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Distinguishing Carmenère from Similar Cultivars by DNA Typing
Abstract
A total of 93 vines from five vineyards in Chile that were originally planted as Merlot, four vines from a cultivar collection in Chile, and two vines in California were analyzed with SSR DNA markers to confirm their identity. DNA profiles were compared to those of previously confirmed reference vines. Vines in the Chilean vineyards matched the DNA profiles of either Merlot or Carmenère, consistent with prior visual identification of these vines. The four vines from the cultivar collection matched Carmenère, although they were originally planted as Merlot. Both California vines were confirmed as Carmenère, although one was originally imported as Cabernet franc. Two markers, VVMD28 and VVMD31, are particularly useful for distinguishing Carmenère from Merlot. VVMD31 will also distinguish Cabernet franc from the other two cultivars, as will VVMD27. Although these three cultivars can be distinguished visually, DNA typing is a valuable adjunct for verifying identity, particularly for vines in nurseries and foundation plantings.
... Chile has become a major player in the world concert of the wine industry; it is eighth in the cultivated area, sixth in wine production, and currently fourth in wine exportation [38]. As in other countries, Chilean viticulture is an industry based on a small number of internationally recognized varieties, such as Cabernet Sauvignon, Merlot, Chardonnay, and Sauvignon blanc, plus Carmenère, a rediscovered French variety that has been instituted as the Chilean wine fagship for a couple of decades [39,40]. At the same time, the world is gradually being more receptive to new favors and aromas, for which new or underexploited genotypes would be required. ...
... Any mistake in the identity of a genotype will be maintained for years unless experienced ampelographers can correct it, as happened with Carmenère, indexed as Merlot for many years but properly identifed ca. 30 years ago [39]. Te SAG catalogue, currently including ca. 100 varieties, has several cases of duplicated synonyms, including local and international names, that have been in part rectifed with this and previous work [20,23,39] or are expected to be so in the near future, complementing the use of ampelographic descriptors and molecular markers; these cases include but are not limited to the four Huevo de Gallo synonymies (already mentioned); the tintorial varieties that include over 7,600 ha of "Tintoreras" (probably including Teinturier, VIVC 12304, which was identifed among Maule valley samples); Cargadora, a local synonymy for Cinsaut (VIVC 2672); Corinto, candidate to be the 262 nd synonym of Chasselas (VIVC 2473), or a local mutation such as Chasselas Musque (VIVC 2473); Gewürztraminer vs. Traminer (this latter with a small cultivated area registered); Albilla, a common homonym in many places that needs to be revised; Moscatel de Frontignan, which we demonstrated is a local name for Muscatà petit grains blancs; and "Muscat," a generic name that could correspond to diferent genotypes. ...
... 30 years ago [39]. Te SAG catalogue, currently including ca. 100 varieties, has several cases of duplicated synonyms, including local and international names, that have been in part rectifed with this and previous work [20,23,39] or are expected to be so in the near future, complementing the use of ampelographic descriptors and molecular markers; these cases include but are not limited to the four Huevo de Gallo synonymies (already mentioned); the tintorial varieties that include over 7,600 ha of "Tintoreras" (probably including Teinturier, VIVC 12304, which was identifed among Maule valley samples); Cargadora, a local synonymy for Cinsaut (VIVC 2672); Corinto, candidate to be the 262 nd synonym of Chasselas (VIVC 2473), or a local mutation such as Chasselas Musque (VIVC 2473); Gewürztraminer vs. Traminer (this latter with a small cultivated area registered); Albilla, a common homonym in many places that needs to be revised; Moscatel de Frontignan, which we demonstrated is a local name for Muscatà petit grains blancs; and "Muscat," a generic name that could correspond to diferent genotypes. Te other relevant aspect of the SAG catalogue is that it is restricted to varieties which are ofcially authorized for wine production; as the inclusion of a new variety into the catalogue requires an ad hoc application including a complete ampelographic, productive, and enological description, small winegrowers would only exceptionally be able to follow this procedure, and consequently they declare those "atypical" vines as if they were the most common ones present in the zone, being phenotypically similar. ...
Genetic erosion of grapevine is becoming a serious worldwide problem and preserving its germplasm is an urgent task. Chile is not an exception to this scenario, where diverse factors led by the replacement of traditional varieties by renown cultivars have reduced the vineyard diversity. Therefore, discovering and characterizing minor varieties were the main objectives of this study. In addition to the official catalogue of around 100 grapevine varieties used to produce wine, we present here 89 genotypes not previously described as present in Chile, widely dispersed in the main wine valleys, which were retrieved from vineyards, small collections, and city gardens. Strikingly, we found 49 grapevines with allele patterns not described before. Ten of these may be old European varieties; the other 39 have allele patterns similar to Criolla varieties (e.g., descendants from the crossing of European varieties born in America). A parentage analysis performed with 15 SSR markers in these 39 N.N. samples revealed that most of them are descendants of the so-called foundational varieties Listán Prieto and Muscat of Alexandria, plus others in lower proportion such as Mollar Cano and Muscat à petit grains blancs, as well as known Criollas varieties such as Italiona and Moscatel Rosado. The 89 genotypes not described in Chile structured in three distinguishable groups: Criolla varieties, Central European varieties, and a third group enriched in Iberic varieties. The prevalence of each Criolla accession was quite variable, some corresponding to a single or a few vines found at a single place, while others were spread in several vineyards, even in different valleys, suggesting they were positively selected and propagated at some time. All the new Criolla varieties were different than the recently described Criollas found in Argentina. The discovery, rescue, and documentation of this rich heritage of Vitis vinifera L. genotypes found in Chile is the first step of a long-term work that should be continued with the evaluation of their enological characteristics, yield, and optimal management systems, aiming for the diversification of the local wine industry.
