BACKGROUND
micrornas  are 20 ~  <dig> nucleotide  non-coding small rnas .
most primary mirnas  are transcribed from mirna genes by rna polymerase ii , while others are transcribed by rna polymerase iii   <cit> .
in plants, the nascent pri-mirna transcripts are first processed into  <dig> to  <dig> nt pre-mirnas by dicer-like <dig>   <cit> .
then, the pre-mirnas undergo a second cleavage in the nucleus, releasing an rna duplex containing the mature mirna and the mirna* sequences.
to maintain the correct size and/or to protect them from polyuridylation  <cit> , the duplexes are methylated at the 3′ ends by hua enhancer  <cit>  and then transported into the cytoplasm by hasty  <cit> .
the mature mirna is incorporated into an rna-induced silencing complex  that contains the argonaute  protein.
the complex recognizes partially complementary sequences in the target messenger rna  where it binds, inducing its target mrna’s degradation or a combination of both degradation and translation inhibition  <cit> .
the mirna* is released and rapidly degraded   <cit> .
the presence of the mirna* has been regarded as a good standard way to reliably annotate a novel mirna  <cit> .
mirtrons are a new, recently discovered, type of mirnas that originate from spliced introns of gene transcripts.
mirtrons reside within the intronic regions of genes and are processed through a drosha-independent pathway, making them quite distinct from the other mirnas.
the mirtron pathway was first identified in drosophila melanogaster, caenorhabditis elegans, and some mammals  <cit>  and has recently been found in arabidopsis thaliana and oryza sativa <cit> .
recent research shows that the mirnas from the intronic regions of genes can survive together with their host genes and support their host genes by mediating synergistic and antagonistic regulatory effects  <cit> .
in plant, mirnas are known to play crucial roles in many developmental events  <cit>  and regulate target transcripts through two modes of action: degradation and translation inhibition  <cit> .
the mirna degradation occurs through mirna-guided transcript cleavage in plants  <cit> .
although the mechanisms involved in the translational inhibition by mirnas are largely unknown, recent studies from xuemei chen’s group show that the mirna translational inhibition occurs at the endoplasmic reticulum  and that altered meristem program <dig>  activity allows for the dissection of mirna-mediated target rna cleavage from mirna-mediated target translation inhibition  <cit> .
with the advent of second generation sequencing technology, the rate of mirna discovery has increased dramatically  <cit> .
in dicotyledonous species,  <dig> mature mirnas in a. thaliana,  <dig> in populus trichocarpa, and  <dig> in nicotiana tabacum have been discovered, and, in monocotyledonous species,  <dig> mature mirnas in o. sativa,  <dig> in saccharum offcinarum,  <dig> in sorghum bicholor,  <dig> in triticum aestivum and  <dig> in zea mays have been reported .
although foxtail millet is an important cereal crop, only a few mirnas in foxtail millet have been reported.
bennetzen et al.
<cit>  reported some known mirnas from the alignment of mature mirna sequences, but they did not investigate whether or not the corresponding precursors existed in the genome.
foxtail millet is a diploid c <dig> panicoid crop species  <cit> .
it is an important grass crop  that has been planted widely in china.
the genome of foxtail millet  has been sequenced  <cit> , making it a possible genetic resource that could be used to investigate plant architecture, genome evolution and physiology in the bioenergy grasses.
because of the important roles that mirnas play in gene regulatory networks  <cit> , it is important to identify the mirnas in foxtail millet and to investigate their potential target genes to gain a better understanding of the biological processes in this plant.
in the present study, we aimed to characterize the mirna repertoire of foxtail millet.
RESULTS
analysis of smrnas in foxtail millet
to identify mirnas in foxtail millet, we sequenced smrnas for shoots  by high throughput illumina sequencing technology  <cit> .
nearly  <dig> million reads of 18 ~  <dig> nt, representing over  <dig> million unique sequences, were generated.
in addition, three smrna datasets  of foxtail millet were downloaded from the comparative sequencing of plant small rnas web site  <cit> .
these datasets contained over  <dig> million smrnas of 18 ~  <dig> nt, representing over  <dig> million unique sequences.
an analysis of the data from the four datasets  revealed that a large number of sequences appeared only once.
the percentages of these singletons were  <dig> %  in shoot,  <dig> %  in leaf,  <dig> %  in flower and  <dig> %  in root.
in previous reports, only 65% and 82% singletons were found in arabidopsis <cit>  and rice  <cit> , respectively, suggesting that the smrnas in foxtail millet are as complex as they are in rice, and more complex than in arabidopsis.
the overall size distribution of the unique reads from four sequencing efforts were very similar, with the  <dig> nt smrnas being the most abundant, followed by the  <dig> nt and  <dig> nt smrnas , which differ from the size distribution of total reads .
a detailed comparison of the smrnas derived from unique reads  or total reads  revealed some features of smrna species.
frist, the  <dig> nt smrnas dominated the pool of unique species in foxtail millet as observed for many other species such as a. thaliana <cit>  and cucumber  <cit> .
second, the  <dig> nt smrnas replicated in even higher frequencies  than  <dig> nt smrnas in all four tissues.
the average times  of  <dig> nt are nearly  <dig> ,  <dig> ,  <dig>  and  <dig>  and the average times of  <dig> nt are nearly  <dig> ,  <dig> ,  <dig>  and  <dig>  in shoot, leaf, flower and root, respectively.
third, mirnas, most of which were  <dig> to  <dig> nt in length, were relatively abundant in root and leaf, while sirnas, most of which were  <dig> nt long, were relatively more prevalent in flower and shoot.
these results were consistent with those reported previously in maize  <cit> .
in foxtail millet, smrna populations in four tissues are extremely complex.
although we have over  <dig> million reads, sequences of smrnas have a limited overlap from the four databases .
only  <dig>  unique sequences appeared in all four tissues and a small portion of the sequences overlapped between two tissues.
the smrnas in each tissue accounted for almost half of all the smrnas in the combined datasets.
