Source: UNIVERSITY OF KENTUCKY submitted to
IMPLICATIONS OF EPIGENETIC REJUVENATION DURING VEGETATIVE PROPAGATION FOR THE PRODUCTION OF LOCALLY ADAPTED PERENNIAL CROP CULTIVARS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1018617
Grant No.
2019-67013-29168
Cumulative Award Amt.
$500,000.00
Proposal No.
2018-06276
Multistate No.
(N/A)
Project Start Date
Feb 15, 2019
Project End Date
Feb 14, 2024
Grant Year
2019
Program Code
[A1141]- Plant Health and Production and Plant Products: Plant Breeding for Agricultural Production
Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001
Performing Department
Horticulture
Non Technical Summary
Grapes are the highest value fruit crop in the US,contributing more than $162 billion to the Nationaleconomy each year and with a 36% of the production beingexported.It is predicted that50-81% of the grapevine growing acreage in the USA will fall out of production due to weather changes by 2040.If this projection becomes a reality it would have devastating effects both on local communities and global wine and grape industry.Moreover,episodes of even relatively mild abiotic stresscan dramatically increase susceptibility of plants to disease, whichadds urgency for a better understanding of plant tolerance to abiotic stress. This urgently demands the breeding of varieties more resilient to periods of environmental stress, without losing fruit quality.To-date progress in this area has been hindered as grapevine, like many other perennial woody crops, imposes protracted(25 to 30 years)and costly breeding programsdue to lengthyevaluation periods in each breeding step.Epigenetic priming (EP) refers to a set of molecular mechanisms by which plants can increase their resilience to stress bygenerating an epigenetic memory of their environmentafter recognition of specific environmental signals. Previous observations both in annual model plants and crops have shown that such memory of stress can last several daysand transmitted over multiple sexual generations. The use ofepigenetic priming has the potential to accelerate the production of locally adapted cultivars of perennial crops. However, for this to be effective, vegetative propagating methodologies must allow the maintenance of epigenetic memory. Unfortunately, the mechanisms regulating epigenetic memory in perennials and the effect that vegetative propagation has on its maintenance are not well understood.Our preliminary resultsindicate that age acquired epialleles can bemaintained or erased during the generation of vegetative propagules depending on the vegetative propagation approach used.This project aims to unravel the processes mediating plant and methylome rejuvenation during vegetative propagation of grapevines.To achieve the goals and objectives of this project,over the next three years, we willusean innovative combination of sexual and vegetative clonal propagation of grapevines, to conduct a multi-omic comparative analysis of epigenetic and gene regulatory mechanisms in tissues derived from each type of propagation at a whole genome level and within the well understood frameworks of flavonoid biosynthesis and water stress response, to uncover the molecular processes underlying plant rejuvenation during vegetative propagation.Unravelling the mechanisms regulating the maintenance of epigenetic memory during vegetative propagation, will allow breeders to generate locally adapted grapevine varieties with higher levels of tolerance to abiotic and biotic stresses. The know-howgenerated during this project will give American breeders, nurseries and growers a commercial advantage by significantly reducing the time needed to generate new perennial crop varieties. The findings of the proposed researchwill provide knowledge transferable to otheragriculturally-importantperennial crops.
Animal Health Component
45%
Research Effort Categories
Basic
55%
Applied
45%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20111311081100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1131 - Wine grapes;

Field Of Science
1081 - Breeding;
Goals / Objectives
This project aims to unravel the molecular processes mediating plant and methylome rejuvenation duringcallused cutting propagation of grapevine. Understanding the mechanisms regulating the maintenance of epigenetic memory in perennial crops, will allow breeders harnessing the potential of plant priming as a mechanism for plant response to abiotic and biotic stresses observed in annual species. Our preliminary data indicates that grapevine's primary and secondary metabolism is affected by epigenetic modifications observed during vegetative propagation, which is supported by previous studies in annual species.This project also aims to provide the basis for the development of novel methods for the propagation,pre-breeding and germplasm enhancement breeding of grapevine varieties more resilient to changing and extreme environments predicted for the near future. Our approach will also allowthe creation of locally adapted cultivarsbased on the historical and predicted conditions of each growing region.This project will increase the competitiveness of the American table-grape and wine industry by enabling the adoption of novel breeding and nursery propagation technologies with the potential to generate improved varieties. Enhanced stress tolerance will also improve vineyard performanceand reduce vineyard input costs promoting sustainability.The ability to use epigenetic memory to improve wine quality could improve the USAs standing in the export wine market.Finally, if, as expected, the findings of the proposed research apply generally, theywill provide tools and knowledge transferable to otheragriculturally-importantperennial crops.Objective 1.Determine if the observed large-scale differences in DNA methylation induced by callused cutting propagation in grapevine are equivalent to the methylome rejuvenation induced by sexual reproduction (Years 1 & 2 of the project, as part of Project Phase I:Multi-omic analyis of the epigenetic resetting during CC propagation of grapevines).Objective 2.Identify the source and nature of the rejuvenating signals involved in such plant/methylome rejuvenation (Years 1 to 3of the project, as part of Project Phase II:Identification of drivers of CC-induced methylome rejuvenation).Objective 3.Determine if endogenous and exogenous drivers of epigenetic differentiation (plant age and water stress respectively) have different effects during the maintenance/erasing of epigenetic marks (Years 2 & 3 of the project, as part of Project Phase III:Effect of the epigenetic variability source on its maintenance during propagation).
Project Methods
Scientific methods:The project's workplan is structured in 3 phases:Phase I.Multi-omic analyis of epigenetic memory resetting during CC propagation of grapevines:This phase will identify differences in smRNA/gene expression, DNA methylation and histone modification patterns between different tissues (leaf and callus) of adult plants, vegetative propagules, and seed propagated plantlets. Well stablished protocols will be used to determine global levels of DNA and histone methylation (EpiGentek ELISA colorimetric analysis), and of smRNAs (Fragment Analyzer) at 12 different stages of development.These results will be used to select 5 time points forin-depth analysis.Samples collected at this time points will then be analysed using:1. Post-bisulfite adaptor-tagging for PCR-free whole-genome bisulfite sequencing to generate individual single base resolution methylation maps.2. Chromatin immunoprecipitation sequencing tostudy plant histone modifications correlated with gene transcription.3. Small RNA and gene expression analysis will be carried using smRNASeq and RNASeq.Phase II.Identification of drivers of CC-induced methylome rejuvenation:This phase aims to identify and characterize the drivers of methylome rejuvenation during vegetative propagation. We will use ELISA to study changes in DNA methylation induced by previously suggested drivers (injury, callus formation, and/or absence of root signals). Plants used here will be propagated/grafted using different approaches to induce combinations of epigenome rejuvenation drivers (e.g. Callused cuttings (CCs) (injury+, callus+, roots-), CCs grafted onto rootstocks (injury+, callus+, roots+), non-callused cuttings grafted onto rootstocks (injury+, callus-, roots+), partially severed layered vines (injury+, callus-, roots+) and layered plants (injury-, callus-, roots+).Next, we will use grafts of callused and non-callused cuttings to validate the existence of a mobile signal. We will graft: 1) non-callused scions onto CCs (with no roots) to validate the putative movement of rejuvenating signals from the callus to the apical meristems; 2) non-callused scions onto non-callused rootstocks to validate the putative movement of adult phase-promoting signals from the root to the apical meristems. Other controls will include grafts of CCs x CCs, non-callused x non-callused, de-rotted seedlings (grafted onto seedling rootstocks and grown in vitro without roots), and reciprocal grafts of two different grapevine genotypes. Global changes in DNA methylation will be assessed using ELISA while the presence of mobile small RNAs will be identified using NGS as in Phase I.Shoots will be cultured in liquid medium containing cocktails of artificially synthesized small RNAs identified as putative molecules preventing/promoting rejuvenation.Phase III.Effect of the epigenetic variability source on its maintenance during propagation:To determine how the source (endogenous (age-acquired) vs exogenous (environmentally induced)) of epigenetic differentiation influences the maintenance/erasing of epialleles during vegetative propagation, mother plants will be exposed to control and drought stress conditions and subsequently propagated using CCs. Methylomes and histone modification maps and whole transcriptome sequencing will be generated for all biological replicates of stressed and control plants as described for Phase I.Data Analysis and Management Plan:Data analysis:DNA methylation, histone modification, smRNA and gene expression analysis will be carried out using freely available and in-house designed bioinformatics pipelines. Finally, multi-omic data integration will be carried out using a network analysis approach.Data management:Primary digital data will be stored on portable drives, purchased for PI, Co-PI. These data will be stored on central computers and a cumulative copy of the total project data will be backed-up on institutional shared drives and reported annually. The university shared drive storage is backed-up to an off-site location daily. At the close of the project, the PI and the university shared drive will all maintain separate copies of the total cumulative data packages for a minimum of five years. We will also strive toward rapid dissemination of results and scientific materials to the greater scientific community, including submission of raw sequencing data, methylomes and transcriptomes to appropriate curated databases (e.g. National Centre Biotechnology Information-GeneBank (https://www.ncbi.nlm.nih.gov/)). All data will be made available upon request to auditors, investigators or interested parties in accordance with institutional data retention, ownership and intellectual property protection policies (https://www.research.uky.edu/office-research-integrity/university-kentucky-data-retention-ownership-policy).In accordance with UKY guidelines, any research manuscripts accepted for publication to peer-reviewed journals will be made available to the interested stakeholders in a public repository prior to final publication.Efforts:To achieve a widespread dissemination, training and adoption, we will deliver the projects recommendations and outputs via a range of mechanisms, including: local, national and international workshops and seminars, updates in industry journals, final report, scientific papers and intellectual property protection.This project will also deliver three highly trained early career researchers who will contribute to build the skill-base in the American horticulture industry.Evaluation:To facilitate evaluation and to provide a means to track progress, a Project Development Plan (PDP) will be stablished. The PDP will be used as a tool to help with assessment, defining short-term and long-term goals, improve efficiency and productivity of the project, and help to structure productive conversations about the Post-Doc career plans and development. It will also include measurable outcomes, such as publications, conference attendance and submission of grant proposals. Updates on progress and achievements will be shared monthly to maintain accountability. In addition, the PI will coordinate General Project Meetings (GPMs) with the rest of co-PIs every four months to track the project's progress, limitations, and results using the project's milestones. During these meetings the PI and Post-Doc will present a progress report. Co-PIs will critically scrutinize the data generated, the rigor of the scientific approaches taken, the completeness of the scientific story and findings' significance.Key milestones for evaluation:Phase I:Comparison of whole genome/transcriptome/histone modification data generated during Year 1. During Year 2 experiments will be replicated to validate the results using targeted approaches (i.e. Differentially methylated regions, Targeted Methylome Sequencing using Illumina MiSeq 600 bp sequencing platform; gene/small RNA expression and histone modifications, QuantStudio Real-Time PCR).Phase II:Validation of methylome rejuvenation induced by each proposed propagation system and by small RNAs identified as differentially expressed during Phases I and II. Methylome rejuvenation validation will be carried out by measuring total DNA levels using ELISA. Validation of the rejuvenation effect of small RNAs will be carried out by culturing grapevine shoots in liquid medium containing cocktails of artificially synthesized small RNAs identified as putative molecules preventing/promoting rejuvenation.Phase III:Determination of the importance of the source of epigenetic variability to its maintenance. Mother vs propagule and controls vs stressed comparisons will be carried out to identify differences in the maintenance/erasing of age and stress induced epigenetic variability. Analysis will be carried out both at a whole genome level and within the well understood frameworks of flavonoid biosynthesis and drought stress response.

