Abstract
Genotypic analysis of ancient grapevine accessions from Argentina, Bolivia, Chile, Peru, and the United States reveals that most correspond to two ancient cultivars still cultivated in Spain: Muscat of Alexandria and Listán Prieto. The latter is grown throughout North and South America under different names such as País, Criolla Chica, Negra Peruana, Misión, and Mission. Most remaining ancient American cultivar genotypes analyzed correspond to hybrid progeny of Muscat of Alexandria, Listán Prieto, or both. We conclude that these two cultivars are major founders of ancient American viticulture.
American viticulture dates back to early 16th-century Spanish colonization. Wine availability became highly relevant in evangelization, as it was used to maintain a drinking diet and for sacramental purposes. Problems with wine preservation during transport to America promoted introduction of European grapevine (Vitis vinifera L.) cultivars to newly conquered territories. Not surprisingly, the first cultivars were introduced by Franciscan and Jesuit friars in Mexico and Peru between 1520 and 1540 (Hidalgo 1999). Grapevine cultivation was greatly associated with the mission system promoted by the Spanish monarchy to Christianize the New World (Kamen 2003). Grapes were planted in mission vineyards and spread with the establishment of new missions. In California, the most common cultivar was Misión (now called Mission). In Peru, a similar cultivar received the name Negra Peruana or Negra Corriente (Truel 1985, del Pozo 1998).
In his 1513 text Agricultura General, agronomist Alonso de Herrera listed 17 widely planted grapevine cultivars in the Castilian Kingdom (Castilla, Extremadura, and Andalucia): Albillo, Torrontés, Moscatel, Cigüente, Jaén, Hebén, Alarije, Malvasía, Lairén, Palomina Negra, Aragonés, Tortozón (today known as Tortosina), Herrial, Vinoso, Castellano Blanco, Castellano Negro, and Uvas Prietas (de Herrera 1988). The latter five cultivars have since been lost in the Iberian Peninsula. By the late 15th and early 16th centuries, numerous varieties were introduced to the Canary Islands, including Moscatel, Torrontés, and Vijariego, as well as Listán Blanco and Listán Negro, synonyms of Palomino Blanco (García Luján et al. 1990) and Palomino Negro or Prieto, respectively (Martínez 1998). The Canary Islands, a useful stop on American trading routes, were a natural source for transported goods.
Today, more than 30 ancient grapevine cultivars, including synonyms and homonyms, are still cultivated in America (Hidalgo 1999), but little is known about their relationships with the Spanish cultivars that established American viticulture. The winegrowing heritage of the Iberian Peninsula changed drastically in the 19th century mainly because of phylloxera, which in 30 years devastated 50% of Spanish vineyards (Hidalgo 1999). The Canary Islands remained free from phylloxera and therefore maintained the original Spainish cultivar diversity.
Several recent studies of ancient American cultivars have revealed that ancient vineyards could consist of nonuniform plantations of grapevines derived from seeds and therefore derived from crosses between V. vinifera cultivars (Agüero et al. 2003, Martínez et al. 2003), as shown for the American Torrontés cultivars. These analyses included the identification of several synonyms and homonyms, but provided little information on the main founder varieties. Our aim was to identify the origin of ancient cultivars grown in North and South America, classify synonymous and homonymous varieties, and establish their genetic relationships.
Materials and Methods
Plant material and DNA isolation.
Seventy-nine grapevine accessions were analyzed in this study, including ancient varieties traditionally used in America, such as País and Criollan cultivars (Table 1⇓). Sixty-eight samples were collected from ancient vineyards at Chilean wine-growing latitudes (30 to 37°S), including three cultivars (Italiona, Huasquina Pisquera, and Torontel) used for production of Pisco, a popular aromatic spirit produced in Peru and Chile. Eleven other accessions were included in the study: eight American accessions from Domaine de Vassal grapevine germplasm bank (INRA, Montpellier, France; www.montpellier.inra.fr/vassal), two from the Department of Tarija (Bolivia), and one from the Department of Viticulture and Enology, University of California (Davis, CA). Seven additional, well-characterized varieties provided by IMIDRA (Instituto Madrileno de Investigación y Desarrollo Rural, Agrario y Alimentario, Alcalá de Henares, Madrid, Spain) were included as control samples in microsatellite analysis: Chenin blanc (14D14), Pinot noir (15C15), Rheinriesling (15F19), Muller Thurgau (15F22), Moscatel Negro (22E60), and Moscatel de Grano Gordo (22C44 and 22C45), a synonym of Muscat of Alexandria. Richter 110, a Vitis berlandieri x V. rupestris hybrid root-stock, was also included in amplied fragment length polymorphism (AFLP) analysis. DNA was extracted from young leaves stored at −80°C, as described elsewhere (Narváez et al. 2001).
