Location: Grape Genetics Research Unit (GGRU)
2019 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.
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 NP301 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, molecular markers and genome editing to accelerate our speed for achieving the research objectives.
We have three 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 six mapping families in FY19 and identified three new resistance loci, two for downy mildew and one for powdery mildew. In addition, we quantified disease severity after controlled inoculations for seven mapping families in FY19. Genetic analyses are in process, including the targeted analysis of a widely used powdery mildew resistance gene (REN3) that is present in grapevine cultivars that are widely planted internationally – Seyval blanc, Chambourcin, Regent, and Villard blanc, for example. The germplasm used in the above mapping families is being evaluated by grape breeders for use in their programs, and the markers for REN3 and other resistance loci are being used nation-wide. 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.
The goals of Objective 2 are to dissect and elucidate the genetic, genomic, and physiological mechanisms of grapevine abiotic stress tolerance and environmental adaptation. Grapevine production that occurs outside of Mediterranean climate regions is limited due to environmental stresses such as cold temperatures. 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. As climate stability impacts sustainability of grape production, uncovering how grapevines survive winter conditions is key to further expansion of this industry in Northern and Eastern areas of the United States. Additionally, the genetics of temperature response will be key to adapting cultivars grown under warmer conditions in the Western United States. In the past year ARS researchers in Geneva, New York have collected and analyzed phenotype data regarding loss of cold hardiness under field and controlled conditions. Thirty-two unique cultivars were evaluated weekly for cold hardiness, chilling requirement, budbreak phenology and deacclimation resistance during the winter of 2018-2019. 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 100,000 dormant buds were screened and measured for these traits. This effort represents the 1st annual replication of this study with the 2nd replication to follow in the coming year. Two manuscripts directly related to this study area were submitted for publication (Log#: 360402, 364709). Phenotypic evaluation of 28 wild and cultivated grape genotypes for deacclimation resistance as it relates to six different temperature exposures were conducted on 35,000 buds to produce empirical data needed to produce a cold hardiness prediction model for the Eastern United States. Significant progress has been made regarding collection and processing of dormant buds needed for ongoing gene expression and methylation studies.
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. Clonal crops such as grapes which are highly heterozygous would greatly benefit from a genome-editing tool like CRISPR-Cas9 for modifying a gene of interest in an elite genetic background. Many traditional grape varieties, especially elite wine grapes such as ‘Chardonnay’ and ‘Pinot Noir’, have been in production and 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. As a result, genetic improvement of 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 CRISPR-Cas9 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. In the past year ARS researchers in Geneva, New York have built a CRISPR-Cas9 construct for modifying the color gene VvMybA1 and transformed it into V. vinifera ‘Chardonnay’ embryogenic callus via Agrobacterium transformation. The efficacy of the editing machinery components in the construct was successfully demonstrated and transgenic callus containing cells with edited color gene VvMybA1 was obtained. This work was the first to demonstrate the feasibility of removing a large piece of DNA from a locus in grapevine. The work continues, and the focus is now on accomplishing the editing by using a non-transgenic approach. The one other key goal for Objective 3 is to elucidate genetic control of red-flesh pigmentation in grape berries through genetic mapping and functional analysis. Toward accomplishing this research goal, ARS researchers in Geneva, New York have phenotyped a population segregating for red flesh by using a leaf-disc assay method and genotyped it by using high throughput illumine sequencing. A candidate QTL has been identified. The work continues, and the focus is to fine map the trait and ultimately understand the molecular mechanism controlling the trait.
Accomplishments
1. Inexpensive, multi-species DNA marker technology. Plant breeders use DNA markers to track traits like disease resistance, yield, and quality, using that information to discard undesirable seedlings. Developing new markers to target each trait is expensive, and in high diversity crops like grapes, markers that return useful information for one breeder often do not work for others. In collaboration with Cornell University, ARS researchers in Geneva, New York developed a core set of 2000 low-cost markers that work in all species of grapes, even for species that diverged 20 million years ago. With this core marker set, we can now predict the same traits in multiple grape breeding. The U.S. grape breeding programs have adopted this core set for their marker-assisted selection. Additionally, other crops have adopted this strategy, and a shared marker platform enables transferability of research results for more rapid and efficient community progress.
