Clonal Selection of the Greek Grape Wine Cultivar Xinomavro

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1 Clonl Selection of the Greek Grpe Wine Cultivr Xinomvro H.C. Spinthiropoulou nd.. Leventkis itro Hells S.A Niseli, Alexndri A.G. Goulioti nd.. Mrinos Ampeloeniki Ltd Technologicl Prk of Thessloniki.. Stvrkkis nd.f. Biniri Lbortory of iticulture Agriculturl University of Athens C.. Dovs nd.. tis Plnt Pthology Lbortory Fculty of Agriculture Aristotle University of Thessloniki Keywords: itis vinifer, Greek cultivr, genetic identifiction, virus detection, enologicl evlution Abstrct Xinomvro is one of the most importnt red-wine grpe cultivrs (itis vinifer L.) of. Different soil, climte nd viticulturl technique interctions, result in gret vribility in respect with morphologicl, mpelogrphicl nd physiologicl chrcters of the specific cultivr. Thus, clonl selection procedures mtch perfectly with Xinomvro, iming to improvement of wine qulity. These procedures re lbour nd time consuming, lsting up to twelve yers. In the present work, clonl selection of Xinomvro ws performed by following combined protocol bsed on the respective ones of Frnce, Itly nd the Lbortory of iticulture (Agriculturl University of ). Clonl selection procedures strted in 1995 ccording to this new protocol (nmed itro ), including ) mrking of existing biotypes grown in different Xinomvro cultivtion centers, b) virus detection (ELISA, multiplex nested RT-PCR nd indexing), c) genetic identifiction nd discrimintion, using moleculr methods (RAPD-PCR), d) estblishment of study collection of ll virus-free biotypes (clones), e) enologicl evlution, f) instlltion of vineyrd for comprtive gronomic nd enologicl evlution nd finlly g) multipliction of the most interesting clones. Only five, out of 20 initilly selected biotypes with stble chrcteristics, seem to ber distinct enologicl fetures nd fulfil requirements for their recognition s clones of Xinomvro. INTRODUCTION Xinomvro is one of the most importnt red-wine grpe cultivrs (itis vinifer L.) of. It is cultivted in Centrl nd West Mcedoni where it prticiptes in the production of some of the most importnt Greek Appelltions of Origin such s Nouss, Goumeniss nd Amyndeon nd mny other ins de pys. Different soil, climte nd viticulturl technique interctions, result in gret vribility in respect with morphologicl, mpelogrphicl nd physiologicl chrcters of the specific cultivr. Thus, clonl selection procedures mtch perfectly with Xinomvro, iming to improvement of wine qulity. This is the first effort to distinguish clones within the popultion of Xinomvro nd produce certified mteril. MATERIALS AND METHODS In the present work, clonl selection of Xinomvro ws performed by following combined protocol bsed on the respective ones of Frnce nd Itly nd the Lbortory of iticulture (Agriculturl University of Athens). This protocol consists of: Loction, Selection nd Observtion of Biotypes Selection strted in 1995, in old vineyrds of Nouss, Goumeniss, Amyndeon, Proc. 1 st IS on Grpevine Eds. Ó.A. de Sequeir & J.C. Sequeir Act Hort. 652, ISHS

