objective 1.B, we completed structural variation (SV) analysis using both long-read and short-read data, detecting and validating SVs across platforms in ruminants. SV, a type of genetic variation that includes gene deletions, insertions, and chromosomal rearrangements, was studied for its population genetics and functional characteristics. We created pangenomes for dairy cattle using Holstein and Jersey breeds and developed a Holstein single-breed pangenome variation reference panel (HsPVRP) to improve genotyping and imputation accuracy. SV-based genome-wide association studies (GWAS) identified associations between SVs and key economic traits. SV analysis has recently been expanded to a herd of 1960’s genetics Holstein with unique immune characteristics. This herd is being tested for resistance to HPAI with the goal of identifying SVs associated with any such resistance. Breed-specific pangenomes enhance SV detection, imputation, and the understanding of ruminant traits. These findings fill critical knowledge gaps and provide a foundation for incorporating SV information into future breeding programs, ultimately leading to healthier and more productive animals for the dairy industry globally. For Objective 1C, Assembling MAGs from the rumen microbiome remains challenging due to high microbial diversity, novel taxa, and strain-level heterogeneity. DFRC scientists aim to develop a reproducible pipeline to enhance genome recovery from rumen metagenomes. Initial tests using nanopore-sequenced rumen liquid samples from Jersey (n=4) and Holstein (n=4) cattle followed a standard pipeline: trimming with Porechop, assembly with metaFlye, Racon polishing, binning (MetaBAT2), and QC (CheckM). Holstein samples produced fewer but higher-quality bins (11 high-quality, 48 medium), while Jersey samples yielded many fragmented bins (n=1349) with no high-quality bins. Reassembling bins improved completeness but increased contamination due to strain heterogeneity. Domain-level classification using GTDB-Tk revealed many unclassified or mixed bins. To address this, the team incorporated a 2 kb read-length filter and pre-assembly domain classification with Kaiju, recovering eight high-quality Jersey bins. Current efforts focus on archaeal/eukaryotic detection, host read removal, bin decontamination (Magpurify, GUNC), domain-specific QC, and packaging the pipeline into a Snakemake framework. For Objective 2.A, over 1,300 whole-genome sequenced goats from 115 global breeds were used to develop an imputation pipeline with Beagle 5.3. The pipeline, scripts, and documentation are available at GitHub. Phased data from this panel was shared with collaborators via the VarGoats Consortium and was featured in the preprint “Inferring domestic goat demographic history through ancient genome imputation” (bioRxiv). The pipeline was validated by imputing whole-genome genotypes from a 50K SNP chip for 3,570 goats from Africa, Europe, West Asia, South America, and the U.S. Current efforts focus on expanding the reference panel with more breeds from India and the Americas. Future plans include integrating pedigree data using the long-range haplotype-based tool findhap.f90. This resource supports fine mapping, population genetics, and genomic prediction, aiming to improve goat production traits for global agriculture. For Objective 2.B, Feed efficiency (FE) is a polygenic trait with moderate heritability and has been widely evaluated and reported in dairy cattle. Since feed costs represent one of the largest expenses on a dairy farm, improving FE is a key target for genetic selection. However, the genetic and biological mechanisms underlying FE in dairy cattle remain largely unknown. To identify genomic regions affecting residual feed intake (RFI) and the biological pathways associated with RFI, we performed a single nucleotide polymorphism (SNP) - based genome-wide association study (GWAS), fitting SNPs as fixed, additive, and dominance effects one locus at a time followed by gene set enrichment and pathway analyses. The data set comprised 7,193 Holstein cows with 73,266 imputed genotypes per animal permitting an unprecedented opportunity to ascertain the genetic architecture of the RFI trait. The study identified 43 SNPs with significant additive effects on RFI (P = 2.74×10-5). The top significant SNPs were clustered across Bos taurus autosomes (BTA) 11, 14, 10, 17, 2, 23, 18, 5, 16, and 25, with BTA11 showing the most significant and dense cluster. Specifically, four significant genomic regions on BTA11 were identified, harboring candidate genes previously associated with RFI in dairy cattle. However, no significant regions were associated with dominance effects. Most notably, this study revealed five genes, members of solute carrier (SLC) families SLC4A1AP, SLC5A6, SLC30A3, and SLC35F6, that were near or underlying the top significant SNPs within the 72.82 to 72.86Mb region on BTA11. The SLC genes are involved in several biological mechanisms that may influence RFI in dairy cattle. Subsequently, the nearest genes (within 1Mb) to all 43 significant SNPs were included for pathway enrichment analysis in public databases. These putative genes were found to be involved in many important immune functions, ubiquitination process, thermotolerance, energy metabolism, glucose metabolism, carbohydrate metabolism, neuroendocrine regulation, and energy homeostasis. The findings support the sufficiency of the inclusion of an additive genetic effects in the model for genetic evaluation of RFI in dairy cattle. However, further research with larger numbers of genotyped animals, more phenotypes, and higher density markers may reveal additional loci with additive or dominance effects associated with the RFI trait and lead to new insights into the biological pathways underlying RFI. For Objective 3.A, we investigated the impact of gastrointestinal nematode infection (Ostertagia ostertagi) on DNAm in Holstein steers. This study revealed tissue-specific differential methylation patterns, suggesting a link between methylation changes and immune responses, enhancing the understanding of epigenetic regulation during infection. Further data analysis has led to a submitted paper on tissue- and sex- specific DNAm patterns, with at least two additional manuscripts in preparation. For Objective 3.B, the FarmGTEx Consortium Sheep/Goat project has been working for the last 2 years to create a comprehensive atlas of tissue- specific gene expression and genetic regulation in sheep and goats. Thousands of whole genome and transcript sequencing datasets from over 30 tissues and cell types across multiple breeds were processed, with cis-eQTL mapping completed. The project aims to provide a detailed transcriptome landscape and identify thousands of variants associated with gene expression and alternative splicing in major tissues for sheep and goats.