this limited overlap indicated that there was a diversity of smrnas in foxtail millet.
identification of known mirnas in foxtail millet
in plants, mirnas are obtained by the precise excision of ~ <dig> nt smrnas from the stem of a single-strand, stem-loop precursor.
as a result, mirnas can be identified by looking for a potential fold-back precursor structure that contains the ~ <dig> nt mirna sequence within one arm of the hairpin.
the hairpin must have the lowest free energy of all the alternative folds for that sequence, as predicted by rna folding programs such as mfold  <cit>  or rnafold  <cit> .
the minimal folding free energies index  can be treated as the main feature to distinguish potential mirna precursors from other rnas.
it has been reported that more than 90% of mirna precursors have a mfei value greater than  <dig> , while the mfei for other rnas  are lower  <cit> .
we used a previously reported workflow  <cit>  to identify mirnas among the smrnas from foxtail millet, but with the mfei values set to  <dig>  rather than to  <dig>  as was done previously.
we identified  <dig> candidate mirnas in the foxtail millet smrna datasets;  <dig> in shoot,  <dig> in leaf,  <dig> in flower and  <dig> in root.
the proportion of mirna reads  in each library as compared to the total reads were  <dig> % in root,  <dig> % in leaf,  <dig> % in flower and  <dig> % in shoot.
among the  <dig> mirnas,  <dig> unique mature mirna sequences belonging to  <dig> mirna families were 100% identical to mature mirnas from  <dig> well-studied species  <cit> .
we defined the  <dig> unique mature mirnas in foxtail millet as known mirnas .
there were  <dig> known mirnas in root,  <dig> in leaf,  <dig> in flower and  <dig> in shoot tissues .
the  <dig> sit-mirnas corresponded to  <dig> pre-mirnas, of which  <dig> had corresponding mirna* forms.
detailed information about the known sit-mirnas is shown in additional file  <dig>
a summary of the total expression profiles for each of the  <dig> mirna families to which the  <dig> sit-mirnas belong is shown in figure  <dig>  the number of mirna reads were enumerated and normalized against the total count of smrna reads, reported as reads per million , for each respective library.
the results showed that the total expression of these  <dig> mirna families was relatively higher in leaf and root than that in shoot and flower.
we noted that the sit-mir <dig>  sit-mir <dig>  sit-mir <dig>  sit-mir <dig> and sit-mir <dig> families showed relatively higher expression  in one or more of the four tissues.
in contrast, the sit-mir <dig>  sit-mir <dig>  sit-mir <dig>  sit-mir <dig> and sit-mir <dig> families showed low expression  levels.
in our samples, the following mirna families showed distinct expression patterns: mir <dig> is barely detected in roots; mir <dig> is practically absent in leaves; mir <dig> and mir <dig> are practically absent in flowers; mir <dig> and mir <dig> are detected only in shoots; and mir <dig> is barely detectable in any of the four tissues.
our data also showed that some members of the same mirna family had different tissue-specific expression patterns .
for example, based on rpm counts, mir156b is highly expressed in flower and shoot but has almost no expression in leaf and root.
while the mir156d is highly expressed in leaf and root but has almost no expression in the flower.
this should be confirmed by other molecular techniques.
together, these results suggest that members from different families and different members from the same family may have greatly different effects on foxtail millet development.
novel mirna identification
our results showed that in addition to the  <dig> known mature sit-mirnas, there were  <dig> novel mirnas  in the smrna datasets that have never been reported in other species.
because some of these mirnas were derived from multiple precursors, these  <dig> nov-sit-mirnas corresponded to  <dig> pre-mirnas, 68% of which were located in intergenic regions.
only a few of the  <dig> pre-rnas were located in introns or in the utrs of coding genes, which is in agreement with the reported statistical characteristics of plant pre-mirnas  <cit>  .
among the  <dig> nov-sit-mirnas, nine were found in all four libraries, nine were in three libraries,  <dig> were in two and  <dig> were in one library.
these potentially nov-sit-mirnas had relatively lower expression levels than the  <dig> known sit-mirnas.
for example, in leaf, the total expression levels of the  <dig> nov-sit-mirnas  were less than 1% of the total expression levels of the  <dig> sit-mirnas  and, in flower, the total expression levels of all the nov-sit-mirnas were only  <dig> rpm.
these results were consistent with a study in rice that found that most of the non-conserved mirnas exhibited tissue-specific expression patterns and had relatively low expression levels compared with known mirnas  <cit> .
for example, nov-sit-mir <dig>  nov-sit-mir <dig> and nov-sit-mir <dig> were expressed only in flowers with expression levels of more than  <dig> rpm, and nov-sit-mir <dig> and nov-sit-mir <dig> were expressed only in roots with expression levels of more than  <dig> rpm .
the conservation of mirna sequences across species could be regarded as powerful evidence to annotate mirna  <cit> .
we identified conserved mirna by allowing no more than three mismatches between the mature mirna sequences in our datasets and the mature mirna sequences from  <dig> well-studied species  <cit>  published in mirbase .
we identified  <dig> nov-sit-mirnas that could be assigned to  <dig> published mirna families based on sequence similarity .
these ‘conserved’ nov-sit-mirnas generally had higher expression levels than the ‘non-conserved’ nov-sit-mirnas.
eight of the nov-sit-mirnas were validated experimentally by stem-loop rt-pcr and sequencing .
the presence of a mirna* could be regarded as additional support for the annotation of the nov-sit-mirnas  <cit> .
we noted that  <dig> of the  <dig> novel pre-mirnas were found having mirna*s. a nucleotide composition analysis showed that 50% of the novel pre-mirnas started with u , which is consistent with the statistical characteristic of mature mirnas  <cit> , and, overall, a higher percentage of a and u compared with c and g was present.
detailed information about novel mirnas was shown in additional file  <dig> and second structure of some novel mirna precursors were presented in additional file  <dig>
to predict mirtrons, we analyzed the secondary structure features of the introns of the foxtail millet genes that were annotated in the phytozome database .
introns can be folded directly to acquire stem–loop structures similar to the pre-mirnas.
using rnafold, we found many introns that had reasonable second structures; that is, a single stem-loop with less than  <dig> nt overhangs at both ends, and a minimum energy of -25 kcal/mol or less.
after mapping the smrnas in our datasets onto the selected introns using the blast algorithm , we found only a few perfectly matched smrnas; therefore, only two mirtron precursor candidates could be identified  .
target prediction and expression validated
mirnas are known to have diverse expression patterns and play regulatory roles in various developmental and physiological processes  <cit> .
in plants, most mirnas are known to regulate development by mediating the destruction of their target mrnas  <cit> .
the target gene has sites that are almost completely complementary to the mirna, so, in plants, mirna target genes can be predicted computationally.
to increase the reliability of our target prediction, we used the modified scoring described by sun et al.
