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Využití Vybraných Regionů Jaderné a Chloroplastové DNA Ke Studiu Fylogeneze Vybraných Zástupců Rodu Lactuca

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Využití Vybraných Regionů Jaderné a Chloroplastové DNA Ke Studiu Fylogeneze Vybraných Zástupců Rodu Lactuca UNIVERZITA PALACKÉHO V OLOMOUCI PŘÍRODOVĚDECKÁ FAKULTA KATEDRA BOTANIKY Využití vybraných regionů jaderné a chloroplastové DNA ke studiu fylogeneze vybraných zástupců rodu Lactuca DIPLOMOVÁ PRÁCE Bc. Denisa Plucnarová Studijní obor: Botanika Forma studia: Prezenční Vedoucí práce: RNDr. Ľuboš Majeský, Ph.D. Konzultant: RNDr. Miloslav Kitner, Ph.D. Olomouc 2018 Prohlašuji, že předložená práce je mým původním autorským dílem, které jsem zpracovala samostatně v průběhu magisterského studia. Veškerou literaturu, z níž jsem čerpala, v práci cituji a je uvedena v seznamu použité literatury. V Olomouci dne ……………………. ……………………….. Bc. Denisa Plucnarová Poděkování: Ráda bych poděkovala vedoucímu mé diplomové práce RNDr. Ľuboši Majeskému, Ph.D. z Katedry botaniky Přf UP za nesmírně cenné rady, věnovaný čas, při zpracování této práce, kritické připomínky a velkou vstřícnost. Také bych mu ráda poděkovala za odborný dohled při zpracování výsledků. V neposlední řadě bych ráda poděkovala RNDr. Miloslavu Kitnerovi, Ph.D a prof. Ing. Aleši Lebedovi, DrSc. za poskytnutí vzorků genomické DNA, bez kterých by tato práce nemohla vzniknout. Bibliografická identifikace Jméno a příjmení autora: Bc. Denisa Plucnarová Název práce: Využití vybraných regionů jaderné a chloroplastové DNA ke studiu fylogeneze vybraných zástupců rodu Lactuca Typ práce: Diplomová práce Pracoviště: Katedra botaniky, PřF UP v Olomouci, Česká republika Vedoucí práce: Vedoucí práce: RNDr. Ľuboš Majeský, Ph.D. Konzultant: Konzultant: RNDr. Miloslav Kitner, Ph.D. Rok obhajoby práce: 2017 Abstrakt Cílem diplomové práce bylo zhodnotit použitelnost vybraných molekulárních markerů k rekonstrukci fylogeneze rodu Lactuca a mezidruhových vztahů mezi vybranými druhy rodu. Pro tento cíl byly zvoleny tři lokusy: jaderný úsek ITS a dva chloroplastové úseky trnL – trnF a ndhF. Vhodnost vybraných markerů k rekonstrukci fylogeneze na nízké taxonomické úrovni byla testována prostřednictvím dvou metod: maximální úspornosti a maximální věrohodnosti. Topologie získaných stromů byla velice podobná napříč hodnocenými markery a také použitými metodami. Na všech zkonstruovaných stromech se vyčlenilo několik monofyletických skupin: "Lactuca sensu lato", skupina asijských taxonů, "afro-americká" skupina, skupina taxonů perennis/undulata a sororia/triangulata. Skupina "Lactuca sensu lato" byla tvořena dvěma příbuznými, dobře odlišenými skupinami "Lactuca sensu stricto" a "Scariola". Vnitřní mezidruhové vztahy v rámci těchto hlavních skupin zůstaly částečně nevyřešené. Nevyřešené zůstaly také fylogenetické vztahy mezi hlavními skupinami. Z práce lze zhodnotit, že použité lokusy jsou dostatečné k vyčlenění hlavních evolučních linií v rámci subtribu Lactucinae, avšak nejsou dostatečné na to, aby dokázaly rozřešit vnitřní vztahy mezi jednotlivými taxony a mezi jednotlivými liniemi. Klíčová slova: DNA, fylogenetické stromy, Lactuca, maximální věrohodnosti, maximální úspornosti, PCR, primer Počet stran/počet příloh: 46/6 Jazyk: český Bibliographical identification: Author’s first name and surname: Bc. Denisa Plucnarová Title: Utilization of selected nuclear and chloroplast DNA markers in the study of phylogeny of selected taxa from the genus Lactuca Type of thesis: Master thesis Department: Department of Botany, Faculty of Science, Palacký University in Olomouc, Supervisor: RNDr. Ľuboš Majeský, Ph.D. Consultant: RNDr. Miloslav Kitner, Ph.D. The year of defense: 2017 Abstract The aim of this master thesis was to evaluate the suitability of selected molecular markers for reconstruction of the phylogeny of the genus Lactuca and to resolve interspecific relationships among selected species of the genus. For this purpose three loci were selected: nuclear locus ITS, and two chloroplast loci trnL – trnF and ndhF. Suitability of the selected markers for reconstruction of phylogeny on the low taxonomic level was tested using two main methods, Maximum parsimony, and Maximum Likelihood. The topology of inferred trees was very similar across all of the tested loci and methods. On all of the inferred trees, several monophyletic groups were consistently present. Namely, the group "Lactuca sensu lato"; group of "Afro- American" taxa, group of "Asian" taxa, perennis/undulata clade, and sororia/triangulata clade. The "Lactuca sensu lato" group consist of two related and clearly separated subgroups, "Lactuca sensu stricto" and "Scariola". Within the inferred groups, the interspecific relationships remained partially unresolved as well as the relationships between the inferred main groups. The results showed that used molecular markers are sufficient for recognition of wider groups/lineages within the subtribe Lactucinae, however, are not sufficient enough for resolving internal relationships within the wider groups and between the main lineages. Keywords: DNA, Lactuca, Maximum Likelihood, Maximum Parsimony, PCR, phylogenetic tree, primer, Number of pages/appendices: 46/6 Language: Czech OBSAH 1 ÚVOD ....................................................................................................................... 