DOI: 10.14720/aas.2016.107.2.19 Agrovoc descriptors: Vitis vinifera, grapevine, grape must, clones, Aromatic compounds, flavour compounds, gas chromatography, mass spectrometry, statistical methods, multivariate analysis, production location Agris category code: F40, Q04, u10 Clone candidates differentiation of grapevine Vitis vinifera'Škrlet bijeli' using aroma compounds detected by gas chromatography-mass spectrometry Ivana Vladimira PETRIC1, Tatjana KOŠMERL2, Ivan PEJIC3, Veronika KUBANOVIC4, Emil ZLATIC5 Received August 01, 2016; accepted September 30, 2016. Delo je prispelo 01. avgusta 2016, sprejeto 30. septembra 2016. ABSTRACT The aim of this work was to investigate existence presence and stability of must specific aroma compounds (monoterpenes C13-norisoprenoids, C6-alcohols, alcohols, esters and carbonyl compounds) and which can be used to establish differences among clone candidates of 'Skrlet bijeli' (Vitis vinifera L.) grapevine variety. The compounds responsible for the varietal aroma profile were determined by gas chromatography- mass spectrometry (GC-MS), in must samples of ten clone candidates grown on two vineyard sites for three consecutive years. Significant variation among clone candidates is shown in 22 out of the total 35 identified aroma compounds. Significant impact of the vineyard site on the clone candidate's aroma profile was identified. Differences in primary aroma compounds responsible for flavour of 'Skrlet bijeli' variety, linalool, terpinolen, nerol and a-terpineol, were not significant among clone candidates, while remarkable differences were established for P-damascenone. Contrary to expectation, monoterpene geraniol was not detected. Other identified aroma compounds (trans-ocimene, 2-methyl-1-butanol, myrcene, a-phelandrene, cis-ocimene and 3-methyl-1-butanol) noticeably less participate in total flavour description, but they still enable notable clone candidates discrimination. Key words: clonal selection, must, aroma compounds, gas chromatography-mass spectrometry (GC-MS), multivariate statistical analysis IZVLEČEK RAZNOLIKOST KLONSKIH KANDIDATOV Vitis vinifera 'ŠKRLET BIJELI' V AROMATIČNIH SNOVEH, DOLOČENIH S PLINSKO KROMATOGRAFIJO-MASNO SPEKTROSKOPIJO Namen tega dela je bil ugotoviti prisotnost in stabilnost specifičnih aromatičnih spojin mošta (monoterpeni C13-norizoprenoidi, C6-alkoholi, alkoholi, estri in karbonilne spojine) ter katere od teh spojin se lahko uporabljajo za razlikovanje klonskih kandidatov sorte Vitis vinifera 'Škrlet bijeli'. V ta namen smo spojine, ki so odgovorne za sortni aromatični profil, identificirali s plinsko kromatografijo-masno spektrometrijo (GC-MS), v vzorcih mošta desetih klonskih kandidatov, ki rastejo na dveh lokacijah, v treh zaporednih letnikih. Za določanje razlik med kloni in opredelitev spojin, ki so odgovorne za te razlike, smo uporabili multivariatne statistične metode (analizo glavnih komponent in linearno diskriminantno analizo). Značilne razlike med klonskimi kandidati so se pokazale v 22 od skupno 35 identificiranih aromatičnih spojin. Za aromatski profil klona smo ugotovili prevladujoč vpliv lokacije vinograda. Razlike v primarnih aromatičnih spojinah, odgovornih za aromo 'Škrlet Bijeli', linaloola, terpinolena, nerola in a-terpineola, niso bile statistično značilne med klonskimi kandidati, medtem ko so bile določene pomembne razlike v 0-damascenonu. V nasprotju s pričakovanji, monoterpena geraniola nismo določili. Druge določene aromatične spojine (trans-ocimen, 2-metil-1-butanol, mircen, a-felandren, czs-ocimen in 3-metil-1-butanol) občutno manj sodelujejo pri skupnem opisu sortne arome, vendar še vedno omogočajo razlikovanje klonskih kandidatov. Ključne besede: klonska selekcija, mošt, aromatične spojine, plinska kromatografija-masna spektrometrija (GC-MS), multivariatna statistična analiza 1 dr., Croatian Center for Agriculture, Food and Rural Affairs, Institute of Viticulture and Enology, Jandriceva 42, Zagreb, Croatia, e-mail: ivana.petric@hcphs.hr 2 prof. dr., University of Ljubljana, Biotechnical Faculty Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia, e-mail: tatjana.kosmerl@bf.uni-lj.si 3 prof. dr., University of Zagreb, Faculty of Agriculture, Svetošimunska 25, 10000 Zagreb, Croatia, e-mail: ipejic@agr.hr 4 dr., same address as 1, e-mail: veronika.kubanovic@hcphs.hr 5 dr., same address as 2, e-mail: emil.zlatic@bf.uni-lj.si This article is a part of doctoral thesis entitled »Evaluation of clonal candidates Škrlet bijeli (Vitis vinifera L.) based on indicators of productivity and grape quality«, issued by Ivana Vladimira Petric, supervisor Prof. Ivan Pejic, Ph. D. / Prispevek je del doktorske disertacije Ivane Vladimire Petric z naslovom »Ovrednotenje klonskih kandidatov sorte 'Škrlet bijeli' (Vitis vinifera L.) na osnovi kazalnikov rodnosti in kakovosti grozdja«, mentor prof. dr. Ivan Pejic Acta agriculturae Slovenica, 107 - 2, september 2016 str. 483 - 482 Ivana Vladimira PETRIC et al. 1 INTRODUCTION Individual clonal selection is the method most commonly used for genetic improvement of autochthonous grapevine varieties, which results by divergent clones. Usually the selection is performed on grape quality parameters such as sugars and acids content, while aroma profile is checked in late selection stages. How the wine aroma profile is important for wine identity and quality and as aroma precursors originated from must, it is very important to include monitoring of must aroma compounds in early stages of clonal selection. Specific varietal wine aroma originates from volatile compounds such as monoterpenes, norisoprenoids, aliphatic compounds, phenylpropanoids, methoxypyrazine and volatile sulfur compounds synthetized in grapes, which in numerous combinations make a unique, distinctive, typical varietal aroma (Coombe and McCarthy, 1997; Ebeler and Thorngate, 2009). Listed compounds can be used for varietal identification (Marais, 1983; Rapp and Mandery, 