Source: UNIV OF IDAHO submitted to NRP
DEVELOPMENT OF THE OVINE PANGENOME
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1024811
Grant No.
2021-67016-33416
Cumulative Award Amt.
$500,000.00
Proposal No.
2020-02844
Multistate No.
(N/A)
Project Start Date
Jan 1, 2021
Project End Date
Dec 31, 2024
Grant Year
2021
Program Code
[A1201]- Animal Health and Production and Animal Products: Animal Breeding, Genetics, and Genomics
Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803
Performing Department
(N/A)
Non Technical Summary
The human and plant research communities have identified substantial numbers of genes, not present, in the reference genome, demonstrating the need for development of pangenome resources. Available sequence data in sheep suggest that diversity of ovine genomes among existing global populations will exceed that of the human species. This project aims to deliver high-quality genomes, using the recently describe trio binning approach, for eight breeds/species of sheep selected for their divergent economically important traits which will serve as the basis of the ovine pangenome.This research effort will leverage complementary efforts including genome assembly, transcriptome, and functional annotation efforts, and provide training for students in the field of animal genomics. Further, it will enhance efforts to identify structural and sequence variants that influence phenotypic variation in economically important traits of sheep. The ovine pangenome project will increase our understanding of how different genomes and gene products from diverse breeds of sheep affect a variety of important biological phenotypes, supporting more accurate prediction of traits and improving breeding strategies. The pangenome will contribute to comparative studies across species, while complementing and leveraging the pangenome being constructed for cattle.
Animal Health Component
0%
Research Effort Categories
Basic
85%
Applied
0%
Developmental
15%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3043610108075%
3033699108125%
Knowledge Area
303 - Genetic Improvement of Animals; 304 - Animal Genome;

Subject Of Investigation
3610 - Sheep, live animal; 3699 - Sheep and wool, general/other;

Field Of Science
1081 - Breeding; 1080 - Genetics;
Goals / Objectives
The goals of this project are to: 1) Deliver high-quality genomes, using the recently described trio binning approach, for eight breeds/species of sheep selected based upon their divergence for economically important traits which will serve as the basis of the ovine pangenome. As a part of this goal we will characterize genomic regions of different ovine breeds/species, thereby identifying breed specific defining genomic regions associated with a range of phenotypically distinct and important biological traits. The assembly of multiple genomes will allow us to compare and contrast genomes that are known to result in phenotypic traits of importance. 2) Facilitate functional annotation of transcript expression in fetal tissues from F1 crosses of genetically divergent sheep breeds. Here we will deliver fetal transcriptomic data across a range of tissues for comparative developmental transcriptomic analyses and characterize the ovine transcriptome and the expression signatures of fetal compared to adult tissues. 3) Deliver genetic resources as a tool for studying the ovine genome through online databases, including those supported by the international FAANG Consortium and during this process, train the next generation of animal scientists.
Project Methods
For this project we will employ the previously proven trio binning method using four crosses. For each of the designated crosses, mature ewes will be estrous synchronized and mated using standard breeding protocols, of either natural service or with semen from the specified ram. To reduce the potential of failure to generate offspring a minimum of two ewes of the same breed will be mated to an individual designated breeding ram to generate a cross-bred offspring.At 100 days post insemination and subsequent to ultrasound confirmed pregnancy, the ewes will be humanely euthanized by a veterinarian and the fetuses will be retrieved. Fetal tissues will be dissected, collected in sterile cryotubes and frozen in liquid nitrogen. While we will collect 20 tissues four (lung, liver, muscle and whole brain) will be used in this project. The same tissues as those collected from the fetuses will also be collected from the ewes and immediately placed in cryotubes and frozen in liquid nitrogen storage for subsequent DNA extraction, sequencing and analyses.