2 Oss, elvendos, Sitist nd Rpsni, centers of Xinomvro cultivtion. It ws bsed minly on grpe chrcteristics nd especilly on desirble chrcteristics of this vriety (medium to smll size of cluster, smll, drk berries). Ampelogrphicl description, mpelometric mesurements of leves, gronomic chrcters were observed nd registered for three yers period. Plnt mteril ws collected nd kept in vivo in pots nd in vitro. Appliction of Serologicl (ELISA), Moleculr (multiplex nested RT-PCR), nd Biologicl Methods for Testing the Selected Biotypes Phloem grpevine tissue (corticl scrpings) ws tested serologiclly by ELISA by using commercilly vilble dignostic kits, for the presence of Grpevine fnlef virus (GFL), Tomto blck ring virus (TBR), Arbis mosic virus (ArM), six different closteroviruses Grpevine lefroll-ssocited virus -1,-2,-3,-5,-6,-7 ( -1,-2,-3,-5,- 6,-7) nd two vitiviruses (Grpevine A, B, GA, GB). The sme smples were lso tested by spot nested multiplex RT-PCR by using degenerte primers, for the simultneous detection of viruses belonging to the genus Closterovirus (Dovs nd Ktis 2003). Grft-trnsmission ws lso performed for limited number of the bove smples onto specific indictor plnts of the Genus itis (LN 33, Ripri Gloire de Montpellier, Rupestris St George, Cbernet frnc, nd Richter 110). Genetic Identifiction-Discrimintion The identifiction nd the discrimintion of different biotypes of grpe cultivr Xinomvro ws mde by the RAPD-PCR nlysis ccording to Stvrkkis et l 1997; Stvrkkis nd Biniri, This method, bsed on rndom mplified polymorphic DNA obtined by PCR nlysis llows the direct comprisons of the genetic mteril of grpe cultivrs. DNA moleculr mrkers hve been used successfully to revel genetic vrition between nd within Greek cultivrs (Stvrkkis et l. 1997, Stvrkkis nd Biniri 1998) Grpevine DNA ws extrcted from young nd fully expnded leves ccording to Thoms et l. (1993) with minor modifictions. Amplifiction rections were performed in 25 µl contining 60 ng of genomic DNA,10 mm TRIS-Cl ph 8.8, 1.5 mm MgCl 2, 50 mm KCl, 0.1% Triton X-100, 200 µm ech of datp, dgtp, dctp, dttp, 50 ng primer nd 1 unit of Tq DNA polymerse (Qigen). Eight rndom decmer oligonoucleodides were used s primers (Tble 2) for the mplifiction of RAPD sequences. Amplifiction ws performed in Perkin Elmer DNA Therml Cycler After 5 min t 94 o C, 34 cycles of PCR were performed, (1 min t the 94 o C, 1 min t 44 o C, 2 min t 72 C) followed by 10 min t 72 o C for extension. Aliquotes of the RAPD products were nlysed in 2.0% grose gel electrophoresis in TAE buffer (40 mm Tris-cette nd 1mM EDTA, ph 8). After stining in ethidium bromide (1µg.l -1 ) the gels were photogrphed on Gel Doc 1000 (Biord). All of the rections were repeted t lest twice with independently isolted genomic DNA s templtes. Estblishment of Study Collection irus-free biotypes were plnted in study collection with 2 replictions of 15 plnts ech nd cultivted under the sme viticulturl techniques. Oenologicl Evlution Grpes of ech biotype were vinified under the sme vinifiction protocol ( ) nd wines produced were evluted by group of enologists in April nd September. Clones Performnce Evlution irus-free biotypes were plnted in two vineyrds in the zone of Appelltion of Origin Nouss for comprtive evlution s well s their evlution with the verge 46

