Research Project: Grapevine Genetics, Genomics and Molecular Breeding for Disease Resistance, Abiotic Stress Tolerance, and Improved Fruit Quality

Location: Grape Genetics Research Unit (GGRU)

2021 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 8060-21220-007-00D “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 6 mapping families and laboratory disease ratings from 2 mapping families in FY21. 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, and we developed rhAmpSeq markers to track 18 disease resistance loci and 7 fruit quality traits. Over 1000 powdery mildew isolates were collected from commercial and research vineyards to investigate how fungicides and resistance genes impact pathogen genetics. 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. In that CRADA, we have imaged the effects of 587 double-stranded RNA sequences at over 86,000 timepoints to identify sequences that effectively control powdery mildew, which is a huge scale enabled by automation and artificial intelligence. 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 (2020-2021) ARS researchers in Geneva, New York, have completed the third year of annual collections of 31 locally important grapevine cultivars, screening them weekly for cold hardiness, deacclimation resistance, and budbreak. This effort represents the final annual replication of the study and data analysis is underway for publication. A third year of collections was also completed for a separate, but similar evaluation of these traits in wild grapevine germplasm held at the cold hardy grapevine germplasm. This study examined deacclimation resistance at 6 different temperatures and provides the empirical data necessary for constructing cold hardiness prediction models for future breeding efforts. Deacclimation resistance was also evaluated in three grapevine genetic mapping populations to determine the extent of phenotypic variation. Vine size remains an issue for conducting these studies as the quantity of dormant bud material is limiting, preventing multiple replications during a single winter season. COVID lab shutdowns and continuation of minimum staffing has prevented any progress on studies designed to examine methylation aspects of dormancy. 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 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 continued the effort of evaluating various configurations of CRISPR-Cas9 constructs for modifying the grape color gene VvMybA1 in V. vinifera ‘Chardonnay’ embryogenic callus via Agrobacterium and biolistic transformation. A dozen of transgenic vines with editing changes of the color gene VvMybA1 were obtained. Molecular analysis of these vines is in progress. Preliminary data showed that editing efficiencies varied significantly among different target sites and were very low when two different target sites were considered jointly. For a given target locus, most often only one copy of the alleles (monoallelic) was edited. We also observed that some transgenic vines were derived from non-edited cells. Because we used a constitutive promoter for Cas9, editing would continue in a transgenic cell, regardless whether or not its progenitor cell was edited or not. As a result, we often observed some transgenic vines heterogenous for the editing alleles. We are pursuing several optimizations of the editing constructs and protocols to overcome this problem. 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, continued the evaluation of non-transgenic approaches for editing grapevine genes. While several successful methods were reported in literature in creating non-transgenic editing plants, none of them have been demonstrated for practical uses, including our effort for grapevine. We will continue exploring this research subject in FY2022. 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 and now been investigating the molecular mechanism for controlling the red-flesh trait. We have developed a hypothesis which is being evaluated. All subordinate projects for this parent project are making good progress.

Accomplishments
1. Predicting grapevine cold hardiness and bud break phenology. When low temperature events happen, grape growers use predictive models to know how their crop will be affected, but current models do not perform well in the Eastern U.S. The collection and processing of three years of weekly cold hardiness, deacclimation response, and bud break phenology measures has produced a cold hardiness prediction model with high accuracy for the New York climate. These data have been shared at multiple meetings and workshops and a publication is in preparation.

2. Modifying berry color through gene editing. Lack of an effective genetic tool for precisely modifying a gene of interest has been a significant challenge for improving elite grape cultivars. Recent development of the CRISPR-Cas9 gene editing technology, however, may offer a potential solution to the problem. To explore the gene editing technology for grape improvement, ARS researchers in Geneva, New York, successfully demonstrated the feasibility of editing the grape color gene VvMybA1 in V. vinifera ‘Chardonnay’ embryogenic callus. A dozen of transgenic vines were obtained with the color gene edited. This work demonstrated the feasibility for editing a color gene in an elite grape cultivar and provided a significant milestone for exploring gene editing technologies in grapevine improvement.

