BACKGROUND
small rna  has direct and versatile involvement as regulator of gene expression in many eukaryotes  <cit> .
this srna consists of small endogeneous rnas, 20– <dig> nucleotides  in length, grouped into two major classes: microrna  and small interfering rna   <cit> .
the biosynthesis of plant mirna starts with the transcription of mir genes by rna polymerase ii  into primary mirna , generating a long-single strand rna that has partial self-complementary, forming a characteristic hairpin structure.
in arabidopsis, this stem-loop precursor is processed by dicer-like protein , hyponastic leaves  <dig>  and serrata resulting in a duplex mirna-mirna*.
the duplex is methylated by hua enhancer  <dig>   <cit>  and transported from the nucleus to the cytoplasm by hasty  <cit> .
finally, one strand of the duplex, the mature mirna, is inserted into a ribonucleoprotein complex, called risc, containing the argonaute <dig>  protein  <cit>  that binds to its target messenger rna by sequence complementarity.
mirna regulates the expression of mrna target by mrna target cleavage, and through translational or transcriptional repression  <cit> .
however, in plants, the most frequent mechanism of mirna regulation is direct cleavage of mrna-target by near-perfect complementarity  <cit> .
several studies have reported that mirna-guided gene regulation is essential for developmental processes  <cit>  and for tolerance to biotic and abiotic stresses  <cit> .
due to the importance of mirna in regulation of gene expression, extensive investigation aiming at the discovery of new micrornas in several plant species has been carried out and the number of known mirnas is increasing with over  <dig>  plant-microrna deposited sequences in the mirbase  <cit> .
most of these mirnas are conserved between all plants  <cit> ; however, there are also several mirnas that are not.
some examples of this are: the mir <dig>  which was identified only in eudicots, and the mir <dig> and mir <dig> that are monocots specific  <cit> .
the process of microrna discovery is becoming faster and an increasingly larger number of novel mirnas 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  <cit> .
according to lu et al.
<cit> , nonconserved mirnas are found at low expression levels.
next generation sequence technologies have accelerated the process of small rna discovery 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  <cit> .
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  <cit> .
the commercial varieties belong to the genus saccharum l, which consists of hybrids derived from interbreeding between saccharum officinarums.
sinenses.
barberis.
robustum and s. spontaneum species  <cit> .
sugarcane has one of the most complex plant genomes, compounded by a variable ploidy number  <cit> .
yields of sugarcane crop can be reduced dramatically due to the influence of environmental factors such as salinity, drought, fungal and bacterial disease  <cit> .
a priority for breeders is to obtain varieties with increased tolerance to abiotic and biotic stresses.
because of the reduced genetic variability in saccharum hybrids, caused by recent speciation, the introduction of biotechnology tools in breeding programs can significantly contribute to the production of improved cultivars  <cit> .
recent research has shown that manipulation of mirna-guided gene regulation can help in the engineering of stress-resistant-plant  <cit> .
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  <dig> sugarcane mirnas are deposited at the mirbase database current version .
in this context, we carried out a comprehensive analysis to discover novel sugarcane mirnas 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 discovered a total of  <dig> new mature mirnas candidates in  <dig> srna libraries and identified up to  <dig>  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 information for future studies.
RESULTS
computational identification of mirnas candidates from srna libraries data
in order to explore the mirna diversity in sugarcane, we constructed and sequenced  <dig> srna libraries using rna isolated from different cultivars of sugarcane submitted to different stresses .
a total of  <dig> , <dig> reads were obtained from illumina-based sequencing, and these were used for computational identification of new mirnas .
first, we processed raw sequence reads to remove the 3’ adaptor, “n” bases, and low complexity and sno/t/rrna  sequences, and grouped the remaining reads according to the number of unique sequences, resulting in  <dig> , <dig> unique sequences between 20– <dig> nucleotides  .
as shown in figure  <dig>  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  <cit> .
the second step was to search for mirnas candidates using mircat pipeline, mapped to the sorghum bicolor  genome.
sorghum genome  was used as a reference because s. bicolor is the most phylogenetically related species that has a genome completely sequenced   <cit> , while genomic resources for sugarcane consist only of  <dig> mbases of est sequences.
we detected  <dig> mirna unique sequences mapping to the sorghum genome, and these mirnas were classified as known mirnas  and novel mirnas candidates  based on blast search against the mirbase and pmrd  databases .
overall information of all and filtered reads and numbers of mirnas for each small rna library are summarized in additional file 1: table s <dig>  most mature mirnas are evolutionarily conserved between plant lineages  <cit> .
this information enabled us to computationally predict new mirna homologs or orthologs in different plant species.
after the mirbase-based classification, we selected the sugarcane mirnas conserved in sorghum.
the information of precursors that matched the criteria described in material and methods is showed in additional file 1: table s <dig>  the length of mirnas precursors ranged from  <dig> to  <dig> nt and their precursors fold-back structures have mfe  - <dig> to −174 kcal/mol.
the mfei  is another parameter that was used to evaluate the novel mirnas precursors.
as reported previously by zhang et al.
