CENTER FOR VITICULTURE AND SMALL FRUIT RESEARCH: EMPOWERING FLORIDA AGRICULTURE AND TRAINING TECHNOLOGY READY PROFESSIONAL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: EVANS-ALLEN
• Reporting Frequency: Annual
• Accession No.: 1019285
• Project Start Date: Apr 3, 2019
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
FLORIDA A&M UNIVERSITY
(N/A)
TALLAHASSEE,FL 32307

Performing Department
Agricultural Research

Non Technical Summary
Florida wine and grape industry has been one of the nation's great agricultural success stories capable to generate economic impact close to 1 billion ("The Economic Impact of the Wine and Grape Industries in Florida", 2010 Report of the Stonebridge Research Group LLC) . In comparison the national grape industry in the United States is valued at more than 40 billion making it the highest value fruit crop in the country.Ever increasing demand for grape products offers a significant economic opportunity for small farmers, especially in the South.Pierces disease is the key limitation for majority of the Southeastern U.S. and commercial grape growing is solely based on native muscadines and Florida hybrid bunch grapes.Florida is currently the 7th producing wine state in the nation and the 2nd largest wine market in the country. This situation offers great economic potential and opportunities for the state to extend the viticulture industry. Because of Pierce's disease caused by the bacterium Xylella fastidiosa, it is not economically feasible to grow the European grapes (Vitis vinifera) such as Merlot, Chardonnay, Syrah, and Cabernet Sauvignon that flourish in California and other major wine growing areas. Only those grape species such as muscadines (Muscadinia rotundifolia) and Florida hybrid bunch grapes (Vitis sp.) that are tolerant to the disease are able to thrive in the hot and humid conditions of Florida and the southeastern region. The Florida grapes (muscadines and Florida hybrids) and wines are unique with their own taste, flavor and aroma that are different from the traditional European grapes. Over the years, breeding and research have resulted in new cultivars with improved fruit and wine quality that has helped the industry grow. However, in spite of these improvements, the industry still faces major challenges that need to be addressed to sustain growth and development. Industry/stakeholder needs to be addressed are as follows:Development of muscadine cultivars with superior characteristics - size, improved taste, color andshelf-life for fresh fruit and wine.Development of Florida hybrid bunch cultivars for red wine with improved vinification qualities (color stability).Enhancement of nutraceutical properties and utilization of value-added products from muscadine grapes.Identification of suitable small fruits as alternative crops for small farmers in North Florida.Identification of best management practices for grapes and small fruits that will help to improve production efficiency and fruit quality.

Animal Health Component

25%

Research Effort Categories

Basic

50%

Applied

25%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1139	1080	40%
202	1199	1081	40%
205	1139	1160	20%

Knowledge Area
205 - Plant Management Systems; 202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1199 - Deciduous and small fruits, general/other; 1139 - Grapes, general/other;

Field Of Science
1160 - Pathology; 1081 - Breeding; 1080 - Genetics;