Review Publications
Sun, X., Jiao, C., Schwaninger, H., Chao, T., Ma, Y., Duan, N., Khan, A., Xu, K., Cheng, L., Zhong, G., Fei, Z. 2020. Phased diploid and pan-genomes of cultivated and wild apples unravel genetic basis of apple domestication. Nature Genetics. https://doi.org/10.1038/s41588-020-00723-9.
Migicovsky, Z., Gardner, K., Richards, C.M., Chao, T., Schwaninger, H., Fazio, G., Zhong, G., Myles, S. 2021. Genomic consequences of apple improvement. Horticulture Research. https://doi.org/10.1038/s41438-020-00441-7.
Bryson, A.E., Brown, M.W., Mullins, J., Dong, W., Bahmani, K., Bornowski, N., Chiu, C., Engelgau, P., Gettings, B., Gomezcano, F., Gregory, L.M., Haber, A.C., Hoh, D., Jennings, E.E., Ji, Z., Kaur, P., Rafu Kenchanmane, S.K., Long, Y., Lotreck, S.G., Mathieu, D.T., Ranaweera, T., Ritter, E.J., Sadohara, R., Shrote, R.Z., Smith, K.E., Teresi, S.J., Venegas, J., Wang, H., Wilson, M.L., Tarrant, A.R., Frank, M.H., Migicovsky, Z., Kumar, J., Vanburen, R., Londo, J.P., Chitwood, D.H. 2020. Composite modeling of leaf shape across shoots discriminates Vitis species better than individual leaves. Applications in Plant Sciences. 1.
Yin, L., Karn, A., Zou, C., Cadle Davidson, L.E., Underhill, A.N., Atkins, P., Voytas, D., Treiber, E., Clark, M. 2021. Genetic mapping and fine mapping of leaf trichome density in cold-hardy hybrid wine grape populations. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2021.587640.
Gur, L., Reuveni, M., Cohen, Y., Cadle Davidson, L.E., Kisselstein, B., Ovadia, S., Frenkel, O. 2021. Population structure of Erysiphe necator on domesticated and wild vines in the Middle East sheds a new light on the origins of the grapevine powdery mildew pathogen. Molecular Ecology. https://doi.org/10.1111/1462-2920.15401.
Zou, C., Massonnet, M., Minio, A., Patel, S., Llaca, V., Avi, K., Gouker, F.E., Cadle Davidson, L.E., Reisch, B., Fennell, A., Cantu, D., Sun, Q., Londo, J.P. 2021. Multiple independent recombinations led to hermaphrodism in domesticated grapevine. Nature Genetics. https://doi.org/10.1073/pnas.2023548118.
Wang, Y., Xin, H., Fan, P., Zhang, J., Liu, Y., Dong, Y., Wang, Z., Yang, Y., Zhang, Q., Ming, R., Zhong, G., Li, S., Liang, Z. 2020. The genome of Shanputao (Vitis amurensis) provides a new insight into cold tolerance of grapevine. Plant Journal. https://doi.org/10.1111/tpj.15127.
Musich, R., Cadle Davidson, L.E., Osier, M.V. 2021. Comparison of short-read sequence aligners indicates strengths and weaknesses for biologists to consider. Frontiers in Plant Science. 12:657240. https://doi.org/10.3389/fpls.2021.657240.
Weldon, W.A., Marks, M.E., Gevens, A.J., D'Arcangelo, K., Quesada-Ocampo, L.M., Parry, S., Gent, D.H., Cadle Davidson, L.E., Gadoury, D.M. 2021. A comprehensive characterization of ecological and epidemiological factors driving perennation of Podosphaera macularis chasmothecia. American Phytopathological Society. https://doi.org/10.1094/PHYTO-11-20-0492-R.
Weldon, W.A., Knaus, B.J., Grunwald, N.J., Havill, J.M., Block, M.H., Gent, D.H., Cadle Davidson, L.E., Gadoury, D.M. 2020. Transcriptome-derived amplicon sequencing (AmpSeq) markers elucidate the U.S. podosphaera macularis population structure across feral and commercial plantings of Humulus lupulus. Phytopathology. 111:194-203. https://doi.org/10.1094/PHYTO-07-20-0299-FI.