<cit> , known plant mirna precursors have mfei value higher than others rna .
in our analysis, more than 86% of new mirnas had mfei value higher than  <dig> , suggesting that they are most to be likely mirna precursors.
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.
based on the hairpin structure profile, our sequence analysis revealed  <dig> precursors of novel mirnas candidates .
after selection of these novel mirna candidates, all  <dig> mature mirnas were provisory nominated like sof-mir-seq <dig>  due to the high frequency of mis-annotated mirna, the plant small rna research community has established a set of criteria for correct annotation of mirna  <cit> .
according to these criteria, the class i mirna precursors have mature mirna and mirna* sequence found in srna libraries.
we identified  <dig> bona fide precursors with mirna/mirna* complementarity, of which  <dig> were unique mature mirnas sequences.
these set of micrornas were considered high confidence and these sequences were deposited in the mirbase database.
the correspondent name of these sequences in the mirbase is available in the additional file 1: table s <dig>  figure  <dig> shows the example of  <dig> precursors and their mfe values.
hairpin structures that have the lowest mfe showed the mfei higher than  <dig> ; and this is strong evidence that the candidate sequence is a mirna precursor.
the structures of bona fide precursors are available in additional file 1: figure s <dig>  however, we cannot dismiss the other mirnas candidates that do not fit these criteria.
as demonstrated by zhang et al.
<cit>  there are some mirna* that are low expressed and do not necessarily appear in sequenced libraries, but the complementary mature mirna sequences were in fact mirna.
next, we analyzed the distribution of reads by the first nucleotide at 5’end .
our results showed that the majority of new mirnas candidates with  <dig> nt have uracile  at the 5’ end and the new mirnas with  <dig> nt have adenine  in the same position.
recent studies showed distinct preference of ago for small rnas with a different 5’- terminal nucleotide  <cit> .
moreover, four osagos that are related to atago <dig> have conserved histidine residue in the  <dig> position that is critical for slicer activity of mirna:ago complex  <cit> .
analysis of small rna sequences obtained by immunoprecipitation assays with anti-ago <dig> antibodies revealed the preferential association of ago <dig> with small rnas containing 5’- terminal uridine  <cit> .
similar experiments with anti-ago <dig> and anti-ago <dig> antibodies showed an enrichment of small rnas bearing a 5’ - terminal adenosine bound to ago <dig>  and ago <dig> associated with small rnas with a 5’ - terminal cytosine  <cit> .
based in the sequence similarity of the sugarcane ago genes to those of other plant species , it is possible that a similar nucleotide preference may exist on sugarcane, and the results in figure  <dig> may indicate that the majority of the new  <dig> 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  <cit> .
in this study, we used  <dig> srna libraries generated from plants of three different experiments .
a drought stress induced by withholding watering.
b salt stress induced by 170 mm solution of nacl.
c pathogen infection with a. avenae.
after trimming of sequence reads, mircat pipeline and blastn search, we identified  <dig> novel mature mirnas candidate sequences in those libraries, as described above.
the abundance of different mirnas can be inferred 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 approach .
the read counts for mirnas vary highly according to the kind of stress.
as showed previously for soybean  <cit> , new and known mirnas were regulated in water deficit and pathogen assays.
analyzing the abundance of mirnas arising from precursor class i  we found  <dig> new mirnas assay-specific and seven mirnas with abundance higher than  <dig> normalized reads counts .
mirnas sof-mir-seq <dig>  sof-mir-seq <dig>  sof-mir-seq <dig>  sof-mir-seq <dig>  sof-mir-seq <dig> and sof-mir-seq <dig> 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 .
furthermore, we observed exceptionally high abundance of sof-mir-seq <dig> and sof-mir-seq513* sequences .
we confirmed the high expression of this novel mirna and its mirna* in saline treatment assay sample treated for 1 h .