3 wine produced in this zone. Multipliction of the Most Interesting Clones Clones judged s the more commercilly interesting will be propgted nd used for the estblishment of new qulittive virus-free vineyrds. RESULTS AND DISCUSSION 20 biotypes with distinguished chrcteristics were mrked in The next two yers 10 of these biotypes kept their chrcters for which they were chosen, nd continue to keep them till now, while only 5 of them nd more specificlly Xinomvro number 6 nd 11 from Nouss region (X6, X11), number 3 from elvendos (X3), number 1 from Oss (X1) nd number 2 from Rpsni region (X2) were fount to be without the presence of the viruses reported (tble1). Eight single, rbitrry 10-mer oligonucleotide primers were used to mplify genomic DNA from six biotypes of grpe cultivr Xinomyro. Ech primer provided t lest 7 frgments- bnds. Over thn 95 reproducible frgments were generted by this method. The primers OPM 04, 1224 nd 1226 proved much more useful becuse they hve generted more polymorphic DNA frgments (Tble 2). Exmples of RAPD ptterns mplified with primers 1226 nd 1227 re shown in Fig. 1 (,b). The identicl ptterns, s detected electrophoreticlly, between the different biotypes of grpe cultivr Xinomvro suggested tht, t lest for the primers studied, there is no genetic vrition. The sme degree of genetic similrity indictes tht the vines studied derive form the sme mother plnt by vegettive propgtion nd the morphologicl, mpelogrphicl nd biochemicl differences re due to environment nd culturl fctors. intge nd vinifiction of the five bove mentioned biotypes reveled differences between produced musts nd wines (Tble 3). Fermenttion rom ws further investigted. Wines produced by the biotypes X6, X11 nd X3 were chrcterised by higher concentrtions of ethyl esters nd cettes of higher lcohols. The lter enhnced more intense, hrmonious nd complex vinifiction roms, thn did the wines produced by the biotypes X2, nd X1 which received inferior notes by sensory evlution s they were less romtic. Flvour precursor nlysis showed the existence of different romtic potentil between the biotypes. The musts coded s X6, X11 nd X3 hd common concentrtions in glycosidiclly bound substituted benzene nd norisoprenoid derivtives (fig.2). Flvour precursor nlysis of the biotype X2 must reveled high concentrtions of benzene derivtives but t the sme time, smll mounts of norisoprenoid compounds. The X1 biotype must ws rich in glycosidiclly bound norisoprenoid components. The exploittion of this flvour potentil could be possible by the use of enzymes during the vinifiction process nd it could led in the elbortion of new improved wine-product. Constntly, during the 4 yers of experimenttion, the enologists prticipting in the sensory evlution of the produced wines prefered wines from clones X6 nd X3. They gree tht the clone X6 is more typicl of Xinomvro, while clone X3 produces more romtic nd less tnnic wine. The five virus-free biotypes isolted nd studied till now (X6, X11, X3, X1, X2) present little differences in mpelogrphic chrcters but gret ones concerning the qulity of their wines, so they could be considered s clones of Xinomvro. It is need to be used greter number of primers for the complete definition of polyclonl synthesis of Xinomvro popultion. Comprtive evlution of clones nd propgtion of the most interesting ones hs to be finished for this procedure to be completed. Literture Cited Dovs, C.I., Ktis, N.I. 2003b. A spot multiplex nested RT-PCR for the simultneous nd generic detection of viruses involved in the etiology of grpevine lefroll nd rugose 47

4 wood of grpevine. J. irol. Meth. 109: Stvrkkis, M.N.nd Biniri, K Genetic study of grpe cultivrs belonging to the Musct fmily by rndom mplified polymorphic DNA mrkers. itis 37 (3) : Stvrkkis, M.N, Biniri, K. nd Htzopoulos, P Identifiction nd discrimintion of eight Greek grpe cultivrs (itis vinifer L.) by rndom mplified polymorphic DNA mrkers. itis 36 (4) : Thoms, M.R., Mtsumoto, S., Cin, P. nd Scott, N.S Repetitive DNA of grpevine clsses present nd sequences suitble for cultivr identifiction. Theor. App. Genet. 86: Tbles Tble 1. irus testing results.. G A G B GL R GFk X X2 X3 X4 + X5 + X6 X7 + X8 + + X9 + X X11 X X13 + X X15 X16 + X X18 + X X GFL ArM T B R Tble 2. Primers used, nucleotide sequence, totl number of frgments mplified nd origin of them Primer 5 3 Number of frgments mplified Origin* OPM-01 GTTGGTGGCT 10 OPM-04 GGCGGTTGTC 16 OPM-07 CCGTGACTCA GTGTGCCCCA AGGTGACCGT 7 - OPF-05 CCGAATTCCC CGCAGGATGG CAGGCCCTTC : IBBM, University of Crete, : Operon Technologies Inc. Almed CA, USA 48

5 Tble 3. Chrcteristics of vintge (verge ) Clone Nr X1 X2 X3 X6 X11 Production per plnt (Kg) 5 4,8 5,5 5 5,5 Sugr content (g/l) ph 3,65 3,64 3,55 3,56 3,61 Acidity (g/l) 6 6,5 7,3 7,3 6,3 Figures b Fig. 1. Amplifiction ptterns of polymorphic DNA from 6 biotypes of grpe cultivr Xinomvro generted by primers 1226 () nd 1227 (b) '. Aliphtic compounds '. Benzene derivtives '. Monoterpenes '. orisoprenoids '. Nitrogen compounds '. Ogygene compounds '. Sulfur compounds 10 0 X6 EH X3 EH X11 EH X2 EH X1 Fig. 2. Distribution percentge of free nd enzyme hydrolysis (EH) voltile compounds identified in musts from the different clones of.. vr. Xinomvro 49