Keywords

grape

muscadinia

breeding

genomics

proteomics

in vitro

ipm

nutraceuticals

flavonoids

Goals / Objectives
Breeding southern grapes for new superior quality native cultivars The subproject under Dr. Islam El-Sharkawy will carry the following specific objectives:1a. Development of muscadine cultivars with superior characteristics - size, improved taste, color and shelf-life for fresh fruit and wine;1b. Development of Florida hybrid bunch cultivars for red wine with improved taste color, and shelf-life.Capitalizing on our close industry clientele partnership we are aiming to develop a model of economically feasible and beneficial breeding pipeline evaluation trial and to screen and compare 11 advance breeding lines with currently cultivated standard cultivars under diverse growing conditions (the state and the region).Enhancement of nutraceutical properties and utilization of value-added products from muscadine grapesThere is no hypothetical limit to the amount of value that can be added to a crop. Crop plants produce large amounts of biomass at low cost and require limited facilities, and are well suited as bioreactors to produce compounds that can be modified for safe, nutritional or low-cost therapeutic use. Our specific objectives are:2a. To establish a feasible technology for molecular farming of pharmaceutically important bioflavonoids by cell cultures of two native North American grape species: Muscadinia rotundifolia and Vitis aestivalis.2b. To apply integrated bioprocess engineering/genomics/molecular engineering approach to study in details and to improve the metabolic pathway that governs secondary metabolites biosynthesis in submerged grape cell and to develop laboratory technology for production of American native grapes biomass with improved nutraceutical value.Improvement of disease tolerance, physiological, enological and therapeutic values of southern grapes.The subproject under Dr. Mehboob Sheikh: The specific objectives of this research are to:3a. Determine the synthetic and accumulation patterns of berry components associated with superior berry and product characteristics to identify components missing, under- and over-expressed in muscadine grapes compared to bunch grapes.3b. Determine differences in expression of genes, proteins and metabolites between anthracnose- and PD-tolerant and -susceptible genotypes.3c. Determine the influences of location, environment and genotype on genes, proteins andmetabolites related to water use efficiency for developing grape genotypes with high water use efficacy.3d. Determine the identity, genetic variation, and cultural and environmental effects on the content and composition of nutraceutical phytochemicals among muscadine and hybrid bunch grape genotypes.3e. Evaluate muscadine grape genotypes to determine efficacy, genetic variation and molecular mechanisms associated with their cytotoxic activity.3f. Determine the ability of biotic and abiotic agents to enhance expression of nutraceutical- phytochemicals for warranting grape berry nutraceuticals content and health value.3g. Determine the fate of berry components during vinification of juice to define interrelationship between berry metabolite content and composition, and its wine quality.3h. Develop diverse functional foods and agro-pharmaceuticals from muscadine/hybrid bunch grapes for use as therapeutic food supplement to increase their utilization, diversification and market value.4. Identification of suitable small fruits as alternative crops for small farmers in North Florida.The fruit and vegetable production in North Florida specifically for small farmer has not achieved its full potential due to a lack of research on the interaction of location, environment, soil, cultural and management practices as they relate to chemical composition of different fruit varieties and dissemination of new and improved farm technology including the availability of new and improved cultivars. In an effort led by Dr. Islam El-Sharkawy to address this problem(s), the project objectives are designed to:4a.Evaluate economical values and recommend improved fruit and vegetable cultivars for adaptability to North Florida growing conditions that are more appealing to our youth and young adults.4b. Train small farmer, extension agent and students in horticultural best management practices through workshops and field days.4c. Conduct On-Farm demonstrations of improved technology and profitability.4d. Provide experiential learning to K12 campuses through horticultural demonstrations to create interest in and appreciation for fresh fruits and vegetables.5. Identification of best management practices for grapes and small fruits that will help to improve production efficiency and fruit quality.The research is focusing on evaluating the impact of trellis systems and management practices such as pruning, spacing and rootstock performance on yield and fruit quality of southern grapes (muscadine and Florida hybrid varieties).