id: identification of sequences; ct: non inoculated plants; aa: under a. avenae infection; t 0 h and t 24 h: tolerant genotypes in control and drought stress conditions, respectively; s 0 h and s 24 h: sensitive genotypes in control and drought stress conditions, respectively; 0 h: plant without salt stress; 1 h, 6 h, 24 h: plants after salt treatment
the number of mature mirna reads were normalized by transcripts per million.
the mature mirna and mirna* sequences were also shown.
the analysis of mirnas arising from all precursor classes revealed differential accumulation of certain new mirna in the context of a particular stress .
only sof-mir-seq <dig> was induced constitutively in all libraries .
the biotic stress library showed higher exclusive expression of new mirnas .
figure 4a shows the distribution of the  <dig> novel sugarcane mirnas found in either treatment  or control samples  or in both .
because the control libraries were constructed with three types of tissues of different genotypes cultivated in vitro, hydroponic and soil condition, we analyzed the new mirnas distribution in all control conditions .
only one novel mirna candidate were shared between all control libraries.
in control plants of pathogen infection assay  were found specifically  <dig> new mirnas candidates and in control plants of salt stress assay   <dig> were found.
control plants of the water deficit assay  shared  <dig> new mirnas, in contrast with control plants of the pathogen infection and salt stress assays that are from the same genotype and had five new mirnas in common.
these results showed that plants grown under the same condition , independently of their genotype, share similar numbers of detectable new mirna.
many genome regions are silenced through dna methylation, histone modifications and small rna-direct dna methylation, while others are activated by the up- or down-regulation of the same epigenetic mechanisms  <cit> .
these modifications in chromatin structure may be activating much of the new mirnas detected in the present study.
interestingly, plants of the same genotype  firstly grown in vitro followed by cultivation in a hydroponic system , do not show a higher number of novel mirnas candidates .
nevertheless, these control plants showed higher amount of new mirnas than control plants of water deficit assay .
in general, the new mirnas expression in control plants seems to be genotype-tissue-culture dependent.
in order to find new sugarcane mirnas that may be involved in the regulation of the plant’s responses to stress we investigated the distribution of mirnas sequences candidates in the srna libraries from treated samples .
the number of shared new mirnas was increased  in all stressed libraries in contrast to all control libraries .
plants under pathogen infection showed the highest number of novel mirna .
in the drought stress, tolerant genotypes showed an increase of the new mirnas number  and sensitive genotypes remained unchanged.
however, in sensitive genotypes under drought stress the number of exclusive new mirnas decreased weakly .
in addition, we observed a high induction of novel exclusive mirnas candidates in the salt stress libraries, where we identified twice as many novel mirna expressed under the stress condition than in the control samples .
plants contain a complex network of small rna pathways.
the canonical pri-mirna is cleaved by dcl <dig> and results in mature mirna 21nt in size.
however, some researches described a novel class of bona fide mirna  <cit> .
this class was denominated long-mirna  and their precursors are processed by dcl <dig> as well as sirna.
contrary to sirnas that require poliv and rdr <dig> to be processed, stem-loop precursors of long-mirna are originated from polii and do not require rdr <dig>  <cit> .
the other characteristic of long-mirna is the mechanism of regulation.
recent study in rice described that long-mirna mediated dna methylation by ago <dig>  <cit> .
canonical mirnas and long-mirnas comprised more than 91 % of all mirnas species characterized in this study.
we detected different distribution of those species in stresses libraries.
in biotic stress we observed a reduction of 21-nt-species and increase in 24-nt-species, similar to salt stress and drought stress sensitive genotypes libraries .
instead, drought stress tolerant genotypes libraries showed an increase of 21-nt-species and a reduction of 24-nt-species.
these analyses suggest that biotic, salt stress and drought stress  may be regulating pathways by mirnas from the same species .
target prediction and function analysis
in plants, mirna regulate gene expression by either cleavage of mrna or translation repression targets  <cit> .
in addition, it has been described recently that some mirna may regulate gene expression by dna methylation  <cit> .
computational identification of plant mirna-target is possible due the perfect or near-perfect match between mirna/target in complementary sites  <cit> .
using two published methods  <cit>  we predicted mirna targets.
we first searched for targets of new mirnas candidates in a sugarcane database  and then in the sorghum databases .
the results of these analyses are available in additional file 1: table s <dig> and additional file 1: table s <dig>  we identified  <dig>  putative target genes for  <dig> new mirnas, with an average of  <dig> targets per mirna candidate.
in sorghum, the average of gene targets per mirna was lower than sugarcane , showing  <dig> targets for  <dig> 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  est assembly discovered  <dig> new mirnas candidates targets that had versatile functional annotation and  <dig> were of unknown function.
