Abstract
Two hundred accessions from the Movera Grapevine Germplasm Bank in Aragón, Spain, were examined. Molecular profiles at six SSR loci allowed confirmation of the trueness-to-type of 86 accessions, correct names were assigned to 33 misnamed accessions, and 43 “unknown” accessions were identified. Thirty-eight accessions, which gave 24 molecular profiles, may be regarded as belonging to autochthonous varieties. Results offer new SSR allelic profiles of ancient Spanish varieties to form part of the genetic heritage of grapevine and reveal the existence of some synonymies, homonymies, and discrepancies in the published database from the El Encín Grapevine Germplasm Bank in Madrid, Spain.
Grapevine diversity today is largely preserved in existing germplasm collections. In Spain there are 13 varietal collections of grapevine (Chomé et al. 2003), situated in the main wine-producing regions. El Encín Germplasm Bank (El Encín collection), in Alcalá de Henares (Madrid), and Rancho de la Merced, in Jerez (Cádiz), established in 1914 and 1940, respectively, are the main national collections, including most of the autochthonous varieties. The Movera Grapevine Germplasm Bank (Movera collection), in Movera (Zaragoza), Aragón, was created in 1990 to preserve the varietal diversity of the region, which was threatened by the abandonment of ~50,000 ha of vineyards in the past 25 years (Lorente 2001). The Movera collection comprises 540 accessions, predominantly winegrapes, many found in very old vineyards or near ancient monasteries. Collections formed using primarily morphological characters tend to contain numerous synonyms, homonyms, and errors, which are difficult to resolve by simple ampelography.
A large set of SSR markers for grape genotyping is now publicly available (Thomas and Scott 1994, Bowers et al. 1996, Sefc et al. 1999, Scott et al. 2000), recently amplified by 169 SSR markers with a wide range of allele sizes (Merdinoglu et al. 2005) and another 38 markers with a high power of discrimination and uniform genome distribution (Cipriani et al. 2008). Nine SSR markers (VVS2, VVMD5, VVMD7, ssrVrZAG21, ssrVrZAG47, ssrVrZAG62, ssrVrZAG64, ssrVrZAG79, and ssrVrZAG83) were selected on the strength of results from studies of European grapevines, indicating that these markers are highly informative and hence constitute a useful set for genetic characterization of cultivars (Sefc et al. 2000). A comparative study among 10 different laboratories suggested six markers—VVS2, VVMD5, VVMD7, VVMD27, ssrVrZAG62, and ssrVrZAG79—as the minimal standard set to create uniform, easily comparable SSR-marker databases for identification of unknown or unconfirmed accessions in international grapevine germplasm collections (This et al. 2004). As ssrVrZAG47 presented some reproducibility problems, it was replaced by VVMD27, which is 20 bp longer than ssrVrZAG47. The potential of SSR analysis has been demonstrated in several genetic studies on Spanish germplasm collections (Ibáñez et al. 2003, Martín et al. 2003, Núñez et al. 2004, Fernández-González et al. 2007) and other national collections, such as in Portugal (Lopes et al. 1999), Greece (Lefort and Roubelakis-Angelakis 2000), Italy (Sefc et al. 2000), and Tunisia (Zoghlami et al. 2009). As a result of systematic research, a robust genetic database for most of the Spanish varieties was created at the El Encín collection (www.sivvem.monbyte.com/sivvem.asp).
This work focuses on the identification and discrimination of cultivars from the Movera collection using six SSR microsatellite markers. Results will enrich existing databases with molecular profiles of ancient Spanish varieties and contribute to their inclusion in the El Encín collection to form part of genetic heritage of grapevine for future plant-breeding programs.
Materials and Methods
This study includes 200 accessions, representing 72% of the black berry varieties in the Movera collection (Table 1⇓). The set encompasses mainly small berry varieties, which are of interest for the wine industry. DNA was extracted using the Master Pure Plant Leaf DNA Purification Kit (Epicentre Technologies, Madison, WI), following the manufacturer’s protocol. Some minor changes were introduced in the protocol to enhance the extraction: the volume of extraction solution was increased to 800 μL, ground tissue was incubated at 62°C for 45 min, and DNA was precipitated by adding an equal volume of isopropanol to 300 μL clarified supernatant. However, not all samples extracted by the kit gave good quality DNA for amplification. For these samples, an alternative DNA extraction method (Doyle and Doyle 1990) was applied, with some modifications. Isopropanol was used for DNA precipitation and the pellet was washed with 70% ethanol containing 0.3 M sodium acetate and 0.01 M magnesium acetate, followed by a wash with 70% ethanol.
