Progress 01/01/24 to 12/31/24
Outputs
Target Audience:Our work's target audience spans basic science researchers all the way to producers. One of the most under-emphasized tools for improving beef cattle productivity is utilizing heterosis. As such, we undertook this somewhat basic project to help explain to producers how the phenomenon works mechanistically. Producers are willing to adopt new (or in the case of heterosis, old) approaches when they area explainable and understandable. Our work in this controlled system is working to help explain the "how" and "why" for procuers. Additionally, this work is of immediate interest to a wide range of the scientific community. Limited work has occured on the mechanisms of heterosis in animal systems, so this work will be of interest to a wide range of the scientific community. Researchers working on understanding gene expression variation will be interested in the datasets created by this project that span two subspecies of cattle at an understudied timepoint of mammalian development. Animal breeders who design crossbreeding programs may also find this work useful as they understand how the directionality of crossing may vary, or the prevalence of parent-of-origin effects. Because we are performing this work in a lab setting, it allows us to remove much of the environmental noise that can mask some of these effects in organism-level studies. The big picture trends identiifed in this work can lay the foundation for further work across species and related to more specific phenotypes. Changes/Problems:No major changes to the project. A no-cost extension has been granted already that will extend the project through the end of 2025. What opportunities for training and professional development has the project provided?This project has involved a variety of graduate and undergraduate students in its execution. Three graduate students have worked directly on data collection, generation, and analysis. Elements of this project will serve as the major focus of one MS degree and one PhD. Two of these students have presented elements of this research at international conferences (Plant and Animal Genome, AGBT-Ag). Additionally, the project has served as a great training opportunity for undergraduate researchers. At least three undergraduate students have played important roles in the project's data generation and analysis. It has been a fantastic teaching tool, and great way to engage students in genomics research. How have the results been disseminated to communities of interest?We have delivered multiple presentations on the ongoing developments of this research. Students presented results of this work at the Plant and Animal Genome (PAG) meeting which includes a wide variety of interested scientists from both the plant and animal genetics communities. Dr. Rowan has also included some of the high-level findings in his extension programming related to crossbreeding. This has reach producer audiences around the US. He has also presented findings form this project to multiple university groups at invited seminars (Kansas State University, Purdue University, University of Guelph). What do you plan to do during the next reporting period to accomplish the goals?The next reporting period will tie up final data generation (parental WGS) and wrap up the final elements of Objective 3 (eQTL mapping). We have extracted DNA and are working to prepare libraries for sequencing of parental WGS data. Following sequencing, embryo imputation will occur and we'll work to finalize our eQTL mapping. Once this final element is finished we will work to tie together each element of this work into a cohesive story for presentation and guidance for future exploration. The other major focus will be on publishing final papers and presenting result of this work to relevant scientific audiences. We will continue presenting at national and international conferences. At least two publications are currently in progress and we anticipate having them submitted by September 2025.
Impacts
What was accomplished under these goals?
We generated 306 total embryos (purebred Angus, purebred Brahman, and reciprocal F1s) that allow us to probe gene expression differences that may be the result of heterosis. We collected > 1000 oocytes from purebred Brahman and Angus cows and then used in vitro fertilization with 3 sires from each breed to make these pools of embryos. The number of individuals in each class was approximately the same, and developmental rate and embro quality were largely the same. During this reporitng period we were able to generate whole transcriptomes for each embryo, averaging ~25 million reads per sample. We have modeled these differences and identified hundreds of genes that show non-additive expression (i.e. significant F1 deviations from parent average). Finally, we have extracted DNA from parental samples that will be used to generate WGS data for imputation of embryo transcriptomes. This will enable the mapping of additive and non-additive molecular QTL.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
GL Franklin, TB Seay, JL Edwards, RR Payton, JE Beever, TN Rowan. 2024. Using Embryos to Dissect Gene Expression Heterosis in Beef Cattle. Plant and Animal Genome Conference/PAG 31 (January 12-17, 2024).
- Type:
Theses/Dissertations
Status:
Awaiting Publication
Year Published:
2024
Citation:
GL Franklin. 2024. Using Embryos to Dissect Molecular Gene Expression Heterosis in Beef Cattle. University of Tennessee MS Thesis.
