Thiebaut et al. BMC Genomics 2012, 13:290 http://www.biomedcentral.com/1471-2164/13/290 between sRNA and target (G-U bases count as 0.5 mismatches); ii) no more than two adjacent mis- matches in the miRNA/target duplex; iii) no adjacent mismatches in positions 2–12 of the miRNA/target duplex (5' of miRNA); iii) no mismatches in positions 10–11 of miRNA/target duplex; no more than 2.5 mismatches in positions 1–12 of the of the miRNA/ target duplex; iv) and Minimum free energy (MFE) of the miRNA/target duplex should be > = 74% of the MFE of the miRNA bound to its perfect complement. All putative targets regulated by new sugarcane miR- NAs were subjected to gene ontology analysis. We extracted the unique IDs of targets and compared them with GO annotations of S. bicolor gene models (available at http://genome.jgi.doe.gov/Sorbi1/Sorbi1. download.ftp.html), S. bicolor TC annotations (avail- able at http://compbio.dfci.harvard.edu/cgi-bin/tgi/ tc_ann.pl?gudb=sorghum) and Saccharum officinarum TC annotations (available at http://compbio.dfci.har- vard.edu/cgi-bin/tgi/tc_ann.pl?gudb=s_officinarum). The GO numbers of targets were subjected to agriGO [54]. The singular enrichment analysis (SEA) was per- formed to find enriched GO terms within annotated miRNA targets. Validation of miRNA expression and target by qRT-PCR The expression profiles of nine sugarcane’s new mature miRNAs were assayed by stem–loop reverse transcription- PCR [67,68]. Total RNA extracted from leaves, roots and whole plants using in the small RNA libraries construction was treated with DNaseI (Promega). Total RNA is then re- verse transcribed into cDNA using Super-ScriptIII reverse transcriptase (Invitrogen) using in the same reaction RT primers specific for all miRNAs analyzed (Additional file 1: Figure S5). To analyze the expression profile of mature miRNA and MADS2 target, qRT-PCR was used with SYBR Green PCR Master Mix (Applied Biosystems). To each well, 1 μL of first strand cDNA, 5 μL of SYBR Green solution, 2 μL of the forward primer (10 μM) and 2 μL of reverse pri- mer (10 μM) were added (Additional file 1: Figure S4). U6 and GAPDH were used as the internal control of miRNA and MADS2 expression, respectively. qRT-PCR was per- formed using Applied Biosystems 7500 Real-Time PCR Systems. Additional file Additional file 1: Table S1. Summary of results obtained after computational data mining for each small RNA library. Overall information of all and filtered reads, numbers of miRNAs detected, known and novel miRNAs candidates. Table S2. miRCat analyses results of new miRNAs precursors. Overall information of precursors that matched to the criteria for miRNA prediction. Table S3. Correspondence among names of high confidence new microRNAs. List of names used in this paper, and the new names of microRNAs after deposit in Page 12 of 14 the miRBase. Figure S4. Predicted precursor structure of high confidence novel miRNAs (class I) identified. The sugarcane mature miRNA (red), miRNA* (pink) were illustrated in pre-miRNA with chromosome and locus information based in sorghum genome. All novel miRNAs sequences were denominated sof-miR-Seq following the number. Figure S5. Confirmation of new miRNA expression. Validation by stem-loop RT-PCR of eight new miRNAs chosen from our libraries. Primers used were listed in the table. Z. Table S6. Electronic Northern of all new miRNAs candidates in each library. The mature new miRNA reads were normalized to transcripts per million. Table S7. Putative targets of all new miRNAs candidates in sugarcane. EST sugarcane data from Gene Index version 3.0 was used to search for potential new miRNA targets by UEA sRNA toolkit-Plant target prediction pipeline. Table S8. Putative targets of all new miRNAs candidates in sorghum. EST sorghum data from Gene Index version 9.0 and Sorghum gene models (JGI v.1.0) were used to search for potential new miRNA targets by UEA sRNA toolkit- Plant target prediction pipeline. Figure S9. Relative expression profile of MADS Box. Relative expression of putative targets of sof-miR-seq09 and sof-miR-seq19 estimated within control and 1 h after saline treatment. Competing interests The authors declare that they have no competing interests. Acknowledgements We are grateful to Centro de Tecnologia Canavieira (CTC) for providing the drought experiment. Research by our group is supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Instituto Nacional de Ciência e Tecnologia em Fixação Biológica de Nitrogênio (INCT), Financiadora de Estudos e Projetos (FINEP), Fundação de Amparo à Pesquisa do Rio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We thank André H Ferreira for copy editing the manuscript. Author details 1Laboratorio de Biologia Molecular de Plantas, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rua Rodolpho Paulo Rocco s/n°, CCS, Bl.B-33A, Cidade Universitária 21941-590, Rio de Janeiro, RJ, Brazil. 2Universidade Federal da Integração Latino-Americana, Av. Tancredo Neves, 6731, Bl.4, 85867-970, Foz do Iguaçu, PR, Brazil. 3Cold Spring Harbor Laboratory, 1 Bungtown RD, Cold Spring Harbor, NY 11724, USA. 4School of Biological Sciences, The University of Queensland, St Lucia, QLD 4072, Australia. 5Fasteris SA, 1228-Plan-les-Ouates, Switzerland. Authors’ contributions FT, CG, MC-B and CAR authors contributed equally to this work; FT and CG wrote the manuscript and carried-out most of bioinformatics analyzes; MC-B performed real-time quantification RT-PCR; FT, MC-B and CAR carried-out the plant experiments and RNA extraction; PCGF and ASH coordinated the study; MT, RAM and LF participated in the libraries construction and sequencing; MT and PCGF critically revised the article. All authors read and approved the final manuscript. Received: 2 January 2012 Accepted: 2 May 2012 Published: 2 July 2012 References 1. Finnegan EJ, Matzke MA: The small RNA world. J Cell Sci 2003, 116:4689– 4693. 2. 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