The Kingroup of the Cultivar Chardonnay Revealed M. Di Vecchi Staraz 1,2, V. Laucou 2, J.-M. Boursiquot 2, T. Lacombe 2, D. Varès 3, S. Gerber 4, M. Boselli 1 and P. This 2 1 Dipartimento di Ortoflorofrutticoltura, Università degli Studi di Firenze, Viale delle Idee, 30, Sesto F.no, Italy 2 UMR Diversité et Génomes des Plantes Cultivées, INRA-Equipe Génétique de la Vigne, 2, Place Viala, Montpellier, France 3 Unité Expérimentale du Domaine de Vassal, INRA, Route de Sète, Marseillan plage, France 4 UMR 1202, BIOGECO, Equipe Génétique, INRA, Site de Recherche Forêt, Bois de Pierroton, 69 route d Arcachon, Cestas Cedex, France Keywords: microsatellite, Vitis vinifera L., parentage analysis Abstract Despite the importance of grapevine (Vitis vinifera L.), relatively few parentages were revealed until now because small databases were analyzed. In the present work, we developed a useful and readable routine for the analysis of large data set, integrating FaMoz software. The routine was applied to a data set consisting of 2,704 individuals of the French national INRA germplasm collection of grapevine, analyzed using 20 SSRs markers well scattered over the genome. The large-scale parentage analysis enabled the identification of several kin relationships. The kingroup of Chardonnay, one of the most celebrated international grapevine cultivars, was then revealed. INTRODUCTION Vitis vinifera L. is a vegetatively propagated crop, rich in clonal populations (it is submitted to farming practices which are interested in cultivar maintaining). Recent analyses of genetic diversity of V. vinifera by molecular markers demonstrated that V. vinifera evolves through natural crosses between cultivars (Bowers and Meredith, 1997; Bowers et al., 1999; Dettweiler et al., 2000; Sefc et al., 1998; Vouillamoz et al., 2003). But until now, a relatively small number of parentages involving few cultivars have been revealed and only relatively small genetic databases were analyzed. Due to the increased study of genetic diversity in grape, and since SSRs data can be compared between laboratories (This et al., 2004), large databases are gradually becoming available (Laucou et al., 2006). Furthermore, a useful method to perform parentage analysis has been recently set up that has no limit with respect to the size of the database (Di Vecchi Staraz et al., 2006). In the present paper, we describe a large scale parentage analysis involving 2,704 cultivars aimed at the identification of kin relationships for one of the most celebrated international grapevine cultivars: Chardonnay. MATERIALS AND METHODS Plant Material and Microsatellite Markers The study is based on 2,704 Vitis vinifera L. unique genotypes (Laucou et al., 2006), from the Domaine de Vassal INRA (Institut National de la Recherche Agronomique, France) grape repository. All material analyzed was previously selected and identified by ampelographic study, in order to guarantee the trueness-to-type of genetic samples (Laucou et al., 2006). The database includes individuals deriving from 38 countries, among them, especially France, but also Italy and Spain are by far the most represented countries. The multilocus unique genotypes are based on 20 microsatellite loci (nssr): VVMD5, VVMD7, VVMD21, VVMD24, VVMD25, VVMD27, VVMD28, VVMD32 Proc. IX th Intl. Conf. on Grape Genetics and Breeding Eds.: E. Peterlunger et al. Acta Hort. 827, ISHS 2009 233
