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.
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.
We continued to add markers to a genetic linkage map of Vitis vinifera. During the past year we added primarily microsatellite (SSR) markers. We have now mapped 216 markers (94 AFLP and 122 SSR) and an additional 91 markers have been analyzed but the linkage analysis is not yet complete. The mapped markers form 19 linkage groups, the number expected in grape. Several fruit and cluster characteristics have also been analyzed and they map to three of the 19 linkage groups. New SSR markers are being developed within the Vitis Microsatellite Consortium, 20 cooperating research groups in 11 countries. The genetic map being created will be a resource that can be used by many viticulture researchers. It will facilitate the isolation of individual grape genes so that we can learn how the genes work and how their functions are influenced by external factors, such as vineyard cultural practices.
Many of the crucial branch points in phenolic biosynthesis occur at the level of the coenzyme A (CoA) esters of the hydroxycinnamic acids, and formation of the CoA esters is a critical step in the biosynthesis of virtually all classes of phenolics in grape. Research on hydroxycinnamate CoA ligases in grape has been very limited. Purification of the enzyme has not been accomplished, and research has been hampered because cDNA probes are not available to study expression of the genes. In the first year of this project we were able to extract and stabilize cinnamoyl CoA ligase (4CL) from grape tissues. We adapted a procedure for enzyme extraction and assay that was described for 4CL from aspen (Populus tremuloidies) so that it can be used with grape tissues and we studied the substrate specificity of the enzyme from leaf and green shoot tissue. In leaves we found that the enzyme showed much more activity with caffeate than with 4-coumaric acid. Activity was also observed with ferulate and sinapate in these extracts. This result is notable because the enzyme from other plants typically shows more activity with 4-coumaric acid than with caffeate. The total enzyme activity extracted from developing shoots was much lower than from leaves and showed substrate specificity more typical of enzymes from other sources, preferring 4-coumaric acid over caffeic acid. Also, with shoot extracts no activity was observed when sinapic acid was used as substrate. These results are significant because they might suggest that there are different isoenzymes present with different substrate specificities in different tissues. Thus, extraction and assay of the enzyme has been successful and we have found that best source of the enzyme from grapevine appears to be developing leaf tissue. We obtained two cDNA clones of 4CL from poplar from a laboratory in British Colombia. These cDNAs were labeled with 32P and used to screen a grape cDNA phage library prepared from mRNA obtained from grape berries at the beginning of ripening. We isolated six strongly positive plaques. The DNA from the positive phage were amplified and have been sent for DNA sequencing. We will know very soon whether or not we have obtained clones of 4CL from grape berries. This would be important because we could then use the grape 4CL clones to study expression of the respective genes in grapevine.
We continued to investigate possible ways to distinguish clones within important winegrape varieties. We tested a method called ISSR and were able to detect some differences in Chardonnay and Pinot noir clones but they were not sufficiently reproducible to be useful. We also tried a selection of new SSR markers developed within the Vitis Microsatellite Consortium and found that 8 of 12 markers we tried could detect some differences between clones of Chardonnay or Pinot noir or both. We obtained and analyzed 150 accessions of Plavac Mali from Croatia for comparison with Zinfandel and found that Plavac Mali is not Zinfandel, contrary to some opinions. Surprisingly, we also found that Plavac Mali is not a polyclonal variety as we had presumed and that the accessions were almost completely uniform in their DNA profile.
The goal of this project is to develop a basic genome map for grape (Vitis vinifera) that will allow us to begin to locate the genes that control important viticultural and enological characteristics, such as disease resistance and fruit composition. This will not only allow us to ultimately move these genes from one variety to another, whether by traditional or biotechnological means, but it will also facilitate the study of how these genes work and how they are affected by environmental and cultural conditions. The development of a genome map requires a population of progeny individuals derived from a cross between two disparate parents. We have used Cabernet Sauvignon and Riesling, two quite different wine grape cultivars, and have a population of 116 seedling vines derived from this cross that is now 4 years old. We have now obtained 178 DNA “markers” that we are placing in positions along the different grape chromosomes. The DNA markers are like signposts along a road. We have located some of these markers on specific chromosomes but others are not yet assigned to a chromosome. Cluster structure (e.g., compactness) is among the many characteristics that are under genetic control. Tight clusters are prone to rot. Loose clusters tend to have smaller berries, which are often preferable for winemaking. It is likely that many genes are involved in determining cluster structure. Some may determine the number of flowers that form on a cluster; others may determine the maximum berry size; and others may determine the length of the pedicel or the branching pattern of the rachis. We are trying to sort out these various components of cluster structure, to determine how many genes control them and to find the genome location for these genes. We are collecting data on 9 berry and cluster characteristics from all of our seedling vines in our mapping population but, because the vines are still quite young, we have only 1 year of data and will need several more before we can begin to interpret this data. Most of the DNA markers that we have been using for our genome map are of the type called AFLP markers. It is relatively easy to generate large numbers of these markers, but they have some limitations and information gained with these markers cannot always be shared with other researchers who are working with different mapping populations. Microsatellite markers, on the other hand, are more powerful and can be used on any mapping population. Unfortunately, these markers are much harder to come by and their discovery and development is very laborious. In order to obtain a large number of microsatellite markers, we have formed an international Vitis Microsatellite Consortium in which researchers in several countries will share in the effort to develop new grape microsatellite markers and will then share in the benefits. After about 10 months of correspondence, organization and the negotiation of a written agreement, the consortium is now underway and up to 20 grape research groups in 10 countries are expected to ultimately join in the effort.
