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Metadata of the article that will be visualized in OnlineFirst Please note: Image will appear in color online but will be printed in black and white. 1 Article Title Molecular Markers for Assessing Must Varietal Origin 2 Article Sub- Title 3 Article Copyright - Year 4 Journal Name Food Analy tical Methods 5 Springer Science+Business Media, LLC 2012 (This will be the copyright line in the final PDF) 6 Particle 7 Given Name Paula Martins-Lopes 8 Suffix 9 Corresponding Organization University of Trás-os-Montes and Alto Douro (CGB-IBB/UTAD) 10 Division Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology 11 Address P.O. Box 1013, Vila Real 5000-911, Portugal 12 e-mail plopes@utad.pt 13 14 Particle Pereira 15 Given Name Leonor 16 Suffix 17 Organization University of Trás-os-Montes and Alto Douro (CGB-IBB/UTAD) 18 Division Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology 19 Address P.O. Box 1013, Vila Real 5000-911, Portugal 20 e-mail 21 22 Particle Batista 23 Given Name Cláudia 24 Suffix 25 Organization University of Trás-os-Montes and Alto Douro (CGB-IBB/UTAD) 26 Division Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology
27 Address P.O. Box 1013, Vila Real 5000-911, Portugal 28 e-mail 29 30 Particle Zanol 31 Given Name Geni C. 32 Suffix 33 Organization National Institute of Biological Resources/National Institute of Agrarian Research (INRB/INIA Dois Portos) 34 Division 35 Address Quinta d Almoinha, Dois Portos 2565-191, Portugal 36 e-mail 37 38 Particle Clímaco 39 Given Name Pedro 40 Suffix 41 Organization National Institute of Biological Resources/National Institute of Agrarian Research (INRB/INIA Dois Portos) 42 Division 43 Address Quinta d Almoinha, Dois Portos 2565-191, Portugal 44 e-mail 45 46 Particle Brazão 47 Given Name João 48 Suffix 49 Organization National Institute of Biological Resources/National Institute of Agrarian Research (INRB/INIA Dois Portos) 50 Division 51 Address Quinta d Almoinha, Dois Portos 2565-191, Portugal 52 e-mail 53 Eiras-Dias 54 Particle 55 Given Name José E. 56 Suffix
57 Organization National Institute of Biological Resources/National Institute of Agrarian Research (INRB/INIA Dois Portos) 58 Division 59 Address Quinta d Almoinha, Dois Portos 2565-191, Portugal 60 e-mail 61 62 Particle Guedes-Pinto 63 Given Name Henrique 64 Suffix 65 Organization University of Trás-os-Montes and Alto Douro (CGB-IBB/UTAD) 66 Division Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology 67 Address P.O. Box 1013, Vila Real 5000-911, Portugal 68 e-mail 69 70 Schedule Revised Received 3 November 2011 71 Accepted 19 January 2012 72 Abstract Wine quality and market v alue greatly depend on the grapev ine v arietal composition, which may be characteristic of specif ic regions. In order to def end the distinct regions, Denominations of Origin were def ined to protect against f raudulent practices. In this study, we ev aluated the ef f iciency of two microsatellite-based sy stems (microsatellite (SSR) and intermicrosatellite (ISSR)) f or must v arietal composition determination and their potential role in certif ication purposes. Elev en Vitis vinifera L. v arieties f rom leaf and monov arietal must DNA samples were screened with six SSR and 14 ISSR primers to discriminate poly morphisms. Principal coordinates analy sis was perf ormed with DCENTER on the resultant data using unweighted pair group mathematical av erage and rev ealed that ISSRs markers were not suitable f or certif ication procedures, whereas nuclear SSR markers presented a complete correspondence between leaf and must samples, demonstrating that they were adequate f or traceability purposes. 73 Keywords separated by ' - ' 74 Foot note information Certif ication - Must - DNA extraction - Grapev ine identif ication - SSR - ISSR
