En esta tesis doctoral han sido analizadas alrededor de 5.000 plantas para evaluar un sistema de 9 microsatélites, tanto en la protección (incluyendo exámenes DHE-Distinción, Homogeneidad y Estabilidad), como en la defensa legal (incluyendo estudios de Identificación y establecimiento de la derivación esencial) de variedades pertenecientes a la especia Vitis vinífera L. Los 9 marcadores microsatélites seleccionados son: ZAG29, ZAG62, ZAG67, ZAG83, ZAG112, VVMD5, VVMD27, VVMD28 y VVS2. La optimación del sistema de genotipado incluyó el diseño de una PCR múltiple, electroforesis capilar y análisis de fluorescencia en un analizador genético. En relación con los exámenes DHE, el análisis de más de 1000 accesiones ha permitido establecer la distancia mínima para establecer la Distinción en 2 alelos. La Homogeneidad y Estabilidad han sido demostradas mediante el estudio de casi 3.700 plantas de 19 variedades distintas de vid. En el análisis de identificación, se ha establecido la significación del análisis molecular con un análisis matemático de la coincidencia, evaluando asimismo la influencia de estructura genética en la colección de 495 genotipos usada como referencia. Este análisis es igualmente válido para el establecimiento de la derivación esencial. En esta tesis se ha elaborado una propuesta de utilización del sistema y toma de decisiones tanto para la protección como para la defensa legal de variedades de vid. Además de forma análoga a la OIV, se han elaborado nuevos descriptores microsatélite para los 5 loci para los que no existían previamente.
Grapevine is one of the oldest cultures in the world. Their plants are woody, and asexually multiplied through cuttings. There are a lot of varieties in the world (between 5,000 and 10,000) and many of them have been cultured several centuries ago. Most are local varieties, and there are numerous synonyms (one variety having different names) and homonyms (different varieties having the same name) within and between countries. There are two clearly distinguishable markets for grapevine: wine and table. The first one is much more important economically, and stable, regarding the varieties used, because in many cases the wine producers of a given place have to use certain varieties to get a “quality” label. So, breeding efforts are mainly focused on clone selection. The table market is much more dynamic and most of the varieties being cultivated now are different from those cultured 30 years ago. Crossbreeding is much more active for table grapes, and new interesting varieties are quickly spread to other producer countries. As a consequence, the issue of variety legal protection (similar to a patent or to intellectual property) in grapevines affects mainly to these table varieties. In the grapevine species, a new variety can come from a sexual cross, where an embryo is produced, or from an established variety through a somatic mutation, and then the new variety is called ‘sport’ (or Essentially-Derived Variety, EDV, within a legal scope). This, in addition to the already mentioned existence of synonyms and homonyms, has made of the identification of grapevine varieties a difficult issue along the time. Competent organisms, and especially OIV, have been working to improve the system, which is based mainly on morphological characteristics. In 1997, the book “Descriptors of grapevine” was published jointly by IPGRI, UPOV, and OIV. Besides the ‘characterization descriptors’, based on the study of morphological characteristics, there were ‘evaluation descriptors’, of lesser importance, which included microsatellites among the molecular markers. The situation today, ten years later, has changed considerably: many grapevine institutes use microsatellite markers to identify their varieties, in addition to, or instead of, the morphological descriptions. The plant variety legal protection requires keeping certain conditions. That requirement is evaluated by means of an exam that determines if the variety, besides new, is distinct, uniform and stable (DUS). ‘The variety shall be deemed to be distinct if it is clearly distinguishable from any other variety whose existence is a matter of common knowledge at the time of the filing of the application’. ‘The variety shall be deemed to be uniform if, subject to the variation that may be expected from the particular features of its propagation, it is sufficiently uniform in its relevant characteristics’. ‘The variety shall be deemed to be stable if its relevant characteristics remain unchanged after repeated propagation or, in the case of a particular cycle of propagation, at the end of each such cycle’. Generally, DUS test is based exclusively on morphological markers and, sometimes, biochemical markers. In Vitis vinifera L., exists a scientific discipline, the Ampelography, which comprises the grapevine plant morphologic description; in spite of being essential, the Ampelography suffers from some characteristics that limit its effectiveness with regard to others more recent techniques. Among them, DNA markers show numerous advantages against morphologic markers and could complement them in many situations, making DUS exams faster and more objective. Their incorporation into the official system as characterization descriptors must be seriously considered in a next, close, future, but it is necessary a previous, thorough study to evaluate their usefulness for that specific purposes. The main purpose of this study is to show the evaluation of 9 microsatellites through the study of a huge number of plants, and how the conclusions reached allow establishing rules that could be useful for different issues related to grapevine varieties: from legal protection, in the DUS test, including both studies of Distinctness, and of the Uniformity and Stability, to legal defence, including identification of varieties and essential derivation establishment.
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