Breeding, Genetics, and Germplasm Evaluation

The overall objective of this research project is to develop and apply a genome editing tool for trait improvement in grape cultivars. During 2018-2019, we built a CRISPR-Cas9 construct for modifying the color gene VvMybA1 and transformed it into V. vinifera ‘Chardonnay’ embryogenic callus via Agrobacterium transformation. We successfully demonstrated the efficacy of the editing machinery components in the construct and obtained transgenic callus containing cells with edited color gene VvMybA1. A 10-kb-long Gret1 transposon fragment in the promoter region of VvMybA1 in ‘Chardonnay’ was removed in the edited cells. Further analysis revealed 20-60 bp deletions at or near the target sites. The overall frequencies of these deletions/insertions were generally low, with the maximum being about 0.5%. From this work, we were the first to demonstrate the feasibility of removing a large piece of DNA from a locus in grapevine. We also demonstrated the technical feasibility for modifying the color gene, an important fruit quality trait. In parallel, we tried two new delivery methods for editing VvMybA1 and VvPDS genes in callus: a) bombardment of plasmid DNA for transiently expressing both Cas9 and gRNA components in grape embryogenic callus; and b) bombardment of in vitro preassembled Cas9–gRNA ribonucleoproteins into grape embryogenic callus. Our results showed that the direct delivery methods didn’t worked well, and the successful editing rate was too low to have any practical utility. This is not a surprise as no successful edited vines have been produced by direct delivery methods due to tremendous technical challenges. We will propose new approaches for improving the direct delivery methods in the project year 2019-2020.

Evaluation of grapevine rootstock selections

The purpose of this project is to identify selections from a USDA rootstock breeding program that might warrant release as commercial stocks, and to develop useful data on the performance of recently released rootstocks from other breeding programs to aid growers in selecting appropriate stocks for their vineyards. For the past several years Dr. Gan-Yuan Zhong, USDA-ARS, screened seedling selections for resistance to aggressive strains of root knot nematodes and shipped cuttings of resistant selections to the UC Kearney Agricultural Center in Parlier, CA, where they were rooted and planted into a vineyard for observation. Thirty selections were sent in 2016, 40 in 2017, and the remaining 112 selections in 2018. The vines will be grown at Kearney for three years before being screened for their potential as rootstock mother vines. Cuttings of vines with excellent traits will be challenged with a multi-species inoculum of plant parasitic nematodes, in collaboration with Dr. Andreas Westphal. Genotypes showing excellent mothervine traits and superior resistance to root knot nematode and/or broad resistance to multiple parasitic nematode species, will be advanced to replicated field trials as grafted vines. Stocks that show excellent potential as grafted vines will be referred to the USDA for consideration of release.

Currently five selections (PC0333-5, PC0349-11, PC0349-30, PC04153-4, and PC0597-13), have advanced to a replicated trial in a commercial table grape vineyard in Delano, and six selections (the same five from Delano plus PC0495-51) are in a replicated trial in a commercial winegrape vineyard in Merced. The vines in Delano were planted in summer, 2016, and grafted to Autumn King in 2017, but many of the PC selections failed after budding and were thus replanted in 2018 and are becoming established. The same rootstock selections were planted in Merced in September 2016, but will not be grafted until 2019. The replanting in Delano, and the grafting in Merced, should resolve questions about the potential, if any, of these selections as young grafted vines. The Merced trial is adjacent to another rootstock trial planted by former UCCE advisor Lindsay Jordan in 2016. The second Merced trial includes full rows of 1103P, and more recently released stocks including RS3, RS9, GRN2, GRN3, GRN4, and GRN5 grafted to Malbec, and replicated four times. However, most vines on GRN5 failed, so GRN5 was eliminated from the trial. Vine training in this trial was mostly completed in 2018, and petiole and nematode samples were collected. These preliminary data suggest the various stocks may affect vine nutrition and that they might differ with respect to nematode resistance. It is anticipated that the first fruit quality and yield data will be collected from the Merced trial in 2019.