The limitations of petiole nitrate-N as a criteria for vine N status are widely recognized. The purpose of this study is to search for improved N tissue sampling and analytical methods which can be used for many wine cultivars under different growing conditions. Total-N and nitrate-N levels are being compared in leaf petiole and blade samples taken at bloom, veraison and harvest in 7 cultivars – French Colombard, Chenin blanc, Ruby Cabernet, Barbera, Grenache, Chardonnay, and Cabernet Sauvignon. All of the trial blocks are located at the UC Kearney Agricultural Center except for Chardonnay and Cabernet Sauvignon which are on the Central Coast. A wide range of N fertilizer treatment is being imposed in order to establish large differences in vine N status and potential plant response. Fertilizer treatment was initiated one year ahead of the beginning of data collection to provide carry-over N in the vines. Significant differences in N determinations for each tissue and sampling stage from N fertilizer treatment were found in 5 cultivars at Kearney, with the exception of bloom blade total-N. This tissue and stage was not significantly different for total-N in 4 cultivars ? Barbera, Grenache, French Colombard and Chenin blanc. Thus far, bloom blades have shown the least promise as an indicator of differences in N status. There was a tendency for the veraison and harvest samples to show greater significant differences in N status as compared to bloom sampling. As expected, nitrate-N showed the greatest range in values from the low to the high N treatments. However, total-N showed as much statistical separation as nitrate-N by the Duncan=s Multiple Range Test. This suggests that there are good possibilities in developing useful critical values for total-N, as well as the traditional nitrate-N. Also, petioles tend to show as good, if not better, statistical separation for total-N as compared with blades. This is encouraging, as it would be very useful to be able to use petioles rather than blades because of value of petiole samples for other nutrient determinations. Some vine yield and fruit composition components showed significant differences due to fertilizer treatment. This should provide the opportunity to correlate vine response with leaf tissue N values. Correlation and regression analyses will be performed after 2 full years of vine and laboratory data. The goal is to develop some tentative critical values for total-N and/or nitrate-N for important wine cultivars. The one year of data from the Chardonnay and Cabernet Sauvignon trials show minor or no differences. Thus, they are too preliminary for any conclusions at this time. Two more years of data collection will be necessary to develop treatment differences. This is due to the typically delayed and carry over effect of N treatment as demonstrated in the completed trials. The Barbera, Grenache, French Colombard, and Chenin blanc trials are now complete. Ruby Cabernet will be studied for one more year to complete 2 full years of data collection.
Biology and Genetics of Rootstock Resistance to Grape Phylloxera
DNA marker development is complete. Nineteen markers that were developed in this project, along with six developed in Australia, will provide more than enough for variety identification. These markers have now been characterized and the number of forms in which they exist has been determined. The theoretical maximum number of different DNA profiles that can be distinguished with these markers is over 1039, or more than a trillion times a trillion times a trillion. A set of just the six most informative markers can theoretically produce more than 1 trillion different profiles. The cultivar database, essential both to provide references for identification and also to provide the critical statistical foundation on which estimates of the probability of a particular DNA profile are based, was expanded from 47 to 72 cultivars, all of which have been typed with at least 19 markers. Vines from eight different Petite Sirah vineyards were analyzed and found to be a mixture of Durif, Peloursin and several other varieties, confirming that the name Petite Sirah is used for more than one variety in California. Efforts to promote the use of our grape DNA markers in other countries have been very successful. They are now being used in nine other countries and we hope to be able to exchange valuable information with researchers who have access to highly reliable European variety collections. The modified AFLP approach by which we tried to differentiate Chardonnay and Pinot Noir clones revealed some differences but they were neither sufficiently numerous or reproducible to be of use.
DNA marker development is now complete. Of the 29 markers we have developed, 15 are sharp and informative enough to be used for cultivar identification. In addition, we can use several markers developed in Australia. This is more than enough to identify any grape cultivar. Our database of cultivar profiles now contains DNA types of 77 cultivars for 8 DNA markers. The database is essential not only as a reference for identification but also to provide the statistical foundation necessary to validate this method. The database must now be expanded to include all of the 15 best markers and a larger number of cultivars. Since the group of 77 cultivars that we have so far analyzed contains a disproportionately high number of French wine grapes and Greek table grapes, additional cultivars will have to be added to make it more representative and thus more accurate. We had originally hypothesized that SSR DNA markers might be able to distinguish clones. After surveying 15 Chardonnay clones, 15 Pinot Noir clones and single-vine samples from a century-old Zinfandel vineyard, we have detected only one DNA difference in one Chardonnay clone. This leads us to think that SSR DNA differences among clones are more rare than we originally hypothesized. Our results suggest that SSR DNA markers could distinguish clones, but that a much larger number of markers would be required than we now have. Such a large number of markers would be very expensive to develop (but not to use) and thus might not be practicable. We are now investigating another DNA-based approach, AFLP analysis, that has promise for identifying clones.