NATIONAL ANIMAL GENOME RESEARCH PROGRAM

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1018159
• Multistate No.: NRSP-8
• Project Start Date: Nov 5, 2018
• Project End Date: Sep 30, 2020
• Program Code: [(N/A)]- (N/A)

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

Performing Department
Animal Science

Non Technical Summary
Epigenetic modifications have the potential to influence variation in economically important phenotypes of agricultural species.The effects of 5-methylcytosine (5-mC) on economically important traits in livestock has been characterized but 5-hydroxymethylation remains largely unexplored. Hydroxymethylation is a recently discovered epigenetic modification that occurswhen 5-mC is oxidized, is prevalent in brain tissues and promotes transcription. The overarching intent for this project is togenerate a profile of 5-hydroxymethylation in three tissues of the bovine brain and investigate differential hydroxymethylationbetween two groups of cattle with extreme measures of docility. To accomplish this goal we will produce genome widehydroxymethylation profiles in the amygdala, cingulate gyrus and hippocampus of the brain via reduced representationhydroxymethylation profile (RRHP) sequencing for each of eight cattle. RRHP is a novel technique that involves β-glucosyltransferase for identification of hydroxymethylcytosine followed by next generation sequencing. Bowtie will be employedfor quality trimming and aligning sequence reads to the bovine reference genome. Patterns of nucleotide hydroxymethylation willbe determined and differentially hydroxymethylated regions will be identified between two groups of animals with extrememeasures of docility. This innovated work will be the first of its kind with regard to an economically important trait in anylivestock species.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

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

bovine

hydroxymethylation

epigenetic

behavior

Goals / Objectives
Advance genome-to-phenome prediction by implementing strategies and tools to identify and validate genes and allelic variants predictive of biologically and economically important phenotypes and traits. The goals of this Objective are to advance genome-to-phenome prediction by facilitating translation of genomic findings into biologically relevant information for genetic improvement of all species. To achieve this goal, genome-phenome association studies, functional validation methods, deep phenotype collection, comparative genomics, metagenomics and phenomics are important approaches that will produce valuable insights. In addition, analytic tool development to effectively leverage all information as well as implementation will be critical for agri-animal research communities to realize the potential of these findings to simultaneously develop agri-animals as biological model systems while maximizing the economic gains from genetic and genomic selection.

The specific aims for this objective include:
1. Exploit the power of high-resolution SNP-chips, genome re-sequencing, and genotyping-by-sequencing in genome-phenome association studies for detection and validation of genomic variants that are predictive of economically important phenotypes.
2. Advance strategies, models, algorithms, pipelines and analytic tools to facilitate the identification, validation and incorporation of novel genetic elements and causal and/or highly predictive variants to allow the most accurate prediction of phenotypic performance based on genotypes. Newly identified variants will be used in Objective 1 to further annotate genomes.
3. Develop and adopt various approaches, including (e.g., CRISPR/Cas9 genome editing) for functional analysis, and evaluation and verification of functional allelic variants of causal genes important for production traits.
4. Support deep phenotyping of important traits at the molecular, cellular, tissue and organismal levels, including the use of high-throughput technologies such as transcriptome sequencing, proteomics and metabolomics studies, whole-animal parameter monitoring, and in vitro gene mutagenesis screening analyses and other tools to support precision monitoring.
5. Use comparative approaches to identify genetic variation within and across species that is associated with phenotypic variation that results from common treatments or environments, or between natural and domesticated populations.
6. Support comparative phenomics, with an emphasis on the use of farm-animal models that mimic human processes to benefit human health.
7. Develop well-characterized animal populations/genetic lines/models that allow for study of biology of various economically important phenotypes and traits.
8. Advance metagenomic studies to help in discovery of novel pathogens, understanding host-pathogen interaction and determining the role of microbiota in agri-animal nutrition, health and reproductive performance.
9. Train the next generation of animal breeders in applying and developing new methods based on high-throughput genomic data to make genetic progress.

Project Methods
Objective 1: Generation of a bovine brain hydroxymethylome map.The experimental steps required to complete Objective #1 are:1. Brains from Red Angus X Simmental steers were collected at time of slaughter in July, 2014. Selected tissues have beendissected, and extracted DNA will undergo reduced representation hydroxymethylation profiling (RRHP).2. Reduced representation hydroxymethylation profiling sequencing will be undertaken for the Amygdala, Cingulate Gyrusand Hippocampus in each of 8 cattle. These animals have been chosen from the extreme tails of the phenotypic distribution,thus 4 animals will have low measures of docility and 4 animals will have high measures of docility.3. Sequences from each animals, for each tissue will be aligned to the UMD3.1 bovine reference genome assembly usingBowtie (Langmead et al., 2009).4. Custom scripts will be developed to generate a list of hydroxymethylated CpG sites and calculate the level ofhydroxymethylation for each cytosine in the genome.5. Patterns of nucleotide hydroxymethylation will be identified using DMEAS (http://sourceforge.net/projects/dmeas/)Objective 2: Overlay RRHP sequencing with WGBS to elucidate the methylation or hydroxymethylation status of cytosines.The experimental steps required to complete Objective #2 are:1. Exact locations of 5-mC and 5-hmC for each animal and each tissue will be generated from existing WGBS data andnewly generated RRHP data.2. A custom script will be developed to determine genomic locations of specific cytosines that were mistakenly identified as5-mC as opposed to 5-hmC when WGBS was performed.Objective 3: Elucidate the relationship between bovine brain hydroxymethylation and docility, an economically importantbehavioral phenotype in cattle.The experimental steps required to complete Objective #3 are:1. SAMTools, GATK and custom software will be used to identify which of the hydroxymethylated CpGs are differentiallyhydroxymethylated between groups within tissues2. Genes located within the differentially hydroxymethylated regions will be identified and undergo pathway analysis usingPanther (Mi et al., 2017) to elucidate underlying biological networks.

