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Analysis of grapevine (Vitis vinifera L.

) varieties by molecular markers


1

Jahnke Gizella, 2Korbuly Jnos, 1Mjer Jnos, 1 Lakatos Anita, 1 Gyrffyn Molnr Jlia
1

FVM Research Institute for Viticulture and Enology, Badacsony


H-8261 Badacsonytomaj, Rmai t 165. e-mail: vitihill@mail.iif.hu; Tel: 36.87-532-200
2

National Institute for Agricultural Quality Control


H-1024 Budapest, Keleti Kroly u. 24. Hungary

Keywords: Vitis vinifera L., isoenzyme, microsatellite, SSR, taxonomy, cultivar identification
Summary
The aim of our work was to investigate the genome of grapevine with molecular markers
(isoenzyme, SSR). The isoenzyme patterns of 4 enzyme systems (CO, GOT, AcP, PER) and
the microsatellite profile in 6 loci (VVS2, VVS16, VVMD7, VMC4A1, VMC4G6, VrZag79)
of 48 grapevine varieties were analysed. In the case of the CO, GOT, AcP and PER enzymes
the results were reproducible and the patterns of the woody stems were independent from the
time of sampling in the resting period of the grape. Based on the isoenzyme patterns of these
4 enzymes the most of the investigated 48 varieties (40 varieties) were identifiable. A
correlation was found between the isoenzyme patterns and the pertain to convarietas of the
varieties. It was established, that while the varieties of the convarietas pontica differentiate
from the varieties of the convarietas orientalis and occidentalis, the last two groups dont
differentiate strongly from each other. A special acid phosphatase isoenzyme banding pattern
was identified, which is characteristic for the pontican cultivars, but it seldom appears in other
two groups. Based on the microsatellite analyses 46 varieties from the 48 investigated ones
were identifiable. It was possible to differentiate 2 cultivars of the Pinot conculta, the Pinot
blanc and the Pinot gris by the microsatellite analyses in the VMC4A1 locus.
Introduction
The grapevine (Vitis vinifera L.) is one of the most important crop plant of the world. Grapes
were grown in Europe even before the Roman age, and there are many traditional and newly
bred cultivars all over the world. In the last decades it has become imperative to handle the large
germplasm of grapevine as well, and to properly identify the different cultivars. The selection of
true-to type material seems to have high importance.
The other problem of germplasm management of the grape is the large number of synonyms
and homonyms. It means, that there are a lot of different cultivars mentioned under the same
name (homonyms), and there are cultivars, what has more than one name (synonyms).
Morphological characters, as the shape of the leaves for example, was used for cultivar
identification, but it sometimes led to unreliable determinations due to environmental influences
(BRETTING and WIRDRLECHNER, 1995). The molecular markers provides significant advantage
for grape cultivar identification, because unlike other vegetatively propagated, plants grapes has
a quite high degree of genetic variability (OLMO, 1976).
The isoenzyme analyses for grape cultivar identification and genetic characterisation was
widely used in 20-30 years (SCHWENNESEN et al., 1982; CRESPAN et al., 1999). In most cases
starch gel electrophoresis was used to separate the isoenzymes. Recreantly poliacrylamide-gel
electrophoresis was used for isozyme investigation (SNCHEZ-ESCRIBANO et al. ,1998) and for
isoelectric focusing as well (PAAR et al., 1999, STEFANOVITS-BNYAI et al., 2002). The most