mads-box transcription factor mads <dig> and 60 s acidic ribosomal protein p2b  were overrepresented.
mads <dig> is a transcription factor that regulates development  <cit>  and rpp2b plays an important role in disease resistance to bacterial pathogens  <cit> .
the expression of mads <dig> was verified using qrt-pcr and an induced profile was shown after 1 h of saline treatment related to control .
this result suggests a regulation of mads <dig> by sof-mir-seq <dig> and sof-mir-seq <dig> where the new mirnas candidates were repressed after 1 h of saline treatment.
most targets annotated as mads <dig> were recognized by 21nt mirna candidate.
similarly, the majority of micrornas conserved among species target transcription factors  <cit> , however, the majority of new sugarcane mirnas candidates that regulate rpp2b have  <dig> nt in length.
according to wu et al.
<cit> , long-mirna may direct dna methylation around their recognition site in the target loci.
this type of mirna may act in their own precursors in cis and also in their targets in trans to guide dna methylation  <cit> .
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,  <dig> new mirnas candidates did not have predicted targets, of which  <dig> have  <dig> 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 possibility 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 profile was observed and the higher amount of mirnas candidates that did not have predicted targets were 24-nt-species .
the sugarcane targets regulated by high confidence new mirnas arising from the precursor class i were listed in table  <dig>  and the conserved mirna targets sites in sorghum are highlighted.
thirty three new mirnas had targets in sugarcane and  <dig> in sorghum.
targets annotated, as serine/threonine kinase, myb protein, mads-box, zinc finger protein-like were potentially regulated by different new mirnas class i. in this set, targets annotated like amp-binding protein were overrepresented.
this is a defense related protein that is involved in the regulation of defense response  <cit> .
a identification of sugarcane targets.
mirnas that only have singletons as targets were identified by no tc found.
mirnas that did not have predicted targets were identified by no target found.
b plus and minus signals indicate presence or absence  of sorghum genes containing conserved mirna recognition sites.
next, sugarcane and sorghum identified targets were subjected to gene ontology analysis.
we extracted the unique ids of targets and compared them with go annotations of s.bicolor gene models, and s.bicolor and sugarcane tc annotations.
the go numbers of targets were subjected to agrigo toolkit  <cit> .
among the distribution of go annotation of the targets, only mirna class i targets were represented in figure  <dig>  the most representative go was the metabolic process .
the enrichment of this go may be consistent with the fact that six libraries of srna were constructed from plants cultivated in vitro, which may have had their development accelerated by combination of plant hormones  <cit> .
in addition, genes involved in immune system processes and cellular responses to stresses were present there, presumably due to the stress treatments.
conservation of novel mirnas in diverse plant species
flowering plants comprise approximately  <dig>  species and originated around  <dig> million years ago  <cit> .
phylogenetic analyses have recently resolved major relationships among angiosperm group using both molecular and morphological information  <cit> .
the wide variability of the angiosperms allow their adaptation to diverse environmental conditions and also their domestication  <cit> .
the estimate of divergence time, acquired using plastid exons and rdna revealed that monocots and eudicots diverged about  <dig> million years ago  <cit> .
within monocots; rice, maize, sugarcane and sorghum  originated approximately  <dig> million years ago  <cit> .
early in the radiation of eudicots plants, the arabidopsis family  diverged   <cit> .
phylogenetic conservation analysis of novel mirnas between closely related species provides supporting evidence and has been used to annotate new mirnas  <cit> .
based on mircat pipeline we searched for the precursors of the novel sugarcane mature mirnas candidates in two monocots  and arabidopsis.
we did not identified conserved precursors of new sugarcane mirnas candidates in arabidopsis.
in rice, we identified  <dig> homologs of these mature mirnas, while in maize, the closest relative to sorghum and sugarcane of the three reference species, we identified phylogenetic evidence for  <dig> novel mirnas .
in polyploid genomes, genetic and epigenetic modifications can quickly change the structure and function of genomes  <cit> .
vincentz et al.
<cit> , 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 segmental duplication  <cit> .
they are fast-evolving sequences that may present a divergence degree reflecting the phylogenetic divergence between species  <cit> .
as mentioned above, the most phylogenetic related species showed more conserved mirna .
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 technologies 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 evolution.
our findings provide the first large scale mirna discovery in sugarcane and help to clarify about potential mirna roles in regulatory pathways of this and other crops.

