Bulletin UASVM Horticulture, 67(1)/2010 Print ISSN 1843-5254; Electronic ISSN 1843-5394

Identification of Romanian Vitis vinifera L. Cultivars in Must Using Nuclear Microsatellite Markers
Monica HARTA, Doru PAMFIL, Rodica POP, Iulia Francesca POP
Faculty of Horticulture, University of Agricultural Sciences and Veterinary Medicine, 3-5 Manastur Street, 400372, Cluj-Napoca, Romania; monica_bodea@hotmail.com Abstract. Accurate identification of grapevine cultivars is important for Romanian winemakers and the extrapolation of the grapevine identification methods into musts is particularly relevant in controlling the quality and authenticity of aromatic and high quality red wines. In this study, molecular SSR (Simple Sequence Repeats) analysis of Vitis vinifera L. cultivars (Busuioac de Bohotin, Tmioas Romneasc, Negru Aromat, Feteasc Neagr, Negru de Drgani, Amurg and Novac) was applied in order to detect and correctly identify the grapevine cultivars present in monovarietal musts. DNA provided from each analyzed cultivar was successful extracted from leaves and must at the beginning of the fermentation (day 1) using CTAB method and amplicons generated from eight SSR primers (VVS2, VVS5, VVS29, MD5, MD7, ZAG 47, ZAG 62, ZAG 79) were analyzed. Results shows that, at molecular level, there were no differences between the corresponding leaf and varietal must profiles and SSR markers can be successful used for characterisation and differentiation of analyzed grapevine cultivars identified in musts. Keywords: grape, must, cultivars, DNA, simple sequence repeats markers

INTRODUCTION Romania is one of the worlds largest wine producers and in recent years it has attracted many European business people and wine buyers due to the affordable prices of both vineyards and wine, compared to others wine producing countries. In these conditions it is necessary to develop efficient and reliable methods for the accurate identification of autochthonous and non autochthonous grapevine cultivars (Gheorghe et al., 2010). Several works concerning characterisation and differentiation of grapes in must and wine have been based primarily on the analysis of chemical and biochemical parameters but these methodologies are time-consuming and for this reason molecular markers techniques, based on DNA samples PCR amplification, have been optimized for this analysis (BaleirasCouto and Eiras-Dias, 2006). Microsatellite sequences, also called simple sequence repeats, are regions of tandem repeats of two to five nucleotides and have been proved to be useful for grapevine cultivars analysis. The detection and identification are performed using several SSR markers accepted internationally as a reference for grapevine genotyping (Faria et al., 2000; Bowers et al., 1996; di Gaspero et al., 2000; Jung et al., 2004). Taking into account the above mentioned consideration, microsatellite markers were those chosen for this work, in an attempt to apply this methodology in varietal characterization of some currently used cultivars for port wine production. The aim of this study was to identify the Romanian grape varieties present in monovarietal musts and to compare with corresponding SSR (Simple Sequence Repeats) leaf profiles. Experiences will be focused in the near future to optimize residual DNA isolation protocol in wines obtained from analyzed grape cultivars and their SSR fingerprinting 198

MATERIALS AND METHODS The seven grapevine cultivars used in this study, namely Busuioac de Bohotin, Tmioas Romneasc, Negru Aromat, Feteasc Neagr, Negru de Drgani, Amurg and Novac provided from the grapevine collection maintained at USAMV Ion Ionescu de la Brad Iai. Two types of samples were analyzed: young leaves collected in the spring (May, 2009) and immediately stored at -80 C and fresh must obtained in autumn after grape crushing, containing the skin and the solid parts of the flesh. Total DNA was extracted, both leaves and must varieties, using the protocol developed by Lodhi et al. (1994) and modified by Pop et al. (2003). Leaf tissue were ground to fine powder in liquid nitrogen in an Eppendorf tube and 700 L of 65 C preheated extraction buffer [100 mM Tris-HCl, 20 mM sodium EDTA, pH=8.0, 1.4 M NaCl, 2% (w/v) CTAB, 2% PVP, 5 mM ascorbic acid and 4 mM DIECA, the last three components being added to the extraction buffer just before the heating at 65 C on the water bath] were then added to the tube and the mixture was incubated at 65 C for 25 min. The lysate was extracted with 700 L of chloroform/isoamyl alcohol (24:1) and centrifuged for 15 min at 11000 rpm in a microcentrifuge. In order to precipitate the nucleic acids, the aqueous fraction was mixed with an equal volume of 5 M NaCl and then with 600 L of ice cold 96% ethanol. The nucleic acid precipitate was washed two times in 76% ethanol and air dried before being resuspension in 50 L TE buffer (10 mM Tris-HCl, pH=8.0, 1 mM disodium EDTA). Must DNA extractions were performed using basically the same protocol, although some modifications had to be implemented due to the sample nature. One milliliter of each homogenized must sample was transferred into a 2 mL Eppendorf tube with added 700 L extraction buffer and three times subjected to freezing (-20 C )/ thawing (65 C ) successive for 25 minutes. After centrifugation the supernatant was discarded and the resulting pellet was washed two times with 700 L of chloroform/isoamyl alcohol (24:1), centrifuged for 15 min at 11000 rpm in a microcentrifuge and the extraction protocol proceeded as described above. The concentration and purity of extracted DNA were determined using a Nanodrop ND-1000 Spectrophotometer (Thermo Scientific). Eight microsatellite loci were analyzed: VVS2, VVS5, VVS29, VVMD5, VVMD7, ZAG 47, ZAG 62 and ZAG 79. PCR amplifications were performed in a 96 Well Gradient Palm-Cycler CG1-96 (Corbett Research) in 20 L reactions consist of 1.5 mM MgCl2, dNTP mix (Promega)100 M, 1 x Buffer 5 X Go Taq Buffer, 1 M of each primer, 0.5 U Go Taq Polymerase (Promega), H2O nuclease free water (Promega) and 5 L DNA (50 ng/L). The following amplification protocol was optimized for the analyzed loci: 1 cycle of 4 min at 95 C, followed by 30 cycles (DNA from leaves) and 40 cycles (musts DNA samples) of 1 min at 95 C, 1 min at X C gradient (value of X depends of the each pair primers annealing temperature) and 1 min at 72 C. After a final extension for 7 min at 72 C the samples were stored at 4 C prior to analysis. The forward primer (Integrated DNA Technologies, USA), in each pair, was labelled with 5 WellRed TM fluorescent dyes: D2 (black), D3 (green) or D4 (blue). To estimate amplification efficiency, 10 L of the PCR reaction was run on a 1,5 % agarose gel stained with ethidium bromide. Each sample (1 L volume) of PCR products were diluted with sample loading solution (30 L), followed by the addition of Genomelab DNA Standard Kit-400 (0.3 L) and electrophoresed in the CEQ 8800TM capillary DNA analysis system (Beckman Coulter, Fullerton, CA, USA). Allele sizes were determined for each SSR locus using the Beckman CEQ fragment analysis software. The analyses were repeated at least twice to ensure reproducibility of the results.