Microsatellite analysis.
Nine microsatellite loci, previously selected to construct the European Vitis database (Sefc et al. 2000), were used to genotype all accessions: VVS2 (Thomas and Scott 1993), VVMD5, VVMD7 (Bowers et al. 1996), VrZAG21, VrZAG47, VrZAG62, VrZAG64, VrZAG79, VrZAG83 (Sefc et al. 1999). Polymerase chain reaction (PCR) was carried out in a final volume of 10 μL containing 20 ng total DNA, 10 mM Tris HCl, 50 mM KCl (pH 8.3), 2 mM MgCl2, 200 mM of each dNTP, 5% DMSO, 0.2 μM [γ-33P]-forward primer, 0.2 μM reverse primer, and 0.4 U Taq DNA Polymerase (Perkin Elmer 5U/μL). Amplification was carried out in a Perkin Elmer 9700 thermocycler (Boston, MA), using a touchdown amplification program, which consisted of: 1 cycle of 30 sec at 94°C, 45 sec at initial annealing temperature (ta, see below), and 45 sec at 72°C, followed by 14 cycles in which the annealing temperature decreased 0.2°C per cycle, followed by 20 cycles of 30 sec at 94°C, 45 sec at ta−3°C, and 45 sec at 72°C, followed by a final extension of 5 min at 72°C. Initial annealing temperatures were 50°C (VrZAG21, VrZAG79, VrZA G83, and VVMD5), 51°C (VVMD7, VVDM47), 59°C (VVS2), and 61°C (VrZAG62, VrZAG64). Subsequently, 25 additional microsatellite markers were used to validate genotypes: VVMD6, VVMD14, VVMD24, VVMD25, VVMD26, VVMD27, VVMD31, VVMD32, VVMD34, VVMD36, VVMD37 (Bowers et al. 1996, 1999), VMC4f2-1, VMC4c6, VMC6g1, VMC7h2, VMC8g9, VMC2a3, VMC4f8, VMC5e9, VMC5h5, VMC9a2-1, VMC9h4-2, VMC4h6, VMC5g8, and VMC2g2. VMC microsatellites were developed by the Vitis Microsatellite Consortium, coordinated by Agrogene SA, Moissy Cramayel, France. PCR amplification of the additional 25 markers was performed using 55°C as ta.
AFLP analysis.
AFLP analysis was performed on 42 out of the 79 accessions, including 24 known as País, synonym accessions with the same allele combination at the nine microsatellite loci analyzed and Criollan accessions. Accessions differing by only a single allele were also included, as well as three accessions corresponding to the varieties Moscatel de Grano Gordo (22C44 and 22C45) and Moscatel Negro (22E60), maintained at the El Encín, IMIDRA, germplasm bank. Richter 110 was used as out-group in the AFLP analysis to apply threshold values previously determined for varietal discrimination and parentage analysis in grapevine (Cervera et al. 2001). AFLP reactions and polyacrylamide gel electrophoresis were performed as described elsewhere (Cervera et al. 1998) using two primer combinations: 2 EcoRI (+ACC, +ACT) / MseI +CAT and 2 EcoRI (+ACC, +ACT) / MseI +CTG). Amplified fragments were independently scored by two persons and used to obtain a binary matrix of presence and absence. Independently harvested leaf samples from the same accessions were analyzed to resolve inconsistent AFLP fragments.
Statistical analysis.