<cit> .
we used the sit-mirna and nov-sit-mirna sequences in our datasets to search for complementary annotated foxtail millet transcript genes in the phytozome database  <cit>  and identified  <dig> candidate target genes for  <dig> of the foxtail millet mirnas .
we randomly picked six target genes to validate using 5′race and four were successfully validated  .
the si016508m protein, which is encoded by one of the predicted target genes, is a gras  transcription factor that has been reported to be involved in development and other processes  <cit> .
the si016508m gene is potentially targeted by five of the sit-mirnas .
the si005991m, si016509m and si034525m protein, which are encoded by a predicted target gene , is known to have three different domains: a b <dig> dna-binding domain, an aux/iaa-arf dimerization motif, and a dna-binding pseudobarrel domain.
previous studies have suggested that for efficient cleavage by mirnas, base pairing between bases  <dig> and  <dig> is essential  <cit> .
the 5′race results showed that the si016508m gene had two breakpoints .
the predicted target sites of these genes showed that sit-mir171a and sit-mir171b guided si016508m cleavage at 1085 bp, while nov-sit-mir <dig>  nov-sit-mir <dig> and nov-sit-mir <dig> guided si016508m cleavage at 1082 bp .
to show that these mirnas, which have validated their targets, are actually expressed, we used the stem-loop rt-pcr method of detection.
we detected mirna <dig>  mirna171a, nov-sit-mir <dig>  nov-sit-mir <dig> and nov-sit-mir <dig> successfully.
however, we failed to detected mirna171b .
mapping of mrna cleavage sites by rna ligase-mediated 5′ race.
the complementary pattern of mirna sequences and partial sequences of the target mrnas are shown in the table.
all predicted mismatch base parings are shown in bold letters.
the positions inferred as 5′ ends of mirna-guided cleavage products, as revealed by 5′ race, and the numbers of sequenced 5′ race clones corresponding to each site are indicated by vertical arrowheads.
about 79%  of the predicted target genes had functional annotations in interpro, the integrated resource for protein families, domains, and functional sites .
a large proportion of the predicted targets were annotated as transcription factors; for example, the ap2/erf domain, homeodomain, and ccaat-binding transcription factors.
in addition, some of the predicted targets were annotated as being involved in various classes of molecular functions, including binding proteins , calcium ion transport proteins, and enzymes .
the functions of predicted mirna targets indicated that the mirnas may play multiple roles in gene regulation networks.
to better understand the functional roles of the predicted mirna target genes in foxtail millet, we analyzed the functional enrichment for all the mirna targets by gene ontology   <cit> .
the mirna predicted targets showed enrichment in go terms from the molecular function and biological process categories, while no enrichment in go terms was observed in the cellular component category.
we found  <dig> go terms in the biological process category that showed strong enrichment in biosynthetic process, cellular process, macromolecule metabolic process, regulation of metabolic process and regulation of cellular process.
in the molecular function category, the enriched go terms included binding, nucleic acid binding, and protein-binding.
the go enrichment analysis showed that the predicted targets of the mirnas were involved in a wide range of regulatory functions as well as some specific biological processes like metabolism, biosynthesis, and gene expression/transcription .
to analyze whether or not mirnas in different tissues have different functions, go enrichment analyses were conducted for the predicted mirna targets in each of the four tissues.
we found that the functions of the targets were similar in all four tissues; the targets were all enriched in biological regulation, biosynthetic process, macromolecule metabolic process and cellular process.
in addition, some of the predicted targets had annotations in the cellular component category, including cell, and cell part in leaf and flower.
in the root dataset, the target genes were annotated with significantly fewer go terms than the target genes in the other three tissues.
details of the go annotations for the target genes in the four tissues are available in additional file  <dig>
to confirm the accuracy and reliability of the smrna-seq results, we randomly chose ten of the mirnas  for stem-loop rt-pcr validation .
the results showed that the stem-loop rt-pcr expression profiles of most mirnas match the smrna-seq data closely.
while there were three mirnas expression profiles  that were not very consistent.
conservation and synteny with sorghum
previous reports in arabidopsis <cit>  and in rice  <cit>  have shown that mirna gene families evolved from segmental, tandem repeat and whole-genome duplication events.
a detailed description of the fate of mirna genes after whole-genome duplication in maize and sorghum has been published  <cit> .
to date, mirnas expression and function has been well studied in the major food and feed crops, such as sorghum, maize and rice  <cit> .
foxtail millet shares a common ancestor with sorghum and maize .
this ancestry is more recent than with rice   <cit> .
sorghum has experienced one whole genome duplication and maize was experienced two whole genome duplications since they diverged   <cit> .
to investigate the evolution of foxtail millet mirnas, we used sorghum as the reference comparison strain.
the syntenic analysis was performed between the foxtail millet and sorghum genes.
based on the total number of genes in foxtail millet and in sorghum, we found  <dig> syntenic blocks containing  <dig>  pairs of syntenic genes between the two species.
in total,  <dig>  foxtail millet and  <dig>  sorghum genes, and  <dig> foxtail millet and  <dig> sorghum pre-mirnas, were found within the syntenic regions.
