Location: Grape Genetics Research Unit (GGRU)
2023 Annual Report
Objectives
Objective 1: Characterize host and pathogen genetic factors applicable to grapevine disease management, with primary emphasis on powdery mildew.
Sub-objective 1.A. Elucidate the genetic basis of host resistance via QTL mapping and genome editing.
Sub-objective 1.B. Identify and target pathogen genes required for infection of grapevine for improved disease management.
Objective 2: Dissect and elucidate the genetic, genomic, and physiological mechanisms of grapevine abiotic stress tolerance and environmental adaptation.
Sub-objective 2.A. Elucidate the physiological basis of temperature sensing in grapevine and develop a rigorous set of phenotypes for cold hardiness and chilling requirement traits.
Sub-objective 2.B. Determine the genetic architecture of winter survival mechanisms in grapevine through genetic mapping, gene expression, and candidate gene studies.
Objective 3: Generate new germplasm, tools, and strategies for improving grapevine fruit quality and other traits.
Sub-objective 3.A. Develop the CRISPR-Cas9 based genome editing tool for improving fruit quality and other traits in elite grape cultivars.
Sub-objective 3.B. Elucidate genetic control of red-flesh pigmentation in grape berries through genetic mapping and functional analysis.
Objective 4: Intergrate key tratis and QTLs into breeding germplasm. Objective 4 will be coordinated with research on genetics/genomics of host-plant resistance to disease and plant tolerance to abiotic stress for an integrated, systems approach to grapevine improvement. Anticipated products include trait selection for resistance to powdery mildew disease; tolerance to stress from adverse drought and cold grape growing conditions; and understanding genetic factors affecting grape quality.
Approach
Sub-objective 1.A. Collect multi-year vineyard foliar ratings and conduct detailed analysis by controlled inoculation for representative populations. The isolate-specific, quantitative resistance data will improve the reproducibility and precision of QTL mapping, uncovering novel resistance and susceptibility QTL. Pursuit of clonal improvement of existing varieties by editing two powdery mildew susceptibility genes: MLO and a Pectate lyase-like (PLL) gene.
Sub-objective 1.B. Characterize how powdery mildew adapts resistance to fungicides and Candidate Secreted Effector Proteins (CSEPs) that may interact with R-genes released in future cultivars. Use AmpSeq primers for the multiplexed genotyping of known fungicide resistance gene target sites in E. necator. Sequencing of the mating type loci to confirm that selective advantages are occurring with even distribution across mating types and sequence SSRs to monitor for shifts in the population biology of the fungus.
Sub-objective 2.A. Develop new methods of phenotyping supercooling ability, acclimation/de-acclimation, and chilling requirements using a combination of studies in programmable chambers and under field conditions, as well as through deployment of replicated, winter-kill experiments with mapping populations made between highly cold-resistant and cold-sensitive grapevine genotypes. Assay traits using dormant buds collected from field grown vines and potted greenhouse plants. Total vine cold hardiness assayed as winter survival by planting mapping populations constructed between highly tolerant and highly sensitive cultivars. Sub-objective 2.B. Search for genetic loci associated with supercooling, rapid acclimation, delayed de-acclimation, and budburst control through the use of mapping populations and QTL analysis. Examine genome patterns of methylation, differential gene expression analysis of phenotypically diverse “sensitive” and “resistant” phenotypes to identify pathways and downstream candidate genes. Use transgene technology to overexpress and delete the function of key cold stress response genes.
Sub-objective 3.A. Use of a VvMybA gene as a target to develop a CRSPR-Cas9 genome editing tool for grapevine improvement. Adaptation of existing and/or develop new protocols for generating embryogenic callus from target varieties, building various configurations of expression vectors, transforming these vectors into embryogenic callus, and evaluating the transformed cells for successful editing. Pursuit of two additional approaches to generate genome edits without stable integration: a) bombard plasmid DNA transiently expressing both CRISPR and Cas9 components in grape cells to facilitate the editing process; and b) deliver in vitro preassembled complexes of both components (Cas9–gRNA ribonucleoproteins) into grape cells to execute genome editing activities. Sub-objective 3.B. Conduct QTL mapping in bi-parental populations segregating for flesh color, RT-PCR analysis of expression profiles of VymybA genes in skin and flesh tissues of developing berries, and functional analysis of allelic sequence variation in the promoter region of the key VvmybA gene responsible for red flesh.
Progress Report
This report is for the Project 8060-21220-007-000D “Grapevine Genetics, Genomics and Molecular Breeding for Disease Resistance, Abiotic Stress Tolerance, and Improved Fruit Quality”, which addresses NP301 Action Plan Component 2 “Plant and microbial genetic resource and information management." This research project aims to provide genetic solutions to some of these challenges. Specifically, we focus on gene and trait discovery and development for resistance to powdery mildew, tolerance to cold stress, and improvement of fruit quality. In parallel, we develop enabling technologies, including molecular markers and genome editing, to accelerate achieving the research objectives. We have three project objectives in this research.
