FACILITATING THE GENERATION OF A BOVINE METHYLATION ARRAY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1008414
• Grant No.: 2016-67016-24766
• Project No.: VT-0074CG
• Proposal No.: 2015-07167
• Program Code: A1201
• Project Start Date: Jan 15, 2016
• Project End Date: Jan 14, 2020
• Grant Year: 2016

Project Director
McKay, S.

Recipient Organization
UNIVERSITY OF VERMONT
(N/A)
BURLINGTON,VT 05405

Performing Department
Sponsored Project Administrati

Non Technical Summary
Genetic and epigenetic factors contribute towards phenotypic variation in complex traits. Methylation is an epigenetic modification of DNA that regulates gene transcription and has been shown to influence variation in economically important phenotypes in agricultural species. The proposed project is designed to facilitate the generation of a bovine methylation array by discerning similarities and differences of nucleotide methylation between breeds of cattle. To accomplish this goal, we shall have genome-wide methylation sequencing performed on DNA extracted from muscle tissue of 18 cattle from the Angus and Hereford breeds (9 per breed). Subsequently, we will identify differentially methylated regions that are conserved between two breeds of cattle. Our long term goal is to utilize the knowledge and data gained from the proposed work to generate a bovine methylation array similar to the Illumina Infinium Human Methylation 450K BeadChip. This tool will enable genome wide epigenetic surveys to be performed in order to determine the epigenetic contribution towards phenotypic variation in complex traits and diseases. The proposed research employs an innovative epigenetic approach that focuses on application of genome-wide methods for identification of gene regulatory regions.

Animal Health Component

100%

Research Effort Categories

Basic

50%

Applied

(N/A)

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
304	3399	1080	100%

Knowledge Area
304 - Animal Genome;

Subject Of Investigation
3399 - Beef cattle, general/other;

Field Of Science
1080 - Genetics;

Keywords

methylation

sequencing

array

bovine

Goals / Objectives
The overall goal of this proposal is to facilitate the generation of a bovine methylation array by discerning similarities and differences of nucleotide methylation between breeds of cattle. In order to achieve this goal the following specific aims will be accomplished: 1. Whole genome bisulfite sequence DNA extracted from muscle tissues of 18 cattle from the Angus and Hereford breeds (9 cattle per breed). 2. Examine conservation and variation of methylation between Angus and Hereford cattle.

Project Methods
The methods for each objective is as follows: 1. Genomic DNA will be extracted from the muscle tissue of eighteen cattle. The DNA methylation profiles will be obtained through commercially out-sourced, genome-wide, bisulfite sequencing analysis. This sequencing platform is expected to target the detection of 8-9 million unique CpG sites at a 5-10X coverage. This sequencing methodology includes coverage of a majority of genetic regulatory elements, gene bodies, repeat DNA sequences, CpG sites and CpG islands. Once, the sequence reads are aligned to the reference genome, methylated CpG sites will be identified and levels of methylation will be calculated for each cytosine in the genome. 2. Analysis will be undertaken to epigenetically characterize breed similarities and differences. These analyses include the identification of differentially methylated regions, and characterizing patterns of nucleotide methylation. Regions of differential methylation will be identified using the list of methylated CpG sites previously identified and a combination of calling software: SAMTools, GATK and custom software. DMEAS will be used to analyze the distribution of DNA methylation patterns. Previously identified differentially methylated regions will be analyzed to determine if conservation exists between Angus and Hereford cattle. An in-house script will be generated to identify regions of overlap between breeds of cattle that contain differentially methylated regions. These regions will be considered conserved.