2. Link between loss of cold hardiness and bud break in grapevine uncovered. Loss of cold hardiness during midwinter warm events reduces grapevine dormant bud freeze resistance and increases risk of lethal bud freezing. It was discovered that loss of cold hardiness is the result of greater adaption to high temperature in wild species. ARS scientists at Geneva, New York compared the cold hardiness of two wild and two cultivated species. This high temperature adaptation in turn may increase the risk of cold damage in hybrid grape cultivars that use specific wild grapevine germplasm in their pedigrees. These findings are important to future development of cold-tolerant grape cultivars.
3. Genes that regulate 'foxy' aroma in 'Concord' grape discovered. ‘Concord’ is the most well-known juice grape cultivar V. labrusca with the characteristic ‘foxy’ aroma. ‘Foxy’ aroma results from the accumulation of the compound methyl anthranilate. The ‘foxy’ aroma is ‘concord’ grape which is very popular in non-fermented grape juice and jellies. However, the ‘foxy’ odor is too strong for the production of popular wines with concord grapes, which is the reason European grape species (V. vinifera) are used for wines. ARS researchers in Geneva, New York uncovered two major structural changes in a gene, named as anthraniloyl-CoA:methanol acyltransferase (AMAT), responsible for different levels of ‘foxiness’, in different grape species. The knowledge from this study can now help wineries to create ‘foxiness’-free ‘Concord’ grapes into wine.
Review Publications
Londo, J.P., Kovaleski, A.P. 2019. Deconstructing cold hardiness: Supercooling ability and chilling requirements in the wild North American grapevine vitis riparia. Australian Journal of Grape and Wine Research. 25:276-285.
Fresnedo-Ramirez, J., Yang, S., Sun, Q., Karn, A., Reisch, B., Cadle Davidson, L.E. 2019. Computational analysis of AmpSeq data for targeted, high-throughput genotyping of amplicons. Frontiers in Plant Science. 10:599. https://doi.org/10.3389/fpls.2019.00599.
Hall, M.E., O'Bryon, I., Osier, M.V., Wilcox, W.F., Cadle Davidson, L.E. 2019. The epiphytic microbiota of sour rot-affected grapes differs minimally from that of healthy grapes, indicating causal organisms are already present on healthy berries. PLoS One. https://doi.org/10.1371/journal.pone.0211378.
Saptoka, S., Chen, L., Yang, S., Hyma, K.E., Cadle Davidson, L.E., Hwang, C. 2018. Construction of a high-density linkage map and QTL detection of downy mildew resistance in Vitis aestivalis-derived ‘Norton’. Theoretical and Applied Genetics. 132:137-147.
Hall, M.E., Loeb, G.M., Cadle Davidson, L.E., Evans, K.J., Wilcox, W.F. 2018. Grape sour rot: a four-way interaction involving the host, yeast, acetic acid bacteria, and insects. Phytopathology. https://doi.org/10.1094/PHYTO-03-18-0098-R.
Kovaleski, A.P., Londo, J.P. 2019. Tempo of gene regulation in wild and cultivated Vitis species shows coordination between cold deacclimation and budbreak. Plant Science. 287:110178. https://doi.org/10.1016/j.plantsci.2019.110178.
Zong, X., Zhang, Y., Walworth, A., Tomaszewski, E.M., Callow, P., Zhong, G., Song, G. 2019. Constitutive expression of an apple FLC3-like gene promotes flowering in transgenic blueberry under nonchilling conditions. International Journal of Molecular Sciences. 20(11):2775. https://doi.org/10.3390/ijms20112775.
Arro, J., Yang, Y., Song, G., Zhong, G. 2019. RNA-Seq reveals new DELLA targets and regulation in transgenic GA-insensitive grapevines. Biomed Central (BMC) Plant Biology. 9(1):80. https://doi.org/10.1186/s12870-019-1675-4.
