New insight into evaluation of DNA methylation levels with CRED-RA technique in the genome of Lycopersicum esculentum

Abstract

Levels of methylcytosine in the genome can be detected with multiple approaches from several standpoints, as there are many techniques, such as coupled restriction enzyme digestion-random amplification CRED-RA , available for determining methylation rates in the genome. CRED-RA, which is a powerful technique for studying the genome methylation status, uses methylation-sensitive enzymes, such as HpaII and MspI. In the present study, we used the CRED-RA technique to determine DNA methylation changes in Lycopersicum esculentum L. subjected to 100 mM NaCl and Polyethylene glycol PEG for different time intervals 0 h, 6 h, 9 h, 12 h, and 24 h . DNA band variations due to both stressors were revealed by Random Amplified Polymorphic DNA RAPD analysis. The DNA band changes obtained by the RAPD analysis at 12 h response to both stressors PEG and NaCl indicated that this was the critical point for demethylation/hypomethylation activity. According to the results of the CRED-RA analysis, the PEG treatment led to greater variation in DNA methylation than NaCl in the tomato genome. For both stressors, the maximum decrease in total methylation levels occurred at 9 h compared to the control plants, indicating that this is the critical point for demethylation/hypomethylation activity. In conclusion, the present study showed that DNA methylation changes can be easily observed with a straightforward technique, CRED-RA, in plants subjected to abiotic stress conditions, such as drought and salinity

Keywords

• Methylation,CRED-RA,salinity,tomato,drought

Tuz ve kuraklığa maruz kalmış domates genomunda Lycopersicum esculentum L. DNA metilasyon seviyesinin CRED-RA tekniği ile araştırılması

Abstract

Genomdaki metilsitozin seviyesi veya metilasyon oranı enzimle kesim-rastgele amplifikasyon CRED-RA tekniğininde içerisinde yer aldığı bir çok yöntem ile belirlenebilir. CRED-RA HpaII ve MspI gibi metilasyon duyarlı enzimlerin kullanıldığı önemli bir tekniktir. Bu çalışma kapsamında CRED-RA tekniği farklı zaman aralıklarında 0, 6, 9, 12 ve 24 saat 100 mM NaCl ve Plietilen glikol PEG uygulanmış domates Lycopersicum esculentum L bitkisinde DNA metilasyon değişikliklerinin belirlenmesi amacı ile kullanılmıştır. DNA band değişiklikleri Rastgele Çoğaltılmış Polimorfik DNA RAPD analizi aracılı ile ortaya konmuştur. 12 saat süre ile NaCl ve PEG uygulanmış örneklerden RAPD analizi ile elde edilen DNA bant değişikliklerinin de-metilasyon/hipometilasyon açısından kritik olduğu görülmüştür. CRED-RA analizi verilerine göre, PEG uygulamasının NaCl uygulamasına oranla domates genomundaki DNA metilasyon seviyesinde daha fazla değişikliğe sebep olduğu görülmüştür. Her iki stres koşulu içinde toplam metilasyon seviyesindeki maksimum düşüşün 9h uygulamasında olduğu belirlenmiştir. Sonuç olarak; bu çalışma ile CRED-RA tekniğinin kuraklık ve tuz gibi abiotik stres koşullarına maruz kalmış bitkilerde DNA metilasyon seviyesindeki değişikliklerin belirlenmesinde kullanılabileceği görülmüştür

Keywords

• metilasyon,CRED-RA,tuzluluk,domates,kuraklık

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