Thiebaut et al. BMC Genomics 2012, 13:290 http://www.biomedcentral.com/1471-2164/13/290 Page 6 of 14 Figure 4 Global distribution analyses of new mature miRNAs candidates. Venn diagram comprising the new miRNAs candidates distribution in each condition: A) Comparison of all control and all stresses libraries; miRNAs distribution in control sRNA libraries (B), and pathogen infection, drought and salt stressed libraries (C); D) Distribution of canonical and long-miRNAs in each libraries. CT: non inoculated plants; AA: under A. avenae infection; T 0 h and T 24 h: tolerant genotypes in control and drought stress conditions, respectively; S 0 h and S 24 h: sensitive genotypes in control and drought stress conditions, respectively; 0 h: plant without salt stress;1 h, 6 h, 24 h: plants after salt treatment. with three types of tissues of different genotypes culti- vated in vitro, hydroponic and soil condition, we ana- lyzed the new miRNAs distribution in all control conditions (Figure 4B). Only one novel miRNA candi- date were shared between all control libraries. In con- trol plants of pathogen infection assay (in vitro cultivation) were found specifically 201 new miRNAs candidates and in control plants of salt stress assay (hydroponic cultivation) 59 were found. Control plants of the water deficit assay (tolerant and sensitive) shared 27 new miRNAs, in contrast with control plants of the pathogen infection and salt stress assays that are from the same genotype and had five new miRNAs in common. These results showed that plants grown under the same condition (plants of water def- icit assay grown in soil), independently of their geno- type, share similar numbers of detectable new miRNA. Many genome regions are silenced through DNA methylation, histone modifications and small RNA-direct DNA methylation, while others are activated by the up- or down-regulation of the same epigenetic mechanisms [40,41]. These modifications in chromatin structure may be activating much of the new miRNAs detected in the present study. Interestingly, plants of the same genotype (SP70-1143) firstly grown in vitro followed by cultivation in a hydroponic system (control plants of salt stress assay), do not show a higher number of novel miRNAs candi- dates (76 against 214 in control plants of pathogen infec- tion assay). Nevertheless, these control plants showed higher amount of new miRNAs than control plants of water deficit assay (tolerant and sensitive genotypes). In general, the new miRNAs expression in control plants seems to be genotype-tissue-culture dependent. In order to find new sugarcane miRNAs that may be involved in the regulation of the plant’s responses to stress we investigated the distribution of miRNAs sequences