Thiebaut et al. BMC Genomics 2012, 13:290 http://www.biomedcentral.com/1471-2164/13/290 closest relative to sorghum and sugarcane of the three reference species, we identified phylogenetic evidence for 69 novel miRNAs (Figure 6). In polyploid genomes, genetic and epigenetic modifica- tions can quickly change the structure and function of gen- omes [62]. Vincentz et al. (2004) [59], using Arabidopsis and rice genomes and sugarcane transcriptome, showed that some genes are monocot-specific, diverging from eudicots. The most accepted hypothesis for miRNA origin is duplication of their target genes, auto-, tandem or seg- mental duplication [63]. They are fast-evolving sequences that may present a divergence degree reflecting the phylo- genetic divergence between species [63]. As mentioned above, the most phylogenetic related species showed more conserved miRNA (Figure 6). However, since the rate of evolution is different between species, many miRNA are not conserved and are, therefore, species-specific. The data suggest that miRNA evolution is on-going process and some of miRNA may be originated in a species during plant evolution. Conclusions We have shown that next-generation sequencing technolo- gies have a substantial impact on miRNA discovery of non- model plants. In our study, numerous small RNA libraries were constructed from sugarcane plants submitted to drought and salt stress, or to pathogen infection. By using bioinformatics analysis, we detected a large number of new sugarcane miRNAs candidate sequences and inferred about their possible biological importance analyzing their deep profiling in different genotypes, tissues and conditions, and also through the analysis of their putative target genes. As regulatory molecules with ancient origin, new sugarcane miRNAs shared greater homology with their monocot- related species, indicating that miRNA are fast-evolving sequences whose origin is closely related with plant evolu- tion. Our findings provide the first large scale miRNA dis- covery in sugarcane and help to clarify about potential miRNA roles in regulatory pathways of this and other crops. Methods Plant material and experimental procedure Water deficit assay Stalks of sugarcane cultivars, with different drought sensitivities, were provided by the Centro de Tecnolo- gia Canavieira (CTC). Based on chlorophyll and water Page 10 of 14 content measurements, cultivars CTC15, CTC6, SP83- 2847 and SP83-5073 and CTC9, CTC13, SP90-1638 and SP90-3414 are considered as drought tolerant and sensitive, respectively. Stalks were germinated and grown in 5 L pots in a greenhouse at 28°C. After three months, the plants were exposed to drought stress by withholding watering. Treated and control roots were harvested at 0 and 24 hours of treatment, respectively. Four small RNA libraries for deep se- quencing were constructed from RNA pools of sensi- tive and tolerant sugarcane cultivars submitted to drought stress and control plants. Salt stress assay In vitro-grown sugarcane plantlets (Saccharum sp. Cv SP70-1143) were rooted in Murashige and Skoog media supplemented with sucrose (2%), citric acid (150 mg/L), kinetin (0,1 mg/L), and IBA (0,2 mg/L). Plants were maintained at 110 mE m-2 s- luminosity, 12 h photoperiod, at 28°C. After the development of a root system (nearly a month) plantlets were transferred to hydroponic system compound in plastic containers (16 liters) supplemented with Hoagland solution [64]. Plantlets were acclimated dur- ing two weeks in a greenhouse at 28°C and then NaCl 170 mM solution was added. Control plants were main- tained in distillated water. Leaves of treated and control plantlets were harvested at 0, 1, 6 and 24 hours after treat- ment. A set of five plants was collected for each time point of the experiment, and the pooled material was used in the construction of four small RNA libraries. Pathogen infection assay Acidovorax avenae subsp avenae obtained from the Cul- ture Collection of the Instituto Biológico (IBSBF) was grown in medium (beef extract 3 g/L; Peptone 5 g/L; NaCl 5 g/L) at 28°C. In vitro-grow sugarcane plantlets were cultivated as described in the saline stress experiment. After the development of a root system, vigorous and pathogen-free plants were divided in two halves with a scalpel. One half was inoculated immersing the root sys- tem for 5 minute in a suspension of A. avenae in distilled water (106 CFU mL-1) and then washed twice with dis- tilled water in order to eliminate superficial bacteria. The other half was used as control, immersing the root system in distilled water for 5 minutes and then washed twice with distilled water. Inoculated and control plants were transferred to MS media and kept for seven Figure 6 Partial conservation of new sugarcane miRNAs candidates between rice and maize. Boxes are highlighting the presence of new miRNA precursor identified by miRCat pipeline in theses plant species.