Caracterização Molecular de Variedades de Nogueira (Juglans regia L.) Portuguesas

Abstract

The genus Juglans contains about twenty species, all producing edible nuts. Among those, the English or Persian walnut, Juglans regia L., is the most widely cultivated species and the most economically important member of the genus Juglans. The fact that fruit production in walnut starts several years after its planting, makes its identification and characterization important for designing objective and repeatable criteria to breeders and farmers in crop improvement programs. Therefore, the development of rapid, reliable and sensitive methods for the identification and verification of cultivars is essential. Several techniques using molecular markers have been used to examine genetic diversity and relationships among cultivars of Juglans regia L., including isozymes, RFLP, RAPD and ISSR markers. However, the need for accurate cultivar identification and verification of paternity and genealogy, led to the application of SSR or microsatellite markers that are well suited to meet these needs. Microsatellite DNA analysis is generally considered the most powerful method for a number of reasons, including maximum precision and reproducibility. Beside those facts, such techniques are useful for understanding the origins and relationships of germplasms and for providing genetic fingerprints that can be used to test or confirm the identity of plant materials and for food authenticity studies. The present study was undertaken to analyze the effectiveness of microsatellite markers in molecular characterization of three Portuguese cultivars of Juglans regia L. and to assess the genetic relationship between those cultivars and nineteen international ones. Also, this study was designed to check walnuts harvested at Soure, commercial walnuts (labelled with reference to the variety) as well as they by-products authenticity, with SSR molecular markers applied in the genetic characterization. For that purpose, three DNA extraction methods were assayed: CTAB, GenElute and Wizard. SSR markers used in this study had disclosed to be efficient in the distinction between the cultivars studied, and showed up a synonymy case, between cultivars Franquette and Ronde de Montignac. Throughout the analysis of the obtained dendrogram, it was possible to associate the Portuguese cultivar Rego with the group of French origin and Samil with the North-American ones. The other Portuguese cultivar, Arco, showed up to be genetically distinct from the overall genotypes. Concerning the international cultivars, its distinction in three main groups became evident (rectangular dendrogram). The group of North-American origin, the group of French origin and the third one that includes two cultivars of French origin, Fernette and Lara, two cultivars of North-American origin, Chico and Chandler and one of Portuguese origin, Samil. The obtained dendrogram show the presence of four cultivars clearly distinct from the overall population (Arco, Meylannaise, Hartley and Parisienne). In what it concerns to the authenticity study, there was a clear difference between the 3 chosen extraction methods. After the choice of the best extraction method, the samples were amplified with 3 primers previously selected. The results showed that the commercial walnuts as well as the walnuts harvested at Soure amplified, but the same fact wasn t noted with walnut processed by-products. It was possible to check, through molecular techniques, the information described in the labels of commercially walnuts (in what concerns the variety type) for most of the samples.