Annual Project REEport 2018-19

PREFACE

This report serves as the summary of second year project updates for VitisGen2. The following is an excerpt from the annual report submitted to the USDA-NIFA Research, Extension, and Education Project Online Reporting system (REEport) for the project entitled “VitisGen2: Application of Next Generation Technologies to Accelerate Grapevine Cultivar Development”. The report is cumulative from all project team members and covers the period from September 1, 2018 through August 31, 2019 and was submitted on November 30, 2019. The report has been reformatted for readability prior to distribution to project directors, collaborators, and the advisory panel.

PROJECT ACCOMPLISHMENTS

Summary

Recent analyses have emphasized the economic and environmental importance of developing new grape cultivars with high fruit quality (FQ) and resistance to powdery mildew (PM). An Advisory Panel of table, juice, raisin and wine grape industry
members concurs that high-quality, PM-resistant cultivars are a top priority. The proposed project (VitisGen2) will i) expand on VitisGen progress in developing novel economic, phenotyping and genetics knowledge and tools related to new grape cultivars,
and ii) translate these and previous VitisGen innovations into new applications for improving grape breeding programs and managing existing vineyard plantings. The Economics team will evaluate the consequences of introducing new grape traits,
including impacts upon cost, yield, revenue, profit, pesticide use, and the environment. The Trait Evaluation team will develop novel high-throughput methods, and apply these along with proven approaches to characterize a range of phenotypes, such as
PM resistance durability and undesirable fruit qualities, as well as locally important traits. The Genetics team will couple phenotyping results with high-resolution genetic maps, which can be combined with genome assembly and RNA-Seq analyses to develop inexpensive, high-resolution markers spanning key genes. Finally, the Outreach team will communicate scientific opportunities and discoveries, and provide stakeholders with knowledge of the benefits of adopting new high-quality PM resistant cultivars along with new tools for characterizing PM and FQ in their existing plantings. The proposed work utilizes plant breeding and genomics approaches to improve grape characteristics and enhance the economic and environmental sustainability of grape production.

Major Project Goals

Powdery mildew (PM) resistance consistently ranks as a top research priority facing the U.S. grape industry, based on more than a dozen stakeholder meetings focused on research priorities since 2005 and numerous industry surveys. PM is the most significant grape disease in California – and likely worldwide – in terms of expenses for control and losses in quality and yield. In an international survey of grape scientists, PM was statistically significant in being both the most important and the most genetically tractable of twelve key traits. However, breeding for PM resistant cultivars via traditional methods frequently results in negative fruit quality trait introgression from wild Vitis. Winemakers report that poor sensory attributes and color are major challenges to using interspecific hybrids, particularly the presence of high acidity, low tannin and color stability, and off aromas. To address the need for PM resistant cultivars with desirable fruit quality, the long-term objectives of the project are listed in the next section.

Accomplishments

Objective 1: Technological Innovations Driven by Trait Economics

  • Integrate genome-wide data with innovations in phenotyping powdery mildew (PM) resistance, and table and wine grape quality for genetic characterization of high-priority traits. Specific goals focus on gene discovery and trait assays: de novo annotation of relevant wild genomes; RNASeq of all VitisGen parents; low-cost AmpSeq marker implementation; automated PM quantitation without staining; multiple high throughput phenotyping screens for key fruit quality traits, and identification of candidate genes for PM resistance and fruit quality.
  • Complete and disseminate economic analysis of several key agronomic and quality traits to drive research and breeding

To support genome-guided improvement strategies in grape, the ongoing development and testing of the core rhAmpSeq probe set for Vitis continued this year. Twelve genomes of Vitis were sequenced and assembled, then used to identify core conserved sequences, resulting in a 2000 amplicon set, with over 95% of the markers returning useful data across multiple families. An additional 55 markers were added to the 2000 core genome amplicons for marker-assisted selection. Multiplexing of these amplicons led to a reduction in cost per sample from ~$20 to under $10 USD. We are integrating the rhAmpSeq and the previous genotype-by-sequencing (GBS) data from VitisGen1 into a single reference set of genomic coordinates.

This year, 41 QTL were mapped across 9 mapping families, new linkage maps constructed, and marker-assisted seedling selection for disease resistance (RUN, REN and RPV loci), muscat flavor, seedlessness and seed trace size were carried out with the previously validated markers. A total of 165 markers (multiple markers per trait) were assessed across 6,425 seedlings. All markers are being converted for use with rhAmpSeq techniques.

Rapid phenotyping of economically important traits remains a high priority. A robotic arm was developed for leaf disk sampling and arraying preceding mildew inoculation, capable of ~2800 samples per day. Four Blackbird imaging robots, commercial versions of the prototype developed earlier in the project, were installed at NY-USDA (processes ~10,000 samples per day). Two new convolutional neural network algorithms were developed for rapid data analyses and quantification of grape powdery mildew (PM) hyphae and conidia. A total of 3413 genotypes were assayed for PM or downy mildew (DM) reactions to support the ongoing genetic mapping, marker validation, gene stacking, fine mapping, and race-specificity testing. Leaf tissue samples were received from USDA-Parlier, NY-USDA, MO, SDSU, and NY-Cornell. Ca. 2000 vines were screened to identify recombinants between the REN3 and REN9 PM resistance loci. Initial testing of a field-based imaging system for field based phenotyping using an ATV-mounted camera began in 2019. A total of 150,000 and 500,000 images were generated from the Blackbird and vineyard imaging, respectively. New collaborations to develop an image-based analysis for seed trace size and 3D scans of fruit architecture were also initiated.

