Primer Pairs for Rice (Oryza sativa L.) Bisulfite Sequencing Studies

Abstract

Many agriculturally important properties such as heterosis, inbreeding depression, phenotypic plasticity, and resistance for biotic and abiotic stresses are thought to be affected with epigenetic components. New discoveries related with epigenetics are likely to have a major impact on strategies for crop improvement in rice breeding. However, assessing the contribution of epigenetics to heritable variation in plant species still poses major challenges. Methylation of cytosine in DNA is one of the most important epigenetic mechanisms in plants. DNA methylation not only plays significant roles in the regulation of gene activity, but also it is related with genomic integrity. Although most of next generation sequencing (NGS) technologies do not require the use of target specific primer pairs to identify and study DNA cytosine methylation, validation studies of NGS uses selective primer pairs. Bisulfite sequencing technique is a gold method for DNA cytosine methylation studies. However, bisulfite sequencing requires the development of bisulfite primer pairs to selectively study DNA sequences of interest. In this study 9 bisulfite specific primer pairs were identified and validated. These primer pairs successfully amplified bisulfite converted and unconverted genomic DNA extracted from radicle and plumule of rice (Oryza sativa L.) seedlings. Results of the present study clearly revealed the occurrence of CG, CHG and CHH (H stands for C. T or A nucleotides) contents in studied DNA sequence targets were different indicating potential role of DNA cytosine methylation in these genes. Primer pairs reported in this study could be used to detect DNA methylation which is one of the most important epigenetic mechanisms affecting the development, differentiation or the response to biotic and abiotic stress in rice (Oryza sativa L.).