Garnacha and Garnacha Tintorera: Genetic Relationships – 237 Garnacha and Garnacha Tintorera: Genetic Relationships and the Origin of Teinturier Varieties Cultivated in Spain José Antonio Cabezas,1 María Teresa Cervera,1,2 Rosa Arroyo-García,1 Javier Ibáñez,3 Inmaculada Rodríguez-Torres,3 Joaquín Borrego,3 Félix Cabello,3 and José Miguel Martínez-Zapater1* Representative grapevine accessions (Vitis vinifera) cultivated in Spain under the names Garnacha and Garnacha Tintorera, as well as their synonyms, were analyzed to determine genetic diversity and relationships. Both varieties are characterized by high levels of intravarietal morphological variation. Results confirmed the monophyletic origin of the Garnacha variety, which is represented by a main genotype with several phenotypic variants, likely corresponding to somatic mutations. In contrast, Garnacha Tintorera was characterized as a genetically heterogeneous group, which included three different teinturier genotypes. Possible parentage relationships among the teinturier varieties were identified and further confirmed using microsatellites, showing that all are derived from crosses performed in the nineteenth century to improve color intensity of well-known red wine varieties. Key words: Garnacha, Grenache, Garnacha Tintorera, Alicante Bouschet, teinturier, intravarietal diversity, AFLP, microsatellite, morphological variant Garnacha is an ancient grapevine variety. It was first ref- Garnacha Tintorera, despite this variety being clearly distin- erenced in 1312, under the name Varnacie, in a legal docu- guishable morphologically from Garnacha by the small pen- ment of the Paris parliament (Peñín et al. 1997). Garnacha tagonal and five-lobed leaves and red-black berries with col- is characterized by pentagonal three-lobed leaves and round, ored flesh. The Spanish word tintorera refers to the strongly dark, red-violet berries with high sugar content. It is now the colored flesh that characterizes all teinturier varieties. most widely grown red wine variety in the world, with more Garnacha Tintorera is a minor variety, with only 17,100 ha than 419,000 ha (Hidalgo 1999), of which almost half is lo- cultivated in Spain (Registros Viticolas 1999), and therefore cated in Spain. It is also widely cultivated under the name of lesser economic importance. As with other teinturier va- Grenache in other countries, including France, the United rieties, it is not used to produce high-quality wines, but is States, and Australia. There are many synonyms for Garnacha, blended in multivarietal wines to increase color intensity. such as Alicante, Roussillon, Rivesaltes, Bois Jaune, and Some authors consider Garnacha Tintorera a native Spanish Carignane Rousse in France (Galet 2000), Cannonau and Tocai variety (Hidalgo and Galet 1988, Peñín et al. 1997), while Rosso in Italy (Caló et al. 1990), and Garnacho, Aragonés, others consider it a synonym of the French variety Alicante Lladoner, Tinta, and Alicante in Spain (Galet 2000). Garnacha Bouschet (Chirivella et al. 1995, Galet 2000). It can be found identification is further complicated by the high level of mor- cultivated under different names. Most should be considered phological variation found among plants cultivated under this synonyms, such as Alicante, Negral, Tintorera, or Moratón name. This variation has given rise to different morphotypes, (Rodriguez-Torres 2001), but homonyms, such as Garnacha, which have been considered as different grape varieties when or false synonyms, such as Alicante (Garnacha is known as affecting important agronomic or ampelographic traits. This Alicante in France), have also been described. Adding to the is the case of Garnacha Tinta (red), Garnacha Blanca (white), confusion, high levels of morphological variation are also Garnacha Gris or Dorada (gray), or Garnacha Peluda (hairy). found among the teinturier plants grown as Garnacha Tintor- Furthermore, the word Garnacha is also used as homonym era or under related names (Rodriguez-Torres 2001). Whereas for other varieties. One of these well-known homonyms is some authors describe Garnacha Tintorera as a single vari- ety (Hidalgo and Galet 1988), others suggest that more than one variety are cultivated under this name in Spain (García 1Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain, and de los Salmones 1914; Martinez de Toda and Sancha 1996). Departamento de Biotecnología, SGIT, INIA, Ctra. de la Coruña Km 7, 28040 Madrid, Spain; Thus, this morphological variation could either represent 2Centro de Investigaciones Forestales, INIA, Madrid, Spain; and 3Departamento de Agroalimentación, IMIA, Finca El Encín, Apdo 127, 28800 Alcalá de Henares, Madrid, Spain. somatic variants or