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Varieties Using Morphological Data and AFLP Markers

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Varieties Using Morphological Data and AFLP Markers Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.6 No.3, Issue of December 15, 2003 © 2003 by Pontificia Universidad Católica de Valparaíso -- Chile Received Febraury 21, 2003 / Accepted December 5, 2003 RESEARCH ARTICLE Evaluation of diversity among Argentine grapevine (Vitis vinifera L.) varieties using morphological data and AFLP markers Liliana Martínez* Laboratorio de Biología Molecular Departamento de Ciencias Biológicas Facultad de Ciencias Agrarias Universidad Nacional de Cuyo Almirante Brown 500, Chacras de Coria, M5528AHB Mendoza, Argentina Tel: 0054 261 496004 (ext 2032) Fax: 0054-261-4960469 E-mail: [email protected] Pablo Cavagnaro Laboratorio de Biología Molecular Departamento de Ciencias Biológicas Facultad de Ciencias Agrarias Universidad Nacional de Cuyo Almirante Brown 500, Chacras de Coria, M5528AHB Mendoza, Argentina Tel: 0054 261 496004 (ext 2032) Fax: 0054 261 4960469 E-mail: [email protected] Ricardo Masuelli Laboratorio de Biología Molecular Departamento de Ciencias Biológicas Facultad de Ciencias Agrarias Universidad Nacional de Cuyo Almirante Brown 500, Chacras de Coria, M5528AHB Mendoza, Argentina Tel: 0054 261 496004 (ext 2032) Fax: 0054 261 4960469 E-mail: [email protected] José Rodríguez Cátedra de Viticultura Departamento de Producción Agropecuaria Facultad de Ciencias Agrarias Universidad Nacional de Cuyo Almirante Brown 500, Chacras de Coria, M5528AHB Mendoza, Argentina Tel: 0054 261 496004 (ext 2032) Fax: 0054 261 4960469 E-mail: [email protected] Financial support: The present work has been done in the framework of a research programme (1998-2002) funded by SECYT (Secretaría de Ciencia y Técnica) of the National University of Cuyo. Keywords: ampelography, Criollas, genetic variability, molecular markers. Abbreviations: AFLP: Amplified Fragment Length Polymorphism; NTSYS:Numerical Taxonomy and Multivariate Analysis System; O.I.V.: Office International du Vin; PCR: Polymerase Chain Reaction; RAPD: Random Amplified Polymorphic DNA; RFLP: Restriction Fragment Length Polymorphism; SSR: Simple Sequence Repeats. Half of the Argentine grapevine growing area is cultivated with local varieties generically called *Corresponding author This paper is available on line at http://www.ejbiotechnology.info/content/vol6/issue3/full/11 Martinez, L. et al. “Criollas”. These materials differ in morphology, with a Muscat taste (Agüero et al. 2001). The rest of the physiology and enological aptitudes from traditional “Criollas” shows relatively low enology quality, only European varieties. To discriminate among them, we appropriate as table wines. used morphological markers to evaluate the genetic diversity and phenetic relatedness of 9 Criollas, 6 Molecular markers like RFLP (Bowers and Meredith, European and 1 American varieties and compared with 1996), RAPD (Gogorcena et al. 1995; Grando et al. 1995), AFLP markers. Three AFLP primer combinations AFLP (Sensi et al. 1996; Cervera et al. 1998) and SSR generated a total of 111 scorable fragments. (Bowers et al. 1996; Sefc et al. 2000) have been used for Dendrograms obtained with morphologic and AFLP genetic studies in grapevine. These studies have increased markers agreed in clustering the “Criollas” separately the understanding of the relatedness of cultivars within and from the European and American varieties assayed, among regions. The high level of heterozygosis that present except for Muscat d’ Alexandrie and Tempranillo which vegetatively propagated grapevines (Reisch, 2000) allows clustered with Criollas in the case of AFLP. the distinction of the most important cultivars by using Comparison of UPGMA dendrograms of morphological almost any molecular marker technique. The recent and AFLP markers using the Mantel test indicated a availability of these molecular markers has facilitated not significant correlation of r = 0.33. Nevertheless, research in Vitis genetics (Reisch, 2000). AFLP (Vos et al. AFLP and selected morphological characters appear as 1995) is a PCR-based fingerprinting technique that is useful and complementary techniques for grapevine particularly useful for this purpose, since it can detect a identification and for evaluation of genetic large number of polymorphism in a single reaction. It diversity. Among the “Criollas”, AFLP similarities presents a good repeatability generating primarily dominant ranged from 76 to 98% (Dice coefficient), indicating an markers which are distributed throughout the genome important source of genetic diversity that can be (Cervera et al. 1998). The goals of this work were to i- exploited in future breeding programs. To our evaluate the genetic diversity of Argentine grapevine knowledge, this is the