nearly all the pre-mirnas were located in only  <dig>  of the  <dig> syntenic blocks, but within these  <dig> blocks there were  <dig>  syntenic gene pairs corresponding to 45% of the total number of syntenic genes in the two species.
we identified  <dig> pairs of syntenic pre-mirnas, and all the foxtail millet and sorghum chromosomes had blocks that contained at least one of the conserved syntenic pre-mirnas .
we noted that  <dig> of the novel mirnas that we identified in foxtail millet had syntenic pre-mirna sequences and conserved mature mirna sequences .
therefore, in some respects, the  <dig> foxtail millet mirnas are new members of already known mirna families.
for example, nov-sit-mir <dig> and nov-sit-mir <dig> could be considered to be new members of the mir <dig> family; similarly, nov-sit-mir <dig>  nov-sit-mir <dig> and nov-sit-mir <dig> are new members of the mir <dig> family.
discussion
some mirnas are expressed only in a specific tissue or at a specific site, and an understanding of their unique expression pattern may help in discovering the function of a particular mirna.
to assess the tissue specificity of the mirnas in our datasets, we investigated the expression levels of the mirna in the four foxtail millet tissues .
we found that the expression of the sit-mir <dig> family was higher in the adult leaf and flower datasets and lower in the young shoot dataset.
in maize, the mir <dig> that is responsible for the transition from juvenile to adult was reported to have the same expression pattern  <cit> .
in addition, compared with the other sit-mirna families, the sit-mir <dig> family was highly expressed, especially in leaf .
in maize, the target gene of mir <dig> is rolled leaf <dig>  which can regulate leaf morphogenesis  <cit> .
similarly, in foxtail millet, the target gene of sit-mir <dig> was predicted as si034251m, a homolog of rolled leaf <dig>  this finding suggests that there may be a similar mechanism of leaf morphogenesis in foxtail millet as the one reported in maize.
we found that, except for a few of the sit-mirnas that had relatively high expression levels, most of the sit-mirnas, especially the nov-sit-mirnas, had low expression levels in the four studied tissues.
it has been suggested that the normal physical functions of an organism require various mirnas working together for precise regulation and control rather than individual mirnas working separately  <cit> .
if this suggestion is correct, then the mirnas with low expression might have an effect on the growth of foxtail millet as great as the mirnas with high expression.
a previous report indicated that most of the mirna targets in maize are conserved across several plant species  <cit>  and we found that this was also true in foxtail millet.
for example, mir <dig> and mir <dig> were predicted to target genes that encode sbp-box transcription factors  <cit> , and mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir <dig> were reported to target no apical meristem   <cit> , ccaat-binding factor   <cit> , gras transcription factor  <cit> , apetala <dig> ethylene-responsive element binding proteins   <cit>  and teosinite branched, cycloidea, and pcf   <cit> , respectively.
in maize and arabidopsis, mir <dig> and mir <dig> were found to target auxin response factor  <cit> .
in foxtail millet, sit-mir <dig> had  <dig> predicted target genes, six were syg1/pho81/xpr <dig>  proteins, and four were nad -binding proteins, in agreement with a report that, in maize, mir <dig> could target nad -binding and spx proteins  <cit> .
most of the predicted target genes of the sit-mirnas are transcription factors that are known to play important roles in plant growth processes and in the regulation of plant development.
this result is also consistent with previous reports in other species  <cit> .
in a. thaliana, the grf gene family  of proteins that are involved in cell expansion in leaf and cotyledon tissues  <cit>  are regulated by mir <dig>  <cit> .
we found that nov-sit-mir <dig> was conserved to mir <dig> of arabidopsis and that its predicted target gene contained the same characteristic regions  and wrc  domains) as the atgrf proteins.
compared with other novel mirnas, the nov-sit-mir <dig> had a relatively high expression in leaf , suggesting that nov-sit-mir <dig> may play an important role in leaf; however, nov-sit-mir <dig> expression was highest in root  compared with in the other three tissues .
these findings suggest that nov-sit-mir <dig>  not only plays an important role in leaf growth, but may also be involved in the development of root.
the expression pattern of nov-sit-mir <dig> was validated by experiment .
foxtail millet is considered to be a diploid, tractable model for polyploid biofuel crops like switchgrass  and napier grass   <cit> .
the synthesis of cellulose is a complicated process that depends on carbon fixation, sugar metabolism and transit, and fat metabolism  <cit> .
in this study, we also find eight potential mirna targets that may be involved in the biological synthesis of cellulose and seven mirnas were predicted to have an effect on the regulation of cellulose biosynthesis .
for example, sit-mir <dig> potentially targets four different genes involved in carbohydrate metabolic process, suggesting that sit-mir <dig> might play a central role in sucrose metabolism and carbon fixation.
cellulose synthase mrna was predicted to be a target of mir- <dig> in maize  <cit> , and laccase, which is involved in lignin catabolic process in foxtail millet, was one of the predicted targets of sit-mir <dig>
the expression pattern of some sit-mirna families also showed similar trends as the corresponding conserved mirnas in different plants.
for example, the expression levels of sit-mir <dig>  sit-mir <dig>  sit-mir <dig> and sit-mir <dig> were comparatively very high, and sit-mir <dig>  sit-mir <dig>  sit-mir <dig> and sit-mir <dig> were comparatively very low, in agreement with similar findings in other plants  <cit> .
because two of the characteristics  of these mirnas were similar in other plants, we inferred that the regulatory mechanisms and main functions of these mirna target genes would also be similar, perhaps explaining the similar expression patterns of the conserved mirnas in different species.
in this work, we examined the expression profiles of ten mirnas in four tissues, three of which appeared to not be very consistent with the smrna-seq data, suggesting that preprocessing and normalizing the data to help in identifying differentially expressed genes is a process that continues to be developed.
