We report the first comprehensive multilocus molecular characterization of cultivated Ilex paraguariensis plants belonging to a breeding program. Using nuclear and homologous chloroplastidic molecular markers, we have genotyped 158 plants from four plantation sites. Analyses of the nuclear data (187 variable dominant loci) allowed detecting high diversity (0.569), the occurrence of four distinct genetic clusters, and a low but significant differentiation among sites. Additionally, 20 chloroplastidic alleles were identified applying five microsatellite polymorphic markers, and a high chloroplastidic diversity was recognized (0.505); two haplogroups were distinguished amongst the 63 haplotypes detected. Our results from both nuclear and plastidic markers indicate that most genetic variation reside within plantations sites (≥ 95%), and that these plantations were established on highly variable materials (either as seeds or plantlets) derived from, at least, 63 maternal lineages. Moreover, our study suggests that the genetic structure within each plantation site was most likely shaped by past admixture favored by farmers´ practices during the establishment of each plantation. Also, subsequent constraints in gene flow and/or a low level of shared polymorphism among plantations could have contributed to current structure.

Differential epigenetic (DNA cytosine methylation) and gene expression patterns were investigated in reproductive and vegetative organs from Ilex paraguariensis and I. dumosa, at distinct developmental stages. We aimed at contributing towards elucidating major molecular changes underlying the sexual differentiation processes which, in these dioecious species, are completely unknown. Simultaneously, as a first step towards the development of an early sexing system, we searched for promising molecular markers. This was assessed through Methylation Sensitive Amplified Polymorphism (MSAP) and Amplified Fragment Length Polymorphism on cDNA (cDNA-AFLP) techniques, applying discriminant multivariate analyses, and bioinformatic characterization of differential fragments. A significant positive correlation was found between epigenetic and indirect 'genetic' information for both species, indicating influence of the genetic background on the epigenetic variation. 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 floral 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 clearcut distinctive patterns, nonetheless profiles of young leaves and floral 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 differentially methylated and expressed fragments were characterized bioinformatically. Forty-three were annotated in various functional categories; four candidate markers were validated through qPCR, finding statistical differences among organs but not among sexes. The methylation condition of epilocus C13m33 appears as indicative of gender in both species. Thirty-three organ-specific and 34 gender-specific methylated markers were discriminated and deserve further research, particularly those expressed in leaves. Our study contributes concrete candidate markers with potential for practical application.