Characterization of DNA methylation activity by gene expression analysis in Fragaria Vesca

DNA methylation represents one of the key processes that play an important role in the transcriptional control of gene expression. The role of cytosine methylation in plant development has been demonstrated. For this purpose gene expression analysis has been widely applied. The present work was directed to demonstrate how nitrate based manuring could induce DNA methylation in Fragaria vesca. Differential display analysis was used for this purpose. Two Fragaria vesca genotypes were analyzed. For each genotype a total of 40 plants were used. Plants were divided into two groups of twenty plants each. The first group was treated with a concentrated solution of calcium nitrate, the second was treated using an organic nitrogen solution at the same molar concentration. Plant material was sampled six times. The first sampling was before treatment, and subsequent sampling was done respectively 1, 3, 7, 15 and 20 days after the first one. For each genotype, leaves and roots were sampled and stored in liquid nitrogen. Genomic DNA was isolated and analyzed by applying isoschizomers to evaluate the extent of DNA methylation. This showed an increase of the DNA methylation in plants treated with calcium nitrate. Total RNA was separately purified from root and leaf and, in order to isolate differentially expressed genome fractions, cDNA was obtained. To generate differentially expressed sequences between different treatments and between sampling times, random and specific primers were applied. 62 differential fragments were isolated and sequenced. All sequences were compared to the database to evaluate similarity with previously isolated genes. Sequence analysis showed that 55% could be classified as ESTs. The other sequences showed homologies with genes related to abiotic stress. In particular it was possible to show a reduction of transcriptomic activity in the sample treated with calcium nitrate. Sequence analysis demonstrated that gene fractions isolated from plants treated with nitrate showed high homology with glycosylase, a gene related with DNA methylation repair action.