Maria Manuela Veloso^1*, Maria Cecília Almandanim², Margarida Baleiras-Couto³, Helena Sofia Pereira⁴, Luís Cruz Carneiro¹, Pedro Fevereiro², José Eiras-Dias³

¹ INRB – INIA, Oeiras, Quinta do Marquês, 2784 – 505 Oeiras (Portugal)

² ITQB, Universidade Nova de Lisboa, Av. Da República, Apt.127, 2781 – 901 Oeiras, Portugal

³ INRB – INIA, Dois Portos, Quinta da Almoinha, 2565 -191 Dois Portos, Portugal

⁴ Centro de Botânica Aplicada à Agricultura, Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada de Ajuda, 1349-017 Lisboa, Portugal

SUMMARY

Six nuclear microsatellite loci (VVMD 5, VVMD 7, VVMD 27, VrZAG 62, VrZAG 79 and VVS 2) were chosen to study the differentiation of 313 grapevine cultivars officially authorized for wine production in Portugal (Portaria nº 428/2000, Diário da República nº 163, 17^th July 2000). These loci were found to be highly informative and therefore constitute a useful marker set for the discrimination of the targeted cultivars. This study allowed identifying 244 distinct genotypes and seventeen other genotypes represented, each, by 2 to 6 cultivars (synonymous) in a total of 40 cultivars. Additionally, 29 cultivars are distributed in 11 distinct sets, the cultivars of each set having identical SSRs profile but different berry colour and, so, should be considered as distinct cultivars. This work will contribute to improve the management of the Portuguese Grapevine National Collection and will also give a good technical support for the update of the list of cultivars officially authorized for wine production in Portugal (Portaria nº 428/2000). Furthermore, it will also be a useful support, in the future, for the control of plant material.

Key words: autochthonous grapevine, cultivar synonymous, Grapevine National Collection

Base de dados de microsatélites das castas (Vitis vinifera L.) utilizadas na produção de vinho em Portugal

RESUMO

Trezentas e treze castas referidas na Portaria nº 428/2000 foram genotipadas usando seis loci de microsatélites nucleares (VVMD 5, VVMD 7, VVMD 27, VrZAG 62, VrZAG 79 e VVS 2). Concluiu-se que estes loci eram muito informativos, tendo permitido discriminar as cultivares estudadas. Assim, este estudo possibilitou a identificação de 244 genótipos distintos e 17 outros genótipos, cada um representado, por 2 a 6 castas (sinónimos), num total de 40 cultivares. Há ainda 29 cultivares, distribuídas por 11 grupos distintos, que embora tenham perfis de microsatélites idênticos têm a cor do bago distinta pelo que, não foram incluídas no grupo dos sinónimos.

Este trabalho contribuirá para melhorar a gestão da Colecção Ampelográfica Nacional e constituirá uma boa base de apoio técnico à alteração da lista oficial das castas usadas na produção de vinho em Portugal. É também de salientar a importância que estes resultados têm para, no futuro, apoiar o controlo da comercialização do material vegetal.

A base de dados dos microsatélites estará disponível na página web do Instituto Nacional de Recursos Biológicos (INRB) www.inrb.pt

Palavras-Chave: videiras autóctones, sinonímias, Colecção Ampelográfica Nacional

Grapevine (Vitis vinifera L.) is composed of a large number of cultivars and is one of the oldest crops in the world. Grapes have been cultivated in Portugal for a long time, with evidence that Vitis expansion in the region now known as Portugal occurred some 5000 years ago and that the Romans played an important role in the Lusitanian viticulture, namely by the introduction of new cultivars.

Portugal still has a great diversity of autochthonous grapevine cultivars, some of them probably originating from the local wild germplasm (Cunha et al., 2010). Although approximately three hundred cultivars are officially recognized, nowadays many of them are hardly used or risk of extinction (Almandanim et al., 2007). Indeed, less than 15 native cultivars represent the majority of those presently utilised for viticulture, namely Alvarinho, Antão Vaz, Arinto, Fernão Pires, for the green yellow cultivars (25 800 ha) and Baga, Castelão, Tinta Barroca, Tinto Cão, Touriga Franca, Touriga Nacional and Trincadeira, for the blue black cultivars (73 630 ha). Others, not of Portuguese origin, like Aragonez (23 500 ha) are also of great importance.

