Abstract
Twenty-five cultivars grown in Galicia (northwest Spain) and other six international cultivars have been analyzed in order to evaluate the genetic variability and cultivar relationships among them. Statistical analysis of 23 different genotypes found from six microsatellite markers (VVS2, VVMD5, VVMD7, VVMD27, ZAG62, and ZAG79) has shown three different groups. Several new synonymies were found among the minority cultivars analyzed. All the genotypes were compared to other databases and five more synonymies were found. A dendrogram shows the genetic similarities among the accessions and establishes the most probable relationship among them. Results also confirm, complement, and add new information to previous studies that have suggested the use of the variability in the phenolic compounds as a tool to identify phylogenetic relationships among these cultivars.
The European grapevine (Vitis vinifera L.) is one of the oldest and most important crops in the world. Spain is a leading grapegrowing countries, with over 1.150 million ha of V. vinifera grapes in cultivation. The high number of cultivars grown in different regions in Spain has led to numerous names for each variety, and synonymies and homonyms are frequent.
Galicia, an important wine-producing region in northwest Spain with a long winemaking tradition, has five denominations of origin (DO) areas: Ribeiro, Rías Baixas, Monterrei, Valdeorras, and Ribeira Sacra. The different DOs in Galicia have important black and white V. vinifera cultivars, and many minority cultivars in Galicia are denominated with different local names according to the area in which they are grown. These cultivars are well-adapted to their environments and impart to their wines the characteristics of the climate and soil in which they are grown. Some of these varieties could be distinguished by studies on the phenolic and volatile composition by using HPLC and GC-MS methods (Pomar et al. 2005, Vilanova and Sieiro 2006, Masa et al. 2007, Masa and Vilanova 2008).
Several types of DNA molecular markers (RAPD, SCAR, AFLP, and RFLP) have been used in agriculture species for grape cultivar identification (Gogorcena et al. 1993, Vidal et al. 1998, 1999, Blaich et al. 2007), and simple sequence repeat (SSR) markers have been developed by different groups for genetic analysis of grapevine cultivars (Bowers et al. 1996, 1999, Sefc et al. 1999). These SSR markers are codominant, frequent, and evenly distributed throughout the genome, selectively neutral, highly reproducible, and rely on simple polymerase chain reaction (PCR) technology. Microsatellite markers are ideal markers for plant genetic linkage mapping, assessment of genetic diversity, and cultivar identification and discrimination (Morgante and Olivieri 1993, Sefc et al. 1999, Fernández-González et al. 2007).
Previous studies have characterized part of Spanish germplasm (Vidal et al. 1999, Ibáñez et al. 2003, Martín et al. 2003), but some of the cultivars from Galicia have not yet been studied using molecular tools. In the present study, SSR markers were used as a tool to characterize the genetic diversity and cultivar relatedness in cultivars from northwest Spain. The study was conducted using six microsatellite markers described in the bibliography and considered by the European Project GenRes 081 as the most adequate core set for the screening of grapevine collections (http://www.genres.de/eccdb/vitis).
Materials and Methods
Cultivars.
A total of 31 grapevine accessions (25 cultivars grown in Galicia and 6 international cultivars used as references) were analyzed from cultivars grown in collections from Misión Biológica de Galicia–CSIC and Xunta de Galicia (Ribadumia, Pontevedra) and accessions grown in commercial vineyards from different geographic areas in Galicia (Pontevedra, A Coruña, Lugo, and Ourense) (Table 1⇓).
DNA extraction.
DNA extraction was based on a modified CTAB extraction procedure (Steenkamp et al. 1994), without the use of liquid nitrogen. Plant material (young leaf) was mechanically disrupted using the sandpaper method (Nakaune and Nakano 2006). Three replicates of DNA extraction from each variety were performed. DNA was quantified with lambda DNA molecular marker on ethidium bromide stained 0.8% agarose gels.
Microsatellite analysis.
Six previously described microsatellites were amplified through PCR: VVMD5, VVMD7 (Bowers et al. 1996), VVMD27 (Bowers et al. 1999), ZAG62, ZAG79 (Sefc et al. 1999), and VVS2. This set of markers is highly polymorphic and is considered by the European Project GenRes 081 (EU Project GenRes 081 1997) as the most adequate core set for the screening of grapevine collections (http://www.genres.de/eccdb/vitis).
