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2 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 , Portugal 12 plopes@utad.pt 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 , Portugal 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

3 27 Address P.O. Box 1013, Vila Real , Portugal 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 , Portugal 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 , Portugal 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 , Portugal Eiras-Dias 54 Particle 55 Given Name José E. 56 Suffix

4 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 , Portugal 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 , Portugal Schedule Revised Received 3 November Accepted 19 January 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

5 1 3 2 DOI /s 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 # Springer Science+Business Media, LLC 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, Vila Real, Portugal 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, 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

6 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 ; Nakamura et al. 2007; Rodríguez-Plaza et al ; 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 ; Cipriani et al. 1994; Le Cunff et al. 2008; Lopes et al , 2006; Sefcetal. 1998; Thisetal. 2004; Veloso et al ). A European project (GENRES#081, 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 ( 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 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 ,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 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)

7 165 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 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 t1:5 samples 809 (AG) 8 G t1:6 811 (GA) 8 C t1:7 817 (CA) 8 A t1:8 827 (AC) 8 G t1:9 841 (AC) 8 G t1: (CA) 8 RT t1: (GT) 8 YA t1: (AC) 8 YA t1: (AC) 8 YG t1: BDB(CA) t1: DBD(AC) t1:16 B0C/G/T; D0A/G/T; H0A/C/T; 890 VHV(GT) t1:17 R0A/G; V0A/C/G; Y0C/T 891 HVH(TG) t1:18 UBC primers from University of British Columbia Total

8 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 ). 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 (VrZAG79; VVMD7) and (VrZAG62; VVS2) while 239 the H e varied between (VVMD7) and (VrZAG62; 240 VVMD27). Polymorphic information content (PIC) values 241 ranged between (VVMD7) and (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 ; Ibañezetal. 2003; Lopes et al. 1999, 2006; Sefcetal ). 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 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, 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 bp to 2.5 kbp in the leaf DNA samples and 120 bp to kbp in the monovarietal must DNA samples. UBC 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). UBC 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

9 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 VVS t2:4 VVMD t2:5 VVMD t2:6 VVMD t2:7 VrZAG t2:8 VrZAG 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 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