CONCLUSIONS
by smrna sequencing, we identified  <dig> known mirnas and  <dig> novel mirnas in foxtail millet and studied their expression profiles in four different tissues.
potential targets were predicted with strict criteria as described, and four targets were validated by 5′race.
the functional annotation provided a deeper understanding of the transcription and regulation of the target genes, confirmed many known regulatory mechanisms, and provided a window into many more potentially novel pathways.
furthermore, a comparative genomic analysis with sorghum contributed to understanding the evolutionary dynamics of mirna family expansions and will serve as the basis for future scomparative functional genomic studies using syntenic analysis.
the identification and characterization of mirnas from foxtail millet will aid in further research on foxtail millet and other species in the poaceae family of grasses.

 micrornas are 20 ~  nucleotide non-coding small rnas. most primary mirnas are transcribed from mirna genes by rna polymerase ii, while others are transcribed by rna polymerase iii. in plants, the nascent pri-mirna transcripts are first processed into to nt pre-mirnas by dicer-like. then, the pre-mirnas undergo a second cleavage in the nucleus, releasing an rna duplex containing the mature mirna and the mirna* sequences. to maintain the correct size and/or to protect them from polyuridylation, the duplexes are methylated at the 3′ ends by hua enhancer and then transported into the cytoplasm by hasty. the mature mirna is incorporated into an rna-induced silencing complex that contains the argonaute protein. the complex recognizes partially complementary sequences in the target messenger rna where it binds, inducing its target mrna’s degradation or a combination of both degradation and translation inhibition. the mirna* is released and rapidly degraded. the presence of the mirna* has been regarded as a good standard way to reliably annotate a novel mirna. mirtrons are a new, recently discovered, type of mirnas that originate from spliced introns of gene transcripts. mirtrons reside within the intronic regions of genes and are processed through a drosha-independent pathway, making them quite distinct from the other mirnas. the mirtron pathway was first identified in drosophila melanogaster, caenorhabditis elegans, and some mammals and has recently been found in arabidopsis thaliana and oryza sativa. recent research shows that the mirnas from the intronic regions of genes can survive together with their host genes and support their host genes by mediating synergistic and antagonistic regulatory effects. in plant, mirnas are known to play crucial roles in many developmental events and regulate target transcripts through two modes of action: degradation and translation inhibition. the mirna degradation occurs through mirna-guided transcript cleavage in plants. although the mechanisms involved in the translational inhibition by mirnas are largely unknown, recent studies from xuemei chen’s group show that the mirna translational inhibition occurs at the endoplasmic reticulum and that altered meristem program activity allows for the dissection of mirna-mediated target rna cleavage from mirna-mediated target translation inhibition. with the advent of second generation sequencing technology, the rate of mirna discovery has increased dramatically. in dicotyledonous species, mature mirnas in a. thaliana, in populus trichocarpa, and in nicotiana tabacum have been discovered, and, in monocotyledonous species, mature mirnas in o. sativa, in saccharum offcinarum, in sorghum bicholor, in triticum aestivum and in zea mays have been reported. although foxtail millet is an important cereal crop, only a few mirnas in foxtail millet have been reported. bennetzen et al. reported some known mirnas from the alignment of mature mirna sequences, but they did not investigate whether or not the corresponding precursors existed in the genome. foxtail millet is a diploid c panicoid crop species. it is an important grass crop that has been planted widely in china. the genome of foxtail millet has been sequenced, making it a possible genetic resource that could be used to investigate plant architecture, genome evolution and physiology in the bioenergy grasses. because of the important roles that mirnas play in gene regulatory networks, it is important to identify the mirnas in foxtail millet and to investigate their potential target genes to gain a better understanding of the biological processes in this plant. in the present study, we aimed to characterize the mirna repertoire of foxtail millet.  analysis of smrnas in foxtail millet to identify mirnas in foxtail millet, we sequenced smrnas for shoots by high throughput illumina sequencing technology. nearly million reads of 18 ~  nt, representing over million unique sequences, were generated. in addition, three smrna datasets of foxtail millet were downloaded from the comparative sequencing of plant small rnas web site. these datasets contained over million smrnas of 18 ~  nt, representing over million unique sequences. an analysis of the data from the four datasets revealed that a large number of sequences appeared only once. the percentages of these singletons were % in shoot, % in leaf, % in flower and % in root. in previous reports, only 65% and 82% singletons were found in arabidopsis and rice, respectively, suggesting that the smrnas in foxtail millet are as complex as they are in rice, and more complex than in arabidopsis. the overall size distribution of the unique reads from four sequencing efforts were very similar, with the nt smrnas being the most abundant, followed by the nt and nt smrnas, which differ from the size distribution of total reads. a detailed comparison of the smrnas derived from unique reads or total reads revealed some features of smrna species. frist, the nt smrnas dominated the pool of unique species in foxtail millet as observed for many other species such as a. thaliana and cucumber. second, the nt smrnas replicated in even higher frequencies than nt smrnas in all four tissues. the average times of nt are nearly,, and and the average times of nt are nearly,, and in shoot, leaf, flower and root, respectively. third, mirnas, most of which were to nt in length, were relatively abundant in root and leaf, while sirnas, most of which were nt long, were relatively more prevalent in flower and shoot. these results were consistent with those reported previously in maize. in foxtail millet, smrna populations in four tissues are extremely complex. although we have over million reads, sequences of smrnas have a limited overlap from the four databases. only unique sequences appeared in all four tissues and a small portion of the sequences overlapped between two tissues. the smrnas in each tissue accounted for almost half of all the smrnas in the combined datasets. this limited overlap indicated that there was a diversity of smrnas in foxtail millet. identification of known mirnas in foxtail millet in plants, mirnas are obtained by the precise excision of ~ nt smrnas from the stem of a single-strand, stem-loop precursor. as a result, mirnas can be identified by looking for a potential fold-back precursor structure that contains the ~ nt mirna sequence within one arm of the hairpin. the hairpin must have the lowest free energy of all the alternative folds for that sequence, as predicted by rna folding programs such as