first report using AFLP markers germplasm using AFLP and morphological data, ii- to assess genetic variability on these materials. compare the phenetic relationships obtained by both systems of analysis and iii- compare European with Criollas Argentina is one of the largest grape and wine producing varieties. countries in South America. In the last decades its viticulture and enology industries have acquired great MATERIALS AND METHODS importance from an economical point of view. Currently, 45% of the grapevine cultivated area is covered with a Plant material group of varieties generically called “Criollas”, a term given to American-born individuals descendant from Nine “Criollas” varieties: Cereza, Criolla Grande, Criolla European parents, although the possibility that some of Chica, Pedro Giménez, Moscatel Rosado, Moscatel these varieties arrived as seeds cannot be excluded. It is Amarillo, Torrontés Riojano, Torrontés Sanjuanino and likely, that these varieties were introduced in Argentina Torrontés Mendocino, 6 European varieties: Chardonnay, soon after the Spaniard conquerors arrived to the New Syrah, Cabernet Sauvignon, Malbec, Muscat d´Alexandrie World. Settlers began planting vines as early as 1556, at and Tempranillo, and 1 American hybrid rootstock: SO4 Santiago del Estero province (Maurín-Navarro, 1967) and (Vitis berlandieri x Vitis riparia), were assayed. All later in the foothills of the Andes Mountains, in Mendoza accessions were taken from the collection vineyard at the and San Juan provinces. Currently, around 70% of the total INTA Luján de Cuyo and the Agricultural College, area cultivated with “Criollas” is in Mendoza, whereas San Universidad Nacional de Cuyo, Mendoza, Argentina. Juan accounts for 20% of the surface (INV, 2001). Morphological characters analysis Due to their rusticity, the “Criollas” have called the Fifty-three characters (Table 1), analyzed and described by attention of local plant physiologists, who have noticed Alcalde, 1989, using “Criollas” varieties, were numerically significantly higher tolerance to some environmental codified using a qualitative multi-status criterion (from 0 to stresses when compared with European traditional varieties 8, depending on the variables of each character) (Sneath (Kaiser and Cavagnaro, 2001). These varieties can grow in and Sokal, 1973) and used to design a numbered-data soils with low water availability and high salt matrix. The corresponding morphological characters of the concentration, and still maintain their characteristic high European varieties, described by O.I.V. were also included yield and vigour. Characterizing the diversity of local in the analysis. Modal values of morphological descriptors populations would allow a more useful application of these from 15 vines per European and Criollas varieties were materials in breeding programs. analyzed in 20 consecutive years. Some “Criollas” varieties such as Moscatel Amarillo, DNA extraction Criolla Chica, Torrontés Mendocino and Torrontés Riojano, give raise to valuable regional wines. Torrontés Riojano has For each variety, young leaves from 5 vines, were been internationally recognized for originating a dry wine independently collected and used for DNA isolation as 245 Evaluation of diversity among Argentine grapevine (Vitis vinifera L.) varieties using morphological data and AFLP markers reported by Bowers et al. 1993. Three replicates of DNA Morphology analysis extraction from the same varieties were made. DNA was quantified either by visual comparison with lambda DNA The UPGMA dendrogram obtained using morphological on ethidium bromide stained agarose gels or by characters clearly separated the “Criollas”, European and spectrophotometry using a Pharmacia Gene Quant the American varieties (Figure 1a) (group A, B and C). Spectrophotometer (Pharmacia, Biotech, Columbus, OH). Members of the European and “Criollas” groups presented more than 40 and 45% similarity, respectively. These two AFLP analysis clusters diverged at a similarity index of 37% based on the dendrogram. Criolla Grande and Cereza showed a very high AFLP reactions were carried out following the instructions degree of similarity (0.73%) indicating that they are closely supplied with the GIBCO-BRL Life Technologies AFLPTM related varieties (Table 2). On the other hand T. Riojano kit, with minor modifications. 250 ng of genomic DNA was seems to be more related to T. Sanjuanino (0.70%) than to double digested with 1.25 units of each EcoRI and MseI, T. Mendocino (0.43%), despite their similar names. and linked to their respective adapters by using 0.25 unit of Members of each these group of varieties have similar T4 DNA ligase. Digested and ligated DNA fragments were morphology. The three Torrontés (T. Riojano, T. used as templates for the first amplification reaction. For Sanjuanino
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