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.

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.

Breeding Rootstocks Resistant to Aggressive Root-knot Nematodes

The objectives for this study are to breed, evaluate, and introduce rootstocks that are resistant to aggressive root-knot nematodes, resulting in improved varieties with adaptation to California viticulture. To achieve this objective, our goal was to evaluate the root-knot nematode resistance of 12,000 grape rootstock seedlings and select resistant seedlings for advancement to the field. We will make crosses specifically for the breeding of rootstocks resistant to aggressive root-knot nematodes.

The USDA grape rootstock improvement program, based at the Plant Genetic Resources 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 2004 we screened 5124 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 81 nematode resistant selections that will be ready for vineyard planting in spring 2004. In 2004 we planted 89 nematode resistant rootstock selections in the vineyard. These selections were identified in nematode resistance screening in 2003 and 2002. In 2004 we pollinated 1500 clusters of crosses specifically aimed at the breeding of improved rootstocks with resistance to aggressive root-knot nematodes.

Developing a Functional Genomics Approach to Berry Ripening and Defense

Our work with fruit ripening and defense related genes is now complete with regard to collecting EST sequences. With the ones we have already collected and the many thousands that are now planned in efforts on this campus and internationally we feel that it will be possible to clone nearly any grape gene we are interested in by application of sequence information and PCR methods. This means that studies where information about gene expression in grape is needed can be undertaken with confidence that most of the genes of interest can be easily cloned. Nevertheless, the picture that has emerged from our work with the veraison stage library is very informative and is a major research accomplishment and result.

We divided the EST sequences we obtained into three classes. Class one are genes for which the role in fruit development or ripening is already known in other systems (e.g. tomato). An example of class-one genes would be polygalacturonase. The second class includes those genes whose function can be known with a high degree of certainty but where its role in fruit development or ripening is unknown. An example of class-two genes would be a tonoplast intrinsic protein. The third class contains those genes that are unknown (a match was found in the data base but no function has been assigned to the protein) hypothetical proteins (e.g. an open reading frame from Arabidopsis) or no match (i.e. no match at all was found in the data base). In our total EST collection 34{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of the genes were assigned to class 1; 44{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} to class 2; and 22{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} to class 3.

In order to provide a functional classification for the genes in class 1 and class 2 above, we assigned each gene to a functional category. The major group in the functional category contained genes related to stress responses; either oxidative, osmotic or water stress. We found that 22{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of the genes were stress related and 20{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} were related to protein synthesis, processing and degradation. We found that 18{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of the genes to which we could assign a function are related to disease resistance, and 8{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} involved with signal transduction. There were 9{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} related to RNA processing or were known transcription factors. Other groups with about 5{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} each were cell wall chemistry, secondary metabolism and photosynthesis.

The view of grape berry ripening that has emerged from our work is much different than we expected when we began to sequence ESTs from veraison berries. The large number of stress induced genes and disease related genes we found was surprising and unexpected. Nevertheless, this result has drawn our attention to the function of stress responses and plant defense gene expression in fruit ripening and berry composition.

PDF: Developing a Functional Genomics Approach to Berry Ripening and Defense

Enhancement of Stress Tolerance in Vitis vinifera

Abiotic stresses affect important aroma, flavor and color components by altering metabolite composition, improving wine quality and human health benefits. Regulated deficit irrigation has been used successfully to grow grapes with less water, an important feature in arid regions such as Nevada. As a first step toward understanding how growth is affected and wine quality improvements might arise following abiotic stress exposure, we have initiated an expressed sequence tag (EST)-based gene discovery program focused solely on stressed plants. We constructed cDNA libraries from mRNA isolated from leaf and berry tissues of Vitis vinifera cv. Chardonnay, exposed to various abiotic stress conditions. To date, we have sequenced over 3000 leaf ESTs and anticipate completing another 5000 berry sequences over the next few months before funds run out this year. Raw sequence data were processed through an automated EST analysis pipeline (ESTAP) developed at the Virginia Bioinformatics Institute (VBI; Blacksburg, VA) in collaboration with UNR and S.R. Noble Foundation (Ardmore, OK). Initial sequence analysis reveals 36{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} novel genes and a low redundancy of transcripts. All 1878 unique EST data generated to date have been deposited in GenBank and is freely available to the public.

In the context of genetic engineering more cold tolerant grapes, we have successfully transformed and regenerated V. vinifera in our laboratory. Embryo culture was successfully initiated using 0.5 to 1 mm immature anthers that were excised from flowers of Chardonnay plants. The excised anthers were placed on one of the following callus-initiation mediums: NB, PT or PIV medium. Tissues were subcultured to fresh plates every 6-8 weeks. Embryogenic calli suitable for transformation formed on some cultures. Single cell somatic embryos were transformed with CBF1 and CBF3 constructs including the CAMV35S promoter. The CBF/DRE transcriptional activators, CBF1 (DREB1B), CBF2 (DREB1C) and CBF3 (DREB1A) are some of the master switches for drought, salinity and cold tolerance. Eleven transgenic plants have been regenerated after selection on Kanamycin media. Three plants have been positively identified by PCR for transformation with CBF3. Confirmation of transformation by PCR and Southern analysis for the rest of the plants is underway. A second batch of transformed somatic embryos is currently going through the regeneration process. Degenerate primers designed to motifs of the CBF gene family have produced 8 distinct PCR products. These products represent putative grape CBF orthologs. Additional CBF orthologs are expected to be obtained from our on going EST sequencing program.
Must samples were obtained from well-watered and drought-stressed Chardonnay grapes. Standards were developed for gas chromatography/mass spectroscopy in preparation for must analysis.