mfold or rnafold. the minimal folding free energies index can be treated as the main feature to distinguish potential mirna precursors from other rnas. it has been reported that more than 90% of mirna precursors have a mfei value greater than, while the mfei for other rnas are lower. we used a previously reported workflow to identify mirnas among the smrnas from foxtail millet, but with the mfei values set to rather than to as was done previously. we identified candidate mirnas in the foxtail millet smrna datasets; in shoot, in leaf, in flower and in root. the proportion of mirna reads in each library as compared to the total reads were % in root, % in leaf, % in flower and % in shoot. among the mirnas, unique mature mirna sequences belonging to mirna families were 100% identical to mature mirnas from well-studied species. we defined the unique mature mirnas in foxtail millet as known mirnas. there were known mirnas in root, in leaf, in flower and in shoot tissues. the sit-mirnas corresponded to pre-mirnas, of which had corresponding mirna* forms. detailed information about the known sit-mirnas is shown in additional file a summary of the total expression profiles for each of the mirna families to which the sit-mirnas belong is shown in figure  the number of mirna reads were enumerated and normalized against the total count of smrna reads, reported as reads per million, for each respective library. the results showed that the total expression of these mirna families was relatively higher in leaf and root than that in shoot and flower. we noted that the sit-mir sit-mir sit-mir sit-mir and sit-mir families showed relatively higher expression in one or more of the four tissues. in contrast, the sit-mir sit-mir sit-mir sit-mir and sit-mir families showed low expression levels. in our samples, the following mirna families showed distinct expression patterns: mir is barely detected in roots; mir is practically absent in leaves; mir and mir are practically absent in flowers; mir and mir are detected only in shoots; and mir is barely detectable in any of the four tissues. our data also showed that some members of the same mirna family had different tissue-specific expression patterns. for example, based on rpm counts, mir156b is highly expressed in flower and shoot but has almost no expression in leaf and root. while the mir156d is highly expressed in leaf and root but has almost no expression in the flower. this should be confirmed by other molecular techniques. together, these results suggest that members from different families and different members from the same family may have greatly different effects on foxtail millet development. novel mirna identification our results showed that in addition to the known mature sit-mirnas, there were novel mirnas in the smrna datasets that have never been reported in other species. because some of these mirnas were derived from multiple precursors, these nov-sit-mirnas corresponded to pre-mirnas, 68% of which were located in intergenic regions. only a few of the pre-rnas were located in introns or in the utrs of coding genes, which is in agreement with the reported statistical characteristics of plant pre-mirnas. among the nov-sit-mirnas, nine were found in all four libraries, nine were in three libraries, were in two and were in one library. these potentially nov-sit-mirnas had relatively lower expression levels than the known sit-mirnas. for example, in leaf, the total expression levels of the nov-sit-mirnas were less than 1% of the total expression levels of the sit-mirnas and, in flower, the total expression levels of all the nov-sit-mirnas were only rpm. these results were consistent with a study in rice that found that most of the non-conserved mirnas exhibited tissue-specific expression patterns and had relatively low expression levels compared with known mirnas. for example, nov-sit-mir nov-sit-mir and nov-sit-mir were expressed only in flowers with expression levels of more than rpm, and nov-sit-mir and nov-sit-mir were expressed only in roots with expression levels of more than rpm. the conservation of mirna sequences across species could be regarded as powerful evidence to annotate mirna. we identified conserved mirna by allowing no more than three mismatches between the mature mirna sequences in our datasets and the mature mirna sequences from well-studied species published in mirbase. we identified nov-sit-mirnas that could be assigned to published mirna families based on sequence similarity. these ‘conserved’ nov-sit-mirnas generally had higher expression levels than the ‘non-conserved’ nov-sit-mirnas. eight of the nov-sit-mirnas were validated experimentally by stem-loop rt-pcr and sequencing. the presence of a mirna* could be regarded as additional support for the annotation of the nov-sit-mirnas. we noted that of the novel pre-mirnas were found having mirna*s. a nucleotide composition analysis showed that 50% of the novel pre-mirnas started with u, which is consistent with the statistical characteristic of mature mirnas, and, overall, a higher percentage of a and u compared with c and g was present. detailed information about novel mirnas was shown in additional file and second structure of some novel mirna precursors were presented in additional file to predict mirtrons, we analyzed the secondary structure features of the introns of the foxtail millet genes that were annotated in the phytozome database. introns can be folded directly to acquire stem–loop structures similar to the pre-mirnas. using rnafold, we found many introns that had reasonable second structures; that is, a single stem-loop with less than nt overhangs at both ends, and a minimum energy of -25 kcal/mol or less. after mapping the smrnas in our datasets onto the selected introns using the blast algorithm, we found only a few perfectly matched smrnas; therefore, only two mirtron precursor candidates could be identified. target prediction and expression validated mirnas are known to have diverse expression patterns and play regulatory roles in various developmental and physiological processes. in plants, most mirnas are known to regulate development by mediating the destruction of their target mrnas. the target gene has sites that are almost completely complementary to the mirna, so, in plants, mirna target genes can be predicted computationally. to increase the reliability of our target prediction, we used the modified scoring described by sun et al.. we used the sit-mirna and nov-sit-mirna sequences in our datasets to search for complementary annotated foxtail millet transcript genes in the phytozome database and identified candidate target genes for of the foxtail millet mirnas. we randomly picked six target genes to validate using 5′race and four were successfully validated. the si016508m protein, which is encoded by one of the predicted target genes, is a gras transcription factor that has been reported to be involved in development and other processes. the si016508m gene is potentially targeted by five of the sit-mirnas. the si005991m, si016509m and si034525m protein, which are encoded by a predicted target gene, is known to have three different domains: a b dna-binding domain, an aux/iaa-arf dimerization motif, and a dna-binding pseudobarrel domain. previous studies have suggested that for efficient cleavage by mirnas, base pairing between bases and is essential. the 5′race results showed that the si016508m gene had two breakpoints. the