BACKGROUND
non-coding small rnas  are important regulators of gene expression in eukaryotes and influence almost all aspects of plant biology  <cit> .
there are two major classes of endogenous srnas: micrornas  and short-interfering rnas , typically of  <dig> to  <dig> nucleotides in length.
mirnas are the best understood class of srnas.
they differ from other types of srnas in their biogenesis.
in plants, mirnas are generated from primary mirna transcripts  that are generally transcribed by rna polymerase ii and fold back on themselves to form distinctive hairpin structures  <cit> .
primary transcripts are first processed  by dicer-like <dig>  to mirna precursors  in the nucleus, which are exported to the cytoplasm and then further excised by dcl <dig> to generate ~21nt mature mirnas  <cit> .
plant mirnas suppress gene expression mainly by directing cleavage of their highly complementary target transcripts.
recent studies suggest that translational repression may be also common in plants  <cit> .
the number of newly discovered mirnas is growing rapidly  <cit> .
at the time of writing,  <dig>  mature mirnas have been discovered and deposited in the public mirna database mirbase .
these mirnas include  <dig>  mirnas from  <dig> flowering plant species.
the majority were obtained from model species with sequenced genomes such as medicago truncatula , oryza sativa , glycine max , populus trichocarpa , arabidopsis thaliana , physcomitrella patens  and arabidopsis lyrata .
brassica napus  is the third largest oilseed crop in the world, providing approximately 13% of the world's supply of vegetable oil  <cit> .
several recent studies have contributed to the identification of mirnas from b. napus.
xie et al.
<cit>  used computational methods to predict  <dig> potential b. napus mirnas.
wang et al.
<cit>  cloned and identified  <dig> conserved mirna families and examined their expression patterns in five double haploid b. napus lines.
buhtz et al.
<cit>  identified  <dig> mirnas from  <dig> different families and a set of unknown srnas from b. napus phloem sap.
at the time of writing,  <dig> mirnas  corresponding to  <dig> families from b. napus have been deposited in the mirbase database .
during revision of this paper, more mirnas were identified from the early siliques of two b. napus cultivars differing in oil content  <cit> , from b. napus seedlings exposed to heavy metals  <cit>  and from pooled b. napus tissues  <cit> .
to date,  <dig> unique mirna sequences representing  <dig> mirnas from  <dig> families have been deposited in the mirbase .
considering the estimated size of the b. napus genome   <cit>  and the small numbers of identified mirnas so far, it can be assumed that many more b. napus mirnas remain to be discovered, especially in seed.
to understand the regulatory roles of mirnas in b. napus, it is important to expand the collection of mirnas and profile their temporal and spatial expression.
seed maturation covers the period from the end of embryo development to the mature dry seed and occurs from about  <dig> to 45 days after flowering .
it can be partitioned into two over-lapping phases, seed filling followed by seed desiccation.
processes occurring in maturation affect seed size, oil production, protein content and antinutritional accumulation, as well as seedling vigor following imbibition of the dry seed for subsequent plant growth.
recent gene expression studies using microarrays have shown that there are vast gene expression changes during seed filling and desiccation in b. napus.
distinct expression patterns related to carbohydrate metabolism, lipid biosynthesis, and storage protein accumulation have been documented  <cit> .
however, expression and regulatory functions of mirnas in brassica seed development, especially during seed maturation, are unknown.
the high throughput and sensitivity of next generation sequencing  technologies make them powerful tools for both discovery of novel mirnas and genome-wide profiling of conserved mirnas.
to discover and characterize the mirnas during brassica seed development, ngs technologies were used for mirna discovery and profiling during seed maturation in b. napus.
comprehensive analyses of mirnas and their targets identified many new mirnas and delivered new insights into their role in seed development and maturation.
RESULTS
sequencing statistics
for maximum sequencing depth, we used two technologies: sequencing by oligonucleotide ligation and detection  and sequencing by synthesis .
small rna libraries were prepared from  <dig> seed developmental stages  plus floral buds and these libraries were sequenced using solid.
to investigate the temporal and spatial expression of mirnas, we further dissected seeds into embryo , endosperm  and seed coat  tissues at  <dig> selected seed stages ; in addition, the embryo was separated into radicle , hypocotyl  and cotyledon  at  <dig> and  <dig> daf.
these dissected samples were used to prepare  <dig> additional small rna libraries that were sequenced using sbs.
the sequencing results from both solid and sbs are summarized in table  <dig>  in total, we obtained 88 m reads from  <dig> solid libraries  and 295 m reads from  <dig> sbs libraries .
after removing low quality reads and adaptors from the raw sequences, we obtained 234 m sbs and 74 m solid clean sequence reads, among which ~175 m sbs and ~17 m solid sequences were of 19– <dig> nt in length.
notes:
1) *discarded  illumina adaptor: atctcgtatgccgtcttctgcttg .
2) *clipped solid adaptor: cgccttggccgtacagcag .
3) **cleaned single nucleotide repeats .
size distribution of small rnas
as expected, b. napus has a complex small rna population .
the most abundant length of small rnas in the whole seed was 24nt followed by 23nt and 21nt at all development stages .
this is consistent with previous reports from other species  <cit> .
the 24nt population increased in early seed development until 25daf and then declined from 25daf to mature seeds.
the 24nt small rnas were the most abundant in all tissue types and were extremely high in the radicle of the embryo, reaching 76% of the total small rna  reads.
the proportion of  <dig> nt small rnas was lower in endosperm - about 50%.
the 24nt rnas mainly consist of sirnas that are associated with the silencing of repeat sequences and transposons  <cit> .
higher levels of 24nt rnas in the developing and mature embryo  compared to other tissue types suggest that repression of repeats and transposons in the embryo is important during seed development.
identification of conserved mirnas
many mirnas are evolutionarily conserved in the plant kingdom  <cit> .
to identify conserved mirnas in b. napus, all processed  sequences from the  <dig> small rna libraries were pooled and searched for the presence of known mirnas listed in mirbase .
<dig> unique sequences showed perfect matches to known mirnas from  <dig> mirna families  .
<dig> of them are a perfect match to mirna mature sequences and  <dig> of them to mirna star  sequences .
at the time of writing,  <dig> mirna entries  from  <dig> mirna families  in b. napus have been deposited in mirbase .
in this study, we detected  <dig> out of these  <dig> mature mirna sequences, covering  <dig> of  <dig> entries and all of the  <dig> mirna families.
in addition, we found  <dig> mirna orthologs that have never been reported in b. napus.
this has substantially expanded the collection of mirnas in b. napus.
from the  <dig> conserved mirna sequences,  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> were exact matches to mature mirna sequences from a. thaliana , a. lyrata , o. sativa , p. trichocarpa , m. trunculata  and g. max  respectively.
among the  <dig> sequences that matched to a. lyrata,  <dig> were mirna* sequences.
the high proportion of mirna* sequence matches was due to the fact that most mirna* sequences in mirbase are from a. lyrata.
usually the mirna sequences are more abundant than their star sequences.
the large coverage of mirnas in seeds indicates that many mirnas are involved in seed development.
the total number of reads from conserved mirnas in whole seeds  decreased from flower buds to 15daf seeds, but then increased substantially during seed development, especially in seed maturation .
the total numbers of conserved reads from dissected seed parts at  <dig> stages are shown in figure 2b.
mirnas were more abundant in endosperm than in embryo and seed coat at 15daf.
however, at later stages, mirnas were substantially more abundant in embryo than in endosperm or seed coat.
the total number of mirna reads in embryo increased significantly until 35daf and then decreased at 45daf.
within the embryo, mirnas are most abundant in the cotyledons, followed by hypocotyl and radicle .
many additional mature mirnas
in addition to exact matches to mature or mature star sequences annotated in mirbase , there were numerous other abundant mature mirna sequences.
most of these sequences differed from the annotated mirnas by only a single nucleotide and could usually be mapped to the corresponding annotated precursors .
allowing one mismatch with the annotated mirnas in mirbase , we identified  <dig>  mirna-like sequences with at least  <dig> reads.
among them,  <dig>  were exact matches and  <dig>  differed from the annotated mirna or mirna* by one nucleotide .
the read counts of these sequences varied from  <dig> to  <dig> .
while some of these sequences might be mirna sequence variants produced from imprecise cleavage during mirna biogenesis, the high abundance of many of these sequences suggests that they may be either independent mature mirnas or, more likely, that they are the ‘true’ mature mirnas in b. napus, rather than the annotated mirnas from other species.
for example, two sequence variants of mir <dig> , are both more abundant than the sequence that is annotated as ath-mir158a , indicating they both are very likely mir <dig> mature sequences.
furthermore, a variant of mir <dig> has  <dig>  reads while the exact ath-mir <dig> sequence has only  <dig>  reads, indicating the former sequence is more likely the functional mirna in b. napus during seed maturation.
therefore we defined these sequence variants as conserved mirnas and focused on the mirna/variant sequences with > <dig> reads for further profiling analyses .
abundance of conserved mirna families in seeds
the abundance of mirnas varied enormously between mirna families, ranging from less than  <dig> to several million reads .
the most frequently sequenced mirnas  are listed in table  <dig>  the total number of sequence reads from all mirna variants  within each mirna family were pooled for abundance analysis.
within each family, the proportion of the total reads accounted for by the major variants  are shown in figure  <dig> as well as the total number of reads for each family and the number of variants.
the mirna <dig> family was the most abundant in seed followed by the mir <dig>  mir <dig>  mir <dig> and mir <dig> families.
the  <dig> top mirna families accounted for 49%, 18%, 7%, 6% and 5% of the total reads from all conserved seed mirnas, respectively.
the remaining mirna families together account for approximately 15% of total conserved mirna reads.