A multiplex PCR reaction was carried out for five of the analyzed SSRs and a singleplex for VVMD5. All forward primers were labeled to allow detection by using 6-FAM, VIC, PET, and NED fluorescent dyes. Multiplex reactions were prepared in a final volume of 25 μL, containing 50 ng genomic DNA, 1 U BioTaq DNA polymerase (Bioline, London, UK), 0.2 mM of each dNT, 5–7.5 pmol (depending on primer pair) of each forward and reverse primers, 1X PCR buffer, and 2 mM MgCl2. Singleplex were performed in a final volume of 12.5 μL containing 25 ng genomic DNA, 0.5 U Taq polymerase, 0.2 mM of each dNTP, 3 pmol VVMD5 forward and reverse primers, 1X PCR buffer, and 2 mM MgCl2. All PCR reactions were carried out in a GeneAmp PCR 9700 thermocycler (Applied Biosystems, Foster City, CA) using the following conditions: 94°C for 5 min, 30 cycles of 1 min at 94°C, 1 min at 51°C or 49°C (for multiplex or singleplex PCR, respectively), and 1 min at 72°C, with a final extension of 30 min at 72°C. Amplified products were size-separated by capillary electrophoresis performed on an ABI 3130xl Genetic Analyzer together with 600 LIZ size standard internal weight marker (36 fragments from 20 to 600 bp) using Performance Optimized Polymer 7 (POP-7) (Applied Biosystems). Labeled fragments were detected using GeneMapper software (Applied Biosystems). Resulting data were analyzed with Genescan 3.7 for internal standard and fragment size determination.
Data analysis.
The number of alleles, allele frequencies, expected and observed heterozygosity, probability of identity (PI) as the probability of obtaining identical profiles and the probability of null alleles were calculated using IDENTITY 1.0 software (Wagner and Sefc 1999). Relationships between cultivars and dendrogram were calculated with XLSTAT 2007 (Addinsoft, Paris, France).
Results and Discussion
In this work, SSR markers were used to investigate the phylogenetic relationships among a set of minority cultivars from northwest Spain and to identify the presence of possible synonymies or homonyms among the cultivars. The results of genotyping were compared to different database genotypes (Martín et al. 2003, European Vitis database, www.genres.de/eccdb/vitis; Sefc et al. 2000; BIOVID project) and were also examined in consideration of biochemical studies previously performed on some of these varieties.
SSRs analysis.
Thirty-one cultivars (25 minority Galician cultivars and an additional 6 international cultivars used as references) were genotyped using six microsatellite markers and 23 different allelic profiles were found (Table 1⇑). A total of 40 alleles were detected, ranging from 5 for ZAG62 to 9 for VVMD5 (Table 2⇓). The highest allelic frequencies were found for VVMD27-183, VVS2-150, VVMD7-235, and ZAG79-244.
Nine different genotypes were identified for regions ZAG62 and ZAG79, 8 for VVMD27, 10 for VVS2, 12 for VVMD7, and 14 for VVMD5. The more frequent genotypes were 185:191 with 39.1% for region ZAG62, followed by 175:183 (34.8%) for VVMD27, 242:244 (30.4%) for ZAG79, and 142:150 (26.1%) for VVS2. The most informative marker in the studied set of accessions was VVMD5, as previously reported (Ibáñez et al. 2003, Bowers et al. 1996, Sefc et al. 2000) with a PI of 0.10, and the least informative was ZAG79, with PI of 0.23. The cumulative probability for the six SSR markers was 10−5, similar to that previously described (González-Andrés et al. 2007). Chimeras were not found in the analyzed cultivars, as tri-allelic or tetra-allelic genotypes were not present.
The level of polymorphisms found in this study was similar to other reports (Martín et al. 2003, Ibáñez et al. 2003). The observed heterozygosity for the set of cultivars was high and was higher than expected for all loci. The percentage of heterozygosity (mean for all loci) observed in Galician cultivars was 88%, with a variation from 83% (VVMD7, VVMD27, and ZAG79) to 96% (VVS2). This heterozygosity was higher than that previously obtained: 85.5% (Bowers et al. 1996), 70.7% (Ibáñez et al. 2003), 74.4% (Sefc et al. 1999), and 81.8% (Martínez et al. 2006). In the present study VVMD5 marker presented the highest expected heterozygosity.
Synonymies.
In this study several new synonymies were found, with Cascón, Corbillón, Estradeira, Follajeiro, and Verceiruda presenting identical genotypes in all the six microsatellites analyzed. Cajarrento and Ratiño showed the same genotype and Sousón and Tinta país showed the same genotype, indicating that in each case two names are used for one cultivar. Anthocyanin and flavonoid composition of several of these cultivars was previously studied by HPLC (Pomar et al. 2005, Masa et al. 2007). In white cultivars, high heterogeneity was found for all compounds. Except in cultivar Albariño, quercetin-3-O-glucuronide was the major flavonoid in all cases studied, and it was significant that this compound was not found in Ratiño (Masa et al. 2007). For the red cultivars Cascón tinto, Corbillón, Follajeiro, and Verceiruda, a similar phenolic profile was has been described (Pomar et al. 2005). Moreover, Verceiruda/ Follajeiro were characterized by high levels of petunidin- 3-monoglucoside-p-coumarate, peonidin-3-monoglucosidep-coumarate, and malvidin-3-monoglucoside-p-coumarate, and Sousón/Tinta país had high levels of malvidin-3- monoglucoside and peonidin-3-monoglucoside. Our results confirmed by genetic analysis previous studies that have suggested variability based on the phenolic compounds of these cultivars.
