The Identification of the Budai gohér with Morphological and Molecular Markers and the Separation from the Gohér conculta

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1 The Identification of the Budai with Morphological and Molecular Markers and the Separation from the Gohér conculta Varga, Zs. 1 Bényei, F. 1 Bisztray, Gy. D. 2 Bodor, P. Jr. 1 Deák, T. 2 1 Corvinus University of Budapest, Faculty of Horticulture, Department of Viticulture H-1118 Budapest, Villányi út Corvinus University of Budapest, Faculty of Horticulture, Department of Genetics and Plant Breeding Corresponding author: Varga, Zs. (vargazsuzsanna@yahoo.co.uk, ) Keywords: old grapevine cultivars, Budai, Gohér conculta, morphological description, molecular markers SUMMARY Gohér is an old Hungarian group of cultivars, Budai, which can be found only in grapevine collections, despite its name is not a member of this conculta. The aim of our study was to make the morphological description and molecular separation (SSR analysis) of Budai and Fehér (the white-berried member of the Gohér conculta) besides determining their cultivation value. The detailed description of Budai, as an individual cultivar, can not be found in the accessible ampelographies. It occurs as the synonym of the following cultivars: Juhfark, Fehér, Királyszől ő and Demjén. According to the opinion of some authors the attribute budai indicated cultivars with high yield but lower quality in Tokaj wine-region. Based on their short descriptions Budai may be identified as one of these cultivars: Demjén, Som and Sombajom. In 2005 and 2006 the morphological characteristics of each cultivar were registered with the application of the code numbers of the O.I.V. The size, the shape and the colour of the berry are worth to emphasize, since by its larger, ellipsoidal berries, which are greenish even at maturity, Budai can be easily separated from the Gohér conculta. Six SSR loci were enough to separate Budai and Fehér. INTRODUCTION Budai, which can be found only in grapevine collections, despite its name is not a member of Gohér conculta, an old Hungarian group of cultivars. Morphological and molecular methods (SSR analysis) are able to separate Budai and Fehér (the white-berried member of the Gohér conculta). The detailed description of Budai, as an individual cultivar, does not occur in the accessible ampelographies. BALASSA (1991) claims that the attribute budai indicated cultivars with high yield but lower quality in Tokaj. NÉMETH (1970) refers it as the synonym of Juhfark, Fehér and Demjén. In author s Ampelography the above-mentioned Demjén can be found among collected cultivars, according to its short description it is equal with the Budai investigated by us. According to BALASSA (1991) the name Demjén occurs in writing first in 1632, though author also declares, it couldn t be a very widespread cultivar, because after the disaster of phylloxera it could not be found anymore. In the Ampelography by NÉMETH (1970) Demjén also appears amongst the synonyms of Fehér and Királyszől ő. The origin of the name probably can be attached to the patron saint of pharmacists and the supposed medicative effect of the cultivar (LŐRINCZ-BÉNYEI- ZANATHY-VARGA, 2004). BALASSA (1991) also mentions other cultivars, such as

2 Somszől ő, Sombajom and Som. Attribute som, which word exactly means cornel, relates to the shape of the berry, namely it represents a cultivar with longish berries. The word bajom, which has several variants, is of unknown origin. According to some opinions it refers to an early-ripening sort of Gohér with large berries and thick berry-skin. Conculta, which is a taxonomic term, means group of cultivars, its members can be distinguished by the colour of the berry, the autumn colouring of leaves and sometimes by the colour of canes (CSEPREGI-ZILAI, 1988) Fehér (one of its synonyms is Török ) is the white-berried member of the Gohér conculta. Probably it is of Hungarian origin, according to the natural system it belongs to convar. pontica subconvar. balcanica provar. mesocarpa subprovar. Hungarica (NÉMETH, 1970). Two subcultivars of Fehér is known: Sárga (yellow) and Zöld (green) (HAJDU, 2003). High number of its synonyms refers that it may be one of our oldest-grown cultivars (HEGEDÜS-KOZMA-NÉMETH, 1966). It is consumed as both wine grape and fresh grape (MOLNÁR, 1897), it also occurs in the book about table grapes by KOZMA (1961). It used to have significant role primarily in the wine-regions of Tokaj, Pécs and the Balaton Highland. Because of its female flowers it needs allogamy, consequently spread of pure plantations, respectively the low yield derived from it, caused its supplanting (LŐRINCZ-BÉNYEI-ZANATHY-VARGA, 2004). Currently it can be planted for experimental growing in Tokaj again (HARSÁNYI-MÁDI, 2005). Beside morphological methods there are molecular ways for identification, clone selection (VIGNANI et al., 1996; REGNER et al., 2000), creation and review of cultivar collections (LEFORT and ROUBELAKIS-ANGELAKIS, 2001; BOCCACCI et al., 2005), just like checking parent-offspring connections (DETTWEILER et al., 2000). Microsatellites (LITT and LUTTY, 1989) also known as SSR (JACOB et al., 1991) are polymorphic DNA markers, with co-dominant inheritance, according to the Mendelian laws. With special primers, this method could be automatised by PCR (Polymerase Chain Reaction) and the results are easily changeable between laboratories (WEBER and MAY, 1989; YAMAMOTO et al., 2002). THOMAS and SCOTT (1993) was the first who used the repeatable sequences to make distinction between grape cultivars. A few years later, some trends started to be evolved according to the necessity. METHODS AND MATERIALS The investigation was carried out and samples were collected in the vineyard of Tokaj Oremus Kft. in Mandolás-dűl ő (plot of land), west from the village Tolcsva, Hungary from 2004 to Morphological description In 2005 and 2006 the morphological characteristics of each cultivar were registered with the application of the code numbers of the O.I.V. The dates of the phenological stages were also registered. Vegetative and generative production Vegetative production can be characterised by the intensity of shoot-growth. The changes of shoot-length were recorded until the first topping. Regarding generative production weight of berries, sugar content, titrable acidity and ph of the juice were measured weekly during the ripening period, having used the general methods of viticultural examinations, such as titration, determination of reductive sugar content etc. In addition at maturity the distribution of berries regarding their size was measured. It is related to the percentage of berry-set, namely irregularly-fertilized berries are only half or quarter of normal-sized berries originated from double-fertilization. Since investigations of

