Thiebaut et al. BMC Genomics 2012, 13:290 http://www.biomedcentral.com/1471-2164/13/290 [9,10] and for tolerance to biotic and abiotic stresses [11-14]. Due to the importance of miRNA in regulation of gene expression, extensive investigation aiming at the discov- ery of new microRNAs in several plant species has been carried out and the number of known miRNAs is in- creasing with over 3.300 plant-microRNA deposited sequences in the miRBase [15]. Most of these miRNAs are conserved between all plants [16]; however, there are also several miRNAs that are not. Some examples of this are: the miR403, which was identified only in eudicots, and the miR444 and miR528 that are monocots specific [17]. The process of microRNA discovery is becoming faster and an increasingly larger number of novel miR- NAs are being discovered. Furthermore, analysis of the expression of miRNAs revealed that many of them are expressed only in certain tissues and/or cell types, and at specific stages of development [18-20]. According to Lu et al. (2006) [21], nonconserved miRNAs are found at low expression levels. Next generation sequence tech- nologies have accelerated the process of small RNA dis- covery in many plant species and increased the recovery of rare miRNA, which together with the completion of more plant genome sequences, allowed the identification of new and weakly expressed miRNA [22]. Sugarcane is an economically important crop, mainly due to sugar and biofuel production. Sugarcane is an alogamous plant which like rice, maize and sorghum, belongs to the Poaceae family [23]. The commercial var- ieties belong to the genus Saccharum L, which consists of hybrids derived from interbreeding between Sac- charum officinarum, S. sinense, S. barberi, S. robustum and S. spontaneum species [24]. Sugarcane has one of the most complex plant genomes, compounded by a variable ploidy number [24]. Yields of sugarcane crop can be reduced dramatically due to the influence of en- vironmental factors such as salinity, drought, fungal and bacterial disease [25-27]. A priority for breeders is to ob- tain varieties with increased tolerance to abiotic and bi- otic stresses. Because of the reduced genetic variability in Saccharum hybrids, caused by recent speciation, the introduction of biotechnology tools in breeding pro- grams can significantly contribute to the production of improved cultivars [24]. Recent research has shown that manipulation of miRNA-guided gene regulation can help in the engineer- ing of stress-resistant-plant [28,29]. Although high throughput sequencing methods allowed for a better understanding of miRNA from non-model plants, the computational information available about sugarcane miRNA is scarce. To date, only 34 sugarcane miRNAs are deposited at the miRBase database current version (release 18). In this context, we carried out a compre- hensive analysis to discover novel sugarcane miRNAs Page 2 of 14 that may be regulated when subjected to drought or salt stress, and under infection of Acidovorax avenae ssp. avenae. In addition, we searched for conservation of these miRNAs in Arabidopsis, rice and maize. We dis- covered a total of 623 new mature miRNAs candidates in 10 sRNA libraries and identified up to 1,975 genes as potential targets for miRNA regulation in sugarcane. Our study substantially increases the number of known miRNA in sugarcane and it also provides detailed genotype-tissue-culture miRNA expression profile infor- mation for future studies. Results and discussion Computational identification of miRNAs candidates from sRNA libraries data In order to explore the miRNA diversity in sugarcane, we constructed and sequenced 10 sRNA libraries using RNA isolated from different cultivars of sugarcane sub- mitted to different stresses (pathogen, drought and salt). A total of 95,427,068 reads were obtained from Illumina-based sequencing, and these were used for computational identification of new miRNAs (Table 1). First, we processed raw sequence reads to remove the 3’ adaptor, “N” bases, and low complexity and sno/t/rRNA (small nuclear/transporter/ribosomal RNA) sequences, and grouped the remaining reads according to the num- ber of unique sequences, resulting in 19,670,792 unique sequences between 20–24 nucleotides (nt) (Table 1). As shown in Figure 1, the redundant sRNA sequence size distribution after the trimming and filtering procedure showed that the most abundant sRNA in sugarcane are 21nt and 24nt in length, similar to other angiosperms [30]. The second step was to search for miRNAs candi- dates using miRCat pipeline, mapped to the Sorghum bi- color (sorghum) genome. Sorghum genome (JGI, v1.0) was used as a reference because S. bicolor is the most phylogenetically related species that has a genome com- pletely sequenced (700 Mbases available) [31], while gen- omic resources for sugarcane consist only of 88 Mbases of EST sequences. We detected 867 miRNA unique Table 1 Summary of results obtained after computational data mining of small RNA libraries Description All reads Filtered readsa miRNAs detectedb Known miRNAsc Novel miRNAsd Total 95,427,068 39,424,801 2883 1,997 886 Unique 33,574,452 19,670,792 867 244 623 a Filtering for tRNA, rRNA, low-complexity sequence and trimming for “N” bases and 3’ adapters. b All miRNAs detected by miRCat pipeline. c Known miRNAs deposited at miRBase database. d Based on BLAST search.