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1Instituto de Investigaciones Agropecuarias, La Platina, Casilla 439/3, Santiago, Chile;2Department of Viticulture and Enology, University of California, Davis, California, USA; 3UnitéMixte de Recherches 1097, Diversité et Génomes des Plantes Cultivées, Ecole NationaleSupérieure Agronomique, 2 Place Viala, 34060 Montpellier, France; 4Present address: PlantGenome Mapping Laboratory, University of Georgia, Athens, GA 30602, USA.

*Corresponding author: [Email: cpmeredith@ucdavis.edu; Tel. (530) 752-7535; Fax (530) 752-0382]

Acknowledgments: This work was supported by the American Vineyard Foundation, CaliforniaFruit Tree, Nut Tree, and Grapevine Improvement Advisory Board, Fondo Nacional de InvestigaciónCientífica y Tecnológica de Chile, and Fondo de Desarrollo e Innovación de CORFO, Chile.

Manuscript submitted October 2000; revised May 2001

Copyright © 2001 by the American Society for Enology and Viticulture. All rights reserved.

396

The grapes grown in the wine regions of the New World arealmost exclusively the classic European cultivars. When thesecultivars were originally introduced from Europe in the eigh-teenth and nineteenth centuries, there was much less interest inthe identity of specific cultivars than exists today. At that time,European wines were identified by the regions in which theywere made, not by the grape cultivars that went into them. Fur-thermore, introductions of grapevines to New World countriesoften consisted of large batches containing many cultivars. La-bels were undoubtedly occasionally confused or lost. It is notsurprising that European grapes have at times been misidentifiedin some New World countries. In California, for example, Melonwas called Pinot blanc for many years until the error was foundand corrected. Similarly, Semillon in Australia was once con-sidered a type of Riesling, as was Crouchen in South Africa.

Cultivar identity has since become more important. Most NewWorld wines are varietally labeled and a growing number areexported. International trade regulations mandate authenticityin labeling, making it crucial that wines, and the grapes fromwhich they are made, be correctly identified.

Distinguishing Carmenère from Similar Cultivars byDNA Typing

P. Hinrichsen,1 C. Narváez,1 J.E. Bowers,2,4 J.M. Boursiquot,3

J. Valenzuela,1 C. Muñoz,1 and C.P. Meredith2*

A total of 93 vines from five vineyards in Chile that were originally planted as Merlot, four vines from a cultivarcollection 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 vineyardsmatched the DNA profiles of either Merlot or Carmenère, consistent with prior visual identification of thesevines. The four vines from the cultivar collection matched Carmenère, although they were originally plantedas Merlot. Both California vines were confirmed as Carmenère, although one was originally imported asCabernet franc. Two markers, VVMD28 and VVMD31, are particularly useful for distinguishing Carmenèrefrom 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 verifyingidentity, particularly for vines in nurseries and foundation plantings.

Key words: Grape, Vitis, simple-sequence-repeat, SSR, microsatellite, DNA-polymorphism, DNA-typing,cultivar identification, variety identification, Carmenère, Cabernet franc, Merlot, Chile

Chile has become an important exporter of varietally labeledwines, among the most important of which has been Merlot. In1994, some Merlot vines in Chile were discovered to be the oldBordeaux cultivar Carmenère [1,8]. Carmenère is now knownto be widely distributed in Chilean Merlot plantings and mayeven constitute the majority of vines in these vineyards. The re-ported area planted to Carmenère in Chile doubled between 1998and 1999 to 2,300 ha [Servicio Agricola y Ganadero, 2000], re-flecting not only the correction to Carmenère of vineyards pre-viously reported as Merlot but also new plantings of Carmenèrenow available from nurseries.

Carmenère was once widely grown in Bordeaux but is almostnonexistent there today. Although its wine was considered tobe very good, it was abandoned in Bordeaux during the nine-teenth century because of its poor fruit set [12]. In Chile the cul-tivar performs well [2]. In addition to being confused withCarmenère, Merlot has at times been confused with Cabernetfranc in Italy and California [2,6].

In this report, we confirm by DNA profiling the identifica-tion of Carmenère vines in Chilean vineyards that were origi-nally thought to be Merlot. We also report the existence ofCarmenère vines in cultivar collections in both Chile and Cali-fornia.

