Identification of autochthonous grapevine varieties in the germplasm collection at the ITA of ‘ Castilla y León ’ in Zamadueñas Station , Valladolid , Spain

During the last decades a study of the grapevine collection located at the Zamadueñas Station of the Instituto Tecnológico Agrario de Castilla y León (ITACyL) was carried out. Recently, molecular markers have been used for varietal identification and detection of the possible synonymies in the collection. This work shows the results of that characterisation, as well as the detected synonymies and homonymies in the study. The maintenance of the existing plant material is recommended, particularly with reference to the varieties with a marked risk of extinction because they are not presently object of new plantations. Additional key words: ampelography, germplasm bank, microsatellites, molecular markers, STMS.


Introduction
At the present time there are at least ten ampelographic collections of grapevine in Spain plus the Vitis germplasm bank located at 'El Encín' (Alcalá de Henares, Madrid), all of them aiming to conserve the grapevine variability existing in the country (Hidalgo, 1999).The increasing importance of the Denominations of Origin (DO) in Spain, each one including a reduced number of authorised varieties, marks a tendency to the reduction of variability in the grapevine growing regions.
The grapevine collection located at the Instituto Tecnológico Agrario (ITACyL) of Castilla y León in Spanish Journal of Agricultural Research (2006) 4(1), 31-36 Zamadueñas (Valladolid) includes a total of 162 accessions, 30 table grapes and 132 wine grape varieties (Hidalgo, 1999).Forty one of them are autochthonous cultivars that were collected in the region in different prospecting missions carried out in the last decades.These accessions were introduced with the varietal name that was used in their sampling sites.Preliminary morphological characterisation of the plants pointed to possible homonymies or synonymies among the collected plant material.Molecular characterisation of these plants has been carried out in order to detect or confirm the true to typeness of the plants and complete the varietal identification of each accession.
García de los Salmones (1914) mentioned in the two regions corresponding to the present day Castilla y León political division, a total of 162 white and 175 red varieties, although this number very likely includes a high number of synonymies, some of them already detected.Nevertheless, the evolution of the viticulture in the region, with a growing number of plantations and with a reduced number of authorised varieties, is leading to the loss of a high part of the varietal patrimony.
The germplasm bank of the Comunidad de Madrid (BGVCAM) contains 130 accessions from the mentioned region (Cabello, 1995), that include a part of the existing variability of the zone.
The increasing interest in avoiding the loss or extinction of grapevine varieties and the complete identification of the already collected material has motivated the present work, the objective of which is to detect the existing synonymies as well as to identify in all possible cases the correspondence of the accessions to other known varieties.

Material and Methods
Table 1 lists all the studied accessions, with the indication of the colour of the berry and their name in the respective places of collection.Some accessions were named when introduced in the collection, and in some other cases a second name or a number was assigned because of the presumed existence of a homonymy.A minimum of eight plants at a 2.8 ´1.4 m spacing were planted in the collection plot, in a sandy clay loam soil.Cultural practices were the traditional in the zone.
The identification of the studied accessions was carried out on the basis of the molecular analysis, complemented with the ampelographic observations, made on each accession following the Organisation Internationale de la Vigne et du Vin (OIV, International Organisation of Vine and Wine) descriptors, and comparing with the existing databases (Martín et al., 2003).The last column in Table 1 indicates the varietal names for each accession.
Sampling for molecular analysis consisted of young fresh leaves that were collected in the field and kept fresh or after lyophilization at -80°C until analysed.
DNA extraction and amplification were carried out by using the MasterPure TM Plant Leaf Purification Kit (Epicentre Technologies, Madison Wis.), and the following six STMS loci were used: VVS2 (Thomas and Scott, 1993), VVMD5 and VVMD7 (Bowers et al., 1996), and ssrVrZAG47, ssrVrZAG62 and ssrVrZAG79 (Sefc et al., 1999), under the conditions detailed in a previous work (Martín et al., 2003).Polymorphism of the amplified products was detected in an automated DNA sequencer ABI PRISM model 310 (PE Applied Biosystems).As a result of the analysis, genotypes for each variety were obtained for the studied loci.

Results
The following identification results were obtained: Major varieties 1) Temprano blanco, is a variety known under this name in some places like Cigales (Valladolid), and under the name Malvasía in the DO 'Bierzo'.It is the French 'Chasselas doré' (http://www.imianet.org/exploraelencin/web/003/), also collected in Spain with the names Temprana Agosteña (CyL-19), Temprana Media, Temprana Tardía, Tempranillo de Nava, Albillo Negro and Elba.It is present in many places of the region, frequently designed with names that include the term 'Temprano' or 'Temprana', that means early.
2) Tinta del País is the Tempranillo, an already known synonymy.It is also synonymous with Tinta de Nava, that is a red variety included in the collection with the name of the locality of sampling.A grey berry mutation is the Tinta de Toro Blanca, the name of which comes the light colour of the berry; it should be more convenient to call it Tempranillo Gris or Tinta de Toro Gris.
3) Albillo de Nava corresponds to the variety Verdejo.In fact, the name Albillo includes several cases of homonymy, none of which had so far been related with Verdejo.It is recommended not to use in this case the name Albillo, to avoid confusion.4) Several accessions of the varieties Rufete and Prieto Picudo have been detected, that could be clones with some intravarietal variability.It also was in the collection a clone of Mencía, with rather short internodes, with the name Mencía pajaral.
5) The variety Juan García, grown in the 'Arribes del Duero' has been collected in this region as Negrera  (Rodríguez-Torres et al., 2000), has been collected with the name Pirulés and Rojal, being the last one a homonymy also used for other varieties.

