Location: Grape Genetics Research Unit (GGRU)2022 Annual Report
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.
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.
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, we collected vineyard disease ratings from 2 mapping families and laboratory disease ratings from 1 mapping family plus about 800 repository accessions in FY22. Two impressive powdery mildew resistance loci (REN11 and REN12) were discovered, published, and integrated with marker-assisted selection in U.S. breeding programs. We discovered the widespread selection for RPV3-1 resistance to downy mildew in several US breeding programs. The genome-wide rhAmpSeq markers that we developed costing $10/sample 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. 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. 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 most of the past year, the position responsible for this objective has been vacant. Efforts focused on generating new RNASeq data to be ready for the incumbent to analyze, maintenance of existing F1 mapping families and development of novel intercross families designed for analysis of low temperature responses in the next Project Plan. The overall aim of 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. Many economically important white grape cultivars, such as V. vinifera ‘Chardonnay’, have a nonfunctional VvMybA1 gene due to the presence of a 10-kb Gret1 transposon in its promoter and restoration of berry color in these cultivars may provide additional choices for grape industry and consumers. ARS researchers in Geneva, New York, initiated a genome editing project a few years ago for removing the 10-kb Gret1 transposon from and restoring the function of the VvMybA1 gene in V. vinifera ‘Chardonnay’. In the past several years, ARS researchers in Geneva, New York, have generated 80 and 106 transgenic vines via Agrobacterium-mediated and biolistic bombardment transformation, respectively, with 8 different editing constructs. Theses transgenic vines were thoroughly analyzed using various molecular analytical methods in the past year. Successful removal of the 10-kb Gret1 transposon from the promoter and functional restoration of a VvMybA1 allele in Vitis vinifera cv. Chardonnay was demonstrated through transgenic expression of Cas9 and two gRNAs simultaneously targeting two junction sequences between Gret1 LTRs and VvMybA1. While the editing efficiencies were as high as 17% for the 5’ target site and 65% for the 3’ target site, simultaneous editing of both 5’ and 3’ target sites resulting in the removal of Gret1 transposon from the VvMybA1 promoter was 0.5% or less in most transgenic calli and vines, suggesting that these calli and vines had very few cells with the Gret1 removed. Nevertheless, two bombardment-transformed vines were found to have the Gret1 successfully edited out from one of their two VvMybA1 alleles. Precise removal of more than a 10-kb DNA fragment from a gene locus in grape broadens the possibilities of using gene editing technologies to modify various trait genes in grapes and other plants. Molecular and sequencing analyses of the edited events in transgenic calli and vines revealed many interesting features of gene-editing, including large structural changes likely resulting from illegitimate recombination of highly homologous VvMybA genes in the VvMybA complex loci. 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. Toward this research goal, ARS researchers in Geneva, New York have made significant progress in dissecting the fine structure of the promoter region of the VvMybA1 locus in several red flesh grapes. We are in the process of investigating the molecular mechanism(s) for controlling the red-flesh trait. Gene editing to restore dark skin color in the white wine grape ‘Chardonnay’. Grapes lack efficient tools for precise genetic improvement. ‘Chardonnay’ is a well-known white wine grape cultivar, in which the gene controlling skin color was long ago inactivated by a large insertion in its promoter. ARS researchers in Geneva, New York, developed gene editing technologies for grape improvement and successfully removed the promoter insertion to restore dark skin color. This work demonstrates the feasibility of altering the quality of an elite grape cultivar and serves as a significant milestone for gene editing technologies for grapevine genetic improvement.
2. Powdery mildew resistant grapevines. Each year across the U.S., grape production requires 6 to 15 fungicide applications to manage powdery mildew (PM), because nearly all grape varieties are highly susceptible to PM. ARS researchers in Geneva, New York, and Parlier, California, discovered two PM resistances and identified the genetic regions responsible. The researchers used traditional breeding and DNA markers to introduce these PM resistances into table grapes. With additional grape breeding and selection, PM resistant table grape varieties would enable a 90% reduction in pesticide use, which would represent a $287 production savings per acre per year.
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