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:The target audiences for this research project mostly fall into two groups that reflect the applied and basic nature of the work: 1. Beef Cattle Producers - The work performed by this project has been presented at multiple producer field days and events throughout Tennsssee and the rest of the country. 2. Academic Researchers in Livestock Genomics - We have shared various aspects of this work at scientific conferences for agricultural genomics, as well as genomics conferences with broad constituencies (human, model organism, agricultural, etc.) Additional work has included an increased number of undergraduate and graduate students in this project. Changes/Problems:We received a no-cost extension to allow us to carry out the objectives of the project that were delayed due to difficulty in finding purebred Brahman oocytes. This has been accomplished and we are on track to be completed by the end of the year 2024. What opportunities for training and professional development has the project provided?Four graduate students have worked on various aspects of the project. They have received broad exposure to the production of embryos in vitro (something outside the scope of our lab), sequencing approaches, and library preparations, as well as the computational follow-up analyses that make up the crux of this project. They are present and hands-on in all aspects of data creation and analysis. The lead student has presented the results from this project at an international meeting (Gordon Research Conference on Quantitative Genetics and Genomics) where he received excellent feedback from a number of leading researchers across species and genomics disciplines. In addition, this project has provided an opportunity for undergraduate students to become involved in research. They have helped with procedures in the lab during IVP, as well as in assisting in follow-up data analysis. How have the results been disseminated to communities of interest?We have presented preliminary results from this work at the Gordon Research Conference on Quantitative Genetics and Genomics (Ventura, California). Additionally, graduate students have presented at internal events, including the University of Tennessee's Beef and Forage Center Annual Research Meeting. This meeting brings together producers and extension personnel to expose them to all research related to beef and forage production in the state. This work has also been the foundation for multiple producer-focused presentations on crossbreeding and heterosis. While these talks focus on the more applied nature of crossbreeding, the motivations for this work were shared. During the reporting period, five presentations on heterosis were delivered to producers in Tennessee (450+ producers), as well as in Estonia and Finland (~150 producers) during an international visit. What do you plan to do during the next reporting period to accomplish the goals?1. Generate a powerful dataset of purebred and crossbred samples in-vitro to explore gene expression heterosis All data generation is complete 2. Identify genes that deviate from parent-average expression in F1 individuals We are in the process of analyzing these gene expression differences among purebred and crossbred embryos. This will continue throughout the 2024 reporting period. We expect to publish a manuscript describing this cursory analysis of the crossbred/purebred gene expression differences using the320 blastocysts generated by objective 1. 3. Map molecular QTL (additive & non-additive) to identify the genetic variants involved in molecular heterosis. In the next year, we will use whole genome sequence information on our embryo's parents to impute non-genic genomic regions into the transcriptomic data. This will allow us to carry out the proposed eQTL analysis. This work is ongoing.
Impacts
What was accomplished under these goals?
1. Generate a powerful dataset of purebred and crossbred samples in-vitro to explore gene expression heterosis We completed all data generation for the project immediately before Christmas break 2023. Following sire testing in our in-vitro embryo culture system in year 1, we generated the project's final data using abattoir-collected oocytes from known registered Angus (AN) and Brahman (BR) females. We performed in-vitro embryo production with these, resulting in a total of 320 blastocysts (approximately 1/4 of each AN x AN, BR x BR, AN x BR, BR x AN). We have generated whole transcriptomes for each of these animals (~20 million reads/sample). This is 120 more blastocysts than proposed in the initial project, providing further power to identify gene expression heterosis. All data has been processed into filtered and normalized reads for use in Objective 2 and 3's analysis. 2. Identify genes that deviate from parent-average expression in F1 individuals We have begun analyzing data to identify non-additive offspring gene expression. This analysis is ongoing, but all processing pipelines and differential gene expression models have been set up for the previous analysis of testing data. Students and computational research associate have produced flexible workflows that are being used on this data and other RNA-Seq datasets generated by our lab. 3. Map molecular QTL (additive & non-additive) to identify the genetic variants involved in molecular heterosis. We have also generated whole genome sequence on all of the sires and dams of our purebred and crossbred blastocysts. This WGS data is being processed now and will be used to impute non-genic regions in transcriptomes making them useful for eQTL mapping.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Franklin G., Seay T., Edwards J.L., Payton R., Beever J.E., Rowan T.N. 2023. Dissecting Molecular Heterosis in Beef Cattle Using Embryos. Gordon Research Seminar and Conference (Ventura, California).