indicate the presence of different hom- *Corresponding author [Tel: 34-91-5854687; fax: 34-91-5854506; email: [email protected]] onym teinturier varieties mixed because of their colored flesh, Acknowledgments: MTC was funded by a Ministerio de Educación y Cultura contract; IR and JAC were funded by predoctoral fellowships from the European Union and Instituto Nacional a trait that differentiates them from most other grapevine de Investigaciones Agrarias y Agroalimentarias (INIA), respectively. This research was funded varieties. in part by projects INIA SC96-010, SC94-092, and CM 07G-0045-2000. Support of research activity at the Centro Nacional de Biotecnología is provided by a CSIC-INIA specific agreement. In order to understand the origin of the morphological Manuscript submitted May 2003; revised August 2003 variation observed in the Garnacha and Garnacha Tintorera Copyright © 2003 by the American Society for Enology and Viticulture. All rights reserved. 237 Am. J. Enol. Vitic. 54:4 (2003) 238 – Cabezas et al. varieties, we performed molecular characterization of repre- Table 1 Grapevine accessions analyzed, with teinturier sentative accessions. Results indicate that Garnacha acces- indicated in bold. Local name, reference code at the sions are a single main genotype, and the studied morpho- germplasm bank of El Encín (Cabello 1995), and logical variants, even those considered as different varieties, place of origin for each plant are indicated. are somatic variants that appear recurrently in the Garnacha Reference Place of origin genetic background. Analysis of Garnacha Tintorera acces- code Local name (Spain) sions revealed the existence of three different teinturier geno- 22-A-04 Garnacha Álava types. Genetic analysis based on amplified fragment length 22-A-08 Garnacho Blanco Alava 22-A-11 Garnacha Blanca Logroño polymorphism (AFLP) and microsatellite markers showed 22-A-39 Garnacha Tinta Navarra that all accessions derive from documented crosses performed 22-C-56 Moscatel Morisco Málaga in the nineteenth century by Louis and Henri Bouschet (Viala 22-D-06 Garnacha Negra Huesca 22-D-07 Garnacha Basta Huesca and Vermorel 1909). 22-D-21 Garnacha Gorda Huesca 22-D-26 Bernacha Blanca Teruel 22-D-30 Garnacha Fina Teruel Materials and Methods 22-D-34 Garnacha Negra Teruel 22-D-36 Garnacha Peluda Teruel Plant material. Sixty-four accessions representative of 22-D-37 Garnacha Blanca Teruel 22-D-48 Garnacha Francesa Zaragoza the material cultivated in Spain under the names Garnacha 22-D-49 Tintorera Zaragoza and Garnacha Tintorera, as well as associated synonyms, were de Longares analyzed in this study. All had previously been characterized 22-D-50 Garnacha Negra Zaragoza 22-E-36 Giró Palma using ampelographic descriptors (Rodriguez-Torres 2001). The 22-F-32 Garnacha Oviedo interspecific hybrid 110 Richter (Vitis berlandieri x V. r u - 22-F-42 Tinto Madrid Cantabria pestris) was also included as an outgroup sample. Local names, 22-G-33 Garnacha Dorada Barcelona 22-G-41 Garnacha Blanca Gerona codes, and places of origin of the studied material are listed 22-G-43 Lladoner Negre Gerona in Table 1. All accessions belong to the grape germplasm 22-G-49 Garnacha Tinta Lérida collection maintained at El Encín (Instituto Madrileño de 22-H-07 Garnacha Negra Tarragona del País Investigación Agraria y Alimentaria of Comunidad de Madrid, 22-H-19 Garnacha Peluda Tarragona Alcalá de Henares, Spain) (Cabello 1995). 22-H-29 Garnacha Blanca Tarragona 22-H-34 Garnacha Negra Tarragona Molecular analysis. Total DNA was extracted from young 22-H-38 Garnacha Albacete leaves, which had been stored at -80°C, following the pro- 22-H-42 Garnacha Tintorera Albacete tocol described by Dellaporta et al. (1983). One percent poly- 22-H-45 Tintorera Albacete 22-I-08 Garnacha Tintorera Albacete vinylpyrrolidone was added to the extraction buffer to pre- 22-I-12 Tinto Navalcarnero Ávila cipitate polyphenols (Lodhi et al. 1994). 22-I-13 Tinto de Aragón Ávila 22-I-17 Garnacha Avila AFLP analysis was carried out following the protocol de- 22-I-43 Garnacha Cuenca scribed by Vos et al. (1995), with slight modifications 22-J-17 Garnacha Madrid 22-J-30 Negral Madrid (Cervera et al. 1998). In order to compare the results obtained 22-J-31 Garnacha Madrid in different experiments, the AFLP primer combinations, as 22-J-33 Garnacha Tintorera Toledo well as the sample used as outgroup, were the same as those 22-J-34 Garnacha Toledo 22-J-41 Colorina Toledo ones used in previous studies. The primer combination used 22-J-50 Tinto Navalcarnero Burgos in the preamplification was EcoRI +A / MseI +C, while the 22-J-51 Tinto Aragonés Burgos two primer combinations used for selective radioactive am- 22-J-55 Aragón Burgos