Identification of redundant genotypes and pedigree reconstruction based on microsatellite data was determined by comparing microsatellite genotypes with data contained in different microsatellite grapevine databases, which included Spanish (Ibáñez et al. 2003, Martín et al. 2003), European (Sefc et al. 2000), and American (Agüero et al. 2003) varieties. Genotype comparison was carried out using the Microsatellite Toolkit v9.0 software package (Park 2001). Parentage relationships were identified by studying allelic compatibility at nine microsatellite loci (VVS2, VVMD5, VVMD7, VrZAG21, VrZAG47, VrZAG62, VrZAG64, VrZAG79, and VrZAG83) in 181 grapevine varieties: 162 European winegrape varieties (Sefc et al. 2000) and the 19 American genotypes detected in this study. Observed allelic frequencies and their 95% upper confidence limits were calculated using the IDENTITY 1.0 software package (Center for Applied Genetics, University of Agricultural Sciences, Vienna) and used to estimate likelihood ratios by comparing the probability of obtaining the observed genotype of each variety with the proposed progenitors versus the probability of it being derived from other crosses (Bowers and Meredith 1997). Information from the 25 remaining loci was used to further support identified pedigrees.
The AFLP binary matrix was analyzed using Numerical Taxonomy System software (NTSYSpc v.2.02g; Exeter Software, Setauket, NY). Genetic similarities among pairs of genotypes were obtained using the Dice coefficient (Sneath and Sokal 1973) and the resulting clustering, based on the unweighted pair-group method of arithmetic averages (UPGMA), represented as a dendrogram. Hybrid genotypes were confirmed by AFLP pattern comparison (Cabezas et al. 2003).
Results
Microsatellite analysis.
Seventy-nine V. vinifera accessions were genotyped at nine microsatellite loci, and grouped into 19 different genotypes, two of them differing by a single allele, and one by lack of amplification at a specific locus (Table 2⇓). A total of 39 alleles, ranging from 132 (VVS2) to 258 bp (VrZAG79), were observed, with an average of 4.3 alleles per locus. Total probability of identity for all loci was 3.9 x 10−6. Values of observed heterozygosity (Ho) were higher than expected heterozygosity (He) for all loci (Table 3⇓), as usually found in analyses of other grape cultivars (Sefc et al. 2000, and references therein). The estimated average Ho was slightly higher with American (0.897) than with European varieties (0.823). Genotypic comparison demonstrated that 50 accessions, including ancient American cultivars such as País (Chile), Rosa del Perú type 1 (Peru), Negra Corriente (Peru), and Mission (USA), were identical. País 81Q showed identical genotype at the eight loci that could be amplified (VVS2 failed), and Criolla Chica (Argentina) only differed in one allele at the VrZAG83 locus (Table 2⇓). Criolla Mediana and Rosa del Perú type 2 also differed in one allele at locus VrZAG83. Some homonyms of País (accessions 71Lb and 72La) were also detected, associated with the Argentinean variety Torrontés Riojano. The 19 genotypes were compared with those available in three databases: European (nine common microsatellites; Sefc et al. 2000), Spanish (six common microsatellites; Ibáñez et al. 2003, Martín et al. 2003), and American (five microsatellites; Agüero et al. 2003). Five microsatellite loci were common between all databases and this study: VVS2, VVMD5, VVMD7, VrZA G62, and VrZAG79. Some adjustments were required to correct size variation associated with differences of fragment size estimation used to construct the databases. Comparison of the 50 accessions sharing a common microsatellite pattern with known European and American cultivars showed that they were identical to the cultivar Listán Prieto, synonymous with Moscatel Negro in the Canary Islands, Spain (Martín et al. 2003) and Hariri in Morocco (P. Truel, confirmed by V. Laucou and T. Lacombe, unpublished data). Also, two accessions shared a genotype with Muscat of Alexandria, and two accessions, Negra Criolla and Mollar, corresponded to the Spanish variety Mollar Cano (Martín et al. 2003). Twenty-one of the remaining 23 accessions had genotypes consistent with hybrid progeny of Listán Prieto and Muscat of Alexandria (Table 2⇓). The last two accessions, Italiona (52R) and a homonym of País (43H), shared one allele at each locus with Muscat of Alexandria and Listán Prieto, respectively (Table 2⇓). These similarities are consistent with a parent-offspring relationship between Muscat of Alexandria and Italiona on one side, and Listán Prieto and the País homonym on the other (explained below). This analysis confirmed that Torrontés Riojano, represented by two synonyms (71Lb and 72La), is also a progeny of Listán Prieto x Muscat of Alexandria, as shown previously (Agüero et al. 2003).
