BACKGROUND
micrornas  are a class of non-coding small rnas , approximately 20– <dig> nucleotides in length, that post-transcriptionally regulate gene expression.
in plants, highly conserved and species-specific mirnas control a vast array of biological processes, such as leaf polarity, flower development and stress responses  <cit> .
the mirnas are excised from stem-loop structures within larger primary mirna transcripts by dicer-like <dig> , which in each case trims the hairpin structure .
in plants, the mature mirna strands have high complementarity  to their target mrnas and regulate gene expression via mrna cleavage  <cit> .
analysis indicates that many plant mirnas and their targets are evolutionarily conserved from species to species within the plant kingdom.
thus, a mirna in one species may exist as orthologs or homologs in other species  <cit> .
plant mirnas have usually been identified either by prediction through bioinformatics or by experimental methods  <cit> .
however, the bioinformatics-based approaches can only identify mirnas that are conserved amongst organisms, and dna or rna sequence information is required in order to run the programs.
thus, sequencing is the most effective method for plant mirna discovery  <cit> .
to date,  <dig> sequences of mature mirnas have been identified in plants, including  <dig> from a. thaliana,  <dig> from a. lyrata,  <dig> from o. sativa and  <dig> from b. rapa .
the genus brassica is cultivated in most parts of the world.
it includes various crops of agronomic importance, such as broccoli, cauliflower and b. rapa.
broccoli  is very popular with a wide range of consumers because of its flavor, and also on account of its anti-cancer activities, notably in relation to prostate and colorectal cancers  <cit> .
because broccoli, a. thaliana and b. rapa all belong to the same family, the cruciferae, the level of synteny between these species provides a basis for studying the mirnas of broccoli  <cit> .
environmental stresses such as salinity are a worldwide problem in agriculture, diminishing plant growth and yield.
broccoli is moderately tolerant to salinity, and it displays better tolerance than some other common vegetables, such as maize and carrot  <cit> .
although several stress-related mirnas have been identified based on the sequencing of a library of srnas isolated from a. thaliana seedlings, o. sativa, saccharum officinarum l. and from populous euphratica exposed to various stresses  <cit> , there have been few studies that have focused upon mirnas in broccoli, and in particular upon the identification of stress-related mirnas in broccoli.
thus, in the present study, we have identified mirnas and their targets related to salt-stress in broccoli, using high-throughput sequencing methods.
the differential expression of mirnas observed between broccoli grown under standard conditions and broccoli subjected to salt stress provides new insights that will inform the genetic improvement of stress tolerance in plants.
RESULTS
sequence analysis of srnas
in order to identify the mirnas responding to salt stress, we constructed and sequenced srna libraries ranging in size from  <dig> to 30 nt, from both control and salt-stressed broccoli.
a total of  <dig> , <dig> and  <dig> , <dig> reads were obtained from control and salt-stressed broccoli, respectively.
after removing the tags with: any n bases, more than  <dig> bases whose quality score was lower than  <dig> and more than  <dig> bases whose quality score was lower than  <dig>  and those that were too small , as well as the adapter sequences,  <dig> , <dig>  and  <dig> , <dig>  clean reads were obtained , representing  <dig> , <dig>  and  <dig> , <dig>  unique, although sometimes partially overlapping, clean reads from control and salt-stressed broccoli, respectively.
the size distributions of the reads in the two datasets were quite similar, and they were not evenly distributed.
the majority of these unique sequence reads were 24 nt in size, followed by 23 nt, 21 nt and 22 nt, both in control and salt-stressed broccoli, which is similar to the size distribution of srnas in plants .
the overall distribution pattern of srnas  in salt-stressed broccoli was similar to that in control broccoli .
moreover, common and specific tags between the control and salt-stressed broccoli were analyzed.
the results showed that only  <dig> , <dig>  of the unique sequences and  <dig> , <dig>  of the total sequences were shared between the two samples , suggesting that the sequence results were reliably representative of the endogenous srnas in broccoli.figure  <dig>
sequence analysis of srnas.
length distribution of srnas.
the length distribution of high-quality sequences obtained from the two broccoli libraries.
the distributions of the total reads are shown as percentages.
summary of common and specific sequences between control and salt-stressed broccoli libraries.
total srnas and unique srnas are shown in the left panel and the right panel, respectively.
because the whole genome of sequence for broccoli was not available, all clean reads were aligned against the b. rapa genome, using short oligonucleotide alignment program- <dig>   <cit> .
however, only a very small percentage of the unique srna sequences could be matched against the b. rapa genome; only  <dig> , <dig>  of the reads in the control broccoli and  <dig> , <dig>  of the reads in the salt-stressed broccoli could be mapped in this way, representing  <dig> , <dig>  and  <dig> , <dig>  of the total reads in control and salt-stressed broccoli, respectively .
all clean reads were annotated into different categories, including plant mirnas , exons and introns , and non-coding rnas .
in those cases in which srnas were mapped to more than one category, the following priority rule was adopted: rrna > known mirna > exon > intron.
the results indicated that the majority of srnas –  <dig> % of the unique reads in the control group and  <dig> % in the salt-stressed group – remained unannotated.
for the control group, when unique srnas were matched, a small proportion of reads were derived from repeated sequences , and a smaller proportion from rrnas .
however, for the total srna pools, rrnas  were the most abundant sequences, followed by repeated sequences  .
all the unannotated sequences were then used for further analysis.table  <dig>
distribution of genome-mapped sequence reads in the srna libraries for control and salt-stressed broccoli
locus class
control
salt-stressed
unique srna
percent
total srna
percent
unique srna
percent
total srna
percent
identification and expression patterns of salt-stress-induced conserved mirnas in broccoli
conserved mirna families are found in many plant species and play an important role in a diversity of plant processes.
