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Rheum – Identification Using Morphological Traits, Tubulin Based Rheum – Identification using morphological traits, Tubulin based Polymorphism and DNA-Barcoding Rheum – Authentifizierung mittels morphologischen Merkmalen, Tubulin basierter Polymorphismus und DNA-Barcoding Masterarbeit Am BOTANISCHEN INSTITUT 1 ABTEILUNG BIODIVERSITÄT KARLSRUHER INSTITUT FÜR TECHNOLOGIE (KIT) vorgelegt von Thomas Hirth Matrikelnummer 1549782 01.05.15- 31.12.15 Betreuer: Dr. Thomas Horn Erstgutachter: Herr Prof. Dr. Peter Nick Zweitgutachter: Herr Prof. Dr. Burkhard Luy i Erklärung Hiermit erkläre ich, dass ich die vorliegende Masterarbeit abgesehen von den angegebenen Hilfsmitteln selbstständig angefertigt habe. Alle Stellen, die ich wörtlich oder sinngemäß aus anderen Werken übernommen habe, sind als solche kenntlich gemacht. Karlsruhe, den ii Danksagung Ich möchte mich herzlichst bei Herrn Prof. Dr. Peter Nick bedanken, der es mir ermöglich hat diese Arbeit verfassen zu können. Außerdem bin ich Ihnen für die vielen konstruktiven Vorschläge während den unterschiedlichen Besprechung dankbar. Mein besonderer Dank gilt Dr. Thomas Horn für die intensive, ausgiebige Betreuung und die unzähligen Hilfestellungen und Anregungen, die ich im Laufe dieser Masterthesis von ihm erhalten habe. Bedanken möchte ich mich auch bei Herrn Prof. Dr. Burkhard Luy für die Übernahme der Zweitkorrektur dieser Masterthesis. Zu guter Letzt möchte ich mich bei allen Mitarbeiterinnen und Mitarbeitern des Botanischen Instituts 1 und des Botanischen Gartens des KITs für die freundliche Zusammenarbeit bedanken. iii Inhaltsverzeichnis Erklärung ..................................................................................................................................................ii Danksagung ............................................................................................................................................. iii Abkürzungsverzeichnis ........................................................................................................................... vii Tabellenverzeichnis ............................................................................................................................... xiv 1. Zusammenfassung ............................................................................................................................... 1 2. Einleitung ............................................................................................................................................. 2 2.1. Authentifizierung .......................................................................................................................... 2 2.2. Artkonzepte .................................................................................................................................. 3 2.3. Tibetische Hochebene als Diversifikationshotspot ...................................................................... 5 2.4. Rheum .......................................................................................................................................... 6 2.5. DNA Barcoding ............................................................................................................................. 8 2.5.1. psbA-trnH .............................................................................................................................. 9 2.5.2. nrITS ....................................................................................................................................... 9 2.5.3. TBP Tubulin basierter Polymorphismus .............................................................................. 10 2.5.4. Stammbäume ...................................................................................................................... 11 2.6. Chemische Analyse ..................................................................................................................... 12 3. Materialien ........................................................................................................................................ 14 3.1. Pflanzenmaterial ........................................................................................................................ 14 3.2. Chemikalien ................................................................................................................................ 16 3.3. Enzyme und Enzym-Kits ............................................................................................................. 16 3.4. Lösungen und Puffer .................................................................................................................. 16 3.5. Primer ......................................................................................................................................... 17 3.6. Molekulare Leitern ..................................................................................................................... 17 3.7. Verbrauchsgegenstände ............................................................................................................. 18 3.8. Geräte ......................................................................................................................................... 19 3.9. Software und APPS ..................................................................................................................... 20 3.11. PCR Programme........................................................................................................................ 20 4. Methoden .......................................................................................................................................... 22 4.1.Morphologische Analyse ............................................................................................................. 22 4.1.1.Bestimmung der Blühzeiten ................................................................................................. 22 4.1.2. Erstellung der Monographie ................................................................................................ 22 4.1.3.Bestimmung der Akzessionen .............................................................................................. 22 4.1.4.Blattanalyse .......................................................................................................................... 22 4.1.6. Ausgewählte Morphologische Messungen ......................................................................... 23 iv 4.2. Molekulare Analyse .................................................................................................................... 23 4.2.1. DNA-Extraktion .................................................................................................................... 23 4.2.2. Amplifikation von DNA Markern ......................................................................................... 24 4.2.2.1. DNA Barcoding (ITS und psbA-trnH) ................................................................................. 24 4.2.2.2 Stammbaumerstellung ...................................................................................................... 25 4.2.3. Fragmentanalyse TBP (tubulin based polymorphism) ........................................................ 26 5.3. Chemische Analyse ......................................................................................................................... 27 5.3.1. Herstellung der Phosphormolybdänschwefelsäure ............................................................ 27 5.3.2. Färbenachweis nach Fluck ................................................................................................... 27 6. Ergebnisse ......................................................................................................................................... 28 6.1. Morphologische Analyse ............................................................................................................ 28 6.1.1 Blühzeitpunkt ....................................................................................................................... 28 6.1.2. Auszug aus der Monographie .............................................................................................. 29 6.1.3.Morphologische Bestimmung .............................................................................................. 30 6.1.4.Ausgewählte morphologische Messungen .......................................................................... 42 6.2.Molekulare Analyse ..................................................................................................................... 45 6.2.1.psbA-trnH ............................................................................................................................. 45 6.2.2.Internal transcribed spacer (ITS) .......................................................................................... 48 6.2.3.Tubulin basierter Polymorphismus (TBP) ............................................................................. 49 6.3.chemische Analyse ...................................................................................................................... 55 6.3.1.Färbenachweiß nach Fluck ................................................................................................... 55 7.Diskussion ........................................................................................................................................... 57 7.1.Morphologie ...............................................................................................................................
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