AFLP analysis.
Identities and parentage relationships suggested by microsatellite analysis were confirmed with AFLP data. Analysis of 45 accessions using two AFLP primer combinations identified a total of 189 fragments ranging from 100 to 700 nucleotides: 107 fragments for primer combination 2EcoRI (+ACC, +ACT) / MseI +CAT and 82 fragments for 2EcoRI (+ACC, +ACT) / MseI +CTG. Of 189 fragments, 108 (57%) showed clear polymorphisms and were used to construct a binary matrix of presence or absence. A dendrogram was constructed from estimates of genetic similarities (GS) (Figure 1⇓). Correlation between the Dice and its cophenetic matrix was very high (r = 0.98 with an associated one-tailed p-value of 0.002), indicating a very good fit of the cluster analysis. AFLP analysis revealed that accessions with microsatellite genotypes differing by a single allele could be assigned to the same cultivar, so the number of genotypes discriminated by microsatellites was reduced from 19 to 17.
The AFLP dendrogram contains one main cluster, País, which groups 27 accessions at GS ≥ 0.92 (Table 4⇓). The País cluster consists of 19 accessions named País (10F, 37C, 65Y, 66Y, 61P, 80Q, 84CU, 63T, 77Q, 75L, 76Q, 41H, 78Q, 72L, 46H, 48G, 57P, 40H, and 60P), one ampelographic uncertainty (34C), different synonyms collected throughout America (Negra Corriente, from Peru; Criolla Chical, Argentina; and Mission, USA), three Chilean synonyms (Uva Negra Vino, Viña Blanca, and Viña Negra), and one Spanish variety (Moscatel Negro 22E60), which lacks muscat flavor, is considered a homonym of Muscat, and corresponds to Listán Prieto (Jorge Zerolo, personal communication, 2004).
For the remaining accessions, those sharing microsatellite genotypes fell within the same AFLP cluster. All remaining accessions except C32, Moscatel de Grano Gordo (22C44 and 22C45), and Italiona (52R) showed a close relationship with País accessions (Table 4⇑). Accessions 43H, 71La (homonyms of País), 71Lb (synonym of Torrontés Riojano), Torontel (58P), Criolla San Juanina (9F), Viña Antigua Negra (56P), the País homonym 62P, T01 (Torontel, a homonym of accession 58P), Criolla Mediana (13F), Rosa del Perú type 2 (6F), and Cereza (11F) showed high pairwise GS values, suggesting a parentage relationship among them (Cervera et al. 2000). Viña Antigua Negra (56P) and the homonym of País 62P, which show complete identity, are two accessions of the same variety. Criolla Mediana (13F) and Rosa del Perú type 2 (6F) showed high GS (0.95), which is expected for accessions belonging to the same variety. This hypothesis is also supported by microsatellite analysis since their genotypes differ by a single allele. Criolla Mediana (13F) and Rosa del Perú type 2 (6F) showed GS values between 0.87 and 0.88 when compared to Cereza (11F), which suggests a very close relationship between these varieties (Table 4⇑). Uva del Padre (61Pa) and 53R, a homonym of País, showed an identical genotype and therefore are accessions of the same variety (Table 4⇑).
Accession 32C, an ampelographic uncertainty, was grouped at GS > 0.97 with Moscatel de Grano Gordo (22C44 and 22C45), a known synonym of Muscat of Alexandria (Hidalgo and Galet 1988). This result confirmed the genotypic identity found with microsatellite analysis. Muscat of Alexandria was compared with all accessions previously described, showing less genetic similarity with País and accession 43H, a homonym of País, than with the remaining varieties, although in some cases less than expected between a progenitor and its progeny. Accessions 53R (homonym of País), Uva del Padre (61Pa), the unclassified accession 70L, and Italiona (52R) had GS values between 0.71 and 0.79 when compared to Muscat of Alexandria.
Pedigree assignment.
