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Salix Alba & S Türkiye’deki Söğüt Türlerinin Moleküler Filogenetiği ve Ekonomik Açıdan Önemli Olan Türlerin (Salix alba & Salix excelsa) Islahına Yönelik Gen Kaynaklarının Karakterizasyonu Program Kodu: 1001 Proje No: 213O154 Proje Yürütücüsü: Prof. Dr. Zeki Kaya Araştırmacılar: Prof. Dr. Hayri Duman Bursiyerler: Araş. Gör. Funda Özdemir Değirmenci Araş. Gör. Pelin Acar Araş Gör. Asiye Çiftci Burak Yelmen Nisan 2017 ANKARA ii ÖNSÖZ Söğüt cinsi (Salicaceae) dünya genelinde yaklaşık 500 türü kapsamakta olup çok fazla morfolojik farklılıklar göstermektedir. Ülkemizde 27 tür yayılış göstermekte olup teşhislerinde problemler yaşanmaktadır. Özellikle morfolojik ve anatomik karakterler bu cins içinde yer alan türlerin taksonomik tayini ve cins içinde yer alan diğer türlerle olan evrimsel ilişkisini tayin etmek için yeterli olamamaktadır. Bu çalışmada evrimsel olarak korunmuş olan ve moleküler filogenetik çalışmalarda yaygınca kullanılan kloroplast (matK, rbcL, tRNA) ve çekirdek (rDNA ITS) genomlarında bulunan bölgeler karşılaştırmalı olarak araştırılmış ve türlerin moleküler düzeyde nasıl farklılaştığı ortaya konmuştur. İlave olarak Türkiye nehir ekosistemlerinin en önemli türlerinden olan ve habitatları sulama ve elektrik amaçlı yapılan barajlarla parçalanmış ya da tahrip edilmiş ekonomik ve ekolojik öneme sahip Salix alba ve Salix excelsa türlerinin gen kaynaklarının etkin bir şekilde belirlenmesi ve korunmasına yönelik çalışmalar yürütülmüştür. Bu çalışmada bu cinste ileride yapılacak tür teşhisi, gen kaynaklarını koruma ve ıslah çalışmalarını daha etkin ve programlı yapılmasına katkıda bulunulacak veriler üretilmiştir. Kloroplast ve çekirdek DNA bölgeleriyle ayrı ayrı oluşturulan her iki ağaçtaki türlerin konumları arasındaki farklar görülmüştür. Bu da söğüt türlerinde görülen kloroplast paylaşımı ve introgresif melezlenme (gen aktarımına yönelik birçok nesilde benzer birey ve ebeveynler arasında geri çaprazlama) ile türlerin arasında yoğun etkileşim ile açıklanmaktadır. Altcinslerin bu şekilde moleküler veriye göre iki ağaçta da iki ana gruba ayrılması (altcins Salix ve Vetrix) farklı biyocoğraflarda; ılıman (altcins Salix) ve soğuk (altcins Vetrix) yer almalarıyla açıklanabilmektedir. Morfolojik olarak iki farklı tür olarak ifade edilen S. alba –S.excelsa türlerinin kloroplast ve çekirdek DNA bölgeleri ile DNA dizi analizi ile etraflıca çalışılmasına rağmen net bir şekilde ayrışmadığı görülmüştür. S. alba ve S. excelsa türlerinde türleşme devam ettiği için kesin bir tür farklılaşması görülmemiştir. Bu nedenle (S.alba/excelsa) kompleksi olarak örneklenen 10 nehirden toplanan tüm popülasyonlar S.alba- S.excelsa ve melezleri olarak çok fazla iç içe girdiğinden S. alba baskın tür olarak dikkate alınmıştır. Türkiye çapında Salix alba popülasyonları 10 nehirde çalışılmış ve alel paylaşımına dayalı genetik yapılaşma değerlendirmeleri bu türün popülasyonlarının 3 atasal gen havuzundan evrimleştiğini ve türleşmenin devam ettiğini göstermektedir. Popülasyonların genetik benzerliklerine göre yapılan sınıflandırmada ise Anadolu Çaprazı’nın önemli rolü olduğu görülmektedir. Bu çaprazın doğusunda Seyhan, Ceyhan, Fırat, Aras ve Çoruh nehir popülasyonları batısında ise üç alt gruptan oluşan Göksu, Kızılırmak-Ihlara-Aksu ve Susurluk iii yeralmaktadır. Genetik çeşitlilik de uzun ve farklı nehirlerde daha yüksek iken kısa ve daha tahrip görmüş kısa nehirlerde oldukça düşük ve akrabalık dereceside yüksek bulunmuştur. Bu proje kapsamında örneklenen nehirlerin hemen hepsinde doğal nehir ekosistemlerinin ve habitatlarının çeşitli çevresel faktörler yüzünden ya yok olduğu ya da parçalı bir yapıya sahip oldukları görülmüştür. Halbuki söğütler nehir ekosistemlerinin en önemli türlerinden olup, nehirlerin temizlenmesi ve ekosistemin tekrar tamir edilmesi çalışmalarında bütün dünya da en etkili bitkisel çözüm (Phytoremediation) aracı olarak kullanılmaktadır. Bu nedenle, bu projenin verileri ışığı altında, parçalanmış ve tahrib olmuş veya tahribatın devam ettiği nehir ekosistemlere öncelik verilerek Salix alba ile akarsu boyları ekosistemlerin restorasyonuna ve türün gen kaynaklarını korumaya yönelik programların vakit geçirilmeden oluşturulması gerekmektedir. Proje TOVAG- 213O154 numaralı araştırma projesi olup TUBİTAK tarafından desteklenmiştir. Proje sonuçlarının konuyla ilgilenen uygulamacı ve araştırmacılara yararlı olması dileğiyle. 5 Nisan 2017 Prof. Dr. Zeki Kaya ODTÜ. Biyolojik Bilimler Bölümü Ankara iv İÇİNDEKİLER ÖNSÖZ .......................................................................................................................... iii ÖZET ............................................................................................................................... xi ABSTRACT ................................................................................................................... xii PROJEYLE İLGİLİ BİLİMSEL VE TEKNİK SONUÇLAR ............................................ 