the number of mirna variants in each mirna family was highly variable .
for example, there were  <dig>   <dig>   <dig>   <dig> and  <dig> unique variant sequences  in the mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir <dig> families respectively, but only one mirna sequence in the mir <dig>  mir <dig>  mir <dig> and mir <dig> families.
usually, one or two mirna variant sequences predominated within each family .
for example, ath-mir156a, ath-mir159a, ath-mir172c, ath-mir167a and bna-mir158b were the predominant variants in the mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir <dig> families.
this suggests that, in most cases, the regulatory role of each family is mostly performed by the dominant mirna variants.
a conspicuous exception to this relationship appears to be mir <dig> but later mapping analysis revealed this not to be a typical mirna.
sequences were compared to mirbase release  <dig> for homology searches.
temporal expression patterns of conserved mirnas
more than  <dig> conserved mirna variant sequences with at least  <dig> reads were identified from b. napus seed developmental stages and flower buds.
the expression profiles of these mirna/variant sequences in flower buds, seed stages and compartments are listed in additional file 3: table s <dig>  we performed hierarchical clustering analysis on the  <dig> most abundant mirnas in the  <dig> flower bud and whole seed samples  and figure  <dig> shows the resulting expression patterns.
expression of mirnas can be grouped into  <dig> major patterns, a1-a <dig>  the majority of mirnas  increased during seed maturation while a minority  were preferentially expressed at early- and mid- seed development stages.
mirnas in group a <dig> included most variants of mir <dig>  mir <dig> and mir <dig> and were weakly expressed in flower buds.
they showed transient expression at early seed development  then decreased to a low level at 15daf.
after 15daf their expression steadily increased until 50daf.
most group a <dig> mirnas such as mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig> were weakly expressed in flower buds and strongly expressed at later stages of seed development.
the expression of mir <dig> and mir <dig>  were high in both flower buds and seed and had two peaks in expression at 25daf and 50daf.
group a <dig>  including mir <dig>  mir <dig> and mir <dig>  were expressed in both flower buds and during seed development.
their expression increased during seed development until 45daf and then decreased at the end of seed maturation.
mirnas in group a <dig>  represented by mir <dig>  mir <dig>  mir <dig>  mir <dig> and several mir <dig> variants, were preferentially expressed in flower buds and, to a much lesser extent, in seed developmental stages.
tissue-specific expression of conserved mirnas
to compare the expression of mirnas in different seed parts, hierarchical clustering analysis was performed on the normalized read counts of the top  <dig> mirnas in the  <dig> seed part samples .
the results showed strong tissue-specific expression of most of the conserved mirnas .
four distinct expression patterns  were identified.
b <dig> and b <dig> groups were preferentially expressed in embryo.
mirnas in pattern b <dig>  which included most variants of mir <dig>  mir <dig>  mir <dig> and mir <dig>  were preferentially expressed in the cotyledons and hypocotyl while mirnas in pattern b <dig>  which included mir <dig>  mir <dig> and mir <dig> were mostly expressed in the radicle and hypocotyl.
most of the mirnas in b <dig> represented by mir <dig>  mir167and several variants of mir156/ <dig>  were endosperm and seed coat specific.
mirnas in b <dig> including mir <dig> and mir <dig> seemed less tissue-specific but were preferentially expressed at later seed developmental stages.
they were highly expressed in the cotyledons and hypocotyl at later stages and occasionally expressed in endosperm and/or seed coat.
most mirna family members showed similar expression patterns such as mir <dig> , mir <dig> , mir <dig> , mir <dig> , mir <dig>  and mir <dig> .
however, some mirna families showed strikingly different expression patterns among variants.
for example, most of the mir <dig> family  were highly expressed in the embryo cotyledons and their expression increased steadily during seed maturation.
in contrast, some mir <dig> variants  showed high endosperm- and seed coat-specific expression and decreased during seed maturation.
in arabidopsis, many known mirna families are expressed from precursor genes in multiple paralogous loci.
this is also true for b. napus since several mirna members within a family were mapped to multiple loci in the brassica rapa genome  <cit>  in our mapping analysis .
members of mirna families with divergent expression patterns are probably expressed from different paralogous loci.
however, it must be noted that within each family, different loci sometimes produce identical mirnas, therefore the abundance of a mature mirna may not always correspond to expression of a specific precursor.
validation of mirna expression by taqman mirna assay
we employed quantitative taqman pcr  to validate mirna expression determined by deep sequencing.
six moderately to highly abundant mirnas were chosen for validation in seven tissues and stages.
all of the  <dig> selected mirnas were detected in all tissues and stages.
as shown in figure  <dig>  the relative changes in mirna expression calculated from qrt-pcr and sequencing are highly correlated , indicating consistency between both approaches.
identification of putative precursors of b. napus mirna’s using the b. rapa genome as a surrogate for the b. napus a genome
currently, the complete b. napus genome sequence is not available; therefore it is difficult to identify mirna precursor genes since weakly expressed genes are not well represented in est databases and homeologous genes are not reliably distinguished.
it is well-known that b. rapa contributed the a genome to the allotetraploid b. napus containing a and c genomes.
although the b. napus a genome has undoubtedly diverged from the b. rapa reference prior to and after polyploidization, evidence suggests that it retains b. rapa characteristics.
for example, cheung et al.
<cit>  showed that “the a genomes of b. rapa and b. napus show a high degree of similarity along their entire length” and that “only a small number of homeologous recombination events have been observed in oilseed rape cultivars”.
furthermore, when aligned genome segments of b. rapa and the b. napus genome a were compared over  <dig> contigs, a low average snp frequency of  <dig> % in coding regions was observed and  <dig> % overall .
comparison of a specific clone between subspecies b. rapa trilocularis and b. rapa pekinensis revealed a snp frequency of  <dig> %.
when the sequence of the same clone was compared between b. rapa trilocularis and the b napus a genome, the snp rate was still a relatively low  <dig> % .
the snp frequencies suggest that the b. napus a genome and b. rapa have not diverged enough to invalidate sequence-based annotations and predictions based on a b. rapa reference.
therefore, we analyzed the recently released brassica rapa genome  <cit>  for b. napus mirna precursor sequences.
we mapped the sequencing reads, known plant mirna mature sequences and hairpin sequences from mirbase  to b. rapa genome sequences , then merged the  <dig> mapping results to identify the conserved mirna loci.
in total,  <dig> plant mirna hairpin sequences and more than  <dig> mature mirnas from mirbase  were mapped to b. rapa chromosomes and scaffolds.
mapping of the  <dig> known plant mirna hairpin sequences identified  <dig> regions of similarity to the mirna queries in the b. rapa genome  which covered  <dig> mirna families.
in most cases, there were corresponding annotated mature sequences and sequencing reads mapped to these loci and the mapping patterns of sequencing reads to the regions were very typical- as described in kozomara and griffiths-jones  <cit>  – indicating they are orthologous mirna loci in b. rapa .
the mirna-like stem-loop secondary structures further confirmed authentic mirna loci.
examples of typical mapping patterns of mirnas are shown in figure  <dig>  multiple sequences, including the annotated mirna and sequence variants with different sizes , star sequences and variants of star sequences, were mapped to each precursor locus, on both arms of each precursor stem-loop structure.
the ‘true’ mirna sequences are presumably dominant among the sequence variants.
most of the mirnas with stem-loop structures and typical mapping patterns are highly conserved in the plant kingdom including mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir <dig>  <cit> .
however, several regions with similarity to mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir <dig> can be folded into stem-loop secondary structures but have no sequencing reads mapped to the genomic loci, indicating that these loci are not expressed during seed development in b. napus or may not be true mirna loci.
loci with similarity to mir <dig>  mir <dig>  mir <dig> and mir <dig> showed multiple offset reads distributed across the entire gene sequences.
patterns of overlapping reads covering the entire putative locus indicate they are more likely sirna loci rather than mirna loci according to recent mapping guidelines in kozomara and griffiths-jones  <cit> .
examining the mapping patterns of the annotated mirna sequences , conserved new mirna sequences identified in this study, and other sequencing reads, revealed an additional  <dig> mirna loci in the b. rapa genome .
no known orthologous hairpin sequences of these mirnas were mapped to these  <dig> loci in b. rapa, however, the stem-loop structure of the flanking regions and the typical mapping patterns of sequencing reads indicated they are authentic mirna loci according to current guidelines  <cit> .
in these cases, there is low sequence conservation in the b. rapa flanking regions outside the mirna mature sequences.
for example, the mir <dig> mature sequence, its variants and star sequences were cleanly mapped to the b. rapa genome , although the orthologous mir <dig> precursors from other species do not share sequence similarity with the flanking region.
both the mirna-like stem-loop secondary structure and the embedded nature of mirna sequencing reads provided strong evidence that this region is an authentic mir <dig> locus in b. rapa.
the evaluation of mapped sequence reads also provides valuable information about the relative abundance of mature sequences from different arms of stem-loops, isoforms  of mature mirna sequences, and the confidence in a given microrna annotation.
in most cases, the annotated mirna  was the most abundant small rna sequence mapped to an mirna locus.
sometimes, the 22-25nt isoforms of the annotated mirna were more abundant, suggesting they are more likely to be the ‘true’  mirna in b. napus.
in some cases, several annotated mirnas from different species were mapped to the same mirna loci.
for instance, both the annotated bna-mir397a, b  and ath-mir397a, b  were mapped to mir397a.
bna-mir397a, b was substantially more abundant than ath-mir397a, b, indicating it is the ‘true’ mirna in b. napus as annotated .
occasionally, the star sequence or a variant of the star sequence from the opposite hairpin arm of the mirna precursor was more abundant than the annotated mirna.