Progress 01/01/21 to 12/31/24

Outputs
Target Audience:The ovine pangenome project primarily targets animal scientists, students, and innovative producers and industries. This year, the key goal was to create the pangenome by combining de novo ovine haploid genomes with all publicly available high-quality assemblies. The assemblies from the F1 crosses surpassed expectations, with several chromosomes being assembled from telomere to telomere. These data also played a role in launching the ruminant telomere-to-telomere project. We have communicated the project's purpose, goals, and some of the results to fellow scientists through various platforms on multiple occasions. Changes/Problems:Sequencing technologies and genome assembly programs have been progressing rapidly, and it was a challenge to keep our data and analyses current. What opportunities for training and professional development has the project provided?This project has provided the opportunity to train team members and students in proper tissue identification and collection protocols for specific functional assays and sequencing. The tissue collection provided experimental learning opportunities and formal training for veterinarians, graduate and undergraduate students, post-doctoral fellow for experimental assay and analyses. Furthermore, tissues collected for this grant have been leveraged for several additional projects. Furthermore, collaborators from the human Telomere-to-Telomere (T2T) consortium have offered valuable input and contributed by providing additional training in T2T genome assembly to interested members of the project. How have the results been disseminated to communities of interest?This project has been shared with the scientific community in several forms. We have delivered no less than ten invited talks and seminars about the development of the ovine pangenome. The data and analyses of these genome assemblies have been leveraged by the ruminant telomere to telomere consortium and resulted in three workshops centered around the new technologies and advances in genome assemblies. Additionally, no less than ten abstracts and poster presentations have been shared by a variety of graduate students, postdoctoral fellows, and project members. Finally, so far, we have published high profile two manuscripts with another two in various stages of preparation. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1) In this objective we committed to delivering eight new breed/species assemblies. Ultimately, we produced 13 new de novo haploid Ovis genome assemblies. Combined with our previous contributions to Ovis aries, we have now compiled 25 high-quality Ovis genome assemblies, spanning 4 species and 13 within-species (Ovis aries) assemblies, to create the first ovine super pangenome. The assemblies generated using either Verkko or Hifiasm were highly contiguous, with sizes ranging from 78.6 to 111.0 Mb. The total size of these assemblies varied from 2.77 to 3.04 Gb, with some being telomere-to-telomere (T2T). To date the bighorn T2T genome assembly has been submitted and is available on NCBI (ARS-UI_OviCan_v2), and the Ovis aries reference genome assembly has been updated to include the complete Y chromosome. Objective 2) We targeted four tissues (lung, muscle, liver, and spleen) for all four crosses generated under the grant, and collected data for ten RNA Isoseq (Kinnex), along with an additional 12 tissues for one cross. The analysis of these transcript data is currently in its final stages, and we expect this work to result in a manuscript authored by Dr. Emily Clark. Objective 3) An ovine super-pangenome, providing a comprehensive representation of the genetic diversity within the Ovis Genus, was created by combining genomes from a variety of individual sheep across different species (n=4), breeds (n=22), environments, and geographic regions. This pangenome, built using assemblies with greater contiguity than the current sheep reference (ARS-UI_Ramb_v3.0), was constructed through two approaches: Pangenome Graph Builder (PGGB) (a reference-agnostic method) and Minigraph-cactus (a reference-based approach). The super-pangenome can be applied to inform genomic selection, aiding breeders in selecting individuals with superior traits, enhanced production efficiency, and greater environmental adaptability. By developing this super-pangenome, scientists can catalog more genetic variation within sheep species, improving the understanding of how specific genes contribute to important phenotypic traits, such as disease resistance, production characteristics, and climate adaptation to environmental stressors.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Olagunju TA, Rosen BD, Neibergs HL, Becker GM, Davenport KM, Elsik CG, Hadfield TD, Koren S, Kuhn KL, Rhie A, Shira K, Skibiel AL, Stegemiller MR, Thorne JW, Villamediana P, Cockett NE, Murdoch BM* and Smith TPL*. The first complete T2T Assemblies of Cattle and Sheep Y-Chromosomes uncover remarkable divergence in structure and gene content. Nat Comm (2024) Apr 3:rs.3.rs-4033388. DOI: 10.21203/rs.3.rs-4033388/v1.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Kalbfleisch T*, McKay SD*, Murdoch BM*, et al., Smith TPL. RT2T: a global collaboration project to study chromosomal evolution in the suborder Ruminantia. 2024-02-05 | Preprint DOI: 10.21203/rs.3.rs-3918604/v1 Nature Genetics (2024).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Murdoch BM, Olagunju T, Clark E, Archibald A, Mousel M, McKay S, Hagen D, Xie S, Hess A, Kalbfleisch T, Heaton M, Freking B, Murphy T, Bickhart D, Clarke S, Brauning R, Hadfield T, Cockett N, Rosen B, Smith T. The Sheep Pangenome Project. International Sheep Genome Consortium. Jan 13, 2025.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Olagunju T, Clark E, Archibald A, Mousel M, McKay S, Hagen D, Xie S, Hess A, Kalbfleisch T, Heaton M, Freking B, Murphy T, Bickhart D, Clarke S, Brauning R, Hadfield T, Cockett N, Rosen B, Smith T., Murdoch BM. An Ovine Super-Pangenome to Characterize Genetic Diversity in Sheep. International Plant and Animal Genome Conference. Jan 11, 2025
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Olagunju TA, Murdoch BM, Xie S, Clark E, Archibald A, Mousel M, McKay SD, Hagen D, Hess A, Kalbfleisch TS, Heaton MP, Freking B, Murphy T, Bickhart D, Clarke S, Brauning R, Hadfield T, Cockett N, Rosen BD, Smith TPL. An ovine pangenome to characterize genetic diversity in sheep breeds. AGBT Ag. Orlando, Florida, April 17, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Olagunju T, Rosen BD, McKay S, Smith T Murdoch BM. Challenges and Opportunities in Genome Processing Using NCBI Tools. International Plant and Animal Genome Conference Jan 13, 2025.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Murdoch BM, Olagunju T., Rhie A., Koren S., Stegemiller M., Hadfield T., Neibergs H.L., Cockett N.E., Rosen B.D., Smith T.P.L. Centromere content and organization in cattle and Sheep Y chromosomes are considerably different than in the human Y chromosome. ABGT General meeting, Orlando, Florida, February 7, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Murdoch BM. The sheep pangenome project. International Sheep Genome Consortium, San Deigo, California, January 15, 2024
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Olagunju TA, Rosen BD Rhie A, Koren S, Stegemiller M, Shira K, Hadfield T, Neibergs HL, Cockett NE Smith TPL and Murdoch BM. Analysis of T2T Y-Chromosome Assemblies of Cattle and Sheep Uncovers Novel Sex Chromosome-Specific Centromeric Satellite Repeats. The International Plant and Animal Genome. San Deigo, California, January 14, 2024


