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Erschließung Genetischer Ressourcen Der Wiesenrispe Für Die Gräserzüchtung Durch Analyse Wichtiger Merkmalsausprägungen

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Erschließung Genetischer Ressourcen Der Wiesenrispe Für Die Gräserzüchtung Durch Analyse Wichtiger Merkmalsausprägungen Aus dem Institut für Pflanzenzüchtung und Pflanzenschutz Direktor: Prof. Dr. habil. W.E. Weber der Landwirtschaftlichen Fakultät Dekan: Prof. Dr. habil. P. Pickel der Martin-Luther-Universität Halle-Wittenberg Fachgebiet: Pflanzenzüchtung Erschließung genetischer Ressourcen der Wiesenrispe für die Gräserzüchtung durch Analyse wichtiger Merkmalsausprägungen Dissertation Zur Erlangung des akademischen Grades doctor agriculturarum (Dr. agr.) Von Diplomagraringenieur Kalina Andreeva Halle/Saale 2005 urn:nbn:de:gbv:3-000010379 [ http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000010379] Aus dem Institut für Pflanzenzüchtung und Pflanzenschutz Direktor: Prof. Dr. habil. W.E. Weber der Landwirtschaftlichen Fakultät Dekan: Prof. Dr. habil. P. Pickel der Martin-Luther-Universität Halle-Wittenberg Erschließung genetischer Ressourcen der Wiesenrispe für die Gräserzüchtung durch Analyse wichtiger Merkmalsausprägungen Dissertation Zur Erlangung des akademischen Grades doctor agriculturarum (Dr. agr.) vorgelegt von Diplomagraringenieur geb.am 17.11.1973 Kalina Andreeva in Burgas Gutachter: Prof. Dr. habil. A. Graner Prof. Dr. habil. W.E. Weber PD Dr. habil. A. Börner Verteidigung am: 16.01.2006 Halle/Saale 2005 urn:nbn:de:gbv:3-000010379 [http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000010379] Inhaltverzeichnis 1 Einleitung...........................................................................................................................1 1.1 Die Wiesenrispe Poa pratensis L. als Nutzgras und genetisches Objekt............................1 1.2 Diversitätsanalyse mittels Marker.....................................................................................3 1.3 Flow-cytometrischer Samen-Test ...................................................................................10 1.4 Berechnung der morphologischen und genetischen Diversität und graphische Darstellung der Ergebnisse................................................................................................................12 1.5 Populationsgenetische Methoden....................................................................................13 1.6 Ziel der Arbeit................................................................................................................13 2 Material und Methoden ..................................................................................................15 2.1 Pflanzenmaterial.............................................................................................................15 2.2 Bonitur auf züchterisch-relevante Merkmale...................................................................16 2.3 Molekulare Markeranalysen ...........................................................................................21 2.3.1 DNA-Extraktion und Quantifizierung..........................................................................21 2.3.2 AFLP-Analysen...........................................................................................................22 2.4 Durchflusszytometrie .....................................................................................................24 2.4.1 Ermittlung der Art der Samenbildung mittels durchflusszytometrischem Samentest ....25 2.4.2. Untersuchungen zur Bestimmung des Ploidiegrades ...................................................28 2.5. Statistische Auswertung der Ergebnisse.........................................................................29 2.5.1 Morphologisch-phänotypische Daten...........................................................................29 2.5.2 Daten zur Art der Samenbildung..................................................................................29 2.5.3. Daten zur Ploidiestufe.................................................................................................30 2.5.4 Molekulare Daten........................................................................................................30 2.5.5 Korrelationen zwischen den Datensätzen.....................................................................30 3 Ergebnisse........................................................................................................................31 3.1 Feldversuche ..................................................................................................................31 3.1.1 Morphologisch-phänotypische Diversität.....................................................................31 3.1.2 Umwelt .......................................................................................................................37 3.2 Ermittlung der