1 1. GİRİŞ .......................................................................................................................... 1 1.1 Literatüre özeti ................................................................................................................... 2 1.1.1 Söğütlerin ekonomik değeri .................................................................................................................. 3 1.1.2 Söğütlerin taksonomisi .......................................................................................................................... 4 1.1.3 Türkiye’de söğüt cinsi ............................................................................................................................ 4 1.1.4 Moleküler çalışmalar ............................................................................................................................. 7 2. ÇALIŞMANIN AMACI ................................................................................................ 9 3. GEREÇ VE YÖNTEM............................................................................................... 11 3.1 Örnekleme ve bitki materyali (İş paketi-1) ......................................................................... 11 3.2 DNA izolasyonu (İş paketi 1-2) ............................................................................................. 2 3.3 Söğüt türlerinin filogenetiğini (soyağacını) Moleküler yöntemlerle (kloroplast DNA matK, rbcL, tRNA ve çekirdek rDNA ITS) ortaya koyma çalışmaları (İş paketi-1) ..................................... 4 3.3.1 Polimeraz Zincir Reaksiyonu ................................................................................................................. 2 3.3.2 Agaroz jel elektroforez koşulları ........................................................................................................... 5 3.4 Verilerin toplanması ve analiz edilmesi ............................................................................... 6 3.5 Önemli nehir sistemlerinde doğal olarak bulunan Salix alba-S. excelsa tür kompleksinin moleküler karakterizasyonu (İş paketi 1-ek) .............................................................................. 7 3.6 Salix alba populasyonlarının genetik çeşitlilik ve yapılanmasının Türkiye nehir sistemlerinde (havzasında) belirlenmesi (iş Paketi 2) ..................................................................................... 10 3.6.1 Örnekleme ve bitki materyali .............................................................................................................. 10 3.6.2 SSR (Simple Sequence Repeats-Basit dizi tekrarları) DNA belirteçlerin seçimi ve PCR koşullarının optmisazyonu ............................................................................................................................................... 12 3.6.3 Polimeraz Zincir Reaksiyonu (PCR) ...................................................................................................... 14 3.6.4 Agaroz jel elektroforezi ....................................................................................................................... 17 3.6.5 Fragment analizi .................................................................................................................................. 17 3.6. 6 Genetik veri analizleri ........................................................................................................................ 17 4. BULGULAR .............................................................................................................. 20 4.1 Türkiye söğüt türlerinin moleküler filogenetiği ................................................................... 20 4.2 Filogenetik Ağaçların Değerlendirilmesi (trn T-F, matK, rbcL ve rDNA ITS) ........................... 23 4.3 Türkiye nehir sistemlerinde Salix alba-S.excelsa teşhisi (İş paketi 1-ek) .............................. 26 4.4 S. alba-S.excelsa filogenetik ağaçlarının değerlendirilmesi .................................................. 27 4.5 Salix alba populasyonlarının genetik çeşitliliğinin belirlenmesi ve genetik yapılarının karakterizasyonu (İş Paketi 2) ................................................................................................. 33 4.5.1 DNA İzolasyonu ve PCR ........................................................................................................................ 33 4.5.2 Fragment Analizi .................................................................................................................................. 33 4.5.3 Genetik verilerin değerlendirmesi ......................................................................................................
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