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Document 1 : Thesis NFP Noannex.Pdf https://lib.uliege.be https://matheo.uliege.be Characterization of the virome of ancient pome fruit cultivars of Malus Mill. and Pyrus L. using high-throughput sequencing Auteur : Fontdevila Pareta, Núria Promoteur(s) : Massart, Sébastien Faculté : Gembloux Agro-Bio Tech (GxABT) Diplôme : Master en bioingénieur : sciences agronomiques, à finalité spécialisée Année académique : 2019-2020 URI/URL : http://hdl.handle.net/2268.2/9475 Avertissement à l'attention des usagers : Tous les documents placés en accès ouvert sur le site le site MatheO sont protégés par le droit d'auteur. Conformément aux principes énoncés par la "Budapest Open Access Initiative"(BOAI, 2002), l'utilisateur du site peut lire, télécharger, copier, transmettre, imprimer, chercher ou faire un lien vers le texte intégral de ces documents, les disséquer pour les indexer, s'en servir de données pour un logiciel, ou s'en servir à toute autre fin légale (ou prévue par la réglementation relative au droit d'auteur). Toute utilisation du document à des fins commerciales est strictement interdite. Par ailleurs, l'utilisateur s'engage à respecter les droits moraux de l'auteur, principalement le droit à l'intégrité de l'oeuvre et le droit de paternité et ce dans toute utilisation que l'utilisateur entreprend. Ainsi, à titre d'exemple, lorsqu'il reproduira un document par extrait ou dans son intégralité, l'utilisateur citera de manière complète les sources telles que mentionnées ci-dessus. Toute utilisation non explicitement autorisée ci-avant (telle que par exemple, la modification du document ou son résumé) nécessite l'autorisation préalable et expresse des auteurs ou de leurs ayants droit. Characterization of the virome of ancient pome fruit cultivars of Malus Mill. and Pyrus L. using high-throughput sequencing NURIA FONTDEVILA PARETA TRAVAIL DE FIN D’ETUDES PRESENTE EN VUE DE L’OBTENTION DU DIPLÔME DE MASTER BIOINGENIEUR EN SCIENCES AGRONOMIQUES ANNÉE ACADÉMIQUE 2019-2020 PROMOTEUR : PROF. SÉBASTIEN MASSART 1 © Toute reproduction du présent document, par quelque procédé que ce soit, ne peut être réalisée qu’avec l’autorisation de l’auteur et de l’autorité académique1 de Agro-Bio Tech. Le présent document n’engage que son auteur. 1 Dans ce cas, l’autorité acadèmiques est representée par le(s) promoteur(s) membre du personnel(s) enseignant de GxABT. Characterization of the virome of ancient pome fruit cultivars of Malus Mill. and Pyrus L. using high-throughput sequencing NURIA FONTDEVILA PARETA TRAVAIL DE FIN D’ETUDES PRESENTE EN VUE DE L’OBTENTION DU DIPLÔME DE MASTER BIOINGENIEUR EN SCIENCES AGRONOMIQUES ANNÉE ACADÉMIQUE 2019-2020 PROMOTEUR : PROF. SÉBASTIEN MASSART Acknowledgements First of all, I would like to thank my promoter, Prof. Sébastien Massart for giving me the opportunity to be part of his team and their scientific network, and for all his support during this master’s thesis. Without him this project would not be a reality. I would like to specially thank as well Dr Arnaud Blouin for his help during this project, supervising the laboratory work and bioinformatic analysis, for sharing his knowledge in plant virology and molecular biology, and for his immense patience with me and all my questions, Last, but not least, I also want to thank all the virology team members and lab technicians for their support and help when I needed it. Master’s thesis performed at the Integrated and Urban Phytopathology Unit of the ULg-Gembloux Agro-Bio Tech University. Abstract Pome fruit viruses have been mainly identified from commercialised cultivars presenting disease symptoms. Their significant impact on fruit yield and quality triggered their identification and characterization ex post. For example, the infection of certain virulent strains of apple stem pitting virus (ASPV) was detected due to necrosis development between susceptible scions and/or rootstocks (rootstock incompatibility). In this context, the diversity of viruses infecting pome fruit trees is still largely underestimated and it is worth additional investigation. The goal of this project is to evaluate the presence of known and unknown viruses infecting ancient cultivars of apple and pear. Leaf samples were taken from six apple cultivars (‘Gravenstein’, ‘Pomme Pellone’, ‘Délices de Beignée’, ‘Reinette Meurens’, ‘Belle de Boskoop’, and ‘Joseph Musch’) and five pear cultivars (‘Poire Cuisse Madame’, ‘Jeanne d’Arc’, ‘Poire Rougette’, ‘Bronzée d’Enghien’, and ‘Colmar du Mortier’). Total RNA was analysed from ‘Joseph Musch’ (tree Q9) after high throughput sequencing. Double-stranded RNA (dsRNA) and virion-associated nucleic acids (VANA) preparation protocols were applied on the other samples individually (dsRNA) or pooled (dsRNA and VANA). After sequencing, the