... So, the morphological description of vine varieties could be used to prevent possible mistakes in plantations, nurseries or in vine growers, being later verified by the microsatellite technique. The combination of molecular markers and ampelographic descriptions irrefutably establishes varietal identity in a more thorough way and leads to more reliable and objective results (HINRICHSEN et al., 2001;SANTIAGO et al., 2005). ...
... On the opposite, Callet and Manto Negro varieties were grouped at a similarity level of 0.93 in the two years of the study, however, they belong to different varieties according to the microsatellite analysis. Similar cases have been described in literature and they point towards a parentage relationship between them (HINRICHSEN et al., 2001). This hypothesis has been probed, since due to their particular geographic location (both are only found in the Balearic Islands), these varieties are the result of natural cross sharing the same mother parent (GARCÍA-MUÑOZ et al., 2011). ...
Aims: We report the genetic, phenological, agronomic and ampelographic characterization of 27 minor grapevine accessions (Vitis vinifera L.) from the Balearic Islands (Spain). The influence of occasional climatic phenomena (hailstorm) and the ampelographer’s experience on these characteristics was studied.
Methods and results: In 2006 and 2007, grapevine accessions were analyzed using 57 OIV (Office International de la Vigne et du Vin) qualitative and quantitative descriptors and six microsatellite loci (SSR). Ampelography is a good preliminary technique for the identification of plant material, since the ampelographic description carried out showed a high degree of similarity between the repetitions of one single accession (higher than 90%), confirming the microsatellite results. The colour of the young leaf’s upper side (OIV-051), the juiciness of the flesh (OIV-232) and the firmness of berry flesh (OIV-235) were the most difficult characters to distinguish by ampelographers. In spite of the greater similarity found among the varieties studied, there were a few strong discriminant characters (OIV-225, OIV-084, OIV-053, and OIV-004). In addition, the ampelographic descriptions, agronomic parameters and phenology were influenced by hailstorm.
Conclusions: The combination of morphological and molecular characterizations of 27 accessions collected in the Balearic Islands (Spain) allowed their classification into 17 different vine varieties. The genetic analysis identified Beba blanca as a possible somatic mutant derived from Beba roja. The hailstorm increased the vegetative period and mostly affected mature leaves, bunches, agronomic characteristics and must composition.
Significance and impact of the study: The present work characterizes, for the first time, the ampelographic and molecular profiles of these minor varieties. It is provide agronomic information about old cultivars never published before which could be interesting for the grape growers.
... Morphological descriptions are useful to prevent possible mistakes in plantations but later need verifcation by molecular methods. Te combination of ampelographic descriptions and molecular markers indeed establishes varietal identity and leads to reliable and objective results [14,15]. Since 1993, diferent types of DNA-based markers have been illustrated for grape molecular characterization such as RFLPs [16,17]; and [18], RAPDs [19][20][21]; and [22], AFLPs [23,24], SSRs [13,[25][26][27][28], and SNPs [29][30][31][32][33]. Yet, the great revolution in grape molecular genetics did not take place until the advent of SSR markers and later SNPs markers that allowed the discrimination between wild and cultivated populations and the study of its genetic relationships [34]. ...
Background and Aims. Vitis vinifera L. is a major global horticulture crop which holds historical connections contributing to the development of human culture. The main objective of the study was to explore the genetic diversity and structure of grapes curated at the germplasm bank of Aragón and link them to cultivar utilization, putative geographic origin, and historical events. Methods and Results. The genetic diversity of the 411 accessions of the Grapevine Germplasm Bank was assessed using 26 SSR markers. SSR markers VVIP31, VMC4F3-1, VVIV67, and VVS2 distinguished the 156 nonredundant genotypes found in the collection. The profiles were compared to the El Encin database, and 105 were identified as known cultivars, of which 93 were Spanish, 12 were from other European origins, and 51 others were not known. The 51 profiles, as they were all collected in Spain, were designated as Spanish unknown for further analysis. To establish a comparative study for principal coordinate analysis (PCoA) and structure, we kept 113 identified genotypes from this collection but added 61 representative genotypes with diverse European and oriental origins. Bayesian analysis and PCoA showed four distinct groups of grape cultivars: (1) traditional Spanish wine grape from Aragón or cultivated along the Ebro River, (2) Spanish wine grape, (3) Spanish and oriental table grape, and (4) Italian, Portuguese, French, and German-Slovenian wine grape varieties. Conclusions. The 51 unknown Spanish genotypes were not registered yet in any European commercial list and are of special interest. These genotypes could be ancient cultivars adapted to local climatic and environmental conditions and probably resilient to face the new conditions derived from climate change.