1 3 2 DOI 10.1007/s12161-012-9369-7 4 Molecular Markers for Assessing Must Varietal Origin 5 Leonor Pereira & Paula Martins-Lopes & 6 Cláudia Batista & Geni C. Zanol & Pedro Clímaco & 7 João Brazão & José E. Eiras-Dias & 8 Henrique Guedes-Pinto 9 Received: 3 November 2011 / Accepted: 19 January 2012 10 # Springer Science+Business Media, LLC 2012 11 12 Abstract Wine quality and market value greatly depend on 13 the grapevine varietal composition, which may be charac- 14 teristic of specific regions. In order to defend the distinct 15 regions, Denominations of Origin were defined to protect 16 against fraudulent practices. In this study, we evaluated the 17 efficiency of two microsatellite-based systems (microsatel- 18 lite (SSR) and intermicrosatellite (ISSR)) for must varietal 19 composition determination and their potential role in certi- 20 fication purposes. Eleven Vitis vinifera L. varieties from leaf 21 and monovarietal must DNA samples were screened with 22 sixssrand14issrprimerstodiscriminatepolymor- 23 phisms. Principal coordinates analysis was performed with 24 DCENTER on the resultant data using unweighted pair 25 group mathematical average and revealed that ISSRs 26 markers were not suitable for certification procedures, 27 whereas nuclear SSR markers presented a complete corre- 28 spondence between leaf and must samples, demonstrating 29 that they were adequate for traceability purposes. Q1 Q2 30 Keywords Certification. Must. DNA extraction. 31 Grapevine identification. SSR. ISSR L. Pereira : P. Martins-Lopes (*) : C. Batista : H. Guedes-Pinto Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro (CGB-IBB/UTAD), P.O. Box 1013, 5000-911 Vila Real, Portugal e-mail: plopes@utad.pt G. C. Zanol : P. Clímaco : J. Brazão : J. E. Eiras-Dias National Institute of Biological Resources/ National Institute of Agrarian Research (INRB/INIA Dois Portos), Quinta d Almoinha, 2565-191 Dois Portos, Portugal Introduction European grapevine (Vitis vinifera L.) is an economically important crop, grown for both table grapes and for winemaking. The Portuguese National Ampelographic Collection contains about 760 accessions, mainly Portuguesespecific. However, the number of distinct varieties may be significantly lower, since there are probably duplicates related to regional denomination (Lopes et al. 2006). A large number of V. vinifera varieties can be used in wine production, although only a small number are commercially important. Portuguese legislation allows 341 varieties for wine production (Baleiras-Couto and Eiras-Dias 2006). Grapevine varieties deeply influence the wine quality and therefore have a direct impact on wine s market price, particularly in referenced market segments such as Denomination of Origin (DO) wines. For that reason, these highly quoted wines are the preferential target for fraudulent practices. Thus, wine authenticity is important in protecting the reputation of DO wines and ensuring consumers confidence in quality control. The control process by grapevine varietal identification should comprise all stages of the vinification process starting with the cellar reception and ending with bottled wine. This control may be more accurate and efficient when DNA-based methodologies are used, once they are independent from environmental conditions. Currently, DNA-based techniques are being widely used for food traceability purposes (Agrimonti et al. 2011; Baleiras-Couto and Eiras-Dias 2006; Doveri and Lee 2007; Faria et al. 2008;Intrierietal. 2007;Jérômeetal. 2008; Martins-Lopes et al. 2008; Pafundo et al. 2007; Vietina et al. 2011). DNA extraction from grapevine vegetative material is well established (Lodhi et al. 1994). However, efficient 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64