for example, the mir2111* sequence was more abundant than mir <dig> in bra-mir2111a, b, c and d , suggesting mir2111* is the true mirna mature sequence in b. napus.
this is also true for bra-mir391a and bra-mir <dig>  the sequence of mir391*  was more abundant than the annotated mir <dig>  and a variant of mir408*  is considerably more abundant than mir <dig>  and mir408*  , indicating that they are most likely the dominant mature mirna sequences in b. napus and therefore are named as bna-mir391a and bna-mir <dig> .
mapping of small rna sequencing reads and known mirna sequences  to the b. rapa genome revealed several chromosomal regions with clusters of mirna genes.
these included a cluster containing three mir <dig> genes  on chromosome a <dig> within a 1000 bp region; a mir <dig> cluster as well as a mir <dig> cluster, also on chromosome a <dig>  in addition, we also found that some mirna precursors gave rise to two or more distinct mirna/mirna* duplexes from different positions.
as shown in additional file 5: figure s <dig>  the mir <dig>  mir <dig> and mir <dig> precursor loci as well as newly identified putative novel mirna loci mir <dig> and mir <dig>  can be folded into a long stem-loop structure and generated multiple distinct srnas.
mirna target prediction
mirnas regulate gene expression through targeting transcripts for cleavage, translational repression or mrna decay.
therefore, it is important to identify mirna targets to understand mirna function.
in plants, all known mirna targets have perfect or near-perfect complementary sequences to cognate mirnas  <cit> .
this has allowed many computational approaches to predict mirna targets  <cit> .
we used targetfinder   <cit> , to search against brassica est unigenes  for the targets of mirnas with more than  <dig> reads.
we predicted  <dig>  mirna-target pairs including  <dig>  unique b. napus mirna targets  for  <dig> unique mirnas .
among these predicted targets,  <dig>  est contigs are annotated as homologs of arabidopsis genes by blast-based analysis of sequence homologies in tair <dig> transcripts and protein datasets, while  <dig> of them have no arabidopsis homologs .
these non-annotated est contigs might represent genes specific to b. napus.
to understand the biological roles of the mirna-target pairs in seed development, we performed mirna-pathway analysis by mapping the arabidopsis homologs of the  <dig> target ests to mapman pathways  <cit> .
the functional classification based on mapman terms using the classification superviewer tool w/bootstrap  revealed that these homologs of target genes were highly enriched in pathways of rna, dna, protein, s-assimilation, development, signaling and redox .
detailed inspection of the results revealed that some mirnas function in the same pathway by targeting different genes in the same family.
previous reports grouped mir <dig> and mir <dig> as one mirna family  due to their high sequence homology and co-targeting to squamosa-promoter binding proteins  or sbp-like proteins .
similarly, mir159/ <dig>  mir165/ <dig> and mir170/ <dig> were also grouped together based on shared targets  <cit> .
in addition, we also predict that mir <dig>  mir <dig> and mir <dig> "co-target" pentatricopeptide  repeat-containing proteins; mir <dig>  mir <dig>  mir <dig> "co-target" f-box family proteins and mir <dig>  mir <dig>  mir <dig> and mir <dig> "co-target" various auxin response factors .
therefore, many mirnas may function together via co-targeting to regulate functionally related genes or pathways.
go term analysis using the bingo plugin tool in cytoscape  <cit>  on the arabidopsis homologs of the  <dig> unique target sequences  revealed that these genes are highly overrepresented in regulation, development and stress responses .
in arabidopsis, a majority of mirna targets have been identified and experimentally validated.
many homologs of these arabidopsis mirna target genes can be found from the predicted targets, confirming that conserved mirnas also have conserved targets across species and likely have similar functions in b. napus and arabidopsis.
this is consistent with previous studies in other species  <cit> .
in addition to the conserved targets, we also predicted new targets for the conserved b. napus mirnas.
for example, in addition to members of the spl gene family , we also predicted other new mir <dig> targets, such as homologs of arabidopsis vacuolar h-atpases , low cell density <dig> , an f-box family protein , a leucine-rich repeat transmembrane protein kinase , an auxin-responsive gh <dig> family protein  and fusca <dig> , which is a key regulator of seed development  <cit> .
individual mir <dig> variants may have specific additional targets such as the homolog of sec <dig> cytosolic factor  specific for bna-mir156_v <dig>  an arid/bright dna-binding domain-containing protein  specific for bna-mir156_v <dig>  fus <dig> and an enth domain-containing protein  specific for bna-mir156_v <dig>  an isoflavone reductase  and an isocitrate lyase  specific for bna-mir156_v <dig> and brassinosteroid insensitive  <dig>  for bna-mir156_v <dig> and bna-mir156_v <dig>  this suggests the possibility that individual mirna variants have different functions through their specific targets.
expression of putative conserved mirna targets
to compare the expression of the mirnas with that of their targets, we performed global transcript profiling using brassica 90k combimatrix arrays .
the expression of mirna target genes in seed development was extracted from the microarray results and is listed in additional file 7: table s <dig>  the overview of the expression of b. napus genes  is shown in figure  <dig>  the expression changes of mirna targets  were notably smaller than overall changes in gene expression.
detailed comparison of the expression patterns of mirnas and their targets at the corresponding time points  revealed that, although some mirna-target pairs showed the expected negative  correlation, many others showed either no correlation or even displayed a positive correlation .
most mirnas were expressed at low levels .
similarly, many of the mirna target genes were also expressed at relatively low levels during seed development and maturation .
the maximum raw signal intensities of genes in group  <dig> were under  <dig> throughout seed development, indicating that the expression of these target genes might be repressed throughout seed development.
homologous est contigs of many known mirna target genes including most spls , myb <dig> , arf <dig> and arf <dig>  and numerous others either showed negative expression patterns or were expressed at very low levels throughout seed maturation.
identification of putative novel mirnas, precursors and targets
many mirna families are highly conserved in evolution  <cit> .
however, there are also species-specific, recently evolved mirna genes  <cit> .
to identify mirnas specific to brassica, we again used the b. rapa genome  since the complete b. napus genome is not available.
all of the sequences with read counts > <dig> that were not annotated as trna, rrna, snorna, mirna, repeat or cdnas, but mapped to the b. rapa genome were used to detect potentially novel mirna genes.
the flanking genomic sequences of the mapped reads were extracted and searched for regions that could be folded into a stable mirna precursor-like hairpin with strong pairing between the mature mirna and mirna* sequence on the opposite hairpin arm.
this analysis employed novomir, a program for the identification of plant mirna genes  <cit> .
we identified  <dig> putative novel mirna sequences which mapped to  <dig> genomic loci in b. rapa.
to eliminate sirnas from repeat sequence classes and ensure that they were from good quality single-stranded hairpins, reads that mapped more than  <dig> times to the reference genome or mapped to a bidirectional small rna cluster were also discarded.
the mapping patterns of sequence reads in each genomic locus were manually examined and the loci that didn't meet the consensus set of guidelines for mirna annotation  <cit>  were filtered out.
loci with an embedded known mirna were identified as conserved mirnas and therefore also eliminated.
following the above filtering, we identified  <dig> putative novel mirna loci for  <dig> out of the  <dig> presumed mirna sequences and several conserved mirna variants including mir <dig>  mir <dig> and mir <dig>  these putative novel mirna sequences and their hairpin structures, as well as the mapping patterns of sequencing reads in the hairpin regions, are presented in additional file 9: table s <dig> and additional file 6: figure s <dig>  several putative mirna sequences were mapped to the same loci.
for example,  <dig> putative novel mirna sequences were embedded in the mir5802; of these ngs_90350641_x <dig> is the most abundant sequence and most likely the true mirna mature sequence  and ngs_93017147_x <dig> is the corresponding star sequence  while ngs_93017180_x <dig> is a variant of the same mirna* .
previous studies indicated that non-conserved mirnas are usually weakly expressed with a tissue-specific pattern  <cit> .
due to the unavailability of b. napus genome sequences  and the fact that the small rna libraries were mostly constructed from seeds, mirnas identified in this study might represent only a fraction of the total number of novel mirnas in b. napus.
we also predicted targets for the novel mirna candidates using targetfinder software .
in total we predicted  <dig> brassica contigs as targets of the  <dig> putative mirnas .
based on the annotations of their arabidopsis homologs, the functions of these predicted novel mirna targets are involved in many pathways including development and signaling.
mir <dig> was the most abundant potential mirna  among all predicted novel mirnas.
it is preferentially expressed in embryo, especially in the cotyledons, and increased substantially during seed maturation.
it was predicted to target homologs of arabidopsis demeter  and at1g <dig> .
the expression of these two genes reciprocally decreased during seed maturation.
mir <dig> , the second most abundant potential mirna, accumulated in different compartments of the seed during seed maturation.
it was predicted to target est contigs homologous to an arabidopsis pentatricopeptide  repeat-containing protein and a sphere organelles protein-related gene.
the expression of the target ppr repeat-containing protein showed consistently low expression from  <dig> to  <dig> daf in seed.
discussion
increase in the number of identified mirnas in b. napus and b. rapa
mirnas are essential regulators of gene expression in all plants and animals.
according to the mirbase database ,  <dig> arabidopsis and  <dig> rice mirnas have been identified respectively.
b. napus is polyploid with a much larger and more complex genome than arabidopsis and rice.
however, there were only  <dig> mirnas  annotated in mirbase .
in this study, we identified more than  <dig> mirnas  including  <dig> known b. napus mirna sequences already in mirbase and  <dig> putative novel mirnas.
the mirna-like hairpin structures of the flanking sequences and the typical mapping patterns of the ngs reads to the precursor regions  provided supporting evidence for the newly discovered mirnas.
in the absence of a b. napus genome sequence, we used the newly released b. rapa draft genome sequence  for identification of the hairpin precursor sequences of the newly identified mirnas.
this led to the discovery and annotation of more than  <dig> mirna hairpin sequences and loci.
to date, there are only  <dig> mirnas and their hairpin precursor sequences from b. rapa deposited in mirbase .
identification of the mirna genes in the b. rapa genome has significantly enriched the repertoire of brassica mirna genes and improved the annotation of the newly sequenced b. rapa genome.
in a report published while this article was in preparation, zhao et al.
