PRECISION BREEDING OF GRAPE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1003965
• Project Start Date: Sep 1, 2014
• Project End Date: Feb 23, 2018
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
Mid-Florida Research and Education Center, Apopka

Non Technical Summary
This project is the continuation of 30 years of continuous research at the University of Florida/IFAS to develop improved cultivars of grapevine that will grow and produce in Florida. Development of improved cultivars suited for Florida will greatly impact other grape-growing regions in the world, sine the cultivars and supporting technology will be attractive there as well. Grapevine is unique among all crops because of its special sensory attributes. A relatively small number of well-known elite cultivars and their landraces account for the majority of world production. They are subject to significant disease pressures, making substantial chemical control and sanitation necessary in many regions. Although better genetic resistance is required to ease production, grapevine is difficult to improve by conventional breeding due to obstacles imposed by its lifecycle. Because of inbreeding depression, self-incompatibility and a long lifecycle, it is not feasible to add specific genetic traits to elite cultivars. However, technology to bypass these obstacles is now available. Initially considered to be highly recalcitrant to advanced genetic engineering, the crucial cell culture and gene insertion systems now are well-established for a wide range of grapevine varieties. The recently-published genomic sequence of 'Pinot Noir' is a significant achievement, providing invaluable insights into grapevine genetics and greatly accelerating the discovery of useful functional genetic elements. Cultivars developed by the PB approach, in which only genetic fragments from sexually-compatible "parents" are utilized, is now attainable and is a logical extension of conventional breeding. A PB approach is more predictable, much less disruptive, and more efficient than that of conventional breeding because only specific traits are transferred and key obstacles are avoided. However, with new varieties under development, substantial field evaluation, as is the norm for conventionally-bred crops, will be required to determine whether cisgenic versions of elite cultivars will possess desirable attributes and/or otherwise be useful.

Animal Health Component

40%

Research Effort Categories

Basic

30%

Applied

40%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1139	1080	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1139 - Grapes, general/other;

Field Of Science
1080 - Genetics;

Keywords

grape

vitis

genetics

precision breeding

breeding

plant improvement

disease resistance

Goals / Objectives
1. To develop and implement a new method for genetic improvement, termed "Precision Breeding", which utilizes only genetic elements from sexually-compatible species. 2.To discover and develop useful grape-derived genetic elements for use in crop improvement, including genes, promoters and other sequences with regulatory functions. 3. To develop acceptable methods to insert these genetic elements into grape plants. 4.Insertselected genetic elements into target varieties and select plants with improved phenotypic changes. 4. To implement objectives 1-3 to develop populations ofdisease-resistant, or otherwise modified, grape plantsvarieties. 5.To conduct screening in the greenhouse and field to select elite individual varieties with desirable phenotypic traits.

Project Methods
In order to actualize a Precision Breeding (PB) approach, we will conductsequence analyses of the grape genome to identify and testantibacterial and antifungal genes, as well as otherDNA elements that control other traits, such as seedless and fruit color. These genetic elements will be isolated from various grape varieties and hybrids that displaydesired traits. Using the novel anthocyanin-based promoter analysis technology that we developed,the transcriptional activity of various promoters will be identified.These promoters will be tested and incorporated in gene insertion experiments in order to replace commonly-used foreign promoters. For disease resistance genes, expression units covering regions of their promoters, protein coding sequences and terminator will be isolated and cloned into DNA insertion vectors using our proprietary high efficiency DNA cloning system. Genes from different varieties may contain functionally important sequence alterations-mutations associated with gain-loss of function and resistance performance. Also, DNA amplification during gene isolation often introduces mismatch mutations. Hence, we will be compelled totest all gene variants that we have isolated to determine their true functionality. So far, over 200 vectors containing different variant genes from thesame and/or different varieties have beenevaluated via a rapid functional assay system using transgenic tobacco. A number of promising genes identified thus far include those encoding VvTL-1, 2S Albumin, Snakin-Defencin, Antimicorbial Peptide-1 (AMP-1), Antimicrobial Peptide-2 (AMP-2), and PR-1 proteins. Semi-PB plants of 'Thompson Seedless' and 'Seyval Blanc' will be produced, screened for trait expression in a greenhouse, then placed in USDA/APHIS/BRS-approved field tests.Extensive functional analysis of these grape genes and their incorporation into the pecision breedingprogram will catapult engineering efforts for achieving enhanced disease resistance, including PD resistance, and other key traits.

Progress 09/01/14 to 02/23/18

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The Project Director, Dr. Dennis Gray has retired from the University of Florida in 2017. We are submitting this Final Report to close the project in REEport.