... 137 In this way, in 2012, around 40 Chilean wineries (about 20% of the total) were making, marketing, and exporting their own brands of luxury Carmenère, defined as retailing at US$50 or more per 750 mL bottle according to the stated by Beverland. 129,130 The reported area of Carmenère has been increasing in recent years, 131,132 and according to the last viticultural report, the Carmenère surface accounted for 10 250 ha, making it the fifth most cultivated variety in Chile after Cabernet-Sauvignon, Chardonnay, Sauvignon Blanc, and Merlot. 83 Although, nowadays, this variety has an important place among Chilean wines, we have included it in this review because Carmenère was believed to be extinct and was rediscovered and revalorized by the Chilean wine industry. ...
In contrast with the general trend of producing wine from the most famous grapevine varieties, associated with the French paradigm, such as Cabernet‐Sauvignon, Merlot, Pinot Noir, Syrah, Sauvignon Blanc, and Chardonnay, there is a tendency to revalorize and preserve minority or autochthonous grapevine varieties worldwide. The South American wine region, where most of the varieties derived from varieties brought after European colonization, is not exempt from this. This has allowed new wines to be provided with distinctive identities that are markedly different from the current homogeneous wine production. Moreover, varietal homogenization increases vineyard genetic vulnerability in relation to the emergence of grapevine diseases, to which the commonly cultivated varieties are not resistant. This review summarizes the oenological potential of minority or autochthonous grapevine varieties cultivated within the South American wine region, focusing on Argentina, Chile, and Bolivia. © 2019 Society of Chemical Industry
... Carmenere were collected from the Central Valley of Chile. Plant identity and purity were determined by ampelographic and PCR-based molecular markers includ-ing microsatellite DNA markers (simple sequence repeats) (Hinrichsen et al. 2001). The clone stocks were planted directly in the soil in the COPEVAL S.A. Research Station located in San Fernando (Colchagua Valley, VI region, Chile) at 1.0 m x 2.0 m spacing and trained in unilateral Guyot. ...
Vitis vinifera cv. Carmenere, a member of the Cabernet family, was recently rediscovered in Chile where it had been misidentified as Merlot. The Carmenere variety produces complex wines, marked with herbaceous and "green" aromas. Results indicate that methoxypyrazines are present in high concentrations in this variety, probably responsible for the strong vegetative character found in Carmenere wines. Wines from Carmenere grapes had high 3-isobutyl-2-methoxypyrazine (IBMP) concentrations (5.0 to 44.4 ng/L), much higher than most Cabernet Sauvignon wines. The genotype dramatically affected the methoxypyrazine content of Carmenere wines, with some clones containing three-fold more than others. Climatic conditions, rather than maturity or terroir, had a critical effect upon IBMP concentration in grapes. 3-Isopropyl-2-methoxypyrazine evolved differently during grape maturation and was less influenced by harvesting year. Copyright © 2007 by the American Society for Enology and Viticulture. All rights reserved.
... One of the main challenges for authentication of foods and beverages is the need for accurate reference materials and standards that can be used to ensure proof-of-identity. The difficulties in developing these authentic standards are exemplified in the Chilean wine industry where many vineyard plantings of Merlot grapevines actually turned out to be plantings of Carménère (23,24). Because of similarities in the visual appearance of these two varieties, the misidentifications were not recognized until the late 1990's when methods were developed for differentiating different grape cultivars using specific DNA markers. ...
Authentication of foods, wines and beverages for varietal, country-(or region-) of-origin, and processing conditions is becoming of increasing concern to consumers and regulators both in the U.S. and internationally. As markets become more globalized and foods and beverages are sourced from many locations outside of the U.S., the need for information on product authenticity is becoming mandated either by legal regulations or by market demand. However, there are significant chemical and analytical challenges faced in confirming food authenticity and determining food adulteration. Here we highlight some recent cases that emphasize the need for improved analytical information that will limit economic fraud and improve food safety.
... SSRs represent codominant, hypervariable loci that are suitable for discriminating among closely related taxa. They have become the DNA marker of preference for a number of grapevine genetic studies , Bowers and Meredith 1997, Sefc et al. 2000, Hinrichsen et al. 2001, Martín et al. 2002, Agüero et al. 2003, González Techera et al. 2004, Martínez et al. 2006a, 2006b. ...
Bonarda is the second most cultivated red variety in Argentina after Malbec. Morphologically, the Argentine-Bonarda shows strong similarities with three grape varieties from northern Italy, collectively called Bonarda, and the French variety Corbeau. Twelve Argentine-Bonarda accessions, three Bonarda-type cultivars from Italy (Bonarda Piemontese, Croatina, and Uva Rara), and the French variety Corbeau were analyzed at eight microsatellite loci. Overall, the eight SSRs revealed only four distinct genotypes, corresponding to Bonarda Piemontese, Croatina, Uva Rara, and Corbeau. All the Argentine-Bonarda accessions were identical in their SSR profiles to the French Corbeau, indicating, with a high confidence level, that they are the same variety.
... Chloroplast DNA [97,98] Wine Cultivar identification STR Microsatellite DNA [90,[99][100][101][102][103][104][105][106][107][108][109][110][111][112][113][114] Wine Cultivar identification SNP Nuclear genome [115,116] Smoked paprika Cultivar identification RAPD, SSR, and ISSR ITS1-5.8S rDNA-ITS2 [117] Soft fruit Species identification Real-time PCR 5S rRNA and anthocyanidin synthase [118] Durum wheat Cultivar identification STR Microsatellite DNA [119] Potato Cultivar identification STR Microsatellite DNA [120] Sweet cherry Cultivar identification SSR-HRM Microsatellite DNA [121] Tomato, carrot, and celery Species identification Real-time PCR Pru1 and rbcL (RUBISCO) [122] PART II Analytical & Chemometric Methods for Food Protected Designation differentiation of populations can also occur without geographical isolation, such as in the case of domestic breeds that often coexist in the same area but remain isolated by human-controlled breeding. ...