in spite of this, sequence information for broccoli mirnas is absent from both the mirbase database and from the plant mirna database.
furthermore, although mirna families are conserved between closely related species  <cit> , there are only  <dig> known b. rapa mirnas in mirbase.
therefore, the srna sequences were aligned to the mirna precursor/mature mirna sequences of the viridiplantae in mirbase.
ninety-seven putative known mirnas were identified in salt-stressed broccoli, including  <dig> mirnas that were also found in the control group .
in particular, the putative conserved mirnas, mir156a, mir166a and mir168a, which have been found in about  <dig> plant species , were amongst the mirnas that were found in both groups.
a comparison of the mirnas in the two libraries indicated that about  <dig> putative mirnas – including, for example, mir <dig>  mir <dig> and mir <dig> – were less abundant in salt-stressed broccoli than in control broccoli.
conversely,  <dig> mirnas – including, for example, mir157d, mir393b-3p and mir169m – were more abundant in salt-stressed broccoli than in control broccoli .
although the levels of some mirnas belonging to the same mirna gene family, such as mir <dig>  mir <dig> and mir <dig>  were found to be increased and decreased in parallel, others were not; the levels of mir <dig> and mir393a, for example, increased and decreased differently .
however, when the fold-change  criterion between salt-stressed broccoli and control broccoli was set to < − <dig>  or >  <dig> , and the false discovery rate  was set to <  <dig> , the levels of  <dig> mirnas were found to be significantly different between salt-stressed and control plants.
as shown in figure 2b, the levels of mir <dig>  mir <dig>  mir <dig>  mir168a and mir <dig> were all decreased; levels of mir <dig>  mir396a, mir397a and mir164a, on the other hand, were increased under salt stress.
the mirna that showed the greatest decrease under salt stress was mir <dig> and the mirnas that showed the greatest increase were mir165a-5p and mir <dig> figure  <dig>
differential expression of putative conserved mirnas between control and salt-stressed broccoli.
scatter diagram of the differential read counts of known mirnas.
each point in the figure represents a mirna.
red points represent mirnas showing a > 2-fold change of expression; blue points represent mirnas showing 1/2 < fold change ≤ 2; green points represent mirnas showing a fold change ≤ 1/ <dig>   the  <dig> mirnas showing the greatest changes in expression, with fold changes < 1/ <dig> or >  <dig>  decreased  and increased  putative conserved mirnas confirmed by stem-loop real-time rt-pcr, as described in methods.
significant difference between salt-stressed broccoli and control broccoli is indicated by *p <  <dig>  and **p <  <dig> .
real-time quantification of mirnas by stem-loop real-time reverse transcription polymerase chain reaction  is specific for mature mirnas and discriminates amongst related mirnas that differ by as little as one nucleotide.
furthermore, it is not affected by genomic dna contamination  <cit> .
therefore, this technique was used to validate the sequencing results.
to confirm the identity of the individual pcr products, they were firstly confirmed by electrophoretic sequencing and by denaturing gel electrophoresis.
for those mirnas that were less abundant in salt-stressed broccoli than in control broccoli, the results obtained by stem-loop rt-pcr agreed with the results obtained by sequencing.
the greatest degree of down-regulation in response to salt stress was shown by mir <dig> and mir <dig> .
amongst the  <dig> mirnas that were more abundant in salt-stressed broccoli than in control broccoli,  <dig> also showed up-regulated expression when analyzed by stem-loop rt-pcr; the three exceptions were mir <dig>  mir858-3p and mir <dig>  which were not detected by this technique, either in control or in salt-stressed broccoli.
of these  <dig>  mir396a showed the greatest degree of up-regulation, followed by mir838-5p, mir852-5p, mir <dig> and mir393a .
these findings contrasted with the results obtained by sequencing, which indicated that the two most abundant up-regulated mirnas were mir165a-5p and mir <dig>  in summary,  <dig> putative conserved mirnas that were differentially expressed between control and salt-stressed broccoli were revealed by their read counts and confirmed by the use of stem-loop real-time rt-pcr.
identification and expression patterns of salt-stress-induced, non-conserved mirnas in broccoli
in order to identify putative novel mirnas in the broccoli dataset, secondary structures and minimum free-energies were calculated.
in total,  <dig> unique mature mirnas were identified initially in our study, and these candidate mirnas originated from predicted rna hairpins.
with an average of about  <dig> read counts, these putative novel mirnas showed fewer read counts than the putative conserved mirnas.
a comparison of the putative novel mirnas in the two libraries indicated that the levels of about  <dig> of theses mirnas were decreased in response to salt stress.
conversely, the levels of about  <dig> mirnas were increased .
real time rt-pcr was next performed for those mirnas with an average of more than  <dig> read counts either in the salt stressed broccoli or control broccoli, and whose levels were found to be significantly different between salt-stressed and control plants, when the fold-change  criterion between salt-stressed broccoli and control broccoli was set to < − <dig>  or >  <dig> , and the fdr was set to <  <dig> .
the results from the  <dig> mirnas analyzed by real time rt-pcr indicated that only  <dig> of these candidate mirnas were expressed in either the control or the salt-stressed group .
a higher proportion of the sequences were identified from the 5’-ends of the hairpins than from the 3’-ends.
secondary hairpin structures for representative mirnas are shown in figure 3b, and the secondary hairpin structures for all the candidate mirnas are listed in additional file 5: figure s <dig> figure  <dig>
differential expression of putative novel mirnas between control and salt-stressed broccoli.
scatter diagram of differential read counts of putative mirnas.
prediction of secondary structure of representative putative mirnas from broccoli with or without salt treatment.
increased  and decreased  putative novel mirnas confirmed by stem-loop real-time rt-pcr.
