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Tc Balikesir Üniversitesi Fen Bilimleri T.C. BALIKESİR ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ BİYOLOJİ ANABİLİM DALI TÜRKİYE’DE YETİŞEN INULA L. (ASTERACEAE) TÜRLERİNİN MOLEKÜLER SİSTEMATİK ANALİZİ VE EKOLOJİSİ DOKTORA TEZİ EMRE SEVİNDİK BALIKESİR, MAYIS - 2014 T.C. BALIKESİR ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ BİYOLOJİ ANABİLİM DALI TÜRKİYE’DE YETİŞEN INULA L. (ASTERACEAE) TÜRLERİNİN MOLEKÜLER SİSTEMATİK ANALİZİ VE EKOLOJİSİ DOKTORA TEZİ EMRE SEVİNDİK BALIKESİR, MAYIS - 2014 KABUL VE ONAY SAYFASI EMRE SEVİNDİK tarafından hazırlanan “TÜRKİYE’DE YETİŞEN INULA L. (ASTERACEAE) TÜRLERİNİN MOLEKÜLER SİSTEMATİK ANALİZİ VE EKOLOJİSİ” adlı tez çalışmasının savunma sınavı 02.05.2014 tarihinde yapılmış olup aşağıda verilen jüri tarafından oy birliği / oy çokluğu ile Balıkesir Üniversitesi Fen Bilimleri Enstitüsü Biyoloji Anabilim Dalı Doktora Tezi olarak kabul edilmiştir. Jüri Üyeleri İmza Danışman Yard.Doç.Dr. Fatih COŞKUN Üye Prof.Dr. Gülendam TÜMEN ............ Üye Prof.Dr. Ali ÇELİK ................ Üye Prof.Dr. Ahmet DURAN ................ Üye Doç.Dr. Ekrem DÜNDAR .................. Jüri üyeleri tarafından kabul edilmiş olan bu tez BAÜ Fen Bilimleri Enstitüsü Yönetim Kurulunca onanmıştır. Fen Bilimleri Enstitüsü Müdürü Prof. Dr. Cihan ÖZGÜR ............................................. Bu tez çalışması Balıkesir Üniversitesi Bilimsel Araştırma Projeler Birimi tarafından BAP 2013/92 nolu proje ile desteklenmiştir. ÖZET TÜRKİYE’DE YETİŞEN INULA L. (ASTERACEAE) TÜRLERİNİN MOLEKÜLER SİSTEMATİK ANALİZİ VE EKOLOJİSİ DOKTORA TEZİ EMRE SEVİNDİK BALIKESİR ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ BİYOLOJİ ANABİLİM DALI (TEZ DANIŞMANI: YARD. DOÇ. DR. FATİH COŞKUN) BALIKESİR, MAYIS - 2014 Asteraceae bir bitki grubu olarak, doğal kimyası, çiçeklenme morfolojisi ve habitata adaptasyonu ile kozmopolit bir familyadır. Çalışma materyali olan Inula, Asteraceae familyasının Inuleae tribusune aittir. Bu çalışma, Türkiye’de yayılış gösteren Inula L. cinsine ait türlerin moleküler sistematik analizini ve ekolojisini içermektedir. Bu çalışmada fenol-kloroform-izoamil alkol ve ticari kit (Sigma) metodu kullanılarak Türkiyede’ki Inula taksonları iç grup, Carlina lanata ve Carlina vulgaris taksonları dış grup seçilerek DNA izolasyonu yapıldı. Literatürde sıklıkla kullanılan ve güvenirliliği kanıtlanmış ITS (Internal Transcribed Spacer) nükleer ribozomal DNA (nrDNA) bölgesinin dizileri, trnL-F ve ndhF (cpDNA) dizileri moleküler işaretleyici olarak kullanılması ile Inula taksonlarının moleküler sistematik analizi yapıldı. Filogenetik analiz için edilen dizilerin işlenmesi için Bioedit, FinchTV ve Sequencher 4,10,1 programları, dizilerin hizalanması için ClustalW programı ve filogenetik analiz için PAUP 4,0b10 programı kullanılmıştır. Karakter temelli yöntemlerden Maksimum Parsimoni kriteri kullanılarak Heuristic Search ağaçları oluşturulmuştur. Yine parsimoni kriteri kullanılarak Boostrap analizi yapılmıştır. Mesafe temelli yöntemlerden ise UPGMA (Unweighted Pair-Group Metod of Arithmetic Avarage) ve NJ (Neighbour Joining) analizi yapılmıştır. Sonuç olarak daha önce Inula üzerine yapılan filogenetik çalışmalar bizim çalışmalarımızı desteklemiştir. Ekolojik çalışmalarda ise, değişik lokalitelerden alınan toprak örneklerinin pH’ına, tekstür, organik madde, E.C (tuz), CaCO3, P, K, Zn, Fe, Cu ve Mn değerlerine bakılmıştır. Taksonların çoğu, nötr, tuzsuz, killi, killi-tınlı, potasyumca eksik ve iz elementlerce yeterli toprakları tercih etmektedirler. ANAHTAR KELİMELER: Inula, ITS/ndhF/trnL-F, Sistematik, Ekoloji, Türkiye i ABSTRACT MOLECULAR SYSTEMATIC ANALYSIS AND ECOLOGY INULA L. (ASTERACEAE) TAXA DISTRIBUTED IN TURKEY PH.D THESIS EMRE SEVİNDİK BALIKESIR UNIVERSITY INSTITUTE OF SCIENCE BIOLOGY (SUPERVISOR: ASSIST. PROF.DR. FATİH COŞKUN ) BALIKESİR, MAY 2014 Asteraceae, as a plant group, is a cosmopolitan family in terms of natural chemistry, flowering morphlogy and adaptation to habitat. Study material under consideration Inula belongs to the tribe Inuleae of the family Compositae. In this study, molecular systematic analysis and ecology features of genus Inula L. was investigated in Turkey. DNA Isolation was performed using phenol- chloroform- isoamylalcohol and commercial cit (Sigma) slecting Inula as ingroup taxa Carlina lanata and Carlina vulgaris as outgroup taxa ITS (Internal Transcribed Region) of the nuclear ribosomal DNA (nrDNA), trnL-F region and ndhF (cpDNA) DNA sequences which were used as molecular markers, taxa belonging to the genus Inula distributed in Turkey were analyzed molecular phylogenetically. Before the phylogenetic analysis, DNA sequences were aligmend using Bioedit, FinchTV and sequences 4.10.1 the sequences were aligment using the ClustalW and the phylogenetic analysis was performed using the PAUP 4.0b10 sofware. The