Progress 11/05/18 to 09/30/20

Outputs
Target Audience:This research has provided an opportunity for graduate students to become engaged in the animal genomicscommunity through learning and performing fundamental epigenetics research. These students will obtain skills both in thelaboratory and in dissemination of knowledge through opportunities to present research findings at national and international conferences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Professional development opportunities for my graduate students includes presenting (oral and poster) at conferences suchas Plant and Animal Genome and attending bioinformatics training workshops such as the Bioinformatics Training Workshopfor Animal Genome Analysis held in San Diego, CA in January 2020. How have the results been disseminated to communities of interest?Results of the bovine brain methylation and hydroxymethylation project have been disseminated through a publication as well as oral and poster presentations. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We utilized a combination of whole genome bisulfite sequencing (WGBS) and oxidative reduced representation bisulfitesequencing (oxRRBS) techniques to identify 5-hydroxymethylcytosine (5-hmC) across the genome at a single nucleotideresolution. Four unique brain tissues (prefrontal cortex, periaqueductal gray, cingulate gyrus, hippocampus) were analyzedfor two groups of steers with extreme measures of docility (n=4 per group). Our findings identify hydroxymethylation in eachtissue and across the genome. However, 5-hmC varied across chromosomes in a tissue-specific manner. Additionally,differential 5-hmC was identified between docile and non-docile animals in three tissues. The amount of differential 5-hmCvaried significantly between tissues, emphasizing the tissue-specific nature of 5-hmC within the brain. Characterization of5hmC in functionally distinct bovine brain tissues provides further functional annotaiton of the bovine genome.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 4. Cantrell, B, S Liu, N Jebbett, RC Switzer III, E Delay, S Zinn, S Aborn, J ONeil, R Funston, R Weaber, G Liu and SMcKay. 2019. A Neuroepigenomic Investigation of DNA Methylation in Cattle with Extreme Measures of Docility.International Plant and Animal Genome XXVI, San Diego, California.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: 3. Cantrell, B., S. Friedman, H. Lachance, C. Bernier, B. Murdoch, S. Frattini, A. Talenti, P. Crepaldi, and S. McKay. 2019.A Novel Understanding of Global DNA Methylation in Bobcat (Lynx rufus) Genome. DOI: 10.1139/gen-2019-0046.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 4. Stassen, E., B. Murdoch, R. Funston, R. Weaber and S. McKay. 2020. Identification of 5-HydroxymethylcytosineMarkers in the Cattle Brain. International Plant and Animal Genome XXVII, San Diego, California.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: 2. Bhattarai, S., B. Cantrell, R. Funston, R. Weaber and S. McKay. 2020. Studying DNA Methylation in the Regions ofStructural Variation in the Limbic System of Cattle. International Plant and Animal Genome XXVII, San Diego, California.

Progress 11/05/18 to 09/30/19

Outputs
Target Audience:The target audience is the global animal genomcis community as well as the global Neuroepigenetics community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Emily Stassen, a PhD student in my lab is leading this project which has permitted development and enhancement of skills including effective collaboration, leadership and effective communication. Additional, Emily was awarded a travel award to present her data at PAG 2020. Finally, Emily attended the FAANG Bioinformatics workshop in San Diego, CA in January 2020. How have the results been disseminated to communities of interest?Results have been disseminated through oral and poster presentations at PAG and the Neuroepigenetics and Neuroepitranscriptomics Conference. What do you plan to do during the next reporting period to accomplish the goals?An additional two tissues from the bovine limbic system for each of 8 cattlewill undergo ox-RRBS. Additional comparison between WGBS and ox-RRBS will be performed and differential hydroxymethylation will be performed for cattle with extreme measures of docility.

Impacts
What was accomplished under these goals? Multiple approaches were implemented for functional analysis. Specifically, global DNA hydroxymethylation was performed and oxidative Reduced Representation Bisulfite Sequencing (ox-RRBS) was performed in two brain tissues for each of 8 animals. A analysis pipeline was established and is being used to analyze ox-RRBS data.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 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. 2020. A Neuroepigenomic Investigation of DNA Methylation in Cattle with Extreme Measures of Docility. 2nd Neuroepigenetic and Neuroepitranscriptomics Conference. Nassau, Bahamas
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Stassen, E., B. Murdoch, R. Funston, R. Weaber and S. McKay. 2020. Identification of 5-Hydroxymethylcytosine Markers in the Cattle Brain. International Plant and Animal Genome XXVII, San Diego, California.