Samenbildung.........................................................................................39 3.3 Ermittlung der Ploidiestufen...........................................................................................41 3.4 Zusammenhang zwischen DNA-Gehalt und morphologischen Merkmalen .....................44 3.5 AFLP-Untersuchungen bei Poa pratensis.......................................................................45 3.5.1 Untersuchungen basierend auf Poa pratensis-Einzelpflanzen.......................................47 3.5.2 Untersuchungen auf Herkunftsebene – Gendiversität innerhalb und zwischen den Ländern.......................................................................................................................48 3.5.3 Hierarchische Populationsstrukturen bei Poa pratensis ................................................52 3.6 Zusammenhang zwischen AFLP- und morphologischen Daten.......................................53 4 Diskussion........................................................................................................................55 4.1 Morphologisch-phänotypische Feldevaluierungen der Primär- und Sekundäranlage .......55 4.2 Einflüsse der Umwelt auf die Merkmalsausprägung .......................................................57 4.3 Ermittlung der Art der Samenbildung.............................................................................58 4.4 Untersuchungen zur Ploidiestufe ....................................................................................60 4.5 Zusammenhang DNA-Gehalt - morphologisch-phänotypische Merkmale.......................61 4.6 AFLP-Untersuchungen bei der Wiesenrispe ...................................................................62 4.6.1 Untersuchungen an Poa pratensis-Einzelpflanzen........................................................63 4.6.2 Gendiversität innerhalb und zwischen den Ländern, Populationsstrukturen..................63 4.7 Zusammenhang zwischen morphologisch-phänotypischen und AFLP-Daten..................69 5 Zusammenfassung/Summary..........................................................................................72 5.1 Zusammenfassung..........................................................................................................72 5.2 Summary........................................................................................................................74 6 Literaturverzeichnis........................................................................................................76 7 Anhang.............................................................................................................................89 Abkürzungsverzeichnis Abb. Abbildung AFLP amplified fragment length polymorphism AMOVA analysis of molecular variance ANOVA analysis of variance APS Ammoniumpersulfat bp Basenpaare °C Grad Celsius DAPI 4,6-diamino-2-phenolindol DNA Desoxiribonukleinsäure DNase Desoxyribonuklease dNTP desoxy-Nukleosidtripohosphat EDTA Ethylendiamin-N,N,N`,N`-tetraessigsäure h Stunde kb Kilobasenpaare mg Milligramm min Minute ml Milliliter µg Mikrogramm µl Mikroliter ng Nanogramm PAA Polyacrylamid PAGE Polyacrylamid-Gelelektrophorese PCR Polymerase-Ketten-Reaktion RAPD random amplified polymorphic DNA RFLP restriction fragment length polymorphism RNA Ribonukleinsäure RNase Ribonuklease rpm rotations per minute/ Umdrehungen pro Minute s Sekunde SNP single nucleotide polymorphism STS sequence tagged site SSR simple sequence repeat Tab. Tabelle TBE Tris, Borat, EDTA TEMED N,N,N´,N´-Tetramethylethylendiamin Tris Tris-hydroxymethyl-aminomethan U Unit (Einheit der Enzymaktivität) UPGMA Unweighted Pair Group Method with Arithmetic Mean UPOV Union for the protection of new varieties of plants Abkürzungen der Länder (IPGRI, ISO 3166-1 Country Codes) BEL Belgien CHE Schweiz CSK Tschechoslowakei DEU Deutschland DNK Dänemark ESP Spanien EST Estland FIN Finnland FRA Frankreich GBR Großbritannien GRL Grönland HRV Bosnien-Herzegowina HUN Ungarn ISL Island ITA Italien LTU Litauen MNG Mongolei NLD Niederlande NOR Norwegen POL Polen PRT Portugal ROM Rumänien RUS Russland SUN Sowjetunion SVK Slowakische Republik SVN Slowenien SWE Schweden TUR Türkei YUG Jugoslawien Abbildungsverzeichnis Abbildung 1: Poa pratensis-Herkünfte im generativen Stadium......................................16 Abbildung 2: Schematische Darstellung der Versuchsanlage „Nachkommen-Diversität“ 19 Abbildung 3: Schematische Darstellung der Versuchsanlage „Nachkommen-Umwelt“...20 Abbildung 4: Aufbau eines Histogrammes: C-DNA-Gehalt im Embryo..........................26 Abbildung 5: Reproduktionswege bei Poa pratensis. ......................................................27 Abbildung 6: Durchflusszytometrische Auswertung des Ploidiegrades. ..........................28 Abbildung 7: Hauptkomponenten-Analyse der 928 Akzessionen polnischer Herkunft ....33 Abbildung 8: UPGMA-Phänogramme von 1583
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