65 DNA extraction and amplification from other matrices 66 such as grape must and wine remain difficult, mainly 67 due to: (a) plant DNA decomposition during the macer- 68 ation process; (b) presence of microorganisms DNA, 69 namely yeasts; (c) DNA polymerase inhibitors such as 70 polysaccharides, tannins, and polyphenols, present in 71 matrices especially further down the processing chain. 72 Nevertheless, several reports have been successful using 73 must and experimental wine samples (Baleiras-Couto and 74 Eiras-Dias 2006; Faria et al. 2000; Garcia-Beneytez et al. 75 2002; Nakamura et al. 2007; Rodríguez-Plaza et al. 76 2006; Siret et al. 2000, 2002). 77 Grapevine collections were formerly described using 78 ampelographic descriptions including morphological and 79 phenological aspects (Alleweldt and Dettweiler 1986; 80 Dettweiler 1993; Ortiz et al. 2004; Santiago et al. 2005). With 81 the development of DNA-based markers, grapevine collec- 82 tions have since been characterized at the DNA level. Micro- 83 satellites markers have been the molecular tool selected for the 84 identification and documentation of Vitis gene banks 85 (Almadanim et al. 2007; Botta et al. 1995; Bowers et al. 86 1996; Cipriani et al. 1994; Le Cunff et al. 2008; Lopes et al. 87 1999, 2006; Sefcetal. 1998; Thisetal. 2004; Veloso et al. 88 2010). A European project (GENRES#081, http://www. 89 genres.de/vitis/vitis.htm) selected a set of six microsatel- 90 lite primer pairs (VVS2, VVMD5, VVMD7, VVMD27, 91 VrZAG62, and VrZAG79) as the most adequate for grapevine 92 varietal characterization due to their high polymorphism level. 93 These six SSRs are also included in the OIV descriptor list Q3 94 for grapevine cultivars and Vitis species (OIV 2007). Recent- 95 ly, the GrapeGen06 Project has suggested the use of three 96 more SSRs markers (VVMD25, VVMD28, and VVMD32) 97 for additional genetic data (http://www1.montpellier.inra.fr/ 98 grapegen06). 99 Other molecular markers have been applied to charac- 100 terize grapevine varieties such as Inter-simple Sequence 101 Repeat (ISSR), randomly amplified polymorphic DNA, 102 amplified fragment length polymorphism, and chloroplas- 103 tidial microsatellites (cpssr), among others (Benjak et 104 al. 2005; Cunhaetal.2009; Fanizza et al. 2003; Herrera 105 et al. 2002; Moreno et al. 1998). ISSR markers are 106 frequently used on genetic diversity, phylogeny, gene 107 tagging, genome mapping, and evolutionary biology 108 studies (Godwin et al. 1997; Guptaetal.1994; Reddy 109 et al. 2002; Zietkiewicz et al. 1994). However, they have 110 been limitedly applied for food certification purposes 111 (Martins-Lopes et al. 2008). 112 The need of certifying methods to determine grapevine 113 varieties used in the winemaking process leads to the main 114 goal of the present study which focuses on the possibility of 115 using ISSR and SSR molecular markers for must varietal 116 authentication, using, as reference, leaf DNA samples, and 117 their possible role for certification purposes. Materials and Methods Grapevine Material Eleven V. vinifera L. varieties, used in Portuguese wine production, were selected. Young leaf samples from six white grapevine varieties (Alvarinho, Loureiro, Fernão Pires, Moscatel Galego, Malvasia Fina, and Viosinho) and five red grapevine varieties (Touriga Nacional, Touriga Franca, Tinto Cão, Aragonez, and Cabernet Sauvignon) were collected (field collection of the University of Trás-os- Montes and Alto Douro, in Vila Real, Portugal) and immediately stored at 80 C. Monovarietal musts were prepared at National Institute of Biological Resources in Dois Portos, using freshly harvested grapes from the same grapevine varieties. All must samples were collected after maceration and immediately frozen at 20 C. DNA Extraction 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 verified on a 0.8% (w/v) agarose gel stained in 7 μg ml 1 Genomic DNA was extracted from leaves using the cetyl 135 trimethylammonium bromide (CTAB) method described by 136Q4 Doyle and Doyle (1987). Must DNA extractions were per- 137 formed using an adapted version of Doyle and Doyle 138 (1987), due to