 small rna has direct and versatile involvement as regulator of gene expression in many eukaryotes. this srna consists of small endogeneous rnas, 20– nucleotides in length, grouped into two major classes: microrna and small interfering rna. the biosynthesis of plant mirna starts with the transcription of mir genes by rna polymerase ii into primary mirna, generating a long-single strand rna that has partial self-complementary, forming a characteristic hairpin structure. in arabidopsis, this stem-loop precursor is processed by dicer-like protein, hyponastic leaves and serrata resulting in a duplex mirna-mirna*. the duplex is methylated by hua enhancer and transported from the nucleus to the cytoplasm by hasty. finally, one strand of the duplex, the mature mirna, is inserted into a ribonucleoprotein complex, called risc, containing the argonaute protein that binds to its target messenger rna by sequence complementarity. mirna regulates the expression of mrna target by mrna target cleavage, and through translational or transcriptional repression. however, in plants, the most frequent mechanism of mirna regulation is direct cleavage of mrna-target by near-perfect complementarity. several studies have reported that mirna-guided gene regulation is essential for developmental processes and for tolerance to biotic and abiotic stresses. due to the importance of mirna in regulation of gene expression, extensive investigation aiming at the discovery of new micrornas in several plant species has been carried out and the number of known mirnas is increasing with over plant-microrna deposited sequences in the mirbase. most of these mirnas are conserved between all plants; however, there are also several mirnas that are not. some examples of this are: the mir which was identified only in eudicots, and the mir and mir that are monocots specific. the process of microrna discovery is becoming faster and an increasingly larger number of novel mirnas 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. according to lu et al., nonconserved mirnas are found at low expression levels. next generation sequence technologies have accelerated the process of small rna discovery 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. 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. the commercial varieties belong to the genus saccharum l, which consists of hybrids derived from interbreeding between saccharum officinarums. sinenses. barberis. robustum and s. spontaneum species. sugarcane has one of the most complex plant genomes, compounded by a variable ploidy number. yields of sugarcane crop can be reduced dramatically due to the influence of environmental factors such as salinity, drought, fungal and bacterial disease. a priority for breeders is to obtain varieties with increased tolerance to abiotic and biotic stresses. because of the reduced genetic variability in saccharum hybrids, caused by recent speciation, the introduction of biotechnology tools in breeding programs can significantly contribute to the production of improved cultivars. recent research has shown that manipulation of mirna-guided gene regulation can help in the engineering of stress-resistant-plant. 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 sugarcane mirnas are deposited at the mirbase database current version. in this context, we carried out a comprehensive analysis to discover novel sugarcane mirnas 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 discovered a total of new mature mirnas candidates in srna libraries and identified up to 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 information for future studies.  computational identification of mirnas candidates from srna libraries data in order to explore the mirna diversity in sugarcane, we constructed and sequenced srna libraries using rna isolated from different cultivars of sugarcane submitted to different stresses. a total of, reads were obtained from illumina-based sequencing, and these were used for computational identification of new mirnas. first, we processed raw sequence reads to remove the 3’ adaptor, “n” bases, and low complexity and sno/t/rrna sequences, and grouped the remaining reads according to the number of unique sequences, resulting in, unique sequences between 20– nucleotides. as shown in figure 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. the second step was to search for mirnas candidates using mircat pipeline, mapped to the sorghum bicolor genome. sorghum genome was used as a reference because s. bicolor is the most phylogenetically related species that has a genome completely sequenced, while genomic resources for sugarcane consist only of mbases of est sequences. we detected mirna unique sequences mapping to the sorghum genome, and these mirnas were classified as known mirnas and novel mirnas candidates based on blast search against the mirbase and pmrd databases. overall information of all and filtered reads and numbers of mirnas for each small rna library are summarized in additional file 1: table s most mature mirnas are evolutionarily conserved between plant lineages. this information enabled us to computationally predict new mirna homologs or orthologs in different plant species. after the mirbase-based classification, we selected the sugarcane mirnas conserved in sorghum. the information of precursors that matched the criteria described in material and methods is showed in additional file 1: table s the length of mirnas precursors ranged from to nt and their precursors fold-back structures have mfe - to −174 kcal/mol. the mfei is another parameter that was used to evaluate the novel mirnas precursors. as reported previously by zhang et al., known plant mirna precursors have mfei value higher than others rna. in our analysis, more than 86% of new mirnas had mfei value higher than, suggesting that they are most to be likely mirna precursors. 