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{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} 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{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} 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{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} 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{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} resistant progeny with 50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} 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{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} 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.

Molecular genetic support to optimize the breeding of fanleaf resistant rootstocks

This report presents results on Walker lab efforts to optimize the breeding of fanleaf degeneration (fanleaf) resistant rootstocks through molecular genetic methods. These efforts are two-fold: 1) to understand and utilize O39-16’s (a Muscadinia rotundifolia based rootstock) ability to induce tolerance to fanleaf virus infection in scions; and 2) to understand and utilize resistance from Vitis arizonica to the dagger nematode, Xiphinema index, which vectors grapevine fanleaf virus (GFLV) from vine to vine. We are in the process of repeating and clarifying past mapping and xylem sap analysis. We hope to have the previous work verified and corrected by Summer. The field trials we established to study xylem borne compounds with an influence on fanleaf infection will fruit well for the first time this Summer and we have renewed our efforts to determine the basis of induced tolerance. We also successfully completed a reworking of our fine-scale mapping efforts and that publication is submitted. This work will generate gene candidates for XiR1, the locus we have identified as responsible for X. index, resistance as derived from V. arizonica. This discovery will be followed with transformation experiments to confirm the resistance function of these candidate genes and allow us to use traditional breeding methods more carefully to avoid the breakdown of resistance and might lead to grape rootstocks genetically engineered with grape resistance genes.

Using marker-assisted selection to introgress powdery mildew resistance genes from different Vitis resistance sources into wine grapes

This report presents results on 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 used these genetic markers to evaluate parents and progeny from our crosses. 2) Worked to verify the single dominant gene (locus) nature of resistance from the apparently resistant V. vinifera table grape, Kishmish vatkana, and test its reliability under California environmental conditions; 3) Utilized the above mentioned sources to make crosses that combine resistance from rotundifolia and vinifera selections; 4) Initiated the study of V. cinerea B9 based powdery mildew resistance; 5) Initiated the utilization of powdery mildew resistance from wild Chinese species in collaboration with the USDA; and 6) Investigated the origin powdery mildew resistance in vinifera-based table grape selections and using the Kishmish vatkana allelic profile have searched for other resistant selections that possess this unusual and very valuable source of powdery mildew resistance. 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. This would insure that their resistance is more durable and that it functions under a broad range of environmental conditions to provide low input, environmentally ?green? grapevines that would not require fungicides for powdery mildew.

Accelerating the development of powdery mildew resistant grapevines through marker assisted selection

The aim of this project is to harness molecular biology in the selection and advancement of improved cultivars having resistance to powdery mildew. Segregating populations from three sources of significant powdery mildew resistance (Vitis davidiiV. rotundifolia, and V. aestivalis), each backcrossed to V. vinifera, were previously generated by Dr. David Ramming. The first objective of this proposal is to characterize the plant-pathogen interactions, in terms of race-specificity and microscopic analysis, for each of the three resistance sources in order to inform the second objective, which is the development of molecular markers that co-segregate with powdery mildew resistance in each of these populations for use by grape breeding programs.

Powdery mildew resistance was assessed in 182 progeny from the three populations using three separate pathogen sources in California and New York. The resulting data suggest the presence of multiple, race-specific resistance genes segregating independently in rotundifolia and aestivalis progeny and suggest that some of the resistance genes would be rapidly overcome if inappropriately deployed. However, some progeny were resistant regardless of the pathogen source, suggesting the presence of all parental resistance alleles as a resistance gene pyramid. The stability of resistance in these individuals and the pathogen-dependent resistance of other individuals were confirmed in 2007. The rotundifolia and aestivalis breeding populations underscore one critical application of marker assisted selection – monitoring and pyramiding all functional resistance genes using a simple molecular assay rather than assaying resistance and durability by complex inoculation studies with multiple pathogen sources.

We also confirmed in 2007 that either of the two putative resistance genes from the davidii resistance source is sufficient for resistance regardless of pathogen source; these genes have the added intrigue of providing resistance against the penetration of the fungus (i.e., the pathogen is unable to access the epidermal cells where it must obtain sustenance to survive). Most powdery mildew penetration resistance genes are effective against all races of powdery mildew, and this appears to hold true with davidii.

To address the second objective, we require molecular markers that are polymorphic (appear different between the two parents) to track regions of the genome that were contributed to progeny by the resistant parent. We have identified 157 Simple Sequence Repeat markers (SSRs) that are polymorphic in these populations. Thus far, we have developed multiplexes for 39 SSRs and used them to screen all progeny in the three populations. In addition, we have identified amplified fragment length polymorphism (AFLP) markers associated with resistance in each of the populations. Our preliminary results support the two-gene models suggested by phenotypic data for the davidii and rotundifolia populations. Marker-trait associations in the aestivalis population will require QTL analysis.

Upon confirmation of which polymorphic markers predict disease resistance, we will focus on providing tightly-linked markers flanking disease resistance genes. From crosses representing each resistance source, we have germinated at least 600 seed and will test the utility of our markers for MAS, while using recombinants to more precisely track resistance genes.

Breeding Rootstocks Resistant to Aggressive Root-knot Nematodes

The USDA grape rootstock improvement program, based at the Grape Genetics Research Unit, is breeding grape rootstocks resistant to aggressive root-knot nematodes. We define aggressive root-knot nematodes as those which feed on and damage the rootstocks Freedom and Harmony. In 2006 we screened 3622 candidate grape rootstock seedlings for resistance to aggressive root-knot nematodes. We select only those seedlings which completely suppress nematode reproduction and show zero nematode egg masses. These selected seedlings are propagated and then planted into the vineyard. In 2006 we planted 372 nematode resistant rootstock selections in the vineyard. These selections were identified in nematode resistance screening in 2005 and 2004. In 2006 we pollinated 132 clusters of crosses specifically aimed at the breeding of improved rootstocks with resistance to aggressive root-knot nematodes. We tested the propagation ability of 114 nematode resistant selections. We confirmed the resistance of our rootstock selections to aggressive root-knot nematodes and we identified nematode resistant germplasm that may be parents for rootstock breeding.