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(Lonicera L.) Genties Atstovų Genetinės Įvairovės Ir Filogenetiniai Tyrimai Dnr Ţymenų Metodais

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(Lonicera L.) Genties Atstovų Genetinės Įvairovės Ir Filogenetiniai Tyrimai Dnr Ţymenų Metodais VILNIAUS UNIVERSITETAS Donatas Naugţemys SAUSMEDŢIO (LONICERA L.) GENTIES ATSTOVŲ GENETINĖS ĮVAIROVĖS IR FILOGENETINIAI TYRIMAI DNR ŢYMENŲ METODAIS Daktaro disertacija Biomedicinos mokslai, biologija (01 B) Vilnius, 2011 Disertacija rengta 2006 – 2010 metais Vilniaus universitete. Mokslinis vadovas: prof. dr. Donatas Ţvingila (Vilniaus universitetas, biomedicinos mokslai, biologija – 01 B) Konsultantas: dr. Silva Ţilinskaitė (Vilniaus universitetas, biomedicinos mokslai, biologija – 01 B) 2 TURINYS SANTRUMPOS ..................................................................................................... 5 ĮVADAS ................................................................................................................. 7 I. LITERATŪROS APŢVALGA ......................................................................... 13 1. Sausmedţio genties apţvalga ....................................................................... 13 1.1. Lonicera L. genties sistematikos istorija ir problemos .......................... 15 1.2. Lonicera L. genties kilmė ...................................................................... 21 2. Molekuliniai ţymenys ir augalų filogenetiniai tyrimai ................................ 24 2.1. RAPD metodo taikymas augalų sistematikoje ...................................... 26 2.2. Chloroplastų DNR nekoduojančių specifinių regionų tyrimas sekoskaitos metodu .............................................................................................. 31 2.3. Lonicera L. genties filogenetikos molekuliniai tyrimai ........................ 35 3. Šiuolaikiniai bioinformatikos metodai filogenetikoje .................................. 36 3.1. Duomenų apdorojimas molekulinėje filogenetikoje .............................. 37 3.2. Filogenetinių dendrogramų sudarymas ir principai ............................... 38 3.3. Filogenetinių medţių patikimumo analizė ............................................ 43 3.4. Duomenų bazės...................................................................................... 44 3.5. DNR sekų palyginimas .......................................................................... 46 4. Augalų genetiniai ištekliai, jų tyrimas ir išsaugojimas ................................. 48 II. TYRIMŲ OBJEKTAS IR METODAI ............................................................ 53 1. Tyrimų objektas – Lonicera L. genties augalai ............................................ 53 1.1. VU Botanikos sodo sausmedţių kolekcija ............................................ 53 1.1.1. Lonicera L. genties rūšys................................................................ 54 1.1.2. L. caerulea porūšiai ........................................................................ 55 1.1.3. L. caerulea veislės .......................................................................... 56 1.1.4. L. caerulea genetinės linijos ........................................................... 57 2. Tyrimų metodika .......................................................................................... 59 2.1. Cheminės medţiagos ............................................................................. 59 2.2. Tirpalai ir jų paruošimas ........................................................................ 60 2.3. Lonicera L. genties augalų DNR skyrimo metodikos optimizavimas ... 60 2.4. DNR koncentracijos ir kokybės nustatymas .......................................... 62 2.5. PGR vykdymas ...................................................................................... 63 2.5.1. RAPD pradmenų parinkimas sausmedţio kolekcijos tyrimams..... 63 2.5.2. Specifinių pradmenų sukūrimas Lonicera caerulea L. rūšies identifikavimui ................................................................................................. 64 2.5.3. Chloroplastų DNR nekoduojančių specifinių regionų PGR vykdymas ......................................................................................................... 65 3 2.5.4. Tiriamų cpDNR specifinių regionų trumpas apibūdinimas ............ 66 2.6. RAPD produktų frakcionavimas agarozės gelyje .................................. 68 2.7. cpDNR sekų analizė naudojant klonavimą ............................................ 68 2.7.1. DNR fragmentų išskyrimas ir paruošimas klonavimui .................. 69 2.7.2. Escherichia coli transformacija ir transformantų analizė ............... 69 2.7.3. Klonuoto DNR fragmento nukleotidų sekų nustatymas ir panašių sekų paieška duomenų bazėse ......................................................................... 70 2.8. Duomenų analizė ................................................................................... 