Progress 01/01/23 to 12/31/23

Outputs
Target Audience:The target audience for the ovine pangenome project are primarily animal scientists, students and progressive producers and industries. This year, the main goal was to generate sequence data and assemble the ovine haploid assemblies. The resulting assemblies from the F1 crosses were higher quality than anticipated with several of assembled chromosomes being telomere to telomere. These data aided with the initiation of the ruminant telomere-to-telomer project. We have described the purpose, goals and some of the preliminary results of the project to fellow scientists on numerous occasions. We have given several oral presentations to the National Animals Genome Research Program, the International Sheep Genome Consortium, and the International Society for Animal Genetics. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided the opportunity to train team members and students in proper tissue identification and collection for specific functional assays and sequencing. Furthermore, collaborators from the human telomere to telomere (T2T) consortium have provided beneficial input and helped by providing some additional training in T2T genome assembly to a few members of the investigator team. Tissues collection efforts of this project have provided experimental learning opportunities in proper tissue identification and collection and formal training in tissue sampling to graduate students and analyses for a post doctoral fellow. Furthermore, tissues collected for this grant have been leveraged for several additional projects. How have the results been disseminated to communities of interest?We have used shownbandage plots of sheep haploid assembliesin talks to the scientific community. What do you plan to do during the next reporting period to accomplish the goals?This next year we will finish the genomes assemblies. Additionally, we will finish the RNA sequencing for the designated tissues from each of the crosses. We are in the process of completingthe assemblies andwill begin the comparative pangenome graphs this year.