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:The work performed during this reporting period is focused primarily on members of the beef industry. This has primarily included communicating early results and the concepts of this research to academic audiences interested in animal breeding and genetics. I was invited to speak about our group's research at three separate university seminars (Universities of Florida, Nebraska, and Guelph). These audiences included investigators, students, and postdocs broadly interested in animal genetics. Further, with my 30% Extension appointment, I was also able to deliver information regarding heterosis in beef cattle to a wide variety of producer audiences. This included multiple talks about heterosis during Master Beef Producer meetings in Tennessee and national-level producer meetings. Changes/Problems:To maximize the potential sample size of embryos for our project we opted to use opportunistic collections of ovaries at slaughter facilities for ourin-vitrofertilization procedures. This has allowed us to generate more embryos over shorter amounts of time than performing ovum pick-up on live cows. We have had to do this collection opportunistically, (i.e., find when and where producers are taking cull cows to facility and collect on that schedule). This has made finding large groups of cull Brahman cows more difficult in East Tennessee, but we have identified opportunities to collect and ship oocytes from other states to our location. This shouldn't significantly alter the timeline of the project. What opportunities for training and professional development has the project provided?This project is being executed by two graduate students in the Rowan Lab. Since they are new students the data generated here has allowed them to build data analysis pipelines from the ground up. With assistance from PI Rowan they have developed flexible workflows for analyzing RNA-Seq data. This has also provided opportunities for exposing genetics-trained students to reproductive physiology (i.e.,in-vitroembryo production) and vice-versa (i.e.,heterosis, next-generation sequencing, etc.). Students have had the opportunity to present the preliminaryresults of this work at the UT Beef and Forage Center Research and Recommendations Meeting in December 2022. This allowed them to present both to a scientific audience (other UT faculty) and to members of the wider beef industry (producers and extension agents). How have the results been disseminated to communities of interest?Both PI Rowan and graduate students have shared results from this work with various producer audiences. This includes producers through extension programming like the Tennessee Master Beef Producer Program and UT Beef and Forage Center. Sharing this project has helped producers understand the complex mechanisms that underlie heterosis. While not directly related to the results of this project, Dr. Rowan's Extension program focuses heavily on relaying the importance of crossbreeding and heterosis to producer audiences. Ongoing work will help bolster this arm of his programming. We have also made an effort to share with a wide scientific audience. This has included presentations at multiple university genetics/genomics seminars (University of Nebraska, University of Florida, and University of Guelph). Scheduled Conferencesin 2023 will provide further opportunities to share the concept and results of this research. What do you plan to do during the next reporting period to accomplish the goals?Two major outstanding pieces exist that will need to be addressed in the upcoming reporting period. 1) Completing IVF crosses with Brahman oocytes: Our initial work in testing bulls has focused on fertilizing Angus oocytes with Angus and Brahman semen. Subsequent rounds of IVF will require oocytes from Brahman cows to complete the diallel-like cross proposed. This will allow us to more completely model the various types of non-additive gene expression (dominant, overdominant, parent-of-origin specific, allele-specific expresison,etc.) that we would expect to drive heterosis. 2) Perform molecular QTL mapping: With the full set of embryo crosses made, we will use sequenced parental genomes (from semen and ovary tissues,also ongoing) to impute missing genotypes into the transcriptomes. Using~200 transcriptomes accurately imputed to sequence-level density using parental genomes as references we will perform expression QTL mapping. This will use both additive and non-additivemodels to understand how underlying genetic variants drive gene expression and/or gene expression heterosis. In addition to these two major objectives that will be completed in the upcoming year, we plan to share the results of this work extensively at scientific conferences and in publications.
Impacts
What was accomplished under these goals?
We have made significant progress on the initial steps of identifying patterns of heterotic gene expression in bovine embryos. To simplify the process of generating these crosses we've opted to use abattoir-collected oocytes for in-vitro fertilization (IVF). This has allowed us to substantially increase sample sizes (compared with ovum pickup procedures). We used ovaries from Angus-influenced cull cows to test the efficacy of 10 total sires (5 Angus and 5 Brahman) inCo-PI Edwards' in-vitro embryo culture system. We identified three bulls from each breed with exceptional performance. Ongoing rounds of IVF are utilizing these sires to maximize the number of embryos produced while maintaining appropriate amounts of genetic diversity across fertilizations. We also used embryos from the three rounds of sire testing to demonstrate our ability to generate transcriptomes.To date we have produced transcriptomes for 90 embryos (59 Angus x Angus and 31 Brahman x Angus). These samples all generated adequate amounts of total RNA that was sequenced with an average of 25 million reads. Despite developing over the same time period, ~15% of embryos had developed to only the morula stage (rather than to mature blastocysts). This developmental phenotype will be an important aspect for us to keep track of and appropriately model during differential gene expression analyses. Despite having only "half" of the diallel-designed cross completed, graduate students have built multiple analysis pipelines for processing RNA-Sequencing data and searching for differentially-expressed genes. This pipeline has allowed new data to be integrated seamlessly and is easily modified as modeling becomes more complex. To enable molecular QTL mapping, we are planning to sequence all parental animals to moderate coverage for optimizing the accuracy of imputation of intergenic regionsinto offspring transcriptomes. For all animals tested thus far we have collected tissue samples and extracted DNA. Sequencing of these samples is ongoing.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Rowan TN. 2022. Invited Review: Genetic decision tools for increasing cow efficiency and sustainability in forage-based beef systems. Applied Animal Science. 38 (6), 660-670.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Franklin G, Mohamed R, Seay T, Beever JE, Rowan TN. 2022. Using Embryos to Dissect Gene Expression Heterosis in Beef Cattle. UT Beef and Forage Center Research Report.
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