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T.C. Ercđyes Ü Đversđtesđ Fe Bđlđmlerđ E Stđtüsü

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T.C. Ercđyes Ü Đversđtesđ Fe Bđlđmlerđ E Stđtüsü T.C. ERCĐYES ÜĐVERSĐTESĐ FE BĐLĐMLERĐ ESTĐTÜSÜ TÜRKĐYE ECHIOPS L. CĐSĐ RITRODES BUGE SEKSĐYOU TAKSOLARII POGP (PEROKSĐDAZ GE POLĐMORFĐZM) YÖTEMĐ KULLAILARAK ARAŞTIRILMASI Tezi Hazırlayan Serkan KAYA Tezi Yöneten Doç. Dr. Cem VURAL Biyoloji Anabilim Dalı Yüksek Lisans Tezi Ocak 2011 KAYSERĐ T.C. ERCĐYES ÜĐVERSĐTESĐ FE BĐLĐMLERĐ ESTĐTÜSÜ TÜRKĐYE ECHIOPS L. CĐSĐ RITRODES BUGE SEKSĐYOU TAKSOLARII POGP (PEROKSĐDAZ GE POLĐMORFĐZM) YÖTEMĐ KULLAILARAK ARAŞTIRILMASI Tezi Hazırlayan Serkan KAYA Tezi Yöneten Doç. Dr. Cem VURAL Biyoloji Anabilim Dalı Yüksek Lisans Tezi Bu çalışma Erciyes Üniversitesi Bilimsel Araştırma Projeleri Birimi tarafından FBY-08-565 kodlu proje ile desteklenmiştir. Ocak 2011 KAYSERĐ ii TEŞEKKÜR Çalışmalarım boyunca bana göstermiş olduğu yardım ve yönlendirmelerden dolayı değerli hocam sayın Doç. Dr. Cem VURAL’a teşekkürü bir borç bilirim. Laboratuar imkanlarını sağlayan ve malzeme temininde büyük emeği olan değerli hocam sayın Yrd. Doç. Dr. Servet ÖZCAN’a teşekkürlerimi sunarım. Değerli katkıları ve yardımlarını benden esirgemeyen sayın hocam Doç. Dr. Osman GÜLŞEN’e sonsuz teşekkür ediyorum. Laboratuar çalışmalarım boyunca desteklerini hep hissettiğim değerli arkadaşlarım Sezin DEMĐRTAŞ, Güler TOPRAK ve Osman ĐBĐŞ’e yardım ve desteklerinden dolayı, laboratuvar ortamını uyum içinde paylaştığım çalışma arkadaşlarım Şeyda ERDOĞAN, Dilek CEYLAN, Şengül HASKILIÇ, Leyla KIRIM ve Esma ÖZHÜNER’e, dostluğuyla bana güç veren Ersin KARABULUT, Muhammer KARABACAK ve Erman AŞIK’a teşekkürlerimi sunarım. Hayatımın her aşmasında olduğu gibi yüksek lisans döneminde maddi ve manevi desteklerini esirgemeyen aileme sonsuz teşekkürlerimi sunarım. iii TÜRKĐYE ECHIOPS L. CĐSĐ RITRODES BUGE SEKSĐYOU TAKSOLARII POGP (PEROKSĐDAZ GE POLĐMORFĐZM) YÖTEMĐ KULLAILARAK ARAŞTIRILMASI Serkan KAYA Erciyes Üniversitesi Fen Bilimleri Enstitüsü Yüksek Lisans Tezi, Ocak 2011 Tez Danışmanı: Doç. Dr. Cem VURAL ÖZET Türkiye’de doğal yayılış gösteren Echinops (Asteracea) cinsi Ritrodes seksiyonu; Echinops spinosissimus ve Echinops orientalis türleri ile temsil edilmektedir. Bu çalışmada bu iki tür ve tür altı taksonları moleküler tekniklerlerden “Peroksidaz Gen Polimorfizm (POGP) marker tekniği” kullanılarak incelenmiştir. Böylece Ritrodes seksiyona ait taksonlar arasındaki akrabalık ilişkileri araştırılmış ve bu türlerin evrimleriyle ilgili ipuçları elde edilmiştir. Çalışmada Ritrodes seksiyonuna ait 2 tür ve 2 alt tür 42 birey ve 4 dış grup kullanılmıştır. Analizler için 14 POGP primer kombinasyonu kullanılarak 104 polimorfik bant elde edilmiştir. En fazla sayıda bant (15 bant) Pox1F/Pox1R primer kombinasyonlarından elde edilirken, en az sayıda bant (3 bant) Pox11F/Pox11R primer kombinasyonundan elde edilmiştir. Bireyler arasındaki genetik mesafe 0.66 ile 0.96 arasında bulunmuştur. Bu sonuçlar Echinops türlerinin karşılaştırılmasında POGP markırlarının kullanılabileceğini göstermiştir. Anahtar Kelimeler: Asteraceae , Echinops, Ritrodes, POGP, Türkiye. iv IVESTIGATIO OF TAXA OF RITRODES BUGE SECTIO I GEUS ECHIOPS L. I TURKEY BY USIG POGP (PEROXIDASE GEE POLYMOPHISM) Serkan KAYA Erciyes University, Graduate School of atural and Applied Sciences M. Sc. Thesis, January 2011 Thesis Supervisors: Assoc. Prof. Dr. Cem VURAL ABSTRACT Ritrodes section in Genus Echinops (Asteracea) in Turkey is represented by Echinops spinosissimus and Echinops orientalis . The aim of this study to detect the similarity and variety of Ritrodes section of Echinops genus which spread naturally in Turkey by practice on In this study, POGP (Peroxidase gene polymorphism) were studied to determine diversity and relationships among 2 species and 2 subspecis of Ritrodes . Fourty two individuals of Ritrodes and 4 outgroup were used in the current study. The 104 polymorphic bands were produced by using selected 14 combinations of forward and reverse POGP primers. The highest number of bands (15) with primer combinations Pox1F/Pox1R and the lowest number of bands (3) with primer combinations Pox11F/Pox11r were obtained. Genetic distances among individuals were between 0.66 and 0.96. This results indicate that the POGP markers used were highly informative and were discussed in terms of their implications for Echinops species. Keywords: Asteraceae, Echinops , Ritrodes , POGP, Turkey. v KISALTMA VE SĐMGELER AFLP : Amplified Fragment Length Polymorphism (Çoğaltılmış Fragment Uzunluk Polimorfizmi) cpDNA : Kloroplast DNA CTAB : Setil 3-Metil Amonyum Bromid DNA : Deoksiribonükleik asit dH 2O : Distile su dNTP : Deoksiribonükleik asit trifosfat EDTA : Etilen diamin tetra asetikasit gDNA : Genomik DNA HCI : Hidrojen klorür ISSR : Internal Simple Sequence Repeats (Basit Dizi Tekrarları Arası) M : Molar MgCI 2 : Magnezyum klorür mM : Milimolar NaCI : Sodyum klorür PAGE : Poli Akrilamid Jel Elektrforezi (Polyacrylamide Gel Electrophoresis) PCA : Principal Components Analysis (Temel bileşen analizi) PIC : Polymorphism information content (Polimorfizm bilgi içeriği) PZR(PCR) : Polymerase Chain Reaction (Polimeraz Zincir Reaksiyonu) RAPD : Random Amplified Polymorphic DNA (Rastgele Çoğaltılmış Polimorfizm DNA) vi RE : Restriksiyon endonükleaz RFLP :Restriction Fragment Length Polymorphism (Kesilen Parça Uzunluk Polimorfizmi) SDS : Sodyum dodesil sülfat TAE : Tris asetik asit – EDTA TE : Tris – EDTA Tris : Trisodyum tuzu Tm : Çözülme (Melting) sıcaklığı V : Volt UPGMA : Unweighted pair group method using arithmetic averages (ağırlıksız aritmetik ortalamayı kullanan çift grup metodu) vii ĐÇĐDEKĐLER TEŞEKKÜR ...................................................................................................................... ii ÖZET ....................................................................................................................... iii ABSTRACT ..................................................................................................................... iv KISALTMA ve SĐMGELER ............................................................................................ v HARĐTALAR LĐSTESĐ ................................................................................................... ix ŞEKĐLLER LĐSTESĐ ........................................................................................................ x TABLOLAR LĐSTESĐ .................................................................................................... xii 1.BÖLÜM GĐRĐŞ ........................................................................................................................ 1 2.BÖLÜM GENEL BĐLGĐLER .......................................................................................................... 5 2.1. Echinops Cinsinin Taxsonomik Sınıflandırılması ......................................... 5 2.2. Seksiyon Hakkında Bilgiler ........................................................................... 6 2.3. Moleküler Sistematik ..................................................................................... 8 2.4. Türler Hakkındaki Bilgiler ............................................................................. 8 2.4.1. Echinops spinosissimus Turra ........................................................................ 8 2.4.2. Echinops orientalis Trautv. ............................................................................ 8 2.5. Peroksidazlar ................................................................................................ 11 2.6. Moleküler Markırlar ..................................................................................... 12 2.6.1. Moleküler Markır Çeşitleri........................................................................... 13 2.6.1.1. Morfolojik Markırlar .................................................................................... 13 2.6.1.2. Biyokimyasal Markırlar ............................................................................... 13 2.6.1.3. DNA Markırları ............................................................................................ 13 2.6.1.4. RAPD ........................................................................................................... 14 2.6.1.5. RFLP ............................................................................................................ 14 viii 2.6.1.6. AFLP ............................................................................................................ 15 2.6.1.7. Mikrosatellit DNA Markırları (Basit Dizin Tekrarları) ............................... 15 2.6.1.8. SNP............................................................................................................... 16 2.6.1.9. SCAR ........................................................................................................... 16 2.6.1.10. SRAP ............................................................................................................ 16 2.6.1.11. ISSR ............................................................................................................. 17 3.BÖLÜM MATERYAL METOD ................................................................................................... 18 3.1. Bitki Materyalinin Toplanması .................................................................... 18 3.2. POGP Çalışmaları Đçin DNA Đzolasyonu ..................................................... 21 3.3. POGP Analizi ............................................................................................... 23 3.3.1. Primer Özellikleri ......................................................................................... 23 3.3.2. Agaroz Jel Elektroforezi..............................................................................
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