The Cornell Food Quality/Wine Center (CFQC) and Donald Danforth Center in Missouri evaluated samples for quality components. A total of 357 grape samples were processed for pH, Brix, berry weight, and titratable acidity; 403 (malic acid concentration); 315 (acetone-extracted tannin); 179 (ethanol-extracted tannin) at the CFQC; and 165 for metals at the Danforth Center. Mapping populations were from MN, MO, and NY-Cornell, and NY-USDA. In addition, metabolomic analyses were performed on wild V. cinerea, V. riparia accessions and V. vinifera (Finger Lakes, NY) sampled in 2017 and 2018. Marker-assisted selection of progenies segregating for malate was carried out and samples used for bulked segregant analysis. Transcriptomic studies were initiated to further understand the degradation pathway of the fruit quality complex in Vitis. At the Table/Raisin Quality Center, ARS-Parlier, samples were evaluated for ripening time, time to full color, cluster imaging, berry texture, etc., and genotyped with rhAmpSeq. Previously published molecular markers associated with fruit architecture were tested and found to be nontransferable to table grape populations.

A 20-fold variation in acetone-extractable tannin was observed from a mapping population at MO. Another family differed in fruit malate concentrations at both pre- and post-veraison stages. Two preliminary QTLs were detected with 2017 data, although with low statistical significance due to small numbers of fruiting vines. The analysis of 2018 samples is underway. Samples of 20 genotypes were bulked into 4 segregating groups of progenies exhibiting contrasting pre- and post- veraison malate trends. Further metabolomic and transcriptomic studies were initiated in the 4 groups. Time-resolved sampling and analysis of vinifera and wild Vitis spp. grapes showed that wild Vitis accumulate malate at higher levels pre-veraison, and dissimilate less malate post-veraison (ripening). Specific measurements of malate levels in mesocarp tissue showed that these differences are not the results of a higher skin/pulp ratio in wild Vitis, but rather physiological differences in the mesocarp tissue.

Field evaluations for PM resistance were carried out in mapping populations at MO, NY-Cornell, NY-USDA, and MN. Other traits evaluated include bloom date, root architecture, DM, leaf trichomes, botrytis bunch rot, time of bud break, etc. New sources of PM resistance from wild species (V. riparia, V. cinerea, V. vulpina, and V. amurensis) were explored. Notable vines demonstrating PM resistance from a V. amurensis x Valley Pearl population were backcrossed with high-quality table grapes and natural dry-on-vine raisins at USDA-Parlier. The use of RNAseq analysis to understand the functional aspects of powdery mildew resistance was initiated. Pooled samples of resistant and susceptible material will allow the differentiation of gene expression of candidate genes and downstream pathways. The inheritance of leaf variegation is being studied using populations from MN and NY. Additionally, a fine-mapping population (n=1000) at MN was phenotyped for leaf phylloxera resistance, stomatal conductance, and photosynthesis. Genetic analysis of phylloxera resistance resulted in the haplotype marker identification of a locus which agreed with a previously reported QTL in the literature. To study the effect of genotype x environment interactions on important grape traits, a V. riparia x Seyval population is being evaluated at two locations in three replicates.

Previously published flower sex markers do not work well in mapping families that utilize wild grape germplasm. The use of RhAmpSeq markers combined with genome sequencing of diverse germplasm was a powerful tool for complex sex locus studies. We found strong evidence for two specific candidate genes leading to the expression of male and female flowers. Results are being validated but currently support the identification of all three flower types at the seedling stage. To evaluate consumer preferences and responses to new products, the adoption study of grape varieties was initiated. Online consumer surveys to evaluate consumer attitudes towards specific traits/varieties, as well as consumer response to technologies such as gene-editing, was launched and is expected to be administered to over 2,000 participants. A new collaboration with postharvest researchers to link fruit texture values with consumer taste panels was also initiated.

Objective 2: Knowledge Extension and Application

  • Incorporate technological innovations and economics-oriented priorities in the generation of grapevine seedlings in breeding programs and the selection of elite breeding lines. Publicly release grapevines, pollen, and/or seed lots with various combinations of RUN1, REN1, REN2, REN3, REN4, REN6, REN7 and REN10 PM resistance.
  • Demonstrate the impact of VitisGen advances to grape growers, enologists, and specialty crop researchers.

Knowledge extension and application are documented here and in later sections. These included targeting a broad audience of other scientists, growers, industry leaders, and the general public through the project website (Vitisgen2.org), where we post project news, accomplishments, technical advances, and goals of the VitisGen2 project. In addition, webinars, trade publication articles, twitter feed and non-technical summaries of VitisGen2 journal articles are detailed below. Across the breeding programs, the use of marker-assisted selection was widely adopted with validated markers for PM, DM, leaf phylloxera resistance, muscat flavor, seedlessness and seed trace size. There are ongoing efforts to explore more PM resistance genes from the wild in addition to gene stacking efforts for multiple combinations of RUN1, REN1, REN2, REN3, REN4, REN6, REN7, and REN10 PM resistance.

