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(APIACEAE) CİNSİNİN MOLEKÜLER FİLOGENİSİ Büşra TO T.C. SELÇUK ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ SESELI L. (APIACEAE) CİNSİNİN MOLEKÜLER FİLOGENİSİ Büşra TOSUN YÜKSEK LİSANS TEZİ Biyoloji Anabilim Dalını Ocak-2015 KONYA Her Hakkı Saklıdır TEZ KABUL VE ONAYI Büşra TOSUN tarafından hazırlanan “Seseli L. (Apiaceae) Cinsinin Moleküler Filogenisi” adlı tez çalışması 05/02/2015 tarihinde aşağıdaki jüri tarafından oy birliği / oy çokluğu ile Selçuk Üniversitesi Fen Bilimleri Enstitüsü Biyoloji Anabilim Dalı’nda YÜKSEK LİSANS TEZİ olarak kabul edilmiştir. Jüri Üyeleri İmza Başkan Prof. Dr. Ahmet DURAN ………………….. Danışman Prof. Dr. Ahmet DURAN ………………….. Üye Doç. Dr. Gökhan KARS ………………….. Üye Yrd. Doç. Dr. Ebru DOĞAN GÜNER ………………….. Yukarıdaki sonucu onaylarım. Prof. Dr. Aşır GENÇ FBE Müdürü Bu tez çalışması BAP tarafından 14201009 nolu proje ile desteklenmiştir. TEZ BİLDİRİMİ Bu tezdeki bütün bilgilerin etik davranış ve akademik kurallar çerçevesinde elde edildiğini ve tez yazım kurallarına uygun olarak hazırlanan bu çalışmada bana ait olmayan her türlü ifade ve bilginin kaynağına eksiksiz atıf yapıldığını bildiririm. DECLARATION PAGE I hereby declare that all information in this document has been obtained and presented in accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all material and results that are not original to this work. Büşra TOSUN Tarih: 20.01.2015 ÖZET YÜKSEK LİSANS TEZİ SESELI L. (APIACEAE) CİNSİNİN MOLEKÜLER FİLOGENİSİ Büşra TOSUN Selçuk Üniversitesi Fen Bilimleri Enstitüsü Biyoloji Anabilim Dalı Danışman: Prof. Dr. Ahmet DURAN 2015, 86 Jüri Prof. Dr. Ahmet DURAN Doç. Dr. Gökhan KARS Yrd. Doç. Dr. Ebru DOĞAN GÜNER Bu çalışmada Türkiye’nin farklı illerinden toplanan Seseli L. (Apiaceae) cinsine ait 15 taksonun Basit İç Dizi Tekrar dizileri (ISSR) ve İç transkribe olan boşluk (ITS) bölgelerinin moleküler sistematik analizi yapıldı. ISSR ve ITS profillerinin belirlenmesi için her taksonun yaprak ve meyve örneklerinden DNA izolasyonu yapıldı. Taksonlara ait DNA’lar önce ISSR metodu için polimeraz zincir reaksiyonu (PCR) yapıldı ve daha sonra ITS bölgeleri için uygun ITS4 ve ITS5A primerleri kullanılarak PCR ile çoğaltıldı. ISSR yöntemi için elde edilen bantlar skorlandı ve NTSYS-pc paket programı ile filogenetik analiz yapıldı. ITS bölgelerinin baz dizileri elde edilip işlenerek MEGA programı ile filogenetik analizi yapıldı. Değerlendirme sonucu çalışılan türler arasındaki akrabalık ilişkilerini ortaya koyan Neighbor Joining (NJ), Unweighted Pair Group Method Using Arithmetic Average (UPGMA), Maksimum Parsimoni (MP), Maksimum Likelihood (ML) ağaçları elde edildi. ISSR ve ITS çalışmalarından sağlanan verilerin istatistiksel analizi sonucunda Türkiye Seseli taksonları arasındaki filogenetik ilişkiler açıklanmaya çalışıldı. ISSR ve ITS verilerine dayalı oluşturulan farklı dendogramların birbirini desteklediği görüldü. ISSR ve ITS verileri kullanılarak oluşturulan dendogramda Seseli taksonları üç klad altında toplanmıştır. Dış grup taksonları ise ayrı bir kladda yer almıştır. Anahtar Kelimeler: Apiaceae, ITS, ISSR, PCR, Seseli, DNA Dizi Analizi, Umbelliferae iv ABSTRACT MS THESIS Molecular phylogeny of the genus Seseli (Apiaceae) Büşra TOSUN THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCE OF SELCUK UNIVERSITY THE DEGREE OF MASTER OF SCIENCE IN BIOLOGY Advisor: Prof. Dr. Ahmet DURAN 2015, 86 Jury Prof. Dr. Ahmet DURAN Assoc. Prof. Dr. Gökhan KARS Asst. Prof. Dr. Ebru DOĞAN GÜNER In this present study, the regions of Inter Simple Sequence Repeats (ISSR) and nuclear DNA (nrDNA) Internal Transcribed Spacer (ITS) of 15 taxa of Seseli L. (Apiaceae) genus collected from different localities in Turkey, were made molecular systematic analysis. DNA was extracted from leaves and fruits for ISSR and ITS profiles. Firstly, polymerase chain reaction (PCR) were applied all taxa DNA’s for the ISSR methods. For the ITS regions, DNA’s were reproduced by PCR with using ITS4 and ITS5A primers. Bands were scored for the ISSR method, and NTSYS software programme were used for the phylogenetic analysis. Results exhibited that there is a relationships between studied species with respect to obtained Neighbor Joining (NJ), Unweighted Pair Group Method Using Arithmetic Average (UPGMA), Maksimum Parsimoni (MP), Maksimum Likelihood (ML) trees. After the statistical data analysis of ISSR and ITS, phylogenetic relationships were explanied among Turkish Seseli taxa. Dendograms constructed ISSR and ITS parameters supported each other. Seseli taxa cluster three clads in dendogram which was constituted with using ISSR and ITS parameters. Outgroup taxa is placed in the another clad. Key words: Apiaceae, ITS, ISSR, PCR, Seseli, DNA Sequens Analysis, Umbelliferae v ÖNSÖZ Yüksek Lisans çalışmamın başından sonuna kadar her zaman destek veren ve yardımını esirgemeyen saygıdeğer danışmanım Prof. Dr. Ahmet DURAN’a teşekkür ederim. Moleküler çalışmalarda önerilerinden faydalandığım ve Ziraat Fakültesi Biyoteknoloji Laboratuvarında çalışmama imkân sağlayan Doç. Dr. Erdoğan E. HAKKI’ya, tez çalışmam boyunca bana yardımcı olan, bilgi ve görüşlerinden faydalandığım Yrd. Doç. Dr. Meryem ŞEKER ve Araş. Gör. Özlem ÇETİN, Uzm. Mustafa Çelik ve arkadaşlarım Şerife ATİKER ve Yasemin GÜRBÜZ’e, tez çalışması kapsamında kullanmış olduğum materyalleri sağlamasından dolayı Gazi Üniversitesi öğretim üyesi Yrd. Doç. Dr. Ebru DOĞAN GÜNER’e teşekkür ederim. 