Progress 02/15/19 to 02/14/24

Outputs
Target Audience:Teaching and laboratory instruction: · Three graduate students (PhD) have been recruited to this project (Tajbir Raihan, Lakshay Anand, and Jia Tan) and one postdoctoral researcher (Dr. Harshraj Shinde). All three students are enrolled in the Integrated Plant and Soil Sciences graduate program at the University of Kentucky (UKy). Miss Tan's researcher focussedon the analysis of the maintenance of epigenetic memory of stress in primed plants from a gene expression and DNA methylation; Mr Raihan focused on the identification of the molecular signal inducing plant rejuvenation during vegetative propagation; and Mr Anand's thesis aim was the development of bioinformatics tools for the integration of multiomic datasets for grapevine research. Dr. Shinde acted as project manager at greenhouse and laboratory level. All three graduate students defended their PhD theses between 2023 and 2024 (Tan, April 2023;Raihan, June 2023; Anand, January 2024).Tan and Raihan work now postdoctoral researchers for USDA. Anand is a postdoctoral researcher at the Department of Horticulture, UKy. ·Two undergraduate students, Bridget Bolt and Makana Randolph (UKy's Agricultural and Medical Biotechnology undergraduate Program), were recruited as part of this project. Miss Bolt, recruited in 2021,focussed her work towards the development of an in vitro system to induce grapevine seed germination without the need of protracted chilling scarification. After graduation Miss Bolt accepted an paid 6 month intershipat E. & J. Gallo. Bridget is now back in the research group at her second year as a graduate student developiung a gene editing system for grapevine and other perennial crops. Miss Makana Randolph, joined the research group in December 2023 to study the effect of in-vitro rejuvenation of grapevines. ·Graduate student, Alberto Rodríguez-Izquierdo, Universidad Politecnica de Madrid, Spain,contributed to the project during his two placements at UKy as visiting scholar. Dr.Rodríguez-Izquierdo research focussed on vegetative propagation has affected the methylome of grapevine during its domestication. During his visits Rodriguez-Izquierdogenerated a multi-omic database using natural epigenomic variation between wild and cultivated grapevine genotypes andcreated a R package to improve GBS Sequencingin grapevine. · Over the duration of the project, PI Lopez has been invited lecturer to two different courses of the University of Kentucky Undergraduate Program Agricultural and Medical Biotechnology (i.e. ABT 301 "Introduction to Biotechnology", and ABT 201 "Scientific Method In Biotechnology") where he discussed the potential applications of epigenetic memory for crop improvement. Extension and outreach: ·2019:• Invited oral presentation and publication of a Proceedings paper in Southern Nursery Association annual meeting "Harnessing the Power of Epigenetic Priming to Improve Stress Tolerance in Vegetative Propagated Perennial Crops" • Invited presentation at the European Research Council funded COST Action CA17111. The First Annual Meeting of COST Action CA17111 "INTEGRAPE 2019 - Data Integration as a key step for future grapevine research". March 25-28, 2019. Conference Center of Mediterranean Agronomic Institute of Chania, Crete, Greece. Title: Ancient epialleles: a multiomic view on wine quality • Poster Presentation at the National Association of Plant Breeders. August 25-29, 2019. Callaway Gardens - Pine Mountain, Ga. Title: NIFA:2019-67013-29168 Crosses between epigenetically distant parents generate novel epi-alleles in commercial grapevine cultivar Cabernet Sauvignon. ·2020:• Invited speaker at the 2020 Kentucky Fruit and Vegetable Conference/Grape and Wine short Course. Title: Epigenetic Memory of Grapevines. Meeting attendants were mainly grapevine growers, winemakers and extension specilists • Participation at the Round Table Discussion: Grape Pricing, Grower and Winery Connections, and Helpful Tools for The Industry during the 2020 Kentucky Fruit and Vegetable Conference. During this round table, the potential applications of epigenetic priming in grapevines were briefly discussed with participant growers and winemakers. • Poster Presentation at the 2019 Commonwealth Computational Summit. October, 2019. Lexington Kentucky. Title: ChromoMap: An R Tool for Interactive Visualization and Annotation of Chromosomes". ChromoMap was created by PI Lopez PhD student Mr Anand and constitute the first chapter of his thesis. Mr Anad was awarded third place overall https://www.uky.edu/hort/node/2104. • PI Lopez NIFA-USDA award is announced on the University of Kentucky College of Agriculture Food and the Environment Newsletter. https://news.ca.uky.edu/article/university-kentucky-researcher-receives-grant-study-'memory'- grapevines (June 5th). • The Spring Issue of the Ag Magazine features PI Lopez research in grapevine epigenetic memory https://agmagazine.ca.uky.edu/spring-2019/cafe-corner/power-memory.•Invited speaker at the 2020 Annual Meeting of the Argentinian Association of no-till growers (Asociación Argentina de Productores en Siembra Directa - Aapresid) (https://www.aapresid.org.ar/). Title: Epigenetics and Plant Memory •Poster presentation at the 25th Annual Meeting of the RNA Society. Title: Dicistronic tRNA-mRNA Transcript Expression Analysis in Grapevine (Vitis vinifera) Suggests Environmental Regulation and Evolutionary Conservation in Plants •Presentation at the 2020 Kentucky Virtual State Fair. Title: Experimental Vineyard (https://www.youtube.com/watch?v=bu52A4BtCOI&list=RDCMUCjLmlBb3hAPB927mQmijXxw&index=1). ·2021:Invited poster presentations (Shinde and Tan) during the 72nd American Society for Enology and Viticulture, National conference (Virtual), 21-24 June 2021. The American Society for Enology and Viticulture (the sciences of winemaking and grape growing) is a professional society dedicated to the interests of enologists, viticulturists, and others in the fields of wine and grape research and production throughout the world. Its membership includes professionals from wineries, vineyards, academic institutions and organizations. · Invited poster presentation (Anand) during the Bio-IT World 2021 International Meeting · Invited poster presentation (Bolt) during the International Plant Propagator's Society (IPPS) 2021 meeting as a poster and a proceedings paper. IPPS is an international association of plant production professionals, including those involved in horticultural research and education. ·2022:Invited speakers (Shinde and Tan) at the International Conference "Plant and Animal Genomes (PAG)" in San Diego, USA in January 2022. PAG is the largest agricultural genomics conference in the world. Invited speaker (Lopez) at the International Symposium of Neurogastronomy where he 1. Defined the concept of terroir and its contribution to wine aromas, 2. Presented two non-traditional components of terror, vineyard age and microbiomes, and 3. Explained how epigenetic mechanisms contribute to wine terroir through the regulation of gene expression?. ·2023:Invited speaker (Lopez) at the XIII International Symposyum on Grapevine Breding and Genetics (Germany), and the International Conference "Plant and Animal Genomes (PAG)" in San Diego, USA in January 2023. PAG is the largest agricultural genomics conference in the world. · 4 Invited poster presentations (Rodriguez-Izquierdo, Bolt, Shinde, and Raihan) during the XIII International Symposyum on Grapevine Breding and Genetics (Germany), and the "Plant and Animal Genomes (PAG)" in San Diego, USA in January 2023. ·2024: PI Lopez was an invited speaker at the Ohio Valley Grain Exchange Annual meeting where he discussed the molecular origins of flavor in grapevines. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Thre graduate students (PhD) has been recruited to develop their PhD theses using samples, data and resources from this proposal. All students enrolled the University of Kentucky's Integrated Plant and Soil Sciences graduate program under the supervission of PI Lopez, January 2019 (Mr Anand),September 2019 (Mr. Raihan), May 2020 (Miss Jia Tan). Mr Anand PhD focusedon developing bioinformatic tools for the analysis and integration of Vitis vinifera multi-omic data. Mr. Raihan's focused on the identification of the molecular mechanisms regulating the epigenetic memory of stress in Vitis vinifera. In addition to the IPSS program 2019-2020 courses, Mr Anand attended two conferences where he presented: One oral presentation: "Analysis of evolutionary co-appearance of mobile dicistronic tRNA-mRNA transcripts and vasculature in land plants" at the Evolution Conference 2019, June 21-25, Providence, Rhode Island. One poster presentation: "Chromomap: An R Tool for Interactive Visualization and Annotation of Chromosomes" at the 2019 Commonwealth Computational Summit, October 2019, Lexington, Kentucky. Mr Anand was awarded hird place overall for his poster. In addition to the IPSS program 2019-2020 courses, Mr. Raihan attended one conference, Southern Nursery Association 2020 Annual Meeting. Baltimore, January 2020. Where was an invited speaker: "Creation and Characterization of an Epimutant population of Soybean". Project's Posdoc (Dr. Harshraj) and graduate students (Mr. Anand and Mr. Raihan) attended the workshop in downstream analyses of epiGBS data organized by Associate professor Christina Richards, University of South Florida. epiGBS is a reduced representation genome sequecing approach used for the analysis of DNA methylation in large populations. Miss Jia Tan through this project has acquired skills in molecular biology and bioinformatics. Miss Tan has presented the results of her work during Dr Lopez weekly research group meetings and did a complete presentation of her progress at her annual PhD committee meeting which has helped her building her communication skills. Additionally, this project is closely related to USDA-NIFA project "Implications of epigenetic rejuvenation during vegetative propagation for the production of locally adapted perennial crop cultivars" accession Number: 1018617. This project involves the participation of two graduate students and one postdoctoral researcher. It is expected that the results from both will finally be integrated to generate a cohesive piece of research. Collaboration with other lab members for the purpose of this project also has help to enhance her communication skills. In addition to the Integrated Plant and Soil Sciences Graduate Program 2019-2020 courses, Miss Tan attended the METHADA 2020 eHands on - Virtual Training School in Transcriptomic Metadata Handling and Data Analysis (http://www.integrape.eu/index.php/training-schools/methada-virtual) as a registered observer. This training school addresses grapevine genomics data handling and analysis, and it is organized in three modules. 