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Bakalářská Práce Univerzita Palackého v Olomouci Bakalářská práce Olomouc 2013 Michala Chaloupská Univerzita Palackého v Olomouci Přírodovědecká fakulta Katedra botaniky Morfologické a molekulární studium fylogeneze a genetické diverzity druhů Vicia tetrasperma, Lathyrus ochrus a Lathyrus clymenum, nejbližších příbuzných rodu Pisum Bakalářská práce Michala Chaloupská Studijní program: Biologie Studijní obor: Systematická biologie a ekologie Forma studia: Prezenční Olomouc 2013 Vedoucí práce: Ing. Petr Smýkal, Ph.D. Prohlášení Prohlašuji, ţe jsem bakalářskou práci vypracovala samostatně pod vedením Ing. Petra Smýkala, Ph.D. a jen s pouţitím citovaných literárních pramenů. V Olomouci 6.5. 2013 podpis Bibliografická identifikace Jméno a příjmení autora: Michala Chaloupská Název práce: Morfologické a molekulární studium fylogeneze a genetické diverzity druhů Vicia tetrasperma, Lathyrus ochrus a Lathyrus clymenum, nejbliţších příbuzných rodu Pisum Typ práce: Bakalářská práce Pracoviště: Katedra botaniky PřF UP Vedoucí práce: Ing. Petr Smýkal, Ph.D. Rok obhajoby práce: 2013 Abstrakt: Poznání fylogenetických vztahů v rámci čeledi bobovitých (Leguminosae), je velmi důleţité pro porozumění původu a rozrůznění této ekologicky a ekonomicky důleţité čeledi. Tato třetí nejrozsáhlejší čeleď vyšších rostlin, zahrnuje více neţ 850 rodů s přibliţně 20 tisíci druhy, včetně domestikovaných druhů. Bakalářská práce si klade za cíl, literární rešerši rozsáhlého dostupného materiálu a syntézu dostupných poznatků na téma vyuţití DNA a morfologických znaků pro účely taxonomických a fylogenetických studií rostlin čeledi Leguminosae se zaměřením na podčeleď Fabeae/Vicieae. Bude studována morfologická variabilita a genetická diverzita pomocí DNA sekvencí tří vybraných druhů, identifikovaných v předchozím studiu jako fylogeneticky nejbliţších příbuzných rodu Pisum. Klíčová slova: genetická diverzita, molekulární fylogeneze, morfologie, Vicia, Pisum, Lathyrus Počet stran: 53 Počet příloh: 4 Jazyk: Čeština Bibliographical identification Author´s first name and surname: Michala Chaloupská Title: Morphological and molecular analysis of phylogeny and genetic diversity of Vicia tetrasperma, Lathyrus ochrus and Lathyrus clymenum, the Pisum genus closest relatives. Type of thesis: Bachelor thesis Department: Department of Botany, Faculty of Science, Palacký University in Olomouc Supervisor: Ing. Petr Smýkal, Ph.D. The year of representation: 2013 Abstract: Knowledge of phylogenetic relationships within the legume family (Leguminosae) is very important for understanding the origin and diversification of this ecologically and economically important family. This is the third largest family of vascular plants, including more than 850 genera with about 20 thousand species, including domesticated species. Bachelor thesis aims, an extensive literature review and synthesis of available material available knowledge about the use of DNA and morphological characters for taxonomic and phylogenetic studies of plants of the family Leguminosae subfamily focusing on Fabeae / Vicieae. Morphological variability and genetic diversity was studied using DNA sequences of three selected species identified in previous studies as phylogenetically closest relatives of the genus Pisum. Keywords: genetic diversity, molecular phylogeny, morphology, Vicia, Pisum, Lathyrus Number of pages: 53 Number of appendices: 4 Language: Czech Obsah: 1 ÚVOD ............................................................................................................................... 1 1.1 Čeleď Leguminosae ................................................................................................... 1 1.2 Podčeleď Fabeae ....................................................................................................... 3 1.3 Molekulární markery ................................................................................................. 5 1.4 Morfologické znaky................................................................................................... 7 1.5 Popis jednolivých rodů podčeledi Fabeae ................................................................ 8 1.5.1 Rod Vicia (vikev) ............................................................................................... 8 1.5.2 Rod Lathyrus (hrachor) ...................................................................................... 9 1.5.3 Rod Pisum (hrách) ........................................................................................... 11 1.5.4 Rod Vavilovia / druh Vavilovia formosa .......................................................... 13 1.6 Popis druhů pouţitých ve vlastní práci .................................................................... 