1986) because characteristic wine aroma of specific variety is attributed to the aroma compounds of grape. The most important group of grape compounds with the greatest contribution to distinctiveness of aroma variety are terpenes (Marais, 1983; Cámara et al., 2007) to which belongs groups of monoterpene and C13 norisoprenoids (Mateo and Jiménez, 2000). The biggest impact on wine primary aroma provide monoterpenes that are present in wine in free form, while they are present in grape in both free and bound glycoside forms. Monoterpens have very important contribution on white wines aroma of Muscat varieties, but also on other aromatic varieties (Mateo and Jiménez, 2000; del Caro et al., 2012). Free monoterpenes are present in less aromatic and non-aromatic varieties in significantly lower concentration (Iyer et al., 2010; Genovese et al., 2013). Aroma precursor's analysis has been used as a strategy to determine the aroma potential of grape both from aromatic and non-aromatic varieties (Loscos et al., 2009). Monoterpenes have strong impact on wine flavor character that is verified by strong correlation of linalool and a-terpineol content and floral description of wine (Komes et al., 2006; Skinkis et al., 2008; Sánchez-Palomo et al., 2012). Aroma profile is extremely important in clonal selection procedure despite that main subject is always wine aroma profile but not grape aroma. Koch et al. (2010) highlighted a significance of aroma compound 2-methoxy-3-isobutylpyrazine in clonal selection of 'Cabernet Sauvignon' variety as characteristic compound of primary aroma, while Boidron (1995) quoteed that wine of two clones of 'Chardonnay' variety have pronounced Muscat aroma tint in comparison to other studied clones and recognizes this as positive and desirable clone characteristic. Versini et al. (1990) stated that it is for quality of clone grapes comparison necessary to study specific aroma compounds on which content in grapes the effect of environmental is at minimum, they are monoterpenes and by them were defined differences between clones of 'Traminer Red' and 'Chardonnay' variety. Marais and Rapp (1991) concluded that it is possible to distinguish clones based on terpene content. They proved that clones 457/48, 14Gm D35, 925/643 and FR46/106 of 'Gewiirztraminer' variety appeared to have a greater potential to produce aroma-rich and variety-typical wines than N20 Kieselberg. With respect to 'Weisser Riesling', two clones, namely 37 and 327, could possibly be selected as more flavorful than the others. McCarthy (1992) studied grapes of 10 clones 'Muscat a petite grains blanc' variety and found that there was no difference in the free volatile terpene concentration between clones, but there were significant differences in bounded form of monoterpene concentration. In order to assess the suitability of some genotypes for functional genomics studies on terpenol synthesis in grapevine, Duchene et al. (2009) studied two varieties differing in their aromatic pattern: 'Gewurztraminer' and 'Sauvignon Rose' and two clones of 'Chardonnay' (76 and 809). There are evidences that clonal variation, through somatic mutations, can modify the aromatic profile of fruits. Genovese et al. (2013) reported results that showed different aroma profile (free and bound volatile compounds) in 'Aglianica' and 'Uva di Troia' grapes. Various authors are using different methods for determination of terpenes in grape and wines. Setkova et al. (2007) developed a rapid headspace solid-phase micro extraction-gas chromatographic- 484 Acta agriculturae Slovenica, 107 - 2, september 2016 Clones differentiation of cv. 'Skrlet bijeli' (Vitis vinifera L.) ... detected by gas chromatography-mass spectrometry time-of-flight mass spectrometric method for qualitative profiling volatile fraction of wines. Volatile compounds of grapes are responsible of varietal aroma. In order to obtain an appropriate technique to study grape volatile compounds in pulp and skins of 'Muscat' grapes, Sánchez-Palomo et al., (2005) have developed HS-SPME method coupled with GC-MC. Sixteen volatile compounds have been quantified. Prosen et al. (2007) using synthetic solution developed an extraction procedure for the aroma compounds from musts and wines, using solid-phase micro extraction. The method was suitable for analyzing free aroma compounds in must of different varieties and for monitoring of their release after enzymatic or acidic hydrolysis. Coelho et al. (2007) propose headspace-solid phase micro extraction (HS-SPME) for the variety- and pre-fermentation-related volatile compounds of 'Fernao-Pires' (FP) white grape berries. Two C13 norisoprenoids, two aromatic alcohols, two C6 aldehydes, and three C6 alcohols were identified by gas chromatography-quadruple mass spectrometry (GC-qMS). Bordiga et al. (2013) suggest using combination of HS-SPME technique with GCxGC/TOF-MS system for the analysis of wine volatile compounds. In a last few years, not only on Croatian but also on European wine market, there is a growing interest for wines made of local grapevine varieties with distinguish quality. These wines contribute in raising a regional wine identity and tourism potential. Therefore, efficiency of individual clone selection is very important to provide high quality propagating material and revitalization of forgotten local varieties. 'Skrlet bijeli' is autochthonous variety that grows on very limited areas in continental region of Croatia, in Pokuplje, Vukomericke gorice and Moslavina. Vineyards planted with 'Skrljet bijeli' represent only 0.3 % of all Croation vineyard area or 66 ha. It is characterized by discrete aroma and freshness, which consumers recognize and prices. Up to now on propagating material market for this particular variety was available only material of the lowest category (CAC; Conformitas Agraria Communitatis). Final phase of individual clonal selection, where is studying the most perspective clone candidates is in progress. The aim of this work was (1) to investigate the presence and stability of specific aroma compounds (monoterpenes, C13-norisoprenoids, benzenoids, alcohols C6, alcohols, esters and carbonyl compounds) in must of 10 clone candidates of 'Skrlet bijeli' variety applying GC-MS method, with intention of (2) their mutual distinction and identification of clones by multivariate analysis. 