Minor varieties
Some of the accessions have been identified as synonymies of known varieties while others were not previously described.The varieties included in this group have a more or less marked risk of extinction, since only isolated plants were reported in most of the cases.
1) Cañorroyo, a variety existing in the BGVCAM at 'El Encín', was collected with the names Alcazpepita and Hornipepita.
2) Doradilla (CyL-05) was collected with this name.But also plants not corresponding to any known variety have been collected under the same name.
4) Huerta de Rey, also existing in the BGCAM at 'El Encín', is a synonymy of the variety Marfal from Extremadura.
5) Puesto Mayor, a synonymy of the portuguese variety Saborinho, has been localized with the name Verdejo Tinto.
6) Two accessions localized at Nava del Rey and Toro, Marta Nava and Puente correspond to the variety Salvador, also existing in the BGCAM at 'El Encín'.

Endangered varieties
Finally, four of the accessions correspond to varieties from which no previous written references or plant material in other grapevine collections have been found.Consequently they are in a marked risk of extinction.They are: 1) Calagraña, a white wine variety; 2) Pan y Carne (CyL-31) and Cenicienta, two red wine varieties, and 3) a table grape variety known as Juliana, with red berries.This variety was previously named Jami by Rojas Clemente (2002 facsimile edition, original of 1807), and it has been recently identified in the collection of the 'Jardín Botánico de Madrid' (F.Cabello, personal communication).
Table 2 shows the obtained genotypes for each variety.Any of them can be distinguished from the others with the exception of Tempranillo and Tempranillo Gris, that have the same genotype for these loci, since the last one is considered as a mutation of Tempranillo that affects the colour of the berry.In the rest of the cases, any two varieties can be distinguished with only four microsatellites.
The percentages of homozygosity oscillate between 3.8 to 23.1%, with an average of 14.7%.The total number of genotypes ranges from 11 to 17 with an average of 14.5, and the number of alleles from 6 to 10 with an average of 8 (Table 3).
As it can be observed in Table 2, there are 10 varieties in a marked risk of extinction, since only isolated plants have been detected, no new plantations are established with them, and no plant material for propagation is available in commercial nurseries.

Discussion
The six SSR markers used here have been established for characterisation and identification of grapevine varieties in a broad European Vitis germplasm database (http://www.genres.de/eccdb/vitis/), and they are able to uniquely identify most of the grapevine varieties.For this reason, they have been widely used in genotype characterisation, mainly among Vitis vinifera collections of cultivars (Vignani et al., 1996;Sefc et al., 1998;Lopes et al., 1999;Sánchez-Escribano et al., 1999;Sefc et al., 2000;Martín et al., 2003).
With respect to the best known varieties, it is advised to reduce the use of local synonymies in order to avoid confusion in the identification of the varieties.In some cases, the most usual name of the variety depends on the zones, as it is the case of Juan García or Mouratón, Tinta del País or Tempranillo and some others already mentioned in the Results section.The use of homonymies should also be avoided as much as possible, since they frequently lead to errors in the identification of the varieties.This is the case of Albillo among others.
The identification of minor varieties already described and existing in some germplasm bank (Cabello, 1995) is important, since the documentation of this bank indicates that those accessions come from the same geographic region.This is the case of Cañorroyo, Huerta de Rey, Puesto Mayor and Castellana Blanca.These accessions should be studied in order to explore the intravarietal variability for propagation or reintroduction purposes in the region.
There are also some varieties that are in a marked risk of extinction, for which no previous information has been found.These are: i) Calagraña, from Rueda;  ii) the unknown variety that was introduced with the name Doradilla 2 (Table 1), a name that should be changed to avoid the existing homonymy (Doradilla 1); iii) the mutation Tempranillo gris; and iv) Pan y Carne that is considered as the true to type variety (CyL-31, Table 2).All these varieties should be thoroughly characterised.New prospections are under way in order to collect and maintain a maximum of variability for further propagation.
With respect to the genotype variability (Table 3), the homozygosity percentages roughly agree with those obtained in a previous study with more than 300 Spanish varieties (Martín et al., 2003).The number of alleles and genotypes that were obtained was lower than the ones obtained in the mentioned study, as expected by the much smaller number of studied accessions.
As a conclusion of the present work, it is recommended to fully characterize each of the varieties, mainly the ones in risk of extinction.Also the collection of a maximum of intravarietal variability is recommended.The maintenance of this material for future use or reintroduction is of high interest.

Table 1 .
Plant material studied and varietal identification Godello, with the name of Prieto Picudo Blanco; Doña Blanca as Verdegudillo, and Bobal as Tinta Madrid.7) Malvasía Riojana, a synonymy of Alarije from Extremadura, Subirat Parent from Catalonia and Blanquirroja, Rojal or Tobía from La Rioja

Table 2 .
Genotypes of the studied varieties for the six used STMS loci 1 Varieties with a high risk of extinction.

Table 3 .
Genotype variability in the analysed STMS loci