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

AVF Proj. ID: 9902 / /Cat.: ,
Primary Investigator(s): Carole P. Meredithby

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.

Clonal Testing of Wine grapes in the San Joaquin Valley

AVF Proj. ID: 9910 / /Cat.: ,
Primary Investigator(s): Peter Christensenby

This is an ongoing study to evaluate promising, virus disease-free FPMS clonal material for the San Joaquin Valley. Completion dates for past trials were: French Colombard and Chenin blanc (1994), Barbera (1995), and Muscat of Alexandria (1997), Grenache (1998), and Muscat blanc (1998). The Sangiovese trial was completed in 1999 with crop level comparisons. Data collection began with the Chardonnay, Cabernet Sauvignon, Merlot, and Zinfandel/Primitivo trials. These trials were planted with 6 clones each in 1997 in order to evaluate clonal differences in a warm climate region. Sangiovese. Clones 2 and 3 are recommended for warm climate sites, while Clone 4 is not recommended. Clone 4 was consistently of poorer fruit composition (lower T A, higher pH and the most bunch rot), even with an imposed lower crop level; most often it was of lowest yield during the 5-year study period. The differences between Clones 2 and 3 were fairly clear in this study, each having certain advantages and disadvantages. Clone 2 was more fruitful and often ripened later than Clone 3 (-1.3 °Brix at harvest over 4 years). This later ripening can be corrected with crop level adjustment, as demonstrated in the 1999 comparative crop level treatments. Clone 2 had the smallest berries, even with crop level adjustment; Clone 3 berry weights were always heaviest, sometimes with fewer berries per cluster. Thus, mean cluster weights were similar between Clones 2 and 3. Overall, it would appear that Clone 2 is preferable due to its smaller berry size. However, its higher fruitfulness may require more attention to crop level management to assure timely ripening of quality fruit. Cabernet Sauvignon. Berry weight, number of berries per cluster and cluster weight differences significantly influenced clone yield which, in turn, affected fruit soluble solids at harvest. Clones 8 and 22 were highest yielding, while Clones 2 and 24 were lowest; Clones 10 and 21 were intermediate. Clones 2 and 24 had the smallest berries and fewest berries/cluster. Merlot. Vine yields and cluster weights were similar among clones, and fruit composition and berry weight differences were minor. Zinfandel/Primitivo. The Zinfandel clones were all similar except for the smaller berries of Zinfandel 2; Zinfandel 3 had a lower bunch rot incidence as compared to Zinfandel 1 A. The Pnmitivo clones did not differ from one another. However, when compared to Zinfandel the Primitivo clones had smaller berries, fewer berries/cluster, and clusters of lower mass and earlier ripening and less rot (~3x less). Yields of Primitivo tended to be lower than those of Zinfandel. 54 Project Title: Clonal Testing of Wine grapes in the San Joaquin Valley (Continued) Chardonnav. Clone 4 was highest yielding, while Clones 6 and 37 were lowest, mainly the result of number of berries/cluster and cluster weight. Berry weight was highest in Clone 20 and lowest in Clone 4. Clone 4 had lower fruit soluble solids but higher TA and pH at harvest. The 1999 results of the ‘fighting varietals” – Cabernet Sauvignon, Merlot, Zinfandel and Chardonnay are preliminary, as they represent first crop data. However, it is interesting to note that significant clonal differences are demonstrated early in the study when crop levels are adjusted uniformly across clones for each cultivar. Thus, the differences reported are mostly the result of berry weight, berries/cluster and the final cluster weights.

Developing a Functional Genomics Approach to Berry Ripening and Defense

AVF Proj. ID: 9914 / /Cat.: ,
Primary Investigator(s): Douglas O. Adamsby

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.