The goal of Objective 1 is to characterize host and pathogen genetic factors applicable to grapevine disease management, with primary emphasis on powdery mildew. Powdery mildew requires 10 to 15 fungicide applications everywhere grapes are grown, and rapidly evolves to cause disease in the presence of various fungicide chemistries. New resistant varieties and improved management of fungicide applications would have a multi-billion-dollar economic impact. In characterizing grapevine host genetics, ARS researchers in Geneva, New York, germinated a total of 8000 grapevine seedlings segregating for resistance to downy mildew and/or powdery mildew; collected vineyard disease ratings from 3 mapping families; and collected laboratory disease ratings from 19 mapping families (over 100,000 sample images) plus about 800 USDA repository accessions in FY23. The genome-wide rhAmpSeq markers developed in Geneva, New York, continue to be widely used for marker assisted selection in public and private breeding programs, tracking 18 disease resistance loci and 7 fruit quality traits. In FY23, these rhAmpSeq markers were applied to 19,000 grape samples from 11 research programs on three continents. We developed new low-cost DNA markers (KASP) for quick screening of eleven high-priority grapevine traits. A Cooperative Research and Development Agreement (CRADA) with a U.S. private company has enabled us to address Sub-objective 1.B. Identify and target pathogen genes required for infection of grapevine for improved disease management, while there is a vacant scientist position in charge of that sub-objective. A CRADA with the Virginia winegrape industry has enabled the establishment of a new grape breeding program for downy mildew resistance, translating Objective 1A knowledge to applied impact.
The goals of Objective 2 are to dissect and elucidate the genetic, genomic, and physiological mechanisms of grapevine abiotic stress tolerance and environmental adaptation, with special focus on winter survival traits. The genetic architecture of environmentally adaptive traits is complex and requires a deep understanding of physiological mechanisms in order to inform the identification of candidate genes. For the past two years, the position responsible for this objective has been vacant, but an offer has been made to fill this vacancy. In the meantime, ARS researchers in Geneva, New York, generated new RNASeq data to be ready for the incumbent to analyze; maintained existing F1 mapping families; and developed two novel intercross families designed for analysis of low temperature responses in the next Project Plan. In addition, an ORISE postdoc has executed experiments to demonstrate the role of abscisic acid on mid-winter cold hardiness and to develop capacity in grapevine physiology for future investigation of responses to drought, UV, and other stresses.
The overall Objective 3 is to generate new germplasm, tools, and strategies for improving grapevine fruit quality and other traits. One key goal is to develop a clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing tool for improving fruit quality and other traits in grape cultivars. Built upon the success of removing a 10-kb Gret1 transposon from a nonfunctional VvMybA1 gene through CRISPR Cas-9 in the white grape cultivar V. vinifera ‘Chardonnay’, ARS researchers in Geneva, New York, have recently evaluated a new gene editing technology in grapevine, prime editing, which allows precise editing of a single nucleotide in a target gene. Muscat flavor represents a group of unique aromatic attributes found in certain wine and table grapes. Biochemically, grape berries with muscat flavor produce much higher levels of monoterpenes. Monoterpene biosynthesis is mainly through the DOXP/MEP pathway and VvDXS1 encodes the first enzyme for this plastidial pathway of terpene biosynthesis in grapevine. A single point mutation resulting in the substitution of a lysine with an asparagine at position 284 in the VvDXS1 protein has been identified as the major cause of muscat flavor in grapes. ARS researchers in Geneva, New York, have successfully created the same mutation in VvDXS1 alleles through prime editing in the table grape Vitis vinifera cv. Scarlet Royal which has no muscat flavor. The targeted point mutation was detected in most of the transgenic vines. One other key goal for Objective 3 is to elucidate the genetic control of red-flesh pigmentation in grape berries through genetic mapping and functional analysis. ARS researchers in Geneva, New York, have made significant progress in investigating the molecular mechanism(s) for controlling the red-flesh trait.
All subordinate projects for this parent project are making good progress.
Accomplishments
1. Successful development of transgenic herbicide resistance in grapevine. Antibiotic resistance genes from microbes are the most commonly used tool for selection of transgenic plants, but these genes are not acceptable to grape consumers. ARS researchers in Geneva, New York, identified an existing gene in grapevine, acetolactate synthase (VvALS), that could be modified to confer herbicide resistance for callus or in vitro plant selection. Transgenic grapevines with any of three mutations (P191S, P191T, or W568) in VvALS displayed very high resistance to herbicides in the imidazolinone or sulfonylurea families. Our results create a pathway for biotechnological improvement of grapevine via an endogenous selection marker gene.
Review Publications
Song, G., Urban, G., Ryner, J.T., Zhong, G. 2022. Gene editing profiles in 94 CRISPR-Cas9 expressing T0 transgenic tobacco lines reveal high frequencies of chimeric editing of the target gene. Plants. https://doi.org/10.3390/plants11243494.
Lu, L., Yang, Y., Zhong, G., Liang, Z., Cheng, L. 2023. Phytochemical composition and content of Red-Fleshed grape cultivars. Horticulturae. https://doi.org/10.3390/horticulturae9050579.
Song, G., Carter, B., Zhong, G. 2023. Multiple transcriptome comparisons reveal the essential roles of FLOWERING LOCUS T in floral initiation and SOC1 and SVP in floral activation in blueberry. Frontiers in Genetics. https://doi.org/10.3389/fgene.2023.1105519.
Wuddineh, W., Xu, X., Zhong, G. 2023. Amino acid substitutions in grapevine (Vitis vinifera) acetolactate synthase conferring herbicide resistance. Plant Cell Tissue and Organ Culture. https://doi.org/10.1007/s11240-023-02512-8.
Sapkota, S., Zou, C., Ledbetter, C.A., Underhill, A.N., Sun, Q., Gadoury, D., Cadle Davidson, L.E. 2023. Discovery and genome-guided mapping of REN12 from Vitis amurensis, conferring strong, rapid resistance to grapevine powdery mildew. Horticulture Research. https://doi.org/10.1093/hr/uhad052.