3 the former cultivars of Tokaj were carried out in the wine-region, the ratio of sound, rotten and noble-rotted berries were also recorded. DNA extraction and SSR analysis The DNA was extracted from both samples (shoot tip) with the DNEasy Plant Mini Kit (Qiagen), according to the protocol of the manufacturing company. Follow-up the concentrations of the extracted DNA samples were measured by the NanoDrop ND-1000 spectrophotometer (NanoDrop Ltd.). Peltier PTC-200 (PTC200) DNA Engine Thermal Cycler (MJ Research) has used to perform the PCR with the following program: pre-denaturation at 94 C for 10 minutes, 2 minutes denaturation at 94 C, 44 cycles of denaturation 1 min. at 94 C, annealing for 45 sec. at 52 C and extension for 1 min. at 72 C. Post cycle for 7 min. at 72 C. Seven microsatellite primers were used in this work: VrZag21, VrZag62, VrZag79 (SEFC et al., 1999), VvMD5, VvMD7 (BOWERS et al., 1996), VvMD27 (BOWERS et al., 1999), and VvS2 (THOMAS et al., 1993). Fluorescent dye (6-FAM) has applied to detect one of the primers in each primer pairs (Applied Biosystems). Electrophoresis: 1 % gel was made for this step (Sigma A 5093), in TAE buffer the gel in 120 W was electrophoresed. The fragment length-analysis was made with the ABI Prism 3100 fragment-analysator. RESULTS AND DISCUSSION Morphological description In case of morphological description more than 15 differences were found among from about 90 characteristics. Namely Budai had larger leaves, longer tendrils, longer petioles, shorter and smaller bunches, longer internodes of canes. It is oval-berried, it is harder to remove berries from the stalk. However Fehér had almost round, juicier berries with thinner skin. Cultivars also differed regarding the colour of young leaves and the density of prostrate hairs of shoots. Vegetative and generative production There was no significant difference found in case of intensity of shoot-growth in either year. In case of harvests (Table 1.) the sugar density of Budai was at least 2 Brix higher in every vintage. The total acidity of the cultivars didn t differ significantly. Regarding the distribution of different-sized berries (Figure 1. and 2.) because of their female flowers both cultivars had serious setting problems in 2004 and 2005, namely the ratio of berries originated from double-fertilization was lower than 20 %. In 2006 only a moderated ratio of irregularly-fertilized berries was recorded at both cultivars. Significant difference between cultivars was not registered. Only in 2004 a greater development of noble rot (Figure 3.) was recorded, ratio of different berries was similar in case of the cultivars. In 2005 Fehér had more sound berries, though even Budai rather rotted then. In 2006 about the same percentage of berries remained sound. Despite of Fehér produced more noble-rotted berries, The sugar content of Budai was higher. DNA extraction and SSR analysis Table 2. contains the results of SSR analysis. Though the locus VvS2 shows similarity between cultivars, all other loci were different. Consequently six SSR loci were enough to separate Budai and Fehér. REFERENCES