Project Methods
General Scientific Methods / Research Desigh and EvaluationConventional Breeding and DNA Recombinant Technology: Eleven advanced breeding lines including two seedless bunch grape hybrids, three seedless and three seeded muscadine for fresh consumption, and two bunch and one muscadine for wine-making will be aggressively tested in the University's experimental vineyard at the Center for Viticulture and Small Fruit Research and commercial vineyards in FL, GA, AL and TX. A comparable trial analysis with standard cultivars as controls will be performed to ensure that the new varieties are optimized for profitable economic gains to growers in grape and wine industry. Ultimately, this work will build a breeding and evaluation pipeline that is expected to result in quick cultivar release and increased profit for Florida grape growers.Gene expression analysis: RNA will be extracted from different muscadine genotypes and ripening stages. cDNA library will be constructed using True Seq library preparation kit (Illumina) and sequenced using Illumina HiSeq 2000 instrument. FASTA raw files obtained will be trimmed for removing low quality reads and aligned with the V. vinifera sequences using CLC genomics workbench 8.0.Protein expression analysis: Proteins will be extracted from developing and ripening berries of various muscadine genotypes. Protein extracts will be separated by SDS-PAGE and subjected to trypsin digestion. Trypsin digested peptides will be sequenced using Mass spectrometer. The MS/MS Data will be analyzed for protein identification and quantification. Proteins with > 2 fold increase or decrease will be used for determining their functional roles coding for various berry metabolites.Metabolite analysis: Grape berries from black and bronze muscadine grape genotypes will be collected during different developmental and ripening stages. Brix, titratable acidity and pH will be determined using hand held refractometer and pH meter. Total sugar content will be determined by Anthrone method. Total Phenolics, Anthocyanins, Flavonoids, Stilbenes will be extracted from grape berries using published protocols and quantified using known standards by HPLC.Plant material and inoculation studies: Florida hybrid bunch (Vitis sp.), bunch (V. vinifera) and muscadine (V. rotundifolia) grape genotypes with varying levels of disease tolerance level will be used in the study. Pure cultures of E. ampelina (anthracnose) will be sprayed on both sides of leaves to initiate the infection. Leaf samples from healthy and infected grape plants will be collected at regular interval (1h, 2hr, 24h, 48h and 72h) after inoculation. Pure culture of X. fastidiosa (Pierces disease) bacterial culture will be injected into grapevine stem to initiate infection. Xylem tissue and sap samples will be collected from inoculated plants at two day interval until after PD symptoms appear (~15 days). Gene identification and proteome analysis will be performed as reported in section (b) and (c). Sugar content, titratable acidity and pH in berries and xylem sap will be determined by anthrone method and pH meter.Stress treatments: Two-year old plants from selected grape cultivars will be subjected to various levels of water stress by withholding irrigation. Leaf tissue samples from the plants exposed to different stress levels (5, 10, 15, 20, 25 MPa) will be collected at regular intervals, used in analytical studies (section a). Molecular responses of grape plant to water stress will be determined as reported in section (b) and (c) to identify transcripts that are up-regulated or down- regulated to specific stress treatment.Nutraceutical content determination: Grape berries from forty nine muscadine and twelve Florida hybrid bunch grape genotypes will be collected in three replications and transported to the lab on ice. Total phenolics, anthocyanins, flavonoids, stilbenes will be extracted from grape berries using published protocols and quantified using HPLC. Metabolites from grape berries will be measured using known standards.Cytotoxic activity: Ripe grape berry from muscadine genotypes containing higher and lower amounts of stilbenes will be collected from the FAMU vineyard and used in this study. Whole grape berry will be ground in liquid nitrogen and the ground tissue will be extracted using organic solvents (1:10w/v) by homogenizing with a solvent mixture of methanol:ethylacetate. The extracts will be dissolved in DMSO and used for cytotoxic evaluation. The seed and skin tissue of the muscadine genotypes showing high cytotoxic activity will be selected and phytochemicals will be extracted, fractionated and quantified by HPLC.Abiotic and biotic treatment for enhancing nutraceutical content: Ripe grape berries from both muscadine and Florida hybrid grape genotypes will be used for eliciting phytochemicals content employing UV and chemicals (abiotic), and non-pathogenic microbes (biotic) as elicitors. Phytochemicals will be quantified as reported in section (a) for determining the ability of different elicitors to enhance phytochemical content in muscadine and Florida hybrid bunch grape genotypes.Methods to be used to reach direct and indirect contacts with target audience Education Class Workshop Group Discussion One-on-One Intervention Demonstrations Other 1 (Field Days) Other 2 (Harvest Festivals) Public Service Announcement Newsletters TV Media Programs eXtension web sites?