Impacts
What was accomplished under these goals? PacBio and Oxford Nanopore sequence data has been generated from lung tissue for each of the four F1 trio crosses. Parental samples (tissue for the ewes and semen for the rams) have been sequenced using Illumina short read sequencing and parental K mers have been generated for all except the Suffolk and bighorn rams. Furthermore, we have used Hifiasm and Verkko to generate eight breed specific haploid genome assemblies.

Publications


    Progress 01/01/22 to 12/31/22

    Outputs
    Target Audience:The target audience for the ovine pangenome project are primarily animal scientists and students. This year, the main goal was to generate sequence data and assemble the eight haploid assemblies. The resulting assemblies from the F1 crosses were higher quality than anticipated with several of assembled chromosomes being telomere to telomere. These data aided with the initiation of the ruminant telomere-to-telomer project. We have described the purpose, goals and some of the preliminary results of the project to fellow scientists on numerous occasions. We have given several oral presentations to the National Animals Genome Research Program, the International Sheep Genome Consortium, and the International Society for Animal Genetics. Tissues collection efforts of this project have provided experimental learning opportunities in proper tissue identification and collection and formal training in tissue sampling to graduate students. Furthermore, tissues collected for this grant have been leveraged for several additional projects. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided the opportunity to train team members and students in proper tissue identification and collection for specific functional assays and sequencing. Furthermore, collaborators from the human telomere to telomere (T2T) consortium have provided beneficial input and helped by providing some additional training in T2T genome assembly to a few members of the investigator team. How have the results been disseminated to communities of interest?We have used the bandage plots of sheep haploid assembly data in talks to the scientific community. What do you plan to do during the next reporting period to accomplish the goals?This next year we will finish the genomes assemblies. Additionally, we will finish the RNA sequencing for the designated tissues from each of the crosses. Following the completion of the annotated assemblies we will begin the comparative genomic analyses.

    Impacts
    What was accomplished under these goals? PacBio and Oxford Nanopore sequence data has been generated from lung tissue for each of the four F1 trio crosses. Parental samples (tissue for the ewes and semen for the rams) have been sequenced using Illumina short read sequencing and parental K mers have been generated for all except the Suffolk and bighorn rams. Furthermore, we have used Hifiasm and Verkko to generate eight breed specific haploid genome assemblies.

    Publications


      Progress 01/01/21 to 12/31/21

      Outputs
      Target Audience:The target audience for the ovine pangenome project are primarily animal scientists and students. In the first year of this project, we mainly focused on describing the purpose and the goals of the project to fellow scientists. I have given oral presentations to the National Animals Genome Research Program and the International Sheep Genome Consortium at their annual meetings. Multiple efforts towards this project include providing experimental learning opportunities in proper tissue identification and collection and formal training in tissue sampling to graduate students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided the opportunity to train team members and students in proper tissue identification and collection for specific functional assays and sequencing. Collaborators from the human telomere to telomere (T2T) consortium have provided beneficial input and helped by providing some additional training in T2T genome assembly to a few members of the investigator team. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?This next year we will finish the DNA sequencing and complete the genomes assemblies. Additionally, we will finish the RNA sequencing for the designated tissues from each of the crosses. Following the completion of the annotated assemblies we will begin the comparative genomic analyses.

      Impacts
      What was accomplished under these goals? In the first year of this project, we have performed all the specific breeding crosses and have collected all the tissues outlined in the project.We are in the process of conducting the specific DNA sequencing using Pac Bio - HiFi, Oxford Nanopore and Illumina sequencing platforms for the tissues.

      Publications