1. On the first unit, trainees will work to learn how to use the guidelines provided by the INTEGRAPE community to correctly annotate experiments and submit them on public repository. We will explore standards and bio-ontologies for FAIR data annotation using their personal data. 1. The second module will teach the trainees how to use the tools from Sequentia (AIR for transcriptomics). 2. Finally, attendees will be introduced to the GREAT (GRapevine Expression ATlas) platform for transcriptomic metaanalysis. The GREAT platform is a good example of reusing publicly available RNA-seq data to answer new biological questions. Trainees will have the opportunity to explore the GREAT platform with their own list of genes of interest Graduate Student associated to this project Miss Jia Tan, has applied for two competitive grants (Southern SARE Graduate Student Grant program and University of Kentucky College of Agriculture Food and the Environment Graduate Student Research Activity Award). Beyond the obvious benefits of successfully applying for a competitive grant as Graduate Student, both applications help Jia develop grantmanship skills that will be of great use as her career as plant scientist develops. Finally, an undergraduate student was recruited to the project. The recruitment of undergraduate student (Bridget Bolt) has allow her to get training in in-vitro culture techniques she had not been exposed to previously. Also, Bridget decided to attend graduate school (as a Master student) under the advising of project PI Lopez developing a CRISPR/Cas9 system for grapevines and other perennial woddy crops. How have the results been disseminated to communities of interest?2019-2020 • PI Lopez has been invited lecturer to two different courses of the University of Kentucky Undergraduate Program Agricultural and Medical Biotechnology (i.e. ABT 301 "Introduction to Biotechnology", and ABT 201 "Scientific Method In Biotechnology") where he discussed the potential applications of epigenetic memory for crop improvement. Aproximately 150 students/year attended both classes. • Invited oral presentation and publication of a Proceedings paper in Southern Nursery Association annual meeting "Harnessing the Power of Epigenetic Priming to Improve Stress Tolerance in Vegetative Propagated Perennial Crops". This meeting is attended by scientists, graduate and undergraduate students in the field of horticulture and plant propagation, extension specialists and nursery profesionals. • Invited presentation at the European Research Council funded COST Action CA17111. The First Annual Meeting of COST Action CA17111 "INTEGRAPE 2019 - Data Integration as a key step for future grapevine research". March 25-28, 2019. Conference Center of Mediterranean Agronomic Institute of Chania, Crete, Greece. Title: Ancient epialleles: a multiomic view on wine quality. • Poster Presentation at the National Association of Plant Breeders. August 25-29, 2019. Callaway Gardens - Pine Mountain, Ga. Title: NIFA:2019-67013-29168 Crosses between epigenetically distant parents generate novel epi-alleles in commercial grapevine cultivar Cabernet Sauvignon. • Invited speaker at the 2020 Kentucky Fruit and Vegetable Conference/Grape and Wine short Course. Title: Epigenetic Memory of Grapevines. Meeting attendants were mainly grapevine growers, winemakers and extension specilists • Participation at the Round Table Discussion: Grape Pricing, Grower and Winery Connections, and Helpful Tools for The Industry during the 2020 Kentucky Fruit and Vegetable Conference. During this round table, the potential applications of epigenetic priming in grapevines were briefly discussed with participant growers and winemakers. • Poster Presentation at the 2019 Commonwealth Computational Summit. October, 2019. Lexington Kentucky. Title: ChromoMap: An R Tool for Interactive Visualization and Annotation of Chromosomes". ChromoMap was created by PI Lopez PhD student Mr Anand and constitute the first chapter of his thesis. Mr Anad was awarded third place overall https://www.uky.edu/hort/node/2104. • PI Lopez NIFA-USDA award is announced on the University of Kentucky College of Agriculture Food and the Environment Newsletter. https://news.ca.uky.edu/article/university-kentucky-researcher-receives-grant-study-'memory'- grapevines (June 5th). This newsletter is mainly read by University of Kentucky students and staff, state growers andagriculture professionals. • The Spring Issue of the Ag Magazine features PI Lopez research in grapevine epigenetic memory https://agmagazine.ca.uky.edu/spring-2019/cafe-corner/power-memory. This magazine is mainly read by University of Kentucky students and staff, state growers and agriculture professionals. 2020-2021 • Invited speaker at the 2020 Annual Meeting of the Argentinian Association of no-till growers (Asociación Argentina de Productores en Siembra Directa - Aapresid) (https://www.aapresid.org.ar/). Title: Epigenetics and Plant Memory • Presentation at the 2020 Kentucky Virtual State Fair. Title: Experimental Vineyard (https://www.youtube.com/watch?v=bu52A4BtCOI&list=RDCMUCjLmlBb3hAPB927mQmijXxw&index=1). 2021-2022 Results from this project have been presented in the form of oral and poster presentations at four different meetings: Plant and Animal Genomes, San Diego, USA; American Society for Enology and Viticulture, National conference (Virtual); Bio-IT World 2021 International Meeting, Boston, USA; and the International Plant Propagator's Society (IPPS) 2021 meeting. See outcomes for a description. 2022-2023 Results from this project have been presented in the form of oral and poster presentations at three different meetings: Plant and Animal Genomes, San Diego, USA; XIII International Symposium on Grapevine Breeding and Genetics. The International Society of Neurogastronomy Symposium 2022. See outcomes for a description. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Epigenetic regulation of propagule rejuvenation during callused cutting production in grapevines Use of callused cuttings for grapevine propagation reduces DNA methylation to levels like those found in juvenile tissues. Global DNA methylation levels in leaf tissue from grapevine seedlings is significantly lower that from leaf tissue from mature plants. Interestingly, global DNA methylation levels in buds and leaves formed after callus cutting production are significantly lower than that of mother plants and similar to those observed in seedling's leaves. These results experimentally validate our preliminary results obtained using commercially grown grapevines of different ages. Callused cutting propagation induces the expression of genes associated with plant juvenility. Genes AP2 and ARF are found up-regulated in seedlings and in buds and leaves formed after callus cutting production compared to leaves from formed prior to callus cutting production. Previous work indicates that AP2 repression marks the transition from juvenile to adult phase and that ARF expression is significantly different in adult than in rejuvenated tissues. Callused cutting propagation increases the expression of a rejuvenation-associated microRNA. miRNA 164, which has been described as a rejuvenation-associated microRNA, was found to be significantly up-regulated in seedlings and leavesformed after callus cutting production compared to leaves produced before callused cutting production. Callus cells express genes associated with RNA mobility signals. Gene ontology (GO) analysis of genes which expression increased during callus development identified GO terms associated to RNA mobility (e.g., RNA methylation, noncoding RNA metabolic processes, non-coding RNA processing, RNA transport) both within the cell and systemically. These results support our initial hypothesis that an RNA molecule generated in the dedifferentiated callus cells formed during callused cutting production could be acting as the signal for rejuvenation of tissues in the apical meristems. 2. Generation and maintenance of epigenetic memory of stress in grapevines Exposure to abiotic stress induces the establishment of epigenetic memory of stress that is maintained for at least one year. Genes associated with epigenetic modifications were found to be differentially expressed after stress removal, which could be interpreted as the establishment of an epigenetic memory of stress. This was further supported by primed plants showing a small number of differentially expressed genes associated with stress response one year after being exposed to stress and in the absence of a second stress. Additionally, primed plants presented a stronger response than naïve plants when re-exposed to stress one year later (i.e., higher number of differentially expressed genes under stress than naïve plants, and larger change in expression on those genes differentially expressed under stress both in primed and naïve plants). The type of vegetative propagation affects the maintenance of epigenetic memory of stress. Plants propagated from primed mother plants using layering presented more differentially expressed genes than plants propagated using callused cuttings. Also, only primed layered propagules showed differentially expressed genes in the absence of a second stress event, suggesting that the established stress memory is, at least partially, lost during callused cutting propagation. These results experimentally support my previous results suggesting that while vegetative propagation using layering faithfully replicates the DNA methylation profiles of the propagated plants, callused cutting propagation induces large but consistent changes on the methylome of the propagules. Taken collectively, sections a and b contain the first evidence of long-term stress memory establishment in grapevine and lay the foundation for the development of a molecular model explaining propagule rejuvenation and maintenance of epigenetic memory during vegetative propagation of perennial plants like grapevine. Current work is focussed on the identification of potentially mobile RNA molecules produced in callus cells, that could putatively act as a the "rejuvenation" signal during callused cutting propagation. 