Genetic methods are becoming increasingly relevant for addressing food authentication problems and include a wide range of techniques and equipment. DNA is present in all biological tissues and can potentially be recovered from any matrix containing even a very small number of cells or cell debris. Important characteristics that make DNA particularly suitable for the analysis of food products are its high information content and resistance to degradation. The current knowledge of partial or complete DNA sequences in a large number of species already allows for optimization of DNA-based methods with high sensitivity and specificity for the genetic target under analysis. The challenges presented by food products with PDO (protected designation of origin) are diverse and may include the identification of individuals, species, breeds, cultivar, or variety in animals, plants, fungi, and microorganisms. Most of the currently used genetic methods rely on polymerase chain reactions for the survey of informative DNA present in food matrices. Methods that can be applied in automated and high-throughput settings for large-scale screening purposes are particularly important. This review is intended to provide an overview of the DNA-based strategies and analytical methods available for the analysis of food products in the context of PDO authentication.
... Cabernet Franc may also be a parent of Carmenère, which is considered to be a very old winegrape from the Bordeaux area and is almost absent now in France with only 15 ha in 2006 (Institut Français de la Vigne et du Vin et al. 2007 ). This cultivar was the subject of recent attention in Chile after it was determined that Carmenère had been confused with Merlot (Hinrichsen et al. 2001). The results presented here confirm both the proximity and the distinction between Merlot and Carmenère, which share one common parent. ...
Background and Aims: Based on parentage analysis of a large nuclear simple sequence repeat (SSR) marker database of grapevine genotypes, we propose the pedigree of several cultivars from southwestern France including Merlot, one of the world's major black winegrapes.
Methods and Results: The putative mother of Merlot, deduced from inheritance at 55 nuclear and three chloroplast microsatellite loci, is a non-referenced and previously unknown cultivar, first sampled some years ago in northern Brittany where vines were cultivated at the end of the Middle Ages, and then identified in four places in Charentes. Considering both the name used by the growers of this grape and the literature, we have named it Magdeleine Noire des Charentes. The putative father of Merlot is Cabernet Franc, already involved in the parentage of Cabernet-Sauvignon. Further analysis of genetic relationships leads us to propose the kinship group of Merlot composed, among others, of Carmenère (Gros Cabernet × Cabernet Franc), Merlot Blanc (Merlot × Folle Blanche), Cot (Magdeleine Noire des Charentes × Prunelard) and Mourtès (Magdeleine Noire des Charentes × Penouille).
Conclusions: These results shed new light on the origin of Merlot and on the relationships among several cultivars from southwestern France.
Significance of the Study: Our discovery of the key genetic role of a previously unknown cultivar in the origins of some significant cultivars reinforces the importance of deep exploration, before it is too late, to discover original genotypes which have not yet been collected or referenced.
... Suckers arising from the rootstocks have resulted in identity questions for some of the Parlier trees, so we compared these trees to the parent trees in Corvallis. In recent years, microsatellite or simple sequence repeat (SSR) markers have been isolated for many fruit and nut crops and some have been used for identity verification as illustrated in grape (Vitis vinifera L.) (Hinrichsen et al., 2001). In hazelnut, a number of SSRs were recently developed ( Bassil et al., 2005a, b;Boccacci et al., 2005; Gurcan and Mehlenbacher, unpublished) and some have been assigned to linkage groups ( Mehlenbacher et al., 2005). ...
The US Department of Agriculture (USDA), Agricultural Research Service maintains a genebank representing world hazelnut (Corylus L.) diversity. More than 670 clones are preserved as self-rooted trees in a two-hectare field planting in Corvallis, Oregon, with a single tree per accession. In 1996 and 1997, prior to the spread of eastern filbert blight caused by Anisogramma anomala to within 75 kilometers of Corvallis, a backup collection was established in Parlier, California. A core collection of 184 genotypes representing the wide taxonomic, geographic and phenotypic diversity of Corylus was targeted for this second planting. Two trees of each ‘core’ genotype were grafted onto seedling rootstocks over a period of five years and an orchard was established in Parlier. The grafted trees in Parlier are at risk of identity problems due to suckers arising from below the graft union. In May 2007, young leaves were collected from 29 Parlier trees that exhibited uncharacteristic morphological phenotypes. A set of 12 simple sequence repeat (SSR) markers was used to fingerprint trees in the backup collection, and the results were compared to the fingerprints of the same accessions in the Corvallis collection. Based on the results, misidentified accessions will be eliminated and the fingerprinting set will be refined further.