13 1.6.1 Vicia tetrasperma (L). Schreber ....................................................................... 13 1.6.2 Lathyrus ochrus (L). DC .................................................................................. 14 1.6.3 Lathyrus clymenum L. ...................................................................................... 14 2 METODIKA ................................................................................................................... 16 2.1 Rostlinný material ................................................................................................... 16 2.1.1 Podmínky pěstování ......................................................................................... 18 2.1.2 Herbarizace ...................................................................................................... 18 2.2 Skenování, stereomikroskopie- fotografická dokumentace .................................... 18 2.3 Morfometrické hodnocení ....................................................................................... 18 2.4 Analýza DNA .......................................................................................................... 20 2.4.1 Isolace DNA ..................................................................................................... 20 2.4.2 PCR amplifikace .............................................................................................. 21 2.4.3 Sekvenační a biostatické analýzy ..................................................................... 25 3 VÝSLEDKY: .................................................................................................................. 26 3.1 Výsledky z molekulární části práce ......................................................................... 27 3.2 Vyhodnocení morfometrické části. ......................................................................... 34 4 DISKUSE: ...................................................................................................................... 37 5 ZÁVĚR: .......................................................................................................................... 42 6 PŘÍLOHY: ...................................................................................................................... 43 7 CITACE: ......................................................................................................................... 47 vi Poděkování: Největší díky patří mému školiteli Ing. Petru Smýkalovi, Ph.D., který mi vţdy ochotně pomáhal a zasvěcoval do problematiky související s touto prací. Naučil mě orientaci v laboratoři a práci potřebné k vykonávání experimentů. Během naší spolupráce jsme sice narazili na nějaké překáţky, ale vše jsme vţdy vyřešili - dá se říci s úsměvem. Dále bych chtěla poděkovat všem lidem se kterými jsem se během mé práce setkávala v laboratoři. Přestoţe jsme se právě v laboratoři viděli poprvé, kdykoliv jsem něco potřebovala a Dr. Smýkal nebyl na dohled, rádi mi pomohli. Za měření morfometrických částí mé práce vděčím paní Ing. Ivě Smýkalové, Ph.D., které bych alespoň touto cestou chtěla poděkovat. Jedno obrovské díky patří také rodině a přátelům, kteří tolerovali mé výkyvy nálad během psaní. Děkuji Vám. vii 1 ÚVOD 1.1 Čeleď Leguminosae Taxonomie této čeledi je dosud ne zcela jednotná a existující tři alternativní moţnosti zařazení: 1) Leguminosae je chápána jako čeleď a Mimosoideae a Caesalpinioideae jako podčeledi, nebo 2) Leguminosae = Fabaceae, zahrnující tři podčeledi: Faboideae (= Papilionoidae), Mimosoideae a Caesalpinioieae a nebo 3) Fabaceae (= Papilionaceae) je chápána jako čeleď stejně jako Mimosaceae a Caesalpiniaceae (Lewis et al. 2005). Čeleď Leguminosae zahrnuje byliny a dřeviny, s výjimkou rostlin adaptovaných na vodní prostředí. Tato čeleď má hned několik zajímavostí. Rostliny patřící do Leguminosae (Fabaceae) rostou na půdách ochuzených o dusík, proto na svých kořenech mají speciální útvary - hlízky, které jim vyrůstají po infekci nitrogenními bakteriemi rodu Rhizobium. Jedná se o symbiózu, kde bakterie metabolizují atmosférický dusík na amonný iont, který vyuţívá hostitelská rostlina, za úplatek v podobě jednoduchých cukrů, které si naopak bakterie berou od rostliny. Rostliny čeledi Leguminosae mají listy střídavé, nejčastěji sloţené, jednoduše zpeřené nebo dlanitě sloţené, vzácněji jednoduché, palistnaté, výjimečně palisty chybějí. Květenství je hroznovité (rod Vicia), méně často jsou květy jednotlivě (rody Lathyrus, vzácněji Vicia nebo Pisum). Květy jsou oboupohlavné, souměrné, kalich srůstá z pěti lístků, korunu tvoří pět lístků volných rozlišených na pavézu, křídla a člunek, kde pavéza je v rovině souměrnosti. V pupenu pavéza obaluje ostatní části květu (sestupná estivace). Dva sousedící lístky - křídla, a člunek srůstající ze dvou zbylých lístků. Tyčinek
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