Evaluation of Winegrape Clones

AVF Proj. ID: 9923 / /Cat.: ,
Primary Investigator(s): James Wolpertby

Pinot noir and Chardonnay. For Chardonnay, yields in 1999 were consistent with the 2-yr averages, in that Wente was the lowest at just over 3 kg/vine while 352 was among the highest at 5.2 kg/vine. Cluster number, berries per cluster and berry wt all played a role in yield differences. Cluster number varied from a low of 30 (clone 4) to a high of almost 44 (clone 352), berries per cluster from a low of 76 (clone 78) to a high of 107 (clone 4) and berry wt from a low of 0.94 (Wente selection) to a high of 1.59 (clone 4). Harvest in 1999 took place over a 9 day period from September 18 to 27. For Pinot noir, yield ranged from a low of 3.83 kg/vine (clone 13) to a high of 8.18 kg (clone 668). Cluster number varied from a low of 31 (clone 743) to a high of 54 (clone 2A), berries per cluster from a low of 74 (clone 13) to a high of 144 (clone 743) and berry wt from a low of 1.26 (clone 872) to a high of 1.57 (clone 13). Harvest occurred over a 17-day period from September 7 through September 24. Merlot and Malbec. Again in 1999, the major difference among Merlot clones was in the yield component of berries per cluster for clone 8, resulting in a significant decrease of more than 3 tons per acre. This trend corresponds precisely with the 5-year average. Despite the lower Brix of clone 8, it had the lowest Brix and highest pH both in 1999 and as a five-year average. The 1999 vintage was another difficult year for Malbec with yields on average being less than 4 tons/acre. Individually, significant differences could be seen among yields of the clones, with clone 8 showing 6.7 tons/acre versus 2.3 and 1.9 tons for clones 4 and 6, respectively. All yield components were involved including clusters per shoot, berry wt and berries per cluster. In 1999 the 11 OR rootstock showed a significantly greater yield than Malbec on 5C due to more clusters per shoot and heavier berries. There were few differences in fruit chemistry with the exception of significantly more potassium in Malbec 8 and all vines on 5C. Malbec values for the 4-year period show the dramatic yield differences among the years.

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

AVF Proj. ID: 9927 / /Cat.: ,
Primary Investigator(s): James Wolpertby

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

AVF Proj. ID: 9930 / /Cat.: ,
Primary Investigator(s): Douglas O. Adamsby

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.

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

AVF Proj. ID: 9802 / /Cat.: ,
Primary Investigator(s): Carole Meredithby

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.

Alternative Trellising Systems for Chardonnay and Merlot Vineyards in the Central Coast

AVF Proj. ID: 9805 / /Cat.: ,
Primary Investigator(s): Larry Bettigaby

The purpose of this study is to compare the performance of bilateral cordon trained, spur pruned Chardonnay and Merlot grapevines trellised to the vertically shoot positioned trellis system (VSP), the Smart-Henry trellis system (SH) and the Smart-Dyson trellis system (SD) in the Salinas Valley of California. In the first year of the study, VSP vines had less primary, lateral and total leaf area compared to SD and SH vines. Canopies oriented upward on both the SD and SH systems generally had more leaf area than canopies oriented downward. VSP vines had greater leaf layer numbers in the fruiting zone compared to SD and SH vines, and these treatments had greater amounts of sunlight in the fruit zone compared to the VSP treatment. Vine yield components were similar among the treatments in Chardonnay, while SH vines in Merlot produced significantly greater yields compared to the other treatments. In both cultivars, trends toward reduced yields for downward-oriented canopies compared to upward-oriented canopies were observed. Merlot fruit from downward-oriented shoots had lower berry weights, reduced rates of sugar accumulation and higher titratable acidity compared to fruit from upward-oriented shoots. Fruit on downward-oriented shoots on the SH system exhibited this trend more strongly than downward-oriented shoots on the SD system. At harvest, however, no significant difference in combined fruit (upper and lower canopies) soluble solids, titratable acidity and pH were observed among the treatments in either cultivar. Wine lots made from each treatment will be evaluated in late 1999.

