ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en UNIVERSITAT AUTÒNOMA DE BARCELONA Facultat de Biociències Dept. Biologia Animal, Biologia Vegetal i Ecologia Study of the resistance to Cucumber mosaic virus aggressive strains in the melon (Cucumis melon L.) accession PI 161375 Dissertation presented by Jinqiang Yan for the degree of Doctor in Plant Biology and Biotechnology by the Universitat Autònoma de Barcelona (UAB). This work was performed in the Centre for Research in Agricultural Genomics (CRAG). Thesis directors Tutor PhD candidate Dra. Ana Montserrat Dra. Marta Pujol Dra. Ana Montserrat Jinqiang Yan Martín Hernández Abajo Martín Hernández Barcelona, October 2018 Index Index of contents Summary Resumen Resum Abbreviations Introduction 1. Plant viruses ................................................................................................ 3 2. Cucumber mosaic virus (CMV) .................................................................. 3 2.1 Genome organization ................................................................................................3 2.2 Taxonomy and classification.....................................................................................5 2.3 CMV viral cycle ........................................................................................................6 2.3.1 Viral replication .................................................................................................6 2.3.2 Cell-to-cell movement .......................................................................................7 2.3.3 Long-distance movement ..................................................................................8 2.4 Plant host ...................................................................................................................9 2.5 Virus transmission and management ......................................................................10 3. Resistance to CMV ................................................................................... 11 3.1 Natural resistance ....................................................................................................11 3.2 Transgenic plants ................................................................................................... 13 3.2.1 pathogen-derived resistance ............................................................................13 3.2.2 Non-pathogen-derived resistance strategies ....................................................14 4. Melon ....................................................................................................... 14 4.1 Melon taxonomy and diversity................................................................................15 4.2 Genetic and genomic resources in melon................................................................16 4.3 Melon reference genome .........................................................................................19 4.4 Melon diseases and resistance .................................................................................20 4.4.1 Insects .................................................................................................................. 4.4.2 Fungi and bacteria ...........................................................................................20 4.4.3 Virus ............................................................................................................... 21 4.4.4 Resistance to CMV in the melon line PI 161375 ......................................... 21 Objectives ....................................................................................... 23 Material and Methods 1. Pant materials ........................................................................................... 27 1.1 Melon lines ............................................................................................................ 27 1.2 Nicotiana benthanimana and zucchini squash .......................................................28 2. Viruses....................................................................................................... 28 2.1 Viral strains ........................................................................................................... 28 2.2 Viral clones for in vitro transcription and agro-infiltration .................................. 28 3. Bacterial strains ......................................................................................... 29 4. Virus inoculations ..................................................................................... 29 4.1 Sap inoculation ...................................................................................................... 29 4.2 Agro-infiltration and viral RNA inoculation ......................................................... 30 5. DNA isolation ........................................................................................... 30 5.1 Genomic DNA extraction ...................................................................................... 30 5.2 Genomic DNA extraction for re-sequencing melon lines ..................................... 30 5.3 Plasmid DNA extraction ........................................................................................ 31 6. RNA isolation ........................................................................................... 31 6.1 Total RNA extraction using Tri-Reagent .............................................................. 31 6.2 Total RNA extraction using Plant mini kit ........................................................... 31 7. Introgression lines re-sequencing ............................................................ 31 8. In vitro transcription ................................................................................ 32 9. Plasmid construction ................................................................................. 32 10. Virus detection ....................................................................................... 33 10.1 ELISA (Enzyme-Linked Immuno Sorbent Assay) ............................................. 33 10.2 Visual phenotyping ............................................................................................. 33 10.3 Reverse transcription-polymerase chain reaction (RT-PCR) ............................. 34 10.4 Real time quantitative RT-PCR (qRT-PCR) ....................................................... 34 11. Data analysis .......................................................................................... 35 12. Genotyping .............................................................................................. 36 12.1 SNP genotyping (single-nucleotide polymorphism) ........................................... 36 12.2 SSR (Simple Sequence Repeat) ........................................................................... 36 12.3 CAPS (Cleaved Amplified Polymorphic Sequences) ........................................ 36 13. Genetic map construction ....................................................................... 38 14. QTL analysis ........................................................................................... 38 15. Sanger sequencing................................................................................... 38 Results Part I QTL analysis for resistance to the aggressive train CMV-FNY I.1 Screening for CMV-FNY resistance ..................................................................... 43 I.2 Production of F2 populations ................................................................................. 45 I.3 QTL analysis with the F2 population made from the cross DHL142 x DHL2012 .............................................................................................. 46 I.3.1 Phenotypic analysis of CMV-FNY resistance ............................................... 46 I.3.2 Genotyping and construction of genetic map ................................................. 48 I.3.3 QTL analysis ................................................................................................... 50 I.4 QTL analysis with the F2 population made from cross DHL142 x 20-91-15 ....... 51 I.4.1 Phenotypic analysis of CMV-FNY resistance ............................................... 52 I.4.2 Genotyping and construction of genetic map ................................................. 54 I.4.3 QTL analysis ................................................................................................... 56 I.5 QTL analysis with the F2 population from the cross DHL69 x DHL2012 ........... 57 I.5.1 Phenotypic analysis of CMV-FNY resistance ............................................... 57 I.5.2 Genotyping and construction of genetic map ................................................. 59 I.5.3 QTL analysis ................................................................................................... 61 I.6 QTL analysis with F2 population made from cross DHL142 x DHL1046 ........... 62