10 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

11 322 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 Acknowledgements This work was supported by the project 354 Genomics Applied to Portuguese Wine Traceability (PTDC/AGR-ALI/ /2006) and a PhD grant to Leonor Pereira (SFRH/BD/44781/2008) 356 from the Fundação para a Ciência e Tecnologia-Portugal. 357 References 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: Alleweldt G, Dettweiler E (1986) Ampelographic studies to character- 363 ize grapevine varieties. Vignevini 13: Almadanim MC, Baleiras-Couto MM, Pereira HS, Carneiro LC, 365 Fevereiro P, Eiras-Dias JE, Morais-Cecilio L, Viegas W, Veloso MM 366 (2007) Genetic diversity of the grapevine (Vitis vinifera L.) varieties 367 most utilized for wine production in Portugal. Vitis 46: Baleiras-Couto MD, Eiras-Dias JE (2006) Detection and identification 369 of grape varieties in must and wine using nuclear and chloroplast 370 microsatellite markers. Anal Chim Acta 563: Benjak A, Ercisli S, Vokurka A, Maletic E, Pejic I (2005) Genetic 372 relationships among grapevine cultivars native to Croatia, Greece 373 and Turkey. Vitis 44: Botta R, Scott NS, Eynard I, Thomas MR (1995) Evaluation of micro- 375 satellite sequence-tagged site markers for characterizing Vitis vi- 376 nifera cultivars. Vitis 34: Bowers JE, Dangl GS, Vignani R, Meredith CP (1996) DNA Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.). Genome 39: Bowers JE, Dangl GS, Meredith CP (1999) Development and characterization of additional microsatellite DNA markers for grape. Am J Enol Vitic 50: Cipriani G, Frazza G, Peterlunger E, Testolin R (1994) Grapevine fingerprinting using microsatellite repeats. Vitis 33: Cipriani G, Spadotto A, Jurman I, Gaspero GDi, Crespan M, Meneghetti S, Frare E, Vignani R, Cresti M, Morgante M, Pezzotti M, Pe E, Policriti A, Testolin R (2010) The SSR-based molecular profile of 1005 grapevine (Vitis vinifera L.) accessions uncovers new synonymy and parentages, and reveals a large admixture amongst varieties of different geographic origin. Theor Appl Genet 121: Cunha J, Santos MT, Carneiro LC, Fevereiro P, Eiras-Dias JE (2009) Portuguese traditional grapevine cultivars and wild vines (Vitis vinifera L.) share morphological and genetic traits. Genet Resour Crop Evol 56: Dettweiler E (1993) Evaluation of breeding characteristics in Vitis. Influence of climate on morphologic characteristics of grapevines. Vitis 32: Dhanorkar VM, Tamhankar SA, Patil SG, Rao VS (2005) ISSR-PCR for assessment of genetic relationship among grape varieties cultivated in India. Vitis 44: Dimitrovska M, Bocevska M, Dimitrovski D, Murkovic M (2011) Anthocyanin composition of Vranec, Cabernet Sauvignon, Merlot and Pinot Noir grapes as indicator of their varietal differentiation. Eur Food Res Technol 232(4): Doveri S, Lee D (2007) Development of sensitive crop-specific polymerase chain reaction assays using 5S DNA: applications in food traceability. J Agric Food Chem 55: Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Focus 12:13 15 Fanizza G, Chaabane R, Lamaj F, Ricciardi L, Resta P (2003) AFLP analysis of genetic relationships among aromatic grapevines (Vitis vinifera). Theor Appl Genet 107: Faria MA, Magalhães R, Ferreira MA, Meredith CP, Monteiro FF (2000) Vitis vinifera must varietal authentication using microsatellite DNA analysis (SSR). J Agric Food Chem 48: Faria MA, Nunes E, Oliveira MBPP (2008) Relative quantification of Vitis vinifera L. varieties in musts by microsatellite DNA analysis. Eur Food Res Technol 227: Garcia-Beneytez E, Maria VM, Joaquin B, Maria CP, Javier I (2002) Application of a DNA analysis method for the cultivar identification of grape musts and experimental and commercial wines of Vitis vinifera L. using microsatellite markers. J Agric Food Chem 50: Godwin ID, Aitken EAB, Smith LW (1997) Application of intersimple sequence repeat (ISSR) markers to plant genetics. Electrophoresis 18: Gupta M, Chyi Y-S, Romero-Severson J, Owen JL (1994) Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple-sequence repeats. Theor Appl Genet 89: Herrera R, Cares V, Wilkinson M, Caligari PDS (2002) Characterization of the genetic variation and cultivar identification of Vitis vinifera cultivars using RAPD and anchored microsatellites markers. Euphytica 124: Ibañez J, De Andrés MT, Molino A, Borrego J (2003) Genetic study of key Spanish grapevine varieties through microsatellite analysis. Am J Enol Vitic 54:22 30 Intrieri M, Muleo R, Buiatti M (2007) Choloroplast DNA polymorphism as molecular markers to identify cultivars of Olea europaea L. J Horticult Sci Biotechnol 82:

12 443 Jérôme M, Martinsohn JT, Ortega D, Carreau P, Verrez-Bagnis V, 444 Mouchel O (2008) Toward fish and seafood traceability: anchovy 445 species determination in fish products by molecular markers and 446 support through a public domain database. J Agric Food Chem : Le Cunff L, Fournier-Level A, Laucou V, Vezzulli S, Lacombe T, Adam- 449 Blondon A-F, Boursiquot J-M, This P (2008) Construction of nested 450 genetic core collections to optimize the exploitation of natural 451 diversity in Vitis vinifera L. subsp. sativa. BMC Plant Biol 8: Lodhi MA, Ye GN, Weeden NF, Reisch BI (1994) A simple and 453 efficient method for DNA extraction from grapevine cultivars 454 and Vitis species. Plant Mol Biol Rep 12: Lopes MS, Sefc KM, Eiras-Dias JE, Steinkellner H, Câmara Machado 456 ML, Câmara Machado A (1999) The use of microsatellites for 457 germplasm management in a Portuguese grapevine collection. 458 Theor Appl Genet 99: Lopes MS, Rodrigues dos Santos M, Eiras Dias JE, Mendonça D, 460 Câmara Machado A (2006) Discrimination of Portuguese grape- 461 vines based on microsatellite markers. J Biotechnol 127: Martins-Lopes P, Gomes S, Santos E, Guedes-Pinto H (2008) DNA 463 Markers for Portuguese olive oil fingerprinting. J Agric Food 464 Chem 56: Moreno S, Martin JP, Ortiz JM (1998) Inter-simple sequence repeats 466 PCR for characterization of closely related grapevine germplasm. 467 Euphytica 110: Nakamura S, Haraguchi K, Mitani N, Ohtsubo K (2007) Novel prep- 469 aration method of template DNAs from wine for PCR to differ- 470 entiate grape (Vitis vinifera L.) Cultivar. J Agric Food Chem : Ortiz JM, Martin JP, Borrego J, Chavez J, Rodriguez I, Munõz G, 473 Cabello F (2004) Molecular and morphological characterization 474 of a Vitis gene bank for the establishment of a base collection. 475 Genet Resour Crop Evol 51: Pafundo S, Agrimonti C, Maestri E, Marmiroli N (2007) Applicability 477 of SCAR markers to food genomics: olive oil traceability. J Agric 478 Food Chem 55: Pereira L, Guedes-Pinto H, Martins-Lopes P (2011a) An enhanced 480 method for Vitis vinifera L. DNA extraction from wines. Am J 481 Enol Vitic 62(4): Pereira L, Zenol G, Eiras-Dias JE, Guedes-Pinto H, Aranha J, Martins- 483 Lopes P (2011b) Comparison of DNA extraction efficiency in 484 commercial and experimental wines. XXXIV World Congress of 485 Vine and Wine, The wine construction, of June, Porto, 486 Portugal. Section II, ID Reddy MP, Sarla N, Siddiq EA (2002) Inter simple sequence repeat 488 (ISSR) polymorphism and its application in plant breeding. 489 Euphytica 128: Rodríguez-Plaza P, González R, Moreno-Arribas MV, Polo MC, Bravo 491 G, Martínez-Zapater JM, Martínez MC, Cifuentes A (2006) Com- 492 bining microsatellite markers and capillary gel electrophoresis 493 with laser-induced fluorescence to identify the grape (Vitis vinif- 494 era) variety of musts. Eur Food Res Technol 223: Rohlf M (1998) NTSYS-PC. Numerical taxonomy and multivariate analysis system, version 2.02i; Department of Ecology and Evolution State University of New York: Setauket, NY Sabır A, Kafkas S, Tangolar S, Büyükalaca S (2008) Genetic relationship of grape cultivars by ISSR (inter-simple sequence repeats) markers. Eur J Hortic Sci 73:84 88 Santiago JL, Boso S, Martín JP, Ortiz JM, Martinez MC (2005) Characterisation and identification of grapevine cultivars (Vitis vinifera L.) from northwestern Spain using microsatellite markers and ampelometric methods. Vitis 44:67 72 Savazzini F, Martinelli L (2006) DNA analysis in wines: development of methods for enhanced extraction and real-time polymerase chain reaction quantification. Anal Chim Acta 563: Sefc KM, Regner F, Turetschek E, Glössl J, Steinkellner H (1998) Genotyping of grapevine and rootstock cultivars using microsatellite markers. Vitis 37:15 20 Sefc KM, Regner F, Turetschek E, Glössl J, Steinkellner H (1999) Identification of microsatellite sequences in Vitis riparia and their applicability for genotyping of different Vitis species. Genome 42: Sefc KM, Lopes MS, Lefort F, Botta R, Robelakis-Angelakis KA, Ibanez J, Pejic I, Wagner HW, Glossl J, Steinkller H (2000) Microsatellites variability in grapevine cultivars from different European regions and evolution of assignment testing to assess the geographic origin of cultivars. Theor Appl Genet 100: Siret R, Boursiquot JM, Merle MH, Cabanis JC, This P (2000) Toward the authentication of varietal wines by the analysis of grape (Vitis vinifera L.) residual DNA in must and wine using microsatellite markers. J Agric Food Chem 48: Siret R, Gigaud O, Rosec JP, This P (2002) Analysis of grape Vitis vinifera L. DNA in must mixtures and experimental mixed wines using microsatellite markers. J Agric Food Chem 50: This P, Jung A, Boccacci P, Borrego J, Botta R, Costantini L, Crespan M, Dangl GS, Eisenheld C, Ferreira-Monteiro F, Grando S, Ibáñez J (2004) Development of a standard set of microsatellite reference alleles for identification of grape cultivars. Theor Appl Genet 109: Thomas MR, Scott NS (1993) Microsatellite repeats in grapevine reveal DNA polymorphisms when analysed as sequence-tagged sites (STSs). Theor Appl Genet 86: Veloso MM, Almadanim MC, Baleiras-Couto MM, Pereira HS, Carneiro LC, Fevereiro P, Eiras-Dias JE (2010) Microsatellite database of grapevine (Vitis vinifera L.) cultivars used for wine production in Portugal. Cienc Tec Vitivinic 25:53 61 Vietina M, Agrimonti C, Marmiroli M, Bonas U, Marmiroli N (2011) Applicability of SSR markers to the traceability of monovarietal olive oils. J Sci Food Agric 91: Wu Z-L, Fang L-Y, Wang J, Shen Y-J (2009) Analysis genetic diversity of Vitis by using ISSR Markers. Acta Hort 827: Zietkiewicz E, Rafalski A, Labuda D (1994) Genome fingerprinting by simple sequence repeat (FSR)-anchored polymerase chain reaction amplification. Genomics 20:

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