3. Molecular regulation of bud break in grapevine MicroRNAs play a key role in grapevine's bud quiescence maintenance. Our results show that Vvi-miRNA166a and b are highly expressed in quiescent buds, while miRNA159a and miRNA159b exhibit a steady increase in expression during bud break. Previous work has shown that the miRNA166 family negatively regulates the expression of TFs of class III Homeodomain leucine zipper (HD-ZIP III), such as REV, PHAB, and homeobox 8 like, known to play an essential role on the regulation of the differentiation on the vascular system. High expression of miRNA166a and b will therefore result in a repression of the production of new vascular tissue, arresting new growth. Conversely, the major function of miR159 is to mediate strong silencing of GAMYB, which is an inhibitor of growth and development. The observed increase in miRNA159a and b would enable growth initiation during bud break. 2. Genetic and epigenetic insights into the domestication of grapevines Cultivated (Vitis vinifera ssp. vinifera) and wild (Vitis vinifera ssp. sylvestris) grapevines are epigenetically different. Reduced representation bisulfite sequencing analysis of the methylome of 10 cultivated varieties and 8 wild genotypes grown in a common garden experiment showed that the genomes of cultivated vines contain significantly higher levels of DNA methylation. At a gene level, cultivated genotypes contained higher levels of CG, while CHG and CHH context and significantly more methylated in the intergenic regions of wild genotypes. Our results suggest that domestication has had an influence on the methylome of cultivated grapevines. A larger study containing 100 genotypes of wild a cultivated grapevines is currently being carried out to validate these results. 3. Development of bioinformatics tools ChromoMap, an R package for interactive visualization of multi-omics data and annotation of chromosomes. ChromoMap is an R package, developed for the construction of interactive visualizations of chromosomes/chromosomal regions, mapping of any chromosomal feature with known coordinates (i.e., protein coding genes, transposable elements, non-coding RNAs, microsatellites, etc.), and chromosomal regional characteristics (i.e. genomic feature density, gene expression, DNA methylation, chromatin modifications, etc.) of organisms with a genome assembly. ChromoMap can integrate multi-omics data (genomics, transcriptomics and epigenomics) in relation to their occurrence across chromosomes. 4. Additional accomplishment: This project studies if vegetative propagation induces propagule rejuvenation and what are the molecular mechanisms controlling such process. To fully understand this process, comparing the effects of vegetative and sexual propagation is paramount. However, grape seed has physiological dormancy and requires three to four months of chilling stratification. Having a system that could by-pass this stratification time to expedite seedling production will allow sexually propagated material to be readily available without the need go through such a protracted chilling period. The recruitment of an undergraduate student to this project allowed us to investigate the impact of partial seed coat removal andgibberellin treatment on germination of freshly harvested and dry grape seeds. Our results show that grape seeds that have been cut and treated with GA can germinate and transition to seedlings without the need for a chilling treatment, thus reducing the time to produce a seedling compared to traditional stratification treatments.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Transcriptome Analysis Reveals Long-Term Somatic Memory of Stress in The Woody Perennial Crop, Grapevine Vitis Vinifera cv. Cabernet Sauvignon Jia W. Tan, Kiflu Tesfamicael, Yikang Hu, Harshraj Shinde, Everard J. Edwards, Penny Tricker, Carlos Marcelino Rodriguez Lopez bioRxiv 2024.04.06.588326; doi: https://doi.org/10.1101/2024.04.06.588326
  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Grapevine Rootstock and Scion Genotypes Symbiosis with Soil Microbiome: A Machine Learning Revelation for Climate-Resilient Viticulture Lakshay Anand, Thanos Gentimis, Allan Bruce Downie, Carlos M. Rodriguez Lopez bioRxiv 2024.02.25.581926; doi: https://doi.org/10.1101/2024.02.25.581926
  • Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Multi-Omics Insights into Grapevine Ecodormancy to Budburst Transition: Interplay of Gene Expression, miRNA Regulation, and DNA Methylation Harshraj Shinde, Tajbir Raihan, Lakshay Anand, Sharyn E. Perry, Robert L. Geneve, Carlos M. Rodriguez Lopez bioRxiv 2023.10.21.563414; doi: https://doi.org/10.1101/2023.10.21.563414
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Rodriguez-Izquierdo, A., Carrasco, D., Anand, L., Magnani, R., Catarecha, P., Arroyo-Garcia, R., Rodriguez Lopez, C. Epigenetic differences between wild and cultivated grapevines highlight the contribution of DNA methylation during crop domestication. BMC Plant Biol 24, 504 (2024). https://doi.org/10.1186/s12870-024-05197-z
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Bridget Bolt, Anna G Baloh, Roberta Magnani, Marta Nosarzewski, Carlos Rodriguez Lopez, Robert L Geneve. Bypassing Seed Dormancy: Improving Grapevine Seed Germination Rate Using Clipping and Gibberellic Acid. 2023 ASHS Annual Conference
  • Type: Theses/Dissertations Status: Accepted Year Published: 2023 Citation: Tan, Jia Wen, "MOLECULAR ANALYSIS OF EPIGENETIC MEMORY OF STRESS ESTABLISHMENT AND LONG-TERM MAINTENANCE IN A PERENNIAL WOODY PLANT" (2023). Theses and Dissertations--Plant and Soil Sciences. 166. https://uknowledge.uky.edu/pss_etds/166
  • Type: Theses/Dissertations Status: Accepted Year Published: 2023 Citation: Raihan, Tajbir, "MULTI-OMIC ANALYSIS OF VEGETATIVE PROPAGATION INDUCED PLANT REJUVENATION IN GRAPEVINES: IMPLICATIONS FOR THE GENERATION OF LOCALLY ADAPTED CULTIVARS USING EPIGENETICS" (2023). Theses and Dissertations--Plant and Soil Sciences. 169. https://uknowledge.uky.edu/pss_etds/169
  • Type: Theses/Dissertations Status: Accepted Year Published: 2024 Citation: Lakshay Anand, "FROM CODE TO CROPS: HARNESSING BIOINFORMATICS AND ARTIFICIAL INTELLIGENCE (AI) IN AGRICULTURAL OMICS" (2024). Theses and Dissertations--Plant and Soil Sciences.


Progress 02/15/22 to 02/14/23

Outputs
Target Audience:Teaching and laboratory instruction: · Three graduate students (PhD) have been recruited to this project (Tajbir Raihan, Lakshay Anand, and Jia Tan) and one postdoctoral researcher (Dr. Harshraj Shinde). All three students are enrolled in the Integrated Plant and Soil Sciences graduate program at the University of Kentucky. Miss Tan's researcher focusses on the analysis of the maintenance of epigenetic memory of stress in primed plants from a gene expression and DNA methylation; Mr Raihan focuses on the identification of the molecular signal inducing plant rejuvenation during vegetative propagation; and Mr Anand's thesis aim is the development of bioinformatics tools for the integration of multiomic datasets for grapevine research. Dr. Shinde acts as project manager at greenhouse and laboratory level · .Additionally an undergraduate student, Bridget Bolt (Junior at the University of Kentucky Agricultural and Medical Biotechnology Program), was recruited in 2021 as part of this project. Miss Bolt project focussed in development of an in vitro system to induce grapevine seed germination without the need of protracted chilling scarification. · PI Lopez has been invited lecturer to two different courses of the University of Kentucky Undergraduate Program Agricultural and Medical Biotechnology (i.e. ABT 301 "Introduction to Biotechnology", and ABT 201 "Scientific Method In Biotechnology") where he discussed the potential applications of epigenetic memory for crop improvement. Extension and outreach: · Invited speaker (Lopez) at the XIII International Symposyum on Grapevine Breding and Genetics (Germany), and the International Conference "Plant and Animal Genomes (PAG)" in San Diego, USA in January 2023. PAG is the largest agricultural genomics conference in the world. · 4 Invited poster presentations (Rodriguez-Izquierdo, Bolt, Shinde, and Raihan) duringthe XIII International Symposyum on Grapevine Breding and Genetics (Germany), and the"Plant and Animal Genomes (PAG)" in San Diego, USA in January 2023. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The recruitment of undergraduate student (Bridget Bolt) has allow her to get training in in-vitro culture techniques she had not been exposed to previously. Also, Bridget has decided to attend graduate school (as a Master student) under the advising of project PI Lopez developing a CRISPR/Cas9 system for grapevines and other perennial woddy crops. How have the results been disseminated to communities of interest?Results from this project have been presented in the form of oral and poster presentations at three different meetings: Plant and Animal Genomes, San Diego, USA; XIII International Symposium on Grapevine Breeding and Genetics.The International Society of Neurogastronomy Symposium 2022. See outcomes for a description. What do you plan to do during the next reporting period to accomplish the goals?A no cost extension has been requested for the complition of this project. The no-cost extension will allow the investigators to finalize the analysis necessary for the publication of the following research articles: 1.Transcriptome Analysis Reveals Long-Term Somatic Memory of Stress in the Woody Perennial Crop Grapevine 2.Grapevine Callused Cutting Propagules Generated from Adult Vines Show Juvenile-Like DNA Methylation, Protein Coding Gene and microRNA Expression Profiles 3.Effect of stress, priming, and plant development on grapevine methylome: a large scale multi-year analysis 4.Maintenance of Long-Term Somatic Memory of Stress in Perennials is Dependent on the Vegetative Propagation System 5.Multi-omics analysis reveals role of molecular and epigenetic regulators during bud ecodormancy to partial budburst transition in grapevine 6.Does pre-conditioning grapevine's response to combined drought and heat stress increasesits tolerance to individual stresses? 7.Domestication process caused different methylation strategies in grapevine 8.Memory of stress loss and plant rejuvenation: two sides of the same molecular coin?