Determining the role of Auxin-Response Factor 4 in the timing of ripening initiation in Vitis vinifera

The timing of ripening initiation is a major trait for wine grape production. Rapidly
evolving climactic conditions will affect the ripening process of major cultivars in several
growing regions of U.S, which may result in loss of fruit and wine quality. While the
development of improved viticulture practices to mitigate effects of climate change is
critical, the use of complementary approaches must be investigated. The identification of
regulatory genes controlling the timing of ripening initiation is a research avenue to
consider for molecular breeding programs in order to identify clones/cultivars more
suitable to evolving climatic conditions. Innovative molecular practices in the field such as
Spray Induced Gene Silencing have recently received scientific attention and its potential
for rapid industrial application can be seen as alternate solution to traditional and molecular
breeding. Yet, all these translational tools need scientific validation up front to characterize
the cause-to-effect relationship between the gene and/or several genes and the trait of
interest (fruit composition, disease resistance, etc.). The current research project aims to
validate the contribution of a regulatory protein, VitviARF4, to the ripening initiation in
grape berry. Three objectives were designed to achieve this goal; 1) the characterization of
VitviARF4 via genetic engineering and the identification of its potential partners during
the ripening process, 2) the identification of ripening-related genes that are targeted by
VitviARF4, and 3) the evaluation of fruit composition on genetically engineered berries.
After the establishment of the microvine model at OSU to conduct the genetic
engineering experiments, our research efforts were focused this year on several milestones
of the project. For the objective 1, we conducted all the microvine transformations (four
in total, control, two over-expression, and one knock -down) to either turn on or off the
expression of VitviARF4. At least 20 independent transformed plants were selected per
transformation event. We also confirmed protein-protein interactions of VitviARF4 with
other proteins that play a major role in various physiological process of fruit ripening
(sugar, brassinosteroids, ethylene, and epigenetic mechanism). For the objective 2, we
demonstrated that we can induce the conditional expression of the transgene in transgenic
microvine plants. This result was critical to the success of the research outcomes of the
objective 2. For the objective 3, we developed a new analytical method to measure
organic, amino, and phenolic acids, different types of carbohydrates, polyols, and three
classes of flavonoids (anthocyanins, flavonols, and monomer and dimer of tannins). We
have built an in-house library of 95 analytes that were tested against berry extracts from
pericarp samples collected at different stages of grape berry ripening. We are currently
testing the method on mature berries of the microvine and we have identified around 30
analytes covering the major families of compounds existing in grape berry. Dr. Tomasino
is currently optimizing the volatile and aroma analysis on mature fruits of the microvines.

Development of Next Generation Rootstocks for California Vineyards

We are making strong progress in streamlining the assay’s for nematode, salt and phylloxera screening to test new germplasm, existing breeding populations to single out best rootstock selections, and test breeding and mapping populations. We have dynamic duo of Becky Wheeler and post-doc Daniel Pap to greatly accelerate nematode screening efforts. We have built up inoculum to carry out germplasm screening for the dagger nematode. Salt screening of germplasm that was promising in earlier screens was initiated at higher concentrations (75mM) to select optimum accessions that we could use in crosses. At the same time, we are initiating salt screening of breeding populations with different accessions of V. longii and 140Ru to look for segregation in order to carry out marker development process. We are making good progress in better understanding of root architecture in multiple rootstock species including Vitis berlandieri. The key point from different drought screens is that specific root length and root diameter is key features that could be used to test rootstock selections. Trials of selected accessions that pass the screening for horticultural features, nematode, phylloxera and salt tolerance are in pipeline for 2018-2019 funding cycle.

An Integrated Approach to Understanding How Grapevine Root Systems Respond to and Recover from Drought Stress

With our first year of funding from CGRIC, we made significant progress on this project and exciting new discoveries about grapevine fine root responses to drought that shed light on the differences among genotypes/rootstocks. The start date of the project was delayed by ~12months while we waited for the funds to become available, thus we couldn’t justify submitting a renewal in January 2016 for additional funds. We are now submitting a renewal to continue these efforts, which will cover the costs for the second year of the project. Our labs are continuing to work together closely on this and other related projects. In addition to the experimental results described below, we have collected and begun analyzing suberin responses using flurol yellow staining for the following 16 rootstocks/genotypes from a drought experiment run by Dr. Kevin Fort (Walker Lab): 140RU, 101-14, 110R, Riparia Gloire, St. George, 1103P, Dog Ridge, 1616C, Ramsey, Freedom, Schwarzmann, Chardonnay, Cabernet Sauvignon, Colombard, GRN-1, and GRN-2.