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. based on the hairpin structure profile, our sequence analysis revealed precursors of novel mirnas candidates. after selection of these novel mirna candidates, all mature mirnas were provisory nominated like sof-mir-seq due to the high frequency of mis-annotated mirna, the plant small rna research community has established a set of criteria for correct annotation of mirna. according to these criteria, the class i mirna precursors have mature mirna and mirna* sequence found in srna libraries. we identified bona fide precursors with mirna/mirna* complementarity, of which were unique mature mirnas sequences. these set of micrornas were considered high confidence and these sequences were deposited in the mirbase database. the correspondent name of these sequences in the mirbase is available in the additional file 1: table s figure shows the example of precursors and their mfe values. hairpin structures that have the lowest mfe showed the mfei higher than; and this is strong evidence that the candidate sequence is a mirna precursor. the structures of bona fide precursors are available in additional file 1: figure s however, we cannot dismiss the other mirnas candidates that do not fit these criteria. as demonstrated by zhang et al. there are some mirna* that are low expressed and do not necessarily appear in sequenced libraries, but the complementary mature mirna sequences were in fact mirna. next, we analyzed the distribution of reads by the first nucleotide at 5’end. our results showed that the majority of new mirnas candidates with nt have uracile at the 5’ end and the new mirnas with nt have adenine in the same position. recent studies showed distinct preference of ago for small rnas with a different 5’- terminal nucleotide. moreover, four osagos that are related to atago have conserved histidine residue in the position that is critical for slicer activity of mirna:ago complex. analysis of small rna sequences obtained by immunoprecipitation assays with anti-ago antibodies revealed the preferential association of ago with small rnas containing 5’- terminal uridine. similar experiments with anti-ago and anti-ago antibodies showed an enrichment of small rnas bearing a 5’ - terminal adenosine bound to ago and ago associated with small rnas with a 5’ - terminal cytosine. based in the sequence similarity of the sugarcane ago genes to those of other plant species, it is possible that a similar nucleotide preference may exist on sugarcane, and the results in figure may indicate that the majority of the new 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. in this study, we used srna libraries generated from plants of three different experiments. a drought stress induced by withholding watering. b salt stress induced by 170 mm solution of nacl. c pathogen infection with a. avenae. after trimming of sequence reads, mircat pipeline and blastn search, we identified novel mature mirnas candidate sequences in those libraries, as described above. the abundance of different mirnas can be inferred 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 approach. the read counts for mirnas vary highly according to the kind of stress. as showed previously for soybean, new and known mirnas were regulated in water deficit and pathogen assays. analyzing the abundance of mirnas arising from precursor class i we found new mirnas assay-specific and seven mirnas with abundance higher than normalized reads counts. mirnas sof-mir-seq sof-mir-seq sof-mir-seq sof-mir-seq sof-mir-seq and sof-mir-seq 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. furthermore, we observed exceptionally high abundance of sof-mir-seq and sof-mir-seq513* sequences. we confirmed the high expression of this novel mirna and its mirna* in saline treatment assay sample treated for 1 h. id: identification of sequences; ct: non inoculated plants; aa: under a. avenae infection; t 0 h and t 24 h: tolerant genotypes in control and drought stress conditions, respectively; s 0 h and s 24 h: sensitive genotypes in control and drought stress conditions, respectively; 0 h: plant without salt stress; 1 h, 6 h, 24 h: plants after salt treatment the number of mature mirna reads were normalized by transcripts per million. the mature mirna and mirna* sequences were also shown. the analysis of mirnas arising from all precursor classes revealed differential accumulation of certain new mirna in the context of a particular stress. only sof-mir-seq was induced constitutively in all libraries. the biotic stress library showed higher exclusive expression of new mirnas. figure 4a shows the distribution of the novel sugarcane mirnas found in either treatment or control samples or in both. because the control libraries were constructed with three types of tissues of different genotypes cultivated in vitro, hydroponic and soil condition, we analyzed the new mirnas distribution in all control conditions. only one novel mirna candidate were shared between all control libraries. in control plants of pathogen infection assay were found specifically new mirnas candidates and in control plants of salt stress assay were found. control plants of the water deficit assay shared new mirnas, in contrast with control plants of the pathogen infection and salt stress assays that are from the same genotype and had five new mirnas in common. these results showed that plants grown under the same condition, independently of their genotype, share similar numbers of detectable new mirna. many genome regions are silenced through dna methylation, histone modifications and small rna-direct dna methylation, while others are activated by the up- or down-regulation of the same epigenetic mechanisms. these modifications in chromatin structure may be activating much of the new mirnas detected in the