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Thesis Rahim Ahmadvand Keszthely, Hungary 2013 UNIVERSITY OF PANNONIA GEORGIKON FACULTY DOCTOR OF PHILOSOPHY (PhD) THESIS RAHIM AHMADVAND KESZTHELY, HUNGARY 2013 2 UNIVERSITY OF PANNONIA GEORGIKON FACULTY DOCTORAL SCHOOL OF CROP PRODUCTION AND HORTICULTURAL SCIENCES PLANT BREEDING, GENETICS AND AGROBIOTECHNOLOGY PROGRAM HEAD OF THE DOCTORAL SCHOOL PROF. DR. LÁSZLÓ KOCSIS, DSC ANALYSIS OF RESISTANCE GENES IN POTATO WITH SPECIAL ATTENTION TO EXPRESSIONAL APPROACHES DOCTOR OF PHILOSOPHY (PhD) THESIS WRITTEN BY RAHIM AHMADVAND SUPERVISORS DR. ZSOLT POLGÁR, PhD AND DR. JÁNOS TALLER, PhD KESZTHELY, HUNGARY 2013 3 ANALYSIS OF RESISTANCE GENES IN POTATO WITH SPECIAL ATTENTION TO EXPRESSIONAL APPROACHES Written By RAHIM AHMADVAND Written at the University of Pannonia, Doctoral School of Crop Production and Horticultural Sciences, Plant Breeding, Genetics and Agrobiotechnology Program Supervisors: Dr. Zsolt Polgár I propose for acceptance (yes /no) Signature Dr. János Taller I propose for acceptance (yes /no) Signature The candidate has achieved………..% at the comprehensive exam, I propose the thesis for acceptance as the reviewer: Name of reviewer: ………………………………………….yes /no Name of reviewer: …………………………………………..yes /no The candidate has achieved………..% at the public discussion. Veszprém/Keszthely, ….…………………………….. Chairman of the Committee Labeling of the PhD diploma ………………………….. ……………………………… President of the UCDH 4 Table of Contents ABSTRACT ....................................................................................................................................... 9 KIVONAT ....................................................................................................................................... 10 ABSTRAKT .................................................................................................................................... 11 ABBREVIATIONS ......................................................................................................................... 12 1. INTRODUCTION ....................................................................................................................... 14 Research objectives .......................................................................................................................... 17 2. LITERATURE REVIEW ............................................................................................................ 18 2.1. Origin and evolution of potato crop ...................................................................................... 18 2.2. Potato production in Hungary ............................................................................................... 19 2.3. History of potato research at Keszthely, Hungary ................................................................ 19 2.4. Potato viruses ........................................................................................................................ 20 2.4.1. Potato virus X ................................................................................................................. 21 2.4.2. Potato virus Y ................................................................................................................. 22 2.5. Genetics of resistance ............................................................................................................ 24 2.5.1. Structure and function of R genes .................................................................................. 25 2.5.2. Mechanism of virus resistance ....................................................................................... 28 2.5.3. Signaling mediated resistance ........................................................................................ 29 2.5.4. Gene silencing ................................................................................................................ 31 2.5.5. Types of resistance to potato viruses .............................................................................. 32 2.5.5.1. Hypersensitive reaction (HR) .................................................................................. 32 2.5.5.2. Extreme resistance (ER) .......................................................................................... 33 2.5.5.3. Resistance to infection ............................................................................................. 35 2.5.5.4. Resistance to virus accumulation ............................................................................. 36 2.5.5.5. Resistance to virus movement in plants ................................................................... 36 2.5.5.6. Mature plant resistance ............................................................................................ 37 2.5.5.7. Tolerance.................................................................................................................. 37 2.5.5.8. Resistance to virus vectors ....................................................................................... 38 5 2.6. The potato resistance genes Rx1 and Rx2 .............................................................................. 39 2.7. Resistance breeding and molecular markers ......................................................................... 40 2.8. Marker assisted selection ...................................................................................................... 41 2.9. Intron-targeting...................................................................................................................... 42 2.10. Gene expression profiling in plant-virus interactions ......................................................... 43 2.10.1. Subtraction Suppressive Hybridization (SSH) ............................................................. 44 2.10.2. cDNA-AFLP ................................................................................................................. 46 2.10.3. Serial Analysis of Gene Expression ............................................................................. 46 2.10.4. Microarray .................................................................................................................... 47 2.10.5. Transcriptome analysis ................................................................................................. 48 2.10.5.1 De novo transcriptome analysis .............................................................................. 51 2.10.5.2. Transcriptome analysis of the sequenced genomes ............................................... 51 3. MATERIALS AND METHODS ................................................................................................. 54 3.1. Plant materials ....................................................................................................................... 54 3.1.1. Plant materials used in PVX inoculation test ................................................................. 54 3.1.2. Evaluation of the validity of developed specific primers for Rx1 and Rx2 genes .......... 54 3.1.3. Development of Intron targeting markers ...................................................................... 56 3.2. DAS-ELISA test .................................................................................................................... 57 3.3. PVX resistance tests .............................................................................................................. 57 3.3.1. Mechanical inoculation ................................................................................................... 57 3.3.2. Graft inoculation ............................................................................................................. 57 3.4. Genomic DNA isolation ........................................................................................................ 58 3.4.1. Lysis of plant cells and protein denaturation .................................................................. 58 3.4.2. Purification ..................................................................................................................... 58 3.5. Identification of resistance gene to PVX............................................................................... 59 3.5.1. Marker analysis............................................................................................................... 59 3.5.2. Development of specific primers for Rx genes ............................................................... 59 3.6. Development of a multiplex PCR for the Rx genes .............................................................. 60 3.7. Transcriptome analysis .......................................................................................................... 60 3.7.1. Inoculation with pathogens ............................................................................................. 61 6 3.7.1. 1. Inoculation with PVX and PVY ............................................................................. 61 3.7.1.2. Inoculation with Ph. infestans.................................................................................. 61 3.7.2. mRNA isolation .............................................................................................................. 61 3.7.3. mRNA analysis ............................................................................................................... 62 3.7.4. mRNA preparation ......................................................................................................... 63 3.7.5. Suppression subtractive hybridization (SSH) ................................................................
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