Progress 01/15/16 to 01/14/20

Outputs
Target Audience:Data has been reported at scientific meetings to the global animal genomics community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Suraj Bhattarai has been working on this project since 2018. He has learned and developed protocols for COBRA, Bisulfite Sanger Sequencing, and Bisulfite Pyrosequencing. He has also learned the WGBS pipeline that was established. He attended the FAANG Bioinformatics Training Workshop in San Diego, CA in Janurary 2020. Finally, Suraj has attended PAG each year that he has been in the lab and has presented data and networked with the global animal genomics community. How have the results been disseminated to communities of interest?Reuslts have been disseminated to the community via poster and oral presentations at ISAG, ISAFG and PAG. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Whole genome bisulfite sequencing was performed on liver tissues from Angus, Hereford, Nellore . Additionally, WGBS was also performed on muscle tissues from Angus and Hereford cattle. For comparative genomics purposes, tissues from species other than cattle have been sequenced and they include liver, spleen and cerebellum samples from sheep and liver samples from Bison. A data analysis pipeline was established and is in use for processign WGBS data. Differential methyaltion was performed between for liver and muscle tissues in Angus and Hereford sampels. Converation of methyaltion is currently underway with the sheep, nellore and bison.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 3. Cantrell, B, S Liu, N Jebbett, RC Switzer III, E Delay, S Zinn, S Aborn, J ONeil, R Funston, R Weaber, G Liu and S McKay. 2019. A Neuroepigenomic Investigation of DNA Methylation in Cattle with Extreme Measures of Docility. International Plant and Animal Genome XXVI, San Diego, California.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 2. Cantrell, B, S Bhattarai, E Stassen, S Liu, T Buttolph, H Lachance, J ONeil, JF Garcia, B Murdoch, R Schnabel, J Taylor, R Funston, B Weaber, G Liu and S McKay. 2019. DNA Methylation Dynamics Across the Bovine Genome. 37th Annual International Society for Animal Genetics Conference, Lleida, Spain.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 4. Bhattarai, S, F Betancourt, T Buttolph, B Cantrell, S Friedman, S Perlee, H Lachance, JF Garcia, B Murdoch, G Murdoch, J Kim, R Schnabel, JF Taylor, S White and S McKay. 2019. DNA Methylation in the 5 AMPK Gene Family of Cattle and Bison. International Plant and Animal Genome XXVI, San Diego, California
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 1. Bhattarai, S., B. Cantrell, R. Funston, R. Weaber and S. McKay. 2020. Studying DNA Methylation in the Regions of Structural Variation in the Limbic System of Cattle. International Plant and Animal Genome XXVII, San Diego, California.

Progress 01/15/18 to 01/14/19

Outputs
Target Audience:Data has been presented at scientific meetings to the global animal genomics community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Suraj Bhattarai, a new graduate student has been working on this project since January 2018. He has learned and developed protocols for COBRA, Bisulfite Sanger sequencing and Bisulfite Pyrosequencing. His work has been presented at local, national and international conferences. Suraj attended PAG 2019 and was able to meet collaborators and network with the global animal genomics community. How have the results been disseminated to communities of interest?Results have been disseminated at the community via poser and oral presentations at the Canadian Society of Animal Science meeting and Plant and Animal Genome Conference. What do you plan to do during the next reporting period to accomplish the goals?Additional samples will be sequenced and analyzed. We expect multiple manuscripts to be published in the next reporting period.

Impacts
What was accomplished under these goals? Whole genome bisulfite sequencing has been performed on liver and muscle tissues of Angus and Hereford. Additional samples from Nelore and Bison are being sent out for Whole Genome Bisulfite Sequencing. A data analysis pipeline has been established for analysis of WGBS data. Additionally, conservation of methylation has been analyzed in members of the bovine AMPK gene family. These findings were validated with bisulfite pyrosequencing in Angus and Charolais cattle.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: S. McKay, F Betancourt, S Bhattarai, T Buttolph, S White, H Lachance, D Quijada, S Friedman, S Perlee, B Cantrell, B Murdoch; 115 Profiling Conservation of DNA Methylation in Cattle., Journal of Animal Science, Volume 96, Issue suppl_3, 7 December 2018, Pages 370, https://doi.org/10.1093/jas/sky404.812
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Bhattarai, S., T Buttolph, B. Cantrell, F. Betancourt, S. Friedman, S. Perlee, H. Lachance and S.D. McKay. 2018. Conservation of DNA Methylation in the 5 AMPK Gene Family of Cattle and Bison. University of Vermont Student Research Conference, Burlington, Vermont
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 1. Bhattarai, S, F Betancourt, T Buttolph, B Cantrell, S Friedman, S Perlee, H Lachance, JF Garcia, B Murdoch, G Murdoch, J Kim, R Schnabel, JF Taylor, S White and S McKay. 2019. DNA Methylation in the 5 AMPK Gene Family of Cattle and Bison. International Plant and Animal Genome XXVI, San Diego, California
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 1. Liu, Shuli*, B Cantrell*, L Fang, J, Kim, RD Schnabel, JF Taylor, G Liu and S McKay . 2019. Genome-wide DNA methylation analysis associated with feed efficiency in Angus and Herford beef cattle. International Plant and Animal Genome XXVI, San Diego, California. * indicates co-first authors, indicates co-corresponding authors

Progress 01/15/17 to 01/14/18

Outputs
Target Audience:Data has been presented at scientific meetings to the global animal genomics community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?At the University of Vermont, one undergraduate student worked on this project until graduation in May 2017. Darla received a travel grant from the Office of Undergraduate Research at the University of Vermont to present data at PAG 2017. While at PAG Darla was exposted to the globla animal genomic community and explored post-graduate opportunities.Additionaly, a new graduate student Suraj Bhattarai started work on this project in Jan. 2018. How have the results been disseminated to communities of interest?Results have been disseminated to the community via poster and oral presentations at the FAANG and Plant and Animal Genome meetings. What do you plan to do during the next reporting period to accomplish the goals?Additional samples will be sequenced and analyzed. Furthermore, we expect two manuscripts to be submitted in the next reporting period.

