Aus dem Institut für Pflanzenbau und Pflanzenzüchtung der Christian-Albrechts-Universität zu Kiel Hybrid Assembly of Whole Genome Shotgun Sequences of Two Sugar Beet (Beta vulgaris L.) Translocation Lines Carrying the Beet Cyst Nematode Resistance Gene Hs1-2 and Functional Analysis of Candidate Genes der Agrar- und Ernährungswissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von M.Sc. Sarah Christina Jäger aus Gießen Kiel, 06. Dezember 2012 Dekan: Prof. Dr. Rainer Horn 1. Berichterstatter: Prof. Dr. Christian Jung 2. Berichterstatter: Prof. Dr. Andre Franke Tag der mündlichen Prüfung: 31.01.2013 Table of Contents I Table of Contents Table of Contents ...................................................................................................................................... I Abbreviations .......................................................................................................................................... V List of Figures ........................................................................................................................................ XI List of Tables ......................................................................................................................................... XV 1 General Introduction ........................................................................................................................ 1 1.1 Recent Advances in Plant Genomics ......................................................................................... 1 1.1.1 Development of Sequencing Technologies ........................................................................ 1 1.2 The Sugar Beet Crop ................................................................................................................. 6 1.2.1 Organization of the Beta Genome ...................................................................................... 6 1.3 Genomic Research in Sugar Beet .............................................................................................. 7 1.3.1 Genetic Mapping ................................................................................................................ 7 1.3.2 Physical Mapping and Genomic Libraries ......................................................................... 9 1.3.3 Proteomics and Transcriptomics in Sugar Beet ............................................................... 10 1.3.4 Sequence Resources for Sugar Beet ................................................................................. 11 1.4 Plant Response to Pathogens ................................................................................................... 12 1.4.1 Plant–Nematode Interaction ............................................................................................. 13 1.4.2 The Beet Cyst Nematode Heterodera schachtii ............................................................... 17 1.4.3 Transfer of Nematode Resistance from Patellifolia species to Beta vulgaris .................. 18 1.4.4 Candidate Genes Involved in the Interaction between Beta vulgaris and Heterodera schachtii ......................................................................................................................................... 22 1.5 Origin and Features of Chromosomal Translocations ............................................................. 24 1.5.1 The Role of Chromosome Translocations in Humans and Plants .................................... 25 1.5.2 Microscopic and Molecular Analysis of Chromosome Translocation ............................. 26 1.6 Objectives and Hypothesis ...................................................................................................... 27 2 Functional Analysis of ORF 702 as a Candidate for the Beet Cyst Nematode Resistance Gene Hs1-2 ..................................................................................................................................... 29 2.1 Introduction ............................................................................................................................. 29 2.2 Material and Methods .............................................................................................................. 31 II Table of Contents 2.2.1 Gene Structure Analysis of ORF 702 using Rapid Amplification of cDNA Ends (RACE®) .......................................................................................................................... 31 2.2.2 Sequence Analyses ........................................................................................................... 32 2.2.3 RNA Isolation and cDNA Synthesis ................................................................................ 32 2.2.4 Plasmid Construction and Transformation into Agrobacterium Strains .......................... 33 2.2.5 Sugar Beet Hairy Root Transformation ............................................................................ 33 2.2.6 Arabidopsis thaliana Transformation............................................................................... 34 2.2.7 In vitro Nematode Resistance Tests ................................................................................. 34 2.2.8 Expression Analysis ......................................................................................................... 35 2.3 Results ...................................................................................................................................... 36 2.3.1 Defining the Gene Structure of ORF 702 using RACE® ................................................ 36 2.3.2 Functional Analysis of ORF 702 as a HS1-2 Gene Candidate ......................................... 38 2.4 Discussion ................................................................................................................................ 47 2.5 References ................................................................................................................................ 52 3 Re-Sequencing and Hybrid Assembly of Two Nematode-Resistant Beta vulgaris Translocation Lines ........................................................................................................................ 57 3.1 Introduction .............................................................................................................................. 57 3.2 Material and Methods .............................................................................................................. 59 3.2.1 Plant Material ................................................................................................................... 59 3.2.2 Primer Sequences ............................................................................................................. 60 3.2.3 Sequences and Clone Libraries ........................................................................................ 61 3.2.4 Whole Genome Shotgun (WGS) Sequencing .................................................................. 62 3.2.5 Inoculation of Plants with Nematodes .............................................................................. 63 3.2.6 RNA Isolation and cDNA Synthesis ................................................................................ 63 3.2.7 Sequencing of Root RNA ................................................................................................. 63 3.2.8 Bioinformatical Analysis .................................................................................................. 63 3.3 Results ...................................................................................................................................... 65 3.3.1 Whole Genome Shotgun Sequencing and Assembly of the Short Reads ........................ 65 3.3.2 Identification of New Translocation Specific Sequences Anchored to the Physical Map .................................................................................................................... 67 Table of Contents III 3.3.3 Identification of New Translocation Specific Sequences Not Anchored to the Physical Map .................................................................................................................... 72 3.3.4 An Integrated Map of the TR520 Translocation .............................................................. 74 3.3.5 Open Reading Frame Prediction and Annotation ............................................................ 77 3.3.6 Candidate Gene Identification .......................................................................................... 79 3.3.7 Analysis of ORF 803 ........................................................................................................ 85 3.3.8 Analysis of Previously Proposed Nematode Resistance Gene Candidates from the Resistant Translocation Lines .......................................................................................... 90 3.3.9 Sequence Similarity between the P. procumbens Translocations and the Sugar Beet Genome .......................................................................................................... 91 3.4 Discussion ................................................................................................................................ 94 3.5 References ............................................................................................................................. 102 4 Closing Discussion ......................................................................................................................