significant difference between salt-stressed broccoli and control broccoli is indicated by *p <  <dig>  and **p <  <dig> .
putative novel mirnas in the two libraries
novel id
location
strand
energy  were detected only in control broccoli, whilst five  were found only in salt-stressed broccoli.
four other putative novel mirnas  exhibited similar expression levels in both libraries.
although most of the putative novel mirnas were observed in both libraries, their expression could be significantly different; for example, mir <dig> showed four-fold higher expression in salt-stressed broccoli than in control plants.
amongst those that were significantly up-regulated, mir <dig> was up-regulated to the greatest extent, followed by mir <dig>  mir <dig> and mir <dig> .
in contrast, mir <dig> and mir <dig> were the putative novel mirnas that showed the largest down-regulation in response to salt stress .
in summary,  <dig> new candidate mirnas that were differentially expressed between control and salt-stressed broccoli were identified by their read counts and confirmed by the stem-loop real-time rt-pcr.
target prediction and analysis of differential expression of mirnas
to understand the biological mechanisms by which broccoli responds to salt stress, the putative target sites of mirnas were identified by aligning mirna sequences with the expressed sequence tags  of b. rapa, following the rules of target prediction suggested by allen et al.
<cit> .
a total of  <dig> and  <dig> mrnas were predicted as targets of putative conserved mirnas and putative novel mirnas, respectively.
in plants, the identification of mrna targets is straight forward because most mirnas and their target mrnas have exact or nearly exact complementarity.
furthermore, the mirna target sites of plants have been shown to be located primarily in the coding regions  <cit> .
the putative target genes appeared to be involved in a wide variety of biological processes and molecular-genetic functions; therefore, the gene ontology  database and the kyoto encyclopedia of genes and genomes  orthology  database were used to analyze the datasets.
studies have shown that salt stress interferes with cell-cycle regulation at the transcriptional level, resulting in an adaptive growth response.
hormone signal transduction and sulfur metabolism are also vital for the improvement of stress tolerance in crop plants  <cit> .
interestingly, the results we obtained using the ko database indicated that genes involved in a diversity of cellular processes, such as the cell cycle, plant hormone signal transduction, and sulfur metabolism  were up-regulated in response to salt stress.
similarly, pathway annotation for the putative novel mirnas that were up-regulated in response to salt stress indicated involvements in calcium reabsorption and in the cell cycle .
the gene targets of both the putative conserved and the putative novel mirnas were classified into three go categories: cellular location, biological process and molecular function.
for the cellular location category, large numbers of targets for putative conserved and putative candidate mirnas were categorized under ‘cell’ and ‘cell part’, whereas ‘extracellular region’ comprised the smallest proportion .
for the biological process category, the majority were classified under ‘cellular process’, ‘metabolic process’ or ‘response to stimulus’.
under the molecular function category, the two most abundant sub-categories for both putative conserved and putative novel mirnas were ‘binding’ and ‘catalytic activity’.figure  <dig>
functional classification of mirna targets according to the gene ontology  program.
in this ontology, ‘cellular location’, ‘biological process’ and ‘molecular function’ are treated as independent attributes.
go classifications for putative conserved and putative novel mirna targets are shown in the upper panel and lower panel, respectively.
discussion
salinity is an increasingly important agricultural problem.
the metabolism and physiological integrity of plants are affected by salt stress and in recent years many studies have been devoted to understanding the molecular mechanisms of plant salt tolerance.
studies have confirmed that resistance to salt stress is associated with the use of compatible solutes by plants  <cit> , and with ion transporters  <cit> .
mirnas are a class of non-coding srnas that are implicated in many developmental processes and in responses to various abiotic stresses, and which play pivotal roles in plant adaptation  <cit> .
to date, in excess of  <dig>  hairpin sequences and  <dig>  mature sequences have been identified from plants, animals, unicellular organisms and viruses ; however, scant attention has been paid to the identification of mirnas in broccoli.
in the present study, we identified several million srna sequences in salt-stressed plantlets of broccoli, using high-throughput sequencing methods to identify mirnas associated with physiology and metabolism under salt stress.
a total of  <dig> putative known and  <dig> putative candidate mirnas that were differentially expressed between control and salt-stressed broccoli were discovered according to their sequencing-reads numbers and confirmed by real-time rt-pcr.
finally, analysis of the predicted targets of the mirnas using the go and ko databases indicated that a range of metabolic pathways and biological processes known to be associated with salt stress were up-regulated in broccoli treated with salt.
the study of mirnas using traditional methods is complex, and high-throughput sequencing methods now provide a rapid and efficient additional approach by which to identify and profile populations of srnas at different stages of plant development.
large numbers of non-conserved or species-specific mirnas often accumulate in plants at lower levels than conserved mirnas, and are therefore not easily revealed using traditional sequencing methods  <cit> .
in the present study, the results indicated a range of srnas, of length 16–29 nt, in broccoli, with most of the unique sequence reads being 24 nt in length.
several plant species, including a. thaliana, citrus sativus and c. sinensis, had been shown to contain substantially more 24-nt srnas than 21-nt srnas  <cit> ; on the other hand, srnas populations with more 21-nt members than 24-nt were reported in b. juncea and in japanese apricot with imperfect flower buds  <cit> .
b. juncea, b. napus and broccoli all belong to the same genus; however, when broccoli srnas are compared to those of b. juncea, a striking difference exists, whereas little difference is observed when broccoli srnas are compared to those of b. napus  <cit> .
thus, we can conclude that the srna transcriptome is complex, and can be highly variable even between closely related plant species.
generally speaking, length distribution analyses of srnas provide a helpful way to assess the composition of srna samples.
previous studies have shown that dcl <dig> mainly produces srnas that are 18–21 nt long.