Clonal Testing of Wine Grapes in the San Joaquin Valley

AVF Proj. ID: 9813 / /Cat.: ,
Primary Investigator(s): Peter Christensenby

This is an ongoing study to evaluate promising, virus disease-free FPMS clonal material for the San Joaquin Valley. Completion dates for past trials are: French Colombard and Chenin blanc (1994), Barbera (1995), and Muscat of Alexandria (1997). Trial blocks of 3 clones each of Grenache, Sangiovese, and Muscat blanc were established in 1993; data collection was initiated at harvest, 1995. Significant clonal differences have been measured over 3 or 4 years in these cultivars as follows: Muscat blanc. The Milan introductions, clones 3 and 4 have been distinctly better than clone 1, an older introduction into California. Clone 1 had fewer and heavier clusters with larger berries, less fruit yield, and more bunch rot. Overall, Clone 4 would be preferable because of its consistent high fruitfulness (3 years) of smaller clusters (1997 only) with less rot and earlier fruit maturation (compared to Clone 3), Clone 4 should be promoted, while Clone 1 should be discouraged, for all new plantings. Grenache. These were all California selections. The trial included a genetic comparison [Clone 1A (Gallo vineyard) vs. 3 (Jackson vineyard), both non-heat-treated] and a heat treatment comparison (1A vs 2, clone 2 being a 62-day heat treatment of Clone 1A). Genetic differences have been more apparent than heat treatment differences, with clone 3 producing the highest yield, lowest fruit maturation and most bunch rot. Clones 1 and 2 (heat treatment comparisons) were more similar, except for Clone 2’s heavier berries of slightly lower pH and higher TA and more bunch rot in one year (19988). Overall, Clone 1A would appear to be preferable due to its higher cluster numbers with smaller berry size and the least rot potential. For yield alone, Clone 3 would be preferable, but with some delayed fruit maturation and greater susceptibility to bunch rot due to heavier berries and clusters. Sangiovese. This cultivar demonstrated distinct clonal differences, with Clone 4 showing the least promise due to poor fruit composition (low TA and high pH), high bunch rot incidence, and low total anthocyanin content of experimental wine. Clone 2 may be preferred over Clone 3 due to its smaller berries and higher vine fruitfulness and yield. However, crop load management would be more important in Clone 2 as its fruit maturation was delayed as compared to less fruitful Clone 3. Further work is planned to determine if crop load adjustment of Clone 2 favors its fruit composition while retaining smaller berry size as compared to Clone 3. Experimental wine lots made from the clones in 1997 demonstrated minor differences in wine making or quality characteristics. Clone 4’s musts and wines were of lowest acidity and highest pH which were reflective of its harvest berry sample analyses. All wine samples were fairly low in color intensity. The Muscat blanc and Grenache trials are now complete. One more year of study with Sangiovese is anticipated to study crop load comparisons. This will determine under what conditions Clones 2 or 3 should be recommended for the best fruit composition with optimum yields. Chardonnay, Cabernet Sauvignon, Merlot, and Zinfandel/Primitivo trials were planted in 1997 at the request of the AVF Research Committee. Six clones of each cultivar will be studied for comparison under warm climate conditions.

Evaluation of Merlot and Malbec Clones

AVF Proj. ID: 9825 / /Cat.: ,
Primary Investigator(s): James Wolpertby

Data collected in 1998 in Merlot showed similarities to previous years. Clone 08 had the lowest yield (5.3 kg/vine) as a result of fewer berries per cluster and smaller berries. It also had the greatest pruning wt (2.4 kg/vine) as a result of heavier, not more, shoots. Clones 01, 03 and 06 were very similar in yield (about 7 kg/vine) but in growth FPMS 1 had the least at 1.98 kg. With the lower crop and greater pruning wt, clone 08 had a much lower yield:prunings ratio (2.17) versus the other clones (2.9 to 3.7). These values are dramatically different from 1997 when with higher yields and lower growth ratios were in the 5 to 8 range. Wines were made in cooperation with Duckhorn Vineyards. All wines will be initially evaluated informally at the winery. Three clones of Malbec, FPMS 04, 06 and 08, were compared on two rootstocks, 5C and 110R. Malbec had a the disastrous fruitset in 1998 similar to what it had in 1996. Yields in 1998 were 1 to 5 kg/vine. Under normal conditions, clone 08 shows a tendency to over-crop but with the poor set in 1998 it was not thinned. At these extremely low yields, pruning wts ranged from a low of 2.8 kg/vine for 08 to about 3.5 kg for clones 04 and 06. Vines on 11 OR yielded significantly more than 5C (3.4 vs 2.2 kg/vine, respectively), primarily due to more clusters per shoot and more berries per cluster. Even with the better crop load, 110R had a half degree higher Brix than 5C. Only clone 08, especially on 110R rootstock, had economically sustainable yields in 1998 with an average across rootstocks of 4.0 tons per acre versus 1.3 for 04 and 0.8 for 06.