Analysis of allelic segregation at microsatellite loci has been widely used in accurately assessing parentage relationships (Bowers and Meredith 1997, Sefc et al. 1999) and, more recently, comparison of AFLP profiles has proved informative in confirming parent-offspring relationships in V. vinifera (Cabezas et al. 2003). Both techniques have shown 11 (if Criolla Mediana and Rosa del Perú type 2 are considered a single variety) out of the 17 different genotypes identified as compatible with a hybrid origin as a cross between Muscat of Alexandria and País (or Listán Prieto). In fact, the likelihood ratios of obtaining these genotypes from the proposed cross versus the probability of obtaining them from two random varieties range from 2.23 x 105 to 6.80 x 107 (1.10 x 104 to 1.54 x 106 using the 95% upper confidence limits for the allelic frequencies) (Table 2⇑). This hypothesis was further confirmed by analysis of allelic combinations at 25 additional microsatellite loci, although they were not used for statistical calculations because of unknown allelic frequencies. Two of the three remaining accessions, Italiona (52R) and a homonym of País (43H), shared one allele at each locus with either Muscat of Alexandria or Listán Prieto, respectively. These similarities are consistent with a parent-offspring relationship between Muscat of Alexandria and Italiona, and País (or Listán Prieto) and the País homonym (43H) (Table 2⇑). A closer look at the second alleles reveal that they fit with a combination of País and Mollar for Italiona (52R) and Muscat of Alexandria and Mollar for the homonym of País (43H), which suggests that these varieties could be complex hybrids. Comparison of AFLP profiles confirmed the hybrid origin of most ancient American cultivars initially inferred from microsatellite analysis and suggested by AFLP GS values (Figure 2⇓). These hybrids showed AFLP fragments that were also detected in Muscat of Alexandria and/or País (or Listán Prieto) and are thus consistent with crosses between both varieties. AFLP profiles of Italiona (52R) and a homonym of País (43H) also suggested a parent-offspring relationship with Muscat of Alexandria and Listán Prieto, respectively (data not shown).
Discussion
This work required use of four databases to search for the founders of American viticulture. To avoid inaccurate allele designation because of different methods of marker detection, a similar method to that described elsewhere (This et al. 2004) was used to develop a standard set of reference microsatellite alleles. In this way, different standards (representative accessions of databases) were used as internal controls to ensure an accurate allele size typing for database comparison. The use of nine microsatellite loci yielded a combined probability estimate of 3.9 x 10−6; therefore, two accessions showing the same microsatellite genotype can be assigned to the same grapevine variety. AFLP analysis was performed with the same primer combinations and outgroup used in previous studies to apply previously determined threshold values to distinguish Spanish grapevine varieties and infer their parentages (Cervera et al. 1998, 2000). However, it is noteworthy that the number of amplified AFLP fragments detected in this study differed between País (43) and Muscat of Alexandria (31 fragments), which are present in pedigrees of most Criollan varieties. Moreover, 24 fragments were shared between both varieties, requiring a modification of threshold values for Muscat of Alexandria, which led to a reduction in threshold for parentage detection.
Microsatellite and AFLP analysis led to identification of three ancient varieties cultivated in Spain: Listán Prieto, Muscat of Alexandria, and Mollar Cano. Different synonyms and homonyms were detected, associated with their cultivation throughout America. Thus, Moscatel Negro, a Spanish homonym of Muscat and synonym of Listán Prieto (Jorge Zerolo, personal communication, 2004), is known as País, Uva Negra Vino, Viña Blanca, and Viña Negra in Chile, Criolla Chica in Argentina, Rosa del Perú and Negra Corriente in Peru, Misión in Mexico, Mission in the United States, and Hariri in Morocco. Two unclassified accessions based on ampelography were assigned to Muscat of Alexandria. Finally, Negra Criolla and Mollar are synonyms of the Spanish variety Mollar Cano cultivated in Andalucía (García Luján et al. 1990).
Molecular characterization of the remaining V. vinifera accessions revealed that they are hybrid progeny of crosses involving País (synonym of Listán Prieto) and/or Muscat of Alexandria. The same cultivars have also been identified as parents of the Argentinean varieties Torrontés Riojano (also in our samples), Torrontés Sanjuanino, and Moscatel Amarillo (Agüero et al. 2003, This et al. 2006). Criolla Grande and Criolla Mediana (included in our samples) were also likely hybrids from the same parents (Agüero et al. 2003). Another cultivar, Torrontés Mendocino, also showed a parent-offspring relationship with Muscat of Alexandria (Agüero et al. 2003). Although all these hybrid accessions derive from a few European cultivars, none has been found in European viticulture, suggesting that they originated in the New World.
