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.

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.

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.

Using Marker-Assisted Selection to Introgress Powdery Mildew Resistance Genes From Different Vitis Resistance Sources Into Wine Grapes

The overall goal of this project is to identify genetic markers for multiple sources of powdery mildew resistance and use them to combine these resistances into a single background. Combining these resistances cannot be done without genetic markers, since the phenotype (resistance to powdery mildew) is the same from each resistance source. We have made good progress at identifying and using markers to mildew resistance, but we need to understand how the marked genes work and if they are different to gain the greatest advantage in combining the resistances. To enable this improved understanding this project is being melded into a new collaborative project between my lab and that or Prof. Dario Cantu, our recently hired “omics” specialist who has had extensive experience working with disease resistance genes in wheat. Dario’s experience will greatly aid our molecular genetics effort to breed new varieties with durable mildew resistance. We have made good progress in developing and using genetic markers from two rotundifoliabased resistance sources; from the pure vinifera cultivar Karadzhandal; and from two accessions of the Chinese species V. romanetii. We are also beginning to map a new form of resistance from another Chinese species, which is taxonomically distant from V. romanetii and V. davidii and has very strong resistance. We have been mapping and using these resistance sources and have assumed that they work in different ways because the location of their resistance genes differs. Some of the genes may be copies that do the same thing but are located on different chromosomes. Dr. Cantu’s involvement in the project will allow us to better understand how the resistance works and which resistance sources are the most genetically and functionally different so that we have the greatest chance of creating lines with durable resistance from multiple genes. We have advanced resistance through backcrossing and crossing lines with different resistance markers and are now poised to begin combining as many lines with different resistance genes as possible. We have also crossed our advanced Pierce’s disease resistant selections with advanced powdery mildew resistant selections and have many selections with >90%vinifera parentage, good fruit quality and resistance to both powdery mildew and PD.

Using Marker-Assisted Selection to Introgress Powdery Mildew Resistance Genes From Different Vitis Resistance Sources into Wine Grapes

This report presents results on the Walker lab efforts in utilizing molecular breeding tools to pyramid powdery mildew resistance from different genetic backgrounds into V. vinifera-based cultivars. In a short time period of four funding years of this project, we have made quick progress that completely relies on our experience of PD resistant winegrape breeding, in hand diverse genetic resistant material, and our ability of utilizing molecular tools developed in the lab as well as in public domain. So far we have: 1) Examined several sources of powdery mildew resistance from Muscadinia rotundifolia and evaluated parents and progeny via markers that are tightly linked to the resistance, and determined the allelic profiles of markers and alleles that are linked to the resistance for MAS; 2) Developed mapping populations with two different rotundifolia cultivars and mapped two forms of a major locus denoted as Run2.1 and Run2.2 on chromosome 18; 3) Mapped locus Ren4 from Chinese origin species V. romanetii on chromosome 18; 4) Verified the single dominant gene (locus) nature of resistance from the V. vinifera table grape, Kishmish vatkana, and tested its reliability under California environmental conditions; 5) Investigated the origin of powdery mildew resistance in vinifera-based table grape selections using the Kishmish vatkana Ren1 allelic profile, and identified five additional resistant selections that possess this unusual and very valuable vinifera-based source of powdery mildew resistance; 6) Nursery screened all vinifera-based plants that had one or both alleles of Ren1 linked markers as well as other resistant germplasm; 7) Utilized the above mentioned resistance sources to make crosses that combine resistance from rotundifolia and vinifera selections; 8) Developed a breeding population to initiate the study of V. cinerea B9 and Villard blanc base powdery mildew resistance; 9) Expanded a breeding population with powdery mildew resistance from the Chinese V. romanetii and conducted field evaluations; 10) Developed resistant lines that already range up to the 94 -97%vinifera level; 11) Initiated crosses with objective of pyramiding resistant loci into single line; 12) Made crosses to develop homozygous resistant lines for at least 3 resistant loci that could be used to develop 100%resistant progeny with 50%of elite cultivar genome; 13) established in lab leaf disk assay to understand the plant pathogen interaction as well as to screen populations; 14) And finally, we are evaluating these breeding populations for fruit quality traits and disease resistance constantly each year. The knowledge and results gained from this work will lead to the development of wine and table grape selections with multiple powdery mildew resistance genes pyramided into a single line, and environmentally ?green? grapevines that do not require the application of fungicides to control powdery mildew.

Next Generation Markers to Accelerate Grape Cultivar Improvement

Molecular DNA markers can be used to tag traits in the genome, providing optimal selection of parents and early selection of elite progeny with multiple desirable traits and/or multiple resistance genes for improved durability. The purpose of this project was to develop a pipeline for applying molecular markers in public grape breeding programs. In this pilot project, two public breeding programs were invited to submit up to 1000 leaf samples each to the Cadle-Davidson lab for DNA extraction, quality control, and processing with markers already discovered and potentially relevant to their breeding programs. Drs. Ramming and Reisch decided to provide populations focused on powdery mildew resistance and seedlessness for validation of known marker-trait associations. We tested three markers linked with seed development inhibitor (SdI) and found that combining the marker data resulted in enhanced selection of seedlings with little or no seed trace. Using stringent selection in three populations, 100%of progeny with SdI markers were seedless. These markers would enable breeders, depending on their preferred stringency, to discard up to three-quarters of young seedlings solely based on predictions of seedlessness. This would enable the focused evaluation of more crosses and more elite progeny.

Using Marker-Assisted Selection to Introgress Powdery Mildew Resistance Genes from Different Vitis Resistance Sources into Wine Grapes

This report presents results on the Walker lab efforts to use molecular breeding tools to pyramid powdery mildew resistance from different genetic backgrounds into V. viniferabased cultivars. Progress has been made on a number of fronts. We have: 1) Examined several sources of powdery mildew resistance from Muscadinia rotundifolia and evaluated parents and progeny via markers that are tightly linked to the resistance, and determined the allelic profiles of markers and alleles that are linked to the resistance for MAS; 2) Developed mapping populations with two different rotundifolia cultivars and mapped a major locus Run2.1 and Run2.2 on chromosome 18; 3) Mapped locus Ren4 from Chinese origin species V. romanetii on chromosome 18; 4) Verified the single dominant gene (locus) nature of resistance from the V. vinifera table grape, Kishmish vatkana, and tested its reliability under California environmental conditions; 5) Investigated the origin of powdery mildew resistance in vinifera-based table grape selections using the Kishmish vatkana Ren1 allelic profile, and identified five additional resistant selections that possess this unusual and very valuable vinifera-based source of powdery mildew resistance; 6) Nursery screened all vinifera-based plants that had one or both alleles of Ren1 linked markers as well as other resistant germplasm; 7) Utilized the above mentioned resistance sources to make crosses that combine resistance from rotundifolia and vinifera selections; 8) Developed a breeding population to initiate the study of V. cinerea B9 based powdery mildew resistance; and 9) Expanded a breeding population with powdery mildew resistance from the Chinese species, romanetii and conducted field evaluations. The knowledge and results gained from this work will lead to the development of wine and table grape selections with multiple powdery mildew resistance genes pyramided into a single line, and environmentally ?green? grapevines that do not require the application of fungicides to control powdery mildew.