71 III. REZULTATAI ............................................................................................... 73 1. VU Botanikos sodo Lonicera L. kolekcijos tyrimai DNR ţymenų metodais ................................................................................................................................. 73 1.1. Sausmedţio genetinių linijų galimybių įvertinimas veislių genetinei bazei praplėsti ...................................................................................................... 78 1.2. Sausmedţio pavyzdţių atranka filogenetinei analizei ........................... 87 1.3. Chloroplastų DNR specifinių sekų tyrimas sekoskaitos metodu........... 91 2. Lonicera caerulea L. filogenetinių ryšių tyrimas ....................................... 100 2.1. L. caerulea L. filogenetiniai tyrimai RAPD metodu ........................... 101 2.2. Filogenetinių ryšių nustatymas naudojant cpDNR sekoskaitos duomenis ........................................................................................................................... 104 2.3. Filogenetinių ryšių analizė panaudojant originalius ir duomenų bazėse registruotų giminingų taksonų cpDNR sekoskaitos duomenis .......................... 105 IV. REZULTATŲ APTARIMAS ...................................................................... 115 1. Lonicera L. genties pavyzdţių polimorfizmas VU Botanikos sodo kolekcijoje.............................................................................................................. 115 2. Melsvauogio sausmedţio genetinių linijų selekcinės galimybės ............... 117 3. Sausmedţių filogenetiniai tyrimai .............................................................. 122 3.1. RAPD metodo panaudojimas sausmedţių filogenetiniams tyrimams . 122 3.2. Filogenetinė analizė naudojant sekoskaitos tyrimo duomenis ............ 123 IŠVADOS ........................................................................................................... 129 MOKSLINIŲ DARBŲ SĄRAŠAS .................................................................... 131 PADĖKA ............................................................................................................ 134 LITERATŪROS SĄRAŠAS .............................................................................. 135 PRIEDAI ............................................................................................................ 164 4 SANTRUMPOS A, C, G, T – adeninas, citozinas, guaninas, timinas AFLP – amplifikuotų fragmentų ilgio polimorfizmas (angl. Amplified Fragments Length Polymorphism) AGI – augalų genetiniai ištekliai AMOVA – genetinės įvairovės molekulinės variacijos analizė (angl. Analysis of MOlecular VAriance) BLAST – pagrindinis vietinis išlyginamosios paieškos įrankis (angl. Basic Local Alignment Search Tool), naudojamas nukleotidų sekų palyginimui su NCBI duomenų bazėje registruotomis sekomis bp – bazių pora, nukleorūgšties ilgio vienetas, lygus vienai bazių porai BS – Vilniaus universiteto Botanikos sodas cpDNR – chloroplastų DNR DDBJ – DNR duomenų bankas Japonijoje (angl. DNA Data Bank of Japan) DNR – deoksiribonukleorūgštis E tikimybės reikšmė (angl. Expectation value) EMBL-EBI – Europos molekulinės biologijos laboratorija bioinformatikos institutas (angl. European Molecular Biology Laboratory – European Bioinformatics Institute) GDxy – genetinis atstumas tarp individų (angl. Genetic Distance) GRIN – JAV augalų sistematikos duomenų bazė (angl. Germplasm Resources Information Network) GSS – genomo sekoskaitos apţvalgos duomenų bazė (angl. Genome Survey Sequences) IPNI – Tarptautinė augalų vardų bazė (angl. The International Plant Names Index) IPTG – izopropil-β-D-tiogalaktopiranozidas IR – invertuotas pasikartojimas (angl. Inverted Repeat) ISSR – paprastų pasikartojančių sekų intarpai (angl. Inter-Simples Sequence Repeats) ITS – vidinis transkribuotas tarpiklis (angl. Internal Transcribed Spacer) IUPAC – tarptautinė teorinės ir taikomosios chemijos sąjunga (angl. The International Union of Pure and Applied Chemistry) 5 kb – kilobazė, nukleorūgščių ilgio vienetas, lygus 103 bazių porų arba bazių LB – Luria-Bertani mitybinė terpė LSC – chloroplasto genomo didţioji vienkopijinė sritis (angl. Large Single Copy) ME – maţiausios evoliucijos metodas (angl. Minimum Evolution) MEGA – molekulinės evoliucijos genetinės analizės programa (angl. Molecular Evolutionary Genetic Analysis) ML – didţiausio tikėtinumo algoritmas (angl. Maximum Likelihood) MP – didţiausio taupumo algoritmas (angl. Maximum Parsimony) NCBI – nacionalinis biotechnologijos informacijos centras (angl. National Center for Biotechnology Information) NJ – kaimynų grupavimo algoritmas (angl. Neighbor-joining) nt – nukleotidai/nukleotidas OTV – operacinis taksonominis vienetas (angl. Operational Taxonomic Units) PGR – polimerazinė grandininė reakcija (angl. PCR, Polymerase Chain Reaction) RAPD – atsitiktinai
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