Opportunities for training and professional development

The project provided numerous training and professional development opportunities for various partners at different levels within this reporting year. The VitisGen2 scientists participated in notable conferences including the American Society for Enology and Viticulture-Eastern Section (ASEV-ES) and the 2019 North American Grape Breeders conferences where they participated in various scientific deliberations and training on high throughput phenotyping of grapevines. Members were also trained on Mixed modeling using R at the American Phytopathological Society (APS) annual meeting. Other training included: Introduction to Linux; bioinformatics in Linux; RNA-Seq data analysis; genome sequence data analysis; genome annotation and sequence-based gene function prediction at the Cornell University Institute of Biotechnology; Linear and multiple regression; Intermediate data analysis in R at the Cornell University Statistical Consulting Unit; and operator training on Universal Robots at Cornell AgriTech.

At ARS-Parlier, a technician was trained to distinguish between seeded and stenospermic fruits and exposed to the diversity of seed trace sizes present in stenospermic vines and how to differentiate them from seeded accessions. Undergraduate and high school students were trained on the different aspects of in-ovulo embryo culture, aseptic technique, medium preparation, fruit disinfestation, berry sampling and analytical approaches, ovule placement, and embryo identification. Others were trained in data assembly, and analysis, as well as survey development and design. A total of 13 undergraduate and 2 high school students received training from VitisGen2 this current year.

Two graduate students and a post doctoral associate at Sacks’ lab in NY-Cornell received training on experimental design related to understanding differences in metabolite accumulation and degradation between wild Vitis and V. vinifera. Other graduate students were trained in field evaluations of fruit quality, DNA extraction, and other aspects of plant breeding. Some Ph.D. students were added to the project at the University of Minnesota, South Dakota State University and Missouri State University and received training on genetic mapping studies.

Visiting scholars and post-doctoral researchers equally received new skills and were exposed to multidisciplinary research opportunities for grape improvement.

Dissemination of results to communities of interest

VitisGen2 utilized various channels to disseminate research findings and discoveries to various audiences including students, the scientific community, industry, stakeholders, extension agents, and the general public both locally and abroad. The VitisGen2 work was featured in notable magazines such as the Wine Enthusiast magazine, e.g. “How Computers are Being Used to Hack Grapes” by Stacy Brooks on April 2, 2019 (https://www.winemag.com/2019/04/02/computers-hack-winegrapes/). Over 100 attendees were present at the VitisGen2 research presentations at the Minnesota Grape Growers Association Cold Climate Conference of the University of Minnesota Grape Breeding and Enology project. Our scientists were featured at the quarterly board meeting updates of the Minnesota grape industry and at the semi-annual board meeting of the National Grape Research Alliance. Four grower workshops in association with an NC SARE partnership grant on grape growing focused on disease resistance management practices and measuring fruit quality traits to improve harvest, conducted at MN. An open house was held at the Horticultural Research Center, Excelsior, MN in Sept 2018 where presentations and posters on grape tasting for potential new varieties were presented to the public, industry, and stakeholders numbering up to 75. VitisGen2 objectives and results were frequently disseminated in lectures requested by local educators at USDA Parlier. Information on durable PM resistance breeding was made available locally and internationally to many including 8 Ukrainian Ag-Econ. teachers on a Foreign Agricultural Service teacher’s exchange program with California State University, Fresno; 19 summer interns employed at a table grape nursery; and 8 Serbian wine industry professionals on a FAS training program with CSUF.

A field day was organized at Cornell-Geneva for scientists at Ithaca and other localities. This field day included the Breeding insight project team during August 2019. One of the papers on the grape industry was published as a chapter in a book, published by the Giannini Foundation, targeting diverse audiences and available to the general public. The powdery mildew phenotyping laboratory (also known as the Robot Lab) was a frequent site of outreach events focused on communicating the importance of multidisciplinary (biology, informatics, engineering) and multi-institutional research, with about 20 tours provided to scientists and the public, including high school students interested in engineering; graduate students in plant breeding; stakeholders from grape and biocontrol industries; USDA national program leaders; congressional representatives; and economic development experts.

VitisGen2 articles and webinars highlighted the need for new varieties to address genetic weaknesses in current varieties, and how advances in DNA technology have made it possible to map and discover traits for disease resistance, insect resistance, cluster architecture, color, and sex determination. Several such articles in general media outlets (Forbes.com, NPR, Smithsonian) featured contributions from project partners. The rhAmpSeq platform developed by VitisGen2 has received more general interest from geneticists and breeders of other crops

FUTURE DIRECTIONS

For the future, one major goal is to incorporate the rhAmpSeq data into the practical haplotype graph structure used by other breeding programs in the development of genomic selection tools. The key to implementing this future aim is to partner with the Breeding Insight project to genotype all existing USDA Vitis germplasm. Incorporating this genome-wide data for the complete repository system will allow us to fully leverage rhAmpSeq technology to identify key germplasm for important traits. The team will capitalize on the advances of rhAmpSeq markers as well as the ongoing work in RNAseq and genome sequencing. Optical map data of V. piasezkii will be used to improve sequence contiguity. Structural and functional gene annotation of three existing genome assemblies will be performed to serve as a reference for the full sibling transcriptomic analysis. Additional parental genomes will be sequenced. Also, there will be an effort to construct a Chambourcin highresolution linkage map with both SSR and rhAmpSeq-based SNP markers.

