396 — Hinrichsen et al.

Distinguishing Carmenère from Similar Cultivars by

DNA 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 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.

Key words: Grape, Vitis, simple-sequence-repeat, SSR, microsatellite, DNA-polymorphism, DNA-typing,

cultivar identification, variety identification, Carmenère, Cabernet franc, Merlot, Chile

The grapes grown in the wine regions of the New World are
almost exclusively the classic European cultivars. When these
cultivars were originally introduced from Europe in the eigh-
teenth and nineteenth centuries, there was much less interest in
the identity of specific cultivars than exists today. At that time,
European wines were identified by the regions in which they
were made, not by the grape cultivars that went into them. Fur-
thermore, introductions of grapevines to New World countries
often consisted of large batches containing many cultivars. La-
bels were undoubtedly occasionally confused or lost. It is not
surprising that European grapes have at times been misidentified
in some New World countries. In California, for example, Melon
was called Pinot blanc for many years until the error was found
and 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 New
World wines are varietally labeled and a growing number are
exported. International trade regulations mandate authenticity
in labeling, making it crucial that wines, and the grapes from
which they are made, be correctly identified.
1Instituto
de Investigaciones Agropecuarias, La Platina, Casilla 439/3, Santiago, Chile;
2Department of Viticulture and Enology, University of California, Davis, California, USA; 3Unité
Mixte de Recherches 1097, Diversité et Génomes des Plantes Cultivées, Ecole Nationale
Supérieure Agronomique, 2 Place Viala, 34060 Montpellier, France; 4Present address: Plant
Genome 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, California
Fruit Tree, Nut Tree, and Grapevine Improvement Advisory Board, Fondo Nacional de Investigación
Científica 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
Am. J. Enol. Vitic. 52:4 (2001)
Chile has become an important exporter of varietally labeled
wines, among the most important of which has been Merlot. In
1994, some Merlot vines in Chile were discovered to be the old
Bordeaux cultivar Carmenère [1,8]. Carmenère is now known
to be widely distributed in Chilean Merlot plantings and may
even constitute the majority of vines in these vineyards. The re-
ported area planted to Carmenère in Chile doubled between 1998
and 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 Carmenère
now available from nurseries.
Carmenère was once widely grown in Bordeaux but is almost
nonexistent there today. Although its wine was considered to
be 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 with
Carmenère, Merlot has at times been confused with Cabernet
franc 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 of
Carmenère vines in cultivar collections in both Chile and Cali-
fornia.
Materials and Methods
Plant material. We analyzed a total of 93 vines from five
commercial vineyards in Chile. Twenty were analyzed in 1997
in Davis, California, and 73 were analyzed in Santiago, Chile in
1998 and 1999 (Table 1). We also analyzed four vines from the
La Platina research station of the Instituto de Investigaciones
Agropecuarias (INIA) and two California vines, one that had
 Distinguishing Carmenère by DNA Typing — 397