The preservation of these important diversity represented by the Portuguese cultivars started in 1988 with the establisment of the Portuguese Grapevine National Collection with approximately 720 cultivars, including all the autochthonous ones. This Collection is of great significance and important to conduct ampelographic studies for resolution of synonyms and homonyms problems, at national and international levels.

The high number of cultivars and their dissemination all over the country had the consequence that different names were attributed to genetically identical plants (synonymous), which is a problem for viticulture and for the germplasm management.

Traditionally, cultivar characterization relied on plant morphological description (Eiras-Dias et al., 1988). However, these observations are time consuming and error-prone due to environmental variations that may alter the expression of the measured characteristics.

In the last years, DNA analysis has been developed for the discrimination of cultivars and the application of the microsatellite (SSR) fingerprinting in viticulture has become the technique of choice for cultivar identification and distinction (Bowers et al., 1996; Sefc et al., 1999). According to the OIV, SSR are the best markers to discriminate the cultivars.

The present study intended to complement detailed ampelographic characterization of grapevine germplasm resources by using six microsatellite loci to discriminate 313 grapevine accessions. The results allowed the identification of duplications and/or redundancies in the Collection as well as the completion of a public database.

MATERIAL AND METHODS

Three hundred and thirteen accessions of V. vinifera ssp. vinifera L. from the Portuguese Grapevine National Collection (PRT 051), located at Dois Portos (Quinta da Almoínha), were analysed in this study. All these cultivars are officially authorized for wine production in Portugal (Portaria nº 428/2000, Diário da República nº 163, 17^th July 2000). Although the Portaria lists 341 cultivars, 28 were not analysed because they did not integrate in the National Collection. Most of the cultivars studied are autochthonous, but some are of foreign origin.

Table I, Table II and Table III list all the accessions with the indication of their Reference in the National Collection and in the official journal, and the colour of the berry according to the OIV descriptor list for grape varieties and the Vitis species (blue black - N, green yellow - B, rose - Rs).

TABLE I

Genetic profiles, at 6 microsatellite loci, of 244 grapevines officially authorized for wine production in Portugal, for which no synonymous were detected. The cultivars are referred by the official name, the reference number at Colecção Ampelográfica Nacional and the Portaria number. Allele size is given in base pairs. B, N and Rs correspond to green yellow, blue black and rose colour of the berry, respectively.

Tamanho dos alelos para seis loci de microssatélites de 244 castas oficialmente autorizadas para a produção de vinho em Portugal. As castas são referidas pelo nome oficial, referência na Colecção Ampelográfica Nacional e na Portaria nº 428/2000. B, N e Rs correspondem a castas brancas, tintas e rosé, respectivamente.

TABLE II

Genetic profiles, at 6 microsatellite loci, of 40 grapevine cultivars officially authorized for wine production in Portugal for which 17 synonymous were detected. The cultivars are referred by the name, the reference number at Colecção Ampelográfica Nacional and the Portaria number   Allele size are given in base pairs. B, N and Rs correspond to green yellow , blue black and rose colour of the berry, respectively.

TABLE III

Genetic profiles, at 6 microsatellite loci, of 29 grapevine cultivars officially authorized for wine production in Portugal of which 11 have identical SSR profi les but different berry colour. The cultivars are referred by the name, the reference number at Colecção Ampelográfica Nacional and the Portaria number Allele size are given in base pairs. B, N and Rs correspond to green yellow, blue black and rose colour of the berry, respectively.

Tamanho dos alelos para seis loci de microssatélites de 29 castas oficialmente autorizadas para a produção de vinho em Portugal. Nestas castas há 11 que embora tendo o mesmo perfil de SSR têm mutações na cor do bago. As castas são referidas pelo nome oficial, referência na Colecção Ampelográfica Nacional e na Portaria nº 428/2000. B, N e Rs correspondem a castas brancas, tintas e rosadas respectivamente.

Microsatellite analysis

DNA extraction, PCR amplification, electrophoresis and detection of polymorphisms were carried out according to Almandanim et al., (2007). Six microsatellite loci, VVMD 5 and VVMD 7 (Bowers et al., 1996), VVMD 27 (Bowers et al., 1999), VrZAG 62 and VrZAG 79 (Sefc et al., 1999) and VVS 2 (Thomas and Scott, 1993), recommended by the OIV, were successfully amplified. Allelic size determination was carried out through capillary electrophoresis in a CEQ8000 Genetic Analysis System (Beckman Coulter).