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Grape growers and winemakers and industry clientele, college students, academic research scientist and comunity at large. Changes/Problems:Chalanges imposed under the global COVID19 pandemic working conditions has been slowing the execution of the research experimentation and limiting the presence of graduate and undergaraduate reseach students at the Center, but no major changes are needed in the work plan of the project. What opportunities for training and professional development has the project provided?The Center continues to foster national and international research cooperation and facilitate faculty and student's membership in professional organizations (FSHS, FGGA, Florida Academy of Science, ASHS, In Vitro Biology, ASEV, ISHS) and elevate the national and international research recognition of the School and the Program. Enhancing and promoting undergraduate and graduate student research, academics and development: Course Taught: AGG 5322 Plant Breeding AGG2050-Introduction Biotechnology AGG2050L Introduction Biotechnology Lecture AGG4420 Global Seminar in Biotechnology Graduate student research: Jiovan Campbell, MS graduate student, major Plant Science, Title: Characterization of Large Muscadine Population for Diverse Phenological, Vegetative Growth and Reproductive Performance Traits. Raquel Mcarthur, MS graduate student, major Plant Science, Title: Identify physiological and physiochemical traits associated with drought tolerance in bunch grapes. Undergraduate Student Research: Salau Oluwaseyi, Biological Systems Engineering, Senior. "Digital Reference Database for Southern Wines" Graduated Spring 2020. Abigahill Simon, Food Science, Junior, "RT qPCR Virus Diagnostic for Florida Graves". Papa Guye, Agronomy, Senior, " Meristematic Shoot-Tip Micropropagation for Diseases Elimination in Florida Grap[es". How have the results been disseminated to communities of interest?Industry/ Growers Workshops and Activities: Grape Harvest Festival - Center for Viticulture & Small Fruit Research, Tallahassee, FL; August 24, 2019 - Organizer team member. The UF Grape Field Day, July 16, 2019, Citra, FL. Vineyard Management Workshop, February 11, 2020 Muscadine Training and Pruning Demonstration- Frenchtown Urban Farm, Tallahassee, FL, February 11th, 2020.? What do you plan to do during the next reporting period to accomplish the goals?The Center continues to maintain a strong working relationship with Florida growers and private industry through research and outreach activities. Partnering with FGGA and Viticulture Advisory Council we provided research and technical support to enhance grape products, demonstrate health benefits, protect vineyards from pest and disease, and address other issues of importance to the successful expansion of the grape growing industry. We continue to foster national and international research cooperation and facilitate faculty and student exchange under the FIPSE/ Atlantis Program and North American Research Network for Grape Functional Genomics.