the samples nature. Two milliliters of ho- 139 mogenized monovarietal must samples were centrifuged at 140 10,000 rpm (Eppendorf Centrifuge 5430 R) for 5 min. Pellet 141 was recovered, and 1 ml of CTAB extraction buffer, con- 142 taining 2% of polyvinylpyrrolidone (PVP) and1%ofβ- 143Q5 mercaptoethanol, was added. Two extractions with chloro- 144 form/isoamyl alcohol were performed. The final pellet was 145 washed twice with washing buffer (76% EtOH, 10 mm 146 ammonium acetate). DNA was resuspended in 50 μl of 147 distilled water. DNA concentration was determined using 148 NanoDrop ND-1000 spectrophotometer (Nanodrop Tech- 149 nologies, Wilmington, DE, USA), and DNA quality was 150 151 ethidium bromide solution. 152 Simple Sequence Repeat Grapevine varieties and correspondent must samples were genotyped at six SSR loci: VVS2 (Thomas and Scott 1993), VVMD5 and VVMD7 (Bowers et al. 1996), VVMD27 (Bowers et al. 1999), VrZAG62, and VrZAG79 (Sefc et al. 1999). Forward primers were purchased from Sigma Aldrich (Sigma, St. Louis, MO, USA) and labeled with specific fluorochrome for the automatic sequencer (Beckman Coulter Sequencer, Beckman Coulter, Inc, Fullerton, USA). Polymerase chain reactions (PCR) and conditions were performed as described by Baleiras-Couto and Eiras-Dias (2006). 134 153 154 155 156 157 158 159 160 161 162 163 164
165 Amplicon separation was carried out through capillary 166 electrophoresis on an automated sequencer, and fragment 167 size was established with the help of internal size standards 168 (CEQ TM DNA Size Standard Kit-400) using the software 169 package CEQ TM 8000 Fragment Analysis System. 170 Inter-simple Sequence Repeat computations employed the appropriate procedures within NTSYS.pc v2.02 (Rohlf 1998). Results and Discussion DNA Extraction and Quantification 191 192 193 194 171 ISSR amplifications were tested using 14 University of British 172 Columbia (UBC) primers (Table 1). Each reaction consisted 173 of 30 and 50 ng of genomic leaf and must DNA sample, 174 respectively. PCR reactions were performed at the conditions 175 described by Martins-Lopes et al. (2008). 176 Amplicons were separated by electrophoresis onto 1.5% 177 (w/v) agarose gels (Seakem LE Agarose) in 1 Tris/borate/ Q6178 EDTA buffer at 80 V for 150 min, after which the gels were 179 stained in 7 μg ml 1 ethidium bromide solution and digital 180 image was obtained directly under UV light. Each DNA 181 sample was independently amplified at least twice with each 182 primer for each DNA extraction, and only reproducible 183 amplified products were scored. 184 Data Analyses 185 The PCR fragments were scored for the presence (1) or 186 absence (0) of equally sized bands, and two matrices of the 187 different ISSR and SSR markers were assembled and used in 188 the statistical analysis. These matrices were used to perform a 189 principal component analysis (PCA) based on the ISSR data 190 performed with DCENTER (double-centering analysis). All The CTAB method was found to be suitable for must samples. The DNA extracted from monovarietal must samples was of high quality (A 260 /A 280, 1.7 to 2.0), which ran as high-molecular-weight band, comparable to the leaf DNA samples in agarose gel (Fig. 1). The must DNA sample concentrations ranged from 211 to 401 ng μl 1. The must DNA yields were higher than those reported by other authors using similar samples (Faria et al. 2000, 2008; Garcia-Beneytez et al. 2002; Siret et al. 2002), ranging from 10 to 20 μg ml 1 of starting material. One of the reasons that may explain the higher yields obtained may be the fact that we have used immediately frozen fresh must samples, which preserved high-quality DNA. Another reason could rely on the DNA extraction protocol, which was slightly different from the previous described. The inclusion of PVP in the extraction buffer helped to eliminate possible PCR inhibitors, such as polysaccharides, polyphenols, and