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.

Evaluation of Zinfandel Heritage Selections

The 2007 growing season was more moderate, without the heat wave of 2006. The result was far less sunburn in 2007. Harvest was compressed with two harvests only two days apart, Sept 11 and Sept 13. Yield varied from a low of 3.7 kg per vine to a high of 6.6 kg. Regarding yield components, berry wt was a very tight range, differing by only 0.2 g, from 1.5 g to 1.7 g per berry. The yield component clusters per vine, as in previous years, was very close among clones due to crop control via pruning. Cluster wt, also following previous the years? pattern differed widely from a low 174 g to almost 300 g. Given the similarity of berry wt, berries per cluster mirrored the difference patterns of cluster wt, with heavy clusters having the most berries and light clusters the least, a range of 108 to 183 berries. Berry samples for comparing selections were taken on a single date just prior to harvest. It shows a range of 24.1 to 26.6 Brix. Values of pH were very low, as is the usual case for Zinfandel at the Oakville Station, and in a relatively tight range, 3.10 to 3.18. Titratable acidity values were also relatively close, ranging from 7.32 to 8.07 (g/L). The patterns of difference in the 2007 data, are similar to those in the 2005 ? 2007 averages. Ripening was a little more uniform in 2007. Cluster numbers are greater in 2007 but this is due to an increase shoot number over year 2005, and a decrease in sunburn loss over year 2006.

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.

Development of a National Expert System for Diagnosis of Grape Problems

Version 1.0 of the diagnosis expert system has been developed with a database containing information on 161 grape problems including 55 arthropods, 48 pathogenic diseases, 25 phytotoxicity problems, 13 nutritional disorders, 9 abiotic stress disorders, 6 wildlife damage, and 5 physiological disorders. Additional problems will be entered into the database in 2007. The system is currently undergoing debugging and refinement prior to evaluation by a test panel. Production of graphics was initiated in 2006 and draft images have been completed for all first-level menu items.

The diagnosis system uses a symptom-based approach that guides the user through a series of directed questions leading to a ranked list of the most probable problems. The user answers questions based on their observations of symptoms and signs of the problem. Questions are organized in hierarchal levels, enabling the software to analyze responses and select the next level of questions on-the-fly based on the previous responses. This approach streamlines the diagnosis process by focusing on discriminating questions and avoiding extraneous ones.

Each vineyard problem is characterized with diagnostic keywords and a Problem Profile. The system uses the keywords to conduct a sorting routine to identify and display a probability-ranked list of possible problems. The Problem Profile contains text and photographs to assist with diagnosis of the problem.

A database was created to hold Diagnostic Keywords and Problem Profiles for known grape problems caused by pathogens, arthropods, vertebrate and other pests, abiotic stresses, nutritional disorders, chemical phytotoxicity, and physiological disorders. Database entries were created based on the personal experience of the principal investigators and from several standard references including the Compendium of Grape Diseases, 1988 APS Press and Grape Pest Management, 1992, University of California, Division of Agriculture and Natural Resources.

The technical accuracy of database entries will be validated by an editorial review process involving experts from around the U.S. Participation in the project by experts will be facilitated by the previous creation of draft versions of Diagnostic Keywords and Problem Profiles, which will minimize the time commitment of reviewers. Experts will be requested to review and edit selected problems. Reviewers will begin testing version 1.0 of the Expert System in 2007 and will be provided password-protected access to the Problem Profile Editor database interface. Reviewers will be credited for their work and will be invited to contribute new problems based on their experience. High-quality photos of symptoms and signs will also be solicited from experts and photo credits acknowledged. The diagnosis expert system will be validated through test use of version 1.0 and 2.0 by a panel comprised of vineyard managers, Extension educators, and students.

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 2005 we screened 6201 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. We have 367 nematode resistant selections that will be ready for vineyard planting in spring 2006. In 2005 we planted 38 nematode resistant rootstock selections in the vineyard. These selections were identified in nematode resistance screening in 2004 and 2003. In 2005 we pollinated 857 clusters of crosses specifically aimed at the breeding of improved rootstocks with resistance to aggressive root-knot nematodes.

Development, Testing and Introduction of Grape Rootstocks with Broad and Durable Nematode Resistance

We continue toward our goal of developing and releasing grape rootstocks with broad and durable resistance to nematode species that are important in California vineyards. In previous years, we have screened rootstock candidates against the root-knot nematode (Meloidogyne incognita race 3), two strains of root-knot nematode that overcome the resistance of Harmony rootstock (Meloidogyne arenaria strain A and Meloidogyne incognitastrain C), and the dagger nematode (Xiphinema index). Fourteen rootstock candidates exhibit broad resistance to those nematodes. This year, we continued to test the breadth of that resistance beyond the range of the primary screen species by evaluating the resistance of the 14 candidates to the ring nematode, Mesocriconema xenoplax, in the presence of other nematode species.

We also evaluated ring nematode resistance in the parents of the current rootstock candidates and in some other Vitis sources. Only two of the rootstock candidates exhibit any resistance to the ring nematode and that may not be durable when other nematodes are present. We continue to seek new sources of resistance. We also continued to test the durability of nematode resistance of the rootstock candidates when they are exposed to combinations of nematode species by determining the durability of resistance at different temperatures. Resistance of the parents of the rootstock candidates to several root-knot nematode variants was compromised at soil temperatures of 30°C and above but not below 27°C. However, some of the rootstock parents maintained resistance to even the virulent Meloidogyne arenaria strain A at high temperatures, indicating that there is durability to temperature among the parentage.

Field testing of the rootstock candidates continues in fields that were heavily infested with root-knot nematodes. Nematode population levels are declining in the root-zones of all rootstock candidates, indicating that reproduction of root-knot nematodes is not occurring. However, population levels of ring nematodes at the field site are high on most of the selections, underscoring the need for obtaining new sources of resistance to that nematode.

Expression of Anti-microbial Genes in Transgenic Grapevines for Enhanced Disease Resistance

Grapevines are susceptible to numerous diseases harming both plants and profits. Transgenic grapevines that resist disease would provide better disease control as well as economic benefits from the reduction in spray applications. Our overall goal has been to research and develop methods to create transgenic selections of elite cultivars with improved resistance to diseases. The transgenic strategy is especially appropriate for clonally-propagated crops, such as grapevines, where the wine industry is rooted in traditional European grapes with strong name recognition and very high disease susceptibility. During the past year, we screened six different antimicrobial peptides, which are small proteins known to be inhibitory to a range of bacteria and fungi, to determine which might best provide resistance to bunch rot (Botrytis) and crown gall (Agrobacterium vitis). These same peptides are also being tested for their effects on germinationof powdery mildew conidia. Based on the incoming results from peptide screening, development of new gene constructs is underway. These constructs will be inserted into Chardonnay and the resulting vines will be tested for improvements in disease resistance.