Impacts
What was accomplished under these goals? · Develop a breeding platform utilizing the unique beneficial qualities of the Muscadine for securing the lead and enable breeding programs in the US via routinely applying modern genomics and genetics tools to efficiently deliver cultivars with producer-required disease resistances and market-essential fruit/vinification qualities. · Enhance Florida grape industry through identifying new bunch grape and muscadine genotypes suitable for wine production and fresh consumption, adapted to Florida condition. This includes the release of two red muscadine cultivars suitable for wine production 'Floriana' and fresh consumption 'Onyx', as well as start the process for the release of two new bunch grape cultivars suitable for white wine production 'Blanc du Soliel' and 'Blanc de Leon'. · Identified and functionally associated Muscadine Whole Genome Sequences (WGS) with the predominant V. vinifera genome. Identified muscadine grape genotypes with particular antioxidant activity and therapeutic properties for breast cancer (please see list of publications). Completed the evaluation of three self-pollinated advanced bunch grape selections; A24-6-6, O47-14-1, and B18-2-2 suitable for white- and red-wine production; for two consecutive years 2018/2019. Completed the evaluation of two self-pollinated advanced muscadine selections; A27-10-10 and O19-19-1 suitable for white- and red-wine production; for three consecutive years 2018 - 2020. Completed the first chromosome-level assembly of the Muscadinia rotundifolia genome. We assembled each of Muscadinia 20 chromosomes from end-to-end with few assembly gaps and unplaced scaffolds (Fig.1) By combining Illumina sequencing, Pacbio sequencing, and Hi-C, we generated a highly accurate, nearly complete draft genome, enabling genetic, genomic, transcriptomic, and proteomic studies. Synteny analysis revealed highly conserved chromosome structure compared to Vitis genomes. Hi-C analysis on adult leaf tissue revealed semi-discrete topologically associated domains, and evidence for intertelomeric contacts during interphase. We provide a web-based genome browser to access, download, and browse the Muscadinia genome and transcriptome data that will be publicly available to enable future studies. The resources developed here enable Muscadinia rotundifolia as a model species to understand the evolution of grapevine chromosomes, biotic/abiotic stress resistance, and antioxidant production. · Identified muscadine grape genotypes with particular antioxidant activity and therapeutic properties for breast cancer. · Under MTA generated between Center for Viticulture/FAMU and Department of Horticulture/UF, we have received UF muscadine and bunch grape germplasm (Table 1 and 2) to maintain under FAMU grape breeding program at the Center for Viticulture. · Seventy bunch grape genotypes (Blanc Du Bois, Cynthiana and Conquistador cultivars along with 67 breeding lines), 305 muscadines (58 cultivars and 247 breeding lines), and 52 Muscadinia x Vitis hybrids were comprehensively evaluated for phenological characteristics to determine phenology-related traits, vegetative growth performance, and reproductive growth quality that includes traits related to fertility, cluster/berry characteristics, yield, and biochemical properties. Identified and characterized sucrose phosphate synthase (SPS), sucrose synthase invertase and invertase inhibitor gene families of high-sugar and low-sugar containing muscadine grapes. We are also working on identification and expression analysis of SWEET family of sugar transporters with the goal of identifying factor/s limiting sugar accumulation in muscadine berry. Quantified differential expression of genes associated with anthracnose DISEASE tolerance (Chalcone Isomerase, Chalcone Synthase, Stilbene Synthase, WRKY transcription factor, Chitinase, Pathogen Resistance gene1 and Dihydro flavonol-4-reductase genes) between anthracnose-tolerant and -susceptible hybrid Bunch grape cultivars. Once the target gene/s is identified and validated for disease tolerance it can be edited to induce anthracnose resistance in Muscadine grapes. Developed various whole berry and juice-based food products to serve as nutraceutical functional foods and supplements for promoting consumer health. Some of these prototypes include Grape Honey, Musca Delights, Flavored Beverages, Sweetener, Dessert Toppings, Jelly, Juice, Vinegar, Breakfast bars, Snacks, Popsicle, Grape nuts, Preserves, Musca Bites, etc. We are optimizing processing protocols to retain bioactive compounds content and properties to warrant product health values. · Total of 23 wines entered into the Digital Reference Database for Southern Wines. · Total of 9 eligible wine entries received from the industry partners which have been analyzed, input into database, and reported back to clientele. · Continued analysis of research wines produced by the CVSFR from vintages 2018, 2019, and 2020. · New experimental varieties of grapes into wine (A-24, O-47, A27, C-11, 'Floriana' cv.). Analysis will be done and added to the Database once winemaking is complete. · Wine Analytical Lab is increasing analytical capacity; utilization of the phenolic classes assay gives us rapid method for estimating total phenols, flavonols, hydroxycinnamic acids, and anthocyanins. During the reporting period, 185 gallons of wine was bottled which produced 1,359 bottles or 113 cases. The quantification of each wine is shown in table 2. This is perhaps the most productive year in terms of wine production to date for the CVSFR. This was the result of many factors, including the utilization of two winemakers and motivated farm workers, favorable growing conditions, and the employment of new enological techniques. The Southeastern NCPN Center for Grape at FAMU evolved further by expanding and including the working group from Texas A&M University/Horticulture Department in College Station and Micropropagation and Repository Unit at North Carolina State University (NCSU). In season 2020 a total of 2,776lb/ 1261kg grapes have been harvested for processing and vinification.This year, cryomaceration was used on varieties such as C30-5-1, C30-7-1, 'Mid-South', 'Conquistador,' 'Floriana', and 'Noble' cvs. After the wines are complete, each will be analyzed and input into the Digital Reference Database for Southern Wines. The addition of these 19 from 2020 as well as the 25 from 2019 will result in 44 more wines added to the Digital Reference Database for Southern Wines bringing the total to over 100 wine entries.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: El-Sharkawy I, Ismail A, Darwish A, El Kayal W, Jayasankar S. Sherif S. (2020) Functional characterization of a gibberellin F-box protein, PslSLY1, during plum fruit development. Journal of Experimental Botany, https://doi.org/10.1093/jxb/eraa438.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ismail A, El?Sharkawy I, Sherif S. (2020) Salt stress signals on demand: cellular events in the right context. International Journal of Molecular Sciences 21:3918.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Georgiev,V., Ananga, A., Dincheva,I., Badjakov,I., Gochev,V., and V. Tsolova, 2019: Chemical Composition, In Vitro Antioxidant Potential and Antimicrobial Activities of Essential Oils and Hydrosols from Native American Muscadine Grapes, Molecules, 24 (18), 3355, http://dx.doi.org/10.3390/molecules24183355
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Darwish AG, Mendonca P, Tsolova V, Soliman KFA, El-Sharkawy I. (2019) The anticancer and antioxidant effects of muscadine grape extracts on racially different triple-negative breast cancer cells. Anticancer Research 39: 4043-4053.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Li C, Dougherty L, Coluccio AE, Meng D, El-Sharkawy I, Borejsza-Wysocka E, Liang D, Pineros MA, Xu K, Cheng L. (2020) Apple ALMT9 requires a conserved C-terminal domain for malate transport underlying fruit acidity. Plant Physiology 182: 9921006.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Humphries, F., (2020) Review of sulfur dioxide alternatives for small and large-scale wineries, FWGGA Newsletter, September 25th, 2020
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Salau Oluwaseyi, Frank Humphries and Violeta Tsolova, 2020: Digital Reference Database for Southern Wines, Annual Conference of FWGGA, DeLand, FL, January 17, 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Abighahill Simon and Violeta Tsolova, 2020: Molecular Diagnostic of Muscadine Grape Viruses, Annual Conference of FWGGA, DeLand, FL, January 17, 2020.