We originally hypothesized that embolism formation would be would of the primary limiting factors reducing fine root conductivity (Lpr) under drought stress. Instead, we discovered that embolism formation is preceded by cortical lacuna formation (i.e. the fine root cortex is tearing apart under mild drought stress for tissue just behind the root tip), which coincides with a precipitous drop in Lpr. In a series of follow-up experiments using neutron radiography, we compared the growth dynamic responses of 110R (drought resistant) and 101-14 (drought susceptible) during drydown and recovery and also linked this to lacuna formation and Lpr. Surprisingly 110R exhibited more lacuna under mild drought stress that was linked to lower Lpr for this tissue despite less suberization and greater root elongation rates after drought stress. A preliminary similar patterns has been found in a comparison of Ramsey and Riparia Gloire. We now suspect that slower suberization rates for fine roots of drought resistant rootstocks may contribute to their greater susceptibility to drought induced cortical-lacuna, but this response could provide the adaptive advantage of ensuring that the root tip is receiving adequate water and other resources (i.e. internal carbon supply). In other words, the dysfunction created by the cortical lacuna (not in the tip, but in tissues starting ~2cm behind the tip would reduce the competing surface area of the root cylinder and ensure that any water that is available is being dedicated to continued growth at the tip. We will continue to pursue this and other related hypotheses with the proposed renewal submitted along with this report.

Determining the Role of Auxin-Response Factor 4 in the Timing of Ripening Initiation in Vitis Vinifera

The timing of the ripening initiation is a major trait for wine grape production. Rapidly evolving climactic conditions might affect the ripening process of major cultivars in several growing regions of U.S resulting in loss of fruit and wine quality. While the development of improved viticulture practices to mitigate the effects of climate changes is critical, the use of complementary approaches should be investigated. The identification of genetic markers associated with the timing of ripening initiation will be valuable to develop targeted breeding programs aimed to tackle grapevine production in the context of climate changes. Innovative molecular practices in the field such as Spray Induced Gene Silencing have recently paid scientific attention and is seen as a potential industrial application. Yet, all these translational tools need a scientific study up front to validate the relationship between a gene and/or several genes with the trait of interest (fruit composition, disease resistance, etc.). The current research project aims to validate the role of a regulatory protein, VviARF4, on the timing of ripening initiation in grape berry. Three objectives were designed to achieve this goal; 1) the characterization of VviARF4 through genetic engineering and the identification of VviARF4 interactors during the ripening process, 2) the identification of ripening-related genes directly targeted by the activity of VviARF4, and 3) the evaluation of fruit composition on berries for which VviARF4 activity has been modified through the genetic engineering procedure.

Since June 2017 we focused our research efforts on several milestones of the current project. We established the microvine culture at OSU part of the objective 1. We conducted our first attempt for genetic engineering of the microvine. We successfully
generated embryogenic cells from immature floral buds, which is necessary to perform genetic transformation. For the same objective, we finalized our different genetic constructs to induce and silence VviARF4 in the microvine. Using a protein-protein interaction test, we identified 170 potential protein partners to VviARF4 and we are in the process of confirming some of these interactions by additional in vitro assays. Some of these interactors are very interesting because they are associated with ABA, ethylene
and sugar signaling pathway that are known to be important in the ripening process. For the objective 3, we adapted a new analytical method to measure metabolites associated with organic, amino, and phenolic acids, different types of carbohydrates, polyols, and three classes of flavonoids (anthocyanins, flavonols, and monomer and dimer of tannins). For those metabolites, we have built an in-house library of 95 analytes. We tested this library using berry extracts from pericarp samples collected at different stages of grape
berry ripening. We were able to identify around 30 analytes covering major families of compounds existing in grape berry. These include tartrate, malate, glucose, fructose, sucrose, and several polyphenol-related compounds. We will be using this analytical method for the aim 3 of this research proposal.