Publications

Progress 09/01/14 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems:I experienced extreme bullying and harassment during 2014 - present from the UF/IFAS Horticulture Departmernt Chair, the Dean for Research and the Senior Vice President. Defending myself against such viscious attacks caused this project to grind to a stop. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Resources to accomplish precision breeding continue to be studied

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Li, Z.T., Hopkins, D. L., Gray, D. J. 2015. Overexpression of lytic peptides protects grapevine from Pierces disease under greenhouse but not field conditions. Transgenic Research, 24: 821-836.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Gray D. J., Z. T. Li, T. N. L. Grant, D. A. Dean, R. N. Trigiano and S. A. Dhekney. 2015. The Application of Precision Breeding (PB) for Crop Improvement is Fully Consistent with the Plant Life Cycle: The Utility of PB for Grapevine. ACTA Hort. (in press).
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Gray D. J., Z. T. Li, T. N. L. Grant, D. A. Dean, R. N. Trigiano, D. R. Bergey, S. A. Dhekney. 2016. La s�lection v�g�tale de precision pour la mise au point de nouveaux c�pages: Une nouvelle technologie g�n�tique dam�lioration des plantes, enti�rement en accord avec le cycle de vie de la vigne. Revue des Oenologues No 157 sp�cial. January 2016. (Translation of The application of precision breeding (PB) for crop improvement is fully consistent with the plant life cycle: the utility of PB for grapevine).
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Awondo, S. N., G.E. Fonsah and D. J. Gray. Incorporating Structure and Stochasticity in Muscadine grapes Enterprise Budget and Investment Analysis. Hortscience (submitted).
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Brown, K., C. Sims, A. Odabasi, L. Bartoshuk, P. Conner, D. Gray. 2015. Acceptability of muscadine grapes and those characteristics related to consumer acceptance. J. Food Science (submitted)
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Edenbrandt, A., L. House, Z. Gao, M. Olmstead, and D. Gray. 2015. Consumer acceptance of cisgenic food and the impact of information and reference points. Agricultural Economics (submitted)
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Dean, D., Gray, D.J, Li, Z.T., Grant, T.N.L.., Dhekney, S.A., and Trigiano, R.N. 2015. The advent of precision breeding: An advanced method of plant genetic improvement and its utility for Vitis. Plant & Animal Genome XXIII Conference, San Diego, CA January 10-14.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Gray, D. J., Z. T. Li, T. N. L. Grant, D. A. Dean, S. A. Dhekney. 2015. Precision breeding technology for the genetic improvement of grapevine. Annual meeting Soc. In Vitro Biol., Tucson, AZ, May 30  June 3. In Vitro Cell. Dev. Biol. 51: S38.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Alhasan, A. S., D. R. Bergey, S. L. Bieber, V. Sitther, Z. T. Li, D. J. Gray, S. A. Dhekney. 2015. Studying micropropagation response of Mentha x piperita in solid and liquid culture systems. Annual meeting Soc. In Vitro Biol., Tucson, AZ, May 30  June 3. In Vitro Cell. Dev. Biol. 51: S61
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Dhekney, S. A., R. Kandel, D. R. Bergey, H. Shafer, V. Sitther, Z. T. Li, D. J. Gray. 2015. Studying micropropagation response of cold-hardy grapes. Annual meeting Soc. In Vitro Biol., Tucson, AZ, May 30  June 3. In Vitro Cell. Dev. Biol. 51: S62.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Gray, D. J., Z. T. Li, S. Stringer, TW Zimmerman, T. Grant, D. Dean 2015. Progress in precision breeding for genetic improvement of muscadine grapevine. 2015 American Society for Horticultural Science ASHS Annual Conference, New Orleans, LA, August, 2015. Abstract #21677 for oral presentation.
• Type: Book Chapters Status: Published Year Published: 2015 Citation: Trigiano, R. N., J. A. Franklin and D. J. Gray. 2015. A brief introduction to plant anatomy and morphology, Chap. 2. In: Beyl, C. and Trigiano, R. N., (eds.), Plant Propagation Concepts and Laboratory Exercises,2nd edition, 15-27 pp.
• Type: Book Chapters Status: Awaiting Publication Year Published: 2016 Citation: Dhekney, S. A., Z. T. Li, T. N. L. Grant and D. J. Gray. 2015. Somatic embryogenesis and genetic modification of Vitis. In: Methods in Molecular Biology. (In press).
• Type: Other Status: Published Year Published: 2015 Citation: US Plant Patent: Li, Z. T. and D. J. Gray, Antimicrobial peptides and uses thereof, US Patent No. 9074218, 2015