Akış sitometri yöntemiyle yabani Ayçiçeği türlerinde çekirdek DNA ile Ploidi analizi ve Orobanşa dayanımı için seçici moleküler Markırların araştırılması
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Tarih
2022
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Yayıncı
Trakya Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Ülkemizde yağlı tohum üretimi yeterli olmayıp, bu eksiklik yüksek oranda döviz ödeyerek ithalatla giderilmektedir. En önemli yağ bitkisi olan ayçiçeğinde verim ve kaliteyi etkileyen en önemli etkenlerden biri de orobanş parazitidir. Ülkemizin hemen hemen tüm ekim alanlarında giderek yaygınlaşmakta olan orobanş paraziti, sürekli yeni ırklar meydana getirerek genetik dayanıklılığı kırmaktadır. Bundan dolayı yeni geliştirilecek ayçiçeği hibritlerinin orobanşa dirençli olması gereklidir. Yabani ayçiçeği türleri orobanşın yeni ırklarına dayanıklı pek çok gen kaynağına sahiptir. Bu direnç genlerinin kültür ayçiçeğine aktarılması devamlı bir dayanıklılık için çok önemlidir. Islah çalışmalarında moleküler metotların kullanılması, hem etkin ve doğru bir seleksiyon, hem de çalışmalara zaman tasarrufu sağlayarak ıslah süresini kısaltmaktadır. Ülkemizde özellikle de orobanşa dayanıklılık konusunda ayçiçeğinde moleküler markırlarla ilgili araştırmaların sayısı oldukça azdır. Markır analizleri için dayanıklı ve hassas ebeveyn hatlar ile bunların melezlenmesi sonucu elde edilen F2 genotipler kullanılmıştır. Dayanıklı ve hassas F2 genotiplere ait gDNA’lar eşit miktarda bir araya getirilerek dayanıklı F2 bulk (DB) ve hassas F2 bulk (HB)’lar oluşturulmuştur. Tez çalışması için bölgedeki orobanş ırklarına dayanıklılık sağlayan Or5 geni hedef alınmış, literatürde bu gen ile bağlantı gösteren ve seleksiyon için kullanılabilir olduğu ifade edilen markırlar seçilmiştir. Bunun yanısıra çeşitli bitkilerin dayanıklılık genlerinden geliştirilen 48 markırıda bu amaçla kullanılmıştır. Yabani türlerden önce bu melezlenmiş ve kendilenmiş ayçiçeği hatlarında markır araştırması yapılmış, fakat orobanş dayanımının seleksiyonda kullanılabilir bir markır tespit edilememiştir. Bu nedenle yabani ayçiçeği türlerinde, orobanşa dayanıklılıkla ilgili moleküler markır taraması kapsamında bir uygulama yapılamamıştır. Moleküler markır çalışmasında başarı sağlanamaması, fenotipik analizlerde kullanılan orobanş parazitinin saf bir ırk olmayıp popülasyon olarak kullanılmasına bağlanmıştır. Moleküler markır çalışmalarıyla birlikte, çekirdek DNA analiz ve ploidi düzeylerini belirlemek için akış sitometrisi kullanılmıştır. Araştırmamızda kullanılan tür ve alt türlerle birlikte 52 yabani ayçiçeği (Helianthus spp.) aksesyonu Amerika’da Ames, IA, USA’da bulunan Kuzey Merkez Bölge İstasyonu’ndan alınmıştır. Akış sitometrisi ile çekirdek DNA içerikleri (bazıları ilk kez) ve ploidi düzeylerini belirleyerek aksesyonların tür farklılıkları gözlenmiştir. Çalışmada örnekler genç ve sağlıklı bitkilerden alınan taze yaprak dokuları, floresan boya olarak propidium iodide ve internal standard olarak fiğ bitkisi kullanılarak hazırlanmıştır. Elde edilen verilere göre; çekirdek DNA içerikleri sonuçlarındaki değişimlerin yabani ayçiçeği türlerinin istatistiki olarak önem taşıdığı belirtilmiştir. Kullanılan yabani ayçiçeği türlerinin 2C çekirdek DNA içerikleri 5.72 pg (H.porteri) ile 27.11 pg (H.tuberosus) arasında değişim göstermiştir. Tezde kullanılan türlerden daha önce çalışılmış olan türlerde yapılan DNA içerik sonuçları benzerlik göstermiştir. Bu çalışmada daha önceki çalışmalarda olmayan birçok yeni türün çekirdek DNA içerikleri ve ploidi düzeyleri ilk kez belirlenmiştir. Yapılan literatür araştırmaları ve kromozom sayılarına göre 52 yabani türün yaklaşık %78’inin (41 tür) (15 Tek yıllık, 26 çok yıllık) diploid (2n) oldukları ortaya konulmuştur. Çalışılan diğer %12’lik türlerin ise ploidi düzeyleri farklılık göstermiştir. 3 türün tetraploid (4n), 8 türün hekzaploid (8n) olduğu belirlenmiştir. Çalışmamızda akış sitometrisi analizinin, ıslah ve taksonomik denemelerde morfolojik gözlemlerle zor olan tür karışıklıklarının tespit edilmesinde ve heterojen yapıda olanların belirlenmesinde kullanılabileceği ortaya çıkmıştır.