Evaluation of Grapevine Rootstock Selections

The primary purpose of this project is to identify selections from a USDA rootstock breeding program that might warrant release as commercial stocks. Dr. Gan-Yuan Zhong screened seedling elections for resistance to aggressive strains of root knot nematode shipping 30 such selections to Kearney in 2016, 40 in 2017, with the remaining 100 selections to be shipped in 2018. Selections are  rooted and outplanted at Kearney and, when established, will be screened for their potential as rootstock mother vines. Those with excellent traits will be advanced to replicated field trials as grafted vines. Any stocks that show excellent potential as grafted vines will be referred to the USDA for consideration of release. Currently five selections (PC0333-5, PC0349-11, PC0349-30, PC04153-4, and PC0597-13), have advanced to a replicated trial in a commercial table grape vineyard in Delano, and six selections (the same five from Delano plus PC0495-51) are in a replicated trial in a  commercial winegrape vineyard in Merced. The vines in Delano were planted in summer, 2016, and grafted to Autumn King in 2017, but many of the PC selections failed after budding. It is not clear whether the failures were due to the stocks per se, or for some other reason. Vines on Freedom and own rooted vines in that trial have done much better than the vines on PC stocks, but the vines on Freedom were planted at a different time, and not budded in the field. Therefore, the PC selections will be replaced in 2018, and more closely monitored. The same rootstock selections were planted in Merced in September 2016, but will not be grafted until 2018. The replanting in Delano, and the grafting in Merced, should resolve questions about the potential, if any, of these selections as young grafted vines. The Merced trial is adjacent to another new rootstock trial planted by Lindsay Jordan in 2016. The second Merced trial includes full rows of 1103P, RS3, RS9, GRN2, GRN3, GRN4, and GRN5 grafted to Malbec, and replicated four times. Jordan’s resignation in 2017 could have led to the abandonment of the trial. We collected petiole data from those vines last year, as Jordan planned, and decided to include the trial in this proposal, at least for the 2018 season. If this additional objective is supported, it will provide an opportunity to acquaint Karl Lund, Jordan’s replacement, with the trial. It will be decided later if it should be separated from the others. Petiole analyses from the Malbec trial revealed that the stocks differed with respect to uptake of nitrate, phosphorus, potassium, boron, calcium, zinc, and magnesium. It was also observed that most vines on GRN5 failed, whereas most of the other vines are becoming established.  Therefore, GRN5 will be eliminated from the trial. It is anticipated that preliminary fruit quality and yield data may be collected from the second trial in Merced in 2018.

Deep Sequencing for Trunk Disease Diagnostics

The aim of this multi-year project is to develop rapid and cost-effective diagnostic methods based on Next Generation Sequencing (NGS) technologies for detection, identification and quantification of trunk pathogens in asymptomatic and symptomatic grape wood. In the 1st year of the project (2015 – 2016) we collected diseased wood material from commercial vineyards and characterized the associated fungal pathogen species using traditional methods, such as morphological and sequence-based identification of purified fungal colonies (see progress report for the 2015-2016 funding cycle). We used these samples to determine how effective ITS-sequencing, meta-genome sequencing and metatranscriptome  sequencing approaches are in identifying and quantifying pathogenic species directly in planta. Data simulations allowed us to determine what mapping algorithm was the most specific and sensitive in detecting trunk pathogens both qualitatively and
quantitatively. All NGS methods we tested were in agreement with traditional diagnostic methods, but also allowed us to detect simultaneously multiple pathogen species with no
need of hands-on sample culturing and colony purification. Additionally, unlike traditional diagnostics, which are strictly qualitative, NGS approaches allowed us to determine the
relative abundances of the different infecting species. Among all methods tested, ITS-seq is still the most cost-effective until library preparation costs for RNA and DNA-seq do not decline significantly. For this reason, ITS-seq was chosen for further protocol optimization.
Both sensitivity and specificity of the ITS-seq approach remain to be improved for diagnostics purposes. In the second year of the project (2016-2017), we (a) confirmed that NGS allows the detection with high specificity of actively infecting pathogens when vines
are experimentally infected with individual pathogen strains; (b) established that NGS detection is quantitative and allows to differentiate between diseased and healthy vines; (c)
developed a protocol for testing dormant cuttings and started testing cuttings provided by a commercial nursery. In the 2016-2017 funding cycle, we also developed a new DNA extraction protocol that reduced the time required for processing and the amounts of
sample, reagents and waste. In the current funding cycle (2017-2018), we have (a) completed the analysis of trunk pathogen presence in propagation material obtained from nurseries; (b) began a collaboration with Dr. Urbez-Torres at Agriculture and Agri-Food Canada to expand the sampling of propagation material to two additional nurseries and compare directly our approach with their DNA-macroarray method and (c) developed a new set of optimized primers for ITS-seq designed specifically to target the ITS of grapevine trunk pathogens.