tannins that are present in high concentrations in these types of samples. The fact that the DNA extracted from these samples presented a high quality can anticipate PCR amplification success. A similar approach was shown to be efficient when dealing with wine samples (Pereira et al. 2011a). t1:1 Table 1 Total number of bands, t1:2 polymorphic bands, and Primer Sequence5-3 No. of bands No. of Leaf % percentage of polymorphism coincident polymorphic Polymorphism t1:3 obtained per each ISSR L M Total bands bands primer among grapevine leaf (L) t1:4 and monovarietal must (M) 807 (AG) 8 T 17 12 18 11 10 59 t1:5 samples 809 (AG) 8 G 19 17 19 17 14 74 t1:6 811 (GA) 8 C 10 12 12 10 7 70 t1:7 817 (CA) 8 A 8 9 9 8 3 38 t1:8 827 (AC) 8 G 16 18 19 15 12 75 t1:9 841 (AC) 8 G 14 12 15 11 8 57 t1:10 846 (CA) 8 RT 16 17 22 11 15 94 t1:11 849 (GT) 8 YA 12 11 12 11 7 58 t1:12 856 (AC) 8 YA 13 15 16 12 10 77 t1:13 857 (AC) 8 YG 16 18 21 12 12 75 t1:14 888 BDB(CA) 7 21 21 21 21 9 43 t1:15 889 DBD(AC) 7 23 21 23 21 15 65 t1:16 B0C/G/T; D0A/G/T; H0A/C/T; 890 VHV(GT) 7 22 22 22 22 12 55 t1:17 R0A/G; V0A/C/G; Y0C/T 891 HVH(TG) 7 23 19 23 19 16 70 t1:18 UBC primers from University of British Columbia Total 229 224 252 201 150 65 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217
Q7 Fig. 1 DNA extracted from leaves and monovarietal musts. Uppercase corresponds to leaf, and lowercase corresponds to monovarietal must samples: lanes: AR/ar Aragonez; CS/cs Cabernet Sauvignon; TC/ tc Tinto Cão; TF/tf Touriga Franca; TN/tn Touriga Nacional; AL/al 218 Nuclear SSR 219 The six nuclear markers selected were revealed as suitable 220 and sufficient for differentiating the varieties studied. All 221 monovarietal must samples were amplified by the six micro- 222 satellite primers, opposite to what was observed by Baleiras- 223 Couto and Eiras-Dias (2006) and Garcia-Beneytez et al. 224 (2002) where some must samples failed to amplify. Apply- 225 ing 45 cycles in the PCR reaction instead of 35 cycles used 226 for leaf DNA samples increased the signal intensity in must 227 DNA samples. The microsatellite alleles size obtained in 228 monovarietal must DNA samples was in accordance 229 with the leaf DNA samples (Fig. 2) and with those 230 referred to in the literature for the grapevine varieties 231 under study (Almadanim et al. 2007; Veloso et al. 232 2011). The six nuclear SSR markers revealed a high 233 level of polymorphism, with a total of 42 alleles, an average 234 of seven alleles per primer (Table 2). The number of alleles per 235 locus ranged from five (VrZAG79) to nine (VrZAG62), which 236 is in accordance with the average number of alleles expected 237 per locus (Cipriani et al. 2010). The H o varied between 0.636 238 (VrZAG79; VVMD7) and 0.909 (VrZAG62; VVS2) while 239 the H e varied between 0.684 (VVMD7) and 0.861 (VrZAG62; 240 VVMD27). Polymorphic information content (PIC) values 241 ranged between 0.622 (VVMD7) and 0.800 (VrZAG62), 242 VrZAG62 being the highest discriminative marker. All genet- 243 ic parameters obtained by SSR data analysis were in accor- 244 dance to literature (Almadanim et al. 2007; Cipriani et al. 245 2010; Ibañezetal. 2003; Lopes et al. 1999, 2006; Sefcetal. 246 2000). 247 In this study, profiles generated by all microsatellite loci 248 matched between samples from the same genotypes, rein- 249 forcing the view that nuclear SSR markers are suitable for 250 certification purposes, as suggested by Baleiras-Couto and Alvarinho; LO/lo Loureiro; FP/fp Fernão Pires; MF/mf Malvasia Fina; MG/mg Moscatel Galego; and VI/vi Viosinho and MM molecular marker GeneRuler TM DNA ladder Mix 10 kbp (MBI Fermentas, Burlington, ON, Canada) Eiras-Dias (2006), Savazzini and Martinelli (2006), and Siret et al. (2000, 2002). Inter-SSR 251 252 253 and (GA) n repeats was approximately 70%, whereas (CA) n The selection of the UBC primers was based on literature 254 that referred to these primers as the most suitable for grape- 255 vine amplification (Herrera et al. 2002; Moreno et al. 1998). 