14201009 numaralı projeyle, gerekli teçhizatı, kimyasalları ve malzemeleri temin etmemizi sağlayan BAP’a teşekkürlerimi sunarım. Çalışma konusu olan bitki örneklerini inceleme imkânı sağlayan GAZI, KNYA, HUB herbaryumlarının yetkililerine teşekkür ederim. Tez çalışmam boyunca benden maddi manevi desteklerini esirgemeyen aileme ayrıca teşekkür ederim. Büşra TOSUN KONYA-2015 vi İÇİNDEKİLER ÖZET ......................................................................................................................... iv ABSTRACT .................................................................................................................v ÖNSÖZ ...................................................................................................................... vi İÇİNDEKİLER ........................................................................................................ vii ÇİZELGELERİN LİSTESİ ...................................................................................... ix ŞEKİLLERİN LİSTESİ ............................................................................................ ix SİMGELER VE KISALTMALAR ........................................................................... xi 1. GİRİŞ .......................................................................................................................1 2. KAYNAK ARAŞTIRMASI ....................................................................................6 2.1. Apiaceae familayasını Morfolojik Özellikleri ......................................................7 2.2. Seseli Cinsi Üzerine Yapılan Moleküler, Morfolojik, Anatomik, Biyokimyasal ve Karyolojik Çalışmalar................................................................................................8 2.2.1. Seseli Cinsinin Taksonomik Gelişimi ......................................................... 10 2.2.2. Selineae Tribusunun Özellikleri ................................................................. 15 2.2.3. Selineae Tribusunun Moleküler Çalışmalara Göre Taksonomik Yeri .......... 17 3. Moleküler Markerlar ve Önemi ............................................................................ 25 3.1. Moleküler Markırların Özellikleri ..................................................................... 26 3.1.1. Moleküler Sistematikte Kullanılan Yöntemlerden Bazıları ......................... 26 3.2. ISSR (Inter Simple Sequence Repeats) Basit İç Dizi Tekrarları ......................... 26 3.3. ITS (Internal Transcribed Spacer) ve rDNA ...................................................... 27 3.2.1. ITS’nın Genel Özellikleri ........................................................................... 28 3.2.2. ITS Bölgesinin Filogenetik Çalışmalarda Kullanılışı .................................. 28 3.2.3. Filogenetik Analiz ve Ağaç Oluşturma ....................................................... 29 4. MATERYAL VE YÖNTEM ................................................................................. 32 4.1. Materyal ........................................................................................................... 32 4.1.1. Çalışmada Kullanılan Bitki Örnekleri ......................................................... 32 4.1.2. Kullanılan Cam Malzeme Ve Plastik Malzemelerin Sterilizasyonu............. 33 4.1.3. Moleküler Çalışmalarda Kullanılan Kimyasal Maddeler............................. 33 4.1.4. Moleküler Çalışmalarda Kullanılan Tampon ve Çözeltiler ......................... 33 4.1.5. Genomik DNA İzolasyonunun Yapılması .................................................. 33 4.1.6. Seseli Cinsine Ait Örneklerin ISSR Primerleriyle PCR Amplifikasyonları . 36 4.1.7. Seseli Cinsine Ait Örneklerin ITS Primerleriyle PCR Amplifikasyonları .... 37 4.1.8. Agaroz Jel Elektroforezi ve Görüntüleme ................................................... 37 4.1.9. Dizileme ve Dizi Analizi ............................................................................ 38 4.1.10. Filogenetik Analiz .................................................................................... 38 vii 5. ARAŞTIRMA SONUÇLARI VE TARTIŞMA ...................................................
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