We will continue to focus on phenotypic evaluations and understanding the biology of key traits especially powdery mildew and fruit quality. An effort will be made to encourage the widespread distribution of Blackbird imaging robots to scientists at other institutions and locations as well as transferring resistance gene stacks (vines with multiple resistance genes) to Report Date 11/27/2019 Page 4 of 18 United States Department of Agriculture Progress Report Accession No. 1013060 Project No. NYG-632586 repositories and/or the national clean plant network (NCPN) for public distribution. Attention will be given to stacks of known resistance loci to investigate phenotypic effects, downstream RNA pathways in single-gene lines, and how those pathways are affected in stacked lines. On the other hand, for unknown resistance loci, we will generate phenotypic data for genetic mapping/QTL discovery. There are plans to expand RNAseq isolation, sequencing, and analysis efforts to explore differences in gene expression between stacked and unstacked resistant grapevine genotypes. This is to better understand how resistance genes network, or interact, in stacked backgrounds and potentially to identify optimal stacking designs to increase the durability of disease resistance.

The analyses of the fruit chemistry of samples submitted by our VitisGen2 collaborators will continue at the CFQC. This will include two new populations harvested in 2019, from NY-Cornell (NY84.0101.03 x V.amurensis), and NY-USDA (V. doaniana 588149 x Chardonnay). Also, 2019 tannin analysis submitted from Missouri will be carried to further investigate tannin QTL in hybrid grapes. We will also complete volatile analyses, which were delayed due to challenges with the GC-MS instrument. The time-resolved studies of malic acid and minerals in vinifera and wild Vitis will continue. RNA libraries will be created and RNAseq analysis conducted on previously collected samples to evaluate if phenotypes can be explained by differences in gene expression. We will also complete metabolomic analyses on 2018 samples. We will phenotype the Horizon X Illinois population at pre- and post-veraison stages for an additional year (2019), to verify QTL identified for malate in 2018. In addition, we will verify the correct selection of segregating groups and target them for further metabolomic and transcriptomic analysis. Based on these analyses and subsequent results, we plan to highlight candidate genes that are regulating malate levels in the grape. Efforts to understand malic acid degradation difference between wild and cultivated varieties are also planned for year 3 of the grant. The use of x-ray in addition to 3D RGB imaging will be deployed to evaluate grape clusters at USDA-Parlier. We have also identified new validation populations from more established populations to offset the small population numbers in our validation populations.

The initiated consumer survey will be completed and analyzed and research outcomes pertaining to new varieties, as well as research findings, will be disseminated to the general public and the necessary academic audiences. The project will continue to expand on current outreach programs using webinars, spotlight profiles, “Research In Plain English” summaries, trade articles and journals in communicating research outputs. Also, field days will be used to strengthen collaborations with growers and industries. A field day in the Central Valley of California in association with Keith Striegler, EJ Gallo is expected in 2020

PUBLICATIONS

Peer-reviewed journal articles

  • Fresnedo-Ramírez, J., Yang, S., Sun, Q., Karn, A., Reisch, B. I., & Cadle-Davidson, L. (2019). Computational analysis of AmpSeq data for targeted, high-throughput genotyping of amplicons. Frontiers in plant science, 10, 599.Doi:10.3389/fpls.2019.00599
  • Andrew Bierman, Tim LaPlumm, Lance Cadle-Davidson, David Gadoury, Dani Martinez, Surya Sapkota, and Mark Rea. 2019. A High-Throughput Phenotyping System Using Machine Vision to Quantify Severity of Grapevine Powdery Mildew. Plant Phenomics 2019:92097272019. doi:10.34133/2019/9209727.
  • Barba, P., Loughner, R., Wentworth, K., Nyrop, J.P., Loeb, G.M. and Reisch, B.I. 2019. A QTL associated with leaf trichome traits has a major influence on the abundance of the predatory mite Typhlodromus pyri in a hybrid grapevine population. Horticulture Research 6, 87 https://doi.org/10.1038/s41438-019-0169-8
  • Burzynski-Chang, E.A., I. Ryona, B.I. Reisch, I. Gonda, M.R. Foolad, J.J. Giovannoni, and G.L. Sacks. 2018. HS-SPMEGC-MS Analyses of volatiles in plant populations – Quantitating compound x individual matrix effects. Molecules. 23(10):2436 https://doi.org/10.3390/molecules23102436
  • Elizabeth A Burzynski-Chang, Elizabeth J Brown, Noam Reshef, Gavin L Sacks. Malate Content in Wild Vitis spp. Demonstrates a Range of Behaviors during Berry Maturation. American Journal of Enology and Viticulture, ajev. 2019.19015.
  • Sambucci, Olena, Julian M. Alston, Kate B. Fuller and Jayson Lusk. 2019. “Pecuniary and non-Pecuniary Costs of Powdery Mildew Management: Evidence From California.” American Journal of Enology and Viticulture 70(2): 177–187.
  • Surya Sapkota, Shanshan Yang, Li-Ling Chen, Lance Cadle-Davidson and Chin-Feng Hwang (2019). 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.
  • Demmings, E.M., B. Williams, C.-R. Lee, P. Barba Burgos, S. Yang, C.-F. Hwang, B.I. Reisch, D.H. Chitwood, and J.P. Londo. 2019. QTL analysis of leaf morphology indicates conserved shape loci in grapevine. Front. Plant Sci
  • Yin, L., Clark, M.D., Burkness, E.C., & Hutchison, W.D. (2019) Grape phylloxera (Hemiptera: Phylloxeridae), on cold-hardy hybrid wine grapes (Vitis spp.): A review of pest biology, damage, and management practices. J. Integrated Pest Management 10(1): 16 https://doi.org/10.1093/jipm/pmz011