256 For ISSR analyses, all reproducible amplicons from leaf and 257 monovarietal must DNA samples were considered. All se- 258 lected ISSR primers contained dinucleotide repeats. Consid- 259 ering only the bands present in the leaf samples, the average 260 percentage of polymorphism obtained with (AC) n,(tg) n, 261 262 and (GT) n repeats presented an inferior percentage of poly- 263 morphism of 58% and 57%, respectively (Table 1). A total 264 of 252 reproducible ISSR fragments were scored; however, 265 we only considered the leaf samples to calculate the per- 266 centage of polymorphism. The bands size ranged from 267 300 bp to 2.5 kbp in the leaf DNA samples and 120 bp to 268 1.5 kbp in the monovarietal must DNA samples. UBC889 269 and UBC891 primers presented the maximum number of 270 bands (23), whereas primer UBC817 presented the lowest 271 band number (eight). The number of polymorphic bands 272 ranged from three (UBC817) to 16 (UBC891). UBC846 273 primer presented the highest percentage of polymorphism 274 (94%), whereas the lowest percentage was found for 275 UBC817 primer (38%). 276 The different profiles obtained through this molecular 277 marker system can result in the DNA contamination from 278 microorganisms, resulting in different bands amplified. An- 279 other constraint may be linked to the presence of PCR 280 inhibitors that may favor certain fragments over others. 281
Q8 Fig. 2 Plots of the dye signal traces provided by CEQ 8000 Fragment Analysis Software for microsatellite amplification of DNA at VVMD5 (a, b) and VVMD7 (c, d) loci using Tinta Roriz samples: (a, c) leaf and (b, d) monovarietal must 282 Based on the data, a principal component analysis was 283 performed (Fig. 3). It was not possible to find a total corre- 284 spondence between leaf and monovarietal must samples due 285 to the presence of high-molecular-weight bands amplified in 286 leaf DNA samples but absent in must DNA samples. t2:1 Table 2 Genetic parameters calculated on data of six SSR loci in 11 samples considering grapevine leaf and monovarietal must t2:2 Locus Samples N o PIC H e H o t2:3 VVS2 11 7 0.759 0.827 0.909 t2:4 VVMD5 11 7 0.770 0.835 0.727 t2:5 VVMD7 11 7 0.622 0.684 0.636 t2:6 VVMD27 11 7 0.799 0.861 0.818 t2:7 VrZAG62 11 9 0.800 0.861 0.909 t2:8 VrZAG79 11 5 0.640 0.727 0.636 PCR inhibitors as do red must samples. When A 260 /A 230 Nevertheless, the presence of low-molecular-weight bands 287 in monovarietal must samples was also observed. The dif- 288 ferent behavior of the samples according to their origin lead 289 to a distribution in the PCA graphic in four main groups: (a) 290 leaf white grapevine variety samples, (b) leaf red grapevine 291 variety samples, (c) white monovarietal must samples, and 292 (d) red monovarietal must samples (Fig. 3). However, re- 293 garding white grapevine varieties, some of the high- 294 molecular-weight bands were present in both leaf and mono- 295 varietal must samples, which justifies the higher proximity 296 found between groups a and c (Fig. 3). The fact that the 297 white must samples produce high-molecular-weight bands 298 may be linked to the fact that they do not present as many 299 300 ratio is considered, a difference between white and red must 301 samples is evident; white must samples present generally 302 higher ratio values (1.51 to 2.26) than red must samples 303 (1.40 to 2.13). Another interesting observation is that the 304 A 260 /A 230 ratio values obtained in different DNA extraction 305 from the same variety present similar values. This could be 306 N o number of alleles, H o observed heterozygosity, H e expected heterozygosity, PIC polymorphic information content