Samples were genotyped using the same six SSR microsatellite markers (VVS2, VVMD5, VVMD7, ssrVrZAG47, ssrVrZAG62, and ssrVrZAG79) as in a previous study (Martín et al. 2003). Multiplex PCRs were carried out in a GeneAmp PCR system 2700 (Applied Biosystems; Foster City, CA), following a previous protocol (Martín et al. 2003) with slight modifications in the concentrations of some primer pairs (0.1 μM each VVS2 and VVMD7 primer). Amplified products were separated by capillary electrophoresis in an ABI PRISM 310 Genetic Analyzer and identified by GeneScan analysis software (Applied Biosystems), following previous procedures (Moussaoui 2005). When some overlapping of the peaks was observed, the primers were run independently.
Results and Discussion
Genetic analysis revealed that all six SSR loci analyzed were multiallelic (data not shown). The average number of alleles per locus (11.5) was high compared to the values reported earlier (Ibáñez et al. 2003, Núñez et al. 2004), which indicates the high diversity of the studied collection.
Analysis of the 200 accessions at six SSR loci allowed us to identify duplicates of the same variety and to detect synonyms or homonyms in the collection. Sixty-nine different molecular patterns were obtained, which constitute an independent source of genetic variation. The entry names of 86 accessions were confirmed, correct names were assigned to 33 misnamed accessions, and 43 “unknown” accessions were identified, based on the coincidence of their molecular profiles with published databases and further morphological observations carried out in the field (Table 1⇑). For 38 accessions, which gave 24 different genetic profiles, no coincidence was found with any published database and so far they are considered as “unknown” (see varietal name, Table 1⇑). Most could belong to ancient autochthonous varieties, well adapted to grow under extreme conditions, which is important considering current climate changes. To advance their identification, they are being further characterized at the molecular level using more SSR loci and by detailed ampelographic description. An enological study of these varieties is under way to determine the distinctive aromatic profiles of the wines derived from this plant material. Identified accessions are reliable germplasm material that should be preserved in the El Encín collection for exchange with other collections and for breeding programs.
Some hybrids, such as Caladoc and Ruby Cabernet, were identified from obtained microsatellite profiles. Caladoc is a cross of Malbec x Garnacha, and Ruby Cabernet is a cross of Cabernet Sauvignon x Mazuela. SSR analysis at the six loci confirmed the progenitors of these two crossings and their molecular profiles were included in the El Encín database (www.sivvem.monbyte.com/sivvem.asp).
Results revealed some synonymies and homonymies as well as some discrepancies with published database (Martín et al. 2003) based on the El Encín collection. We found the same genotypes and the same ampelographic characteristics for the following pairs of accessions (the first listing in pairs is the name from the Movera collection and the second is from the El Encín collection): Macicillo and Morate; Blasco and Tinto Velasco; Graciano de Alfaro and Trepat; Negro Samuel and Sumoll; and Negro S. Marcos and Sumoll.
In the Movera collection, cultivar Benedicto had a molecular profile (Moussaoui 2005) different at three of the six loci studied (Table 2⇓) than the cultivar Benedicto in the El Encín collection (accession BGVCAM0878; Martín et al. 2003) and identical at all six loci to the cultivar named Tinta Femia de Aldán (accession BGVCAM1589; Martín et al. 2003). The Movera collection accession Benedicto is considered true-to-type, since the name and the cultivar originate from this region, while both accessions BGVCAM0878 and BGVCAM1589 in the El Encín collection seem to be erroneous and should be corrected. Another study on grapevine varieties from northwestern Spain, including Tinta Femia de Aldán, which originates from this same region, also revealed a different molecular profile than the accession with the same name in El Encín collection (BGVCAM1589), where it is likely misnamed (Santiago et al. 2005). Accessions named Miguel de Arco in the Movera collection showed different molecular and phenological characteristics than the Miguel de Arco in the El Encín collection (accession BGVCAM1189; Martín et al. 2003), suggesting that they are homonyms, and in consequence should have different names (Table 2⇓). The name Miguel de Arco de Aragón is proposed for the variety at the Movera collection.