Impacts
What was accomplished under these goals? 1. Epigenetic regulation of propagule rejuvenation during callused cutting production in grapevines Use of callused cuttings for grapevine propagation reduces DNA methylation to levels like those found in juvenile tissues. Global DNA methylation levels in leaf tissue from grapevine seedlings is significantly lower that from leaf tissue from mature plants. Interestingly, global DNA methylation levels in buds and leaves formed after callus cutting production are significantly lower than that of mother plants and similar to those observed in seedling's leaves. These results experimentally validate our preliminary results obtained using commercially grown grapevines of different ages. Callused cutting propagation induces the expression of genes associated with plant juvenility. Genes AP2 and ARF are found up-regulated in seedlings and in buds and leaves formed after callus cutting production compared to leaves from formed prior to callus cutting production. Previous work indicates that AP2repression marks the transition from juvenile to adult phase and that ARF expression is significantly different in adult than in rejuvenated tissues. Callused cutting propagation increases the expression of a rejuvenation-associated microRNA. miRNA 164, which has been described as a rejuvenation-associated microRNA, was found to be significantly up-regulated in seedlings and leaves formed after callus cutting production compared to leaves produced before callused cutting production. Callus cells express genes associated with RNA mobility signals. Gene ontology (GO) analysis of genes which expression increased during callus development identified GO terms associated to RNA mobility (e.g., RNA methylation, non-coding RNA metabolic processes, non-coding RNA processing, RNA transport) both within the cell and systemically. These results support our initial hypothesis that an RNA molecule generated in the dedifferentiated callus cells formed during callused cutting production could be acting as the signal for rejuvenation of tissues in the apical meristems. 2. Generation and maintenance of epigenetic memory of stress in grapevines Exposure to abiotic stress induces the establishment of epigenetic memory of stress that is maintained for at least one year. Genes associated with epigenetic modifications were found to be differentially expressed after stress removal, which could be interpreted as the establishment of an epigenetic memory of stress. This was further supported by primed plants showing a small number of differentially expressed genes associated with stress response one year after being exposed to stress and in the absence of a second stress. Additionally, primed plants presented a stronger response than naïve plants when re-exposed to stress one year later (i.e., higher number of differentially expressed genes under stress than naïve plants, and larger change in expression on those genes differentially expressed under stress both in primed and naïve plants). The type of vegetative propagation affects the maintenance of epigenetic memory of stress. Plants propagated from primed mother plants using layering presented more differentially expressed genes than plants propagated using callused cuttings. Also, only primed layered propagules showed differentially expressed genes in the absence of a second stress event, suggesting that the established stress memory is, at least partially, lost during callused cutting propagation. These results experimentally support my previous results suggesting that while vegetative propagation using layering faithfully replicates the DNA methylation profiles of the propagated plants, callused cutting propagation induces large but consistent changes on the methylome of the propagules. Taken collectively, sections a and b contain the first evidence of long-term stress memory establishment in grapevine and lay the foundation for the development of a molecular model explaining propagule rejuvenation and maintenance of epigenetic memory during vegetative propagation of perennial plants like grapevine. Current work is focussed on the identification of potentially mobile RNA molecules produced in callus cells, that could putatively act as a the "rejuvenation" signal during callused cutting propagation. 3. Molecular regulation of bud break in grapevine MicroRNAs play a key role in grapevine's bud quiescence maintenance. Our results show that Vvi-miRNA166a and b are highly expressed in quiescent buds, while miRNA159a and miRNA159b exhibit a steady increase in expression during bud break. Previous work has shown that the miRNA166 family negatively regulates the expression of TFs of class III Homeodomain leucine zipper (HD-ZIP III), such as REV, PHAB, and homeobox8 like, known to play an essential role on the regulation of the differentiation on the vascular system. High expression of miRNA166a and b will therefore result in a repression of the production of new vascular tissue, arresting new growth. Conversely, the major function of miR159 is to mediate strong silencing ofGAMYB, which is an inhibitor of growth and development. The observed increase in miRNA159a and b would enable growth initiation during bud break. 2. Genetic and epigenetic insights into the domestication of grapevines Cultivated (Vitis vinifera ssp. vinifera) and wild (Vitis vinifera ssp. sylvestris) grapevines are epigenetically different. Reduced representation bisulfite sequencing analysis of the methylome of 10 cultivated varieties and 8 wild genotypes grown in a common garden experiment showed that the genomes of cultivated vines contain significantly higher levels of DNA methylation. At a gene level, cultivated genotypes contained higher levels of CG, while CHG and CHH context and significantly more methylated in the intergenic regions of wild genotypes. Our results suggest that domestication has had an influence on the methylome of cultivated grapevines. A larger study containing 100 genotypes of wild a cultivated grapevines is currently being carried out to validate these results. 3. Development of bioinformatics tools ChromoMap, an R package for interactive visualization of multi-omics data and annotation of chromosomes. ChromoMap is an R package, developed for the construction of interactive visualizations of chromosomes/chromosomal regions, mapping of any chromosomal feature with known coordinates (i.e., protein coding genes, transposable elements, non-coding RNAs, microsatellites, etc.), and chromosomal regional characteristics (i.e. genomic feature density, gene expression, DNA methylation, chromatin modifications, etc.) of organisms with a genome assembly. ChromoMap can integrate multi-omics data (genomics, transcriptomics and epigenomics) in relation to their occurrence across chromosomes. 4. Additional accomplishment: This project studies if vegetative propagation induces propagule rejuvenation and what are the molecular mechanisms controlling such process. To fully understand this process, comparing the effects of vegetative and sexual propagation is paramount. However, grape seed has physiological dormancy and requires three to four months of chilling stratification. Having a system that could by-pass this stratification time to expedite seedling production will allow sexually propagated material to be readily available without the need go through such a protracted chilling period. The recruitment of an undergraduate student to this project allowed us to investigate the impact of partial seed coat removal and gibberellin treatment on germination of freshly harvested and drygrape seeds. Our results show that grape seeds that have been cut and treated with GA can germinate and transition to seedlings without the need for a chilling treatment, thus reducing the time to produce a seedling compared to traditional stratification treatments.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Bolt, Bridget, Roberta Magnani, Marta Nosarzewski, Carlos Rodr�guez L�pez and Robert Geneve (2021). Gibberellin and Clipping Promote Germination in Fresh Grape Seed. Combined Proceedings of the International Plant Propagators Society 70:1
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Bolt, B., Baloh, A., Magnani, R., Nosarzewski, M., L�pez, C. R., & Geneve, R. (2023). Clipping and Gibberellin Treatments Promote Germination in Dormant Grape Seeds, HortTechnology, 33(2), 157-160.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Tan J.W., Shinde H., Tesfamicael K., Hu Y., Fruzangohar M., Tricker P., Baumann U., Edwards E.J., Rodr�guez L�pez C.M. (2023). Global transcriptome and gene co-expression network analyses reveal regulatory and non-additive effects of drought and heat stress in grapevine. Front Plant Sci. 2;14:1096225. doi: 10.3389/fpls.2023.1096225. PMID: 36818880; PMCID: PMC9932518.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Anand, L., Rodriguez Lopez, C.M. (2022). ChromoMap: An R package for Interactive Visualization of Multi-Omic Data and Annotation of Chromosomes, BMC Bioinformatics. 11;23(1):33. doi: 10.1186/s12859-021-04556-z. PMID: 35016614; PMCID: PMC8753883.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Rodriguez Lopez C. M., (July 12, 2022). Influence of vegetative propagation on epigenetic rejuvenation and its effect on vine response to stress: a multi-omic study. XIII International Symposium on Grapevine Breeding and Genetics. International Organization of Vine and Wine, Landau, Germany.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Rodriguez Lopez C. M., (April 20, 2022). Bugs, epialleles and ancient vines: a multifocal view on wine quality. The International Society of Neurogastronomy Symposium 2022, The International Society Of Neurogastronomy, Bonita Springs, FL, United States.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Rodriguez Lopez C. M., (January 9, 2022). Influence of Vegetative Propagation on Methylome Rejuvenation and its Effect on Gene Expression in Vitis vinifera cv. Cabernet Sauvignon. Plant and Animal Genome Conference, San Diego, CA, United States. Invited.
  • Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Tan, J., Rodriguez Lopez C. M. Epigenomics: A New Tool for the Generation of Climate Resilient Grapevines. Under review in Front. Hortic. - Viticulture, Pomology, and Soft Fruits


Progress 02/15/21 to 02/14/22

Outputs
Target Audience:Teaching and laboratory instruction: · Three graduate students (PhD) have been recruited to this project (Tajbir Raihan, Lakshay Anand, and Jia Tan) and one postdoctoral researcher (Dr. Harshraj Shinde). All three students are enrolled in theIntegrated Plant and Soil Sciences graduate program at the University of Kentucky. Miss Tan's researcherfocusses on the analysis of the maintenance of epigenetic memory of stress in primed plants from a gene expression and DNA methylation; Mr Raihan focuses on the identification of the molecular signalinducing plant rejuvenation during vegetative propagation; and Mr Anand's thesis aim is the development of bioinformatics tools for the integration of multiomic datasets for grapevine research.Dr. Shinde acts as project manager at greenhouse and laboratory level · .Additionally an undergraduatestudent, Bridget Bolt(Junior at the University of Kentucky Agriculturaland MedicalBiotechnology Program), was recruited in 2021 as part of this project. Miss Bolt project focussed indevelopment ofan in vitro system to induce grapevine seed germination without the need of protracted chilling scarification. · PI Lopez has been invited lecturer to two different courses of the University of Kentucky Undergraduate Program Agricultural and Medical Biotechnology (i.e. ABT 301 "Introduction to Biotechnology", and ABT 201 "Scientific Method In Biotechnology") where he discussed the potential applications of epigenetic memory for crop improvement. Extension and outreach: · Invited speakers (Shinde and Tan) at the International Conference "Plant and Animal Genomes (PAG)" in San Diego, USA in January 2022. PAG is the largest agricultural genomics conference in the world. · Invited poster presentations (Shinde and Tan) during the 72nd American Society for Enology and Viticulture, National conference (Virtual), 21-24 June 2021. The American Society for Enology and Viticulture (the sciences of winemaking and grape growing) is a professional society dedicated to the interests of enologists, viticulturists, and others in the fields of wine and grape research and production throughout the world. Its membership includes professionals from wineries, vineyards, academic institutions and organizations. · Invited poster presentation (Anand) during the Bio-IT World 2021 International Meeting · Invited poster presentation (Bolt) during the International Plant Propagator's Society (IPPS) 2021 meeting as a poster and a proceedings paper. IPPS is an international association of plant production professionals, including those involved in horticultural research and education.? Changes/Problems:Unexpected delays during the hiring process of project personnel (Post-Doctoral Researcher) at the beginning of the project (Not effective until December 2019), together with the recent restrictions to laboratory and greenhouse facilities access due to the COVID-19 pandemic impacted the normal progress of the project. For this reason we haverequested a no-cost extension to the project. What opportunities for training and professional development has the project provided?The recruitment ofundergraduate student (Bridget Bolt) has allow her to get training in in-vitro culture techniques she had not been exposed to previously. Also, Bridget has decided to attend graduate school (as a Master student) under the advising of project PI Lopez. Bridget has applied for multiple fellowships to cover the cost of her grad student fees and stipend. How have the results been disseminated to communities of interest?Results from this project have been presented in the form of oral and poster presentationsat four different meetings: Plant and Animal Genomes, San Diego, USA; American Society for Enology and Viticulture, National conference (Virtual); Bio-IT World 2021 International Meeting, Boston, USA; and the International Plant Propagator's Society (IPPS) 2021 meeting. See outcomes for a description.? What do you plan to do during the next reporting period to accomplish the goals?A no cost extension has been requested for the complition of this project.The no-cost extension will allow the investigators to generate Next Generation Sequencing data from chromatin immunoprecipitation libraries created during 2021, and to integrate such results to those obtained from whole transcriptome, whole methylome and small RNA Sequencing data generated from the same plants (i.e., mother plants, vegetatively propagated plants and seed propagated plants). Additional time will also be used to Sequence the methylome of a second generation of grapevines propagated from mother plants grown under control and stress condition. Finally, this time will also be used to validate Next Generation Sequencing results using the targeted methodologies described in the proposal. Unexpected delays during the hiring process of project personnel (Post-Doctoral Researcher) at the beginning of the project (Not effective until December 2019), together with the recent restrictions to laboratory and greenhouse facilities access due to the COVID-19 pandemic impacted the normal progress of the project, warranting this request.