Progress 04/03/19 to 09/30/19

Outputs
Target Audience: Changes/Problems:Recovering operations in the experimental vineyards and pendingrepairs of experimental screen houses post huricane "Michael" . What opportunities for training and professional development has the project provided?The Center continues to foster national and international research cooperation and facilitate faculty and student's membership in professional organizations (FSHS, FGGA, Florida Academy of Science, ASHS, In Vitro Biology, ASEV, ISHS) and elevate the national and international research recognition of the School and the Program. Enhancing and promoting undergraduate and graduate student research and development: Graduate student research: Pranavkumar D. Gajjar, MS graduate student, major Plant Science, graduated in August 2018, Title: Plant Performance and Nut Characteristics of Chestnut (Castanea Spp.) Grown in North Florida. Patricia Akingboye, MS graduate student, major Plant Science, graduated in June 2019, Title: Genotypic Variation in Vinification Qualities and Antioxidant Properties of Muscadine Wines in Florida. Jiovan Campbell, MS graduate student, major Plant Science, Title: Characterization of Large Muscadine Population for Diverse Phenological, Vegetative Growth and Reproductive Performance Traits. Raquel Mcarthur, MS graduate student, major Plant Science, Title: Identify physiological and physiochemical traits associated with drought tolerance in bunch grapes. Undergraduate Student Research: Abigahil Simon, 2019 and 2020 FAMU "World Scholar Program"-Project Title: Microbiomic Analyssis of Muscadinia rotundifolia and Vitis sp. using qPCR., PI Dr. Violeta Tsolova Papa Gueye, Project Title: Project: "Meristematic Shoot Tip Culture and Grapevine Virus Free Clean Plant Material, PI: Dr. Violeta Tsolova Salau Oluwaseyei-Digital Reference Database of the Vinification and Healthy Qualities of Southern Wines., PI Dr. Violeta Tsolova Victor Cupp - Project Title: Phenological, vegetative and reproductive growth characterization of large bunch/muscadine grape populations to identify high quality advance selections meeting the diverse Florida grape industry demands., PI Dr. Islam El-Sharkawy Anthony Enriquez - Project Title: Evaluation of pollen viability in a small Vitis x Muscadinia grapevine hybrids population., PI Dr. Islam El-Sharkawy Bilal Albibi - Project Title: Phenological, vegetative and reproductive growth characterization of large bunch/muscadine grape populations to identify high quality advance selections meeting the diverse Florida grape industry demands., Dr. Islam El-Sharkawy Ms. Alexandra Reid, Project Title-Phytochemical extraction methods and human cell culture techniques., FAMU 2019 Summer Research Scholars Training Program., PI-Dr. Mehboob Sheikh How have the results been disseminated to communities of interest? Industry/ Growers Workshops and Activities: 2019 Grape Harvest Festival, August 24, 2019, Tallahassee, FL. The UF Grape Field Day, July 16, 2019, Citra, FL. Summer Growers IPM Field Day, Centre for Viticulture & Small fruit Research, May 1, 2019. Vineyard BMP Workshop, February 13, 2019, Tallahassee, FL. Florida Wine & Grape Growers Association (FWGGA) Annual Conference, Deland, FL, January 17-19, 2019 Winemaking Workshop, 2019 Grape Harvest Festival, Center for Viticulture and Small Fruit Research, August 26th, 2019 Muscadine Pruning Demonstration- Fairview Middle School, February, 2019 What do you plan to do during the next reporting period to accomplish the goals?The Center continues to maintain a strong working relationship with Florida growers and private industry through research and outreach activities. Partnering with FGGA and Viticulture Advisory Council we provided research and technical support to enhance grape products, demonstrate health benefits, protect vineyards from pest and disease, and address other issues of importance to the successful expansion of the grape growing industry. We continue to foster national and international research cooperation and facilitate faculty and student exchange under the FIPSE/ Atlantis Program and North American Research Network for Grape Functional Genomics.