Impacts
What was accomplished under these goals? Whole genome bisulfite sequencing has been performed on liver and muscle tissues in the Angus and Holstein breeds. A data analysis pipeline has been established and preliminary data was presented at the January 2018 FAANG meeting. Additional tissues samples from additional breeds have been gathered and are being whole genome bisulfite sequenced.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Quijada, Q., F. Betancourt, S. Friedman, S. Perlee, H. Lachance and S.D. McKay. 2017. 5 AMPK Gene Family: Exploration of Conserved DNA Methylation. International Plant & Animal Genome XXV, San Diego, California. https://pag.confex.com/pag/xxv/meetingapp.cgi/Paper/24073
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Betancourt, F., S. Friedman, S. Perlee, H. Lachance and S.D. McKay*. 2018. Examining conserved DNA methylation in the bovine 5 AMPK gene family. International Plant & Animal Genome XXVI, San Diego, California.

Progress 01/15/16 to 01/14/17

Outputs
Target Audience:Data has been presented at scientific meetings to the global animal genomics community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?At the University of Vermont, two undergraduate students have been working on this project. Each student received grants from the Office of Undergraduate Research at the University of Vermont. Grant money, totaling $1,500 was used foreither data generation or for travel to Plant and Animal Genome to present data. Each undergraduate student presented a poster at PAG. While there, they were exposed to the global animal genomics community and explored post-graduate opportunities. How have the results been disseminated to communities of interest?Results have been disseminated to the community via poster and oral presentations at thePlant and Animal Genome Conferenceas well as the International Society of Animal Genetics Meeting. What do you plan to do during the next reporting period to accomplish the goals?Additional funds were secured from the University of Vermont College of Agriculture and Life Sciences to expand the scope of the project. Additionally, funds have been secured to bring an additional graduate student on board and a potential graduate student has been identified.DNA is being sent out for whole genome bisulfite sequencing in April 2017 and analysis will ensue.

Impacts
What was accomplished under these goals? The first major goal of the project was to whole genome bisulfite sequence DNA extracted from muscle tissues from Angus and Hereford breeds. DNA from these animals has been gathered and is being sent out for whole genome bisulfite sequencing. This project has been expanded to include whole genome bisulfite sequencing of four tissues from three different breeds of cattle (Angus, Holstein and Nellore). For each tissue, two biological replicates will be sequenced and the same tissue from the bovine reference genome animal will be sequenced as well. DNA is being sent out for sequencing in April 2017 and data analysis pipelines have been explored in preparation of impending sequence data.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Betancourt, F., S. Friedman, S. Perlee, H. Lachance and S.D. McKay. (2016). P2014 Examining conserved DNA methylation in the bovine 5 AMPK gene family. Journal of Animal Science 94(7supplement4), 44-45. doi: 10.2134/jas2016.947supplement444a
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Betancourt F, S Friedman, S Perlee, H Lachance and S McKay. 2016. Exploring DNA Methylation in the Bovine 5 AMPK Gene Family. International Plant & Animal Genome XXIV, San Diego, California. https://pag.confex.com/pag/xxiv/webprogram/Paper21045.html
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Quijada, Q., F. Betancourt, S. Friedman, S. Perlee, H. Lachance and S.D. McKay. 2017. 5 AMPK Gene Family: Exploration of Conserved DNA Methylation. International Plant & Animal Genome XXV, San Diego, California. https://pag.confex.com/pag/xxv/meetingapp.cgi/Paper/24073
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: McKay, SD, GE Liu, Y Zhou, R Tearle, M Del Corvo, E Capra, B Cantrell, B Rosen, JL Williams, P Ajmone Marsan and A Stella. 2017. Whole Genome Bisulfite Sequencing of Three Tissues Related to the Bovine Reference Genome. International Plant & Animal Genome XXV, San Diego, California. https://pag.confex.com/pag/xxv/meetingapp.cgi/Paper/26225
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Zhou, Y, R. Tearle, M. Del Corvo, E.. Capra, B. Cantrell, B. Rosen, A. Stella, J.L. Williams, P. Ajmone Marsan, G.E. Liu and S.D. McKay. 2017. Whole Genome Bisulfite Sequencing of Three Tissues Related to the Bovine Reference Genome. International Plant & Animal Genome XXV, San Diego, California. https://pag.confex.com/pag/xxv/meetingapp.cgi/Paper/24291