<cit>  analyzed small rna expression profiles of b. napus siliques at early embryonic developmental stages  in two cultivars with differing oil contents.
they identified  <dig> conserved mirnas of which  <dig> were identical to and  <dig> highly homologous to known mirnas in mirbase.
in addition, they predicted  <dig> new mirnas.
this study provides a more comprehensive temporal and spatial profiling of mirnas focused on post-embryonic development and seed maturation.
by employing greater sequencing depth, we identified many more mirnas including conserved and novel putative mirnas with high confidence and investigated their specific expression patterns during seed development.
to provide a more complete picture of mirnas in b. napus seed development we provide a detailed comparison of our data with that of zhao et al.
<cit>  .
we also identified almost all of the conserved mirnas that they reported.
for the  <dig> predicted new mirnas, we identified the corresponding precursor loci in the b. rapa genome and examined the patterns of sequences that mapped to each region .
among the  <dig> new mirnas predicted by zhao et al.
<cit>  bna-mir <dig>  bna-mir <dig> and bna-mir <dig> were also predicted in this study and bna-mir <dig> was predicted to be a star sequence.
we did not detect reads corresponding to the exact sequences of bna-mir2206a, b , bna-mir <dig> and bna-mir <dig>  the sequences of bna-mir <dig>  bna-mir2206a, b and bna-mir <dig> occurred in the b. rapa genome  <dig>   <dig> and  <dig> times respectively, suggesting that they are unlikely to be authentic mirnas.
the observed pattern of overlapping reads mapping to a broad region of the flanking sequences of mir <dig>  mir <dig> and mir <dig> indicates that they are more likely sirna loci .
the number of sequence reads for bna-mir <dig>  and bna-mir <dig>  were low and therefore excluded from our prediction.
due to the inconsistency of mirna names in zhao et al.
<cit>  and mirbase,  we retain our temporary mirna names for all mirna/variants with reads > <dig> in each family according to their abundance.
these names are subjected to change when the b. napus whole genome sequence becomes available.
very recently, xu et al.
identified  <dig> conserved and  <dig> brassica-specific candidate b. napus mirnas from pooled b. napus tissues using b. napus, b. rapa and b. oleracea genomic survey sequences  databases and ests as references  <cit> .
we detected  <dig> out of the  <dig> available sequences they identified, of which  <dig> had > <dig> total reads, mostly conserved mirnas although a few of them are actually the same as the new conserved or putative novel mirnas we identified .
for instance, the sequence of bna-mirc17a- <dig> is identical to our new conserved bna-mir158_v <dig> , the sequence of bna-mirc <dig> is annotated as bna-mir319_v <dig>  and bna-mirc <dig> is the same as our predicted mir5807a/b .
the remaining  <dig> sequences with no reads or total reads less than  <dig> were excluded from our analysis.
zhou et al.
<cit>  identified  <dig> conserved and non-conserved mirnas  from cd-treated and non-treated b. napus seedlings using the b. napus cdna database and tentative consensus sequences of the b. napus gene index, leaving  <dig>  mirna homologues with high similarity to mirnas from other species  not validated due to lack of a reference sequence.
comparison of the expression of the  <dig> known mirnas in zhou et al.
<cit>  and this study revealed many commonly expressed mirnas despite the very different conditions and stages, especially for the most highly expressed mirnas such as mir <dig>  mir <dig>  mir <dig> and mir <dig>  however,  <dig> of the  <dig> previously annotated mirnas were not expressed under any tissues/stages/conditions in either study , indicating that some of the "known" mirnas are likely not true mirnas .
in this study, we identified many more mirnas with a much more stringent cutoff at > <dig> reads using the b. rapa genome as reference.
with our deep sequencing data, we were able to follow the latest microrna annotation guidelines  <cit>  to integrate sequencing data with mirna annotation and exclude many false mirna loci which are more likely sirna loci based on read mapping patterns.
many of the mirnas identified in this study are now validated by recent reports   <cit> .
the most abundant mirna in seeds of b. napus relative to other species
mir <dig>  is the most abundant mirna in b. napus seeds, accounting for more than 48% of total mirna reads.
in monocotyledonous plants, mir168a was reported as the most abundant mirna in rice  <cit> , brachypodium distachyon <cit> , barley  <cit>  and wheat  <cit>  while mir <dig> is the second most abundant mirna in these species.
our data showed mir <dig>  to be preferentially expressed in the cotyledons of embryo and mir <dig>  to be preferentially expressed in endosperm at a much lower level .
but mir <dig> is the most abundant mirna not only in monocot seeds  but also in other monocot tissues .
therefore the extraordinarily high level of mir <dig> in monocots is not due to the larger portion of endosperm in grass seeds but represents a significant regulatory difference between monocots and dicots.
it has been reported that mir <dig> targets argonaute <dig>  in arabidopsis, rice, and wheat.
ago <dig> is involved in mirna biogenesis and mir168-mediated regulation of ago <dig> mrna has been reported to be important for proper plant development  <cit> .
it will be interesting to further compare the regulation of mir168-ago <dig> in monocots and dicots as more data becomes available.
mirna targets
various prediction approaches have been applied successfully for the identification of mirna targets in plants  <cit> .
in b. napus, xie et al.
<cit>  predicted  <dig> potential target genes for  <dig> newly identified mirnas.
huang et al.
<cit>  identified several mir <dig> target genes.
since few mirnas targets have been identified in b. napus, we may assume that the majority of mirna targets have not been discovered.
in this study, we predicted  <dig>  mirna-target pairs including  <dig>  unique b. napus mirna targets  for  <dig> unique mirnas using targetfinder .
very recently, xu et al.
<cit>  experimentally identified  <dig> non-redundant mrna targets of  <dig> conserved brassica mirnas and  <dig> new non-redundant mrna targets of novel brassica-specific mirnas by sequencing the mrna degradome.
zhou et al.
<cit>  identified  <dig> targets for  <dig> mirna families by deep sequencing of four degradome libraries from cd-treated and control  roots and shoots of b. napus seedlings.
comparing our putative targets to xu et al.
<cit>  and zhou et al.
<cit> , we found significant overlap among them based on the annotations of target genes, including mir <dig> targets , mir <dig> targets , mir <dig> targets , mir <dig> targets , mir <dig> targets  and mir <dig> targets .
detailed comparison revealed that  <dig> out of the  <dig> category i mirna targets from zhou et al.
<cit>  were also predicted as targets of the same mirna families in this study .
mirnas with reads <  <dig> counts such as mir <dig> and mir <dig> were excluded from our target prediction.
this might explain some of the differences between two studies.
in addition, different b. napus est datasets were used in the two studies which might also contribute to differences.
expression patterns of mirna’s and their putative targets
a typical mirna acts to downregulate expression of its target gene by directing cleavage of its highly complementary target transcripts.
this implies that expression of mirnas and their targets should be negatively correlated.
however, as others have noted  <cit> , the expected inverse correlation was often not observed.
in this study, we found that the top  <dig> most abundant mirnas represented 85% of all mirna sequence reads and that most mirnas were expressed at low levels .
similarly, many of the mirna target genes were expressed at relatively low levels during seed development and maturation.
for example, mir <dig> expression increased substantially but spls  were expressed at low levels from  <dig> to  <dig> daf .
in arabidopsis, the expression of spls significantly decreased from the globular to heart stage of seed development and remained low throughout the rest of seed development .
thus, in b. napus, it is likely that expression of spls was already suppressed by mir <dig> before 10daf.
we also observed that typically, the range of expression changes exhibited by the mirna targets during seed development were smaller than the overall range of gene expression changes .
this observation is consistent with previous reports that mirna-mediated post-transcriptional regulation might contribute to buffering changes in target gene expression by establishing a threshold for target-triggered regulatory effects within a complex regulatory network  <cit> .
the buffering function of mirna might be critical to prevent the fluctuation of target gene expression levels and therefore prevent aberrant development events.
the occurrence of mirna multiple targeting and co-targeting also complicates the relationship between mirna and their targets.
given the complexity of gene regulatory networks, an individual mirna target might also be regulated by various factors in addition to the cognate mirnas and one of the other factors may be predominant in a given tissue or stage.
in addition, recent studies have shown that plant mirnas can repress target genes at the level of translation with little or no influence on the mrna abundance  <cit> .
the roles of mirnas in seed development and maturation
plant mirnas are important for fine-tuning plant development including organ identity, patterning, polarity, developmental phase transitions and development of cellular organelles.
previous reports suggested the potential involvement of mir <dig>  mir <dig>  mir <dig>  and mir <dig> in seed development, dormancy, and germination  <cit> .
however, the specific functions of mirnas in seed maturation are largely unknown.
mir <dig>  mir <dig> and their targets  are key players in coordinating plant phase transitions, from juvenile to adult and from the vegetative to the reproductive phase, during post-embryonic development  <cit> .
in arabidopsis and maize, mir <dig> maintains the juvenile phase and prevents precocious flowering while mir <dig> acts downstream of mir <dig> and promotes flowering by repressing apetala 2-like repressors of flowering locust .