Accomplishments
1. Construction of complete, gapless genome assemblies through the Ruminant Telomere-to-Telomere Consortium (RT2T). Reference genomes are the digital standardized representation of an organism’s genetic instructions. Breeding better livestock using genomics requires informative reference genomes. Current reference genomes contain errors and missing sequences. This deprives researchers and breeders of important information about variation in many complex genomic regions. Led by ARS scientists in Beltsville, Maryland, and Clay Center, Nebraska, the RT2T is developing new and improved genome assemblies. The Ruminant T2T team has generated nearly complete reference genome assemblies for cattle, sheep, and goats with over 100 times fewer errors and without the numerous gaps present in current genome assemblies. These new references include a complete Y chromosome for the very first time, now available through National Center for Biotechnology Information. Farmers, producers, industry partners, researchers, and policymakers will benefit from these advances through improved breeding tools, healthier herds, and more sustainable livestock production.


Review Publications
Li, C., Xu, J., Zhang, Y., Ding, Y., Zhou, X., Su, Z., Qu, C., Liang, J., Han, Y., Wang, D., Shi, Y., Li, C., Liu, G., Kang, X. 2024. Alternative polyadenylation landscape of longissimus dorsi muscle with high and low intramuscular fat content in cattle. Journal of Animal Science. https://doi.org/10.1093/jas/skae357.
 Fang, L., Teng, J., Lin, Q., Bai, Z., Liu, S., Guan, D., Li, B., Gao, Y., Hou, Y., Gong, M., Pan, Z., Yu, Y., Clark, E., Smith, J., Rawlik, K., Xiang, R., Chamberlain, A.J., Goddard, M.E., Littlejohn, M., Larson, G., Machugh, D.E., O’Grady, J.F., Sorensen, P., Sahana, G., Lund, M., Jiang, Z., Pan, X., Gong, W., Zhang, H., He, X., Zhang, Y., Gao, N., He, J., Yi, G., Liu, Y., Zhao, P., Zhou, Y., Wang, X., Young, R.S., Xia, C., Cheng, H., Ma, L., Cole, J.B., Baldwin, R.L., Li, C., Van Tassell, C.P., Lunney, J.K., Liu, W., Guan, L., Zhao, X., Ibeagha-Awemu, E., Luo, O., Lin, L., Canela-Xandri, O., Derks, M., Crooijmans, R., Godia, M., Madsen, O., Groenen, M., Koltes, J.E., Tuggle, C.K., Mccarthy, F.M., Rocha, D., Amills, M., Clop, A., Ballester, M., Tosser-Klopp, G., Li, J., Fang, C., Fang, M., Wang, Q., Hou, Z., Wang, Q., Zhao, F., Jiang, L., Zhao, G., Zhou, Z., Zhou, R., Liu, H., Li, M., Mo, D., Liu, X., Chen, Y., Yuan, X., Li, J., Zhao, S., Ding, X., Sun, D., Sun, H., Li, C., Jiang, Y., Wu, D., Wang, W., Fan, X., Zhang, Q., Li, K., Yang, N., Hu, X., Liu, G. 2025. The farm animal genotype-tissue expression (FarmGTEx) project. Nature Genetics. https://doi.org/10.1038/s41588-025-02121-5.
 Azam, S., Sahu, A., Pandey, N.K., Neupane, M., Van Tassell, C.P., Rosen, B.D., Gandam, R.K., Rath, S.N., Majumdar, S.S. 2025. Advancing the Indian cattle pangenome: characterizing non-reference sequences in Bos indicus. Journal of Animal Science and Biotechnology. https://doi.org/10.1186/s40104-024-01133-1.
 Gao, Y., Yang, L., Kuhn, K.L., Li, W., Zanton, G.I., Bowman, M.E., Zhao, P., Zhou, Y., Fang, L., Cole, J.B., Rosen, B.D., Ma, L., Li, C., Baldwin, R.L., Van Tassell, C.P., Zhang, Z., Smith, T.P., Liu, G. 2025. Long read and preliminary pangenome analyses reveal breed-specific structural variations and novel sequences in Holstein and Jersey cattle. Journal of Advanced Research. 79:137-150. https://doi.org/10.1016/j.jare.2025.04.014.
