Diferential DNA methylation and gene expression during development of reproductive and vegetative organs in Ilex species

Abstract

Diferential epigenetic (DNA cytosine methylation) and gene expression patterns were investigated in reproductive and veg- etative organs from Ilex paraguariensis and I. dumosa, at distinct developmental stages. We aimed at contributing towards elucidating major molecular changes underlying the sexual diferentiation processes which, in these dioecious species, are completely unknown. Simultaneously, as a frst step towards the development of an early sexing system, we searched for promising molecular markers. This was assessed through Methylation Sensitive Amplifed Polymorphism (MSAP) and Amplifed Fragment Length Polymorphism on cDNA (cDNA-AFLP) techniques, applying discriminant multivariate analyses, and bioinformatic characterization of diferential fragments. A signifcant positive correlation was found between epigenetic and indirect ‘genetic’ information for both species, indicating infuence of the genetic background on the epigenetic varia- tion. Higher epigenetic than genetic diversities were estimated. Our outcomes showed up to 1.86 times more representation of mCG subepiloci than mCCG in all organs sampled. Along the maturing stages of foral buds, the frequency of mCG evidenced an incremental trend, whereas mCCG and unmethylated conditions showed opposite tendencies. Reproductive and vegetative samples tended to cluster apart based on epigenetic patterns; at gene expression level, organs exhibited clear- cut distinctive patterns, nonetheless profles of young leaves and foral primordia resemble. Epigenetic and expression data allowed discrimination of I. dumosa ́s samples according to the gender of the donor; more elusive patterns were observed for I. paraguariensis. In total, 102 diferentially methylated and expressed fragments were characterized bioinformatically. Forty-three were annotated in various functional categories; four candidate markers were validated through qPCR, fnding statistical diferences among organs but not among sexes. The methylation condition of epilocus C13m33 appears as indica- tive of gender in both species. Thirty-three organ-specifc and 34 gender-specifc methylated markers were discriminated and deserve further research, particularly those expressed in leaves. Our study contributes concrete candidate markers with potential for practical application.