2 MATERIALS AND METHODS 2.1 Samples Ten clone candidates of 'Skrlet bijeli' variety produced by propagation of elite vines, selected in process of mass positive clonal selection, and planted in two sites, Popovaca and Repusnica, during three vintages were investigated. Both locations are in viticulture region of continental Croatia, sub-region Moslavina. Production viticulture zone is B (Winkler et al., 1974). Both field trails were planted in period from year 2001 to 2004. Clone candidates coded as SK-07, SK-11, SK-29, SK-32, SK-33, SK-57, SK-60, SK-69, SK-74, SK-77 represent progeny of individual elite vine selected from old vineyards. All clone candidates originated from mass clonal selection of 'Skrlet bijeli' which was performed on agricultural traits (yield and sugar accumulation) as well as on good vigour of mother plants. In the field trails, each clone candidate was represented with 3-5 vines planted in same row, that were grafted, at the place, with gem originated from elite vine by method „green on green" on virus-free rootstock: Leaf Roll Virusis.(LR1 and LR3) and Raspberry Ringspot Virus (RRSV). Harvest date was determined based on phenotype evaluation and refractometry tracking of sugar accumulation dynamics and was harvested at the same date for all clone candidates at about 85 °Oe as it is common sugar content at harvest of 'Skrlet bijeli'. The basic agricultural traits (yield and sugar accumulation) are listed in Annexes 1-2. Sampling for aroma compound analysis carried out according to the plan: first vintage year 2006, the average samples of must for four clone candidates SK-29, SK-33, SK-57 and SK-69; second (2007) Acta agriculturae Slovenica, 107 - 2, september 2016 Ivana Vladimira PETRIC et al. and third (2008) vintage year, the average samples of must for ten clone candidates SK-29, SK-32, SK-33, SK-60, SK-69, SK-74, SK-77, SK-07, SK-11, SK-57 were prepared from both location. Must samples were frozen at - 28 °C and defrost right before analysis. 2.2 Head-space SPME extraction The SPME extraction conditions were 10 ml of sample that was spiked with internal standard of 3-octanol in concentration of 0,0844 (g l-1 (Sigma-Aldrich, St. Louis, MO, USA) in a 20 ml glass headspace vials, with addition of 1,5 g NaCl, extraction time of 45 min and extraction temperature of 50 °C under stirring at 350 r min-1. The headspace was sampled using a 50/30 (m divinylbenzene-carboxen-poly(dimethylsiloxane) (DVB-CAR-PDMS) coated fiber in a Supelco fiber holder (Bellefonte, PA, USA). After equilibration, the fiber was removed from the sample and the analytes were thermally desorbed in the injector port of the GC. 2.3 GC-MS A multipurpose autosampler MPS2 (Gerstel GmbH, Germany) with an agitator and SPME fiber conditioning station was used to extract the volatiles from sample vial headspace. All chromatographic analysis was performed using an Agilent 7890A Series GC system with an Agilent 5975C Mass Selective Detector (Agilent, Palo Alto, USA). The apparatus used was equipped with split/split less injector, J&W DB-Wax column (60 m length x 0.32 i.d. x 0.25 (m film thickness (J&W Scientific, Folsom, CA, USA). The temperature program used was 40 °C for 5 min; 4 °C min"1 to 230 °C; 20 min at maximum temperature. Carrier gas (He) flow was 1.2 ml min" 1. Injections of 1 (j,l were performed in split less mode while the injector port and the ion source were maintained at 230 °C and 250 °C, respectively. Positive electron impact spectra were recorded at 70 eV in a range m/z 30 - 250. Mass spectrometric information of each chromatographic peak was compared to NIST (National Institute for Standards and Technology, USA) mass spectra library. Data is given as relative peak area (RPA) ± standard deviation (SD) presented ratio of area peak of identified compound and peak area of internal standard. 2.4 Statistical analysis Significant differences between clones of 'Šklet bijeli' were determined on the basis of the most abundant aroma compounds: linalool, P-damascenone, terpinolen, nerol and a-terpineol by one-way analysis of variance using software package Statistics (version 8.0, Statsoft Inc., Tulsa, USA), while differences between averages of RPA by Student-Newman-Keul test. Principle component analysis (PCA) and linear discriminant analysis (LDA) were performed to classify the grapevine clone candidates regarding to vineyard site and vintage. A total of 35 major aroma compounds were included in analysis. PCA was performed to provide a data structure study over a reduced dimension, covering the maximum amount of the information present in the basic data. It was conducted using software Statistical Package for the Social Sciences (version 15.0 for Windows; SPSS Inc., Chicago, USA) and statistics software (version 8.0; Statsoft Inc., Tulsa, USA). Annex 1: Yield (kg of grape per stock) of clone candidates in investigated vintages and vineyard sites Priloga 1: Pridelek (kg grozdja po trsu) klonskih kandidatov v proučevanih letnikih in lokacijah Clone candidate 2006 2007 2008 Popovača Repušnica Popovača Repušnica Popovača Repušnica SK-07 3.40 2.21 5.88 2.38 3.70 3.95 SK-11 2.87 0.76 3.82 2.91 3.76 3.39 SK-29 4.04 1.40 4.63 3.73 2.79 3.30 SK-32 3.34 1.78 2.34 1.40 3.67 1.67 SK-33 2.20 1.55 4.04 3.62 3.00 2.10 SK-57 2.82 1.41 2.32 2.91 3.09 2.13 SK-60 3.89 1.37 2.29 2.62 3.57 3.32 SK-69 2.44 1.62 3.80 4.95 3.27 4.09 SK-74 4.20 1.58 2.25 4.33 2.96 2.13 SK-77 4.20 1.50 2.87 4.29 3.89 3.84 Average 3.34 1.50 3.32 3.22 3.33 3.02 486 Acta agriculturae Slovenica, 107 - 2, september 2016 Clones differentiation of cv. 