Impacts
What was accomplished under these goals? The 1st version of the whole-genome sequencing and assembly of the muscadine grape, Muscadinia rotundifolia cv. Noble. PCR-free libraries of total genomic DNA were generated and used to produce 200 million paired-end reads (~100 Gb) using different sequencing strategies, including Illumina HiSeq 2500 (produced 80M reads), Chicago (produced 181M reads) and Hi-C (produced 223M reads). Analysis of muscadine genome assembly statistics revealed an estimated whole genome size of 414 Mb. Due to high heterozygosity (1.47%), we generated an assembly representing 400.26 Mb of the estimated 414 Mb genome (96.6%) using Discover De Novo for primary assembly construction and Redundans for consolidating redundant contigs. However, the contig and scaffold N and L values indicated a high contig and scaffold assembly. Assessment of muscadine genome assembly and gene set using Benchmarking Universal Single-Copy Ortholog (BUSCOs) suggests highly complete representation of protein-coding genes. A total of 1440 BUSCO groups has been searched, among them 1382 were complete BUSCOs (96%) [1339 complete and single-copy BUSCOs (93%), and 43 complete and duplicated BUSCOs (3%)], 17 fragmented BUSCOs (1.2%), and 41 missing BUSCOs (2.8%). Comparison of M. rotundifolia and V. vinifera genomes revealed that muscadine genome size is only 82% of Vitis genome size. The two genomes display high colinearity with some muscadine scaffolds that that entirely align with Vitis chromosomes. However, the extra Muscadinia chromosome is composed of two large segments homologous to Vitis chromosome 7. Currently, we are generating a full transcriptome sequence data using RNA-seq and PacBio-seq in order to annotate the muscadine genome for final submission at Phytozome and NCBI gene bank. Develop a breeding platform utilizing the unique beneficial qualities of the Muscadine for securing the lead and enable breeding programs in the US via routinely applying modern genomics and genetics tools to efficiently deliver cultivars with producer-required disease resistances and market-essential fruit/vinification qualities. Enhance Florida grape industry through identifying new bunch grape and muscadine genotypes suitable for wine production and fresh consumption, adapted to Florida condition. This includes the release of two red muscadine cultivars suitable for wine production (Floriana) and fresh consumption (FL Onyx), as well as start the process for the release of two new bunch grape cultivars suitable for white wine production (Blanc du Soliel and Blanc de Leon). Identify muscadine grape genotypes with particular antioxidant activity and therapeutic properties for breast cancer. Breeding program in season 2019: have been completed 13 cross-pollinations trials and collected 4053 seeds (3438 for bunch and 615 for muscadine). These seeds are under cold storage for germination process. Identified high stilbene producing and low stilbene containing Muscadine grape cultivars by assessing the pattern from three consecutive years. As stilbenes are the major bioactive metabolites in Muscadine berries, this research will have impact on breeding programs, nutraceutical development and fresh fruit market. Developed a post-harvest stilbene induction technique to enhance its berry content for increasing nutraceutic potency as well as improve shelf-life of muscadine grape products. The efficacy of muscadine berry phytochemicals against a variety of cancers (liver, pulmonary and mammary) was determined and the elite varieties with potent anti-cancer activity against specific tumors have been identified and validated. Molecular targets of muscadine grape phytochemicals have been identified in mammary and pulmonary cancer using gene expression studies. Additional studies showed that the phytochemicals profile and cytotoxic potential of muscadine berry changes during berry development and dictate their cytotoxic potency. Developed various whole berry and juice-based food products to serve as nutraceutical functional foods and supplements for promoting consumer health. Some of these prototypes include Grape Honey, Musca Delights, Flavored Beverages, Sweetener, Dessert Toppings, Jelly, Juice, Vinegar, Breakfast bars, Snacks, Popsicle, Grape nuts, Preserves, Musca Bites, etc. We are optimizing processing protocols to retain