Other synonymies have been found in other studies. Castellana shows the same genotype to Rufete (Martín et al. 2003). The same genotype was found for Mouratón and Touriga Nacional (European Vitis database; Sefc et al. 2000), yet was different than the Mouratón described elsewhere (Martín et al. 2003, Sefc et al. 2000), which indicates a possible homonym. Sousón was synonymous to Vinhao (Sefc et al. 2000), Tinta da Zorra to Negrón de Aldán and Petit Bouschet (BIOVID project; Martín et al. 2003), and Retinto to Borraçal and Caiño Tinto (BIOVID project).
We were unable to differentiate two pairs of samples: Pinot noir/Pinot blanc and Cascón tinto/Cascón blanco. Each showed the same allelic combinations for all the markers analyzed. The unique ampelographic difference was berry color, which could be explained by the occurrence of somatic variants in the same cultivar, such as single variation or mutation in specific genes (Bowers et al. 1996, Walker et al. 2007).
Genetic distance.
A dendrogram representing the genetic similarities among the accessions was constructed to investigate possible parentage relationships among samples (Figure 1⇓). The UPGMA dendrogram revealed three groups by clustering cultivars with 10% similarity (Figure 1⇓, Table 3⇓). One group (C1) formed by 17 cultivars showed 22% similarity: Albarello, Blanco lexítimo, Blanco XR, Cabernet Sauvignon, Cajarrento, Castellana, Colgadeira, Collón de mico, Couxo, Cumbrao, Chavacana, Mouratón, Pinot, Retinto, Sauvignon blanc, Sousón, and Tinta da zorra. A second group (C2) was composed of five cultivars and presented 30% similarity: Albariño, Blanca mar, Caiño blanco, Cascón, and Loureira. The third group (C3) contained only Riesling.
In the C1 group, the centroid was represented by Mouratón, with an average distance among cultivars of 2.336. In C2, the centroid was Blanca mar; the other cultivars showed a minor distance to the centroid with an average distance of 2.173. The Chavacana and Colgadeira cultivars from group C1 showed the highest similarity (90%), as they only differed in one allele for the marker VVMD5, indicating that those cultivars could be related. In some cases, slight differences at only one or two different alleles allow us to consider the cultivars as clones (Ibáñez et al. 2000, Lefort et al. 2001, Regner et al. 2000). These differences are probably due to the relatively high probability of mutation that occurs in this type of sequence and/or to the age of many cultivars of grapevine (Ibáñez et al. 2003). Retinto and Collón de mico from group C1 showed a similarity of 62%, with four different alleles in VVS2, VVMD7, and ZAG62. Cascón and Blanca mar from group C2 showed a similarity of 59% and also differed in four alleles sited in the loci for VVMD5, VVMD7, VVMD27, and ZAG62. In general, no clear separation according to geographical origins was observed.
The use of classical ampelographic methods has not been able to establish phylogenetic relationships among the minority cultivars from Galicia, fundamentally for the identification of synonymies and homonyms among the studied cultivars. Microsatellite DNA analysis has demonstrated to be a powerful and reliable technique for the characterization of these grapevine cultivars.
Conclusion
Several new synonymies among plants were identified in this study: Cascón tinto, Cascón blanco, Corbillón, Estradeira, Follajeiro and Verceiruda; Cajarrento and Ratiño; and Sousón and Tinta país. The Chavacana and Colgadeira cultivars only differed in one allele and showed the highest similarity (90%), indicating that they could be related. Information on the genetic relationships among minority cultivars of Galicia may be used to identify their origin and will be helpful in the analysis of cultivars for the production of new wines.
Footnotes
Acknowledgments: M. Vilanova and M. de La Fuente are grateful to Consellería de Innovación e Industria from Xunta de Galicia for financing. The authors thank DO Ribeira Sacra, especially Ana López Pardo, for the localization of minority cultivars and thank J.M. Martínez-Zapater and J.R. Vidal for their assistance.
- Received September 2008.
- Revision received January 2009.
- Accepted February 2009.
- Published online June 2009
- Copyright © 2009 by the American Society for Enology and Viticulture