Oilseed production is not sufficient for Turkey, and this deficiency is compensated by importing with paying a high rate of foreign currency. One of the most important factor affecting the yield and quality of sunflower which is the most important oil plant in Turkey, is the broomrape parasite. Orobanche parasite which is becoming more and more common in almost all cultivation areas of our country with breaking the genetic resistance by constantly creating new races, so new developed sunflower hybrids must be resistant to broomrape. Wild sunflower species have many sources of genes resistant to new races of broomrape. The transferring of these resistance genes to the cultivated sunflower is so great issue for obtaining a continuous resistance in sunflower production. The use of molecular methods in the breeding studies shortens the breeding period by providing an effective and accurate selection, as well as saving time. In Turkey, the number of studies on molecular markers in sunflower is quite low especially for obtaining resistance breeding. Resistant and sensitive parent lines and F2 genotypes obtained by crossing them were used for marker analysis in our study. Resistant F2 bulk (DB) and sensitive F2 bulk (HB) were formed by combining gDNAs belonging to resistant and sensitive F2 genotypes in equal amounts. For the thesis study, the Or5 genes, which provides resistance to the orobanche races in the region, was targeted and the markers that showed a connection with this gene and were stated to be usable for selection were selected in the literature. In addition, the 48 markers developed from the resistance genes of various plants were used fort his purpose. Marker research was carried out in these hybridized and inbred sunflower lines before wild species to obtain a usable selection of orobanche resistance but any suitable marker could not be detected in the study. Therefore, the molecular markers could not be performed within the scope of molecular marker screening related to resistance to orobanche in wild sunflower species. The failure to success in the molecular marker study was attributed to the fact that the broomrape parasite used in phenotpic analysis was not a pure race and was used as a population. Along with molecular marker studies, core DNA analysis and flow cytometry were used to determine ploidy levels. 52 wild sunflower (Helianthus spp.) accessions along with the species and subpecies used in our study obtained from the North Central Regional Plant Introduction Station in Ames, IA, USA. It is to determine the core DNA contents by flow cytometry (some of them for the first time) and ploidy levels were determined by determining the type differences of the accessions. In the study, the samples were prepared using fresh leaf tissues from young and healthy plants, propidium iodide as fluorescent dye and vetch plant as internal standart. According to the research data, the changes between the core DNA contents of sunflower species were determined statistically significant. The 2C core DNA contents of wild sunflower species used varied between 5.72 pg (H. porteri) and 27.11 pg (H. tuberosus). The DNA content result of the previously studied species from the species used in the thesis were similar. In this study, nuclear DNA contents and ploidy levels of many new species that were not present in the previous studies were determined for the first time. According to the obtained results, it was revealed that approximately 78% (41 species) of 52 wild species (15 annual, 26 perennial) were diploid (2n). The ploidy levels of the other 12% studied species differed that 3 species were tetraploid (4n) and 8 species were hexaploid (8n). It has been stated that flow cytometry analysis could be used in breeding and taxonomic studies to detect species confusions that are difficult with morphological observations and to identify heterogeneous ones.