RESULTS AND DISCUSSION

Table I lists 244 cultivars which, by the SSR profiles, correspond to distinct genotypes. The remaining 69 cultivars include 40 which are officially registered under different names, but that, correspond only to 17 distinct genotypes due to the presence of synonymous (Table II). In this group a single cultivar is registered under two to six distinct names as, for example, Ramisco-Tinto, Rabo-de-Ovelha-Tinto, Saborinho, Molar, Tinto-de-Porto-Santo e Tinta-Negra, which unambiguously refer to one single genotype. Síria and Sabro were also considered as synonymous, although they have one allele (VVS2) with a reproducible difference on two bases. As they have the same ampelographic characteristics, we considered this difference as a mutation. The other 29 cultivars can be distributed in 11 distinct sets, the cultivars of each set having identical SSRs profile but different berry colour (Table III). For instance, Fernão-Pires and Fernão-Pires-Rosado have the same SSR profile but Fernão-Pires have green yellow berries while Fernão-Pires-Rosado has rose berries.

In fact, the berry colour types are indistinguishable by microsatellite analysis as already reported for some of the Portuguese cultivars (Lopes et al., 1999). According to the Portuguese official legislation they should be considered as distinct cultivars, as consequence of the difference in berry colour.

Concerning the origin of the distinct cultivars described in the present work, we could arrange them in four groups: 1) autochthonous to Portugal (almost 50%); 2) hybrids obtained at Estação Agronómica Nacional by the researcher Leão Ferreira de Almeida, corresponding to 29 cultivars; 3) common to other countries, as for example Alicante-Branco, Aragonez, Moscatel-Galego-Branco, Moscatel-Graúdo, Tália and Tinta-Miúda; 4) a group that includes the 37 varieties that have recently been introduced (mainly from France and Germany) in the national viticulture, as for example, Cabernet-Franc, Sauvignon, Syrah and Riesling.

CONCLUSIONS

The preservation of grapevine genetic resources is of prime importance. However, the management of a grapevine germplasm collection is expensive, requiring a considerable land area. So, it is important to eliminate repetitions and misclassifications.

The microsatellite analysis was very useful in the identification and discrimination of all the studied cultivars. Investigation of putatively synonymous among the cultivars has resulted in the confirmation of 15 synonymous. We consider that this work contributed to improve the management of the Portuguese Grapevine National Collection.

Furthermore, the present study will give a good technical support for the update of the list of the cultivars officially authorized for wine production in Portugal (Portaria nº 428/2000, Diário da República nº 163, 17^th July 2000), and will also be a support for the trade control of plant material.

The SSR database will be available at the web page of Instituto Nacional de Recursos Biológicos (www.inrb.pt). This database will be regularly updated, what means that new information will be uploaded when necessary.

Financial support from Project AGRO 187; grants to M. M. Baleiras-Couto (FCT - BPD/18959/2004), H. S. Pereira (FCT - SFRH/BPD/4549/2003) and M. C. Almandanim (AGRO Project 187). The collaboration of Celina Matos on the preparation of the data tables is acknowledged.

REFERENCES

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Cunha J., Teixeira-Santos M., Veloso M., Carneiro L., Eiras-Dias J. Fevereiro P. 2010. The Portuguese Vitis vinífera L. Germplasm: Genetic Relations between Wild and Cultivated Vines. Ciência e Téc. Vitiv. 25, 25-37.        [ Links ]

Eiras-Dias J. E., Pereira C. A., Baptista da Cunha J. P. 1988. Catálogo das Castas: Região do Ribatejo, Oeste e Península de Setúbal. Instituto da Vinha e do Vinho. Estação Vitivinícola Nacional.

Lopes M. S., Santos M. R., Eiras-Dias J. E., Mendonça D., Câmara Machado A. 2006. Discrimination of Portuguese grapevines based on microsatellite markers. Journal of Biotechnology, 127, 34-44.

Sefc K., Regner F., Turetschek E., Glossl J., Steinkellner H. 1999. Identification of microsatellite sequences in Vitis riparia and their applicability for genotyping of different Vitis species. Genome 42, 367-373.

Thomas M., Scott N. 1993. Microsatellite repeats in grapevine reveal DNA polymorphisms when analyzed as sequence-tagged sites (STSs). Theor. Appl. Genet. 86, 985-990.

* Corresponding author (+351 21 4403500), email: mveloso.inrb@gmail.com

(Manuscrito recebido em 10.11.10 . Aceite para publicação em 17.12.10)