DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Andrea Arbeiter :
High-resolution mapping and molecular analysis of the region around the resistance locus RPB1 on chromosome 1 of Arabidopsis thaliana (L.) Heynh.
Hochauflösende Kartierung und molekulare Analyse der Region um den Resistenzlocus RPB1 auf Chromosom 1 von Arabidopsis thaliana (L.) Heynh.

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Abstract

The resistance gene RPB1 is located on chromosome 1 of Arabidopsis thaliana and confers a dominantly inherited resistance to the obligate biotrophic pathogen Plasmodiophora brassicae. The objective of this work was to narrow down the region where the RPB1 gene had been mapped in preceding studies as far as possible, in order to isolate a DNA fragment carrying the resistance gene, which could then be used for the transformation of susceptible plants. To achieve this, high resolution mapping studies had to be done in the RPB1-region. The first step was the construction of a BAC contig that spans the resistance locus and was used to identify new, closely linked markers. Two BAC clones which span the RPB1 locus were identified. The published DNA sequence of the smaller one of these two clones, BAC T12O21, was used for further molecular analysis of this region. Since the BAC clones carry DNA of the susceptible Arabidopsis ecotype Columbia, a cosmid library of the resistant ecotype Tsu-0 (the ecotype used for building up the mapping population) was created. The existing mapping population was expanded from 900 to 4230 plants. Two allelspecific PCR markers were established that permitted a quick preselection of plants with recombination events near the RPB1 locus. Altogether 17 new closely linked RFLP and PCR markers were mapped in a section of 200 kb around the resistance locus. In the last part of the mapping studies some BAC fragments showing cosegregation with the resistance locus in a large section of approximately 29 kb could be mapped. According to these mapping data the RPB1-region is a section on chromosome 1 with a reduced number of recombination events (cold spot). The RPB1 locus was narrowed down to a region of approximately 71 kb according to the Col sequence. In this section three pseudogenes and 13 coding sequences were located. Six of these 13 candidate genes were placed in the region that showed cosegregation with RPB1. The other seven genes were placed in the regions to the left and to the right of this cosegregating region and have to be classified as candidate genes too. One cosmid clone was isolated from the Tsu library that carries three of these candidate genes. Two of the six genes in the cosegregating section are very probably Col specific, two other genes are duplicated on chromosome 5. None of the 13 coding sequences match known genes or proteins or have similarities to previously identified resistance genes. Therefore these genes were classified as hypothetical or unknown genes in the databases. In cDNA analyses 10 of these 13 coding sequences were confirmed to be expressed in at least one of the two ecotypes Tsu (resistant) and Cvi (susceptible). One gene (CDS9) was exclusively expressed in the resistant ecotype Tsu in both infected and non-infected plants. A high rate of polymorphism with a large number of single-nucleotide-polymorhisms between the individual ecotypes could be detected by sequence comparison of the two ecotypes Tsu and Col or PCR analyses of the four ecotypes Tsu, Cvi, RLD and Col. The high resolution mapping and the molecular analysis of the RPB1-region can now serve as the basis for subsequent complementation studies that will be necessary for isolating the RPB1 gene.

Table of Contents

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0. Titelblatt, Inhaltsverzeichnis
1. Einleitung 1
1.1. Abwehrmechanismen bei Pflanzen 1
1.2. Strukturmerkmale und Funktionen von Resistenzgenen 3
1.3. Isolierung von Genen 5
1.4. Arabidopsis thaliana 7
1.5. Genetik der Wirt-Pathogen-Interaktion 8
1.6. Das Krankheitsbild von Plasmodiophora brassicae 9
1.7. Die Interaktion von Plasmodiophora brassicae und Arabidopsis thaliana 10
1.8. Identifizierung und Kartierung des Resistenzgens RPB1 11
1.9. Zielsetzung der Arbeit 12
2. Material und Methoden 14
2.1. Material 14
2.2. Methoden 20
3. Ergebnisse 48
3.1. Identifizierung und Charakterisierung von BACs in der RPB1-Region zur Erstellung eines BAC-Contigs 48
3.2. Molekulare Analyse in der RPB1-Region anhand veröffentlichter BAC-Sequenzen 56
3.3. Herstellung einer Cosmidbibliothek des Arabidopsis-Ökotyps Tsu 58
3.4. Durchmusterung von Cosmidbibliotheken mit BAC-Fragmenten 59
3.5. Hochauflösende Kartierung in der RPB1-Region 63
3.6. PCR-Analysen zur Expression von Genen in der RPB1-Region 70
3.7. Untersuchungen zur genetischen Diversität zwischen den Arabidopsis-Ökotypen 74
4. Diskussion 79
4.1. Auswertung der Kartierungsdaten im Hinblick auf Interferenzen und Rekombinations-cold-spots in der RPB1-Region 79
4.2. Abgleich der Daten der charakerisierten BACs mit publizierten Ergebnissen 83
4.3. Eingrenzung des RPB1-Locus und Identifizierung entsprechender Kandidatengene im Ökotyp Col 84
4.4. Untersuchungen zur Expression der Kandidatengene in den Ökotypen Tsu und Cvi 86
4.5. Die RPB1-vermittelte Resistenz: Eine bekannte Abwehrreaktion, aber keine Hinweise auf einen typischen Resistenzmechanismus 87
4.6. Bedeutung der Isolierung des RPB1-Gens für die Resistenzzüchtung 88
4.7. Ausblick auf weiterführende Arbeiten 90
5. Zusammenfassung 93
5a. Summary 95
6. Literaturverzeichnis 96
7. Anhang 104

More Information:

Online available: http://www.diss.fu-berlin.de/2002/217/indexe.html
Language of PhDThesis: german
Keywords: Arabidopsis thaliana, Plasmodiophora brassicae, resistance, mapping
DNB-Sachgruppe: 32 Biologie
Date of disputation: 12-Apr-2002
PhDThesis from: Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee: Prof. Dr. Maria Dolores Sacristán
Second Referee: Prof. Dr. Thomas Schmülling
Contact (Author): arbeiter@zedat.fu-berlin.de
Contact (Advisor): sacris@zedat.fu-berlin.de
Date created:25-Oct-2002
Date available:01-Nov-2002

 

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