Microsatellite markers. The markers

Table 1 Sampled vines and their identity.
used are presented in Table 2 and have been
Vineyard Name when No. of vines Marker DNA described elsewhere [4,5,10,11]. After this
Location code planted tested set useda identification
work was completed, VrZAG47 was found
Chile, Valle de Casablanca A Merlot 10 III Merlot to be the same locus as VVMD27 [J.E.
5 III Carmenère
Bowers, unpublished results], so no sepa-
B, Block 1 Merlot 5 III Carmenère rate results are reported for this marker. All
B, Block 2 Merlot 5 II Merlot
vines tested in Davis were analyzed with
C Merlot 35 I Carmenère all 14 markers. Vines tested in Chile were
D Merlot 7 II Carmenère analyzed with subsets of either 6 or 9 of
Carmenère 6 II Carmenère these markers (see Table 1).
Chile, Valle del Maipo E Carmenère 10 I Carmenère Preliminary screening of PCR reac-
Merlot 10 I Merlot tions. Five µL from each reaction was
Chile, INIA La Platina Merlot 4 III Carmenère mixed with 10 µL of 2x loading buffer for
California, Napa Valley Carmenère 1 III Carmenère agarose gels [9] and separated in a 2.5%
agarose gel prepared in TAE 1x and con-
University of California FPMS Cabernet franc 1 III Carmenère
taining EtBr (5 mg/mL). The gel (20 x 20
a
Set I: 6 markers (VVMD5, VVMD6, VVMD7, VVMD24, VVMD25, VVMD31); Set II: 9 markers (Set cm) was run at 250 V for approximately 1
I plus VVMD 27, VVMD28, VVMD32); Set III: 14 markers (Set II plus VVMD34, VVMD36, VVS2,
VVS29, VrZAG47). hr, and the amplified DNA bands visual-
ized over an UV transilluminator and pho-
tographed with a Polaroid system, film type
been directly imported from France as Carmenère, growing in a 667. When the amplified products were too faint, less template
commercial vineyard, and one that had been imported from Italy DNA was used, which usually rendered a better result.
as Cabernet franc, growing in the Foundation Plant Materials Identification of alleles with polyacrylamide sequencing
Service (FPMS) vineyard at the University of California, Davis. gels. The reaction products were separated in sequencing gels
All sampled commercial vineyards in Chile except one were [9] of 6% polyacrylamide, 8 M urea. The gels were 40 x 30 cm
originally planted as Merlot and were already suspected to con- and 0.4 mm deep. The amplification products were analyzed in
tain mixtures of Carmenère and Merlot based on visual exami- duplex combinations such that the products from each primer
nation. Vineyard E, in Valle del Maipo, was deliberately planted differed in size by at least 30 base pairs. The duplexes were
to both cultivars.
VVMD5/VVMD6, VVMD7/VVMD27, VVMD24/VVMD25,
Reference cultivars. Identification was by comparison with VVMD31/VVMD32, VVMD34/VVS2, VVMD36/VVS29, and
the DNA profiles of previously authenticated vines at FPMS VVMD28/VrZAG47. The samples (15 µL remaining after the
(Cabernet franc and Merlot) and the Institut National de la Re- initial screening) were mixed with 15 µL of sequencing loading
cherche Agronomique (INRA) collection at Domaine de Vas- buffer. Of this mixture, 5 µL from one reaction was combined
sal, Montpellier, France (Carmenère). with 5 µL of the other member of the duplex pair, heated to 80°C
DNA isolation. Samples were obtained from young leaves. for 2 to 5 min, and loaded onto the gel. The gels were preheated
Some DNA preparations were performed by the method of Lodhi and then run for 2.5 to 3 hr at 1,800 to 2,000 V, until the xylene
et al. [7], but most were as described in Bowers et al. [3], except
that the protocol was performed at approximately 1/20th scale
to reduce it to a volume suitable for microcentrifuge tubes. The
Table 2 Microsatellite markers used in this study.
DNA concentration was measured by fluorimetry and adjusted
to 2.5 ng/µL. Both protocols produced DNA that amplified well. Marker Reference

PCR amplification. Each reaction contained grape genomic VVMD5 4

VVMD6
DNA (10 ng in 4 µL), 1.0 mM MgCl2, 20 pmoles of each primer, VVMD7
0.5 unit AmpliTaq polymerase (Perkin-Elmer, Foster City, CA)
VVMD24 5
and 2 µL 10x PCR Buffer II (Perkin-Elmer), 2.5 mM each dNTP VVMD25
and water to a volume of 20 µL. Reactions were covered with a VVMD27
drop of mineral oil. The only exceptions to these conditions were VVMD28
for marker VrZAG47, in which case MgCl2 was adjusted to 2 VVMD31
VVMD32
mM, in the presence of AmpliTaq Gold from Perkin-Elmer. The VVMD34
40 amplification cycles were performed with a Perkin-Elmer 480 VVMD36
thermocycler, as follows: 92°C 1 min, 56°C 1 min, and 72°C 2 VVS2 11
min, except for VVMD6, VVMD7, VVS2, and VrZAG47, for
VVS29 *
which the annealing temperature was 52°C, and VVS29, for
which the annealing temperature was 50°C. For all markers, a VrZAG47 10
final extension of 7 min at 72°C was employed. *M. Thomas, personal communication.