Peer-reviewed journal article (submitted)

  • Fresnedo-Ramírez, J.,Yang, S., Sun, Q., Karn, A., Reisch, B., Cadle-Davidson, L. Computational analysis of AmpSeq data for targeted, high-throughput genotyping of amplicons. Frontiers in Plant Science. (Submitted July 5, 2018).
  • Avinash Karn, Cheng Zou, Dave Manns, Qi Sun, Jason Londo, Lance Cadle-Davidson, Anna Katharine Mansfield, Bruce I. Reisch, and Gavin Sacks (2019) Genetic Basis of Acylated Anthocyanins Variation in North-American Interspecific Grapevine Hybrids. Molecular Breeding (In process)
  • Avinash Karn, Rachel Naegele, Siraprapa Brooks, Jonathan Fresnedo Ramirez, David Ramming, Craig Ledbetter, and Lance Cadle-Davidson (2019) Genetic analysis of powdery mildew resistance QTL from Vitis hybrid ‘Tamiami’. Phytopathology (In process)
  • Cheng Zou, Avinash Karn, Bruce Reisch, Allen Nguyen, Yongming Sun, Yun Bao, Michael S. Campbell, Deanna Church, Stephen Williams, Xia Xu, Craig A Ledbetter, Sagar Patel, Anne Fennell, Jeffrey C. Glaubitz, Matthew Clark, Doreen Ware, Jason Londo, Qi Sun and Lance Cadle-Davidson (2019) A rhAmpSeq haplotype strategy targeting the collinear core genome improves marker transferability across a diverse genus. Nature (In process)

Newsletters, trade and grower magazines

  • Martinson, T., Q. Sun, C. Zou, and L. Cadle-Davidson. (2019). Grape breeders search for reliable DNA markers: Why the Pinot Noir PN40024 reference genome is not enough. Submitted to Wine Business Monthly, July 2019.
  • Moyer, M. M., T. Martinson and L. Cadle-Davidson (2019). Disease-resistant varieties are on the way: Can we ensure they last? Wine Business Monthly, June 2019. 76-83. Online at: https://cpb use1.wpmucdn.com/blogs.cornell.edu/dist/c/7890/files/2019/08/Moyer-martinson Disease-Resistant-Varieties-on-theWay.pdf.
  • Martinson, T. and L. Cadle-Davidson (2018). The Phenotyping Bottleneck: How grape breeders link desired traits to DNA markers. Wines and Vines, December 2018. 142-145. Online at: https://cpb use1.wpmucdn.com/blogs.cornell.edu/dist/c/7890/files/2019/01/The-Phenotying Bottleneck-_-Wines-and-Vines-Dec-2018- 1w6gc3o.pdf
  • Martinson, T. and C. Ledbetter and R. Naegele (2019). “Marker-Assisted Selection” Makes Efficient Table Grape Breeding. American Vineyard, March 2019. 10-12. Online at: https://cpb use1.wpmucdn.com/blogs.cornell.edu/dist/c/7890/files/2019/03/AV201903 Breeding-2a0n2qj.pdf.
  • Michelle M. Moyer, Timothy Martinson and Lance Cadle-Davidson. 2019. Disease resistant Varieties are on the Way: Can we ensure they last? Wine Business Monthly, July 2019: pp. 76-83.

Book Chapters

  • Clark, M.D. 2019. Development of Cold Climate Grapes in the Upper Midwestern U.S.: The Pioneering Work of ElmerSwenson. In Plant Breeding Reviews Vol 43. Ed. I.  Goldman. p. 31-59
  • Julian M. Alston and Olena Sambucci. 2019. “Grapes in the World Economy.” In D. Cantu and M.A. Walker, eds, The Grape Genome, Compendum of Plant Genomes. Springer Nature Switzerland AG. In press.
  • Julian M. Alston, James T. Lapsley, and Olena Sambucci. 2018. “Grape and Wine Production in California.” In P.L. Martin, R.E. Goodhue, and Brian D. Wright, eds, California Agriculture: Dimensions and Issues. Giannini Foundation of Agricultural Economics, University of California.
  • Lance Cadle-Davidson, Jason Londo, Dani Martinez, Surya Sapkota, Ben Gutierrez. From Phenotyping to Phenomics: Present and Future Approaches in Grape Trait Analysis to Inform Grape Gene Function. Chapter in The Grape Genome, Eds. Dario Cantu and M.A. Walker.