Fig. 3 Principal component analyses performed with DCENTER among the grapevine leaf and correspondent monovarietal must samples. When an ISSR matrix is factored using the EIGEN program, the elements of the eigenvectors corresponding to positive eigenvalues are interpreted as the coordinates of each point in a Cartesian space (dotted lines represent Eigen-vectors). Uppercase corresponds to leaf, and lowercase corresponds to must samples: AR/ar Aragonez; CS/cs Cabernet Sauvignon; TC/tc Tinto Cão; TF/tf Touriga Franca; TN/tn Touriga Nacional; AL/al Alvarinho; FP/fp Fernão Pires; LO/lo Loureiro; MF/mf Malvasia Fina; MG/mg Moscatel Galego; and VI/vi Viosinho. Groups: a White grapevine leaf varietal samples; b red grapevine leaf varietal samples; c monovarietal white must samples; and d monovarietal red must samples 307 explained by the unique composition of each variety 308 (Dimitrovska et al. 2011). Similar results were observed re- 309 cently in wines (Pereira et al. 2011b). 310 ISSRs have been widely applied to grapevine mainly to 311 detect intravarietal differences and to assess genetic diversi- 312 ty and relationships among grapevine varieties (Dhanorkar 313 et al. 2005; Herrera et al. 2002; Moreno et al. 1998; Sabır et Q9314 al. 2008; Wu et al. 2009; Zietkiewicz et al. 1994). To our 315 knowledge, for certification purposes, this marker system has only been applied in olive oil (Martins-Lopes et al. 2008), this study being the first approach to applying ISSR markers in monovarietal musts for this specific goal. Conclusions Our results showed that a simple DNA extraction method with minor modifications may be applicable for both must 316 317 318 319 320 321
322 and leaf samples. Genomic DNA extracted from monovar- 323 ietal must samples was amplifiable with SSR and ISSR 324 molecular markers. 325 In this work, SSR analysis was successfully applied for 326 the varietal identification in monovarietal must samples. The 327 existence of a complete correspondence between must and a 328 leaf samples for the same grapevine variety indicates that 329 these nuclear markers may be used for certification purpo- 330 ses. This procedure allows not only the identification of the 331 presence or absence of a certain variety, but also a positive 332 detection of other varieties that may exist simultaneously in 333 the must. This approach may be easily applied in must 334 samples from particular demarcate regions, where only a 335 limited number of varieties are authorized, reducing the 336 number of expected alleles. Therefore, this technique can 337 be successfully established in cellars during grape reception, 338 contributing to maintenance of the grapevine varietal value 339 used in wine production. This can help avoid potential 340 abusive use of prestigious denominations by adulterated 341 products. 342 Although ISSR markers are efficient for the assessment 343 of genetic relationships among grapevine varieties, the pres- 344 ent study demonstrated that these molecular markers are not 345 suitable for authentication purposes, possibly due to repro- 346 ducibility issues linked to matrix specificity. Undoubtedly, 347 microsatellite DNA analysis is a powerful, objective, and 348 reliable technique for grapevine characterization and conse- 349 quently can be used in certification and traceability purposes, 350 protecting consumers against misleading information and 351 promoting a fair trade. 352 353 Acknowledgements This work was supported by the project 354 Genomics Applied to Portuguese Wine Traceability (PTDC/AGR-ALI/ 355 69516/2006) and a PhD grant to Leonor Pereira (SFRH/BD/44781/2008) 356 from the Fundação para a Ciência e Tecnologia-Portugal. 357 References 358 359 Agrimonti C, Vietina M, Pafundo S, Marmiroli N (2011) The use of 360 food genomics to ensure the traceability of olive oil. Trends Food 361 Sci Tech 22:237 244 362 Alleweldt G, Dettweiler E (1986) Ampelographic studies to character- 363 ize grapevine varieties. 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