character based method parsimony was used as the criterion with a heuristic search option and the Maximum Parsimony criterion was based on the character and the public by using Heuristic Search trees was created. Again using the Bootstrap analysis have been made to the criterion of parsimony. Distance-based methods, the UPGMA (Unweighted Pair Group Method of Arithmetic Average) and NJ (Neighbour Joining) analyses were made. Prior phylogenetic studies on Inula are in parallel with the results of our research. In ecological studies, pH, texture, organic matter, E.C. (salt ratio), CaCO3, P, K, Zn, Fe, Cu and Mn values of soil samples taken from various places were examined. Most of the taxa prefer neutral, non-saline, clayey, clay-loamy, low in potassium and sufficient in trace elements soils. KEYWORDS: Inula, ITS/ndhF/trnL-F, Systematic, Ecology, Turkey. ii İÇİNDEKİLER Sayfa ÖZET ................................................................................................................... i ABSTRACT ....................................................................................................... ii İÇİNDEKİLER ................................................................................................ iii ŞEKİL LİSTESİ ............................................................................................... vi TABLO LİSTESİ ............................................................................................. ix KISALTMALAR .............................................................................................. x ÖNSÖZ .............................................................................................................. xi 1. GİRİŞ ............................................................................................................. 1 1.1 Asteraceae (Compositae) Familyası ile İlgili Genel Bilgiler ............... 2 1.1.1 Inula L. (Anduzotu) Cinsi ile İlgili Genel Bilgiler ....................... 4 1.1.1.1 Inula Örneklerinin Toplandığı ve Yayılış Gösterdiği Lokaliteler 5 1.1.2 Inula Türlerinin Kullanım Alanları .............................................. 20 1.2 Moleküler Markırlar ........................................................................... 21 1.2.1 Morfolojik Markırları .................................................................. 22 1.2.2 Protein Markırları ........................................................................ 22 1.2.3 DNA Markırları ........................................................................... 23 1.2.3.1 Hibridizasyona Bağlı Markırlar .............................................. 24 1.2.3.2 PCR Temelli Markırlar ........................................................... 25 1.2.4 ITS (İç Transkribe Olan Bölgeler) ............................................... 34 1.2.4.1 ITS Bölgesinin Genel Özellikleri ............................................ 35 1.2.4.2 rDNA ve ITS Bölgeleri Arasındaki İlişki ............................... 36 1.2.4.3 ITS Bölgesinin Filogenetikte Kullanımı ................................. 37 1.2.5 trnL-F Bölgesi .............................................................................. 38 1.2.6 ndhF Geni .................................................................................... 39 1.2.7 Moleküler Filogenide Kullanılan DNA Çeşitleri ......................... 40 1.2.7.1 Mitokondriyal DNA ................................................................ 40 1.2.7.2 Kloroplast DNA'sı ................................................................... 42 1.2.7.3 Çekirdek DNA'sı ..................................................................... 44 1.2.8 DNA Dizileme ............................................................................. 45 1.2.8.1 Maxam ve Gilbert’in Kimyasal KırılmaYöntemi ................... 45 1.2.8.2 Sanger Dizileme (Cycle Sequencing) Yöntemi ...................... 48 1.2.9 Filogenetik Analiz ........................................................................ 49 1.2.10 Filogenetik Ağaç .......................................................................... 50 1.2.11 Filogenetik Ağaç Oluşturmada Kullanılan Yöntemler ................ 52 1.2.11.1 Karakter Temelli Yöntemler ................................................... 52 1.2.11.2 Mesafe Temelli Yöntemler ..................................................... 54 1.2.12 Filogenetik Ağaç Programları ...................................................... 55 2. MATERYAL VE METOD ........................................................................ 56 2.1 Arazi Çalışması .................................................................................. 56 2.2 Moleküler Sistematik Çalışmalar ....................................................... 56 iii 2.2.1 Kullanılan Malzemelerin Hazırlaması ......................................... 56
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