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RESULTS AND DISCUSSION Leaf DNA extraction methodology was successfully applied to musts with some modifications as described under Materials and Methods. For example, figure 1 and 2 shows that at Busuioac de Bohotin sample nondegraded DNA was extracted from leaves (2478.5 ng/L) and musts (250.3 ng/L) respectively. DNA concentrations from leaves grapevine cultivars analyzed were between 878.5 ng/L and 2478.5 ng/L with A260/A280 readings between 1.80-2.05. DNA concentration from analyzed musts samples ranged from 130.4 ng/L to 250.3 ng/L with purity between 1.72-2.10.

Fig. 1. Concentration and purity of isolated DNA from Busuioac de Bohotin leaves

Fig. 2. Concentration and purity of isolated DNA from Busuioac de Bohotin must

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Five of the eight nuclear microsatellite markers used in this study are accepted to be universal markers (VVS2, VVS5, ZAG47, ZAG62, ZAG 79) for grapevine genotyping, as demonstrated by the results of the European GENRES-081 research project (www.genres.de/vitis/vitis.htm). Our results showed that all cultivars used were differentiated by nuclear SSR markers (Tab. 1).
Tab. 1 SSR genotypes using eight nuclear loci, expressed as the size of the alleles in base pairs, obtained with DNA from leaves and musts Cultivar Leaves Busuioac de Bohotin Tmioas romneasc Negru aromat Feteasc neagr Negru de Drgani Novac Amurg Monovarietal Must Busuioac de Bohotin Tmioas romneasc Negru aromat Feteasc neagr Negru de Drgani Novac Amurg Microsatellite (SSR) loci VVMD5 VVMD7 ZAG47 226 233 156 234 247 171 226 234 224 237 224 237 226 225 238 230 239 226 234 226 234 224 237 224 237 226 225 238 230 239 242 247 242 238 253 238 247 238 252 238 247 233 247 242 247 242 238 253 238 247 238 252 238 247 156 171 156 171 156 162 156 169 162 158 167 156 171 156 171 156 171 156 162 156 169 162 158 167

VVS2 135

VVS5 110

VVS29 167

ZAG62 198 202 186 198 186 192 198 186 202 198 202 186 198 202 186 198 186 192 198 186 202 198 202 186

ZAG79 248 253 248 253 253 245 253 235 256 241 248 245 253 248 253 248 253 253 245 253 235 256 241 248 245 253

135 135 147 137 145 134 145 137 154 135

109 85 85 152 119 119 110 126 110

167 176 167 167 176 168 176 167 178 167

135 135 147 137 145 134 145 137 154

109 85 85 152 119 119 110 126

167 176 167 167 176 168 176 167 178

As it can be seen in Table 1 the size of microsatellite alleles obtained in must is in accordance with those from leaves. Our results are consistent with other research teams (Faria et al., 2000; Baleiras-Couto and Eiras-Dias, 2006) and shows that SSR analysis can be successfully applied for the authentication of monovarietal fresh musts analyzed (Fig. 3). This type of analysis is particularly important when one needs to know if a must sample contains only one variety included in a group of possibilities (in special for the production of monovarietal wines).

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Fig. 3. Beckman Coulter CEQ 8800 TM fragment analysis image for Busuioac de Bohotin and Negru aromat cultivars at the VVMD7 microsatellite locus. The first cultivar (leaves and must) show a heterozygotic condition (revealed by the presence of two peaks) and Negru aromat cultivar has a homozygotic status (reflected by a single peak)

CONCLUSIONS Leaf DNA extraction methodology was successfully applied to musts with some modifications due to the sample nature and described under Materials and Methods. SSR analysis can be successfully applied for the authentication of monovarietal fresh musts analyzed. Because degradation of DNA in musts in fermentation may leading to erroneous amplification results, it is important to use reference data (amplifying SSRs from young leaves provided by grapevine cultivars). This type of analysis is important when one needs to know if a must sample contains only one grapevine cultivar included in a group of possibilities. Accurate identification of grapevine cultivars is important for Romanian wine-makers and the extrapolation of the grapevine identification methods into musts is particularly relevant in controlling the quality and authenticity of aromatic and high quality red wines.

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Acknowledgments. This study has been financed by the Romanian Education and Research Ministry, through the Second National Plan for Research, Development and Innovation, grant no. 51-003 (2007-2010). REFERENCES
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