Materials and MethodsPlant material. We analyzed a total of 93 vines from five

commercial vineyards in Chile. Twenty were analyzed in 1997in Davis, California, and 73 were analyzed in Santiago, Chile in1998 and 1999 (Table 1). We also analyzed four vines from theLa Platina research station of the Instituto de InvestigacionesAgropecuarias (INIA) and two California vines, one that had

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been directly imported from France as Carmenère, growing in acommercial vineyard, and one that had been imported from Italyas Cabernet franc, growing in the Foundation Plant MaterialsService (FPMS) vineyard at the University of California, Davis.All sampled commercial vineyards in Chile except one wereoriginally planted as Merlot and were already suspected to con-tain mixtures of Carmenère and Merlot based on visual exami-nation. Vineyard E, in Valle del Maipo, was deliberately plantedto both cultivars.

Reference cultivars. Identification was by comparison withthe DNA profiles of previously authenticated vines at FPMS(Cabernet franc and Merlot) and the Institut National de la Re-cherche Agronomique (INRA) collection at Domaine de Vas-sal, Montpellier, France (Carmenère).

DNA isolation. Samples were obtained from young leaves.Some DNA preparations were performed by the method of Lodhiet al. [7], but most were as described in Bowers et al. [3], exceptthat the protocol was performed at approximately 1/20th scaleto reduce it to a volume suitable for microcentrifuge tubes. TheDNA concentration was measured by fluorimetry and adjustedto 2.5 ng/µL. Both protocols produced DNA that amplified well.

PCR amplification. Each reaction contained grape genomicDNA (10 ng in 4 µL), 1.0 mM MgCl2, 20 pmoles of each primer,0.5 unit AmpliTaq polymerase (Perkin-Elmer, Foster City, CA)and 2 µL 10x PCR Buffer II (Perkin-Elmer), 2.5 mM each dNTPand water to a volume of 20 µL. Reactions were covered with adrop of mineral oil. The only exceptions to these conditions werefor marker VrZAG47, in which case MgCl2 was adjusted to 2mM, in the presence of AmpliTaq Gold from Perkin-Elmer. The40 amplification cycles were performed with a Perkin-Elmer 480thermocycler, as follows: 92°C 1 min, 56°C 1 min, and 72°C 2min, except for VVMD6, VVMD7, VVS2, and VrZAG47, forwhich the annealing temperature was 52°C, and VVS29, forwhich the annealing temperature was 50°C. For all markers, afinal extension of 7 min at 72°C was employed.

Microsatellite markers. The markersused are presented in Table 2 and have beendescribed elsewhere [4,5,10,11]. After thiswork was completed, VrZAG47 was foundto be the same locus as VVMD27 [J.E.Bowers, unpublished results], so no sepa-rate results are reported for this marker. Allvines tested in Davis were analyzed withall 14 markers. Vines tested in Chile wereanalyzed with subsets of either 6 or 9 ofthese markers (see Table 1).

Preliminary screening of PCR reac-tions. Five µL from each reaction wasmixed with 10 µL of 2x loading buffer foragarose gels [9] and separated in a 2.5%agarose gel prepared in TAE 1x and con-taining EtBr (5 mg/mL). The gel (20 x 20cm) was run at 250 V for approximately 1hr, and the amplified DNA bands visual-ized over an UV transilluminator and pho-tographed with a Polaroid system, film type

667. When the amplified products were too faint, less templateDNA was used, which usually rendered a better result.

Identification of alleles with polyacrylamide sequencinggels. The reaction products were separated in sequencing gels[9] of 6% polyacrylamide, 8 M urea. The gels were 40 x 30 cmand 0.4 mm deep. The amplification products were analyzed induplex combinations such that the products from each primerdiffered in size by at least 30 base pairs. The duplexes wereVVMD5/VVMD6, VVMD7/VVMD27, VVMD24/VVMD25,VVMD31/VVMD32, VVMD34/VVS2, VVMD36/VVS29, andVVMD28/VrZAG47. The samples (15 µL remaining after theinitial screening) were mixed with 15 µL of sequencing loadingbuffer. Of this mixture, 5 µL from one reaction was combinedwith 5 µL of the other member of the duplex pair, heated to 80°Cfor 2 to 5 min, and loaded onto the gel. The gels were preheatedand then run for 2.5 to 3 hr at 1,800 to 2,000 V, until the xylene

Table 1 Sampled vines and their identity.

Vineyard Name when No. of vines Marker DNALocation code planted tested set useda identification

Chile, Valle de Casablanca A Merlot 10 III Merlot5 III Carmenère

B, Block 1 Merlot 5 III CarmenèreB, Block 2 Merlot 5 II Merlot

C Merlot 35 I Carmenère

D Merlot 7 II CarmenèreCarmenère 6 II Carmenère

Chile, Valle del Maipo E Carmenère 10 I CarmenèreMerlot 10 I Merlot

Chile, INIA La Platina Merlot 4 III Carmenère

California, Napa Valley Carmenère 1 III Carmenère

University of California FPMS Cabernet franc 1 III Carmenère

aSet I: 6 markers (VVMD5, VVMD6, VVMD7, VVMD24, VVMD25, VVMD31); Set II: 9 markers (SetI plus VVMD 27, VVMD28, VVMD32); Set III: 14 markers (Set II plus VVMD34, VVMD36, VVS2,VVS29, VrZAG47).