bioactives content and properties to warrant product health value. Identified and characterized sucrose phosphate synthase (SPS), sucrose synthase invertase and invertase inhibitor gene families of high-sugar and low-sugar containing muscadine grapes. We are also working on identification and expression analysis of SWEET family of sugar transporters with the goal of identifying factor/s limiting sugar accumulation in muscadine berry. Quantified differential expression of genes associated with anthracnose DISEASE tolerance (Chalcone Isomerase, Chalcone Synthase, Stilbene Synthase, WRKY transcription factor, Chitinase, Pathogen Resistance gene1 and Dihydro flavonol-4-reductase genes) between anthracnose-tolerant and -susceptible hybrid Bunch grape cultivars. One the gene/s is identified they can be edited to induce anthracnose resistance in Muscadine grapes.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Balasubramani SP, Rahman MA, Basha SM. Synergistic Action of Stilbenes in Muscadine Grape Berry Extract Shows Better Cytotoxic Potential Against Cancer Cells than Resveratrol alone. Biomedicines. 2019 Dec; 7(4): 96. doi: 10.3390/biomedicines7040096
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: I. El-Sharkawy, A. Darwish, V. Tsolova, 2019: Genotype Variability of Anticancer & Antioxidant Effects in Muscadine Grape, The 2019 North American Grape Breeders Conference, August 1516, 2019, Mountain Grove, MO.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: I. El-Sharkawy, D. Kambiranda, D. Vera, V. Tsolova, 2019: The First Version of the Whole-Genome Sequencing of the Muscadine Grape (Muscadinia rotundifolia cv. Noble), The 19th Biannual Research Symposium  Association of 1890 Research Directors (ARD), March 30  April 3, 2019, Jacksonville, FL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: J. Campbell, V. Cupp, A. Darwish, V. Tsolova, I. El-Sharkawy, 2019: Structure of the Variability of Fruit Phenology, Phenotypical and Biochemical Traits in Large Non-Targeted Population of Muscadinia rotundifolia. The 19th Biannual Research Symposium  Association of 1890 Research Directors (ARD), March 30  April 3, 2019, Jacksonville, FL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: I. El-Sharkawy, 2019: Insights into Muscadine (Muscadinia rotundifolia) Grape Breeding in Florida, The University of Guelph  The Department of Plant Agriculture Seminar, June 26, 2019, Guelph, Canada.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Georgiev,V., Ananga, A., Dincheva,I., Badjakov,I., Gochev,V., and V. Tsolova, 2019: Chemical Composition, In Vitro Antioxidant Potential and Antimicrobial Activities of Essential Oils and Hydrosols from Native American Muscadine Grapes, Molecules, 24 (18), 3355, http://dx.doi.org/10.3390/molecules24183355
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Darwish AG, Mendonca P, Tsolova V, Soliman KFA, El-Sharkawy I. (2019) The anticancer and antioxidant effects of muscadine grape extracts on racially different triple-negative breast cancer cells. Anticancer Research 39: 4043-4053.
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Li C, Dougherty L, Coluccio AE, Meng D, El-Sharkawy I, Borejsza-Wysocka E, Liang D, Pi�eros MA, Xu K, Cheng L. (2019) Apple ALMT9 requires a conserved C-terminal domain for malate transport underlying fruit acidity. Plant Physiology, under revision.
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Dougherty L, Borejsza-Wysocka E, Borejsza-Wysocki W, El-Sharkawy I, Liang D; Cheng L; Kellerhals M, Patocchi A, Xu K. (2019) A deletion lethal to a novel and Rosaceae specific gene Q8 reduces fruit acidity in apple. New Phytologist, under revision.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Kambiranda DM, Basha SM, Stringer SJ, Obuya JO, Snowden JJ. Multi-year Quantitative Evaluation of Stilbenoids Levels Among Selected Muscadine Grape Cultivars. Molecules. 2019 Mar 11;24(5). pii: E981. doi:10.3390/molecules24050981
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Patricia Akingboye, 2019: Genotype and the Vinification and Healthy Qualities of Two Advanced Selection Muscadine Grape (Muscadinia rotundifolia).
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Humphreys, A. and V. Tsolova, 2019:Wine Chemistry 101: Testing Your Wines 2019 FWGGA Annual Conference, January 17-19, 2019 Courtyard by Marriott, Deland Florida.