Targeted Genome Editing for Trait Improvement in Vitis vinifera

Targeted genome editing to modify gene expression has rapidly emerged as an effective tool for cultivar improvement. This approach can result in two types of new grape varieties with one or several trait improvements: 1) Transgenic clones of existing varieties, or 2) Non-transgenic breeding lines to develop new varieties. In this first phase of the project, we aimed to demonstrate this technology by enhancing the powdery mildew resistance of ‘Thompson Seedless.’ Among the approaches that confer powdery mildew resistance in Vitis vinifera, silencing of susceptibility genes has proven to be promising both in terms of efficacy as well as potential durability. We previously showed that reducing expression of grapevine VvPmr6A, VvPmr6B, VvMloA, or VvMloQuadX resulted in up to a 60%reduction of powdery mildew incidence, even though our artificial microRNA approach only partially reduced target gene expression. In this current work we are using a Transcription Activator-like Effector Nuclease (TALEN) strategy to completely knock out our target genes and obtain even stronger disease resistance. In addition, we have identified two additional candidates that are grapevine genes directly related to Arabidopsis Pmr4 and Pmr5, which confer strong powdery mildew resistance in other plants. In this context, in the past year we were successful in assembling 7 TALEN constructs targeting all 6 target genes. The constructs are now being transferred into the final plant destination vector, which is expected to be sent for grape transformation by late February 2015.

Based on our previous results and publications in other plants, we expect to see a significant increase in powdery mildew resistance. This targeted genome editing approach will demonstrate the potential to knock out genes in V. vinifera, establishing a protocol that could be extended to various traits in improving existing or new varieties. In the current and subsequent years, we aim to demonstrate another genome modification technique (dTALE-mediated activation of native gene expression) for increased abiotic stress tolerance in grapevine.

Integrating Systems Biology with Marker Assisted Selection to Guide the Stacking of Powdery Mildew Resistance Genes

The long-term goal of this project is to develop grape varieties that possess effective and durable resistance to powdery mildew (PM). Stacking resistance genes from multiple resistant genetic backgrounds and with the least functional redundancy is a proven breeding strategy to improve both durability and level of resistance. This strategy requires (a) the identification of multiple sources of resistance, (b) the functional characterization of the mechanisms of resistance to prioritize optimal genetic combination, and, finally, (c) marker assisted breeding to introduce the selected genes into elite varieties. The specific objectives of the 2014-2015 funding period consisted of (i) the genetic mapping of the two PM resistance loci in V. piasezkii and development of molecular markers for marker assisted breeding and (ii) the functional characterization of resistance responses in a panel of V. vinifera accessions (Ren1, Ren6, Ren7). We have successfully completed the genetic map of the Chinese species V. piasezkii and identified two new loci, Ren6 and Ren7 that manifest complete and partial resistance to powdery mildew, respectively. Both of these loci are present on two chromosomes that were not reported before in earlier studies. The identification of these two loci has enhanced the repertoire of resistance loci for grape powdery mildew breeding. We have developed closely linked markers that will be used to facilitate the combining of resistance from V. piasezkii to other advance powdery mildew resistant selections in our grape breeding program for durable field resistance. To be able to select which specific accessions to use for further breeding we initiated the characterization of the resistance responses using RNA sequencing approaches (RNA-seq). RNA-seq was successfully applied to study early (1 day post infection, dpi) and late (5 dpi) responses to powdery mildew associated with Ren1 resistance. Comparisons of 7 V. vinifera accessions carrying the Ren1 locus led to the identification of sets of constitutive and inducible defense related genes associated with Ren1-dependent resistance. We also successfully produced replicated PM infections in control environment for individual genotypes segregating for Ren6, Ren7, or both Ren6/Ren7, for which sequencing libraries are being sequenced.