predicted target sites of these genes showed that sit-mir171a and sit-mir171b guided si016508m cleavage at 1085 bp, while nov-sit-mir nov-sit-mir and nov-sit-mir guided si016508m cleavage at 1082 bp. to show that these mirnas, which have validated their targets, are actually expressed, we used the stem-loop rt-pcr method of detection. we detected mirna mirna171a, nov-sit-mir nov-sit-mir and nov-sit-mir successfully. however, we failed to detected mirna171b. mapping of mrna cleavage sites by rna ligase-mediated 5′ race. the complementary pattern of mirna sequences and partial sequences of the target mrnas are shown in the table. all predicted mismatch base parings are shown in bold letters. the positions inferred as 5′ ends of mirna-guided cleavage products, as revealed by 5′ race, and the numbers of sequenced 5′ race clones corresponding to each site are indicated by vertical arrowheads. about 79% of the predicted target genes had functional annotations in interpro, the integrated resource for protein families, domains, and functional sites. a large proportion of the predicted targets were annotated as transcription factors; for example, the ap2/erf domain, homeodomain, and ccaat-binding transcription factors. in addition, some of the predicted targets were annotated as being involved in various classes of molecular functions, including binding proteins, calcium ion transport proteins, and enzymes. the functions of predicted mirna targets indicated that the mirnas may play multiple roles in gene regulation networks. to better understand the functional roles of the predicted mirna target genes in foxtail millet, we analyzed the functional enrichment for all the mirna targets by gene ontology. the mirna predicted targets showed enrichment in go terms from the molecular function and biological process categories, while no enrichment in go terms was observed in the cellular component category. we found go terms in the biological process category that showed strong enrichment in biosynthetic process, cellular process, macromolecule metabolic process, regulation of metabolic process and regulation of cellular process. in the molecular function category, the enriched go terms included binding, nucleic acid binding, and protein-binding. the go enrichment analysis showed that the predicted targets of the mirnas were involved in a wide range of regulatory functions as well as some specific biological processes like metabolism, biosynthesis, and gene expression/transcription. to analyze whether or not mirnas in different tissues have different functions, go enrichment analyses were conducted for the predicted mirna targets in each of the four tissues. we found that the functions of the targets were similar in all four tissues; the targets were all enriched in biological regulation, biosynthetic process, macromolecule metabolic process and cellular process. in addition, some of the predicted targets had annotations in the cellular component category, including cell, and cell part in leaf and flower. in the root dataset, the target genes were annotated with significantly fewer go terms than the target genes in the other three tissues. details of the go annotations for the target genes in the four tissues are available in additional file to confirm the accuracy and reliability of the smrna-seq results, we randomly chose ten of the mirnas for stem-loop rt-pcr validation. the results showed that the stem-loop rt-pcr expression profiles of most mirnas match the smrna-seq data closely. while there were three mirnas expression profiles that were not very consistent. conservation and synteny with sorghum previous reports in arabidopsis and in rice have shown that mirna gene families evolved from segmental, tandem repeat and whole-genome duplication events. a detailed description of the fate of mirna genes after whole-genome duplication in maize and sorghum has been published. to date, mirnas expression and function has been well studied in the major food and feed crops, such as sorghum, maize and rice. foxtail millet shares a common ancestor with sorghum and maize. this ancestry is more recent than with rice. sorghum has experienced one whole genome duplication and maize was experienced two whole genome duplications since they diverged. to investigate the evolution of foxtail millet mirnas, we used sorghum as the reference comparison strain. the syntenic analysis was performed between the foxtail millet and sorghum genes. based on the total number of genes in foxtail millet and in sorghum, we found syntenic blocks containing pairs of syntenic genes between the two species. in total, foxtail millet and sorghum genes, and foxtail millet and sorghum pre-mirnas, were found within the syntenic regions. nearly all the pre-mirnas were located in only of the syntenic blocks, but within these blocks there were syntenic gene pairs corresponding to 45% of the total number of syntenic genes in the two species. we identified pairs of syntenic pre-mirnas, and all the foxtail millet and sorghum chromosomes had blocks that contained at least one of the conserved syntenic pre-mirnas. we noted that of the novel mirnas that we identified in foxtail millet had syntenic pre-mirna sequences and conserved mature mirna sequences. therefore, in some respects, the foxtail millet mirnas are new members of already known mirna families. for example, nov-sit-mir and nov-sit-mir could be considered to be new members of the mir family; similarly, nov-sit-mir nov-sit-mir and nov-sit-mir are new members of the mir family. discussion some mirnas are expressed only in a specific tissue or at a specific site, and an understanding of their unique expression pattern may help in discovering the function of a particular mirna. to assess the tissue specificity of the mirnas in our datasets, we investigated the expression levels of the mirna in the four foxtail millet tissues. we found that the expression of the sit-mir family was higher in the adult leaf and flower datasets and lower in the young shoot dataset. in maize, the mir that is responsible for the transition from juvenile to adult was reported to have the same expression pattern. in addition, compared with the other sit-mirna families, the sit-mir family was highly expressed, especially in leaf. in maize, the target gene of mir is rolled leaf which can regulate leaf morphogenesis. similarly, in foxtail millet, the target gene of sit-mir was predicted as si034251m, a homolog of rolled leaf this finding suggests that there may be a similar mechanism of leaf morphogenesis in foxtail millet as the one reported in maize. we found that, except for a few of the sit-mirnas that had relatively high expression levels, most of the sit-mirnas, especially the nov-sit-mirnas, had low expression levels in the four studied tissues. it has been suggested that the normal physical functions of an organism require various mirnas working together for precise regulation and control rather than individual mirnas working separately. if this suggestion is correct, then the mirnas with low expression might have an effect on the growth of foxtail millet as great as the mirnas with high expression. a previous report indicated that most of the mirna targets in maize are conserved across several plant species and we found that this was also true in foxtail millet. for example, mir and mir were predicted to target genes that encode sbp-box transcription factors, and mir mir mir mir and mir