in contrast, the products of dcl <dig>  dcl <dig> and dcl <dig> are 22 nt, 24 nt and 21 nt long, respectively.
furthermore, mirnas are normally 21 nt or 22 nt long, whereas small interfering rna  are 24 nt long  <cit> .
by this reasoning, therefore, our results imply that the most abundant srnas are mirnas in broccoli and sirnas that have been cleaved by dcl <dig> and dcl <dig>
sequence information for broccoli mirnas is entirely absent from mirbase, and furthermore there are fewer than  <dig> b. rapa mirnas in this database.
therefore, the srna sequences were aligned instead against mirna precursors/mature mirnas of the viridiplantae in mirbase.
a search for conserved mirnas revealed that the majority of these molecules, known already from a. thaliana and other species, were detectable in broccoli and were expressed at relatively high abundance.
the patterns of expression of conserved mirnas are variable in different plants.
previous studies had shown that mir <dig>  mir166a, mir <dig>  mir171f and mir <dig> were all detectable in b.rapa, and with relatively high numbers of reads, whereas members of the mir <dig> family showed low read numbers  <cit> .
the findings of the present study were quite different; mir166a, mir168a and mir157d showed the highest values for copy number in control broccoli.
a previous report suggested that mir <dig>  which was initially identified in a. thaliana and later found in populus trichocarpa, was a dicot-specific mirna and that mir <dig> and mir <dig> might be monocot-specific mirnas  <cit> .
on the other hand, sunkar et al.
indicated that mir <dig> was present in both monocots and dicots  <cit> .
our results provided a large-scale database against which to examine these suggestions.
we detected both mir <dig> and mir <dig> in broccoli, whereas mir <dig> and mir <dig> were not detectable, thereby confirming the previous reports.
furthermore, the mir <dig> and mir <dig> sequences have been considered to be a. thaliana-specific  <cit> ; however, we detected mir158a, mir158b, and mir <dig> in broccoli, each at an abundance of about  <dig>  reads.
although many plant mirnas have been found to be conserved – for example, mir <dig>  mir156/ <dig>  mir <dig>  mir165/ <dig> and mir <dig> have been found in more than  <dig> plant species  <cit>  – studies have indicated that most mirnas can be induced by environmental stresses.
for example, mir <dig> is induced under low-phosphate conditions  <cit> , mir <dig> increases upon sulfate starvation  <cit>  and mir <dig> is up-regulated in response to cold, dehydration, nacl and abscisic acid  stress  <cit> .
a recent study showed that mir <dig> was transiently up-regulated in response to osmotic stress  <cit> .
on the other hand, mir <dig> is down-regulated in response to oxidative stress.
several differentially regulated mirnas have been identified in salt-stressed plants.
for example, mir530a, mir <dig>  mir1446a-e and mir <dig> were down-regulated during salt stress in p. trichocarpa, as measured by microarray analysis of known mirnas, whereas mir <dig>  and mir <dig> were up-regulated  <cit> .
in a. thaliana, mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir <dig> were all up-regulated in response to salt stress, whereas mir <dig> was down-regulated.
furthermore, mir169g and mir169n were also reported to be induced by high salinity  <cit> .
recently, a study of maize roots using mirna microarray hybridization indicated that members of the mir <dig>  mir <dig>  mir <dig>  and mir <dig> families were down-regulated by salt shock, whereas mir <dig> and mir <dig> were up-regulated  <cit> .
our result in broccoli indicated that, amongst the conserved mirnas, mir <dig> and mir <dig> were the most strongly down-regulated in response to salt stress, whereas conserved mir838-5p and mir396a were the most strongly up-regulated.
of the putative novel mirnas, mir <dig> showed the greatest degree of up-regulation in response to salt stress, whereas mir <dig> and mir <dig> showed the greatest degree of down-regulation.
the differences between our findings and the reports relating to salt stress in p. trichocarpa, a. thaliana and maize roots  <cit>  might be explained in part, of course, by differences in behavior between species but they might also reflect the use of different experiment methods: microarray analysis was used in some of the previous studies, whereas in the present study we used sequencing, which is more effective than microarray analysis for the detection of mirnas, including novel mirnas, that are expressed at low levels.
in the case of the human genome, more than a third of the genes have been predicted to be mirna targets, and these targets would appear to be involved in a wide range of biological functions.
in a. thaliana, mirnas may target transcripts that encode proteins and transcription factors  <cit> .
our results indicated that a total of  <dig> and  <dig> mrnas were targets of putative conserved mirnas and putative novel mirnas, respectively.
these targets regulate a range of cellular processes, within the broad categories of cell-cycle regulation, plant hormonal signal transduction, and metabolism.
however, there are several mirnas without target genes; these could be the result of erroneous target predictions, or they might be low-abundance mirnas with limited or no activity.
it is also possible that mirnas might exist that have no targets.
nevertheless, the ko and go analyses revealed that many of the genes targeted by mirnas in broccoli are related to salt stress, supporting the hypothesis that mirnas play an important role in the response of broccoli to salinity.
CONCLUSIONS
a comprehensive study of broccoli mirnas in relation to salt stress has been undertaken.
our results provide new and significant data on the mirna profile of broccoli.
the differential regulation of mirnas between control and salt-stressed broccoli indicates that mirnas have a marked involvement in regulatory networks associated with salt stress.
further studies of gene function and regulation are now required in order to elucidate the precise mechanisms.