frequently used isoenzymes in the genus Vitis were the peroxidase (BACHMANN and BLAICH,
1988), glucose phosphate isomerase and phosphoglucomutase (PARFITT and ARULSEKAR,
1989.). The catechol oxidase and esterase systems were reported to be highly polymorphic.
(SCIENZA et. al., 1994.).
DNA based molecular markers were widely used in the last few years for genetic studies and
cultivar characterisation, identification. RFLP analysis were used for the identification of 16
commercialized rootstock cultivars (BOURQUIN et al., 1992), or RAPD analyses were applied to
investigate the genetic relationships among 14 Muscat grapevines in Apulia (FANIZZA et al.,
2000). Microsatellites were used for the characterisation of cultivars grown in Italy (FATAHI et
al., 2003), and in Hungary (HALSZ et al., 2005, KOCSIS et al., 2005) as well. al.,2005), and for
managing grape germplasm collection as well (DANGL et al, 2001).
The aim of our work was to investigate the genome of grapevine with molecular markers
(isoenzyme, SSR).
Materials and methods
The plant materials (canes) were gathered in the collection of the FVM Research Institutes for
Viticulture and Enology in Badacsony and Pcs, in the resting period of 2003-2004.
The canes were pealed, and the phloem was removed. 150 mg polyclar AT and 1 ml of 4oC
extraction buffer (described by ARULSEKAR and PARFITT, 1986.) were added to about 300 mg of
plant material.
After centrifugation at 4C 20l of clear supernatant was applied for analyses. For further
examination the extracts were frozen and stored at 75C.
Vertical poliacrylamide gel electrophoresis was used for the separation of the isoenzymes of
catechol-oxidase (CO), peroxidase (PER), glutamate-oxalacetate-transaminase (GOT) and acid
phosphatase (AcP) as described by ROYO et al. (1997).
After the electrophoresis the gels were stained for AcP, GOT and PER with the staining
solutions as described by ARULSEKAR and PARFITT (1986); or for CO as described by SNCHEZYLAMO (1992). The isoenzyme pattern obtained in the gel complex at a fixed pH was
evaluated visually.
DNA was extracted from canes with DNA Plant mini kit (Quiagen). Microsatellite (SSR)
analysis was performed in 6 loci (VMC4A1, VMC4G6, VVS2, VVS16, VVMD7, VrZag79).
Polymerase chain reactions were carried out in a total volume of 25 l containing 12,5 l of Hot
Start Master Mix (Quiagen), 0.2 M of each primer, and 50 ng of template DNA, using the
following thermal profile: (1) 94C for 45 min; (2) 94C for 1 min, 50 C (in the case of
VVS16, VVMD7, VMC4G6) or 60 C (in the case of VMC4A1, VrZag79, VVS2) for 1 min,
73C for 1 min per 35 cycles; (3) 73C for 7 min. One primer of each primer pairs were
fluorescently labelled with FAM (6FAM) on the 5 end of the DNA chain. PCR products were
run on a PE-Applied Biosystem 3100 Automated Capillary DNA Sequencer, the length of the
products were determined using GeneScan 2.0 software (Applied Biosystem).
Results and discussion
In the case of the CO, GOT, AcP and PER enzymes the results were reproducible and the
patterns of the woody stems were independent from the time of sampling in the resting period
of the grape. Based on the isoenzyme patterns of these 4 enzymes the most of the investigated
48 varieties (40 varieties) were identifiable. The results of isoenzyme analyses are presented
in table 1.

Table 1: Isoenzim patterns for cathecol-oxidase (CO), acid phosphatase (AcP), glutamateoxalacetete-transaminase (GOT) and peroxidase (PER).
Fajta
Arany srfehr
Badacsony-15
Badacsony-43
Bakator (tdszn)
Bouvier
Budai
Cabernet franc
Cabernet sauvignon
Chardonnay
Chasselas
Cirfandli
Csomorika
Ezerj
Fehr ghr
Furmint
Hrslevel
Juhfark
Kadarka
Kkfrankos
Kkmedoc
Kknyel
Kkoport
Kirlylenyka
Kvrszl
Kvidinka
Lenyka
Olasz rizling
Ottonel muskotly
Picolit
Pinot blanc
Pinot noir
Pintes
Pozsonyi fehr
Rajnai rizling
Rzsak
Srga muskotly
Sauvignon
Semillon
Szrkebart
Tramini
Vulcanus
Zefir
Zeng
Zenit
Zta
Zeus
Zld szilvni
Zld veltelni

1
0
0
0
1
0
0
1
1
0
0
0
1
1
0
0
1
0
1
1
0
0
0
1
0
0
1
0
0
1
0
0
1
1
0
0
1
1
1
0
0
1
0
1
0
0
0
0
0

2
1
0
0
1
1
0
1
1
1
1
1
1
1
0
0
0
1
0
0
0
0
1
0
1
1
0
0
1
0
1
1
1
0
1
0
0
1
1
1
1
1
1
1
1
0
1
1
1

