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Utilizarea Tehnicilor De Analiză Moleculară La Genurile Prunus, Corylus, Juglans Şi Castanea În

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Utilizarea Tehnicilor De Analiză Moleculară La Genurile Prunus, Corylus, Juglans Şi Castanea În UNIVERSITATEA DE ŞTIINŢE AGRICOLE ŞI MEDICINĂ VETERINARĂ CLUJ-NAPOCA ŞCOALA DOCTORALĂ FACULTATEA DE HORTICULTURĂ Ing. Pop Iulia Francesca Utilizarea tehnicilor de analiză moleculară la genurile Prunus, Corylus, Juglans şi Castanea în vederea conservării în bănci de gene (REZUMAT AL TEZEI DE DOCTORAT) Conducător ştiinţific: Prof. univ. dr. Doru Pamfil Cluj-Napoca 2009 CUPRINS teza rezumat INTRODUCERE ....................................................................................................1 1 CAPITOLUL I........................................................................................................5 2 1.1. IMPORTANŢA CONSERVĂRII RESURSELOR GENETICE PENTRU MENŢINEREA BIODIVERSITĂŢII.................................................5 2 1.1.1. Definiţia biodiversităţii ..........................................................................5 1.1.2. Tipuri de biodiversitate ..........................................................................8 1.1.3. Conceptul de biodiversitate....................................................................9 1.1.4. Protecţia şi obiectul conservării biodiversităţii....................................11 1.1.5. Strategia conservării biodiversităţii......................................................12 1.1.6. Istoricul conservării biodiversităţii ......................................................13 1.2. BĂNCILE DE GENE .................................................................................15 2 1.2.1. Generalităţi despre băncile de gene.......................................................15 1.2.2. Categorii de bănci de gene ....................................................................18 1.2.3. Modalităţi de conservare în băncile de gene .........................................18 1.3. CADRUL LEGISLATIV AL PROTECŢIEI NOILOR SOIURI DE PLANTE ............................................................................................................22 2 1.3.1. Legislaţia românească în domeniul protecţiei noilor soiuri de plante...22 1.3.2. Uniunea Europeană ...............................................................................23 1.3.3. Statele Unite ale Americii .....................................................................23 1.3.4. UPOV ....................................................................................................24 1.3.5. Tratatul Internaţional asupra Resurselor Genetice Vegetale (ITPGRFA)......................................................................................................26 1.3.6. Alte tratate internaţionale......................................................................26 1.3.7. Patentarea genetică................................................................................27 1.4. IMPORTANŢA CONSERVĂRII RESURSELOR GENETICE ALE GENURILOR PRUNUS, CORYLUS, JUGLANS ŞI CASTANEA ................28 3 1.4.1. Importanţa culturii genului Prunus .......................................................28 1.4.2. Importanţa culturii genului Corylus ......................................................37 1.4.3. Importanţa culturii genului Juglans ......................................................40 1.4.4. Importanţa culturii genului Castanea....................................................54 I.5. METODE DE AMPRENTARE GENETICĂ .............................................56 4 1.5.1. Generalităţi privind markerii moleculari...............................................56 1.5.2. Clasificarea markerilor moleculari........................................................57 1.5.3. Alegerea tehnicilor moleculare folosite în experiment .........................65 1.5.4. Compararea diferitelor tehnici de marcare moleculară.........................66 1.5.5. Metode utilizate pentru interpretarea statistico-matematică a rezultatelor amprentării genetice la genurile Prunus, Corylus, Juglans şi Castanea..........................................................................................................69 1.5.6. Software pc utilizate pentru interpretarea datelor rezultate în urma analizelor moleculare ......................................................................................73 CAPITOLUL II. OBIECTIVELE CERCETĂRII, MATERIALUL ŞI METODA78 5 2.1. SCOPUL CERCETĂRILOR ......................................................................78 2.1.1. Obiectivele urmărite în cercetările aferente tezei de doctorat...............79 2.2. MATERIALUL BIOLOGIC.......................................................................79 5 2.2.1. Materialul biologic utilizat în experienţele de diferenţiere a accesiunilor cu ajutorul markerilor moleculari ...............................................79 