Investigation of interspecific genome-plastome incompatibility in Oenothera and Passiflora [Elektronische Ressource] / vorgelegt von Jaroslav Mráček

Jaroslav Mracek

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Investigation of interspecific genome-plastome
incompatibility in Oenothera and Passiflora






Dissertation zur Erlangung des Doktorgrades der Fakultät für Biologie
der Ludwig-Maximilians-Universität München







vorgelegt von
Jaroslav Mrá ek
aus Bedihoš , Tschechische Republik


München
2005


























1. Gutachter: Prof. Dr. R. G. Herrmann
2. Gutachter: Prof. Dr. G. Heubl

Datum der mündlichen Prüfung: 24.03.2006 CONTENT I
CONTENT

ABBREVIATIONS ................................................................................................. V

1 INTRODUCTION ............................................................................................... 1

1.1 Oenothera - an ideal system for studying genome-plastome
interactions as well as speciation and evolutionary processes ............... 2
1.1.1 Genetics and chromosome arrangement of Oenothera ..................... 3
1.1.2 Genome-plastome incompatibility in Oenothera ............................ 9
1.1.3 Incompatible genome-plastome combination AA-III ...................... 9
1.2 Passiflora – a new model plant for genome-plastome
incompatibility? .......................................................................................... 11
1.3 Nuclear genome analyses ........................................................................... 11
1.3.1 Expressed Sequence Tag (EST) ........................................................ 11
1.3.2 DNA Fingerprinting and Amplified Fragment Length
Polymorphism (AFLP)...................................................................... 12

2 MATERIAL AND METHODS ................................................................. 16

2.1 Material ....................................................................................................... 16
2.1.1 Plant material .................................................................................... 16
2.1.1.1 Oenothera EST library ........................................................ 16
2.1.1.2 Oenothera genotyping and linkage analysis ....................... 16
2.1.1.3 Passiflora ............................................................................ 16
2.1.2 Chemicals ......................................................................................... 16
2.1.3 Reagents and kits .............................................................................. 16
2.1.4 Devices ............................................................................................. 17
2.1.5 General buffers ................................................................................. 17
2.1.6 Software ............................................................................................ 18
2.1.7 Bacterial strains ................................................................................. 19 CONTENT II
2.1.8 Vectors .............................................................................................. 19
2.1.9 Oligonucleotides ............................................................................... 20
2.1.9.1 Unmodified oligonucleotides .............................................. 20
2.1.9.2 Unmodified oligonucleotides used in the development
of co-dominant markers ...................................................... 20
2.1.9.3 AFLP unmodified primers and adapters ............................. 21
2.1.9.4 AFLP modified primers ...................................................... 22
2.1.10 Enzymes ............................................................................................ 22
2.1.11 Antisera ............................................................................................. 23

2.2 Methods ........................................................................................................ 23
2.2.1 Nucleic acid analysis ......................................................................... 23
2.2.1.1 DNA analysis ...................................................................... 23
2.2.1.1.1 Isolation of total DNA ......................................... 23
2.2.1.1.1.1 Isolation of total DNA from
Oenothera ............................................. 23
2.2.1.1.1.2 Isolation of total DNA from
Passiflora .............................................. 24
2.2.1.1.2 PCR product purification ..................................... 25
2.2.1.1.3 Plasmid transformation ........................................ 25
2.2.1.1.4 Plasmid isolation .................................................. 26
2.2.1.1.5 Southern analysis ................................................. 26
2.2.1.2 RNA analysis ...................................................................... 27
2.2.1.2.1 Isolation of total RNA .......................................... 27
2.2.1.2.2 Isolation of poly(A)+ mRNA ............................... 28
2.2.1.2.3 Northern analysis.................................................. 28
2.2.1.3 Radioactive labeling of PCR products ................................ 29
2.2.1.4 Hybridisation with radioactively labelled probe ................. 29
2.2.1.5 Automated sequencing on the ABI PRISM 377 DNA
Sequencer ............................................................................ 30 CONTENT III
2.2.2 Protein analysis ................................................................................. 31
2.2.2.1 Protein isolation .................................................................. 31
2.2.2.2 Protein gel electrophoresis .................................................. 32
2.2.2.3 Staining of PAA gels .......................................................... 33
2.2.2.3.1 Coomassie Brilliant Blue staining ....................... 33
2.2.2.3.2 Staining with silver nitrate.................................... 33
2.2.2.4 Immunological detection of proteins on membranes .......... 34
2.2.2.4.1 Transfer of proteins onto nitrocellulose
and PVDF membranes ......................................... 34
2.2.2.4.2 Staining of blots with Ponceau S ......................... 35
2.2.2.4.3 Western analysis .................................................. 35
2.2.3 Expressed sequence tags (ESTs) ....................................................... 36
2.2.3.1 Construction of a cDNA library .......................................... 37
2.2.3.2 Computer analyses of EST sequences ................................ 40
2.2.4 Amplified Fragment Length Polymorphism (AFLP) ....................... 40
2.2.4.1 AFLP reactions ................................................................... 41
2.2.4.2 DNA fragment detection ..................................................... 42
2.2.4.3 Computer analysis of genotyping data ................................ 43
2.2.5 Electron microscopy ......................................................................... 43

3 RESULTS .................................................................................................... 46

3.1 ESTs FROM OENOTHERA....................................................................... 46
3.1.1 Preparing of an EST library .............................................................. 46
3.1.2 Sequencing of ESTs .......................................................................... 48
3.1.3 Clustering of the EST sequences....................................................... 48
3.1.4 Distribution of GC content in the EST sequences ............................ 48
3.1.5 Annotation of EST sequences ........................................................... 50

3.2 GENOTYPING AND LINKAGE ANALYSIS ........................................ 55 CONTENT IV
3.2.1 Genotyping analysis .......................................................................... 55
3.2.2 Development of AFLP markers and construction
of linkage maps ................................................................................. 55
3.2.3 Development of co-dominant markers derived from EST
sequencing ........................................................................................64

3.3 DETECTION AND INVESTIGATION OF INTERSPECIFIC
GENOME-PLASTOME INCOMPATIBILITY IN PASSIFLORA ....... 67
3.3.1 Investigation of Passiflora cpDNA .................................................. 68
3.3.2 Ultrastructure of the Passiflora chloroplasts .................................... 73
3.3.3 Northern analysis of Passiflora RNA................................................ 75
3.3.4 Protein analyses of Passiflora .......................................................... 76
3.3.4.1 SDS-PAGE protein gels ........................................................ 76
3.3.4.2 Western analysis ................................................................... 77

4 DISCUSSION .............................................................................................. 79

4.1 Expressed sequence t