4 BALASSA I. (1991): Tokaj-Hegyalja szőleje és bora. Történeti-néprajzi tanulmány. Tokaj- Hegyaljai ÁG. Borkombinát, Tokaj. BOCCACCI, P.; TORELLO MARINONI, D.; GAMBINO, G.; BOTTA, R.; SCHNEIDER, A. (2005): Genetic characterization of endangered grape cultivars of Reggio Emilia Province. Am. J. Enol. Vitic. 56:4 BOWERS, J.E.; DANGL, G.S.; VIGNANI, R.; MEREDITH, C.P. (1996): Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.). Genome 39: BOWERS, J.E.; DANGL, G.S.; MEREDITH, C.P. (1999): Development and characterization of additional microsatellite DNA markers for grape. American Journal of Enology and Viticulture. 50 (3) p. CSEPREGI P. ZILAI J. (1988): Szőlőfajta-ismeret és használat. Mezőgazdasági Kiadó, Budapest. DETTWEILER, E.; JUNG, A.; ZYPRIANI, E.; TÖPFER, R. (2000): Grapevine cultivar Müller-Thurgau and its true to type descent. Vitis 39 (2): HAJDU E. (2003): Magyar szőlőfajták. Mezőgazda Kiadó, Budapest. HARSÁNYI J. MÁDI R.-NÉ szerk. (2005): Szőlő- és gyümölcsfajták. Nemzeti fajtajegyzék. Szaporításra egyedileg engedélyezett fajták jegyzéke. Országos Mezőgazdasági Minősít ő Intézet, Budapest. HEGEDÜS Á. KOZMA P. NÉMETH M. (1966): A szől ő. Akadémiai Kiadó, Budapest. JACOB, H. J.; LINDPAINTNER, K.; LINCOLN, S.E.; KUSUMI, K.; BUNKER, R.K.; MAO, YI-PEI; GANTEN, D.; DZAU, V.J. and LANDER, E.S. (1991): Genetic mapping of a gene causing hypertensive rat. Cell 67: KOZMA P. (1961): A csemegeszől ő. Mezőgazdasági Kiadó, Budapest. LEFORT, F.; ROUBELAKIS-ANGELAKIS K. K.A. (2001): Genetic comparison of Greek cultivars of Vitis vinifera L. by Nuclear Microsatellite Profiling. Am. J. Enol. Vitic. 52: 2. LITT, M.; LUTY, J.A. (1989): A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am J Hum Genet. 44 (3): LŐ RINCZ A., BÉNYEI F., ZANATHY G., VARGA ZS. (2004): A nővirágú fajták a magyar szőlőtermesztésben. Borászati Füzetek, 2004 / 3. (Kutatás) p MOLNÁR I. (1897): Szőlőművelés és Borászat. Budapest. NÉMETH M. (1970): Ampelográfiai album. Termesztett borszőlőfajták 2. Mezőgazdasági Kiadó, Budapest. REGNER, F.; WIEDECK, E.; STADLBAUER, A. (2000): Differentation and identification of White Riesling clones by genetic markers. Vitis 39 (3), p. SEFC, K.M.; REGNER, F.; TURETSCHEK, E.; GLOSSL, J.; STEINKELLNER, H. (1999): Identification of microsatellite sequences in vitis Vitis riparia and their applicability for genotyping of different Vitis species. Genome (42) 1-7 p. THOMAS, M.R.; SCOTT, N.S. (1993): Microsatellite repeats in grapevine reveal DNA polymorphisms when analysed as sequence-tagged sites (STSs). Theor.Appl. Genet. 86: WEBER, J.L.; MAY, P.E. (1989): Abundant class of human DNA polymorhisms which can be typed using the polymerare chain reaction. Am. J. Hum. Genet. 44: VIGNANI, R.; BOWERS, J.E.; MEREDITH, C.P. (1996): Microsatellite DNA polymorphism analysis of clones of Vitis vinifera Sangiovese. Scientia Horticulturae. (65) p. YAMAMOTO, T.; KIMURA, T.; SAWAMURA, Y.; MANABE, T.; KOTOBUKI, K.; HAYASHI, T. (2002): Simple sequence repeat for genetic analysis in pear. Euphytica 124: TABLES AND FIGURES

5 Table 1.: The main qualitative data of vintage of Budai and Fehér (Tolcsva, ) vintage cultivar sugar content (Brix o ) titrable acidity (g/l) Budai 25,1 7 Fehér 23 7,1 Budai 23 8,1 Fehér 20,6 9,7 Budai 25,1 7,1 Fehér 23,2 6,9 Table 2.: Genotypes of the investigated cultivars at six SSR loci in base pairs locus cultivar VvS2 VvMD5 VvMD7 VvMD27 VrZag62 VrZag79 Fehér aspec aspec Budai Figure 1.: Distribution of different-sized berries of Budai (Tolcsva, )

6 Figure 2.: Distribution of different-sized berries of Fehér (Tolcsva, ) 100% 80% 60% sound berries rotten berries noble-rotted berries 40% 20% 0% Budai Fehér Budai Fehér Budai Fehér Figure 3.: Distribution of sound, rotten and noble-rotted berries of Budai and Fehér (Tolcsva, )