Determination of DNA Cytosine Methylation Polymorphism Using RAPD Markers in Some Nigella sativa L. Genotypes

Abstract

Epigenetics is defined as heritable changes in gene expression and function without DNA base changes. Methylation in DNA and chemical modification in histone proteins are the two most studied epigenetic mechanisms in plants. There exist low-throughput and high-throughput DNA methylation detection techniques in epigenetic research. One of the low-throughput techniques is an enzyme-based approach based on the presence or absence of DNA cytosine methylation. Using this approach, DNA cytosine methylation in random genes was investigated for Nigella sativa L. varieties with biological activities and therapeutic potential. This study, touch-down polymerase chain reactions methylation sensitiverandom amplified polymorphic DNA (TD-MS RAPD) technique was used to investigate cytosine methylation differences among genotypes of five different Nigella sativa L. species; Çameli, Eskişehir, Konya, Suriye and Şanlıurfa. Genomic DNA samples extracted from the seedlings of these genotypes were treated with MspI, a relative methylation-insensitive restriction enzyme and HpaII, a methylation-sensitive restriction enzyme before touch-down polymerase chain reactions. Among 8 oligonucleotide primers used, one primer (OPB-12) resulted in methylation polymorphisms among the five genotypes. The TD-MS-RAPD-PCR method is a simple and cost-effective method that requires basic equipment. This method can be easily implemented using a normal DNA thermal cycler and DNA gel electrophoresis system. However, the level of methylation polymorphisms detected with this method is very low in black cumin. It was concluded that there is a low level of polymorphism among five different black cumin genomes, which is thought to be due to the low 5’-CCGG-3’ contents of the black cumin genome.

Keywords

• Black cumin
• Epigenetic
• MspI-HpaII

Bazı Nigella sativa L. Genotiplerinde RAPD Markırları Kullanılarak DNA Sitozin Metilasyon Polimorfizminin Belirlenmesi

Abstract

Epigenetik, DNA baz değişimi olmaksızın gen ifadesi ve fonksiyonundaki kalıtsal değişimler olarak tanımlanmaktadır. DNA'da metilasyon ve histon proteinlerindeki kimyasal modifikasyonlar bitkilerde en çok çalışılan iki epigenetik mekanizmadır. Epigenetik araştırmalarda düşük işlem hacimli ve yüksek işlem hacimli DNA metilasyon saptama teknikleri vardır. Düşük işlem hacimli tekniklerden bir tanesi de enzim tabanlı DNA sitozin metilasyonunun varlığına ya da yokluğuna dayanan bir yaklaşımdır. Bu yaklaşım kullanılarak biyolojik aktiviteleri ve terapötik potansiyeli olan Nigella sativa L. genotipleri için rastgele genlerde DNA sitozin metilasyonu araştırılmıştır. Bu çalışmada, beş farklı Nigella sativa L. türüne ait genotipler (Çameli, Eskişehir, Konya, Suriye, Şanlıurfa) arasındaki sitozin metilasyon farklılıklarını araştırmak için touch-down polimeraz zincir reaksiyonları metilasyon duyarlı-rastgele arttırılmış polimorfik DNA (TD-MS RAPD) tekniği kullanılmıştır. Bu genotipler fidelerinden izole edilen genomik DNA örnekleri touch-down polimeraz zincir reaksiyonlarından önce metilasyona duyarsız olan MspI restriksiyon enzimi ve metilasyona duyarlı HpaII restriksiyon enzimi ile muamele edilmiştir. Kullanılan 8 oligonükleotid primerinden bir primer (OPB-12), beş genotip arasında metilasyon polimorfizmleri ile sonuçlanmıştır. TD-MS-RAPD-PZR yöntemi basit ve temel cihazları gerektiren uygun maliyetli yöntemdir. Bu yöntem normal bir DNA termal döngü cihazı ve DNA jel elektroforez sistemi kullanılarak kolayca uygulanabilmektedir. Ancak bu yöntemle saptanan metilasyon polimorfizmlerinin düzeyi çörek otunda çok düşüktür. Beş farklı çörek otu genomu arasında düşük düzeyde polimorfizm olduğu sonucuna varılmış olup bu durum çörek otu genomunun 5’-CCGG-3’ içeriklerinin düşük olmasından kaynaklandığı düşünülmektedir.

Keywords

• Çörek otu
• Epigenetik
• MspI-HpaII

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