2.2.1.1. Descrierea accesiunilor din genul Prunus luate în studiu ...........80 2.2.1.2. Descrierea accesiunilor din genul Corylus luate în studiu ..........93 2.2.1.3. Descrierea accesiunilor din genul Juglans luate în studiu ..........97 2.2.1.4. Descrierea accesiunilor din genul Castanea luate în studiu......108 2.2.1.6. Amorsele SSR utilizate în amplificarea ADN-ului......................113 2.3. METODE DE LUCRU .............................................................................119 6 2.3.1. Extracţia şi cuantificarea ADN-ului în vederea executării analizelor RAPD şi SSR.................................................................................................119 2.3.1.1.Protocolul extracţiei de ADN ......................................................119 2.3.1.2. Cuantificarea ADN-ului..............................................................121 2.3.2. Metode de lucru aplicate în experienţele de amplificare cu amorsele RAPD ............................................................................................................122 2.3.2.1. Protocolul de amplificare RAPD................................................124 2.3.2.2. Electroforeza în gel de agaroză..................................................127 2.3.2.2.1. Prepararea gelurilor de agaroză.......................................128 2.3.2.2.2. Migrarea elecroforetică şi pregătirea probelor de ADN...129 2.3.2.3. Preluarea imaginilor ..................................................................130 2.3.2.4. Analizarea imaginilor.................................................................130 2.3.3. Metode de lucru aplicate în experienţele de amplificare cu amorsele SSR... ............................................................................................................131 6 2.3.3.1. Protocol de amplificare SSR.......................................................132 2.3.3.2. Electroforeza capilară................................................................133 2.3.3.3. Analiza datelor............................................................................136 2.3.4. Metode statistice de calcul si interpretare a rezultatelor amplificărilor cu amorsele RAPD şi SSR .....................................................136 CAPITOLUL III. REZULTATE ŞI DISCUŢII .................................................141 7 3.1. REZULTATE PRIVIND EXTRACŢIA PROBELOR DE ADN .......141 7 3.1.1. Rezultate privind extracţia probelor de ADN la genul Prunus...........142 3.1.2. Rezultate privind extracţia probelor de ADN la genul Corylus..........144 3.1.3. Rezultate privind extracţia probelor de ADN la genul Juglans ..........145 3.1.4. Rezultate privind extracţia probelor de ADN la genul Castanea .......147 3.2. REZULTATE PRIVIND AMPLIFICAREA ŞI ELECTROFOREZA PRODUŞILOR DE REACŢIE RAPD ....................148 7 3.2.1. Rezultate privind amplificarea şi electroforeza produşilor de reacţie RAPD la genul Prunus..................................................................................153 7 3.2.2. Rezultate privind amplificarea şi electroforeza produşilor de reacţie RAPD la genul Corylus.................................................................................156 8 3.2.3. Rezultate privind amplificarea şi electroforeza produşilor de reacţie RAPD la genul Juglans .................................................................................157 8 3.2.4. Rezultate privind amplificarea şi electroforeza produşilor de reacţie RAPD la genul Castanea ..............................................................................159 8 3.3. REZULTATE PRIVIND ANALIZA IMAGINILOR ŞI INTERPRETAREA DATELOR OBŢINUTE CU AMORSELE RAPD........161 8 3.3.1. Analiza imaginilor şi interpretarea datelor obţinute cu amorsele III RAPD la genul Prunus..................................................................................161 8 3.3.2. Analiza imaginilor şi interpretarea datelor obţinute cu amorsele RAPD la genul Corylus.................................................................................164 10 3.3.3. Analiza imaginilor şi interpretarea datelor obţinute cu amorsele RAPD la genul Juglans .................................................................................167 12 3.3.4. Analiza imaginilor şi interpretarea datelor obţinute cu amorsele RAPD la genul Castanea ..............................................................................170 14 3.4. REZULTATE PRIVIND ANALIZA ŞI INTERPRETAREA DATELOR OBŢINUTE CU AMORSELE SSR.............................................174 16 3.4.1. Analiza şi interpretarea datelor obţinute cu amorsele SSR la genul Prunus.........................................................................................................174 16 3.4.2. Analiza şi interpretarea datelor obţinute cu amorsele SSR la genul Corylus........................................................................................................179
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