were reported to target no apical meristem, ccaat-binding factor, gras transcription factor, apetala ethylene-responsive element binding proteins and teosinite branched, cycloidea, and pcf, respectively. in maize and arabidopsis, mir and mir were found to target auxin response factor. in foxtail millet, sit-mir had predicted target genes, six were syg1/pho81/xpr proteins, and four were nad -binding proteins, in agreement with a report that, in maize, mir could target nad -binding and spx proteins. most of the predicted target genes of the sit-mirnas are transcription factors that are known to play important roles in plant growth processes and in the regulation of plant development. this result is also consistent with previous reports in other species. in a. thaliana, the grf gene family of proteins that are involved in cell expansion in leaf and cotyledon tissues are regulated by mir. we found that nov-sit-mir was conserved to mir of arabidopsis and that its predicted target gene contained the same characteristic regions and wrc domains) as the atgrf proteins. compared with other novel mirnas, the nov-sit-mir had a relatively high expression in leaf, suggesting that nov-sit-mir may play an important role in leaf; however, nov-sit-mir expression was highest in root compared with in the other three tissues. these findings suggest that nov-sit-mir not only plays an important role in leaf growth, but may also be involved in the development of root. the expression pattern of nov-sit-mir was validated by experiment. foxtail millet is considered to be a diploid, tractable model for polyploid biofuel crops like switchgrass and napier grass. the synthesis of cellulose is a complicated process that depends on carbon fixation, sugar metabolism and transit, and fat metabolism. in this study, we also find eight potential mirna targets that may be involved in the biological synthesis of cellulose and seven mirnas were predicted to have an effect on the regulation of cellulose biosynthesis. for example, sit-mir potentially targets four different genes involved in carbohydrate metabolic process, suggesting that sit-mir might play a central role in sucrose metabolism and carbon fixation. cellulose synthase mrna was predicted to be a target of mir- in maize, and laccase, which is involved in lignin catabolic process in foxtail millet, was one of the predicted targets of sit-mir the expression pattern of some sit-mirna families also showed similar trends as the corresponding conserved mirnas in different plants. for example, the expression levels of sit-mir sit-mir sit-mir and sit-mir were comparatively very high, and sit-mir sit-mir sit-mir and sit-mir were comparatively very low, in agreement with similar findings in other plants. because two of the characteristics of these mirnas were similar in other plants, we inferred that the regulatory mechanisms and main functions of these mirna target genes would also be similar, perhaps explaining the similar expression patterns of the conserved mirnas in different species. in this work, we examined the expression profiles of ten mirnas in four tissues, three of which appeared to not be very consistent with the smrna-seq data, suggesting that preprocessing and normalizing the data to help in identifying differentially expressed genes is a process that continues to be developed.  by smrna sequencing, we identified known mirnas and novel mirnas in foxtail millet and studied their expression profiles in four different tissues. potential targets were predicted with strict criteria as described, and four targets were validated by 5′race. the functional annotation provided a deeper understanding of the transcription and regulation of the target genes, confirmed many known regulatory mechanisms, and provided a window into many more potentially novel pathways. furthermore, a comparative genomic analysis with sorghum contributed to understanding the evolutionary dynamics of mirna family expansions and will serve as the basis for future scomparative functional genomic studies using syntenic analysis. the identification and characterization of mirnas from foxtail millet will aid in further research on foxtail millet and other species in the poaceae family of grasses. 
micrornas are a class of short non-coding, endogenous rnas that play key roles in many biological processes in both animals and plants. of the predicted target genes, 79% had functional annotations in interpro and go analyses, indicating the targets of the mirnas were involved in a wide range of regulatory functions and some specific biological processes. potential targets of the foxtail millet mirnas were predicted based on our strict criteria. although many mirnas have been identified in a large number of organisms, the mirnas in foxtail millet have, until now, been poorly understood. in this study, two replicate small rna libraries from foxtail millet shoots were sequenced, and million reads representing over million unique sequences were generated. we identified known mirnas, novel mirnas and mirtron precursor candidates in foxtail millet. a total of pairs of syntenic mirna precursors that were conserved between foxtail millet and sorghum were found. we predicted, for the first time, mirnas and mirna targets in foxtail millet at a genome-wide level. some of the novel foxtail millet mirnas and mirna targets were validated experimentally. the precursors, expression levels, mirna* sequences, target functions, conservation, and evolution of mirnas we identified were investigated. further, eight novel mirnas were validated by stem-loop rt-pcr. additionally, stem-loop rt-pcr was conducted to confirm the tissue-specific expression of some mirnas in the four tissues identified by deep-sequencing. some mirna*s of the known and novel mirnas were detected as well.
 micrornas are a class of short non-coding, endogenous rnas that play key roles in many biological processes in both animals and plants. although many mirnas have been identified in a large number of organisms, the mirnas in foxtail millet have, until now, been poorly understood.  in this study, two replicate small rna libraries from foxtail millet shoots were sequenced, and million reads representing over million unique sequences were generated. we identified known mirnas, novel mirnas and mirtron precursor candidates in foxtail millet. some mirna*s of the known and novel mirnas were detected as well. further, eight novel mirnas were validated by stem-loop rt-pcr. potential targets of the foxtail millet mirnas were predicted based on our strict criteria. of the predicted target genes, 79% had functional annotations in interpro and go analyses, indicating the targets of the mirnas were involved in a wide range of regulatory functions and some specific biological processes. a total of pairs of syntenic mirna precursors that were conserved between foxtail millet and sorghum were found. additionally, stem-loop rt-pcr was conducted to confirm the tissue-specific expression of some mirnas in the four tissues identified by deep-sequencing.  we predicted, for the first time, mirnas and mirna targets in foxtail millet at a genome-wide level. the precursors, expression levels, mirna* sequences, target functions, conservation, and evolution of mirnas we identified were investigated. some of the novel foxtail millet mirnas and mirna targets were validated experimentally. mirnafoxtail milletexpression patternmirtronmirna*targetssynteny 