PDF: Enhancement of Stress Tolerance in Vitis vinifera

A Genetic Map of Vitis Vinifera: A Foundation for Improving the Management of

We have continued to steadily add information to a genetic linkage map of Vitis vinifera. We have now mapped 145 microsatellite DNA markers to 18 linkage groups. New DNA markers have been developed within the Vitis Microsatellite Consortium, 20 cooperating research groups in 12 countries. Our map is now the most advanced genetic map of Vitis vinifera in the world. It is a resource that can be used by many viticulture researchers to facilitate the identification and isolation of individual grape genes so that we can learn how these genes work and how their functions are influenced by external factors, such as vineyard cultural practices. Our map is now being used by research groups in France, Germany, Italy, Spain and Australia to connect genetic maps being developed in their programs and to further gene identification efforts. These groups are also sharing their own results with us. We have also analyzed quantitative fruit and cluster characteristics that contribute to berry size and cluster tightness. Most of these characteristics map to only 3 regions of the grape genome, suggesting that key genes controlling berry size and cluster structure are located there.

Developing a Functional Genomics Approach to Berry Ripening and Defense

For the past year we have focused our work on gene discovery in grape. We are using a gene library from veraison stage fruit as the source of the new genes. We screened more than 700 plasmids to identify candidates for DNA sequencing, and identified 312 that appeared to have inserts large enough to identify the gene once its sequence was obtained. Preparation for sequencing required growing selected E. coli colonies overnight and then extracting plasmid DNA. One round of sequence was obtained using T3/T7 sites on the pBluescript as sequencing primers. After the sequences were acquired they were examined for open reading frames (ORF) and when an ORF was found it was submitted for a BLAST search to determine if there was homology with known genes. We have obtained many important and interesting genes that are expressed during veraison. The results obtained thus far constitute a list of genes that can be organized into related groups. The groups are based on their physiological and biochemical functions. The groups are: l)Protein Synthesis, Processing and Turnover; 2 Abscisic Acid and Water Stress; 3 Oxidative Stress and Redox; 4 Cell Wall and Cell Wall Management; 5 Plant Hormone and Signal Transduction; 6 Transcription Factors; 7 Enzymes of Primary and Secondary Metabolism/Structural Proteins. Several of the genes have very interesting roles and have been shown to be valuable in other systems. For example we found a pectate lyase that has been shown to cause preactivation of defense genes in transgenic potato, providing resistance to the pathogen Erwinia carotovora. However, the role of the pectate lyase in grape berries is unknown. Some of the proteins expressed at veraison seem to be involved with water stress or related to abscisic acid (ABA), the plant hormone commonly associated with responses to water stress. Finding this category was somewhat surprising but is clearly an important area for further study. It has been recognized for some time that ABA has a role in grape ripening, but it may be that its role is associated with water stress experienced by the berry at veraison. We now have several genes related to ABA and water stress with which to address the role of ABA in ripening, and its possible association with water stress. Our results in the past year have pointed out several important features of berry physiology that were unexpected. We have found several genes related to plant hormones such as auxin, ABA and ethylene. It is well known that hormones are important in berry ripening, and we now have clues as to which of these might be important, and tools to study their effect on expression of specific genes. Taken together, the new view of berry ripening that is emerging from our results may perhaps be the most important accomplishment of the 1999 season.

Genetic Transformation: A Means to Add Disease Resistance to Existing Grape

Methods for initiating embryogenic cultures, the starting material for genetic transformation, have been improved and large numbers of embryogenic cultures suitable for transformation have been produced from Chardonnay and Thompson Seedless. A better strain of Agrobacterium (the gene delivery organism) was obtained. Methods for selecting and isolating putatively transformed somatic embryos have been improved. Transformation experiments with liquid cultures have not been successful. Several experiments with solid cultures of Chardonnay, Thompson Seedless and St. George, however, have yielded cultures that appear to be uniformly transgenic in that they express the blue color of the introduced GUS marker gene throughout the tissue. Some of these putatively transgenic embryos are being induced to germinate into plants so that they can be further tested for the presence and expression of the introduced gene.

Identification and Characterization of Genes that Control the Phenolic

We isolated a cDNA clone for grape 4-coumaryl-CoA ligase (4CL) which is nearly full length. This is an enzyme that is positioned at an important branch-point in phenolic metabolism. We inserted the partial cDNA clone into an expression system, and initial experiments indicate that we can obtain sufficient quantities of purified grape 4CL for antibody production. We also inserted a 4CL clones from poplar into an E.coli expression system and demonstrated that permeabilized cells containing poplar 4CL are able to produce caffeoyl-CoA. Although the yield of caffeoyl-CoA is lower than we had hoped, it will be more than adequate for use in the assay of tartrate O-hydroxycinnamoyltransferase from grape. We isolated cDNA clones for grape dihydroflavonol reductase and a glucosyl transferase. The glucosyl transferase may be involved in glycosylation of phenolic compounds in grape, such as glycosylation of anthocyanidins to give the anthocyanins found in the hypodermal cells of the berry skin. The substrate specificity of the glucosyl transferase has not been determined, thus its exact role in phenolic biosynthesis has not been confirmed. The dihydroflavonol reductase is important because the product of the reaction (flavan-3,4-diols )can be directed into several different classes of phenolics in grape berries such as catechin, epicatechin, tannins and anthocyanins. Like the 4CL enzyme described above, the dihydroflavonon reductase occurs at an important branch point in phenolic metabolism in grape.