Chile is part of one of the centers of crop origin identified by Vavilov, e.g., for strawberries and potatoes. It is also a center of diversification of other crop species such as maize, beans and quinoa. It is one of the biodiversity hotspots of the world and several native species have potential for domestication. All of these types of species are considered Plant Genetic Resources for Food and Agriculture (PGRFA). However, the rich plant genetic diversity present in Chile is being lost, mostly due to human activity. Therefore, ex situ and in situ conservation of this diversity are of critical importance. In this review we show the achievements in PGRFA conservation activities in the last 15 yr and in plant breeding for the last 60 yr in this country. Several gene banks exist, administrated by different institutions, with over 48 000 accessions preserved, mostly cereals (65%) and grain legumes (23%). Significant advances were achieved between 2006 and 2020 in the conservation, regeneration, characterization and documentation of PGRFA, but work is still needed to complete a fully operable data base for all collections. Over 16 000 accessions of Chilean origin are also kept in gene banks abroad. Plant breeding programs of several agriculturally important crops have made an outstanding contribution to Chilean agriculture and food security, with more than 375 commercial cultivars developed. More effort needs to be made to strengthen ex situ conservation and the sustainable use of PGRFA under coordinated actions, guided by a national strategy on genetic resources, if significant contributions are to be made in response to climate change.
In this genome report, we describe the sequencing and annotation of the genome of the wine grape Carménère (clone 02, VCR-702). Long considered extinct, this old French wine grape variety is now cultivated mostly in Chile where it was imported in the 1850s just before the European phylloxera epidemic. Genomic DNA was sequenced using Single Molecule Real Time technology and assembled with FALCON-Unzip, a diploid-aware assembly pipeline. To optimize the contiguity and completeness of the assembly, we tested about a thousand combinations of assembly parameters, sequencing coverage, error correction and repeat masking methods. The final scaffolds provide a complete and phased representation of the diploid genome of this wine grape. Comparison of the two haplotypes revealed numerous heterozygous variants, including loss-of-function ones, some of which in genes associated with polyphenol biosynthesis. Comparisons with other publicly available grape genomes and transcriptomes showed the impact of structural variation on gene content differences between Carménère and other wine grape cultivars. Among the putative cultivar-specific genes, we identified genes potentially involved in aroma production and stress responses. The genome assembly of Carménère expands the representation of the genomic variability in grapes and will enable studies that aim to understand its distinctive organoleptic and agronomical features and assess its still elusive extant genetic variability. A genome browser for Carménère, its annotation, and an associated blast tool are available at http://cantulab.github.io/data.
The identity of the Carménère variety was lost in Chile and regarded as a Merlot grape. This grape disappeared in Bordeaux, France, the country of origin of the grape, because of the phylloxera plague. In the present paper, a study on the classification of Chilean Carménère and Merlot wine samples based on chemical parameters was carried out. A total of 64 samples were analysed, and 20 chemical parameters were determined. Forty‐five samples were labelled as “Carménère” and 19 samples as “Merlot” according to the wine label. The samples were preprocessed with sampling algorithms to double the number of Merlot samples to 38 and reduce the Carménère samples to 38. The dataset was analysed with the data mining techniques support vector machines and correlation‐based feature selection. The capability of classifying the samples with all 20 chemical parameters was 86.8% accurate, and when using only the variables a*, total anthocyanins, cyan‐3‐glu, malv‐3‐acetylglu, peon‐3‐acetylglu, and vitisin A, which were selected through correlation‐based feature selection, accuracy increased to 93.4%. Therefore, wine anthocyanins and the parameter a* of wine colour proved useful to discriminate the wines.
'Carmenère' is a fine red wine cultivar (Vitis vinifera L.) that has spread, unrecorded from France to other countries. It probably arrived in Chile before the Phylloxera crisis in Europe where it remained confused with Merlot and other red wine cultivars until the mid 1990s. In this study, genetic diversity among 26 accessions from Chile, France and Italy was analysed using microsatellite (SSR) and AFLP markers. Using 20 SSR markers, a "standard genotype" was established and three different haplotypes were found, presumably arising by a mutation at the WMD7 and VMC5g7 loci. In the case of AFLP, using 11 primer combinations five groups were identified, with one main cluster of 22 accessions not differentiated. Combining both techniques it was possible to identify five out of the 26 accessions analysed. Together, these results suggest that 'Carmenère' exhibits a lower genetic diversity in comparison with other French red wine cultivars. This is a factor to consider when managing a clonal selection assay. Possible causes are discussed.
Numerous chiral compounds occur in foods and beverages. The fact that enantiomeric composition can be used to distinguish biological processes from chemical processes has been recognized for over a century. However, analysis of enantiomeric purity or enantiomeric ratios of specific compounds remained challenging, requiring large samples, derivatization of functional groups, or separations based on physical process such as crystallization. Development of cyclodextrin-based GC stationary phases in the 1970-80's provided the ability to directly separate a large number of underivatized chiral compounds present at low concentrations. Now, chiral analysis is frequently used for authentication of foods and beverages with respect to source ("natural" /synthetic), geographic origin, processing/aging treatments, and formation mechanisms (chemical/ enzymatic). This paper will use selected examples to demonstrate the application of enantiomeric analysis for food and beverage authentication.
Fungal pathogens are major problems for grape growers among which the grey mold, caused by Botrytis cinerea, is the most severe and difficult to control in Chile and other viticultural regions of the world. Botrytis can cause economic loss both pre- and post-harvest and even low levels of infection can negatively impact the value of table grapes. A genetic transformation approach was initiated to improve resistance to fungal pathogens three years ago, based on a combination of Trichoderma harzianum anti-fungal genes and anti-microbial peptide genes introduced into selected table grape cultivars including 'Thompson Seedless'. Transformation of embryogenic cultures was initially optimized using GFP and then combinations of antifungal genes were introduced using Agrobacterium-mediated transformation. More than 140 PCR-tested plants are under evaluation. Preliminary evaluations based on inoculation of leaves with Botrytis indicate a significant reduction in lesion size for some of the transgenic lines. After additional characterization of the transgenic lines we plan to evaluate the resistance of selected lines in field trials under isolation conditions.