Dissertations/Theses

  • Ebennga, D. Drosophila suzukii in wine grapes: Phenology, exclusion netting, and varietal resistance. MSc Thesis, University of Minnesota
  • Elizabeth Burzynski-Chang. “Tools for the improvement of flavor in interspecific hybrid grape varieties: a post hoc analysis method for quantitation of volatiles and a profile of malate during berry maturation across Vitis spp.” PhD Thesis, Cornell University
  • Underhill, A. (2019) Using high-throughput phenotyping to investigate the genetic bases of quantitative traits in hybrid wine grapes (Vitis spp.). University of Minnesota Master’s Thesis

PROJECT OUTPUTS

Webinars

The extension team produced two VitisGen2 videos in collaboration with the Breeding and Genetics Teams highlighting different aspects of the VitisGens project to educate the public and researchers.

  • Martinez, D. (2019). Advanced computer vision techniques: New technologies to streamline grape breeding. VitisGen2 Webinar, live broadcast March 21, 2019. Audience: 32 . Posted at: https://www.youtube.com/watch?v=aDyj5-M20jI. 52 views online. A webinar on the use of computer vision and computer learning to advance phenotyping was broadcast live, and later published on YouTube, to provide information to the general public about how technological advances help breeding research, as well as to provide more technical instruction to other researchers looking to improve phenotyping.
  • Sacks, G. (2019). What the wild things are: Flavor challenges of breeding disease resistant and cold-tolerant grapes using North American Vitis species. VitisGen2 Webinar, live broadcast April 18, 2019. Audience: 72. Posted at: https://www.youtube.com/watch?v=j0HxekCF7NM. 161 views online. A webinar on the flavor attributes (positive and negative) of North American Vitis species used in grape breeding for disease resistance provided information to the general public, to grape breeders, and to winemakers.
  • Part 1: Produced to document the harvests of powdery mildew resistant table grape accessions S7, S8 and T11. The videos were at the request of the California Table Grape Commission to provide interested growers with specific harvest details about these three new accessions. Videos were presented as part of table grape showings during the 2019 harvest season.
  • Part 2: Produced to document the harvests of powdery mildew resistant table grape accessions S7, S8 and T11. The videos were at the request of the California Table Grape Commission to provide interested growers with specific harvest details about these three new accessions. Videos were presented as part of table grape showings during the 2019 harvest season.
  • Part 3: Produced to document the harvests of powdery mildew resistant table grape accessions S7, S8 and T11. The videos were at the request of the California Table Grape Commission to provide interested growers with specific harvest details about these three new accessions. Videos were presented as part of table grape showings during the 2019 harvest season.

Databases and software

  • Sun, Q.: VitisGen database was updated to facilitate new collaborations with USDA Breeding Insight project, to manage the rhAmpSeq data of Germplasm collections.
  • Sun, Q. Software: amplicon.py (https://bitbucket.org/cornell_bioinformatics/amplicon). The old tools for amplicon data analysis are too slow for rhAmpSeq. A new tool was developed for analyzing rhAmpSeq data sets with much improved parallelization and speed.
  • Cantu D.: Libraries for PacBio sequencing for V. piasezkii, V. romanetii and the PS11 breeding line were prepared. 71.5 Gb of data were generated for V. piasezkii (~143x) and assembled into contigs while scaffolding is in progress. 52 Gb of data were generated for PS11 and assembly is in progress. Genomes of V. piasezkii, V. romanetii and the PS11 breeding line were sequenced using Single Molecule Real Time (SMRT; Pacific Biosciences) technology. Sequencing generated 71.5 Gb (~143x), 55 Gb (~110x), and 63.6 Gb (~127x) for V. piasezkii, V. romanetii and the PS11 breeding line, respectively. Long reads (N50 ~ 20 Kb) were assembled in a diploid-aware mode into primary contigs and haplotigs. Diploid assembly of V. piasezkii genome is 1.13 Gb-long, with a primary assembly scaffolded into 619 scaffolds covering 653.7 Mb, and 5,037 haplotigs representing 476.8 Mb. Similarly, V. romanetii and PS11 breeding line are 1.16 and 1.06 13 Gb-long, with 891 and 402 primary scaffolds covering 655.5 and 723.9 Mb, and haplotigs representing 412 and 438 Mb, respectively. Optical maping (BioNano) was performed on V. piasezkii, generating 667 Gb (~1334x).

Protocols

  • NA 2018-2019

Patents

  • NA 2018-2019

Survey instruments

  • NA 2018-2019

Models

  • NA 2018-2019

Educational aids, curricula, training

  • NA 2018-2019

Websites

  • VitisGen2 website (http://www.vitisgen2.org/): New content includes 4 popular press articles, 4 Research in Plain English articles, 6 Staff Spotlight profiles, 2 webinars, and 4 news articles/blog posts. Statistics: 6,238 visits (2638 unique visitors), 13,552 page views, 42% of visits were ‘new visitors’.
  • @VitisGen (https://twitter.com/VitisGen)

Workshops and project meetings

  • A workshop on the grape adaptation of CassavaBase at the annual 2019 VitisGen2 meeting, Jan 11, 2019. Lance CadleDavidson with Qi Sun and Lukas Mueller’s group to familiarize and train the VitisGen co-PIs in using VitisBase online database and FieldBook phenotyping application, to get feedback on what needs to be modified for those who work with grape breeding and genetics programs, and to involve enthusiastic volunteers into further participation.
  • Co-Led Amplicon Sequencing workshop with Lance Cadle-Davidson and Jonathan Fresnedo Ramirez. Hands-On Analysis of Amplicon Sequence (AmpSeq) Data for Targeted Multiplexed Genotyping at ICPP Boston, MA