 micrornas are a class of non-coding small rnas, approximately 20– nucleotides in length, that post-transcriptionally regulate gene expression. in plants, highly conserved and species-specific mirnas control a vast array of biological processes, such as leaf polarity, flower development and stress responses. the mirnas are excised from stem-loop structures within larger primary mirna transcripts by dicer-like, which in each case trims the hairpin structure. in plants, the mature mirna strands have high complementarity to their target mrnas and regulate gene expression via mrna cleavage. analysis indicates that many plant mirnas and their targets are evolutionarily conserved from species to species within the plant kingdom. thus, a mirna in one species may exist as orthologs or homologs in other species. plant mirnas have usually been identified either by prediction through bioinformatics or by experimental methods. however, the bioinformatics-based approaches can only identify mirnas that are conserved amongst organisms, and dna or rna sequence information is required in order to run the programs. thus, sequencing is the most effective method for plant mirna discovery. to date, sequences of mature mirnas have been identified in plants, including from a. thaliana, from a. lyrata, from o. sativa and from b. rapa. the genus brassica is cultivated in most parts of the world. it includes various crops of agronomic importance, such as broccoli, cauliflower and b. rapa. broccoli is very popular with a wide range of consumers because of its flavor, and also on account of its anti-cancer activities, notably in relation to prostate and colorectal cancers. because broccoli, a. thaliana and b. rapa all belong to the same family, the cruciferae, the level of synteny between these species provides a basis for studying the mirnas of broccoli. environmental stresses such as salinity are a worldwide problem in agriculture, diminishing plant growth and yield. broccoli is moderately tolerant to salinity, and it displays better tolerance than some other common vegetables, such as maize and carrot. although several stress-related mirnas have been identified based on the sequencing of a library of srnas isolated from a. thaliana seedlings, o. sativa, saccharum officinarum l. and from populous euphratica exposed to various stresses, there have been few studies that have focused upon mirnas in broccoli, and in particular upon the identification of stress-related mirnas in broccoli. thus, in the present study, we have identified mirnas and their targets related to salt-stress in broccoli, using high-throughput sequencing methods. the differential expression of mirnas observed between broccoli grown under standard conditions and broccoli subjected to salt stress provides new insights that will inform the genetic improvement of stress tolerance in plants.  sequence analysis of srnas in order to identify the mirnas responding to salt stress, we constructed and sequenced srna libraries ranging in size from to 30 nt, from both control and salt-stressed broccoli. a total of, and, reads were obtained from control and salt-stressed broccoli, respectively. after removing the tags with: any n bases, more than bases whose quality score was lower than and more than bases whose quality score was lower than and those that were too small, as well as the adapter sequences,, and, clean reads were obtained, representing, and, unique, although sometimes partially overlapping, clean reads from control and salt-stressed broccoli, respectively. the size distributions of the reads in the two datasets were quite similar, and they were not evenly distributed. the majority of these unique sequence reads were 24 nt in size, followed by 23 nt, 21 nt and 22 nt, both in control and salt-stressed broccoli, which is similar to the size distribution of srnas in plants. the overall distribution pattern of srnas in salt-stressed broccoli was similar to that in control broccoli. moreover, common and specific tags between the control and salt-stressed broccoli were analyzed. the results showed that only, of the unique sequences and, of the total sequences were shared between the two samples, suggesting that the sequence results were reliably representative of the endogenous srnas in broccoli.figure sequence analysis of srnas. length distribution of srnas. the length distribution of high-quality sequences obtained from the two broccoli libraries. the distributions of the total reads are shown as percentages. summary of common and specific sequences between control and salt-stressed broccoli libraries. total srnas and unique srnas are shown in the left panel and the right panel, respectively. because the whole genome of sequence for broccoli was not available, all clean reads were aligned against the b. rapa genome, using short oligonucleotide alignment program-. however, only a very small percentage of the unique srna sequences could be matched against the b. rapa genome; only, of the reads in the control broccoli and, of the reads in the salt-stressed broccoli could be mapped in this way, representing, and, of the total reads in control and salt-stressed broccoli, respectively. all clean reads were annotated into different categories, including plant mirnas, exons and introns, and non-coding rnas. in those cases in which srnas were mapped to more than one category, the following priority rule was adopted: rrna > known mirna > exon > intron. the results indicated that the majority of srnas – % of the unique reads in the control group and % in the salt-stressed group – remained unannotated. for the control group, when unique srnas were matched, a small proportion of reads were derived from repeated sequences, and a smaller proportion from rrnas. however, for the total srna pools, rrnas were the most abundant sequences, followed by repeated sequences. all the unannotated sequences were then used for further analysis.table distribution of genome-mapped sequence reads in the srna libraries for control and salt-stressed broccoli locus class control salt-stressed unique srna percent total srna percent unique srna percent total srna percent identification and expression patterns of salt-stress-induced conserved mirnas in broccoli conserved mirna families are found in many plant species and play an important role in a diversity of plant processes. in spite of this, sequence information for broccoli mirnas is absent from both the mirbase database and from the plant mirna database. furthermore, although mirna families are conserved between closely related species, there are only known b. rapa mirnas in mirbase. therefore, the srna sequences were aligned to the mirna precursor/mature mirna sequences of the viridiplantae in mirbase. ninety-seven putative known mirnas were identified in salt-stressed broccoli, including mirnas that were also found in the control group. in particular, the putative conserved mirnas, mir156a, mir166a and mir168a, which have been found in about plant species, were amongst the mirnas that were found in both groups. a comparison of the mirnas in the two libraries indicated that about putative mirnas – including, for example, mir mir and mir – were less abundant in salt-stressed broccoli than in control broccoli. conversely, mirnas – including, for