The cultivar 'Carmenère' is originated from the Médoc in France. Its presence was dramatically reduced after the phylloxera crisis during the second half of the 19th century. During the last decade no more than 10 ha were registered in France. In contrast, 'Carmenère' was re-discovered in 1994 in Chile, where it was confused with 'Merlot'. In the present study we compared the polymorphic level of Carmenère and Cabernet Sauvignon clones by using microsatellite and AFLP markers. Fifty-six 'Carmenère' clones from clonal evaluation plots in Chile as well as nine clones from France and one from Italy were analyzed. Only two out of 21 microsatellite markers assayed were found polymorphic. VVMD7 locus exhibited two allelic forms, grouping 67% and 33% of the clones, respectively. Instead, VMC5g7 locus showed only one mutated allele in one clone. AFLP analysis of the Chilean Carmenère clones showed 2.3% polymorphism. In contrast, an analysis of 75 'Cabernet Sauvignon' clones with 91 microsatellite markers found 17 polymorphic loci, and an analysis of 46 of these clones with AFLP found 5.9% clonal polymorphism. These results suggest that the genetic diversity in 'Carmenère' clonal germplasm is low, in comparison to the more diverse germplasm of Cabernet Sauvignon clones.
Site evaluation and selection to optimize wine quality in Europe has been a long-term goal of vine scientists seeking to adapt to new production strategies. Denominazione di Origine Controllata (DOC) in viticulture is based on natural environmental conditions and on 'terroir'. Organoleptic characteristics typical of Cabernet Franc were researched because in the past the variety was often confused with Carmenère. The present trial was carried out in order to study its typical characteristics. During the period, 1997-2001, eight different clones of Cabernet Franc were tested in two viticultural areas of Northeastern Italy (Friuli Venezia Giulia and Trentino Alto Adige). The eight clones examined were from French selections (210, 212, 214, 312, 327, 331, 394, and 623). The following quantitative and qualitative parameters were collected annually at harvest: number of buds, number of grapes, bud fertility, yield/vine, sugar content (°Brix), titratable acidity, pH. During winter, pruning wood was measured. Data collected were standardized for removing the "year effect" and were submitted to Analysis of Variance (ANOVA) using the Student-Newman-Keules SNK method for mean separation. They were also analyzed with Wricke's eco-valence using program S116 (Statistical Reasearch Program Librarian Agriculture - Canada). This allowed definition of clonal properties and underlined the factor that different microclimatic conditions are very important in determining grape quality. The two viticultural regions showed great variability in vine behavior and vine balance. In particular, Friuli Venezia Giulia showed vines with less vigor, lower yield and less sugar accumulation than those of Trentino Alto Adige. Among clonal selections under observation, clone 623 demonstrated the highest yield and cluster weight and adequate sugar content. The Wricke test showed that French selections were highly stable depending on the 'terroir'.
Chilean wine exports increased from 52 to 835 millions US-dollars between 1990 and 2004. They mainly consist of the red varieties Cabernet Sauvignon, Merlot and Carmenère. For sustainable long term growth, it is essential to develop a reliable system to verify product authenticity. For varietal authenticity in red wine, various authors proposed the use of the ratio between acetylated and coumaroylated anthocyanins (Rac/cum). In the present paper, the anthocyanin, flavonol, and shikimic acid profiles were determined in wines produced in Chile during the vintages 2001-2004, in order to differentiate between wine varieties. The results of principal component analysis of anthocyanins show that it is possible to differentiate between the three grape varieties using only their anthocyanin profiles. This is confirmed by linear discriminant analysis of these compounds, which classified well a total of 95.8% of the investigated wines. Only by the Rac/cum ratio, it is not possible to differentiate between Merlot and Carmenère, however, this ratio tends to discriminate between Cabernet Sauvignon and the other two varieties. The concentration of shikimic acid improves the differentiation between Cabernet Sauvignon and Merlot or Carmenère, but not between the latter two varieties. With respect to flavonols, our results show that the ratio of total quercetin/ total myricetin (TQ/TM) in combination with peonidin-3-glucoside in a simple ratio or with all anthocyanins determined in a model improves the differentiation between Carmenère and Merlot. The high error of classification in Merlot is in part attributed to the fact that in Chilean vineyards this variety was previously confused with Carmenère.