Listán Blanco has been described as a synonym of Palomino Blanco (García Luján et al. 1990). The Spanish word prieto means dark or black color. Thus, the variety Listán Prieto was probably known as Palomina Negra during the 16th century. This variety was an important cultivar in Castilian viticulture (de Herrera 1988) and was introduced in the Canary Islands at that time (Martínez 1998). It is currently grown in the islands and has disappeared from viticulture in peninsular Spain, likely because of the 19th-century phylloxera plague. Muscat of Alexandria is an ancient cultivar grown throughout the Mediterranean. Cultivation of Muscats in the Iberian Peninsula was reported by Abu Zacaria in the 12th century (Banquero 1988). The early introduction into the New World of both cultivars agrees with a report in 1616 that the Spanish conqueror and missionary, Francisco de Caravantes, introduced the first grapevine plants to Peru from the Canary Islands (Garcilaso de la Vega 1976). Two varieties are described: a black cultivar Uva prieta and Muscat. The Uva prieta variety introduced by missionaries to produce wine was Negra Corriente, also known as Misión, Criolla Chica, and País, as reported previously, which was well adapted to adverse environmental conditions. This black variety was spread by Franciscans throughout Mexico, California, Lima (Peru), southern Maule (Chile), and southern Argentina, and by Jesuits throughout southern Mexico and Central America, southern Brazil, northern Argentina, Mendoza (Argentina), Arequipa (Peru), Santiago, Concepción, and Cauquenes (Chile). Listán Prieto is a black variety that produces red wine with poor color but pleasant taste, as the cultivar Uva Prieta was described (Garcilaso de la Vega 1976). Muscat of Alexandria is a white table grape variety used to produce raisins or white aromatic wines and spirits. Spirit made from muscat, now known as Pisco, was much appreciated by local people in Peru, who would drink chicha, a traditional fermented corn drink, and thus it rapidly became a popular drink (del Pozo 1998). These two complementary cultivars became the progenitors of most current Criollan cultivars and can be considered the founders of ancient American viticulture.
Conclusion
We sought to genetically characterize ancient American grapevine accessions. The combined use of different marker types and public databases allowed the identification of original varieties introduced in America, and revealed close relationships among Criollan cultivars. Microsatellite and AFLP genotyping showed that 52 accessions, including País, Rosa del Perú (type 1), Negra Corriente, Criolla Chica, Misión, and Mission, are synonyms of Listán Prieto, an ancient Spanish variety now restricted to the Canary Islands in Europe. Additionally, two accessions were identified as Muscat of Alexandria, an ancient Mediterranean variety, and two other accessions were synonyms of the ancient Spanish variety Mollar Cano. Finally, all remaining accessions correspond to hybrid progeny of Muscat of Alexandria, Listán Prieto, or both. Together with historical data, molecular characterization conducted in this work has allowed the identification of the origins of ancient American viticulture.
Footnotes
↵3 [current address] Centro de Estudios Avanzados en Zonas Áridas, Benavente 980, La Serena, Chile
↵6 [current address] Departamento de Sistemas y Recursos Forestales, CIFOR, INIA, Ctra. de La Coruña km 7, 28040 Madrid, Spain
Acknowledgments: AMT was funded by a BID-CONICYT-Chile fellowship. JAC was funded by predoctoral fellowship from INIA (Instituto Nacional de Investigaciones Agrarias y Agroalimentarias, Spain). This research was funded in part by project contract ICA4-CT-2001-10065 from UE, and project BIO2002-10134-E from MCYT (Spain).
We thank agronomist Arturo Lavín (Estación Experimental Cauquenes, INIA, Chile) and historian Silvia García-Beneytez for information on viticulture during the 15th and 16th centuries and María-Herminia Castro for technical assistance.
- Received February 2006.
- Revision received November 2006.
- Copyright © 2007 by the American Society for Enology and Viticulture