Table 2 Microsatellite markers used in this study.

Marker Reference

VVMD5 4VVMD6VVMD7

VVMD24 5VVMD25VVMD27VVMD28VVMD31VVMD32VVMD34VVMD36

VVS2 11

VVS29 *

VrZAG47 10

*M. Thomas, personal communication.

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cyanol dye reached the bottom. The DNA fragments were stainedwith the Silver Sequencing kit from Promega (Madison, WI).Allele size was determined by comparison to reference cultivarsrepresenting common alleles that were run in the same gel. Gelswere scanned and stored as digital images.

Results and DiscussionAllele sizes for the three reference cultivars are shown in

Table 3. The results from the commercial Merlot vineyards inChile were as expected. The vines suspected to be Carmenèredid in fact match the DNA profile of the reference Carmenèrevine (Table 1).

The four samples from the INIA collection at La Platina hadbeen planted as Merlot, but the DNA profiles of all four matchedthe Carmenère reference. The identity of these vines had notpreviously been investigated. The California vine that was im-ported directly from France as Carmenère was confirmed asCarmenère. The vine that originally came to FPMS from Italyas Cabernet franc but was suspected to be Carmenère was alsoconfirmed as Carmenère.

Eight of the markers used in this study will each clearly dis-tinguish Carmenère from Merlot (Table 3). Two of them,VVMD28 and VVMD31, are notable because Carmenère andMerlot do not share any alleles at these loci so the cultivars canbe separated by both alleles at either lo-cus. Although VVMD28 and VVMD31do have slightly overlapping allele sizeranges [5], the alleles of Carmenère andMerlot do not overlap; for the purpose ofseparating these two cultivars, thesemarkers could be combined on a singlegel. VVMD31 will, furthermore, separateCabernet franc from the other two culti-vars, as will VVMD27. Two markers notused in this study, VrZAG62 andVrZAG79 [10], will also each separate allthree cultivars. Although one or twomarkers are not sufficient to identify a

Table 3 Microsatellite genotypes of the three reference cultivars.

Marker Cabernet franc Carmenère Merlot

VVMD5 226 240 226 238 226 236

VVMD6 205 211 211 212 205 212VVMD7 239 263 239 263 239 247

VVMD24 210 210 210 214 210 214

VVMD25 243 259 243 259 243 253

VVMD27 181 189 175 189 189 191VVMD28 231 239 239 251 231 237

VVMD31 206 216 206 210 212 216

VVMD32 241 259 241 241 241 241

VVMD34 240 240 240 240 240 240VVMD36 254 254 254 272 254 254

VVS2 139 147 139 147 139 151

VVS29 175 181 175 181 175 181

Figure 1 Leaf shapes of Merlot, Carmenère, and Cabernet franc(scanned from real leaves).

cultivar, they do provide a practical means to screen individualvines in a mixed planting of Carmenère and Merlot.

The morphological similarity among these three cultivars(Figure 1) probably reflects a close genetic relationship, a no-tion that is supported by the considerable allele sharing at the13 loci analyzed in this study as well as 10 additional loci [J.E.Bowers, unpublished results]. Despite their similarity, these threecultivars can be distinguished visually (Table 4), as demonstratedby the preliminary correct visual identification of many of thesamples tested in this study. However, the morphological dif-ferences between these cultivars can be subtle and may be ob-scured by environmental conditions, disease status, or culturalpractices. Thus, DNA typing can be a valuable adjunct for veri-fying the identity of these cultivars, particularly in plantings thatprovide propagation material to growers, such as nurseries, foun-dation plantings, and collections.

Table 4 Morphological distinctions among the leaves of Merlot, Carmenère, and Cabernet franc.

Merlot Carmenère Cabernet franc

Overall leaf shape Cuneiforma Round Pentagonal

Petiolar sinus shape Open U, Lyre, Narrow lyre,never overlapping overlapping slightly overlapping

No. of lobes 5 to 7 5 3 to 5

Young leaves Green Red to orange Bronze

Other leaf features Usually darker Funnel-shaped Edges rolledgreen than the with edges rolled toward upperother two toward underside surface

aShield-shaped, like a square above a triangle.

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