Am. J. Enol. Vitic. 52:4 (2001)

398 — Hinrichsen et al.

Table 3 Microsatellite genotypes of the three reference cultivars.

Marker Cabernet franc Carmenère Merlot

VVMD5 226 240 226 238 226 236

VVMD6 205 211 211 212 205 212
VVMD7 239 263 239 263 239 247
VVMD24 210 210 210 214 210 214
VVMD25 243 259 243 259 243 253
VVMD27 181 189 175 189 189 191
VVMD28 231 239 239 251 231 237
VVMD31 206 216 206 210 212 216
VVMD32 241 259 241 241 241 241
VVMD34 240 240 240 240 240 240
VVMD36 254 254 254 272 254 254
VVS2 139 147 139 147 139 151
VVS29 175 181 175 181 175 181

cyanol dye reached the bottom. The DNA fragments were stained
with the Silver Sequencing kit from Promega (Madison, WI).
Allele size was determined by comparison to reference cultivars
representing common alleles that were run in the same gel. Gels
were scanned and stored as digital images.
Figure 1 Leaf shapes of Merlot, Carmenère, and Cabernet franc
Results and Discussion (scanned from real leaves).

Allele sizes for the three reference cultivars are shown in

Table 3. The results from the commercial Merlot vineyards in cultivar, they do provide a practical means to screen individual
Chile were as expected. The vines suspected to be Carmenère vines in a mixed planting of Carmenère and Merlot.
did in fact match the DNA profile of the reference Carmenère
The morphological similarity among these three cultivars
vine (Table 1).
(Figure 1) probably reflects a close genetic relationship, a no-
The four samples from the INIA collection at La Platina had tion that is supported by the considerable allele sharing at the
been planted as Merlot, but the DNA profiles of all four matched 13 loci analyzed in this study as well as 10 additional loci [J.E.
the Carmenère reference. The identity of these vines had not Bowers, unpublished results]. Despite their similarity, these three
previously been investigated. The California vine that was im- cultivars can be distinguished visually (Table 4), as demonstrated
ported directly from France as Carmenère was confirmed as by the preliminary correct visual identification of many of the
Carmenère. The vine that originally came to FPMS from Italy samples tested in this study. However, the morphological dif-
as Cabernet franc but was suspected to be Carmenère was also ferences between these cultivars can be subtle and may be ob-
confirmed as Carmenère. scured by environmental conditions, disease status, or cultural
Eight of the markers used in this study will each clearly dis- practices. Thus, DNA typing can be a valuable adjunct for veri-
tinguish Carmenère from Merlot (Table 3). Two of them, fying the identity of these cultivars, particularly in plantings that
VVMD28 and VVMD31, are notable because Carmenère and provide propagation material to growers, such as nurseries, foun-
Merlot do not share any alleles at these loci so the cultivars can dation plantings, and collections.
be separated by both alleles at either lo-
cus. Although VVMD28 and VVMD31
do have slightly overlapping allele size
Table 4 Morphological distinctions among the leaves of Merlot, Carmenère, and Cabernet franc.
ranges [5], the alleles of Carmenère and
Merlot do not overlap; for the purpose of Merlot Carmenère Cabernet franc
separating these two cultivars, these Overall leaf shape Cuneiform a
Round Pentagonal
markers could be combined on a single Petiolar sinus shape Open U, Lyre, Narrow lyre,
gel. VVMD31 will, furthermore, separate never overlapping overlapping slightly overlapping
Cabernet franc from the other two culti- No. of lobes 5 to 7 5 3 to 5
vars, as will VVMD27. Two markers not Young leaves Green Red to orange Bronze
used in this study, VrZAG62 and Other leaf features Usually darker Funnel-shaped Edges rolled
VrZAG79 [10], will also each separate all green than the with edges rolled toward upper
three cultivars. Although one or two other two toward underside surface

markers are not sufficient to identify a a

Shield-shaped, like a square above a triangle.

Am. J. Enol. Vitic. 52:4 (2001)


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