Genes Differentially Expressed Under Salt Stress Revealed by a DNA Methylation Approach (MSAP) in Rapeseed (Brassica napus var. oleifera Del.)

Salinity is an important limiting environmental factor for rapeseed production worldwide and can hamper initial developmental stages in Mediterranean climates where the crop is sown in late summer. Since DNA methylation is known to play a crucial role in regulating plant development and tissue differentiation, in this study, we compared the extent and pattern of cytosine methylation in one tolerant (Exagone) and one susceptible (Toccata) rapeseed (Brassica napus) cultivar, germinated in distilled water and grown either in distilled water or in a 100 mM NaCl solution, using the Methylation-Sensitive Amplified Polymorphism (M-SAP). Analysis of amplification products generated by fifteen primer combinations showed that the rapeseed genome is hypermethylated with several polymorphic fragments. Interestingly, salt stress induced demethylation in Exagone loci and hypermethylation in Toccata loci. Twenty-four methylation-related fragments were recovered either from tolerant or susceptible samples, cloned, sequenced and subjected to: i) BLAST (Arabidopsis and Brassica genome), ii) transposon and repetitive search, and iii) Gene Ontology analyses. Some sequences shared high homology with Arabidopsis thaliana genes somehow related to stress tolerance. In particular, 4 of these sequences showed high similarities with trehalose phosphatase/synthase, SH3 domain-containing proteins, glutamine fructose 6 phosphate and LACERATA genes. To correlate the methylation pattern and gene expression of methylated fragments, qRT-PCR analyses were carried out for trehalose phosphatase/synthase and LACERATA genes. Results are reported and discussed.