'Skrlet bijeli' (Vitis vinifera L.) ... detected by gas chromatography-mass spectrometry Annex 2: Sugar content (g l-1) of clone candidates in investigated vintages and vineyard sites Priloga 2: Vsebnost sladkorja (g l-1) klonskih kandidatov v proučevanih letnikih in lokacijah Clone candidate 2006 2007 2008 Popovača Repušnica Popovača Repušnica Popovača Repušnica ŠK-07 205.0 186.8 187.4 229.7 179.4 179.8 ŠK-11 224.3 205.2 224.5 191.2 216.4 221.0 ŠK-29 217.0 188.0 206.9 224.6 223.4 168.2 ŠK-32 215.3 170.7 245.1 215.5 211.5 188.7 ŠK-33 215.8 194.2 206.8 206.0 207.6 208.2 ŠK-57 217.7 185.0 215.3 200.3 189.5 205.7 ŠK-60 196.3 162.8 222.3 186.4 168.3 153.5 ŠK-69 213.0 181.3 192.5 196.1 207.0 173.4 ŠK-74 196.0 190.3 221.5 201.7 209.0 182.4 ŠK-77 186.7 196.3 201.9 194.5 217.7 177.4 Average 208.8 186.3 213.5 204.6 204.8 188.6 3 RESULTS AND DISCUSSION 3.1 Free terpenes content in grape must In grape must of ten clone candidates 'Skrlet bijeli', the most abundant aroma compounds were as followed: linalool, P-damascenone, terpinolen, nerol and a-terpineol. However, monoterpene geraniol was not identified. According to the literature, the most usually analyzed aroma compounds in grape must generally are linalool, geraniol, nerol, a-terpineol and P-damascenone (Sánchez-Palomo et al., 2012; Gómez García-Carpintero et al., 2011). In order to determine if there are differences among clones on the level of free terpene compounds linalool, P-damascenone, terpinolen, nerol and a-terpineol expressed as mean RPA values for all ten clones, the belonging rank of significant differences was determined by analysis of variance. The results are presented in Table 1. Acta agriculturae Slovenica, 107 - 2, september 2016 Ivana Vladimira PETRIC et al. Table 1 The influence of clone candidates of 'Škrlet bijeli' on the content of linalool, p-damascenone, terpinolen, nerol and a-terpineol (mean value of RPA ± SD) with belonging rank of significant differences, for both vineyard sites through two (n = 4) or three (n = 6) years, respectively. Preglednica 1: Vpliv klonskih kandidatov 'Škrlet bijeli' na vsebnosti linaloola, p-damascenona, terpinolena, nerola in a-terpineola (povprečna vrednost relativne ploščine vrha ± SD) s pripadajočim rangom značilnih razlik, za obe lokaciji tekom dveh (n = 4) oziroma treh (n = 6) let. Clone Linalool P-Damascenon Terpinolen Nerol (RPA / 106) a-Terpineol ŠK-07 4 57440 ± 36976 a* 10621±4117 ab 1863±557 a 1599±2137 a 1257±1126 a ŠK-11 4 32705 ± 38096 a 12198±7591 ab 1522±1311 a 869 ±1645 a 754±1144 a ŠK-29 6 61376± 88124 a 8479±4461 a 2436 ± 2744 a 2543 ± 4030 a 1523 ±2311 a ŠK-32 4 98760 ± 91769 a 14019±7740 ab 6518±6917 a 3168±3799 a 2696 ± 2929 a ŠK-33 6 96225 ± 108533 a 11924±3845 ab 5593±4995 a 2627 ± 4793 a 2270±3104 a ŠK-57 6 94418± 90699 a 16135±6800 b 9573 ± 12949 a 2396 ± 4452 a 2836±4030 a ŠK-60 4 89147 ± 95225 a 16826 ± 9992 b 7078 ± 7568 a 2033±3101 a 2983 ± 3279 a ŠK-69 6 56035 ± 37655 a 9442 ± 2654 a 4693 ± 4096 a 1152±1597 a 1356 ±1404 a ŠK-74 4 61547±54192 a 13340±5299 ab 4455±4217 a 1656 ± 2006 a 1779±2001 a ŠK-77 4 52773 ± 36890 a 10821±3911 ab 4373 ± 4546 a 1092 ± 1194 a 1726 ±1970 a *Student-Newman-Keul test; values in column marked by the same letter are not significantly different (p < 0.05) n Acta agriculturae Slovenica, 107 - 2, september 2016 Clones differentiation of cv. 'Skrlet bijeli' (Vitis vinifera L.) ... detected by gas chromatography-mass spectrometry Neither linalool, with the highest RPA values which ranged from 32705 to 98760, nor terpinolen (RPA ranged from 1522 to 9573), nerol (RPP ranged from 869 to 3168) nor a-terpineol (RPP ranged from 754 to 2983) content were not statisticaly significantly different among clone candidates. RPA values of norisoprenoid compound P-damascenone were statistically different and clone candidats SK-29 and SK-69 had lower RPA values then clones SK-57 and SK-60). These results were expected because the study was done on progeny obtained by vegetative propagation of the same plant, and selection of ten clone candidates that were subject of this study based only on phenotype selection of clones from population, selection was not include preliminary aroma compounds analysis. The impact of vintage and vineyard site on the RPA values of linalool, P-damascenone, terpinolen, nerol and a-terpineol in grape must of clone candidates of 'Skrlet bijeli' with the belonging rank of significant differences was determined by analysis of variance. The results were presented in Table 2 and 3. Acta agriculturae Slovenica, 107 - 2, september 2016 Ivana Vladimira PETRIC et al. Table 2: The influence of vintage on the content of linalool, P-damascenone, terpinolen, nerol and a-terpineol (mean value of RPA ± SD) in must with belonging rank of significant differences for 10 clone candidates of 'Škrlet bijeli' for both sites. Preglednica 2: Vpliv letnika trgatve na vsebnosti linaloola, P-damascenona, terpinolena, nerola in a-terpineola (povprečna vrednost relativne ploščine vrha ± SD) v moštu s pripadajočim rangom značilnih razlik za 10 klonskih kandidatov 'Škrlet bijeli' za obe lokaciji. Linalool P-Damascenon Terpinolen Nerol a-Terpineol Year n (RPA / 106) 2006 8 58837±31215 a* 9362±2961 a 4701±2467 a 246 ± 134 a 938±544 a 2007 20 19730 ± 12942 a 9765 ± 4492 a 1073 ± 622 a 141±338 a 310±385 a 2008 20 127626±77314 b 15849±6167 b 8898 ± 7756 b 4460 ± 3445 b 3950±2631 b F exp 24.23 8.29 13.67 23.41 2711 Pr > F < 0.0001 0.0009 < 0.0001 < 0.0001 < 0.0001 *Student-Newman-Keul test; values in column marked by the same letter are not significantly different (p < 0.05); p-value is the significance; Pr > F - This is the p-value associated with the F-statistic. It is used in testing the null hypothesis that all of the model coefficients are 0; F - This is the F-statistic is the mean square model divided by the mean square error; F exp - this is the variance between treatments devided by the variance