obtained data were analysed with Geneious Prime to identify the viruses present in the sampled trees and to reconstruct their genomes. In addition, two other bioinformatics pipelines were tested on the generated data (Kaiju and Kraken). The nearly complete genome sequence of seven new isolates from several known viruses was reconstructed. All the samples were infected by at least one virus, the most prevalent was ASPV. Interestingly, the detection of Apple rubbery wood virus-1 (ARWV-1), Apple luteovirus-1 (ALV-1) and Apple hammerhead viroid-like RNA (AHVd-like RNA) corresponded to the first detection in Europe. Primer and RT-PCR protocols were designed. The presence of ARWV- 1 was detected by RT-PCR, confirming the first detection of this virus in Europe. Further studies need to be carried out to assess the distribution of ARWV-1 within the germplasm collection and to confirm the detection of ALV-1 and AHVd. The analysis of the local prevalence of these viruses will be a first step to evaluate the biological risk they can pose for European production. Keywords: pome fruit, apple (Malus Mill.), pear (Pyrus L.), plant virus, high-throughput sequencing (HTS), virome characterization, germplasm resources, Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple luteovirus-1 (ALV-1), Apple rubbery wood virus-1 (ARWV-1), Apple hammerhead viroid-like RNA (AHVd-like RNA) Résumé Historiquement, les virus des pommiers et poiriers ont été principalement identifiés à partir de plantes cultivées présentant des symptômes de maladie. Leurs impacts significatifs sur le rendement et la qualité des fruits a déclenché leur identification et leur caractérisation. Par exemple, l'infection de certaines souches virulentes du virus « Apple stem piting virus » (ASPV) ont été détectées en raison du développement de la nécrose entre les scions et/ou les porte-greffes sensibles. Dans ce contexte, la diversité des virus qui infectent les pommiers et poiriers est encore largement sous-estimée et il convient de poursuivre les recherches. L'objectif de ce projet est d'évaluer la présence de virus connus et inconnus infectant d'anciens cultivars de pommiers ("Gravenstein", "Pomme Pellone", "Délices de Beignée", "Reinette Meurens", "Belle de Boskoop" et "Joseph Musch") et de poiriers ("Poire Cuisse Madame", "Jeanne d'Arc", "Poire Rougette", "Bronzée d'Enghien" et "Colmar du Mortier"). L'ARN total a été séquencé et analysé à partir de feuilles de "Joseph Musch" (arbre Q9). Des protocoles de préparation d'ARN double brin (dsRNA) et d'acides nucléiques associés à des virions (VANA) ont été appliqués aux autres échantillons individuellement (dsRNA) ou en pool (dsRNA et VANA). Après le séquençage, les données obtenues ont été analysées avec Geneious Prime pour identifier les virus présents dans les arbres échantillonnés et pour reconstruire les génomes des isolats présants. En outre, deux autres pipelines bioinformatiques ont été testés sur les données générées (Kaiju et Kraken). La séquence du génome presque complète de sept nouveaux isolats de plusieurs virus connus a été reconstruite. Tous les échantillons ont été infectés par au moins un virus, le plus répandu étant l'ASPV. Il est intéressant de noter que la détection de « Apple rubbery wood virus-1 » (ARWV-1), « Apple luteovirus-1 » (ALV-1) et « Apple hammerhead viroid-like RNA » (AHVd-like RNA) correspond à la première détection en Europe. Les amorces ont été conçues pour confirmer la présence de l'ARWV-1 par RT-PCR, confirmant la première détection de ce virus en Europe. D'autres études doivent être menées pour évaluer la distribution de l'ARWV-1 au sein de la collection de germoplasme et pour confirmer la détection de l'ALV-1 et de l'AHVd. L'analyse de la prévalence locale de ces virus sera une première étape pour évaluer le risque biologique qu'ils peuvent représenter pour la production européenne. Mots-clés: pommiers (Malus Mill.), poiriers (Pyrus L.), virus des plantes, séquençage à haut débit (HTS), virome, ressources génétiques, Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple luteovirus-1 (ALV-1), Apple rubbery wood virus-1 (ARWV-1), Apple hammerhead viroid-like RNA (AHVd-like RNA) TABLE OF CONTENTS 1 Introduction .................................................................................................................................... 1 2 Bibliography ................................................................................................................................... 2 2.1 Rosaceae Family ..................................................................................................................... 2 2.1.1 Growth and development of pome fruit trees .................................................................. 2 2.1.2 Vegetative propagation of pome
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