CO
3 4
0 1
0 1
0 1
0 0
0 1
1 0
0 0
0 0
0 0
0 1
0 1
0 0
0 0
0 0
1 0
1 0
0 1
1 0
1 0
0 0
1 0
0 1
0 1
1 0
1 0
0 1
0 1
1 0
1 0
0 1
0 1
0 0
0 0
0 0
0 1
1 0
0 0
0 0
0 1
0 1
0 0
0 0
0 1
0 0
0 1
0 0
0 1
0 1

5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

6
0
0
0
1
1
0
1
1
1
1
1
0
0
0
0
1
1
1
0
1
0
0
0
1
0
0
0
0
1
1
1
1
1
1
0
0
0
0
1
0
1
0
1
1
1
1
1
1

1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

GOT
2 3
1 1
1 1
1 1
0 1
1 0
1 0
1 0
1 0
1 1
1 1
1 0
1 1
1 1
1 0
1 0
1 1
1 1
1 1
1 1
0 1
1 1
1 0
1 1
1 1
1 1
1 0
1 0
1 0
1 0
1 1
1 1
1 0
1 0
1 0
1 0
1 1
1 1
1 1
1 1
1 0
1 0
1 0
1 1
1 1
1 0
1 0
1 0
1 1

4
1
1
1
1
0
0
0
0
1
1
0
1
1
0
0
1
1
1
1
1
1
0
1
1
1
0
0
0
0
1
1
0
0
0
0
1
1
1
1
0
0
0
1
1
0
0
0
1

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

2
1
0
1
1
1
1
1
1
1
1
1
1
0
1
0
1
1
1
1
1
1
0
1
1
1
0
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1

3
1
1
0
0
0
1
1
1
0
1
1
0
1
0
1
0
0
1
1
1
0
1
0
1
0
1
0
0
1
0
0
0
1
1
0
1
1
1
0
0
0
0
1
0
1
1
1
0

AcP
4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

6
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

7
1
0
0
1
0
0
0
0
0
0
1
1
0
1
1
1
0
1
0
0
0
0
0
1
1
0
0
0
1
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0

1
0
1
1
1
0
1
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
1
0
0
0
0
1
1
0
1
1
0
0
1
1
0
0
0
0
1
0

2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

PER
3 4
1 0
1 0
1 0
1 1
1 0
1 0
1 0
1 0
1 1
1 1
1 0
1 0
1 1
1 0
1 0
1 0
1 0
1 0
1 1
1 0
1 0
1 0
1 0
1 0
1 1
1 0
1 0
1 0
0 0
1 0
1 0
1 0
1 0
0 0
0 1
1 0
1 1
1 1
1 0
1 1
0 1
1 0
1 0
1 0
1 0
1 0
1 0
1 1

5
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

6
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

A special banding pattern of the acid phosphatase could be observed of the woody stems in the
case of 14 cultivars (see Table 1-with bold letters). This characteristic pattern has not been
reported in the literature yet. These cultivars have four bands in the faster migrating region of

the gel, while the others have only three bands (Figure 1). Analysis of leaf extracts of these
cultivars resulted in one additional band in their zymogram as well. It may mean, that this
additional form of acid phosphatase is controlled by an additional locus. 12 of these 14 cultivars
are described by morphological features as pontican cultivars.
From the 17 pontican cultivars (with italic letters in Table 1) involved in the analysis 13
showed this pattern. The newly bred grapevine cultivar Zta originates from the cross Bouver
(occidentalis) x Furmint (pontica) produces the special acid phosphatase isoenzyme band. The
only one, which has this special pattern but does not belong to the pontican cultivars, and is not
an intraspecific hybrid of a cultivar of pontican origin is the Cirfandli.

1
2
3
4
5
6
7

Figure 1: Acid phosphatase isoenzyme patterns of 10 Vitis vinifera L. varieties (Zld


veltelni, Zld szilvni, Bouvier, Zta, Chasselas, Juhfark, Pintes, Lenyka, Kkmedoc,
Kkoport respectively)
The results of microsatellite analysis are presented in table 2.
Based on the microsatellite analyses 46 varieties from the 48 were identifiable. Based on the
results of the misrosatellite analyses with the VMC4A1 primers it was possible to differentiate
2 cultivars of the Pinot conculta, the Pinot blanc and the Pinot gris (see Table 2 and
Figure 2).