Four accessions identified as Ambrosina in the Movera collection originated from vineyards that have disappeared, planted between 1920 and 1930 in Cariñena and Ainzón (Zaragoza). The name, phenotype, and molecular profile were not found in the El Encín or any other European database. Ambrosina shows three unique genotypes in the loci VVMD7 (251/255), ssrVrZAG62 (193/199), and ssrVrZAG79 (249/249) (Table 2⇑). This cultivar is likely an autochthonous variety cultivated in Aragón before the appearance of the phylloxera and abandoned later (M. Lorente, author’s unpublished data, 2005). Researchers note that certain varieties with cultural importance before the outbreak of phylloxera, cited in 19th-century ampelographic publications, either are no longer found in the vineyards or only exist as “traces” or “impurities” in very old plots (Martínez de Toda and Sancha 1997).
Ten ancient accessions with the same molecular profile (and not yet published in El Encín collection) received the double name of Cadrete/Parrel (Table 2⇑). The name Cadrete has been used in the Huerva Valley and La Almunia regions of Zaragoza (Aragón) and is likely from the town of Cadrete, close to where this variety had been abundantly cultivated. In recent years, the last remaining vineyard in this area was eradicated. Parrel was mentioned in the 18th century (de Asso y del Rio 1784) and its description is very similar to the Parrel accessions present today in the Movera collection. Parrel was referred to as a very common local variety, still used for winemaking and table grapes, with a name used only in the Somontano area (Huesca, Aragón) (M. Lorente, author’s unpublished data, 2005). Observations by field experts revealed that these accessions displayed small differences in various ampelographic characters, such as cluster form, leaf color, and berry size, which suggest they might be different clones, which could not be discriminated by SSR analysis. One possibility to differentiate these clones would be to use a larger set of SSR markers, as in previous studies (Hocquigny et al. 2004, Moncada et al. 2006), or other more powerful molecular markers (Gogorcena et al. 1993).
Some differences were detected regarding the microsatellite profiles published in an earlier study, which included two Parraleta accessions and two Graciano accessions from the same Movera collection (Montaner et al. 2004). Molecular analysis carried out in this study demonstrated that accession 17-15, named Graciano de Alfaro in the Movera collection, had the profile of Trepat instead of Graciano or Rojal and that accession 20-4, named Parraleta in the collection, displayed a molecular profile identical to Mandón (Table 2⇑), which was confirmed by field observations. Parraleta is an autochthonous Spanish cultivar grown in the Somontano area (Huesca, Aragón) and authorized by the European Union to obtain “Quality Wines Produced in Determined Regions.” We identified 10 more accessions in the Movera collection (Table 2⇑) in addition to accession 7-5 studied elsewhere (Montaner et al. 2004). Three of the accessions had entry names different from Parraleta in the original list and five were within the group of the “unknown” varieties (Table 1⇑).
Conclusion
Genetic analysis at six SSR loci enabled us to distinguish 69 different molecular patterns in 200 accessions, 45 of which were identical to existing profiles in published databases. For 24 of the microsatellite profiles, no coincidence was found in any published database. These results facilitate the management of the cultivar collection by reducing the number of accessions and guarantee the authenticity of plant material for duplication and conservation in the El Encín collection.
Footnotes
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Acknowledgments: This work was supported by the Spanish Institute for Agricultural Research grant RF02-004-C5-5; Regional Government of Aragón grant A44 and grant CONSID-DGA, PM071/2007; and Spanish Ministry of Science and Innovation grant AGL-2008-00283, cofunded by FEDER.
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The authors thank Felix Cabello Sáenz de Santa María for helpful assistance in the ampelographic evaluation of cultivars in the field.
- Received November 2009.
- Revision received February 2010.
- Revision received June 2010.
- Accepted July 2010.
- Published online December 1969
- Copyright © 2010 by the American Society for Enology and Viticulture