within treatments Table 3: The influence of vineyard site on the content of linalool, P-damascenone, terpinolen, nerol and a-terpineol (mean value of RPA ± SD) with belonging rank of significant differences for 10 clone candidates of 'Škrlet bijeli', for both vineyard sites through three years. Preglednica 3: Vpliv lokacije vinograda na vsebnosti linaloola, P-damascenona, terpinolena, nerola in a-terpineola (povprečna vrednost relativne ploščine vrha ± SD) s pripadajočim rangom značilnih razlik za 10 klonskih kandidatov 'Škrlet bijeli' za obe lokaciji. Linalool P-Damascenon Terpinolen Nerol a-Terpineol Location n (RPA / 106) Popovača 24 78129±90991 a* 11826±6206 a 6288±8190 a 2539±3921 a 2469±3082 a Repušnica 24 59333±42874 a 12639 ± 5606 a 3587±2764 a 1377±1778 a 1393±1414 a F exp 1.45 0.11 1.74 1.85 2.97 Pr > F 0.235 0.739 0.194 0.182 0.092 *Student-Newman-Keul test; values in column marked by the same letter are not significantly different (p < 0.05); p-value - this is the significance; Pr > F - This is the p- value associated with the F-statistic. It is used in testing the null hypothesis that all of the model coefficients are 0; F - This is the F-statistic is the Mean Square Model divided by the Mean Square Error; F exp - this is the variance between treatments/variance within treatments Acta agriculturae Slovenica, 107 - 2, september 2016 Clones differentiation of cv. 'Skrlet bijeli' (Vitis vinifera L.) ... detected by gas chromatography-mass spectrometry Results of vintage year and vineyard site influence on RPA values of five the most abundant terpene compounds linalool, P-damascenone, terpinolen, nerol and a-terpineol in must (Table 2 and 3), show during year 2008, significantly higher RPA values for all five dominant aroma compounds which indicates favourable climatological conditions for their individual synthesis. At the same time, RPA values for all five the most abundant terpene compounds were not significantly influenced by vineyard site. It is not possible to make a real judgment on must aroma based on processing data of individual aroma compounds because different combination of compound concentration at the end bring different wine olfactory experience (Robinson, 2011; Botelho, 2008). To determine ifthere is stable correlation between clones, vintage years and location and all 35 detected aroma compounds, PCA method was used. Results of PCA analysis imply that the loading values of the variables associated with the first five principal components were as followed: cis-ocimene, trans-ocimene, myrcene, limonene, geranic oxide and hotrienol, were the dominant variables in the first principal component, which accounted for 32 % of the total variance. The 2-methyl-1-butanol, 3-methyl-1-butanol, ethyl acetate, hexyl acetate, isoamyl acetate, acetaldehyde, 2-hexenal and 2-hexene-1-ol (E) dominated the second principal component that explained up to 26 % of the total variance. The first five principal components thus accounted for 83 % (PC3 14 %, PC4 8 % and PC5 5 %) of the variation among the samples analyzed. Out of these 35 parameters, 12 were recognized by PCA as being less important. Therefore, the remaining 22 parameters were included in the LDA test (Table 4). Acta agriculturae Slovenica, 107 - 2, september 2016 Ivana Vladimira PETRIC et al. Table 4: Content of aroma compounds present in grape musts of 10 clone candidates of 'Škrlet bijeli' Preglednica 4: Vsebnosti aromatičnih spojin, prisotnih v moštu 10 klonskih kandidatov 'Škrlet bijeli' Aromatic compound* Sample A** B C D E F G H I J K L M N O P Q R S T U V P200629*** 432 9285 177 n.d. 810 44 6 76 1878 2722 5 19 34 300 60 142 21 49090 591 24 156 P200633 355 2946 134 96 83 474 24 40 697 424 727 59 141 368 2693 67 83 29 38395 1352 105 697 P200657 373 7243 235 105 122 442 21 36 685 506 837 60 148 328 2361 45 119 25 48568 1396 115 728 P200669 491 11215 178 110 217 536 29 57 815 979 2180 76 133 421 3232 57 136 21 46481 621 138 918 P200707 328 2002 3732 81 86 414 47 52 571 298 551 115 1299 159 1182 6 80 42 61337 12181 n.d. 474 P200711 237 2242 2004 3 56 60 n.d. n.d. 72 65 145 9 1049 23 117 5 46 26 52399 17743 n.d. 217 P200729 413 25950 2319 n.d. 1364 16 n.d. n.d. 31 539 1120 n.d. 725 4 22 3 420 26 77309 8422 n.d. 80 P200732 393 8537 4659 6 103 112 4 7 93 291 590 18 1475 38 246 4 185 32 80501 7636 n.d. 341 P200733 465 38886 1751 35 2854 175 9 16 235 1154 1810 32 339 79 484 4 648 24 65414 1070 45 544 P200757 450 3459 2668 53 155 185 10 16 242 486 826 35 741 83 503 3 155 32 67941 2637 53 526 P200760 392 12400 1733 17 1221 152 7 10 193 1001 2152 24 368 68 387 7 245 34 58110 440 n.d. 322 P200769 245 1328 980 19 118 90 2 6 142 279 574 14 426 43 223 6 80 16 47649 2976 n.d. 263 P200774 402 9047 2701 7 159 96 3 7 115 366 694 15 915 42 243 6 194 28 62514 3801 n.d. 227 P200777 226 2191 1786 37 67 304 19 26 326 196 384 51 912 132 862 4 80 32 54642 11820 46 590 P200807 975 72796 59 12 4385 223 14 22 190 671 1483 33 156 93 756 73 2050 15 56091 3964 26 344 P200811 142 2249 272 14 45 219 94 66 204 22 64 161 1588 89 650 10 85 9 36842 19595 26 418 P200829 588 9445 553 93 675 2067 170 179 1386 914 1471 344 439 571 5238 30 379 34 123024 3368 31 276 P200832 428 6993 44 162 143 1305 128 185 1905 219 509 263 351 692 6338 19 196 12 59320 10002 42 612 P200833 884 92124 120 176 1602 2569 251 304 2390 766 1389 541 190 885 8033 338 929 53 84514 3376 74 919 P200857 295 10949 423 578 133 3320 263 432 4905 83 463 534 1157 1613 12311 34 132 70 42657 17773 67 672 P200860 203 1654 707 349 46 2318 178 243 2522 35 235 362 1174 935 7460 13 83 42 34643 18186 42 407 P200869 136 1746 716 136 21 817 82 133 1380 26 144 146 1686 452 4067 10 63 8 29348 13378 41 518 P200874 193 2127 448 164 36 915 95 130 1184 72 220 198 1189 384 3581 26 162 18 61264 21516 52 760 P200877 151 2039 813 203 28 928 88 128 1559 32 160 179 1938 499 4139 8 64 7 26104 13920 58 870 R200629 360 7802 411 9 542 108 91 62 140 1065 2007 132 156 78 669 64 234 11 59173 2626 