Figure 2: Varieties of the Pinot conculta: Pinot blanc, Szrkebart (Pinot gris) and
Pinot oir respectively

Table 2: Microsatellite analysis results of 48 varieties in 6 loci


Fajta neve
Arany srfehr
Badacsony-15
Badacsony-43
Bakator (tdszn)
Bouvier
Budai
Cabernet franc
Cabernet sauvignon
Chardonnay
Chasselas
Cirfandli
Csomorika
Ezerj
Fehr ghr
Furmint
Hrslevel
Juhfark
Kadarka
Kkfrankos
Kkmedoc
Kknyel
Kkoport
Kirlylenyka
Kvrszl
Kvidinka
Lenyka
Olasz rizling
Ottonel muskotly
Picolit
Pinot blanc
Pinot noir
Pintes
Pozsonyi fehr
Rajnai rizling
Rzsak (B-36)
Srga muskotly
Sauvignon
Semillon
Szrkebart
Tramini
Vulcanus (B-38)
Zefir
Zeng
Zenit
Zta
Zeus
Zld szilvni
Zld veltelni

VMC4A1
259
261
265
275
269
271
259
261
265
267
271
275
269
271
267
271
271
275
273
275
265
267
261
275
265
267
261
261
269
271
267
271
263
263
259
261
277
279
275
275
273
275
265
267
269
271
267
267
259
261
269
271
271
279
273
275
265
267
267
275
267
275
265
267
259
261
269
271
275
275
267
279
267
267
265
267
267
275
265
273
265
267
269
271
267
267
265
265
265
267
267
271
265
267
265
267
269
271

VVS2
140
150
140
148
134
150
134
140
130
148
140
150
136
144
136
148
134
140
130
140
130
130
130
130
130
140
130
130
130
150
130
142
132
142
130
140
140
142
142
150
130
148
142
150
128
130
130
142
130
132
130
130
132
148
130
140
132
136
134
148
134
148
136
150
132
152
140
150
148
150
130
130
130
148
130
130

VrZag79
232
238
234
246
232
232
244
246
234
246
246
246
242
254
242
242
238
240
246
254
240
246
232
254
232
244
244
254
232
244
232
246
230
242
246
252
232
246
244
248
246
246
242
252
242
244
232
246
242
244
232
246
244
246
248
252
234
254
234
240
234
240
238
240
244
246
238
240
246
246
246
250
240
242
242
246

VMC4G6
122
126
122
124
122
122
122
130
122
122
136
138
128
132
122
132
122
126
132
138
122
122
120
124
120
126
122
126
128
138
122
128
120
128
130
140
122
128
122
132
120
128
122
128
126
126
130
140
128
128
122
124
122
122
122
132
120
120
122
122
122
122
122
122
124
128
122
126
128
128
122
138
122
124
122
138

VVMD7
237
251
235
241
237
247
237
251
241
241
247
247
237
261
237
237
237
241
237
245
241
251
237
247
237
237
237
247
237
247
237
245
237
245
237
247
237
247
241
245
237
241
241
253
245
247
237
253
245
253
247
251
245
255
237
241
243
243
237
241
237
241
237
255
247
253
247
255
237
247
231
247
237
255
237
255

VVS16
291
291
285
285
285
285
285
285
285
285
285
291
283
285
285
285
285
291
285
285
263
285
289
291
285
285
285
289
285
291
285
285
285
291
263
291
263
289
285
285
285
285
263
285
285
291
285
291
285
285
263
285
285
285
285
285
285
285
285
285
285
285
285
285
285
289
285
291
285
285
285
285
285
285
285
285

134

148

234

240

122

122

237

241

285

285

148
134
146
130
130
130
130
150
130

150
150
148
140
148
130
140
150
150

238
232
234
232
232
234
232
242
240

244
246
246
246
246
244
246
244
244

122
122
122
120
122
122
120
122
122

122
138
122
122
126
138
122
128
128

241
237
231
237
237
241
237
241
245

255
247
241
241
241
247
241
245
255

285
285
285
285
285
285
285
285
285

285
291
285
285
285
291
285
291
285

Literature
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BACHMANN O., BLAICH R. 1988. Isoelectric focusing of grapevine peroxidases as a tool for
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grapevine rootstocks by RFLP and RFLP analysis of nuclear DNA extracted from the wood.
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BRETTING P. K., WILDRLECHNER M. P. 1995. Genetic Markers and Horticultural Germplasm
Management. HortScience 30(7) 1349-1356. p.
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