Impacts
What was accomplished under these goals? At this stage, milestones from all three objectiveswere projected to be initiated. These milestones included: Phase I: Multi-omic analysis of the epigenetic resetting during CC propagation of grapevine 1.3 Tissue/time-point global levels of DNA and histone methylation and small RNAs estimation - Accomplished January 2021 1.4 Preliminary analysis of Next Generation Sequencing (NGS) results - Accomplished June 2021 1.5 Generation of CCs propagules from dormant mother plants - Accomplished August 2021 1.6 Sample collection from MPs, CCs, and SPPs, and total nucleic acid extraction - Accomplished September 2021 1.7 Finalize analysis of NGS data and comparison with targeted approach results - (May 2021) DELAYED Phase II: Identification of drivers of CC-induced methylome rejuvenation 2.1 Bioinformatic identification of best cultivars for grafting experiment - (May 2020) DELAYED 2.2 Generation of CCs propagules, reciprocal grafts, and layered plants from dormant mother plants and seed scarification.. . .(October 2020) DELAYED 2.3 Sample collection and total nucleic acid extraction - (March 2021) DELAYED 2.4 Small RNA library preparation and sequencing - (April 2021) DELAYED 2.5 Analysis of global levels of DNA methylation and small RNA NGS results - (June 2021) DELAYED 2.6 In vitro culture of green cuttings (GCs) supplemented with small RNA cocktails.(August 2021) DELAYED 2.7 Analysis of global levels of DNA methylation and small RNA NGS results from CCs - November 2021 Phase III (Effect of the source of epigenetic variability on its manteinance during propagation) 3.3 Sample collection from MPs and CCs, and total nucleic acid extraction - Achieved March 2021 3.4 NGS library preparation and sequencing - Achieved April 2021 3.5 Finalize analysis of NGS data - June 2022 Summary of main achievements accomplished during 2021: Phase I: Callused cuttings (CC) (n=144) and seed propagated plantlets(SPPs) (n=12) were grown under control conditions during 2020. Bud samples were collected from mother plants. Bud, callus and leaf samples were collected from 12 different time points during bud break in callused cuttings. Also, first leaf after bud break was collected from one year old CC plants (2021). Finally, first true leaf was collected from seed derived plants on year one and year 2 (2020 and 2021). Information on callus growth, root production, and first leaf emergence was collected from all CCs. Global DNA methylation levels were measured using a methylated DNA methylation levels was determined for callus, leaf, and bud samples at all time points and sample types described above. Analysis of this data allowed for the selection of the most informative sampling times to be analysed using next generation sequencing for each sample type. DNA and RNA was extracted from 3 samples per time point/sample type for genome methylation analysis (whole genome bisulfite sequencing (WGBS)), protein coding gene expression analysis (RNASeq), small RNA expression analysis (smRNASeq), and lysine 4 di-methylation, lysine 9 tri-methylation and 27 tri-methylation Histone modification analysis (ChIPSeq). WGBS, RNASeq, smRNASeq, and ChIPSeq next generation sequencing (NGS) libraries were prepared for each type of sample at all time points and sent for sequencing. To date, the generated data has been analysed to answer two questions relevant this projecto: What are the molecular regulators of budburst in grapevine? Measurement of global levels of DNA methylation in buds during dormancy breaking indicates that DNA methylation increases as domanrcy break progresses. Additionally, two microRNAs (miRNA159a and miRNA159b) showed an increase in expression as dormancy break progresses. 3399 protein coding genes showed a decreasing trend in expression as dormancy breaks. Finally, 137 genes were identified as targets of miRNA159a and miRNA159b. We are currently finalizing the drafting of a manuscript summarizing these results for publication. Does callused cutting propagation induce epigenetic rejuvenation (i.e., reduction of global levels of DNA methylation) in the resultant propagules? Briefly, our results indicate that global DNA methylation levels in leaf tissue from SSPs is significantly lower that from leaf tissue from CCs. Also, global DNA methylation levels do not change significantly in bud and leaf samples collected from CCs when compared to those from mother plants. This is not surprising, since such tissues are already formed prior the CCs induction. However, DNA methylation levels in leaves from 1 year old CCs is lower than that of mother plants and similar to that observed in leaves from SPPs. Leaves produced by one year old CCs are formed after the cutting is generated (that is, they are not present in the dormant buds present when callus is induced in year 1). This result support our hypothesis that a mobile signal generated. RNASeq and smRNASeq data generated from callus, bud and leaf tissue is currently being mined for the identification of potential mobile RNA species (small RNAs, dicistronic tRNA-mRNA molecules, and other previously published mobile RNAs in grapevine). We are expecting to prepare a manuscript from these results to be submitted for peer-review by the end of 2022. Phase III: The physiological response and gene expression patterns of grapevine plants grown under control or stress conditions (i.e., drought, heat, drought and heat) were compared during the period of stress and once the plants recovered from environmental stress. We identified large clusters of genes which expression correlated with change in leaf temperatures and stem water potential. These results suggest that the effect of combined stresses on vine physiology and gene expression was synergistically more severe than for individual stresses, and that epigenetic chromatin modifications may play an important role in grapevine responses to combined drought and heat stress. Interestingly, genes associated with epigenetic mechanisms genes (e.g., histone-lysine methyltransferase) appeared to be exclusively differently expressed only after the stress conditions were imposed for multiple days or after stress removal, suggesting the establishment of an epigenetic memory of stress. To validate the results suggesting the establishment of an epigenetic memory of stress in grapevine, leaf samples were collected from the same plants one year after the initial experiment. Our hypothesis here was that primed plants (plants exposed to stress the year before) will show differences in gene expression compared to naïve plants (plants that were not exposed to stress). This hypothesis was supported by primed plants showing a small number of differentially expressed genes associated with stress response even in the absence of a second stress. These results are currently been prepared for publication as two independent manuscripts. Additional accomplishment: This project studies if vegetative propagation induces propagule rejuvenation and what are the molecular mechanisms controlling such process. To fully understand this process, comparing the effects of vegetative and sexual propagation is paramount. However, grape seed has physiological dormancy and requires three to four months of chilling stratification. Having a system that could by-pass this stratification time to expedite seedling production will allow sexually propagated material to be readily available without the need go through such a protracted chilling period. The recruitment of an undergraduate student to this project allowed us to investigate the impact of partial seed coat removal and gibberellin treatment on germination of freshly harvested grape seeds. Our results show that grape seeds that have been cut and treated with GA can germinate and transition to seedlings without the need for a chilling treatment, thus reducing the time to produce a seedling compared to traditional stratification treatments.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Raihan, Tajbir, Robert L. Geneve, Sharyn E. Perry, and Carlos M. Rodriguez Lopez. 2021. "The Regulation of Plant Vegetative Phase Transition and Rejuvenation: miRNAs, a Key Regulator" Epigenomes 5, no. 4: 24. https://doi.org/10.3390/epigenomes5040024
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Pastor Jullian Fabres, Lakshay Anand, Na Sai, Stephen Pederson, Fei Zheng, Alexander A Stewart, Benjamin Clements, Edwin R Lampugnani, James Breen, Matthew Gilliham, Penny Tricker, Carlos M Rodr�guez L�pez, Rakesh David, Tissue and regional expression patterns of dicistronic tRNAmRNA transcripts in grapevine (Vitis vinifera) and their evolutionary co-appearance with vasculature in land plants, Horticulture Research, Volume 8, 2021, 137, https://doi.org/10.1038/s41438-021-00572-5
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Anand, L., Rodriguez Lopez, C.M. ChromoMap: an R package for interactive visualization of multi-omics data and annotation of chromosomes. BMC Bioinformatics 23, 33 (2022). https://doi.org/10.1186/s12859-021-04556-z
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhou J, Cavagnaro TR, De Bei R, Nelson TM, Stephen JR, Metcalfe A, Gilliham M, Breen J, Collins C and Rodr�guez L�pez CM (2021) Wine Terroir and the Soil Bacteria: An Amplicon SequencingBased Assessment of the Barossa Valley and Its Sub-Regions. Front. Microbiol. 11:597944. doi: 10.3389/fmicb.2020.597944
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: POSTER - Shinde, H., Tesfamicael, K., Rodriguez Lopez, C. Co-expression network-based analysis of genes associated with leaf temperature, stomatal conductance and stem water potential in grapevine. 72nd American Society for Enology and Viticulture, National conference (Virtual), 21-24 June 2021. DOI: 10.13140/RG.2.2.22795.62247
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Invited speaker - Jia Tan. Plant propagation and epigenetic priming modulates grapevine response to subsequent stress: A transcriptomic study. Plant and Animal Genome Conference, San Diego, USA. January 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Invited speaker - Harshraj Shinde. Influence of vegetative propagation on methylome rejuvenation and its effect on gene expression in Vitis vinifera cv. Cabernet Sauvignon. Plant and Animal Genome Conference, San Diego, USA. January 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Lakshay Anand, Carlos Rodriguez Lopez. ChromoMap: An R Tool for Interactive POSTER - Genomic Visualization of Multi-Omics Data. Poster presentation at bioITworld 2021 at Boston Massachusetts (https://www.bio-itworldexpo.com/poster-presentations)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: POSTER - Bolt, B., Roberta Magnani, Marta Nosarzewski, Carlos Rodr�guez L�pez and Robert Geneve. Gibberellin and Clipping Promote Germination in Fresh Grape Seed. International Plant Propagator's Meeting. September 28-October 1, 2021, Chicago, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: POSTER - Tan, J., Shinde, H., Tesfamicael, K., Hu, Y., Tricker, P., Baumann, U., Edwards, E., Rodriguez Lopez, C. Differentially-Expressed Genes in Grapevine Associated with Epigenetic Changes Identified under Combined Stress. 72nd American Society for Enology and Viticulture, National conference (Virtual), 21-24 June 2021. DOI: 10.13140/RG.2.2.12254.51529


Progress 02/15/20 to 02/14/21

Outputs