example, mir157d, mir393b-3p and mir169m – were more abundant in salt-stressed broccoli than in control broccoli. although the levels of some mirnas belonging to the same mirna gene family, such as mir mir and mir were found to be increased and decreased in parallel, others were not; the levels of mir and mir393a, for example, increased and decreased differently. however, when the fold-change criterion between salt-stressed broccoli and control broccoli was set to < − or > , and the false discovery rate was set to < , the levels of mirnas were found to be significantly different between salt-stressed and control plants. as shown in figure 2b, the levels of mir mir mir mir168a and mir were all decreased; levels of mir mir396a, mir397a and mir164a, on the other hand, were increased under salt stress. the mirna that showed the greatest decrease under salt stress was mir and the mirnas that showed the greatest increase were mir165a-5p and mir figure differential expression of putative conserved mirnas between control and salt-stressed broccoli. scatter diagram of the differential read counts of known mirnas. each point in the figure represents a mirna. red points represent mirnas showing a > 2-fold change of expression; blue points represent mirnas showing 1/2 < fold change ≤ 2; green points represent mirnas showing a fold change ≤ 1/ the mirnas showing the greatest changes in expression, with fold changes < 1/ or >  decreased and increased putative conserved mirnas confirmed by stem-loop real-time rt-pcr, as described in methods. significant difference between salt-stressed broccoli and control broccoli is indicated by *p <  and **p < . real-time quantification of mirnas by stem-loop real-time reverse transcription polymerase chain reaction is specific for mature mirnas and discriminates amongst related mirnas that differ by as little as one nucleotide. furthermore, it is not affected by genomic dna contamination. therefore, this technique was used to validate the sequencing results. to confirm the identity of the individual pcr products, they were firstly confirmed by electrophoretic sequencing and by denaturing gel electrophoresis. for those mirnas that were less abundant in salt-stressed broccoli than in control broccoli, the results obtained by stem-loop rt-pcr agreed with the results obtained by sequencing. the greatest degree of down-regulation in response to salt stress was shown by mir and mir. amongst the mirnas that were more abundant in salt-stressed broccoli than in control broccoli, also showed up-regulated expression when analyzed by stem-loop rt-pcr; the three exceptions were mir mir858-3p and mir which were not detected by this technique, either in control or in salt-stressed broccoli. of these mir396a showed the greatest degree of up-regulation, followed by mir838-5p, mir852-5p, mir and mir393a. these findings contrasted with the results obtained by sequencing, which indicated that the two most abundant up-regulated mirnas were mir165a-5p and mir in summary, putative conserved mirnas that were differentially expressed between control and salt-stressed broccoli were revealed by their read counts and confirmed by the use of stem-loop real-time rt-pcr. identification and expression patterns of salt-stress-induced, non-conserved mirnas in broccoli in order to identify putative novel mirnas in the broccoli dataset, secondary structures and minimum free-energies were calculated. in total, unique mature mirnas were identified initially in our study, and these candidate mirnas originated from predicted rna hairpins. with an average of about read counts, these putative novel mirnas showed fewer read counts than the putative conserved mirnas. a comparison of the putative novel mirnas in the two libraries indicated that the levels of about of theses mirnas were decreased in response to salt stress. conversely, the levels of about mirnas were increased. real time rt-pcr was next performed for those mirnas with an average of more than read counts either in the salt stressed broccoli or control broccoli, and whose levels were found to be significantly different between salt-stressed and control plants, when the fold-change criterion between salt-stressed broccoli and control broccoli was set to < − or > , and the fdr was set to < . the results from the mirnas analyzed by real time rt-pcr indicated that only of these candidate mirnas were expressed in either the control or the salt-stressed group. a higher proportion of the sequences were identified from the 5’-ends of the hairpins than from the 3’-ends. secondary hairpin structures for representative mirnas are shown in figure 3b, and the secondary hairpin structures for all the candidate mirnas are listed in additional file 5: figure s figure differential expression of putative novel mirnas between control and salt-stressed broccoli. scatter diagram of differential read counts of putative mirnas. prediction of secondary structure of representative putative mirnas from broccoli with or without salt treatment. increased and decreased putative novel mirnas confirmed by stem-loop real-time rt-pcr. significant difference between salt-stressed broccoli and control broccoli is indicated by *p <  and **p < . putative novel mirnas in the two libraries novel id location strand energy were detected only in control broccoli, whilst five were found only in salt-stressed broccoli. four other putative novel mirnas exhibited similar expression levels in both libraries. although most of the putative novel mirnas were observed in both libraries, their expression could be significantly different; for example, mir showed four-fold higher expression in salt-stressed broccoli than in control plants. amongst those that were significantly up-regulated, mir was up-regulated to the greatest extent, followed by mir mir and mir. in contrast, mir and mir were the putative novel mirnas that showed the largest down-regulation in response to salt stress. in summary, new candidate mirnas that were differentially expressed between control and salt-stressed broccoli were identified by their read counts and confirmed by the stem-loop real-time rt-pcr. target prediction and analysis of differential expression of mirnas to understand the biological mechanisms by which broccoli responds to salt stress, the putative target sites of mirnas were identified by aligning mirna sequences with the expressed sequence tags of b. rapa, following the rules of target prediction suggested by allen et al.. a total of and mrnas were predicted as targets of putative conserved mirnas and putative novel mirnas, respectively. in plants, the identification of mrna targets is straight forward because most mirnas and their target mrnas have exact or nearly exact complementarity. furthermore, the mirna target sites of plants have been shown to be located primarily in the coding regions. the putative target genes appeared to be involved in a wide variety of biological processes and molecular-genetic functions; therefore, the gene ontology database and the kyoto encyclopedia of genes and genomes orthology database were used to analyze the datasets. studies have shown that salt stress interferes with cell-cycle regulation at the transcriptional level, resulting in an adaptive growth response. hormone signal