present study. interestingly, plants of the same genotype firstly grown in vitro followed by cultivation in a hydroponic system, do not show a higher number of novel mirnas candidates. nevertheless, these control plants showed higher amount of new mirnas than control plants of water deficit assay. in general, the new mirnas expression in control plants seems to be genotype-tissue-culture dependent. in order to find new sugarcane mirnas that may be involved in the regulation of the plant’s responses to stress we investigated the distribution of mirnas sequences candidates in the srna libraries from treated samples. the number of shared new mirnas was increased in all stressed libraries in contrast to all control libraries. plants under pathogen infection showed the highest number of novel mirna. in the drought stress, tolerant genotypes showed an increase of the new mirnas number and sensitive genotypes remained unchanged. however, in sensitive genotypes under drought stress the number of exclusive new mirnas decreased weakly. in addition, we observed a high induction of novel exclusive mirnas candidates in the salt stress libraries, where we identified twice as many novel mirna expressed under the stress condition than in the control samples. plants contain a complex network of small rna pathways. the canonical pri-mirna is cleaved by dcl and results in mature mirna 21nt in size. however, some researches described a novel class of bona fide mirna. this class was denominated long-mirna and their precursors are processed by dcl as well as sirna. contrary to sirnas that require poliv and rdr to be processed, stem-loop precursors of long-mirna are originated from polii and do not require rdr. the other characteristic of long-mirna is the mechanism of regulation. recent study in rice described that long-mirna mediated dna methylation by ago. canonical mirnas and long-mirnas comprised more than 91 % of all mirnas species characterized in this study. we detected different distribution of those species in stresses libraries. in biotic stress we observed a reduction of 21-nt-species and increase in 24-nt-species, similar to salt stress and drought stress sensitive genotypes libraries. instead, drought stress tolerant genotypes libraries showed an increase of 21-nt-species and a reduction of 24-nt-species. these analyses suggest that biotic, salt stress and drought stress may be regulating pathways by mirnas from the same species. target prediction and function analysis in plants, mirna regulate gene expression by either cleavage of mrna or translation repression targets. in addition, it has been described recently that some mirna may regulate gene expression by dna methylation. computational identification of plant mirna-target is possible due the perfect or near-perfect match between mirna/target in complementary sites. using two published methods we predicted mirna targets. we first searched for targets of new mirnas candidates in a sugarcane database and then in the sorghum databases. the results of these analyses are available in additional file 1: table s and additional file 1: table s we identified putative target genes for new mirnas, with an average of targets per mirna candidate. in sorghum, the average of gene targets per mirna was lower than sugarcane, showing targets for 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 est assembly discovered new mirnas candidates targets that had versatile functional annotation and were of unknown function. mads-box transcription factor mads and 60 s acidic ribosomal protein p2b were overrepresented. mads is a transcription factor that regulates development and rpp2b plays an important role in disease resistance to bacterial pathogens. the expression of mads was verified using qrt-pcr and an induced profile was shown after 1 h of saline treatment related to control. this result suggests a regulation of mads by sof-mir-seq and sof-mir-seq where the new mirnas candidates were repressed after 1 h of saline treatment. most targets annotated as mads were recognized by 21nt mirna candidate. similarly, the majority of micrornas conserved among species target transcription factors, however, the majority of new sugarcane mirnas candidates that regulate rpp2b have nt in length. according to wu et al., long-mirna may direct dna methylation around their recognition site in the target loci. this type of mirna may act in their own precursors in cis and also in their targets in trans to guide dna methylation. 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, new mirnas candidates did not have predicted targets, of which have 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 possibility 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 profile was observed and the higher amount of mirnas candidates that did not have predicted targets were 24-nt-species. the sugarcane targets regulated by high confidence new mirnas arising from the precursor class i were listed in table and the conserved mirna targets sites in sorghum are highlighted. thirty three new mirnas had targets in sugarcane and in sorghum. targets annotated, as serine/threonine kinase, myb protein, mads-box, zinc finger protein-like were potentially regulated by different new mirnas class i. in this set, targets annotated like amp-binding protein were overrepresented. this is a defense related protein that is involved in the regulation of defense response. a identification of sugarcane targets. mirnas that only have singletons as targets were identified by no tc found. mirnas that did not have predicted targets were identified by no target found. b plus and minus signals indicate presence or absence of sorghum genes containing conserved mirna recognition sites. next, sugarcane and sorghum identified targets were subjected to gene ontology analysis. we extracted the unique ids of