Presentations (including seminars, lectures, and conference talks/posters)

  • Chin-Feng Hwang, Li-Ling Chen, Bryce Bentley and Sadie Land (2019). Optimization of Chambourcin Grape Breeding Using Molecular Genetic Approaches. Abstract for the 2019 Show Me Grape and Wine Conference and Symposium.
  • Chin-Feng Hwang, Li-Ling Chen, Sadie Land and Bryce Bentley (2019). Optimization of Chambourcin Grape Breeding Using Marker-Assisted Selection. Abstract for the 44th Annual Conference of the American Society for Enology and Viticulture (ASEV)-Eastern Section.
  • Sapkota, S. D. Martinez, B. Reisch, D. Gadoury, and L. Cadle-Davidson. 2019. Combined effects of stacking multiple resistance genes upon grapevine powdery mildew (Erysiphe necator). International Plant and Animal Genome Conference XXVII. 12-16 January 2019. San Diego, California, USA. (abstr.)
  • Sapkota, S. D. Martinez, B. Reisch, D. Gadoury, and L. Cadle-Davidson. 2019. Do resistance genes act synergistically against grapevine powdery mildew (Erysiphe necator)?. Plant Health 2019, APS Annual Meeting, 3-7 August 2019, Cleveland, OH, USA (abstr.)
  • Gavin L. Sacks. “Not to everyone’s taste – an update on research in the Sacks Lab”. Cornell Recent Advances in Viticulture and Enology (CRAVE). Ithaca, NY. Dec 11, 2018.
  • Noam Reshef, Burzynski-Chang EA, Avinash Karn, Jason Londo, Bruce Reisch, Gavin L. Sacks. Elucidating the molecular regulation of grape sourness by integrating QTL mapping with RNAseq and metabolomics of non-domesticated grapevine species. Society of Experimental Biology Meeting. Seville, Spain.
  • Terry L Bates, Madeleine Y Bee, Xuefei Kuang, and Gavin L Sacks GL. “Simultaneous Measurement of Key Odorants at their Sensory Thresholds in Juice Grapes”. 67th Annual Conference of the American Society for Mass Spectrometry. Atlanta, GA.
  • Martinson, T. E. 2019 “VitisGen2: Application of next generation technologies to accelerate grapevine cultivar development”, Presentation, 2019 Eastern New York Fruit and Vegetable Conference Albany, NY, February 21, 2019. Audience = 20 grape producers and winery representatives from Eastern New York.
  • Zou, C. High-fidelity rhAmpSeq™ primers targeting the core genome enables shared exploration of highly diverse and heterozygous species at Grape Genome Initiative workshop, International Plant & Animal Genome XXVII, January 12-16, 2019 – San Diego, CA, USA
  • Belton, J., Kisselstein, B. M., Gadoury, D. M. Mating Type Distribution and Formation of Chasmothecia on Grapevine by Erysiphe necator in the Northeastern United States. Summer Research Scholars Poster Session in Geneva, NY. July 2019.
  • Cheng Zou, Avi Karn, Qi Sun, Lance Cadle-Davidson, et al. High-fidelity rhAmpSeq™ primers targeting the core genome enables shared exploration of highly diverse and heterozygous species. Grape Genome Initiative workshop, International Plant & Animal Genome XXVII, January 12-16, 2019 – San Diego, CA, USA.
  • Cheng Zou, Lance Cadle-Davidson, Qi Sun, Avi Karn, et al. rhAmpSeq™ 2000-Plex Amplicon Sequencing Panel Targeting Core Genome in Vitis vinifera shows Elevated Marker Transferability, Increased Specificity and Applicability to Diverse Organisms. Integrated DNA Technologies workshop, International Plant & Animal Genome XXVII, January 12-16, 2019 – San Diego, CA, USA.
  • Dani Martinez, Andrew Bierman, Timothy Plummer, Lance Cadle-Davidson, David Gadoury, Surya Sapkota. Blackbird: A novel high-throughput laboratory phenotyping system to quantify incidence and severity of powdery mildews. Poster at Plant Health 2019 in Cleveland, OH, August 3-7, 2019.
  • Dani Martinez, Surya Sapkota, Andrew Bierman, Timothy Plummer, David Gadoury, Mark Rea, Lance Cadle-Davidson. 2019. Blackbird: A next-gen imaging system for high-throughput laboratory phenotyping. Talk at Phenome 2019 in Tucson, AZ, February 6-9, 2019.
  • Kisselstein, B.M., Cadle-Davidson, L., and Gadoury, D.M. Genetic diversity of Erysiphe necator populations in a center of diversity: a case study of select vineyards in New York State. American Phytopathological Society Annual Meeting in Cleveland, OH. August 3-7, 2019.
  • Lance Cadle-Davidson. Computer vision for high-throughput phenotyping of powdery mildew resistance, presented at Phenome in Tucson, Arizona. February 9, 2019.
  • Moreira, L.S., Underhill, A., and M. Clark. Postharvest evaluation of cold-hardy table grape breeding lines. XII International Conference on Grapevine Breeding and Genetics. .doi:10.17660/ActaHortic.2019.1248.15
  • Surya Sapkota, Dani Martinez, David Gadoury, and Lance Cadle-Davidson. 2019. Phenotyping grapevine powdery mildew: an update from VitisGen2 phenotyping center. Talk at National Grape Breeders Conference, in Mountain Grove, MO, August 15-16, 2019.