(Bowers et al., 1996, 1999); VVIn16, VVIv67, VVIv37, VVIq52, VVIp60, VVIh54, VVIb01, VVIn73, VVIp31 (Merdinoglu et al., 2005); VVS2 (Thomas and Scott, 1993) and VMC1b11, VMC4f3 (Vitis Microsatellite Consortium). Parentage Analysis Parentage analysis and handling of dataset were performed as described by Di Vecchi Staraz et al. (2006), using FaMoz software (Gerber et al., 2003). This method involves calculating a logarithm of the likelihood ratio, Log of odds ratio (LOD-score), a threshold for parentage assignment and several exclusion probabilities. The LOD-score, with a value greater than zero, gives a statistical significance to any potential parentage relationship (single parent/parent pair). RESULTS AND DISCUSSION Exclusion Probabilities The paternity exclusion probability for single parent exclusion with 20 markers reaches 0.999999. A probability of cumulative exclusion of 1 was reached using only six markers for pair exclusion and 11 markers for paternity exclusion. These probabilities are largely significant to assess pedigree reconstruction. The threshold is >8 to identify a valid potential single parent and parent pair with 20 SSRs. Pedigree and Kingroup Reconstruction Searching for the kingroup of Chardonnay, we identified 32 direct relatives (Table 1 and Fig. 1). Two are the parents of Chardonnay, and we confirmed the parent pair Pinot noir x Gouais blanc, observed by Bowers et al. (1999), with high LODscore (29.03) and high CLR (X x Y=1,44x10 10 ; Pinot noir x X=6,56x10 5 ; Gouais blanc x X=2,31x10 10 ). Furthermore, we identified the parent pairs of 30 cultivars composing the Chardonnay kingroup, except for the parents Pinot noir and Gouais blanc (Table 1). The kingroup is composed of either full-sibs or offsprings of the cultivar. Twenty-two individuals originating from the Pinot noir x Gouais blanc cross were identified. Some have already been identified by Bowers et al. (1999), but seven of these are additional ones: François noir, Gros bec, Konigstraube, Mezi, Romaine, Rubi, and Troyen. The Gros bec is not a synonym of Dameron as declared before (Galet, 2000). All are from Central Europe (from the East of France to Austria). Four of them are currently grown, while Mezi, François noir, and Gros bec have disappeared from farming. The other eight kin are offspring of Chardonnay. All the identified offspring were expected, because they are all recent controlled crosses. One is a self-fertilization ( S.F. Chardonnay ). Two are recent INRA official breeds: Liliorila ( Baroque x Chardonnay ) and Perdea ( Raffiat de Moncade x Chardonnay ). Charmont and Doral are selected breeds from the cross Chasselas x Chardonnay obtained by Jacquinet in Switzerland in 1965. Faberrebe was obtained in 1929 by Scheu, from the cross Chardonnay x Müller-Thurgau, and not from the presumed cross Pinot blanc x Müller-Thurgau (Galet, 2000). Soukholimansky bely ( Chardonnay x Plavaï ) was obtained in Ukraine in 1949. Finally, Bourdier 2-04-58 is a selected breed from Chardonnay x Chenin. We confirmed that the largest parentage identified between grapevine cultivars is from the Pinot noir x Gouais blanc cross (Bowers et al., 1999; Boursiquot et al., 2004). We also proved that grapevine evolve by sexual reproduction from spontaneous or controlled crosses. It is important to point out that descendants of this cross have hermaphrodite flowers logically, but at least one of them have female flowers ( François noir ). The possible male descendants were not selected for cultivation and they have quickly disappeared. 234
CONCLUSIONS In the present work, we performed a parentage analysis routine to identify kinship relationships in a large dataset consisting of 20 SSR markers and data for 2,704 unique genotypes. Up to now, this is the biggest database analyzed for grapevine. We identified a very large kingroup for well-known cultivar Chardonnay. We confirmed that the cross Pinot noir x Gouais blanc has the largest and successfully descent of grapevine, and that a large data set is fundamental to assess genealogy reconstruction. Literature Cited Bowers, J.E., Dangl, G.S., Vignani, R. and Meredith, C.P. 1996. Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L). Genome 39:628-633. Bowers, J.E. and Meredith, C.P. 1997. The parentage of a classic wine grape, Cabernet Sauvignon. Nature Genetics 16:84-87. Bowers, J.E., Boursiquot, J.