 Liu, L., Yi, G., Yao, Y., Liu, Y., Li, J., Yang, Y., Liu, M., Fang, L., Mo, D., Zhang, L., Liu, Y., Niu, Y., Wang, L., Qu, X., Pan, Z., Wang, L., Chen, M., Fan, X., Chen, Y., Zhang, Y., Li, X., Wang, Z., Tang, Y., Huang, H., Yuan, P., Liao, Y., Li, X., Yin, Z., Liu, D., Zhang, D., Zhou, Q., Wu, W., Jiang, J., Gao, Y., Liu, G., Wang, L., Chen, Y., Li, K., Groenen, M.A., Tang, Z. 2024. Multi-omics analysis reveals signatures of selection and loci associated with complex traits in pigs. iMeta. https://doi.org/10.1002/imt2.250.
 Boschiero, C., Beshah, E., Zhu, X., Tuo, W., Liu, G.E. 2024. Profiling genome-wide methylation patterns in cattle infected with Ostertagia ostertagi. International Journal of Molecular Sciences. 26(1).Article e89. https://doi.org/10.3390/ijms26010089.
 Hu, Z., Boschiero, C., Neupane, M., Bhowmik, N., Yang, L., Kilian, L., Dejarnette, M., Sargolzaei, M., Harstine, B., Li, C., Tuo, W., Baldwin, R.L., Van Tassell, C.P., Sattler, C.G., Liu, G.E. 2025. Exploring tissue- and sex-specific DNA methylation in cattle using a pan-mammalian infinium array. International Journal of Molecular Sciences. 26(9).Article e4284. https://doi.org/10.3390/ijms26094284.
 Boschiero, C., Neupane, M., Yang, L., Schroeder, S.G., Tuo, W., Ma, L., Baldwin, R.L., Van Tassell, C.P., Liu, G. 2024. A pilot detection and associate study of gene presence-absence variation in Holstein cattle. Animals. https://doi.org/10.3390/ani14131921.
 Wang, X., Zhou, X., Li, C., Qu, C., Shi, Y., Li, C., Kang, X. 2024. Integrative analysis of whole genome bisulfite and transcriptome sequencing reveals the effect of sodium butyrate on DNA methylation in the differentiation of bovine skeletal muscle satellite cells. Genomics. https://doi.org/10.1016/j.ygeno.2024.110959.
 Gao, Y., Liu, G., Ma, L., Fang, L., Li, C., Baldwin, R.L. 2024. Transcriptomic profiling of gastrointestinal tracts in dairy cattle during lactation reveals molecular adaptations for milk synthesis. Journal of Advanced Research. https://doi.org/10.1016/j.jare.2024.06.020.
 Niu, Q., Wu, J., Wu, T., Zhang, T., Wang, T., Xu, Z., Zhao, Z., Xu, L., Wang, Z., Zhu, B., Zhang, L., Gao, H., Liu, G., Li, J., Xu, L. 2025. Comprehensive multi-omics analysis of regulatory variants for body weight in cattle. Genomics, Proteomics and Bioinformatics. https://doi.org/10.1093/gpbjnl/qzaf067.
 Cai, J., Yang, L., Gao, Y., Liu, G., Da, Y., Ma, L. 2025. Selection signature analysis of whole-genome sequences to identify genome differences between selected and unselected Holstein cattle. Animals. https://doi.org/10.3390/ani15152247.
 Gu, L., Peng, C., Chen, A., Chen, K., Zheng, X., Yu, D., Wang, Z., Fang, L., Liu, G., Zhao, P. 2025. Haplotype-resolved genome and pan-genome graphs reveal the impacts of structural variation on functional genome and feather colors in chickens. iMetaOmics. https://doi.org/10.1002/imo2.70027.
 Dai, S., Zhao, P., Li, W., Peng, L., Jiang, E., Du, Y., Zhang, W., Dai, X., Yang, L., Li, Z., Xu, L., Lan, X., Lyu, W., Yang, L., Fang, L., Liu, G., Zhou, Y. 2025. Global pangenome analysis highlights the critical role of structural variants in cattle improvement and identifies a unique event as a novel enhancer in IGFBP7+ cells. Molecular Biology and Evolution. https://doi.org/10.1093/molbev/msaf205.
 Han, B., Li, H., Zheng, W., Zhang, Q., Chen, A., Zhu, S., Shi, T., Wang, F., Zou, D., Song, Y., Ye, W., Du, A., Fu, Y., Jia, M., Bai, Z., Yuan, Z., Liu, W., Tuo, W., Hope, J.C., MacHugh, D.E., O’Grady, J.F., Madsen, O., Sahana, G., Luo, Y., Lin, L., Li, C., Cai, Z., Li, B., Huang, J., Liu, L., Zhang, Z., Ma, Z., Hou, Y., Liu, G., Jiang, Y., Sun, H., Fang, L., Sun, D. 2025. A multi-tissue single-cell expression atlas in cattle. Nature Genetics. https://doi.org/10.1038/s41588-025-02329-5.