targets and compared them with go annotations of s.bicolor gene models, and s.bicolor and sugarcane tc annotations. the go numbers of targets were subjected to agrigo toolkit. among the distribution of go annotation of the targets, only mirna class i targets were represented in figure the most representative go was the metabolic process. the enrichment of this go may be consistent with the fact that six libraries of srna were constructed from plants cultivated in vitro, which may have had their development accelerated by combination of plant hormones. in addition, genes involved in immune system processes and cellular responses to stresses were present there, presumably due to the stress treatments. conservation of novel mirnas in diverse plant species flowering plants comprise approximately species and originated around million years ago. phylogenetic analyses have recently resolved major relationships among angiosperm group using both molecular and morphological information. the wide variability of the angiosperms allow their adaptation to diverse environmental conditions and also their domestication. the estimate of divergence time, acquired using plastid exons and rdna revealed that monocots and eudicots diverged about million years ago. within monocots; rice, maize, sugarcane and sorghum originated approximately million years ago. early in the radiation of eudicots plants, the arabidopsis family diverged. phylogenetic conservation analysis of novel mirnas between closely related species provides supporting evidence and has been used to annotate new mirnas. based on mircat pipeline we searched for the precursors of the novel sugarcane mature mirnas candidates in two monocots and arabidopsis. we did not identified conserved precursors of new sugarcane mirnas candidates in arabidopsis. in rice, we identified homologs of these mature mirnas, while in maize, the closest relative to sorghum and sugarcane of the three reference species, we identified phylogenetic evidence for novel mirnas. in polyploid genomes, genetic and epigenetic modifications can quickly change the structure and function of genomes. vincentz et al., 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 segmental duplication. they are fast-evolving sequences that may present a divergence degree reflecting the phylogenetic divergence between species. as mentioned above, the most phylogenetic related species showed more conserved mirna. 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.  we have shown that next-generation sequencing technologies 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 evolution. our findings provide the first large scale mirna discovery in sugarcane and help to clarify about potential mirna roles in regulatory pathways of this and other crops. 
our findings contribute to knowledge of mirna roles in regulatory pathways in the complex, polyploidy sugarcane genome. deep sequencing analyses accelerate the process of small rna discovery in many plants and expand our understanding of mirna-regulated processes. the biological function of the new mirnas candidates was assessed by analyzing their putative targets. a large scale investigation of srna in sugarcane using a computational approach has identified a substantial number of new mirnas and provides detailed genotype-tissue-culture mirna expression profiles. we therefore undertook small rna sequencing of sugarcane mirnas in order to understand their complexity and to explore their role in sugarcane biology. a bioinformatics search was carried out to discover novel mirnas that can be regulated in sugarcane plants submitted to drought and salt stresses, and under pathogen infection. by means of the presence of mirna precursors in the related sorghum genome, we identified candidates of new mature mirnas in sugarcane. additionally, a mads-box transcription factor and an rpp2b protein, which act in development and disease resistant processes, could be regulated by cleavage and dna methylation, respectively. microrna-regulation of gene expression plays a key role in the development and response to biotic and abiotic stresses.
 microrna-regulation of gene expression plays a key role in the development and response to biotic and abiotic stresses. deep sequencing analyses accelerate the process of small rna discovery in many plants and expand our understanding of mirna-regulated processes. we therefore undertook small rna sequencing of sugarcane mirnas in order to understand their complexity and to explore their role in sugarcane biology.  a bioinformatics search was carried out to discover novel mirnas that can be regulated in sugarcane plants submitted to drought and salt stresses, and under pathogen infection. by means of the presence of mirna precursors in the related sorghum genome, we identified candidates of new mature mirnas in sugarcane. of these, were classified as high confidence mirnas. the biological function of the new mirnas candidates was assessed by analyzing their putative targets. the set of bona fide sugarcane mirna includes those likely targeting serine/threonine kinases, myb and zinc finger proteins. additionally, a mads-box transcription factor and an rpp2b protein, which act in development and disease resistant processes, could be regulated by cleavage and dna methylation, respectively.  a large scale investigation of srna in sugarcane using a computational approach has identified a substantial number of new mirnas and provides detailed genotype-tissue-culture mirna expression profiles. comparative analysis between monocots was valuable to clarify aspects about conservation of mirna and their targets in a plant whose genome has not yet been sequenced. our findings contribute to knowledge of mirna roles in regulatory pathways in the complex, polyploidy sugarcane genome. small rnabiotic stressabiotic stressdeep sequencing 