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Nepenthes Mit Primern Nach White Et Al

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Nepenthes Mit Primern Nach White Et Al Molekular-Systematische Untersuchungen an den Familien Nepenthaceae und Ancistrocladaceae sowie verwandter Taxa aus der Unterklasse Caryophyllidae s. l. Inaugural-Dissertation zur Erlangung des Doktorgrades der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Harald Meimberg aus München November 2002 1. Berichterstatter: Prof. Dr. Günther Heubl 2. Berichterstatter: Prof. Dr. Peter Dittrich Tag der mündlichen Prüfung: 11.2.2003 Verzeichnis der Abkürzungen und Fachausdrücke AAdenin ATP Adenosin-5‘-triphosphat bp Basenpaare CCytosin CI Consistency Index CTP Cytosin-5‘-triphosphat ddNTP 2`,3`- Didesoxynukleosidtriphosphat DNA Desoxyribonukleinsäure dNTP 2`-Desoxynukleosidtriphosphat ETS external transcribed spacer G Guanin GTP Guanosin-5‘-triphosphat H2Obid bidestilliertes Wasser H2Odest destilliertes Wasser IInosin IGS intergenic spacer ISSR inter simple sequence repeat ITS internal transcribed spacer kult. kultiviert LMM Laser-Microbeam-Microdissection LPC Laser-Pressure-Catapulting Ma vor Millionen Jahren matK Gen der MaturaseK N unbekanntes Nukleotid, NCBI National Center for Biotechnology Information NP Nationalpark NTP Nukleotidtriphosphat p.A. per analysis PCR polymerase chain reaction (Polamerase-Kettenreaktion) RAPD random amplified polymorphic DNA rbcL Gen der Ribulose-1,5-Bisphosphat-Carboxylase rDNA für ribosomale RNA kodierende DNA RI Retention Index RNA Ribonucleinsäure rRNA ribosomale RNA RT Raumtemperatur S Sedimentationskoeffizient SSR simple sequence repeat T Thymin TBR tree-bisection-reconnection Tm Schmelztemperatur tRNA Transfer-RNA trnF Gen der Transfer-RNA für Phenylalanin trnK Gen der Transfer-RNA für Leucin trnL Gen der Transfer-RNA für Leucin trnT Gen der Transfer-RNA für Threonin TTP Thymidin-5‘-triphosphat Upm Umdrehungen pro Minute I Inhaltsverzeichnis Inhaltsverzeichnis Abbildungsverzeichnis ...........................................................................................................VI Tabellenverzeichnis ................................................................................................................IX 1 Einleitung .......................................................................................................................1 1.1 Die Unterklasse Caryophyllidae..................................................................................2 1.2 Die Familie Nepenthaceae............................................................................................5 1.3 Hypothesen zur Evolution der Nepenthaceae..........................................................11 1.4 Die Familien Dioncophyllaceae und Ancistrocladaceae..........................................12 1.5 Aufgabenstellung ........................................................................................................18 2 Material und Methoden ..........................................................................................19 2.1 Untersuchtes Pflanzenmaterial .................................................................................19 2.2 Isolierung von Gesamt-DNA......................................................................................26 2.2.1 CTAB-Isolierung nach Doyle und Doyle (1987) .................................................27 2.2.2 DNA-Isolierung mit Nucleospin-Plant-Kit ..........................................................28 2.2.3 Isolierung von DNA aus altem Herbarmaterial....................................................29 2.3 Amplifikation ..............................................................................................................30 2.3.1 Inverse PCR..........................................................................................................33 2.3.2 ISSR-PCR.............................................................................................................34 2.4 Durchführung einer Single-Chloroplasten-PCR .....................................................35 2.4.1 Chloroplasten-Isolierung ......................................................................................35 2.4.2 Separierung von Chloroplasten durch Laser Pressure Catapulting (LPC) ...........36 2.4.3 Amplifikation aus separierten Chloroplasten .......................................................37 2.5 Reinigungsverfahren für PCR-Produkte .................................................................38 2.6 Klonierung...................................................................................................................40 2.6.1 Ligation und Transformation................................................................................40 2.6.2 Kolonie-PCR von E. coli......................................................................................41 2.7 Amplifikations- und Sequenzierungsstrategien.......................................................42 2.7.1 Ableitung von Primern .........................................................................................42 2.7.2 Strategien zur Amplifikation und Sequenzierung ................................................44 2.7.2.1 ITS ........................................................................................................................45 2.7.2.2 Amplifikation von DNA aus Herbarmaterial .......................................................46 2.7.2.3 Rpl2 ......................................................................................................................46 2.7.2.4 TrnK......................................................................................................................47 2.7.2.5 TrnL......................................................................................................................50 2.8 Sequenzierung.............................................................................................................50 II Inhaltsverzeichnis 2.8.1 Sequenzierung mit dem GATC-Direct-Blotter.....................................................51 2.8.2 Sequenzierung mit dem ABI 377 .........................................................................54 2.8.3 Auswertung der Sequenzdaten .............................................................................55 2.8.4 Identifizierung paraloger Sequenzen durch Allelsubtraktion...............................56 2.9 Bearbeitung von Sequenzdaten .................................................................................57 2.9.1 Alinierung und Vorbereitung der Datenmatrizen.................................................57 2.9.2 Distanzverfahren...................................................................................................57 2.9.3 Phylogenetische Rekonstruktionsverfahren .........................................................59 2.9.3.1 Maximum Parsimony Analyse .............................................................................59 2.9.3.2 Maximum Likelihood Analyse.............................................................................62 3 Ergebnisse ....................................................................................................................64 3.1 Überprüfung der Verwandtschaftsverhältnisse der carnivoren Taxa der Caryophyllidae............................................................................................................64 3.1.1 Die Phylogenie der Caryophyllidae s. l. auf der Basis vergleichender Sequenzanalysen des Maturase K-Gens (matK) ..................................................64 3.1.1.1 Alinierung der matK Daten ..................................................................................65 3.1.1.2 Phylogenetische Analyse partieller matK-Daten..................................................67 3.1.1.3 Bestimmung der Leserichtung der Topologie ......................................................68 3.1.2 Phylogenetische Analyse von trnK-Intron Sequenzdaten ....................................72 3.1.3 Phylogenetische Rekonstruktionen veröffentlichter Sequenzdaten der Caryophyllidae s. l................................................................................................73 3.1.3.1 Phylogenetische Analyse von rbcL-Sequenzen....................................................74 3.1.3.2 Kombination der rbcL- und matK-Daten .............................................................76 3.1.3.3 Vergleich der 18S rDNA- und matK-Phylogenie.................................................78 3.1.3.4 Vergleich der Variabilität von 18S rDNA, rbcL und matK .................................80 3.1.4 Deletion des rpl2-Introns ......................................................................................80 3.2 ITS-Polymorphismus bei Nepenthaceae und Dioncophyllaceae............................83 3.2.1 Sequenzierung der ITS-Region bei Nepenthes mit Primern nach White et al. (1990) ....................................................................................................................84 3.2.2 Amplifikation der ITS-Region mit für Angiospermen spezifischen Primern ......87 3.2.3 Nachweis von ITS-Pseudogenen in Nepenthes ....................................................91 3.2.3.1 Phylogenetische Analyse der 5,8S rDNA.............................................................91 3.2.3.2 Sekundärstruktur von ITS2...................................................................................94 3.3 TrnK-Polymorphismus bei Nepenthaceae ...............................................................98 3.3.1 Entwicklung einer Single-Chloroplasten-PCR.....................................................99 3.3.1.1 Isolierung
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