8 2 LITERÁRNÍ PŘEHLED ...................................................................................... 11 2.1 Charakteristika a morfologický popis rodu locika (Lactuca) ........................... 11 2.1.1 Geografické rozšíření rodu ........................................................................ 11 2.1.2 Taxonomie rodu ........................................................................................ 12 2.2 Molekulární metody používány ve studii fylogeneze rodu Lactuca ................ 15 3 MATERIÁL A METODY .................................................................................... 20 3.1 Rostlinný materiál ............................................................................................ 20 3.2 Izolace DNA ..................................................................................................... 22 3.2.1 Měření koncentrace a stanovení míry integrity vyizolované DNA .......... 22 3.3 Amplifikace vybraných oblastí cp a nrDNA pomocí PCR .............................. 22 3.4 Pročištění vzorků před sekvenací ..................................................................... 24 3.5 Zpracování sekvencí a fylogenetické analýzy .................................................. 25 4 VÝSLEDKY .......................................................................................................... 27 5 DISKUZE ............................................................................................................... 34 6 ZÁVĚR ................................................................................................................... 38 SEZNAM POUŽITÉ LITERATURY ......................................................................... 39 SEZNAM POUŽITÝCH ZKRATEK ......................................................................... 45 7 PŘÍLOHY .............................................................................................................. 46 SEZNAM TABULEK Tabulka 1 Seznam použitých rostlin, jejich původ a počet jedinců z každého druhu. .. 20 Tabulka 2 Složení reakční směsi pro PCR reakce ......................................................... 23 Tabulka 3 Program pro PCR reakci pro testované lokusy ............................................. 23 Tabulka 4 Testované DNA markery .............................................................................. 25 Tabulka 5 Vybrané modely u metody maximální věrohodnosti, použité v setu A a v setu B u jednotlivých lokusů k rekonstrukci fylogeneze ................................................ 26 Tabulka 6 Charakteristika parsimonických analýz a použitých aligmentů ................... 27 SEZNAM OBRÁZKŮ Obrázek 1 Fylogenetické stromy vytvořené z jaderného úseku ITS ............................. 30 Obrázek 2 Fylogenetické stromy vytvořené z chloroplastového úseku ndhF.. ............. 31 Obrázek 3 Fylogenetické stromy vytvořené z chloroplastového úseku trnL – trnF.. .... 32 Obrázek 4 Fylogenetické stromy vytvořené ze spojených chloroplastových úseků trnL – trnF a ndhF.. ......................................................................................................... 33 SEZNAM PŘÍLOH Tabulka 7 Seznam haplotypů pro lokus trnL – trnF ...................................................... 46 Tabulka 8 Seznam haplotypů pro lokus ndhF ............................................................... 47 Tabulka 9 Seznam ribotypů pro lokus ITS .................................................................... 48 Tabulka 10 Seznam vzorků stažených z databáze GenBank ......................................... 49 Seznam 1 Použité přístroje a laboratorní vybavení ........................................................ 51 Seznam 2 Použité chemikálie a roztoky ......................................................................... 52 Obrázek 5 Fylogenetický strom setu B vytvořený z chloroplastového úseku trnL – trnF metodou maximální věrohodnosti Obrázek 6 Fylogenetický strom setu B vytvořený z jaderného úseku ITS metodou maximální úspornosti 1 ÚVOD Rod Lactuca L. patří do jedné z největších a evolučně nejúspěšnějších rostlinných čeledí Asteraceae. Do daného rodu se řadí jeden kulturní druh L. sativa L., který má mnoho variet či kultivarů různých barev a tvarů listů, má také hospodářský význam a pěstuje se jako listová zelenina. Mnoho planě rostoucích druhů se vyskytuje po celém světě a to zejména v Evropě, Asii, Severní a Střední Americe a Africe a Australii (Lebeda et al., 2007). Biogeograficky
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