Evaluation of New Pinot Noir and Chardonnay Clones for Sparkling Wine

Only the 13 Chardonnay clones were harvested, as the Pinot noir vines were too variable to include in 1998. Chardonnay clones were harvested over a 7-day period from September 7 through September 14. Juice yield (adjusted for the differing amounts of fruit) ranged from 135 gallons per ton (Clone 132) to 173 gallons (Clone 4 and Wente). Fruit yields in the difficult fruit-set year of 1998 were similar among clones, varying only from about 5.1 to 6.4 kg/vine, except for Wente which yielded only 3 kg/vine. With the exception of cluster number, Wente was the most radically different in other yield components, having the lightest cluster (75 g), 40 to 50%lighter than other clones. This resulted from having somewhat fewer berries per cluster (89) and 40%smaller berries (84 g per 100 berries). Among the remaining clones, cluster weights generally fell into two groups, one at about 150 g (4, 75, 96, 121) and another at about 130 g (76, 78, 118, 134, 130, 131, 132). Triplicate fermentations of each Chardonnay clone were made at UC Davis and will be further evaluated in 1999.

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

The aim of this project is to develop a means by which genes can be introduced into existing grape varieties. The genes being used at this early stage are model genes that are easy to detect in the laboratory but ultimately genes will be introduced that are intended to confer new characteristics without otherwise altering the distinctive characteristics of the variety. Genes conferring resistance to specific diseases and pests are of particular interest. During 1998-99, six transformation experiments were initiated and monitored for 6 to 9 months each. The experiments involved Chardonnay, Thompson Seedless, St. George and 11 OR and the experimental factors tested in the experiments included the culture medium, the gene vector and the selection conditions. Two of the experiments have yielded both somatic embryos and a few plantlets that developed after co-cultivation with the gene vector and exposure to selective pressure designed to allow only cells carrying the introduced genes to survive. Some of the embryos exhibit the blue color that indicates expression of the introduced GUS marker gene. However, none of the three plantlets that were tested expressed GUS. Several additional transformation experiments are in progress and it is still too early to evaluate them. During April and May 1999, approximately 8,000 immature grape flowers were dissected in order to culture their anthers to produce new embryogenic cultures. We initiate new cultures each spring so as to always have a supply of relatively young cultures for transformation experiments. Older cultures may accumulate genetic errors and can lose their ability to regenerate plants. Several research groups in other countries and in private companies are already well along in this kind of research, but public research in California has lagged behind because funding was not available for such long-term research. Now that financial support for viticulture research has increased, public research in this field can be undertaken at the University of California. It may lead to better pest and disease management and ultimately to better control of fruit composition.

Identification and Characterization of Genes that Control the Phenolic

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.

Identifying Varieties and Clones by DNA Typing

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.

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

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.

Identifying Varieties and Clones by DNA Typing

In 1997 we conceived and founded the Vitis Micro satellite Consortium, a group of 18 cooperating grape research groups in 10 countries that are sharing the costs and labor to develop 100 to 200 new grape SSR DNA markers. Although we already have enough DNA markers for routine grape variety identification, these additional markers will be used for genome mapping and may also be of value for distinguishing closely related varieties and possibly for identifying clones. We were not able to initiate a proposed new approach to clone discrimination until late in the 1997-98 period because of insufficient funding but will actively pursue this avenue during 1998-99. Our cultivar database has continued to grow and now contains 111 cultivars that have been typed with 13 to 18 DNA markers. This geographically balanced database not only provides us with references against which to compare unknowns, but is also the basis for estimating the statistical probability that a DNA-based identification is correct. We have also developed a database of 300 French cultivars that we will keep separate for statistical purposes so as not to bias the original database geographically. This database will be useful for verifying the identity of new French vine importations and for identifying unknowns in California vineyards (see Carmenere below). In order to continue to investigate the hypothesis that Zinfandel is the Croatian variety Plavac Mali, we obtained three accessions of this variety from the Croatian island of Brae. As we had earlier found with two Plavac Mali accessions growing in our UC Davis collection, none of these three is identical to Zinfandel. They are, however, very closely related to Zinfandel. We now know that the variety Plavac Mali is not genetically uniform and contains several related sub-types. (We have detected three.) We think that we have not yet seen all the Plavac Mali sub-types and that analysis of additional Plavac Mali samples is warranted. If Zinfandel is found in Croatia, then we may have a source of needed clonal diversity for California Zinfandel. If Zinfandel is not found, then any attempts to market Croatian Plavac Mali wine as Zinfandel can be countered with strong scientific evidence. In collaboration with a visiting Chilean researcher, Dr. Patricio Hinrichsen, we investigated the identity of some vines in Merlot vineyards in Chile that have been identified ampelographically as the old Bordeaux variety Carmenere, as well as an FPMS vine originally labeled as Cabernet Franc but also suspected to be Carmenere. All were confirmed as true Carmenere by comparison to an authentic DNA reference obtained from Montpellier France (see French database above).

Improved Tissue Analysis Methods for Nitrogen Assessment of Wine Grape

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

Biology and Genetics of Rootstock Resistance to Grape Phylloxera

Evaluation of Merlot and Malbec Clones

Five Merlot clones were compared for a second year. In 1996, clone 8 was the lowest yielding clone and clone 3 was the highest. Pruning wts. were the converse, with 3 being lowest and 8 being highest. Shoot numbers did not differ, indicating that average shoot wt was the critical factor. Clone 8 also had the lowest cluster wt and fewest berries per cluster. Cluster numbers did not differ in 1995 but in 1996 clone 8 had the lowest number while others did not differ. Clones 8 and 9 were higher in Brix than the other three clones, although TA did not differ. K+ levels tended to mirror pH values with higher K+ being associated with higher pH. With respect to wines, no significant differences were seen with regard to anthocyanins. Wine TA was lower for clone 8. Wine pH differences were similar to those for juice values. VA was well below threshold sensory values. Ethanol values were small, as predicted given the small Brix differences seen at harvest. Significant differences were seen in all co-pigmentation values including the amount of anthocyanin in copigment form, amount of free anthocyanin and amount of polymeric anthocyanin, with clone 8 being highest in copigment and free while being lowest in polymeric form. Malbec clones were not harvested in 1996. The rains and cool weather during bloom, as well as severe pruning designed to correct some training errors, resulted in disastrous fruitset. Only about 400 lbs of fruit were harvested from the entire 1.4 acres. Therefore, the data were meaningless. Initial observations in 1997 seem to mirror those in 1995 with clone 8 have the appearance of more and heavier clusters than that of clone 4 or 6.

Identifying Varieties and Clones by DNA Typing

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.