mir <dig> targets and represses the expression of spl genes that are positive regulators of mir <dig>  the levels of mir <dig> and mir <dig> exhibit contrasting age/development-specific expression patterns: mir <dig> levels decline during vegetative development whereas mir <dig> levels correspondingly increase  <cit> .
as noted earlier, the mir <dig> family is the most abundant mirna in seed development.
ath-mir156a, the most abundant variant of the mir <dig> family, is preferentially expressed in embryo and increased 113-fold from 15daf to 50daf.
ath-mir172c, the most abundant variant of the mir <dig> family, was preferentially expressed in endosperm in early seed development and decreased substantially during subsequent maturation, showing the opposite expression pattern to mir <dig>  the complementary tissue- and stage-specific expression patterns of mir <dig> and mir <dig> are analogous to the contrasting age/development-specific expression patterns of mir <dig> and mir <dig> during the transition from the vegetative phase to flowering.
this suggests that indirect repression of mir <dig> by the highly expressed mir <dig> is important for embryo maturation and that the mir156-spls-mir <dig> regulatory cascade also plays an important role during seed development and germination.
nodine and bartel  <cit>  proposed that mirnas enable proper embryonic patterning by preventing precocious expression of genes normally expressed during seed maturation, partially through mir156-mediated repression of spl transcripts .
however, the substantial increase of mir <dig> during seed maturation and very low expression of the target spl genes in late seed development indicate that the mir156-spl regulatory cascade is not likely to suppress the expression of maturation genes.
indeed it seems intuitively more likely that mir <dig> represses floral identity  in order to allow vegetative identity to become established during maturation.
the plant life-cycle is characterized by two major developmental phase transitions: germination  and flowering .
recent studies indicate that genes involved in flowering and other phase transitions also regulate the transition from dormancy to germination, suggesting that conserved mechanisms control all plant phase transitions.
a study of global transcriptional interactions  revealed coordinated regulation of plant cellular phase transitions in seed germination.
an intermediate transition phase between dormancy and germination is enriched with genes involved in cellular phase transitions such as early bolting in short days , flowering locus c , flowering locus t  and abscisic acid insensitive  <dig>  <cit> .
the hyper-abundance of mir <dig> in mature seeds and the presence of common regulators of phase changes in seeds and flowering suggest that the mir156-spl regulatory cascade might control the developmental phase transition from embryonic to seedling stage, analogous to shoot maturation and the transition from the vegetative state to flowering  <cit> .
high levels of mir <dig> and reduced spls and mir <dig> in the mature embryo may repress the developmental transition and keep seeds in the maturation/dormant state.
ath-mir <dig> is the second most abundant mirna during seed development and maturation.
the expression pattern of mir <dig> is very similar to that of ath-mir <dig>  in a study by reyes and chua, mir <dig> was induced by aba via the transcription factor abi <dig> and cleaves myb domain protein  <dig>  and myb domain protein  <dig>  transcripts, which were described as positive regulators of aba signaling during germination  <cit> .
however, this study is inconsistent with other papers that provide evidence that gamb-like genes myb <dig>  myb <dig> and myb domain protein  <dig>  mediate ga effects.
for example, mir <dig> was implicated in floral and anther development by targeting the expression of myb <dig> and myb <dig>  which were shown to be involved in ga-promoted activation of leafy <cit> .
recently, alonso-peral et al.
<cit>  demonstrated that in mir <dig> mutants , deregulation of myb <dig> and myb <dig> in vegetative tissues resulted in up-regulation of genes that are highly expressed in the aleurone and induced by ga during germination, suggesting that mir <dig> acts as a molecular switch confining the expression of myb <dig> and myb <dig> to anthers and seeds.
our data shows that mir <dig> is preferentially expressed in the embryo, presumably allowing expression of myb <dig> and myb <dig> in the endosperm to facilitate storage product hydrolysis and later programmed cell death  .
furthermore, within the embryo, mir <dig> is preferentially expressed in the cotyledons and hypocotyl, perhaps repressing specific ga processes to allow aba-associated maturation  to be maximized.
aba regulates many aspects of plant seed development such as seed desiccation tolerance, dormancy, and the inhibition of the phase transitions from embryonic to germinative growth  <cit> .
gas are antagonists of aba and release seed dormancy, promote germination and induce flowering  <cit> .
taken together, we speculate that mir <dig> restricts specific ga effects in the embryo, primarily affecting cotyledon and hypocotyl tissues.
several mirnas that target components of the auxin response were identified during seed development.
mir <dig> and mir <dig> are predicted to target auxin response factors , specifically arf <dig>  arf <dig> and arf <dig>  mir <dig> and mir165/ <dig> are predicted to target transcription factors nac and hd-zip respectively, which also play an important role in early auxin response  <cit> .
our data showed very specific expression patterns of these auxin responsive mirnas.
mir <dig> is one of the most abundant mirnas during seed development and is preferentially expressed in the seed coat and endosperm.
there are two peaks of mir <dig> reads at  <dig> and  <dig> daf and expression of mir <dig> was negatively correlated with that of its targets arf <dig> and arf <dig>  mir <dig> was preferentially expressed in the embryo cotyledons and mir165/ <dig> was preferentially expressed in the hypocotyl.
mir <dig> was preferentially expressed in early embryo development .
the expression of the mirna targets arf <dig>  phv and nac <dig> were negatively correlated with expression of the corresponding mirnas.
the mir166/ <dig> group and its target genes have been reported to regulate apical and lateral meristem formation, leaf polarity, and vascular development  <cit> .
the key role of auxin in plant embryogenesis has been well described  <cit>  and a proper distribution and activity of auxin are crucial for embryo development.
the high abundance and specific expression of mir165/ <dig>  mir <dig>  mir <dig> and mir <dig> indicate that these mirnas play important roles in maintaining proper auxin signaling homeostasis in seed development.
one of the predicted novel mirna, mir <dig>  also the most abundant putative mirna sequence with  <dig>  total reads, was predicted to target homologs of arabidopsis demeter .
dme and dem-like  proteins are required for appropriate distribution of dna methylation marks, endosperm gene imprinting and seed viability in arabidopsis <cit> .
recently, kim et al.
<cit>  reported that mir <dig> affects seed germination of arabidopsis thaliana under stress conditions via targeting demeter-like protein <dig> mrna .
it is hypothesized that the induction of mir <dig> by stress guides cleavage of dml <dig>  which in turn maintains dna methylation in genes that play a negative role in seed germination.
lu et al.
<cit>  found both methylation and demethylation events are detected during seed germination in b. napus.
our data showed high level of mir <dig> and low level of dme in developing and mature seeds, suggesting that mirnas contribute to the epigenetic regulation of genes during seed development, maturation and germination through regulation of dme or dme-like genes.
CONCLUSIONS
large numbers of mirnas with diverse expression patterns, multiple-targeting and co-targeting of many mirnas, and complex relationships between expression of mirnas and targets were identified in this study.
development is regulated by complex networks consisting mainly of interactions between tfs, micrornas and hormones.
the relative abundance as well as specific temporal and spatial expression patterns of these mirnas and their targets suggested that mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir <dig> are the major contributors to the network controlling seed development and maturation through their pivotal roles in plant development.
our data suggests a possible role for mirna <dig> in regulating the phase transition from seed  to vegetative state  that is analogous to its role in the vegetative to floral transition.
altogether, the large diversity of conserved and novel mirnas identified in b. napus seed development, provide new perspectives on the regulation of gene expression networks and on developmental timing by mirnas during seed development and maturation.

