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European Mountain Ash Ringspot-Associated Virus (Emarav) in Sorbus Aucuparia

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European Mountain Ash Ringspot-Associated Virus (Emarav) in Sorbus Aucuparia UNIVERSIDAD POLITÉCNICA DE MADRID ESCUELA TÉCNICA SUPERIOR DE INGENIERÍA AGRONÓMICA, ALIMENTARIA Y DE BIOSISTEMAS GRADO EN BIOTECNOLOGÍA DEPARTAMENTO DE BIOTECNOLOGÍA BIOLOGÍA VEGETAL European mountain ash ringspot-associated virus (EMARaV) in Sorbus aucuparia. Studies on spatial distribution, genetic diversity and virus-induced symptoms TRABAJO FIN DE GRADO Autor: Héctor Leandro Fernández Colino Tutor académico: Fernando García- Arenal Rodríguez Tutora profesional: Susanne von Bargen Febrero 2020 UNIVERSIDAD POLITÉCNICA DE MADRID ESCUELA TÉCNICA SUPERIOR DE INGENIERÍA AGRONÓMICA, ALIMENTARIA Y DE BIOSISTEMAS GRADO DE BIOTECNOLOGÍA EUROPEAN MOUNTAIN ASH RINGSPOT-ASSOCIATED VIRUS (EMARAV) IN SORBUS AUCUPARIA. STUDIES ON SPATIAL DISTRIBUTION, GENETIC DIVERSITY AND VIRUS-INDUCED SYMPTOMS TRABAJO FIN DE GRADO Héctor Leandro Fernández Colino MADRID, 2020 Tutor académico: Fernando García-Arenal Rodríguez Catedrático de Universidad Departamento de Biotecnología-Biología Vegetal Tutora profesional: Susanne von Bargen Doctor Division Phytomedicine, Albrecht-Daniel Thaer Institute, Humboldt University Berlin II TITULO DEL TFG- EUROPEAN MOUNTAIN ASH RINGSPOT- ASSOCIATED VIRUS (EMARAV) IN SORBUS AUCUPARIA. STUDIES ON SPATIAL DISTRIBUTION, GENETIC DIVERSITY AND VIRUS-INDUCED SYMPTOMS Memoria presentada por HÉCTOR FERNÁNDEZ COLINO para la obtención del título de Graduado en Biotecnología por la Universidad Politécnica de Madrid Fdo: Héctor Leandro Fernández Colino Vº Bº Tutores D. Fernando García-Arenal Rodríguez Catedrático de Universidad Departamento de Biotecnología-Biología Vegetal Centro: ETSIAAB - Universidad Politécnica de Madrid D.ª Susanne von Bargen Doctor Division Phytomedicine Centro: Albrecht-Daniel Thaer Institute - Humboldt University of Berlin Madrid, 03 febrero 2020 III Agradecimientos – Special thanks A la doctora Susanne von Bargen, por aconsejarme siempre con paciencia y hacer de mi debut en el verdadero trabajo científico algo tan agradable y memorable. To doctor Susanne von Bargen, for always patiently providing me with advice and making my debut in true scientific work something so nice and memorable. A Renate Junge y Andrea Klinke, que siempre estuvieron ahí cuando tuve dudas y sin las cuales nunca habría conseguido mi soltura e independencia en el trabajo de laboratorio. To Renate Junge and Andrea Klinke, who were always there for me when I had doubts and without whom I would have never got such skills and independence in lab work Al profesor Fernando García-Arenal por haberme ayudado a tallar minuciosamente este trabajo y convertirlo en algo para recordar toda la vida. To professor Fernando García-Arenal for helping me sculpt so carefully this piece of work and turning it into something to remember my whole life. A mi amigo y compañero investigador Thomas Bleß por hacer mis días de trabajo mucho más llevaderos, por muchos más paseos y cafés compartidos. To my dear friend and fellow researcher Thomas Bleß for having made my workdays so much more bearable, to many more shared walks and coffees. A mi familia y en especial a mi padre Javier y mi madre María Victoria por haberme ayudado en la recta final, pero también por haber estado ahí durante todo el camino, lejos pero también muy cerca. A mi abuela María Victoria, que conoció el comienzo, pero no el final. To my family and specially to my father Javier and mother María Victoria for having helped me in the final stretch, but also for having been there all the way, far away but also close to me. To my grandmother María Victoria, who knew the beginning but not the end. A mi querida hermana Irene por haberme visitado, apoyado y hecho descubrir tantas cosas nuevas. To my dear sister Irene, for having visited, supported and made me discover many new things. A la profesora Carmen Büttner y a todo el departamento de Fitomedicina de la Universidad Humboldt de Berlín, que me dieron la oportunidad de empezar esta aventura y que aún hoy me acogen tan cálidamente. To professor Carmen Büttner and all the Phytomedicine division of the Humboldt University of Berlin, who gave me the opportunity of starting this adventure and who still today take me in so warmly. Al resto de mis amigos, de cerca y de lejos, que me mantuvieron el ánimo alto. To the rest of my friends, far and near, who kept my mood high. A mí mismo por haber sido capaz de llevar este barco a buen puerto. To myself for having been able to come through. Héctor Leandro Fernández Colino, European mountain ash ringspot-associated virus (EMARaV) in Sorbus aucuparia. Studies on IV spatial distribution, genetic diversity and virus-induced symptoms (UPM-ETSIAAB, 2020) Table of Contents Agradecimientos – Special thanks ................................................................................................ IV TABLE INDEX ................................................................................................................................ VII FIGURE INDEX ............................................................................................................................. VIII ABBREVIATIONS ........................................................................................................................... XI ABSTRACT .................................................................................................................................... XII CHAPTER 1. INTRODUCTION ......................................................................................................... 1 1.1. Relevance of virus diseases in trees. .................................................................................. 1 1.2. Genus Sorbus and the European mountain ash. ................................................................ 1 1.3. European mountain ash ringspot disease. ......................................................................... 3 1.4. Emaraviruses. ..................................................................................................................... 4 1.5. EMARaV. ............................................................................................................................. 5 1.6. Objectives. .......................................................................................................................... 7 CHAPTER 2. MATERIALS AND METHODS ...................................................................................... 8 2.1. Sampling of plant material. ................................................................................................ 8 2.1.1. Sampling of dormant Sorbus intermedia buds. .......................................................... 8 2.1.2. Sampling of Sorbus aucuparia leaves showing little to no symptoms of EMARaV infection. ............................................................................................................................... 8 2.1.3. Controls. .................................................................................................................... 10 2.2. Total RNA extraction from plant material. ....................................................................... 10 2.2.1. Nucleic acid concentration measurement. ............................................................... 11 2.3. Electrophoresis of total RNA extraction results. .............................................................. 11 2.4. Total cDNA synthesis. ....................................................................................................... 12 2.5. Control PCR of Nad5 gene. ............................................................................................... 12 2.6. Detection PCR of viral genome segments. ....................................................................... 13 2.7. Analytical electrophoresis of PCR results. ........................................................................ 14 2.7.1. Electrophoresis of larger DNA fragments (>250 bp). ................................................ 15 2.7.2. Electrophoresis of smaller DNA fragments (<250 bp). .............................................. 15 2.7.3. UV spectroscopy of electrophoresis gels. ................................................................. 15 Héctor Leandro Fernández Colino, European mountain ash ringspot-associated virus (EMARaV) in Sorbus aucuparia. Studies on V spatial distribution, genetic diversity and virus-induced symptoms (UPM-ETSIAAB, 2020) 2.8. PCR product purification. ................................................................................................. 15 2.9. Sequencing. ...................................................................................................................... 16 CHAPTER 3. RESULTS ................................................................................................................... 17 3.1. Detection of EMARaV in buds of EMARaV-infected S. intermedia. ................................. 17 3.1.1. Total RNA extraction from S. intermedia buds. ........................................................ 17 3.1.2. Control of cDNA synthesis from S. intermedia buds’ extracted RNA ........................ 17 3.1.3. Detection RT-PCR of EMARaV genome fragments. ................................................... 18 3.2. Virus presence in Sorbus aucuparia leaves. ..................................................................... 18 3.2.1. Symptom distribution in S. aucuparia trees. ............................................................. 18 3.2.2. RT-PCR detection of EMARaV...................................................................................
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