n.d. 330 R200633 538 78267 145 51 4485 543 36 69 687 1731 2985 82 25 412 3791 67 828 8 41660 526 91 853 R200657 833 69384 154 85 4046 783 59 82 918 1767 2771 134 28 533 4702 113 1054 11 48843 1000 119 1007 R200669 518 90488 260 36 7057 499 28 56 538 1987 3779 66 22 327 3078 146 1624 23 32974 882 110 900 R200707 415 62791 914 19 4099 180 210 189 157 1153 1900 294 487 86 1095 36 1513 24 64440 3738 50 639 R200711 224 13746 909 8 1861 57 21 22 73 873 1195 33 475 35 378 12 1376 53 63692 3819 22 203 R200729 195 26649 1334 n.d. 722 39 1515 782 40 418 725 1775 862 17 271 19 472 22 65137 10472 n.d. 221 R200732 468 97154 1834 6 7985 146 1952 1025 120 1326 2109 2433 666 73 841 103 3489 31 74030 1305 33 380 R200733 400 38595 1285 4 2517 101 40 33 97 663 1323 54 433 56 518 21 2002 24 59930 3558 21 297 R200757 386 60035 840 7 4709 146 108 79 129 1570 2030 129 246 78 748 17 2036 25 80490 1710 27 351 R200760 297 29803 922 17 1588 140 39 34 123 468 904 60 525 73 611 12 1860 25 66148 11042 30 460 R200769 196 2093 837 4 394 69 3 9 68 876 1200 10 365 35 331 9 431 21 92184 4165 21 180 R200774 295 11357 1045 18 1087 139 9 20 156 492 787 23 972 80 699 10 764 19 65886 12623 38 313 R200777 300 21365 2484 4 1422 75 4 7 79 1026 1350 16 957 32 227 13 516 26 74599 10441 18 202 R200807 1522 103793 173 28 6620 635 127 92 398 1936 2760 239 361 161 1595 81 2693 24 79533 2899 40 418 R200811 1166 49889 109 66 2100 568 139 112 549 1231 1993 251 78 174 1734 30 1103 23 83628 2525 35 425 R200829 715 136871 75 38 22703 811 1194 813 656 2814 4301 1725 145 228 2398 59 2050 18 53826 1804 58 326 R200832 1935 164594 73 103 7920 1184 188 205 1026 1910 3172 354 51 431 4276 40 3388 45 86157 1857 97 1430 R200833 730 24316 75 85 976 676 480 370 811 532 1062 805 231 263 2605 35 866 27 73882 9542 57 612 R200857 350 4684 996 106 269 705 110 135 975 863 1103 211 752 315 2938 117 278 23 83945 7493 69 850 R200860 1403 68263 162 396 3655 757 70 100 1830 1050 1745 164 81 424 3486 19 826 34 66365 4360 107 1121 R200869 188 2782 988 42 198 534 61 68 637 298 487 121 1059 228 1985 7 126 14 41303 15781 35 259 R200874 427 13648 99 87 835 721 172 164 933 761 1204 306 220 336 2803 18 426 17 65548 9222 64 703 R200877 305 22093 63 56 1805 492 54 68 695 948 1281 110 23 236 2020 11 410 10 40714 505 42 370 *mean values of aroma compounds expressed in relative peak area (x106) calculated for three replicates; ** aroma com pounds: A=acetaldehyde, B=ethyl acetate, C=hexanal, D=geranic oxide, E=isoamyl acetate, F=myrcene, G=a-felandren, H=a-terpinene, I=limonene, J=2-methyl-1-butanol, K=3-methyl-1-butanol, L=p-phellandrene, M=2-hexenal, N=trans-ocimene, O=cz's-ocimene, P=cinnamen, Q=hexyl acetate, R=6-methyl-5-hepten-2-one, S=1- hexanol, T=2-hexene-1-ol (E), U=Z-linalool oxide, V= hotrienol; ***sample code: location (P-Popovaca, R-Repusnica), vintage year (2006-2008), code of clones; n.d.=indication that aroma compound is at level lower to detection limit 492 Acta agriculturae Slovenica, 107 - 2, september 2016 Clones differentiation of cv. 'Skrlet bijeli' (Vitis vinifera L.) ... detected by gas chromatography-mass spectrometry 3.2 Influence of vineyard site and clone candidates on aroma compounds content Using LDA method, six parameters were selected as the most discriminating variables: trans -ocimene, 2-methyl-1-butanol, myrcene, a-phelandrene, cis-ocimene and 3-methyl-1-butanol. The other five parameters isoamyl acetate, acetaldehyde, Z-linalool oxide, ethyl acetate and limonene also contribute significantly to better separation among the samples. When the LDA was Figure 1: Projection of the scores of the samples (right) and parameters (left) for 48 must samples depending on vineyard site and clone candidates in the plane defined by the two standardized canonical discriminant function coefficients. Slika 1: Projekcija rezultatov vzorcev (desno) in parametrov (levo) za 48 vzorcev mošta v odvisnosti od lokacije vinograda in klonskih kandidatov v ravnini, ki ju določata standardizirani funkciji diskriminantnih koeficientov. From Figure 1 it can be seen, that only two clone candidates (ŠK-69 and ŠK-33) on two vineyard sites (Popovača and Repušnica) do not show differences in analyzed parameters. applied to the data (48 samples, 22 variables), three discriminate functions explained 80 % of the total variance. Function 1 explains 43.8 % of the total variance, function 2 explains 23.4 %. The scores of the samples and parameters for these first two functions are plotted on Figure 1. As it can be seen, the samples are well separated depending on vineyard site and clone candidates. The accuracy of the placement of each sample into 20 groups was 100 %. 3.3 Influence of vintage year on aroma compounds in vineyard site Popovaca Using LDA method, four parameters were selected for vintage year of grape production and clone candidate as the most discriminating variables: myrcene and a-phelandrene, as well as trans-ocimene and cis-ocimene. The other ten parameters: (2-hexene-1-ol (E), a-terpinene, acetaldehyde, 2-hexenal, 3-methyl-1-butanol, 2-methyl-1-butanol, isoamyl acetate, hexanal, ethyl acetate and geranic oxide also contribute significantly to better separation among the samples. When the LDA was applied to the data (24 samples, 22 variables), three discriminate functions explained 84.6 % of the total variance. Function 1 explains 48.0 % of the total variance, function 2 explains 26.1 % and function 3 10.5 %. The scores of the samples and parameters for these first two functions are plotted on Figure 2. As it can be seen, the samples are well separated depending on grape production year. The accuracy of the placement of each sample into 10 groups was 1007%. Clone candidates' SK-60, SK-69, SK-77, SK-29, SK-11 and SK-07 during three or two vintage years did not show differences in analyzed parameters. Acta agriculturae Slovenica, 107 - 2, september 2016 Ivana Vladimira PETRIC et al. Figure 2: Projection of the scores of the samples (right) and parameters (left) for 24 must samples from vineyard site Popovača depending on vintage year and clone candidates used in the plane defined by the two standardized canonical discriminant function coefficients. Slika 2: Projekcija rezultatov vzorcev (desno) in parametrov (levo) za 24 vzorcev mošta iz lokacije vinograda Popovača v odvisnosti od letnika trgatve in klonskih kandidatov v ravnini, ki ju določata standardizirani funkciji diskriminantnih koeficientov. 