 non-coding small rnas are important regulators of gene expression in eukaryotes and influence almost all aspects of plant biology. there are two major classes of endogenous srnas: micrornas and short-interfering rnas, typically of to nucleotides in length. mirnas are the best understood class of srnas. they differ from other types of srnas in their biogenesis. in plants, mirnas are generated from primary mirna transcripts that are generally transcribed by rna polymerase ii and fold back on themselves to form distinctive hairpin structures. primary transcripts are first processed by dicer-like to mirna precursors in the nucleus, which are exported to the cytoplasm and then further excised by dcl to generate ~21nt mature mirnas. plant mirnas suppress gene expression mainly by directing cleavage of their highly complementary target transcripts. recent studies suggest that translational repression may be also common in plants. the number of newly discovered mirnas is growing rapidly. at the time of writing, mature mirnas have been discovered and deposited in the public mirna database mirbase. these mirnas include mirnas from flowering plant species. the majority were obtained from model species with sequenced genomes such as medicago truncatula, oryza sativa, glycine max, populus trichocarpa, arabidopsis thaliana, physcomitrella patens and arabidopsis lyrata. brassica napus is the third largest oilseed crop in the world, providing approximately 13% of the world's supply of vegetable oil. several recent studies have contributed to the identification of mirnas from b. napus. xie et al. used computational methods to predict potential b. napus mirnas. wang et al. cloned and identified conserved mirna families and examined their expression patterns in five double haploid b. napus lines. buhtz et al. identified mirnas from different families and a set of unknown srnas from b. napus phloem sap. at the time of writing, mirnas corresponding to families from b. napus have been deposited in the mirbase database. during revision of this paper, more mirnas were identified from the early siliques of two b. napus cultivars differing in oil content, from b. napus seedlings exposed to heavy metals and from pooled b. napus tissues. to date, unique mirna sequences representing mirnas from families have been deposited in the mirbase. considering the estimated size of the b. napus genome and the small numbers of identified mirnas so far, it can be assumed that many more b. napus mirnas remain to be discovered, especially in seed. to understand the regulatory roles of mirnas in b. napus, it is important to expand the collection of mirnas and profile their temporal and spatial expression. seed maturation covers the period from the end of embryo development to the mature dry seed and occurs from about to 45 days after flowering. it can be partitioned into two over-lapping phases, seed filling followed by seed desiccation. processes occurring in maturation affect seed size, oil production, protein content and antinutritional accumulation, as well as seedling vigor following imbibition of the dry seed for subsequent plant growth. recent gene expression studies using microarrays have shown that there are vast gene expression changes during seed filling and desiccation in b. napus. distinct expression patterns related to carbohydrate metabolism, lipid biosynthesis, and storage protein accumulation have been documented. however, expression and regulatory functions of mirnas in brassica seed development, especially during seed maturation, are unknown. the high throughput and sensitivity of next generation sequencing technologies make them powerful tools for both discovery of novel mirnas and genome-wide profiling of conserved mirnas. to discover and characterize the mirnas during brassica seed development, ngs technologies were used for mirna discovery and profiling during seed maturation in b. napus. comprehensive analyses of mirnas and their targets identified many new mirnas and delivered new insights into their role in seed development and maturation.  sequencing statistics for maximum sequencing depth, we used two technologies: sequencing by oligonucleotide ligation and detection and sequencing by synthesis. small rna libraries were prepared from seed developmental stages plus floral buds and these libraries were sequenced using solid. to investigate the temporal and spatial expression of mirnas, we further dissected seeds into embryo, endosperm and seed coat tissues at selected seed stages; in addition, the embryo was separated into radicle, hypocotyl and cotyledon at and daf. these dissected samples were used to prepare additional small rna libraries that were sequenced using sbs. the sequencing results from both solid and sbs are summarized in table  in total, we obtained 88 m reads from solid libraries and 295 m reads from sbs libraries. after removing low quality reads and adaptors from the raw sequences, we obtained 234 m sbs and 74 m solid clean sequence reads, among which ~175 m sbs and ~17 m solid sequences were of 19– nt in length. notes: 1) *discarded illumina adaptor: atctcgtatgccgtcttctgcttg. 2) *clipped solid adaptor: cgccttggccgtacagcag. 3) **cleaned single nucleotide repeats. size distribution of small rnas as expected, b. napus has a complex small rna population. the most abundant length of small rnas in the whole seed was 24nt followed by 23nt and 21nt at all development stages. this is consistent with previous reports from other species. the 24nt population increased in early seed development until 25daf and then declined from 25daf to mature seeds. the 24nt small rnas were the most abundant in all tissue types and were extremely high in the radicle of the embryo, reaching 76% of the total small rna reads. the proportion of nt small rnas was lower in endosperm - about 50%. the 24nt rnas mainly consist of sirnas that are associated with the silencing of repeat sequences and transposons. higher levels of 24nt rnas in the developing and mature embryo compared to other tissue types suggest that repression of repeats and transposons in the embryo is important during seed development. identification of conserved mirnas many mirnas are evolutionarily conserved in the plant kingdom. to identify conserved mirnas in b. napus, all processed sequences from the small rna libraries were pooled and searched for the presence of known mirnas listed in mirbase. unique sequences showed perfect matches to known mirnas from mirna families. of them are a perfect match to mirna mature sequences and of them to mirna star sequences. at the time of writing, mirna entries from mirna families in b. napus have been deposited in mirbase. in this study, we detected out of these mature mirna sequences, covering of entries and all of the mirna families. in addition, we found mirna orthologs that have never been reported in b. napus. this has substantially expanded the collection of mirnas in b. napus. from the conserved mirna sequences, and were exact matches to mature mirna sequences from a. thaliana, a. lyrata, o. sativa, p. trichocarpa, m. trunculata and g. max respectively. among the sequences that matched to a. lyrata, were mirna* sequences. the high proportion of mirna* sequence matches was due to the fact that most mirna* sequences in mirbase are from a. lyrata. usually the mirna sequences are more abundant than their star sequences. the large coverage of mirnas in seeds indicates that many mirnas are involved in seed development. the total number of reads from conserved mirnas in whole seeds decreased from flower buds to 15daf seeds, but then increased substantially during seed development, especially in seed maturation. the total numbers of conserved reads from dissected seed parts at stages are shown in figure 2b. mirnas were more abundant in endosperm than in embryo and seed coat at 15daf. however, at later stages, mirnas were substantially more abundant in embryo than in endosperm or seed coat. the total number of mirna reads in embryo increased significantly until 35daf and then decreased at 45daf. within the embryo, mirnas are most abundant in the cotyledons, followed by hypocotyl and radicle. many additional mature mirnas in addition to exact matches to mature or mature star sequences annotated in mirbase, there were numerous other abundant mature mirna sequences. most of these sequences differed from the annotated mirnas by only a single nucleotide and could usually be mapped to the corresponding annotated precursors. allowing one mismatch with the annotated mirnas in mirbase, we identified mirna-like sequences with at least reads. among them, were exact matches and differed from the annotated mirna or mirna* by one nucleotide. the read counts of these sequences varied from to. while some of these sequences might be mirna sequence variants produced from imprecise cleavage during mirna biogenesis, the high abundance of many of these sequences suggests that they may be either independent mature mirnas or, more likely, that they are the ‘true’ mature mirnas in b. napus, rather than the annotated mirnas from other species. for example, two sequence variants of mir, are both more abundant than the sequence that is annotated as ath-mir158a, indicating they both are very likely mir mature sequences. furthermore, a variant of mir has reads while the exact ath-mir sequence has only reads, indicating the former sequence is more likely the functional mirna in b. napus during seed maturation. therefore we defined these sequence variants as conserved mirnas and focused on the mirna/variant sequences with > reads for further profiling analyses. abundance of conserved mirna families in seeds the abundance of mirnas varied enormously between mirna families, ranging from less than to several million reads. the most frequently sequenced mirnas are listed in table  the total number of sequence reads from all mirna variants within each mirna family were pooled for abundance analysis. within each family, the proportion of the total reads accounted for by the major variants are shown in figure  as well as the total number of reads for each family and the number of variants. the mirna family was the most abundant in seed followed by the mir mir mir and mir families. the top mirna families accounted for 49%, 18%, 7%, 6% and 5% of the total reads from all conserved seed mirnas, respectively. the remaining mirna families together account for approximately 15% of total conserved mirna reads. the number of mirna variants in each mirna family was highly variable. for example, there were and unique variant sequences in the mir mir mir mir and mir families respectively, but only one mirna sequence in the mir mir mir and mir families. usually, one or two mirna variant sequences predominated within each family. for example, ath-mir156a, ath-mir159a, ath-mir172c, ath-mir167a and bna-mir158b were the predominant variants in the mir mir mir mir and mir families. this suggests that, in most cases, the regulatory role of each family is mostly performed by the dominant mirna variants. a conspicuous exception to this relationship appears to be mir but later mapping analysis revealed this not to be a typical mirna. sequences were compared to mirbase release for homology searches. temporal expression patterns of conserved mirnas more than conserved mirna variant sequences with at least reads were identified from b. napus seed developmental stages and flower buds. the expression profiles of these mirna/variant sequences in flower buds, seed stages and compartments are listed in additional file 3: table s we performed hierarchical clustering analysis on the most abundant mirnas in the flower bud and whole seed samples and figure  shows the resulting expression patterns. expression of mirnas can be grouped into major patterns, a1-a the majority of mirnas increased during seed maturation while a minority were preferentially expressed at early- and mid- seed development stages. mirnas in group a included most variants of mir mir and mir and were weakly expressed in flower buds. they showed transient expression at early seed development then decreased to a low level at 15daf. after 15daf their expression steadily increased until 50daf. most group a mirnas such as mir mir mir mir mir were weakly expressed in flower buds and strongly expressed at later stages of seed development. the expression of mir and mir were high in both flower buds and seed and had two peaks in expression at 25daf and 50daf. group a including mir mir and mir were expressed in both flower buds and during seed development. their expression increased during seed development until 45daf and then decreased at the end of seed maturation. mirnas in group a represented by mir mir mir mir and several mir variants, were preferentially expressed in flower buds and, to a much lesser extent, in seed developmental stages. tissue-specific expression of conserved mirnas to compare the expression of mirnas in different seed parts, hierarchical clustering analysis was performed on the normalized read counts of the top mirnas in the seed part samples. the results showed strong tissue-specific