The asociation of a grape variety with a growing area is something considered desirable and usual in viticulture. Several examples of this kind exist in the world, however that had not been posible in Chile until 1994 when Carménère was rediscovered. It was the "Pontificia Universidad Católica de Chile" that proposed Carménère as chilean's emblematic variety. This paper describes how Carménère was discovered, a variety that according to literature was at the origin of the fame of the wines of Medoc in France, before philoxera in the XIX century made it disapear from french viticulture. An ampelographic description of Carménère is done and a discusion of its cultivation and agronomic requirements, the sensitivity to pests and diseases and its technological potential
In the world, table grape production represents 27% of the 750.000 hectares planted with this species. Global production reaches over 66 millions tons. In spite of the fact that table grape production in North and South America represents only 18% of the total world production, America accounts for almost 50% of the global exports. Main exporters are Chile and Italy, followed by the USA, South Africa, and Mexico. Of the thousands of existing cultivars, only about 20 are the most important for fresh consumption, with 'Sultanina' ('Sultani', 'Sultana', 'Kishmish' or 'Thompson Seedless') representing about 40% of the grapes grown for fresh consumption. This cultivar has also been used extensively as a parent for the development of new cultivars, such as 'Flame Seedless' and 'Crimson Seedless'. These varieties, together with 'Red Globe' (a very important seeded cultivar because of its excellent post-harvest life, productivity and public acceptance) are some of the most cultivated worldwide. Most breeding programs were initially publicly funded, but now-a-days many of them are privately owned. Also, most new cultivars are now protected by Intellectual Property Rights and, therefore growers need to pay royalties for their use or may no have access to some of the cultivars that are commercialized in close "clubs". Because of this new scenario, many countries and companies started breeding programs, and Chile was no exception.
Wine differentiation is an important issue for the Chilean winemaking industry, especially for the Carménère variety, which was rediscovered in this country around 20 years ago. Authentication parameters are required for this wine due to its frequent confusion with Merlot. The concentration of anthocyanins, shikimic acid, and the principal flavonols found in wine allowed some varietal differentiation between Carménère and Merlot wines. Myricetin and quercetin are the most concentrated flavonols in wine in which they are present in free and conjugated forms. These compounds are responsible for important wine antioxidant properties. In the present work, using only the concentrations of free and conjugated quercetin and myricetin, differentiation between Carménère and Merlot varieties was better achieved. Flavonol profiles of wine produced in Chile were studied with HPLC-DAD-ESI-MS n . An overview of the concentration range of flavonols present in 248 Chilean red wines is presented, finding that the mean concentration of the sum of total myricetin and total quercetin were higher in Carménère (81.5 mgL -1) and Merlot (78.9 mgL -1) than in Cabernet Sauvignon (53.9 mgL -1) wines. These mean levels were higher than the majority of the concentrations reported in the literature. The chemometric analysis shows that the ratio of total quercetin/total myricetin combined with the concentration of free myricetin allowed the varietal differentiation between Carménère and Merlot wines.
The process through which food or beverages is verified as complying with its label description is called food authentication. We propose to treat the authentication process as a classification problem. We consider multivariate observations and propose a multivariate Bayesian classifier that extends results from the univariate linear mixed model to the multivariate case. The model allows for correlation between wine samples from the same valley. We apply the proposed model to concentration measurements of nine chemical compounds named anthocyanins in 399 samples of Chilean red wines of the varieties Merlot, Carmenere and Cabernet Sauvignon, vintages 2001-2004. We find satisfactory results, with a misclassification error rate based on a leave-one-out cross-validation approach of about 4%. The multivariate extension can be generally applied to authentication of food and beverages, where it is common to have several dependent measurements per sample unit, and it would not be appropriate to treat these as independent univariate versions of a common model.
Food and beverage authentication is the process by which foods or beverages
are verified as complying with its label description, for example, verifying if
the denomination of origin of an olive oil bottle is correct or if the variety
of a certain bottle of wine matches its label description. The common way to
deal with an authentication process is to measure a number of attributes on
samples of food and then use these as input for a classification problem. Our
motivation stems from data consisting of measurements of nine chemical
compounds denominated Anthocyanins, obtained from samples of Chilean red wines
of grape varieties Cabernet Sauvignon, Merlot and Carm\'{e}n\`{e}re. We
consider a model-based approach to authentication through a semiparametric
multivariate hierarchical linear mixed model for the mean responses, and
covariance matrices that are specific to the classification categories.
Specifically, we propose a model of the ANOVA-DDP type, which takes advantage
of the fact that the available covariates are discrete in nature. The results
suggest that the model performs well compared to other parametric alternatives.
This is also corroborated by application to simulated data.
The presence of local vines in the Tuscia area (Latium region, Italy) was documented by historical sources. Ampelographic and molecular characterization (35 morphological descriptors and 16 microsatellite loci) was carried out on accessions belonging to the most frequently mentioned local vines. SSR markers have enabled us to distinguish cases of local synonymy and microsatellite profiles have permitted comparison with previously reported profiles indicating synonymy and divergence with vines in other collections. Ampelographic traits showed that some accessions of the same genotype differ in minor characteristics that can have a role in clonal evaluation.
La industria chilena del vino se ha modificado substancialmente en las últimas décadas, predominando la plantación de los denominados "cultivares finos" sobre los tradicionales, como la cepa País. Por otra parte, la globalización de los mercados ha resaltado la necesidad de certificar la identidad genética y pureza de los cultivares, que hasta hace poco tiempo se realizó exclusivamente mediante ampelografía. El análisis directo del ADN ha permitido el desarrollo de nuevas y poderosas herramientas analíticas, como las secuencias de mini- y microsatélites, actualmente usadas como marcadores moleculares para diferentes fines. En este trabajo se presenta la caracterización genética de los cultivares de vides de vinificación más comúnmente usados en Chile. Para este propósito, se ha usado un conjunto de 12 marcadores de microsatélites para caracterizar 20 cultivares de vino tinto y blanco, generando patrones alélicos únicos para cada uno de ellos. La heterocigocidad esperada (He) para cada marcador fue bastante alta y diversa, entre 0,27 y 0,87 (promedio 0,70), y fue tan diversa como el número de alelos detectados con cada marcador, desde sólo cuatro para VVMD-25, VVMD-34 y VVS-29, hasta 10 para VVMD-28. El número de genotipos identificado con cada marcador fue moderado en algunos casos, cuatro en VVMD-34, y hasta 15 con VVMD-28. La combinación de un pequeño número de estos marcadores (por ejemplo, VVMD-5 más VVMD-28) fue suficiente para obtener una completa diferenciación de los cultivares. Considerando estos antecedentes, se propone una combinación de marcadores para ser usada en certificación genética de cepas viníferas.
