Progress 10/01/22 to 09/30/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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, 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. Artificial Intelligence (AI)/Machine Learning (ML) Artificial intelligence (AI) methods were used for this project during FY2023 for computer vision quantification of disease severity from microscope images. These analyses were conducted on local computing hardware primarily along with secondary effort to implement analyses on SCINet⿿s HPC clusters for future collaborations within ARS. This effort has greatly accelerated research progress. In 6 hours, ARS researchers in Geneva, New York, now capture the same quantity of data via automated microscopy that was recently captured by manual microscopy in an entire year. Further, the quality of computer vision data enables better precision and discovery of resistance gene loci that were not detectable using manual microscopy. ACCOMPLISHMENTS 01 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.
Impacts
(N/A)
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.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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 8060-21220-007-00D ⿿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 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 progress 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. ACCOMPLISHMENTS 02 Powdery mildew resistant grapevines.. Each year across the US, 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.
Impacts
(N/A)
Publications
- Yin, L., Karn, A., Cadle Davidson, L.E., Zou, C., Londo, J.P., Sun, Q., Clark, M. 2022. Candidate resistance genes to foliar phylloxera identified at Rdv3 of hybrid grape. Horticulture Research. https://doi.org/10.1093/hr/ uhac027.
- Evans, J.R., Romero Galvan, F.E., Cadle Davidson, L.E., Gold, K.M. 2022. QScout: A QGIS plugin tool suite for georeferencing and analysis of field scouted and remote sensing data. The Plant Phenome Journal. https://doi. org/10.1002/ppj2.20031.
- Reshef, N., Karn, A., Manns, D., Mansfield, A., Cadle Davidson, L.E., Reisch, B., Sacks, G. 2022. Stable QTL for malate levels in ripe fruit and their transferability across Vitis species. Journal of Theoretical and Applied Genetics. https://doi.org/10.1093/hr/uhac009.
- Park, M., Vera, D., Kambrianda, D., Gajjar, P., Cadle Davidson, L.E., Tsolova, V., El-Sharkawy, I. 2022. Chromosome-level genome sequence assembly and genome-wide association study of Vitis rotundifolia reveal the genetics for 12 berry-related traits. Horticulture Research. https:// doi.org/10.1093/hr/uhab011.
- Alahakoon, D., Fennell, A., Helget, Z., Bate, T., Karn, A., Manns, D., Mansfield, A., Reisch, B., Sacks, G., Sun, Q., Zou, C., Cadle Davidson, L. E., Londo, J.P. 2022. Berry anthocyanin, acid, and volatile trait analyses in a grapevine interspecific F2 population using an integrated GBS and rhAmpSeq genetic map. Plants. https://doi.org/10.3390/plants11050696.
- Karn, A., Diaz-Garcia, L., Reshef, N., Yang, S., Zou, C., Manns, D., Sun, Q., Cadle Davidson, L.E., Mansfield, A.K., Reisch, B.I., Sacks, G. 2021. The genetic basis of anthocyanin acylation in North American grapes (Vitis spp.). Genes. https://doi.org/10.3390/genes12121962.
- Brillouet, J., Romieu, C., Bacilieri, R., Nick, P., Trias-Blasi, A., Maul, E., Solymosi, K., Szelak, P., Jiang, J., Sun, L., Ortolani, D., Londo, J.P. , Gutierrez, B.L., Prins, B.H., Reynders, M., Vancaekenberghe, F., Maghradze, D., Marchal, C., Sultan, A., Thomas, J., Scherberich, D., Fulcrand, H., Roumeas, L., Billerach, G., Salimov, V., Musayev, M., Ul Islam Dar, H., Peltier, J., Gaudeul, M., Grisoni, M. 2022. Tannins phenotyping in the Vitaceae reveals constellations of compositions intimately linked to genera and species. Annals Of Botany. https://doi.org/ 10.1093/aob/mcac077.
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Progress 10/01/20 to 09/30/21
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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 8060-21220-007-00D ⿿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. Record of Any Impact of Maximized Teleworking Requirement: For Objective 1, nearly all (about 90%) laboratory phenotyping for genetic analysis of disease resistance was eliminated for the 2020 and 2021 growing seasons during the COVID-19 pandemic. Similarly, disease field ratings could not be taken in Autumn 2020. The combined lab and field impacts likely precluded the discovery and validation of about 8 to 16 disease resistance genes. All wet lab work was paused, resulting in at least a 20-month delay in the functional analysis of known disease resistance genes via RNA sequencing and elimination of gene editing plans. The COVID-19 pandemic also had significant negative impacts on Objective 2b, preventing essentially all progress on this subaim. Efforts were diverted instead to fulfill collections and phenotyping of Objective 2a, and to maintain minimum effort deliverables for extramurally funded projects. Our Objective 3 on generating new germplasm, tools, and strategies for improving grapevine fruit quality and other traits was impacted because most activities for accomplishing this objective require conducting laboratory work. While some of the work, such as tissue culture, was treated as essential function, not being able to work on the project at a full capacity compromised the progress of the research. ACCOMPLISHMENTS 01 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. 02 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.