expression of most of the conserved mirnas. four distinct expression patterns were identified. b and b groups were preferentially expressed in embryo. mirnas in pattern b which included most variants of mir mir mir and mir were preferentially expressed in the cotyledons and hypocotyl while mirnas in pattern b which included mir mir and mir were mostly expressed in the radicle and hypocotyl. most of the mirnas in b represented by mir mir167and several variants of mir156/ were endosperm and seed coat specific. mirnas in b including mir and mir seemed less tissue-specific but were preferentially expressed at later seed developmental stages. they were highly expressed in the cotyledons and hypocotyl at later stages and occasionally expressed in endosperm and/or seed coat. most mirna family members showed similar expression patterns such as mir, mir, mir, mir, mir and mir. however, some mirna families showed strikingly different expression patterns among variants. for example, most of the mir family were highly expressed in the embryo cotyledons and their expression increased steadily during seed maturation. in contrast, some mir variants showed high endosperm- and seed coat-specific expression and decreased during seed maturation. in arabidopsis, many known mirna families are expressed from precursor genes in multiple paralogous loci. this is also true for b. napus since several mirna members within a family were mapped to multiple loci in the brassica rapa genome in our mapping analysis. members of mirna families with divergent expression patterns are probably expressed from different paralogous loci. however, it must be noted that within each family, different loci sometimes produce identical mirnas, therefore the abundance of a mature mirna may not always correspond to expression of a specific precursor. validation of mirna expression by taqman mirna assay we employed quantitative taqman pcr to validate mirna expression determined by deep sequencing. six moderately to highly abundant mirnas were chosen for validation in seven tissues and stages. all of the selected mirnas were detected in all tissues and stages. as shown in figure  the relative changes in mirna expression calculated from qrt-pcr and sequencing are highly correlated, indicating consistency between both approaches. identification of putative precursors of b. napus mirna’s using the b. rapa genome as a surrogate for the b. napus a genome currently, the complete b. napus genome sequence is not available; therefore it is difficult to identify mirna precursor genes since weakly expressed genes are not well represented in est databases and homeologous genes are not reliably distinguished. it is well-known that b. rapa contributed the a genome to the allotetraploid b. napus containing a and c genomes. although the b. napus a genome has undoubtedly diverged from the b. rapa reference prior to and after polyploidization, evidence suggests that it retains b. rapa characteristics. for example, cheung et al. showed that “the a genomes of b. rapa and b. napus show a high degree of similarity along their entire length” and that “only a small number of homeologous recombination events have been observed in oilseed rape cultivars”. furthermore, when aligned genome segments of b. rapa and the b. napus genome a were compared over contigs, a low average snp frequency of % in coding regions was observed and % overall. comparison of a specific clone between subspecies b. rapa trilocularis and b. rapa pekinensis revealed a snp frequency of %. when the sequence of the same clone was compared between b. rapa trilocularis and the b napus a genome, the snp rate was still a relatively low %. the snp frequencies suggest that the b. napus a genome and b. rapa have not diverged enough to invalidate sequence-based annotations and predictions based on a b. rapa reference. therefore, we analyzed the recently released brassica rapa genome for b. napus mirna precursor sequences. we mapped the sequencing reads, known plant mirna mature sequences and hairpin sequences from mirbase to b. rapa genome sequences, then merged the mapping results to identify the conserved mirna loci. in total, plant mirna hairpin sequences and more than mature mirnas from mirbase were mapped to b. rapa chromosomes and scaffolds. mapping of the known plant mirna hairpin sequences identified regions of similarity to the mirna queries in the b. rapa genome which covered mirna families. in most cases, there were corresponding annotated mature sequences and sequencing reads mapped to these loci and the mapping patterns of sequencing reads to the regions were very typical- as described in kozomara and griffiths-jones – indicating they are orthologous mirna loci in b. rapa. the mirna-like stem-loop secondary structures further confirmed authentic mirna loci. examples of typical mapping patterns of mirnas are shown in figure  multiple sequences, including the annotated mirna and sequence variants with different sizes, star sequences and variants of star sequences, were mapped to each precursor locus, on both arms of each precursor stem-loop structure. the ‘true’ mirna sequences are presumably dominant among the sequence variants. most of the mirnas with stem-loop structures and typical mapping patterns are highly conserved in the plant kingdom including mir mir mir mir mir mir mir and mir. however, several regions with similarity to mir mir mir mir and mir can be folded into stem-loop secondary structures but have no sequencing reads mapped to the genomic loci, indicating that these loci are not expressed during seed development in b. napus or may not be true mirna loci. loci with similarity to mir mir mir and mir showed multiple offset reads distributed across the entire gene sequences. patterns of overlapping reads covering the entire putative locus indicate they are more likely sirna loci rather than mirna loci according to recent mapping guidelines in kozomara and griffiths-jones. examining the mapping patterns of the annotated mirna sequences, conserved new mirna sequences identified in this study, and other sequencing reads, revealed an additional mirna loci in the b. rapa genome. no known orthologous hairpin sequences of these mirnas were mapped to these loci in b. rapa, however, the stem-loop structure of the flanking regions and the typical mapping patterns of sequencing reads indicated they are authentic mirna loci according to current guidelines. in these cases, there is low sequence conservation in the b. rapa flanking regions outside the mirna mature sequences. for example, the mir mature sequence, its variants and star sequences were cleanly mapped to the b. rapa genome, although the orthologous mir precursors from other species do not share sequence similarity with the flanking region. both the mirna-like stem-loop secondary structure and the embedded nature of mirna sequencing reads provided strong evidence that this region is an authentic mir locus in b. rapa. the evaluation of mapped sequence reads also provides valuable information about the relative abundance of mature sequences from different arms of stem-loops, isoforms of mature mirna sequences, and the confidence in a given microrna annotation. in most cases, the annotated mirna was the most abundant small rna sequence mapped to an mirna locus. sometimes, the 22-25nt isoforms of the annotated mirna were more abundant, suggesting they are more likely to be the ‘true’ mirna in b. napus. in some cases, several annotated mirnas from different species were mapped to the same mirna loci. for instance, both the annotated bna-mir397a, b and ath-mir397a, b were mapped to mir397a. bna-mir397a, b was substantially more abundant than ath-mir397a, b, indicating it is the ‘true’ mirna in b. napus as annotated. occasionally, the star sequence or a variant of the star sequence from the opposite hairpin arm of the mirna precursor was more abundant than the annotated mirna. for example, the mir2111* sequence was more abundant than mir in bra-mir2111a, b, c and d, suggesting mir2111* is the true mirna mature sequence in b. napus. this is also true for bra-mir391a and bra-mir the sequence of mir391* was more abundant than the annotated mir and a variant of mir408* is considerably more abundant than mir and mir408*, indicating that they are most likely the dominant mature mirna sequences in b. napus and therefore are named as bna-mir391a and bna-mir. mapping of small rna sequencing reads and known mirna sequences to the b. rapa genome revealed several chromosomal regions with clusters of mirna genes. these included a cluster containing three mir genes on chromosome a within a 1000 bp region; a mir cluster as well as a mir cluster, also on chromosome a in addition, we also found that some mirna precursors gave rise to two or more distinct mirna/mirna* duplexes from different positions. as shown in additional file 5: figure s the mir mir and mir precursor loci as well as newly identified putative novel mirna loci mir and mir can be folded into a long stem-loop structure and generated multiple distinct srnas. mirna target prediction mirnas regulate gene expression through targeting transcripts for cleavage, translational repression or mrna decay. therefore, it is important to identify mirna targets to understand mirna function. in plants, all known mirna targets have perfect or near-perfect complementary sequences to cognate mirnas. this has allowed many computational approaches to predict mirna targets. we used targetfinder, to search against brassica est unigenes for the targets of mirnas with more than reads. we predicted mirna-target pairs including unique b. napus mirna targets for unique mirnas. among these predicted targets, est contigs are annotated as homologs of arabidopsis genes by blast-based analysis of sequence homologies in tair transcripts and protein datasets, while of them have no arabidopsis homologs. these non-annotated est contigs might represent genes specific to b. napus. to understand the biological roles of the mirna-target pairs in seed development, we performed mirna-pathway analysis by mapping the arabidopsis homologs of the target ests to mapman pathways. the functional classification based on mapman terms using the classification superviewer tool w/bootstrap revealed that these homologs of target genes were highly enriched in pathways of rna, dna, protein, s-assimilation, development, signaling and redox. detailed inspection of the results revealed that some mirnas function in the same pathway by targeting different genes in the same family. previous reports grouped mir and mir as one mirna family due to their high sequence homology and co-targeting to squamosa-promoter binding proteins or sbp-like proteins. similarly, mir159/ mir165/ and mir170/ were also grouped together based on shared targets. in addition, we also predict that mir mir and mir "co-target" pentatricopeptide repeat-containing proteins; mir mir mir "co-target" f-box family proteins and mir mir mir and mir "co-target" various auxin response factors. therefore, many mirnas may function together via co-targeting to regulate functionally related genes or pathways. go term analysis using the bingo plugin tool in cytoscape on the arabidopsis homologs of the unique target sequences revealed that these genes are highly overrepresented in regulation, development and stress responses. in arabidopsis, a majority of mirna targets have been identified and experimentally validated. many homologs of these arabidopsis mirna target genes can be found from the predicted targets, confirming that conserved mirnas also have conserved targets across species and likely have similar functions in b. napus and arabidopsis. this is consistent with previous studies in other species. in addition to the conserved targets, we also predicted new targets for the conserved b. napus mirnas. for example, in addition to members of the spl gene family, we also predicted other new mir targets, such as homologs of arabidopsis vacuolar h-atpases, low cell density, an f-box family protein, a leucine-rich repeat transmembrane protein kinase, an auxin-responsive gh family protein and fusca, which is a key regulator of seed development. individual mir variants may have specific additional targets such as the homolog of sec cytosolic factor specific for bna-mir156_v an arid/bright dna-binding domain-containing protein specific for bna-mir156_v fus and an enth domain-containing protein specific for bna-mir156_v an isoflavone reductase and an isocitrate lyase specific for bna-mir156_v and brassinosteroid insensitive for bna-mir156_v and bna-mir156_v this suggests the possibility that individual mirna variants have different functions through their specific targets. expression of putative conserved mirna targets to compare the expression of the mirnas with that of their targets, we performed global transcript profiling using brassica 90k combimatrix arrays. the expression of mirna target genes in seed development was extracted from the microarray results and is listed in additional file 7: table s the overview of the expression of b. napus genes is shown in figure  the expression