Target Audience: Teaching and laboratory instruction: • In addition to the 2 graduate students (PhD) recruited to this project during the 2019-2020 period, a third PhD student was recruitedto develop her PhD theses using samples, data and resources from this proposal. Miss Jia Tan enrolled the Integrated Plant and Soil Sciences graduate program at the University of Kentucky, May 2020. Miss Tann is focussing on the analysis of the maintenance of epigenetic memory of stress in primed plants from a gene expression and DNA methylation point of view. • PI Lopez has been invited lecturer to two different courses of the University of Kentucky Undergraduate Program Agricultural and Medical Biotechnology (i.e. ABT 301 "Introduction to Biotechnology", and ABT 201 "Scientific Method In Biotechnology") where he discussed the potential applications of epigenetic memory for crop improvement. Extension and outreach •Invited speaker at the 2020 Annual Meeting of the Argentinian Association of no-till growers (Asociación Argentina de Productores en Siembra Directa - Aapresid) (https://www.aapresid.org.ar/).Title:Epigenetics and Plant Memory •Poster presentation at the25th Annual Meeting of the RNA Society.Title:Dicistronic tRNA-mRNA Transcript Expression Analysis in Grapevine (Vitis vinifera) Suggests Environmental Regulation and Evolutionary Conservation in Plants •Presentation at the 2020 Kentucky Virtual State Fair.Title:Experimental Vineyard (https://www.youtube.com/watch?v=bu52A4BtCOI&list=RDCMUCjLmlBb3hAPB927mQmijXxw&index=1).? Changes/Problems: Major changes/problems Although the project officially started February 15th 2019, it was not scheduled to start until September 2019 due to the predicted availability of plant material. Moreover, the project did fully not start until the 28th of Novemeber. This was due to the time it took to find the right person for the Postdoctoral position included in the application. We found a reallygood candidate in May 2019 and offered him the position but unfortunately, the applicant finally did not accept the job offer. We re-advertised and offered the position to a scholar identified in the second round of advertisement (Dr. Shinde Harshraj Subbash). Dr. Harshraj could not start until the very end of November because of the time it took to get his visa sorted. For this reason, Phase I was not innititated until December 2019, when seeds and vegetative propagules were collected at co-PI Cantu's experimental vineyard at UC-Davis (California). This has resulted in a 10 month over the February offcial project initiation date, and of three months over initiation date included in the project management plan included in the proposal. Such delay got exacerbated by the covid-19 pandemic. During this period our laboratory facilities got to be closed first by direct mandate of the University, and then because of reported cases of covid-19 in the building. Additionally, in order to comply with CDC guidelines, the number of personel in the lab at anytime had to be drastically reduced, which negatively affected productivity. Minor changes/problems The addition of a new PhD student to this project allowed multiple milestones from Phase III to be completed in advance. Very briefly, the comparison of transcriptome data from plants under control andstress (Drought/Heat) conditions indicate thatDEGs that are associated with epigenetic changes during stress and after stress removal, such as histone modification, protein methylation, and protein alkylation. Alteration of the expression of those epigenetic changes-related genes suggests the potential establishment of epigenetic memory after stress removal. Interestingly,analysis of gene expression inprimed and naive plantexposed to subsequent control and stress conditions showthat primed plants present more differentially expressed genes than naive plants when both are under stress. Also, and this is interesting, those genes that are differentially expressed in both stressed primed and stressed naive plants present significantly higher levels of change in primed plants than in naïve ones. What opportunities for training and professional development has the project provided? Miss Jia Tan through this project has acquired skills in molecular biology and bioinformatics. Miss Tan has presented the results of her work during Dr Lopez weekly research group meetings and did a complete presentation of her progress at her annual PhD committee meeting which has helped her building her communication skills. Additionally, this project is closely related to USDA-NIFA project "Implications of epigenetic rejuvenation during vegetative propagation for the production of locally adapted perennial crop cultivars" accession Number: 1018617. This project involves the participation of two graduate students and one postdoctoral researcher. It is expected that the results from both will finally be integrated to generate a cohesive piece of research. Collaboration with other lab members for the purpose of this project also has help to enhance her communication skills. In addition to the Integrated Plant and Soil Sciences Graduate Program 2019-2020 courses, Miss Tan attended the METHADA 2020 eHands on - Virtual Training School in Transcriptomic Metadata Handling and Data Analysis (http://www.integrape.eu/index.php/training-schools/methada-virtual) as a registered observer. This training school addresses grapevine genomics data handling and analysis, and it is organized in three modules. 1. On the first unit, trainees will work to learn how to use the guidelines provided by the INTEGRAPE community to correctly annotate experiments and submit them on public repository. We will explore standards and bio-ontologies for FAIR data annotation using their personal data. The second module will teach the trainees how to use the tools from Sequentia (AIR for transcriptomics). Finally, attendees will be introduced to the GREAT (GRapevine Expression ATlas) platform for transcriptomic meta- analysis. The GREAT platform is a good example of reusing publicly available RNA-seq data to answer new biological questions. Trainees will have the opportunity to explore the GREAT platform with their own list of genes of interest Graduate Student associated to this project Miss Jia Tan, has applied for two competitive grants (Southern SARE Graduate Student Grant program and University of Kentucky College of Agriculture Food and the Environment Graduate Student Research Activity Award). Beyond the obvious benefits of successfully applying for a competitive grant as Graduate Student, both applications help Jia develop grantmanship skills that will be of great use as her career as plant scientist develops. How have the results been disseminated to communities of interest? • Invited speaker at the 2020 Annual Meeting of the Argentinian Association of no-till growers (Asociación Argentina de Productores en Siembra Directa - Aapresid) (https://www.aapresid.org.ar/).Title:Epigenetics and Plant Memory • Presentation at the 2020 Kentucky Virtual State Fair.Title:Experimental Vineyard (https://www.youtube.com/watch?v=bu52A4BtCOI&list=RDCMUCjLmlBb3hAPB927mQmijXxw&index=1). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? At this stage, only objectives 1 and 2 milestones were projected to be initiated. These milestones included: Phase I: Multi-omic analysis of the epigenetic resetting during CC propagation of grapevine 1.1Generation of callused cuttings (CCs) propagules from dormant mother plants and seed scarification. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .(November 2019)Accomplished February 2020 1.2Sample collection and total nucleic acid extraction from mother plants (MPs), CCs, and seed propagated plantlets (SPPs).. . (March 2020)Accomplished November 2020 1.3Tissue/time-point global levels of DNA and histone methylation and small RNAs estimation. . . . . . . . . . . . . . . . . . . . . . . . . . . (March 2020)Accomplished January 2021 1.4Preliminary analysis of Next Generation Sequencing (NGS) results. . . . . .(May 2020)DELAYED 1.5Generation of CCs propagules from dormant mother plants. . . . . . . . . .(October 2020)DELAYED Phase II: Identification of drivers of CC-induced methylome rejuvenation 2.1Bioinformatic identification ofbest cultivars for grafting experiment............(May 2020) DELAYED 2.2Generation of CCs propagules, reciprocal grafts, and layered plants from dormant mother plants and seed scarification....(October 2020) DELAYED 2.3Sample collection and total nucleic acid extraction..................... . . . .(March 2021)DELAYED Thanks to the hiring of a third PhD student, Phase III (Effect of the source of epigenetic variability on its mantenance during propagation), could be initiated during 2020 (instead of during 2021 as originally planned). The following milestones have been accomplished in advance: 3.1Cultivation of mother plants under control/heat stress conditions............ ...(July 2021) 3.2Generation of callous cuttings (CCs) propagules from dormant mother plants... . .......... ............................. ....... ...... .......................(November 2021) 3.3Sample collection fromMPs and CCs, and total nucleic acid extraction...... . .(March 2022) 3.4NGS library preparation and sequencing............................. . . .. .(April 2022) Milestone3.5Finalize analysis of NGS data, is being developed at the time of report submission. We are drafting two manuscripts with these results to be submitted before the end of 2021.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Rakesh David Pastor Jullian Fabres, Na Sai, Stephen Pederson, Fei Zheng, Alexander Stewart, Benjamin Clements, Edwin R Lampugnani, James Breen, Matthew Gilliham, Penny Tricker, C.M. Rodriguez Lopez. Tissue and regional expression patterns of dicistronic tRNA-mRNA transcripts in grapevine (Vitis vinifera) and their evolutionary co-appearance with vasculature in land plants. Accepted Horticulture Research, May 2021
  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2020 Citation: Carlos M. Rodriguez Lopez. Epigenetics and Plant Memory. 2020 Annual Meeting of the Argentinian Association of no-till growers


Progress 02/15/19 to 02/14/20

Outputs
Target Audience:Teaching and laboratory instruction: Two graduate students (PhD) has been recruited to develop their PhD theses using samples, data and resources from this proposal. Both students enrolled the Integrated Plant and Soil Sciences graduate program at the University of Kentucky, January 2019 (Mr Anand) and September 2019 (Mr. Raihan). Mr Anand PhD will focus on developing bioinformatic tools for the analysis and integration ofVitis viniferamulti-omic data. Mr. Raihan's will focus on the identification of the molecular mechanisms regulating the epigenetic memory of stress inVitis vinifera. PI Lopez has been invited lecturer to two different courses of the University of Kentucky Undergraduate Program Agricultural and Medical Biotechnology (i.e. ABT 301 "Introduction to Biotechnology", and ABT 201 "Scientific Method In Biotechnology") where he discussed the potential applications of epigenetic memory for crop improvement. Extension and outreach Invited oral presentation and publication of a Proceedings paper in Southern Nursery Association annual meeting "Harnessing the Power of Epigenetic Priming to Improve Stress Tolerance in VegetativePropagated Perennial Crops" Invited presentation at the European Research Council funded COST Action CA17111. The First Annual Meeting of COST Action CA17111 "INTEGRAPE 2019 - Data Integration as a key step for future grapevine research". March 25-28, 2019. Conference Centerof Mediterranean Agronomic Institute of Chania, Crete, Greece.Title:Ancient epialleles: a multi-omic view on wine quality Poster Presentation at the National Association of Plant Breeders. August 25-29, 2019. Callaway Gardens - Pine Mountain, Ga.Title:NIFA:2019-67013-29168 Crosses between epigenetically distant parents generate novel epi-alleles in commercial grapevine cultivar