  • Diering, A., Tork, D.G., Underhill, A., Freund, D.M., Clark, M.D., and A. Hegeman. HPLC-MS as a detection method for pigments, phenolics, and co regulation in a hybrid wine grape family & Using PCA for QTL mapping to optimize plant breeding. American Society for Mass Spectrometry, Atlanta, GA
  • Moreira, L.S. Hegeman, A.D., Brockman, S.A., Suresh, J., and M.D. Clark. Non destructive field sampling of volatile organic compounds for metabolomics analysis of grape cluster development. ASEV-ES, Geneva, NY
  • Yin, L. Cadle-Davidson, L., Sun, Q., Londo, J., Karn, A., Zou, C., Gouker, F., Fresnedo, J., and M. Clark. Recombinant identification using rhAmpSeq in a hybrid cold-hardy grape population for fine mapping a major resistance QTL to foliar phylloxera. Plant and Animal Genome Conference, San Diego, CA
  • Karn, A, Zhou, C, Alahakoon, D, Fennell, A, Sun, Q, Londo, J, Reisch, B, Cadle-Davidson, L. rhAmpSeq Core Genome Markers Improve the Coverage of a GBS-Based Genetic Map and Marker Transferability to Other Grapevine Populations. INTEGRAPE 2019 Data integration as a key step for future grapevine research, Chania, Crete, Greece Mar. 25-28, 2019.
  • Mcdermaid, A, Monier, B, Wang, C, Zhao, J, Miller, A, Fennell, A, Ma, Q. IRIS-EDA: An integrated RNA-Seq interpretation system for gene expression data analysis. INTEGRAPE 2019 Data integration as a key step for future grapevine research, Chania, Crete, Greece Mar. 25-28, 2019.
  • Sun Qi, Zou Cheng, Karn Avinash, Reisch Bruce, Nguyen Allen, Sun Yongming, Bao Yun, Campbell Michael S., Church Deanna, Williams Stephen, Smith Timothy P. L., Fennell Anne, Clark Matthew, Ware Doreen, Londo Jason, CadleDavidson Lance (2019). Haplotype Markers Developed from the Vitis Core Genome and Their Applications for QTL Mapping and MAS Breeding in Hybrid Grape Populations. INTEGRAPE 2019 Data integration as a key step for future grapevine research, Chania, Crete, Greece Mar. 25-28, 2019.
  • Moreira, L.S. University of Minnesota—State Report. North American Grape Breeders Conference. Columbia, MO
  • Noam Reshef. “The real sour grapes – integrated QTL mapping with omics approaches to elucidate malic acid regulation across grapevine species”. Society of Experimental Biology Meeting. Seville, Spain. July 2019.
  • Moreira. L.S. Using metabolomics analysis to characterize volatile compounds in cold hardy hybrid grapes in Minnesota. ASEV National Conference, Napa, CA
  • Fennell, A. 15th Annual VESTA National Summit and Michigan Wine Consortium Conference, June 6, 2019, Traverse City, MI. “Advancing the American grape industry through research.”
  • Fennell, A. North American Grape Breeders Conference, Aug. 16, 2019, Springfield, Missouri. “Genetic analysis of grapevine root system architecture.”
  • Zou, C. rhAmpSeq™ 2000-Plex Amplicon Sequencing Panel Targeting Core Genome in Vitis vinifera shows Elevated
    Marker Transferability, Increased Specificity and Applicability to Diverse Organisms at Integrated DNA Technologies workshop, International Plant & Animal Genome XXVII, January 12-16, 2019 – San Diego, CA, USA
  • Gavin L. Sacks. “Speeding up SPMESH”. SPME Approaches Applied to Mass Spectrometry Techniques Workshop at the 67th ASMS Meeting. Atlanta, GA. June 3, 2019.
  • Bruce Reisch. North American Grape Breeders Conference, Aug. 16, 2019, Springfield, Missouri. “Perspectives from VitisGen2 project; and updates from Geneva’s grape breeding program”.

CHANGES/PROBLEMS

There were no problems warranting a deviation from the proposed objectives and goals. The few recorded challenges within the reporting period were adequately resolved in keeping with the expected goals. Some issues with DNA extraction quality were identified and later traced to sampling issues. This was adequately addressed in collaboration with the DNA isolation contractor (Intertek), IDT and Cornell sequencing facilities.

There were delays in the volatile phenotyping of VitisGen2 populations due to problems with the main instrument used for the analyses (LECO Pegasus GC-MS). The use of the LECO device was decommissioned and the methods were translated to a different instrument (Shimadzu 8040 GC-MS/MS). This will be used to continue volatile phenotyping in the future. Also, there was a technical challenge with the Orbitrap LC-MS based metabolomics sampling for malic acid metabolism studies. Although negative ion mode analysis was successful, positive ion mode analyses yielded irreproducible results for amino acids and several other metabolites. We are transitioning to the GC-MS platform for future metabolomic analyses. Some of our fruit quality validation populations had low vigor and vine numbers were insufficient for validating markers. Other internal delays were related to hiring which brought some delays in the delivery of a few expected outcomes. For example, the 3D imaging boxes were expected to be available in July, but are not expected until next spring due to manpower.