Thiebaut et al. BMC Genomics 2012, 13:290 http://www.biomedcentral.com/1471-2164/13/290 candidates in the sRNA libraries from treated samples (Figure 4C). The number of shared new miRNAs was increased (1 to 10) in all stressed libraries in contrast to all control libraries (Figure 4B and C). Plants under pathogen infection showed the highest number of novel miRNA (268). In the drought stress, tolerant genotypes showed an increase of the new miRNAs number (68 to 82) and sensi- tive genotypes remained unchanged. However, in sensitive genotypes under drought stress the number of exclusive new miRNAs decreased weakly (27 to 22). In addition, we observed a high induction of novel exclusive miRNAs can- didates in the salt stress libraries, where we identified twice as many novel miRNA expressed under the stress condition than in the control samples (59 to 101). Plants contain a complex network of small RNA path- ways. The canonical pri-miRNA is cleaved by DCL1 and results in mature miRNA 21nt in size. However, some researches described a novel class of bona fide miRNA [7,42]. This class was denominated long-miRNA (24 nt length) and their precursors are processed by DCL3 as well as siRNA. Contrary to siRNAs that require PolIV and RDR2 to be processed, stem-loop precursors of long- miRNA are originated from PolII and do not require RDR2 [7]. The other characteristic of long-miRNA is the mechanism of regulation. Recent study in rice described that long-miRNA mediated DNA methylation by AGO4 [7]. Canonical miRNAs and long-miRNAs comprised more than 91 % of all miRNAs species characterized in this study. We detected different distribution of those spe- cies in stresses libraries. In biotic stress we observed a re- duction of 21-nt-species and increase in 24-nt-species, similar to salt stress and drought stress sensitive genotypes libraries (Figure 4D). Instead, drought stress tolerant gen- otypes libraries showed an increase of 21-nt-species and a reduction of 24-nt-species. These analyses suggest that bi- otic, salt stress and drought stress (in the case of sensitive genotypes) may be regulating pathways by miRNAs from the same species (24-nt). Target prediction and function analysis In plants, miRNA regulate gene expression by either cleavage of mRNA or translation repression targets [10,43]. In addition, it has been described recently that some miRNA may regulate gene expression by DNA methylation [7]. Computational identification of plant miRNA-target is possible due the perfect or near-perfect match between miRNA/target in comple- mentary sites [44]. Using two published methods [45,46] we predicted miRNA targets. We first searched for targets of new miRNAs candidates in a sugarcane database (Saccharum officinarum ESTs – DFCI gene index) and then in the sorghum databases (S. bicolor gene models and ESTs – DFCI gene index). The results of these analyses are available in Page 7 of 14 Additional file 1: Table S7 and Additional file 1: Table S8. We identified 1,975 putative target genes for 483 new miRNAs, with an average of 4 targets per miRNA candidate. In sorghum, the average of gene targets per miRNA was lower than sugarcane (2.4), showing 895 targets for 373 miRNA. Importantly, the results demonstrated that most sugarcane targets have conserved miRNA candidate recognition sites as they are found in sorghum as well as in sugarcane. Due the importance of miRNA in regulating a variety of biological processes, we analyzed the putative miRNA targets in order to understand the biological function of novel miRNAs candidates. Analysis of the sugarcane Tentative Consensus (TC) EST assembly discovered 776 new miRNAs candidates targets that had versatile func- tional annotation and 336 were of unknown function. MADS-box transcription factor MADS2 and 60 S acidic ribosomal protein P2B (RPP2B) were overrepresented. MADS2 is a transcription factor that regulates develop- ment [47,48] and RPP2B plays an important role in dis- ease resistance to bacterial pathogens [49]. The expression of MADS2 was verified using qRT-PCR and an induced profile was shown after 1 h of saline treat- ment related to control (Additional file 1: Figure S9). This result suggests a regulation of MADS2 by sof-miR- Seq09 and sof-miR-Seq19 where the new miRNAs candi- dates were repressed after 1 h of saline treatment. Most targets annotated as MADS2 were recognized by 21nt miRNA candidate. Similarly, the majority of micro- RNAs conserved among species target transcription factors [50,51], however, the majority of new sugarcane miRNAs candidates that regulate RPP2B have 24 nt in length. According to Wu et al. (2010) [7], long-miRNA may direct DNA methylation around their recognition site in the tar- get loci. This type of miRNA may act in their own precur- sors in cis and also in their targets in trans to guide DNA methylation [52]. Because of the features applied on the search of complementary sites of miRNA and mRNA, we have also identified targets that were possibly regulated by DNA methylation. In sugarcane, 140 new miRNAs candi- dates did not have predicted targets, of which 113 have 24 nt length. The sugarcane genome sequence is not available, we cannot exclude the possibility that bona fide targets may exist and they have been not sequenced yet. Another possi- bility is that these miRNAs are targeting their own loci in cis, and that is why we did not detect their targets by the pipeline used. Interestingly, when we searched targets of these novel miRNAs candidates in sorghum, a similar pro- file was observed and the higher amount of miRNAs candi- dates that did not have predicted targets were 24-nt-species (190 in 250 new miRNAs candidates). The sugarcane targets regulated by high confidence new miRNAs arising from the precursor class I were listed in Table 4, and the conserved miRNA targets sites