Cabernet Sauvignon. Invited speaker at the 2020 Kentucky Fruit and Vegetable Conference/Grape and Wine short Course.Title:Epigenetic Memory of Grapevines. Meeting attendants were mainly grapevine growers, winemakers and extension specilists Participation at the Round Table Discussion: Grape Pricing, Grower and Winery Connections, and Helpful Tools for The Industry during the 2020 Kentucky Fruit and Vegetable Conference. During this round table, the potential applications of epigenetic priming in grapevines were briefly discussed with participant growers and winemakers. Poster Presentationat the 2019 Commonwealth Computational Summit. October, 2019. Lexington Kentucky.Title:ChromoMap:An R Tool for Interactive Visualization and Annotation of Chromosomes". ChromoMap was created by PI Lopez PhD student Mr Anand and constitute the first chapter of his thesis.Mr Anad was awarded third place overallhttps://www.uky.edu/hort/node/2104. PI Lopez NIFA-USDA award is announced on the University of Kentucky College of Agriculture Food and the Environment Newsletter.https://news.ca.uky.edu/article/university-kentucky-researcher-receives-grant-study-'memory'-grapevines(June 5th). The Spring Issue of the Ag Magazine features PI Lopez research in grapevine epigenetic memoryhttps://agmagazine.ca.uky.edu/spring-2019/cafe-corner/power-memory. Changes/Problems:Major changes/problems Although the project officially started February 15th 2019, it was not scheduled to start until September 2019 due to the predicted availability of plant material. Moreover, the project did fully not start until the 28th of Novemeber. This was due to the time it took to find the right person for the Postdoctoral position included in the application. We found a really goodcandidate in May 2019 and offered him the position but unfortunately, theapplicant finally did not accept the job offer. We re-advertised and offered the position to a scholar identified in the second round of advertisement (Dr. Shinde Harshraj Subbash). Dr. Harshrajcould not start until the very end of Novemberbecause of the time it took to get his visa sorted. For this reason, Phase I was not innititated until December 2019, when seeds and vegetative propagules were collectedat co-PI Cantu's experimental vineyard at UC-Davis (California). This has resulted in a 10 month over the February offcialproject initiation date, and of three months overinitiation date included in the project management plan included in the proposal. Minor changes/problems Grapevine cultivar PN40024 was proposed as the genotype to be used during this project because at the time it had the best genome assembly available for grapevine. However, since the submission of the grant, co-PI Cantu had generated an assembly for cultivar Cabernet Sauvignon. This new assembly is more complete than that available for PN40024. More importantly, this is the first phasedhaplotypes genome assembly available for grapevine. This means that it provides an independent sequence and assembly forboth chromosomes in each chromosome pair, instead of a single consensus sequence per pair. This is of particular importance in species like Vitis viniferawhich are not only highly heterozygous but also contain a large ammountof structural variantion (indels, duplications, inversions) between their chromosomes, for which consensus sequences are not as informative as phased haplotypessequences. What opportunities for training and professional development has the project provided?Two graduate students (PhD) has been recruited to develop their PhD theses using samples, data and resources from this proposal. Both students enrolled the University of Kentucky'sIntegrated Plant and Soil Sciences graduate program under the supervission of PI Lopez, January 2019 (Mr Anand) and September 2019 (Mr. Raihan). Mr Anand PhD will focus on developing bioinformatic tools for the analysis and integration ofVitis viniferamulti-omic data. Mr. Raihan's will focus on the identification of the molecular mechanisms regulating the epigenetic memory of stress inVitis vinifera. In addition to the IPSS program 2019-2020 courses, Mr Anandattended two conferences where he presented: One oral presentation: "Analysis of evolutionary co-appearance of mobile dicistronic tRNA-mRNA transcripts and vasculature in land plants"at the Evolution Conference 2019, June 21-25, Providence,Rhode Island. One poster presentation:"Chromomap: An R Tool for Interactive Visualization and Annotation of Chromosomes" at the 2019 Commonwealth Computational Summit,October 2019, Lexington, Kentucky. Mr Anand was awardedhird place overall for his poster. In addition to the IPSS program 2019-2020 courses, Mr. Raihanattended oneconference,Southern Nursery Association 2020 Annual Meeting. Baltimore, January 2020. Where was an invited speaker: "Creation and Characterization of an Epimutant population of Soybean". Project's Posdoc (Dr. Harshraj) and graduate students (Mr. Anand and Mr. Raihan) attended the workshop in downstream analyses of epiGBS data organized byAssociate professor Christina Richards,University of South Florida. epiGBS is a reduced representation genome sequecing approach used for the analysis of DNA methylation in large populations. How have the results been disseminated to communities of interest? PI Lopez has been invited lecturer to two different courses of the University of Kentucky Undergraduate Program Agricultural and Medical Biotechnology (i.e. ABT 301 "Introduction to Biotechnology", and ABT 201 "Scientific Method In Biotechnology") where he discussed the potential applications of epigenetic memory for crop improvement. Aproximately 150 students attended both classes. Invited oral presentation and publication of a Proceedings paper in Southern Nursery Association annual meeting "Harnessing the Power of Epigenetic Priming to Improve Stress Tolerance in VegetativePropagated Perennial Crops". This meeting is attended by scientists, graduate and undergraduate students in the field of horticulture and plant propagation, extension specialists and nursery profesionals. Invited presentation at the European Research Council funded COST Action CA17111. The First Annual Meeting of COST Action CA17111 "INTEGRAPE 2019 - Data Integration as a key step for future grapevine research". March 25-28, 2019. Conference Centerof Mediterranean Agronomic Institute of Chania, Crete, Greece.Title:Ancient epialleles: a multi-omic view on wine quality. Poster Presentation at the National Association of Plant Breeders. August 25-29, 2019. Callaway Gardens - Pine Mountain, Ga.Title:NIFA:2019-67013-29168 Crosses between epigenetically distant parents generate novel epi-alleles in commercial grapevine cultivar Cabernet Sauvignon. Invited speaker at the 2020 Kentucky Fruit and Vegetable Conference/Grape and Wine short Course.Title:Epigenetic Memory of Grapevines. Meeting attendants were mainly grapevine growers, winemakers and extension specilists Participation at the Round Table Discussion: Grape Pricing, Grower and Winery Connections, and Helpful Tools for The Industry during the 2020 Kentucky Fruit and Vegetable Conference. During this round table, the potential applications of epigenetic priming in grapevines were briefly discussed with participant growers and winemakers. Poster Presentationat the 2019 Commonwealth Computational Summit. October, 2019. Lexington Kentucky.Title:ChromoMap:An R Tool for Interactive Visualization and Annotation of Chromosomes". ChromoMap was created by PI Lopez PhD student Mr Anand and constitute the first chapter of his thesis.Mr Anad was awarded third place overallhttps://www.uky.edu/hort/node/2104. PI Lopez NIFA-USDA award is announced on the University of Kentucky College of Agriculture Food and the Environment Newsletter.https://news.ca.uky.edu/article/university-kentucky-researcher-receives-grant-study-'memory'-grapevines(June 5th). This newsletter is mainly read by University of Kentucky students and staff, state growers and agriculture professionals. The Spring Issue of the Ag Magazine features PI Lopez research in grapevine epigenetic memoryhttps://agmagazine.ca.uky.edu/spring-2019/cafe-corner/power-memory.This magazine is mainly read by University of Kentucky students and staff, state growers and agriculture professionals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? At this stage, only objective 1 milestones were projected to be innititated.Objective 1 milestones included: 1.1 Generation of callused cuttings (CCs) propagules from dormant mother plants and seedscarification. . . . . . . . . . .. . . . . .(November 2019) Accomplished February2020 1.2 Sample collection and total nucleic acid extraction from mother plants (MPs), CCs, and seedpropagated plantlets (SPPs)].. (March 2020) 1.3 Tissue/time-point global levels of DNA and histone methylation and small RNAs estimation. . . . . . . . . . . . . . . .. . . . . . . . . . . (March 2020) 1.4 Preliminary analysis of Next Generation Sequencing (NGS) results. . . . . . . . . . . . . (May 2020) 1.5 Generation of CCs propagules from dormant mother plants. . . . . . . . . . . . . . . .(October 2020) 1.6 Sample collection from MPs, CCs, and SPPs, and total nucleic acid extraction. . .(March 2021) 1.7 Finalize analysis of NGS data and comparison with targeted approach results. . . . . (May 2021) As explained in the problems and changes section, the current project could not be innitiated until December 2019 which has resulted in a three month delay for all milestones. Project's Posdoc (Dr. Harshraj) and graduate student (Mr. Raihan), under the supervison of co-PI Geneve have innitiated the scarification of grapevine seeds and the production of dormant callused cuttings. We have delivered two outcomes not included in the proposal: 1. Dr. Cantu has generated the first single base resolution methylome for the the grapevine cultivar Cabernet Sauvignon (See other products and changes sections). 2. Graduate student Mr Lakshay Anand, under the supervison of Dr. Lopez, has generated a bioinformatic tool, ChromoMap, that would allow the visualization of multi-omic data in phased haplotypes genome assemblies as that now available for Cabernet Sauvignon (See other products and changes sections).

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Rodriguez Lopez, C.M. Harnessing the Power of Epigenetic Priming to Improve Stress Tolerance in Vegetative Propagated Perennial Crops. SNA Research Conference Proceedings, Plant Breeding and Genetics, 63, 11-14.
  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Corbin, R., Rodriguez Lopez, C.M. Library Preparation for Whole Genome Bisulfite Sequencing of Plant Genomes. In: DNA Methylation Mechanism, Ed. Intech Open.
  • Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Anand, L., Rodr�guez L�pez, C.M. ChromoMap: An R Tool for Interactive Visualization and Annotation of Chromosomes. Archived at BioRXiv https://doi.org/10.1101/605600
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Anand L., Rodr�guez L�pez, C.M. ChromoMap: An R Tool for Interactive Visualization and Annotation of Chromosomes. Commonwealth Computational Summit.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Rodr�guez L�pez, C.M. Ancient epialleles: a multi-omic view on wine quality. First Annual Meeting of COST Action CA17111 "INTEGRAPE 2019 - Data Integration as a key step for future grapevine research".
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Rodr�guez L�pez, C.M., Minio, A., Figueroa-Balderas, R., Cantu, D. NIFA:2019-67013-29168 Crosses between epigenetically distant parents generate novel epi-alleles in commercial grapevine cultivar Cabernet SauvignonPoster Presentation at the National Association of Plant Breeders.