Oilseed production is not sufficient for Turkey, and this deficiency is compensated by importing with paying a high rate of foreign currency. One of the most important factor affecting the yield and quality of sunflower which is the most important oil plant in Turkey, is the broomrape parasite. Orobanche parasite which is becoming more and more common in almost all cultivation areas of our country with breaking the genetic resistance by constantly creating new races, so new developed sunflower hybrids must be resistant to broomrape. Wild sunflower species have many sources of genes resistant to new races of broomrape. The transferring of these resistance genes to the cultivated sunflower is so great issue for obtaining a continuous resistance in sunflower production. The use of molecular methods in the breeding studies shortens the breeding period by providing an effective and accurate selection, as well as saving time. In Turkey, the number of studies on molecular markers in sunflower is quite low especially for obtaining resistance breeding. Resistant and sensitive parent lines and F2 genotypes obtained by crossing them were used for marker analysis in our study. Resistant F2 bulk (DB) and sensitive F2 bulk (HB) were formed by combining gDNAs belonging to resistant and sensitive F2 genotypes in equal amounts. For the thesis study, the Or5 genes, which provides resistance to the orobanche races in the region, was targeted and the markers that showed a connection with this gene and were stated to be usable for selection were selected in the literature. In addition, the 48 markers developed from the resistance genes of various plants were used fort his purpose. Marker research was carried out in these hybridized and inbred sunflower lines before wild species to obtain a usable selection of orobanche resistance but any suitable marker could not be detected in the study. Therefore, the molecular markers could not be performed within the scope of molecular marker screening related to resistance to orobanche in wild sunflower species. The failure to success in the molecular marker study was attributed to the fact that the broomrape parasite used in phenotpic analysis was not a pure race and was used as a population. Along with molecular marker studies, core DNA analysis and flow cytometry were used to determine ploidy levels. 52 wild sunflower (Helianthus spp.) accessions along with the species and subpecies used in our study obtained from the North Central Regional Plant Introduction Station in Ames, IA, USA. It is to determine the core DNA contents by flow cytometry (some of them for the first time) and ploidy levels were determined by determining the type differences of the accessions. In the study, the samples were prepared using fresh leaf tissues from young and healthy plants, propidium iodide as fluorescent dye and vetch plant as internal standart. According to the research data, the changes between the core DNA contents of sunflower species were determined statistically significant. The 2C core DNA contents of wild sunflower species used varied between 5.72 pg (H. porteri) and 27.11 pg (H. tuberosus). The DNA content result of the previously studied species from the species used in the thesis were similar. In this study, nuclear DNA contents and ploidy levels of many new species that were not present in the previous studies were determined for the first time. According to the obtained results, it was revealed that approximately 78% (41 species) of 52 wild species (15 annual, 26 perennial) were diploid (2n). The ploidy levels of the other 12% studied species differed that 3 species were tetraploid (4n) and 8 species were hexaploid (8n). It has been stated that flow cytometry analysis could be used in breeding and taxonomic studies to detect species confusions that are difficult with morphological observations and to identify heterogeneous ones.
Açıklama
Anahtar Kelimeler
Ayçiçeği, Yabani ayçiçeği, Orobanş, Moleküler Markır, Flow sitometri, Sunflower, Wild species, Orobanche, Molecular Markers, Flow cytometry