3.4 Influence of vintage year on aroma compounds in vineyard site Repusnica Using LDA method, six parameters were selected for vintage year of grape production and clone candidate as the most discriminating variables: 3-methyl-1-butanol, ethyl acetate and trans-ocimene, as well as 2-methyl-1-butanol, a-phelandrene and a-terpinene. The other eight parameters: 2-methyl-1-butanol, acetaldehyde, hexanal, geranic oxid, isomyl acetate, myrcene, 2-hexanal and hexyl acetate also contribute significantly to better separation among the samples. When the LDA was applied to the data (24 samples, 22 variables), two discriminant functions explained 82.7 % of the total variance. Function 1 explains 60.8 % of the total variance, function 2 explains 21.9 %. The scores of the samples and parameters for these first two functions are plotted on Figure 3. As it can be seen, the samples from site Repusnica are well separated depending on vintage year. The accuracy of the placement of each sample into 10 groups was 100 %. Clones SK-32, SK-57, SK-77 and SK-29 during three or two production years did not show differences in analyzed parameters. O Mil», M-blUHrt HtMun MtfmM Acdanlv» OMU» """i'" B/Kt» SJIi-UCIKIII O Hrnul o O AfianM o j£t»FlrlHdi*K O Î0WÎK-ÏÎ « MwSiu o Ma 7007AK-» « KIHfSK-14 0 MOftrëK-M MOtJ-iK.JV »î/SK-1! m HWfttKM ¡fS«t1 . „ 0 lawi HS 300 M&AfcM ira»» »08/5* »7/4 (i-« M ioor/itn 0 ÎOOWjK-77 ■m -e -s -t -i o 2 F1 [61%) S 10 12 14 Figure 3: Projection of the scores of the samples (right) and parameters (left) for 24 must samples from vineyard site Repušnica depending on vintage and clone candidates used in the plane defined by the two standardized canonical discriminant function coefficients. Slika 3: Projekcija rezultatov vzorcev (desno) in parametrov (levo) za 24 vzorcev mošta iz lokacije vinograda Repušnica v odvisnosti od letnika trgatve in klonskih kandidatov v ravnini, ki ju določata standardizirani funkciji diskriminantnih koeficientov. If compared both vineyard sites it can be concluded that only two clones ŠK-77 and ŠK-29 during investigated vintage years did not show differences in analyzed parameters. 22 Acta agriculturae Slovenica, 107 - 2, september 2016 Clones differentiation of cv. 'Skrlet bijeli' (Vitis vinifera L.) ... detected by gas chromatography-mass spectrometry 4 CONCLUSION Analyzing free volatile terpene compounds responsible for primary aroma compounds responsible for flavour of 'Skrlet bijeli' such as linalool, terpinolen, nerol, a-terpineol and P-damascenone were detected, while contrary to expectation, monoterpene geraniol was not detected. Differences of RPA values for the first four compounds were not significant among clone candidates. However, remarkable differences of RPA values among clone candidates were established for some less represented compounds, as eg. norisoprenoid compound P-damascenone. It is noteworthy that significantly higher RPA values for all five dominant aroma compounds were established in the 2008 vintage, which indicates favourable climatological conditions for their synthesis and they can be used as quantitate indicator for prediction of wine aroma intensity. At the same time, RPA values for all five the most abundant aroma compounds were not significantly influenced by vineyard site and therefore could not effectively discriminated among them. Meanwhile, other aroma compounds were identified (trans--ocimene, 2-methyl-1-butanol, myrcene, a-phelandrene, cis-ocimene and 3-methyl-1-butanol) that noticeably less participate in total flavour description, but they still enable notable clone candidates discrimination using LDA method according to the individual compounds (only within individual vineyard site). Results of these aroma compounds showed that influence of vineyard site (soil, climate, fertilization and other) was dominant over clone genetic potential when it is placed in other environment (or ecological) conditions, that their RPA values for individual clones and their order (rank) were not consistent on different location. However, within individual site, clones were through three very different vintages retained their mutual relations regarding to aroma compounds synthesis and it was possible to differentiate them. Analysis of must aroma compounds enabled positive discrimination of two clones SK-32 and SK-57 in comparison to others. It is necessary to initiate additional comparative study on must and further the wines of these clones in order to find answer what is must aroma profile analysis can help in clonal selection procedure. 5 REFERENCES Boidron, R. (1995). Clonal selection in France - Methods, organization, and use, Proceedings of the International Symposium of Clonal Selection, American Society of Enology Viticulture, Davis, CA, pp 1-7. Bordiga, M., Rinaldi, M., Locatelli, M., Piana, G., & Travaglia, F. (2013). Characterization of Muscat wines aroma evaluation using comprehensive gas chromatography followed by a post-analytic approch to 2D contour plots comparison, Food Chemistry, 140, 57-67. Doi: 10.1016/j.foodchem.2013.02.051 Botelho, G. M. A. (2008). Characterisation of the aroma components of clonal grapes and wines from Aragonez and Trincadeira Vitis vinifera L. cultivars, Ph thesis, Universidade de tras-os-montes e alto douro. Camara, J. S., Alves, M. A., & Marques, J.C. (2007). Clasiffication of Boal, Malvazia, Sercial and Verdelho wines based on terpenoid patterns, Food Chemistry, 101, 475-484. Doi: 10.1016/j.foodchem.2006.02.004 