transduction and sulfur metabolism are also vital for the improvement of stress tolerance in crop plants. interestingly, the results we obtained using the ko database indicated that genes involved in a diversity of cellular processes, such as the cell cycle, plant hormone signal transduction, and sulfur metabolism were up-regulated in response to salt stress. similarly, pathway annotation for the putative novel mirnas that were up-regulated in response to salt stress indicated involvements in calcium reabsorption and in the cell cycle. the gene targets of both the putative conserved and the putative novel mirnas were classified into three go categories: cellular location, biological process and molecular function. for the cellular location category, large numbers of targets for putative conserved and putative candidate mirnas were categorized under ‘cell’ and ‘cell part’, whereas ‘extracellular region’ comprised the smallest proportion. for the biological process category, the majority were classified under ‘cellular process’, ‘metabolic process’ or ‘response to stimulus’. under the molecular function category, the two most abundant sub-categories for both putative conserved and putative novel mirnas were ‘binding’ and ‘catalytic activity’.figure functional classification of mirna targets according to the gene ontology program. in this ontology, ‘cellular location’, ‘biological process’ and ‘molecular function’ are treated as independent attributes. go classifications for putative conserved and putative novel mirna targets are shown in the upper panel and lower panel, respectively. discussion salinity is an increasingly important agricultural problem. the metabolism and physiological integrity of plants are affected by salt stress and in recent years many studies have been devoted to understanding the molecular mechanisms of plant salt tolerance. studies have confirmed that resistance to salt stress is associated with the use of compatible solutes by plants, and with ion transporters. mirnas are a class of non-coding srnas that are implicated in many developmental processes and in responses to various abiotic stresses, and which play pivotal roles in plant adaptation. to date, in excess of hairpin sequences and mature sequences have been identified from plants, animals, unicellular organisms and viruses; however, scant attention has been paid to the identification of mirnas in broccoli. in the present study, we identified several million srna sequences in salt-stressed plantlets of broccoli, using high-throughput sequencing methods to identify mirnas associated with physiology and metabolism under salt stress. a total of putative known and putative candidate mirnas that were differentially expressed between control and salt-stressed broccoli were discovered according to their sequencing-reads numbers and confirmed by real-time rt-pcr. finally, analysis of the predicted targets of the mirnas using the go and ko databases indicated that a range of metabolic pathways and biological processes known to be associated with salt stress were up-regulated in broccoli treated with salt. the study of mirnas using traditional methods is complex, and high-throughput sequencing methods now provide a rapid and efficient additional approach by which to identify and profile populations of srnas at different stages of plant development. large numbers of non-conserved or species-specific mirnas often accumulate in plants at lower levels than conserved mirnas, and are therefore not easily revealed using traditional sequencing methods. in the present study, the results indicated a range of srnas, of length 16–29 nt, in broccoli, with most of the unique sequence reads being 24 nt in length. several plant species, including a. thaliana, citrus sativus and c. sinensis, had been shown to contain substantially more 24-nt srnas than 21-nt srnas; on the other hand, srnas populations with more 21-nt members than 24-nt were reported in b. juncea and in japanese apricot with imperfect flower buds. b. juncea, b. napus and broccoli all belong to the same genus; however, when broccoli srnas are compared to those of b. juncea, a striking difference exists, whereas little difference is observed when broccoli srnas are compared to those of b. napus. thus, we can conclude that the srna transcriptome is complex, and can be highly variable even between closely related plant species. generally speaking, length distribution analyses of srnas provide a helpful way to assess the composition of srna samples. previous studies have shown that dcl mainly produces srnas that are 18–21 nt long. in contrast, the products of dcl dcl and dcl are 22 nt, 24 nt and 21 nt long, respectively. furthermore, mirnas are normally 21 nt or 22 nt long, whereas small interfering rna are 24 nt long. by this reasoning, therefore, our results imply that the most abundant srnas are mirnas in broccoli and sirnas that have been cleaved by dcl and dcl sequence information for broccoli mirnas is entirely absent from mirbase, and furthermore there are fewer than b. rapa mirnas in this database. therefore, the srna sequences were aligned instead against mirna precursors/mature mirnas of the viridiplantae in mirbase. a search for conserved mirnas revealed that the majority of these molecules, known already from a. thaliana and other species, were detectable in broccoli and were expressed at relatively high abundance. the patterns of expression of conserved mirnas are variable in different plants. previous studies had shown that mir mir166a, mir mir171f and mir were all detectable in b.rapa, and with relatively high numbers of reads, whereas members of the mir family showed low read numbers. the findings of the present study were quite different; mir166a, mir168a and mir157d showed the highest values for copy number in control broccoli. a previous report suggested that mir which was initially identified in a. thaliana and later found in populus trichocarpa, was a dicot-specific mirna and that mir and mir might be monocot-specific mirnas. on the other hand, sunkar et al. indicated that mir was present in both monocots and dicots. our results provided a large-scale database against which to examine these suggestions. we detected both mir and mir in broccoli, whereas mir and mir were not detectable, thereby confirming the previous reports. furthermore, the mir and mir sequences have been considered to be a. thaliana-specific; however, we detected mir158a, mir158b, and mir in broccoli, each at an abundance of about reads. although many plant mirnas have been found to be conserved – for example, mir mir156/ mir mir165/ and mir have been found in more than plant species – studies have indicated that most mirnas can be induced by environmental stresses. for example, mir is induced under low-phosphate conditions, mir increases upon sulfate starvation and mir is up-regulated in response to cold, dehydration, nacl and abscisic acid stress. a recent study showed that mir was transiently up-regulated in response to osmotic stress. on the other hand, mir is down-regulated in response to oxidative stress. several differentially regulated mirnas have been identified in salt-stressed plants. for example, mir530a, mir mir1446a-e and mir were down-regulated during salt stress in p. trichocarpa, as