Thiebaut et al. BMC Genomics 2012, 13:290 http://www.biomedcentral.com/1471-2164/13/290 Page 4 of 14 Figure 3 The percentage of 5’ terminus nucleotide in relation of new miRNAs candidates lenght. The arrow indicates the calculation of first nucleotide percentage in novel miRNAs candidates from each size. A: adenine; U: uracile (timine convertion); C: cytosine; G: guanine. showed distinct preference of AGO for small RNAs with a different 5’- terminal nucleotide [36,37]. Moreover, four OsAGOs that are related to AtAGO1 have con- served histidine residue in the 798 position that is crit- ical for slicer activity of miRNA:AGO complex [38]. Analysis of small RNA sequences obtained by immuno- precipitation assays with anti-AGO1 antibodies revealed the preferential association of AGO1 with small RNAs containing 5’- terminal uridine [5]. Similar experiments with anti-AGO2 and anti-AGO4 antibodies showed an enrichment of small RNAs bearing a 5’ - terminal ad- enosine bound to AGO2, and AGO5 associated with small RNAs with a 5’ - terminal cytosine [5,36]. Based in the sequence similarity of the sugarcane AGO genes to those of other plant species (data not shown), it is pos- sible that a similar nucleotide preference may exist on sugarcane, and the results in Figure 3 may indicate that the majority of the new 21 nt microRNA candidates identified in this work are canonical miRNA. Abundance changes of novel sugarcane miRNAs under biotic and abiotic stresses Many studies have reported the role for miRNA in gene regulation and their involvement in responses to plant stress such as cold, salt, drought and pathogens [11-14]. In this study, we used 10 sRNA libraries generated from plants of three different experiments (Table 2). After trimming of sequence reads, miRCat pipeline and Blastn search, we identified 623 novel mature miR- NAs candidate sequences in those libraries, as described above. The abundance of different miRNAs can be in- ferred from their frequency in the library. To compare the distribution of new miRNAs abundance in drought, salt and pathogen stresses, we normalized the miRNA reads abundance and used the electronic northern ap- proach (methods). The read counts for miRNAs vary highly according to the kind of stress. As showed previ- ously for soybean [39], new and known miRNAs were regulated in water deficit and pathogen assays. Analyzing the abundance of miRNAs arising from pre- cursor class I (high confidence miRNAs sequences) we found 26 new miRNAs assay-specific and seven miRNAs with abundance higher than 50 normalized reads counts (Table 3). miRNAs sof-miR-Seq42, sof-miR-Seq143, sof- miR-Seq488, sof-miR-Seq504, sof-miR-Seq511 and sof- miR-Seq656 were selected for experimental confirmation by stem-loop RT-PCR method. These novel miRNAs gave detectable expression levels in qRT-PCR analysis using controls samples of biotic and abiotic assays (Add- itional file 1: Figure S4). Furthermore, we observed ex- ceptionally high abundance of sof-miR-Seq513 and sof- miR-Seq513* sequences (12,593 and 8,703 reads, re- spectively – Additional file 1: Table S2). We confirmed the high expression of this novel miRNA and its miRNA* in saline treatment assay sample treated for 1 h (Additional file 1: Figure S5). The analysis of miRNAs arising from all precursor classes revealed differential accumulation of certain new miRNA in the context of a particular stress (Additional file 1: Table S6). Only sof-miR-Seq296 was induced constitutively in all libraries (control and treated). The biotic stress library showed higher exclusive expression of new miRNAs (155 sequences). Figure 4A shows the distribution of the 623 novel sugarcane miRNAs found in either treatment (255) or control samples (160) or in both (208). Because the control libraries were constructed Table 2 Summary of experimental procedure for sRNA libraries construction Assay Water deficita Salt stressb Culture condition soil in vitro - hydroponic SP70-1143 Genotype Tolerant - CTC15, CTC6, SP83-2847 and SP83-5073 Sensitive - CTC9, CTC13, SP90-1638 and SP90-3414 Pathogen infectionc in vitro SP70-1143 a Drought stress induced by withholding watering. b Salt stress induced by 170 mM solution of NaCl. c Pathogen infection with A. avenae. Sample root leaf whole plant sRNA libraries Control: T0h and S0h T24h and S24h Control (0 h) 1 h; 6 h; 24 h Control (CT) Inoculated (AA)