Coelho, E., Rocha, S. M., Barros, A. S., Delgadillo, I., & Coimbra, M. A. (2007). Screening of variety and pre-fermentation related volatile compounds during ripening of white grapes to define their evolution profile, Analytica Chimica Acta, 597(2), 257-264. Doi: 10.1016/j.aca.2007.07.010 Coombe, B. G., & McCarthy, M. G. (1997). Identification and naming of the inception of aroma development in ripening grape berries, Australian Journal of Grape and Wine Research, 3 (1), 18-20. Doi: 10.1111/j.1755-0238.1997.tb00111.x del Caro, A., Fanara, C., Genovese, A., Moio, L., Piga, A., & Piombino, P. (2012). Free and enzymatically hydrolysed volatile compounds of sweet wines from Malvasia and Muscat grapes (Vitis vinifera L.) grown in Sardinia, South Africa Journal of Enology and Viticulture, 33(1), 115-121. Duchene, E., Legras, J. L., Karst, F., Merdinoglu, D., Claudel, P., Jaegli, N., & Pelsy, F. (2009). Variation of linalool and geraniol content within two pairs of aromatic and non-aromatic grapevine clones, Australian Journal of Grape and Wine Research, 15(2), 120-130. Doi: 10.1111/j. 1755-0238.2008.00039.x Acta agriculturae Slovenica, 107 - 2, september 2016 Ivana Vladimira PETRIC et al. Ebeler, S. E. & Thorngate, J. H. (2009). Wine chemistry and flavor: Looking into the crystal glass, Journal of Agricultural and Food Chemistry, 57, 8098-8108. Doi: 10.1021/jf9000555 Genovese, A., Lamorte, A. S., Gambuti, A., & Moio, L. (2013). Aroma of Aglianica and Uva di Troi grapes by aromatic series, Food Research International, 53, 1523. Doi: 10.1016/j.foodres.2013.03.051 Genovese, A., Gambuti, A., Lamorte, S. A., & Moio, L. (2013). An extract procedure for studying the free and glycosilated aroma compounds in grapes, Food Chemistry, 136, 822-834. Doi: 10.1016/j.foodchem.2012.08.061 Gómez García-Carpintero, E., Sánchez-Palomo, E., Gómez, Gallego, M. A., & González-Viñas, M. A. (2011). Volatile and sensory characterization of red wines from cv. Moravia Agria minority grape variety cultivated in La Mancha region over five consecutive vintages, Food Research International, 44, 15491560. Doi: 10.1016/j.foodres.2011.04.022 Iyer, M. M., Sacks, G. L., & Padilla-Zakour, O. I. (2010). Impact of harvesting and processing conditions on green leaf volatile development and phenolics in Concord grape juice, Journal of Food Sciences, 75, 297-304. Doi: 10.1111/j. 1750-3841.2010.01559.x Koch, A., Doyle, C. L., Matthews, M. A., Williams, L. E., &Ebeler, S. E. (2010). 2-Methoxy-3-isobutylpyrazine in grape berries and its dependence on genotype, Phytochemistry, 71(17-18), 2190-2198. Doi: 10.1016/j.phytochem.2010.09.006 Komes, D., Ulrich, D., & Lovric, T. (2006). Characterization of odor-active compounds in Croatian Rhine Riesling wine, subregion Zagorje, European Food Research and Technololgy, 222, 1-7. Doi: 10.1007/s00217-005-0094-y Loscos, N., Hernandez-Orte, P., Cacho, J., & Ferreira, V. (2009). Comparison of the suitability of different hydrolytic strategies to predict aroma potential of different grape varieties, Journal of Agricultural and Food Chemistry, 57, 2468-2480. Doi: 10.1021/jf803256e Marais, J. (1983). Terpens in the aroma of grapes and wines: a review, South African Journal of Enology and Viticulture, 4, 49-60. Marais, J., & Rapp, A. (1991). The selection of aroma-rich clones of Vitis vinifera L. cv. gewürtztraminer and weisser riesling by means of terpene analyses, South African Journal for Enology and Viticulture, 12(1), 51-56. Mateo, J. J., & Jiménez, M. (2000). Monoterpene in grape juice and wines, Journal Chromatography A, 881, 557-567. Doi: 10.1016/S0021-9673(99)01342-4 McCarthy, M. G. (1992). Clonal and pruning effects on Muscat á petite grains blanc yield and terpene concentration, American Journal of Enology and Viticulture, 43(2), 149-152. Prosen, H., Janes, L., Strlic, M., Rusjan, D., & Kocar, D. (2007). Analysis of free and bound aroma compounds in grape berries using headspace solid-phase microextraction with GC-MS and preliminary study of solid-phase extraction with LC-MS, Acta Chimica Slovenica, 54(1), 25-32. Rapp, A. (1988). Wine aroma substances from gas chromatographic analysis, In: Wine Analys, Linskens, H. F. and Jackson, J. F. (Eds.), Springer-Verlag, Berlin Heidelberg, p. 29-66. Doi: 10.1007/978-3-642-83340-3_3 Robinson, A. L. (2011). Environmental influences on grape aroma potential, PhD thesis, Murdoch University. Sánchez-Palomo E., Gómez García-Carpintero E., Gómez Gallego M.A., & González-Viñas M. A. (2012). The Aroma of Rojal Red Wines from La Mancha Region -Determination of Key Odorants. Gas Chromatography in Plant Science, Wine Technology, Toxicology and Some Specific Applications, p. 147-170. Doi: 10.5772/32801 Sánchez-Palomo, E., Consuelo Diaz-Maroto, M., & Soledad Pérez-Coello, M. (2005). Rapid determinationof volatile compounds in grapes by HS-SPME coupled with GC-MS, Talanta, 66(5), 11521157. Doi: 10.1016/j.talanta.2005.01.015 Setkova, L., Risticevic, S., & Pawliszyn, J. (2007). Rapid headspace solid-phase microextraction-gas chromatographic-time-of-flight mass spectrometric method for qualitative profiling of ice wine volatile fraction: II: Classification of Canadian and Czech ice wines using statistical evaluation of the data, Journal of Chromatography A, 1147(2), 224-240. Doi: 10.1016/j.chroma.2007.02.052 Skinkis, P. A., Bordelon, B. P., & Wood K. V. (2008). A Comparison of Monoterpene Constituents of Traminette, Gewurztraminer and Riesling Wine Grapes, American Journal of Enology and Viticulture, 59(4), 440-445. Versini, G., Rapp, A., Volkmann, C., & Scienza, A. (1990). Flavour compounds of clones from different grape varieties, In: Proceeding of the 5th International Symposium on Grape Breeding, 513-524, (Special Issue of Vitis) St Martin, Pfalz, Germany. Winkler A. J., Cook J. A., Kliewe, W. M., Lider L.A. (1974). General viticulture. University of California press, Berkeley, Los Angeles, London. 496 Acta agriculturae Slovenica, 107 - 2, september 2016