measured by microarray analysis of known mirnas, whereas mir and mir were up-regulated. in a. thaliana, mir mir mir mir mir mir mir mir mir mir mir mir and mir were all up-regulated in response to salt stress, whereas mir was down-regulated. furthermore, mir169g and mir169n were also reported to be induced by high salinity. recently, a study of maize roots using mirna microarray hybridization indicated that members of the mir mir mir and mir families were down-regulated by salt shock, whereas mir and mir were up-regulated. our result in broccoli indicated that, amongst the conserved mirnas, mir and mir were the most strongly down-regulated in response to salt stress, whereas conserved mir838-5p and mir396a were the most strongly up-regulated. of the putative novel mirnas, mir showed the greatest degree of up-regulation in response to salt stress, whereas mir and mir showed the greatest degree of down-regulation. the differences between our findings and the reports relating to salt stress in p. trichocarpa, a. thaliana and maize roots might be explained in part, of course, by differences in behavior between species but they might also reflect the use of different experiment methods: microarray analysis was used in some of the previous studies, whereas in the present study we used sequencing, which is more effective than microarray analysis for the detection of mirnas, including novel mirnas, that are expressed at low levels. in the case of the human genome, more than a third of the genes have been predicted to be mirna targets, and these targets would appear to be involved in a wide range of biological functions. in a. thaliana, mirnas may target transcripts that encode proteins and transcription factors. our results indicated that a total of and mrnas were targets of putative conserved mirnas and putative novel mirnas, respectively. these targets regulate a range of cellular processes, within the broad categories of cell-cycle regulation, plant hormonal signal transduction, and metabolism. however, there are several mirnas without target genes; these could be the result of erroneous target predictions, or they might be low-abundance mirnas with limited or no activity. it is also possible that mirnas might exist that have no targets. nevertheless, the ko and go analyses revealed that many of the genes targeted by mirnas in broccoli are related to salt stress, supporting the hypothesis that mirnas play an important role in the response of broccoli to salinity.  a comprehensive study of broccoli mirnas in relation to salt stress has been undertaken. our results provide new and significant data on the mirna profile of broccoli. the differential regulation of mirnas between control and salt-stressed broccoli indicates that mirnas have a marked involvement in regulatory networks associated with salt stress. further studies of gene function and regulation are now required in order to elucidate the precise mechanisms. 
finally, analysis of the predicted gene targets of mirnas using the go and ko databases indicated that a range of metabolic and other cellular functions known to be associated with salt stress were up-regulated in broccoli treated with salt. little is known about the role of mirnas in the response of broccoli to salt stress. in this study, broccoli subjected to salt stress and broccoli grown under control conditions were analyzed by high-throughput sequencing. a comprehensive study of broccoli mirna in relation to salt stress has been performed. the differential regulation of mirnas between control and salt-stressed broccoli indicates that mirnas play an integral role in the regulation of responses to salt stress. broccolisalt stresshigh-throughput sequencingmicrornaissue-copyright-statement© the author 2014 furthermore, putative known and putative candidate mirnas that were differentially expressed between control and salt-stressed broccoli were revealed by their read counts and confirmed by the use of stem-loop real-time rt-pcr. the brassica vegetable, broccoli, is very popular with a wide range of consumers, but environmental stresses such as salinity are a problem worldwide in restricting its growth and yield. amongst these, the putative conserved mirnas, mir and mir and two putative candidate mirnas, mir and mir were the most strongly down-regulated when broccoli was salt-stressed, whereas the putative conserved mirna, mir396a, and the putative candidate mirna, mir were the most up-regulated. we report significant data on the mirna profile of broccoli that will underpin further studies on stress responses in broccoli and related species. micrornas are a new class of endogenous regulators of a broad range of physiological processes, which act by regulating gene expression post-transcriptionally. the prediction of mirna targets was undertaken using the kyoto encyclopedia of genes and genomes orthology database and gene ontology -enrichment analyses.
 micrornas are a new class of endogenous regulators of a broad range of physiological processes, which act by regulating gene expression post-transcriptionally. the brassica vegetable, broccoli, is very popular with a wide range of consumers, but environmental stresses such as salinity are a problem worldwide in restricting its growth and yield. little is known about the role of mirnas in the response of broccoli to salt stress. in this study, broccoli subjected to salt stress and broccoli grown under control conditions were analyzed by high-throughput sequencing. differential mirna expression was confirmed by real-time reverse transcription polymerase chain reaction. the prediction of mirna targets was undertaken using the kyoto encyclopedia of genes and genomes orthology database and gene ontology -enrichment analyses.  two libraries of small rnas were constructed and sequenced by high-throughput solexa sequencing. a total of, and, clean reads, representing, and, unique reads, were obtained for control and salt-stressed broccoli, respectively. furthermore, putative known and putative candidate mirnas that were differentially expressed between control and salt-stressed broccoli were revealed by their read counts and confirmed by the use of stem-loop real-time rt-pcr. amongst these, the putative conserved mirnas, mir and mir and two putative candidate mirnas, mir and mir were the most strongly down-regulated when broccoli was salt-stressed, whereas the putative conserved mirna, mir396a, and the putative candidate mirna, mir were the most up-regulated. finally, analysis of the predicted gene targets of mirnas using the go and ko databases indicated that a range of metabolic and other cellular functions known to be associated with salt stress were up-regulated in broccoli treated with salt.  a comprehensive study of broccoli mirna in relation to salt stress has been performed. we report significant data on the mirna profile of broccoli that will underpin further studies on stress responses in broccoli and related species. the differential regulation of mirnas between control and salt-stressed broccoli indicates that mirnas play an integral role in the regulation of responses to salt stress. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords broccolisalt stresshigh-throughput sequencingmicrornaissue-copyright-statement© the author 2014 