A formal compositional study of the proanthocyanidins of Vitis vinifera L. cv. Carménère was conducted in this work. We first characterized the polymeric proanthocyanidins of Carménère skins, seeds, and wines. In addition, the wine astringency was analyzed and compared with Cabernet Sauvignon. Although Carménère wines had a higher proanthocyanidin concentration and mean degree of polymerization than Cabernet Sauvignon wines, the former wines were perceived as less astringent. The low seed/skin proportion in Carménère wines as compared to other varieties, as evidenced by the reduced number of seeds per berry and the higher amount of epigallocatechin subunits of Carménère wine proanthocyanidins, could explain this apparent paradox.
A quick, simple, and reliable method for the extraction of DNA from grapevine species, hybrids, andAmpelopsis brevipedunculata (Vitaceae) has been developed. This method, based on that of Doyle and Doyle (1990), is a CTBA-based extraction procedure
modified by the use of NaCl to remove polysaccharides and PVP to eliminate polyphenols during DNA purification. The method
has also been used successfully for extraction of total DNA from other fruit species such as apple (Malus domestica), apricot (Prunus armeniaca), cherry (Prunus avium), peach (Prunus persica), plum (Prunus domestica), and raspberry (Rubus idaeus). DNA yield from this procedure is high (up to 1 mg/g of leaf tissue). DNA is completely digestible with restriction endonucleases
and amplifiable in the polymerase chain reaction (PCR), indicating freedom from common contaminating compounds.
Four new simple sequence repeat (SSR) loci (designated VVMD5, VVMD6, VVMD7, and VVMD8) were characterized in grape and analyzed by silver staining in 77 cultivars of Vitis vinifera. Amplification products ranged in size from 141 to 263 base pairs (bp). The number of alleles observed per locus ranged from 5 to 11 and the number of diploid genotypes per locus ranged from 13 to 27. At each locus at least 75% of the cultivars were heterozygous. Alleles differing in length by only 1 bp could be distinguished by silver staining, and size estimates were within 1 or 2 bp, depending on the locus, of those obtained by fluorescence detection at previously reported loci. Allele frequencies were generally similar in wine grapes and table grapes, with some exceptions. Some alleles were found only in one of the two groups of cultivars. All 77 cultivars were distinguished by the four loci with the exception of four wine grapes considered to be somatic variants of the same cultivar, 'Pinot noir', 'Pinot gris', 'Pinot blanc', and 'Meunier'; two table grapes that are known to be synonymous, 'Keshmesh' and 'Thompson Seedless'; and three table grapes, 'Dattier', 'Rhazaki Arhanon', and 'Markandi', the first two of which have been suggested to be synonymous. Although the high polymorphism at grape SSR loci suggests that very few loci would theoretically be needed to separate all cultivars, the economic and legal significance of grape variety identification requires the increased resolution that can be provided by a larger number of loci. The ease with which SSR markers and data can be shared internationally should encourage their broad use, which will in turn increase the power of these markers for both identification and genetic analysis of grape. Key words : grape, Vitis, microsatellite, simple sequence repeat, DNA typing, identification.
A Vitis riparia genomic library was screened for the presence of (GA), simple sequence repeats (SSR) and 18 primer pairs yielding amplification products of the expected size were designed. Heterologous amplification with the primer pairs in related species (V. rupestris, V. berlandieri, V. labrusca, V. cinerea, V. aestivalis, V. vinifera, and interspecific hybrids) was successful in most primer-species combinations. Therefore, the new markers are applicable to the genotyping of a range of Vitis species. Variations in the SSR flanking sequence were detected between and within the species. The degree of polymorphism and performance of the markers were determined in up to 120 individuals of V. vinifera. Four of fifteen alleles per locus were detected and expected heterozygosity ranged between 0.37 and 0.88. Null alleles were shown to be present at two loci by a lack of heterozygous individuals and by transmission of the null alleles in a controlled cross. Regular Mendelian inheritance is indicated for all but one loci by a preliminary segregation analysis in 36 offspring. Thirteen of the markers were found suitable for the genotyping of grapevines (V. vinifera).
Microsatellite repeat sequences were investigated as sequenced-tagged site (STS) DNA markers to determine the potential for genetic analysis of the grapevine genome. The PCR-generated markers detect codominant alleles at a single locus or site in the genome. The marker type is very informative detecting high heterozygosity (69%-88%) within individual grapevine cultivars and high genetic variation between cultivars, making it a useful marker type for plant genome mapping and genome typing. For five loci a screening of 26 V. vinifera cultivars found 13, 12, 8, 5, and 4 different length alleles respectively with some alleles more common than others. The genomic DNA sequences surrounding microsatellite sequences were conserved within the genus permitting STS primers to amplify STSs from other Vitis species. These Vitis species were found to have some unique alleles not present in V. vinifera.