Impacts
(N/A)
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.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Accomplishments 01 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. 02 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. 03 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.
Impacts
(N/A)
Publications
- Cadle-Davidson, L.E. 2019. A perspective on breeding and implementing durable powdery mildew resistance. Acta Hortic. 1248:541-548.
- Sapkota, S.D., Chen, L.L., Yang, S., Hyma, K.E., Cadle-Davidson, L.E. and Hwang, C.F. 2019. Quantitative trait locus mapping of downy mildew and botrytis bunch rot resistance in a Vitis aestivalis-derived 'Norton'-based population. Acta Hortic. 1248:305-312.
- Zou, C., Karn, A., Reisch, B., Nguyen, A., Sun, Y., Bao, Y., Campbell, M.S. , Church, D., Williams, S., Xu, X., Ledbetter, C.A., Patel, S., Fennell, A. , Glaubitz, J., Clark, M., Ware, D., Londo, J.P., Sun, Q., Cadle Davidson, L.E. 2020. Haplotyping the Vitis collinear core genome with rhAmpSeq improves marker transferability in a diverse genus. Nature Communications.
- Kovaleski, A.P., Londo, J.P., Finkelstein, K. 2019. X-ray phase contrast imaging of Vitis spp. buds reveals freezing pattern and correlation between volume and cold hardiness. Scientific Reports. 9:14949.
- Demmings, E.M., Williams, B., Lee, C., Barba, P., Yang, S., Hwang, C., Reisch, B.I., Chitwood, D.H., Londo, J.P. 2019. QTL analysis of leaf morphology indicates conserved shape loci in grapevine. Frontiers in Plant Science. 10:1373. doi: 10.3389/fpls.2019.01373.
- Weldon, W., Palumbo, C.D., Kovaleski, A.P., Tancos, K., Gadoury, D.M., Osier, M.V., Cadle Davidson, L.E. 2020. Transcriptomatic profiling of acute cold stress-induced disease resistance (SIDR) genes and pathways in the grapevine powdery mildew pathosystem. Molecular Plant-Microbe Interactions. 33(2):284-295.
- Patel, S., Robben, M., Fennell, A., Londo, J.P., Alahakoon, D., Villegas- Diaz, R., Swaminathan, P. 2020. Draft genome of the Native American cold hardy grapevine Vitis riparia Michx. 'Manitoba 37'. Horticulture Research. doi.org/10.1038/s41438-020-0316-2.
- Yang, Y., Cuenca, J., Wang, N., Liang, Z., Sun, H., Gutierrez, B.L., Xi, X. , Arro, J., Wang, Y., Fan, P., Londo, J.P., Cousins, P., Li, S., Fei, Z., Zhong, G. 2020. A key ⿿foxy⿿ aroma gene is regulated by homology-induced promoter indels in the iconic juice grape ⿿Concord⿿. Horticulture Research. 7(67).
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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 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 01 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. 02 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. 03 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.
Impacts
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Publications
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Since this project started just 3 months ago on April 1, 2018, we have not accumulated enough data to report on milestones and accomplishments. However, ARS researchers in Geneva, New York have made some good progress for meeting the milestone of 2018-2019 for the project. Our Objective 1 is to characterize host and pathogen genetic factors applicable to grapevine disease management, with primary emphasis on powdery mildew. In the past three months since the initiation of this project, we have partnered with the VitisGen2 grape breeding project to implement automated imaging of powdery mildew on grape leaf discs. In collaboration, we collected over 40,000 time-series images for 11,000 leaf discs representing 8 breeding populations and 9 resistance loci. In parallel, we are developing and validating computer vision algorithms for high-throughput quantitative analysis of these images. Upon validation, the images will be used to genetically map the resistance loci, identify candidate genes for resistance, and select desirable seedlings as breeding parents. Our Objective 2 is to dissect and elucidate the genetic, genomic, and physiological mechanisms of grapevine abiotic stress tolerance and environmental adaptation. In the past three months since the initiation of this project, we have continued to process and analyze phenotype data collected regarding loss of cold hardiness under controlled conditions. Two manuscripts were submitted describing the kinetics of deacclimation and the relationship with budburst in cultivated and wild grapevine species. Our Objective 3 is to generate new germplasm, tools, and strategies for improving grapevine fruit quality and other traits. In the past three months the initiation of this project, we have generated embryogenic callus from Vitis vinifera cv. �Chardonnay� and V. vinifera cv. �Thompson Seedless�. These embryogenic callus will be used for developing a genome- editing tool in coming months. In addition, we have identified more DNA polymorphic markers and mapped them to loci closely linked to the red flesh trait in a mapping population.
Impacts
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Publications
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