-M., This, P., Chu, K., Johansson, H. and Meredith, C.P. 1999. Historical genetics: the parentage of Chardonnay, Gamay, and other wine grapes of Northeastern France. Science 285:1562-1565. Boursiquot, J.-M., Lacombe, T. and Bowers, J.E. 2004. Gouais, a key vine of the European wine heritage. Bulletin de l OIV 77:6-19. Dettweiler, E., Jung, A., Zyprian, E. and Töpfer, R. 2000. Grapevine cultivar Müller- Thurgau and its true to type descent. Vitis 39:63-65. Di Vecchi Staraz, M., Laucou, V., Gerber, S., Lacombe, T., Varès, D. and This, P. 2006. Large scale parentage analysis in grapevine (Vitis vinifera L.) using microsatellite markers (submitted for publication). Galet, P. 2000. Dictionnaire encyclopédique des cépages. Hachette edn., Paris, France. Gerber, S., Chabrier, P. and Kremer, A. 2003. FaMoz: a software for parentage analysis using dominant, codominant and uniparentally inherited markers. Molecular Ecology Notes 3:479-481. Laucou, V., Lacombe, T., Dechesne, F., Bruno, J.-P. et al. 2006. High throughput analysis of genetic diversity in grape (submitted for publication). Merdinoglu, D., Butterlin, G., Bevilacqua, L., Chiquet, V., Adam-Blondon, A.-F. and Decroocq, S. 2005. Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex PCR. Mol. Breeding 15 (4):349-366. Sefc, K.M., Steinkellner, H., Glössl, J., Kampfer, S. and Regner, F. 1998. Reconstruction of a grapevine pedigree by microsatellite analysis. Theoretical and Applied Genetics 97: 227-231. This, P., Jung, A., Boccacci, P. et al. 2004. Development of a standard set of microsatellite reference alleles for identification of grape cultivars. Theor. Appl. Genet. 109:1448-1458. Thomas, M.R. and Scott, N.S. 1993. Microsatellite repeats in grapevine reveal DNA polymorphisms when analyzed as sequence-tagged sites (STSS). Theor. Appl. Genet. 86:985-990. Vouillamoz, J., Maigre, D. and Meredith, C.P. 2003. Microsatellite analysis of ancient alpine grape cultivars: pedigree reconstruction of Vitis vinifera L. Cornalin du Valais. Theor. Appl. Genet. 107:448-454. 235
Tables Table 1. Comparison of Relatedness coefficient (R), likelihood of different possible relationship by Kinship and possible parentage with LOD-score calculated by FaMoz for the cultivar Chardonnay of its kin group. Probable single parents Lod-score Parentage S.F. Chardonnay 22.44 OF [Chardonnay x Chardonnay] Knipperlé 18.39 FS [Pinot noir x Gouais blanc] Gros Bec 17.77 FS [Pinot noir x Gouais blanc] Franc noir de la Haute-Saône 17.26 FS [Pinot noir x Gouais blanc] Liliorila 17.08 OF [Baroque x Chardonnay] Gouais blanc 16.41 P [?] Perdea 15.90 OF [Raffiat de Moncade x Chardonnay] Soukholimansky bely 15.67 OF [Chardonnay x Plavaï] Bourdier 2-04-58 15.60 OF [Chardonnay x Chenin] Beaunoir 15.27 FS [Pinot noir x Gouais blanc] Doral 14.84 OF [Chardonnay x Chasselas] Faberrebe 14.55 OF [Chardonnay x Müller-Thurgau] Charmont 14.01 OF [Chasselas x Chardonnay] Pinot noir 12.47 P [?] Batoca blanc 8.79 OF [Chardonnay x Malvar] Romaine - FS [Pinot noir x Gouais blanc] Auxerrois - FS [Pinot noir x Gouais blanc] Königstraube - FS [Pinot noir x Gouais blanc] Dameron - FS [Pinot noir x Gouais blanc] Sacy - FS [Pinot noir x Gouais blanc] François noir - FS [Pinot noir x Gouais blanc] Melon - FS [Pinot noir x Gouais blanc] Gamay blanc Gloriod - FS [Pinot noir x Gouais blanc] Peurion - FS [Pinot noir x Gouais blanc] Gamay - FS [Pinot noir x Gouais blanc] Romorantin - FS [Pinot noir x Gouais blanc] Aligoté - FS [Pinot noir x Gouais blanc] Roublot - FS [Pinot noir x Gouais blanc] Troyen - FS [Pinot noir x Gouais blanc] Bachet - FS [Pinot noir x Gouais blanc] Aubin vert - FS [Pinot noir x Gouais blanc] Mezi - FS [Pinot noir x Gouais blanc] Rubi - FS [Pinot noir x Gouais blanc] 236
Figurese Fig. 1. A simplified pedigree drawing of the Chardonnay kingroup. Every group contains cultivars with the same relationship with Chardonnay. 237
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