Location: Grape Genetics Research Unit (GGRU)2020 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.
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 8060-21220-006-00D “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 will 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 will develop enabling technologies, including molecular markers and genome editing, to accelerate our speed for 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 5 mapping families in FY20 and identified three new resistance loci for powdery mildew and three new resistance loci for downy mildew. Three U.S. grape breeders made cross hybridizations using resistant vines we developed, for which we have markers to help them track resistance introgression into cultivated backgrounds. In addition, we quantified disease severity after controlled inoculations for four mapping families in FY20, and genetic analyses are in process. The genome-wide rhAmpSeq markers that we developed costing $10/sample were implemented this year for marker assisted selection across the U.S. including two U.S. private breeding programs and several international collaborators. In addition, we are transferring this knowledge to an inexpensive $2/sample marker platform for breeders interested in four or fewer genetic loci. In characterizing pathogen genetics, over 1000 powdery mildew isolates were collected from commercial and research vineyards. Analysis of fungicide resistance is underway and will guide grower decisions about what fungicides should be most effective. In April 2020, we established a Cooperative Research and Development Agreement (CRADA) with a U.S. private company, enabling 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. COVID-19 restrictions have kept us from hiring the two staff for that project and initiating the research. Campus policies not to allow new temporary hires will likely delay laboratory progress at least nine months. These barriers have already eliminated the first field season. Further delays to laboratory progress threaten next year's field season, as laboratory data are required for field design. These delays may cause the company to revoke the agreement due to nonperformance, which would eliminate future progress on Sub-objective 1.B. 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. In the past year (2019-2020) ARS researchers in Geneva, New York, have repeated annual collections of 32 locally important cultivated grapevine varieties, screening them for weekly cold hardiness, dormancy response, and budbreak potential. This effort represents the 2nd annual replication of the study and data analysis is underway to determine if subsequent years of collection are needed. Measuring the responses for these traits are critical to understanding the phenotype of cold hardiness in grapevine and in designing a screen for identifying elite germplasm. In total over 200,000 dormant buds have been screened and measured for these traits. Further, phenotypic evaluation deacclimation resistance at six different temperature treatments, for 28 wild and cultivated grape genotypes, was conducted to develop the statistical framework needed to model temperature effects in late winter and develop a cold hardiness prediction model for the Eastern United States. Evaluation of cold hardiness and deacclimation in 96 progeny from two different grapevine mapping families was conducted to attempt to use QTL studies to identify genomic regions important for these traits. In tandem with field collections, dormant bud material and dormant stem material was collected for RNA sequencing and methylome evaluation as it relates to the endodormancy-ecodormancy transition. Preparation of samples and sequencing has been interrupted by the COVID lab shutdown. 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 CRISPR-based genomic editing tool for improving fruit quality and other traits in elite grape cultivars. Many traditional grape varieties, especially elite wine grapes such as ‘Chardonnay’ and ‘Pinot Noir’, have been in production use for hundreds of years and consumers have developed olfactory recognition and preference for them. Such brand recognition will continue to dominate how grape and wine products are perceived and marketed. However, genetic improvement of these elite grape varieties has been limited by the high heterozygosity of grapevine – any modification of a variety through conventional hybridization and selection would unavoidably change the whole genome makeup, or brand identity, of the variety. With the recent development of the CRISPR-Cas9 gene editing technology one can now make a targeted change of a gene of interest for modifying a trait without impacting the rest of the genome, thus keeping the brand identity of a variety intact. To explore the editing technology for grape improvement, ARS researchers in Geneva, New York, in the past year transformed several CRISPR-Cas9 constructs for modifying the grape color gene VvMybA1 into V. vinifera ‘Chardonnay’ embryogenic callus via Agrobacterium and biolistic transformation. Several transgenic vines with expected editing changes of the color gene VvMybA1 were obtained. Molecular analysis of these vines for further confirmation is in progress. The success of using a transgenic approach for editing a grape gene provides us a proof of concept for pursuing this research further. In practical application, the editing must be done through a non-transgenic approach, because any vines modified through traditional transgenic approaches are regarded as GMOs which are not acceptable to growers and consumers anytime soon. In the past year, ARS researchers in Geneva, New York, evaluated two non-transgenic approaches for editing grapevine genes. The preliminary results were encouraging, and several follow-up research experiments are being actively pursued in this area. 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 fine mapped the red flesh trait to the VvMybA1 locus. The molecular mechanism controlling the trait is under active investigation. Furthermore, ARS researchers in Geneva, New York, recently elucidated the genetic control of a ‘foxy’ aroma gene in the leading juice grape ‘Concord’. This work represents a significant genetic breakthrough for understanding genetic regulation of ‘foxy’ aroma, a special attribute of ‘Concord’ and other V. labrusca derived grapes. All subordinate projects for this parent project are making good progress.
1. Artificial intelligence analysis of disease resistance. The vast majority of grapevines grown in the U.S. are highly susceptible to powdery mildew and downy mildew, requiring 10 to 15 fungicide applications each year to produce a healthy crop. ARS researchers in Geneva, New York, are working with United States grape breeders to develop new varieties with disease resistance that would enable a 90 percent reduction in fungicide applications. This automated robotic imaging and artificial intelligence are improving grape genetics and enhancing precision agriculture.
2. Predicting grapevine bud break using winter severity and chilling exposure. Progressive exposure to low, non-freezing temperatures during winter and chilling exposure, primes dormant grapevine buds for rapid loss of cold hardiness when exposed to warm temperatures. Monitoring cold hardiness point and rate of cold hardiness loss throughout the winter has revealed that these two conditions are strongly associated with bud break. Monitoring the severity of winter and the exposure to chilling temperatures strongly predicts the bud break of grapevine.
3. Genomic resources and molecular markers for a key 'foxy' aroma gene developed for the iconic juice grape 'Concord'. Extensive genomic resources have been generated for common wine and table grapes Vitis vinifera L. However, no parallel work has been reported for juice grapes. ‘Concord’ is the most well-known juice grape cultivar with the characteristic ‘foxy’ aroma of North American grape species V. labrusca. It is this ‘foxiness’ that makes ‘Concord’ grape very popular for non-fermented juice and jellies. However, the ‘foxy’ aroma, not desirable for wine, is not present in common wine grapes V. vinifera. The genetic cause for this species-specific difference is unknown. ARS researchers in Geneva, New York, developed a draft genome of ‘Concord’, uncovered two major structural changes in AMAT which is a key gene for controlling presence/absence of ‘foxy’ aroma between different grape species, and developed molecular markers for making the AMAT gene genetically trackable and amenable in grapevine breeding.
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