changes of mirna targets were notably smaller than overall changes in gene expression. detailed comparison of the expression patterns of mirnas and their targets at the corresponding time points revealed that, although some mirna-target pairs showed the expected negative correlation, many others showed either no correlation or even displayed a positive correlation. most mirnas were expressed at low levels. similarly, many of the mirna target genes were also expressed at relatively low levels during seed development and maturation. the maximum raw signal intensities of genes in group were under throughout seed development, indicating that the expression of these target genes might be repressed throughout seed development. homologous est contigs of many known mirna target genes including most spls, myb, arf and arf and numerous others either showed negative expression patterns or were expressed at very low levels throughout seed maturation. identification of putative novel mirnas, precursors and targets many mirna families are highly conserved in evolution. however, there are also species-specific, recently evolved mirna genes. to identify mirnas specific to brassica, we again used the b. rapa genome since the complete b. napus genome is not available. all of the sequences with read counts > that were not annotated as trna, rrna, snorna, mirna, repeat or cdnas, but mapped to the b. rapa genome were used to detect potentially novel mirna genes. the flanking genomic sequences of the mapped reads were extracted and searched for regions that could be folded into a stable mirna precursor-like hairpin with strong pairing between the mature mirna and mirna* sequence on the opposite hairpin arm. this analysis employed novomir, a program for the identification of plant mirna genes. we identified putative novel mirna sequences which mapped to genomic loci in b. rapa. to eliminate sirnas from repeat sequence classes and ensure that they were from good quality single-stranded hairpins, reads that mapped more than times to the reference genome or mapped to a bidirectional small rna cluster were also discarded. the mapping patterns of sequence reads in each genomic locus were manually examined and the loci that didn't meet the consensus set of guidelines for mirna annotation were filtered out. loci with an embedded known mirna were identified as conserved mirnas and therefore also eliminated. following the above filtering, we identified putative novel mirna loci for out of the presumed mirna sequences and several conserved mirna variants including mir mir and mir these putative novel mirna sequences and their hairpin structures, as well as the mapping patterns of sequencing reads in the hairpin regions, are presented in additional file 9: table s and additional file 6: figure s several putative mirna sequences were mapped to the same loci. for example, putative novel mirna sequences were embedded in the mir5802; of these ngs_90350641_x is the most abundant sequence and most likely the true mirna mature sequence and ngs_93017147_x is the corresponding star sequence while ngs_93017180_x is a variant of the same mirna*. previous studies indicated that non-conserved mirnas are usually weakly expressed with a tissue-specific pattern. due to the unavailability of b. napus genome sequences and the fact that the small rna libraries were mostly constructed from seeds, mirnas identified in this study might represent only a fraction of the total number of novel mirnas in b. napus. we also predicted targets for the novel mirna candidates using targetfinder software. in total we predicted brassica contigs as targets of the putative mirnas. based on the annotations of their arabidopsis homologs, the functions of these predicted novel mirna targets are involved in many pathways including development and signaling. mir was the most abundant potential mirna among all predicted novel mirnas. it is preferentially expressed in embryo, especially in the cotyledons, and increased substantially during seed maturation. it was predicted to target homologs of arabidopsis demeter and at1g. the expression of these two genes reciprocally decreased during seed maturation. mir, the second most abundant potential mirna, accumulated in different compartments of the seed during seed maturation. it was predicted to target est contigs homologous to an arabidopsis pentatricopeptide repeat-containing protein and a sphere organelles protein-related gene. the expression of the target ppr repeat-containing protein showed consistently low expression from to daf in seed. discussion increase in the number of identified mirnas in b. napus and b. rapa mirnas are essential regulators of gene expression in all plants and animals. according to the mirbase database, arabidopsis and rice mirnas have been identified respectively. b. napus is polyploid with a much larger and more complex genome than arabidopsis and rice. however, there were only mirnas annotated in mirbase. in this study, we identified more than mirnas including known b. napus mirna sequences already in mirbase and putative novel mirnas. the mirna-like hairpin structures of the flanking sequences and the typical mapping patterns of the ngs reads to the precursor regions provided supporting evidence for the newly discovered mirnas. in the absence of a b. napus genome sequence, we used the newly released b. rapa draft genome sequence for identification of the hairpin precursor sequences of the newly identified mirnas. this led to the discovery and annotation of more than mirna hairpin sequences and loci. to date, there are only mirnas and their hairpin precursor sequences from b. rapa deposited in mirbase. identification of the mirna genes in the b. rapa genome has significantly enriched the repertoire of brassica mirna genes and improved the annotation of the newly sequenced b. rapa genome. in a report published while this article was in preparation, zhao et al. analyzed small rna expression profiles of b. napus siliques at early embryonic developmental stages in two cultivars with differing oil contents. they identified conserved mirnas of which were identical to and highly homologous to known mirnas in mirbase. in addition, they predicted new mirnas. this study provides a more comprehensive temporal and spatial profiling of mirnas focused on post-embryonic development and seed maturation. by employing greater sequencing depth, we identified many more mirnas including conserved and novel putative mirnas with high confidence and investigated their specific expression patterns during seed development. to provide a more complete picture of mirnas in b. napus seed development we provide a detailed comparison of our data with that of zhao et al.. we also identified almost all of the conserved mirnas that they reported. for the predicted new mirnas, we identified the corresponding precursor loci in the b. rapa genome and examined the patterns of sequences that mapped to each region. among the new mirnas predicted by zhao et al. bna-mir bna-mir and bna-mir were also predicted in this study and bna-mir was predicted to be a star sequence. we did not detect reads corresponding to the exact sequences of bna-mir2206a, b, bna-mir and bna-mir the sequences of bna-mir bna-mir2206a, b and bna-mir occurred in the b. rapa genome and times respectively, suggesting that they are unlikely to be authentic mirnas. the observed pattern of overlapping reads mapping to a broad region of the flanking sequences of mir mir and mir indicates that they are more likely sirna loci. the number of sequence reads for bna-mir and bna-mir were low and therefore excluded from our prediction. due to the inconsistency of mirna names in zhao et al. and mirbase, we retain our temporary mirna names for all mirna/variants with reads > in each family according to their abundance. these names are subjected to change when the b. napus whole genome sequence becomes available. very recently, xu et al. identified conserved and brassica-specific candidate b. napus mirnas from pooled b. napus tissues using b. napus, b. rapa and b. oleracea genomic survey sequences databases and ests as references. we detected out of the available sequences they identified, of which had > total reads, mostly conserved mirnas although a few of them are actually the same as the new conserved or putative novel mirnas we identified. for instance, the sequence of bna-mirc17a- is identical to our new conserved bna-mir158_v, the sequence of bna-mirc is annotated as bna-mir319_v and bna-mirc is the same as our predicted mir5807a/b. the remaining sequences with no reads or total reads less than were excluded from our analysis. zhou et al. identified conserved and non-conserved mirnas from cd-treated and non-treated b. napus seedlings using the b. napus cdna database and tentative consensus sequences of the b. napus gene index, leaving mirna homologues with high similarity to mirnas from other species not validated due to lack of a reference sequence. comparison of the expression of the known mirnas in zhou et al. and this study revealed many commonly expressed mirnas despite the very different conditions and stages, especially for the most highly expressed mirnas such as mir mir mir and mir however, of the previously annotated mirnas were not expressed under any tissues/stages/conditions in either study, indicating that some of the "known" mirnas are likely not true mirnas. in this study, we identified many more mirnas with a much more stringent cutoff at > reads using the b. rapa genome as reference. with our deep sequencing data, we were able to follow the latest microrna annotation guidelines to integrate sequencing data with mirna annotation and exclude many false mirna loci which are more likely sirna loci based on read mapping patterns. many of the mirnas identified in this study are now validated by recent reports. the most abundant mirna in seeds of b. napus relative to other species mir is the most abundant mirna in b. napus seeds, accounting for more than 48% of total mirna reads. in monocotyledonous plants, mir168a was reported as the most abundant mirna in rice, brachypodium distachyon, barley and wheat while mir is the second most abundant mirna in these species. our data showed mir to be preferentially expressed in the cotyledons of embryo and mir to be preferentially expressed in endosperm at a much lower level. but mir is the most abundant mirna not only in monocot seeds but also in other monocot tissues. therefore the extraordinarily high level of mir in monocots is not due to the larger portion of endosperm in grass seeds but represents a significant regulatory difference between monocots and dicots. it has been reported that mir targets argonaute in arabidopsis, rice, and wheat. ago is involved in mirna biogenesis and mir168-mediated regulation of ago mrna has been reported to be important for proper plant development. it will be interesting to further compare the regulation of mir168-ago in monocots and dicots as more data becomes available. mirna targets various prediction approaches have been applied successfully for the identification of mirna targets in plants. in b. napus, xie et al. predicted potential target genes for newly identified mirnas. huang et al. identified several mir target genes. since few mirnas targets have been identified in b. napus, we may assume that the majority of mirna targets have not been discovered. in this study, we predicted mirna-target pairs including unique b. napus mirna targets for unique mirnas using targetfinder. very recently, xu et al. experimentally identified non-redundant mrna targets of conserved brassica mirnas and new non-redundant mrna targets of novel brassica-specific mirnas by sequencing the mrna degradome. zhou et al. identified targets for mirna families by deep sequencing of four degradome libraries from cd-treated and control roots and shoots of b. napus seedlings. comparing our putative targets to xu et al. and zhou et al., we found significant overlap among them based on the annotations of target genes, including mir targets, mir targets, mir targets, mir targets, mir targets and mir targets. detailed comparison revealed that out of the category i mirna targets from zhou et al. were also predicted as targets of the same mirna families in this study. mirnas with reads <  counts such as mir and mir were excluded from our target prediction. this might explain some of the differences between two studies. in addition, different b. napus est datasets were used in the two studies which might also contribute to differences. expression patterns of mirna’s and their putative targets a typical mirna acts to downregulate expression of its target gene by directing cleavage of its highly complementary target transcripts. this implies that expression of mirnas and their targets should be negatively correlated. however, as others have noted, the expected inverse correlation was often not observed. in this study, we found that the top most abundant mirnas represented 85% of all mirna sequence reads and that most mirnas were expressed at low levels. similarly, many of the mirna target genes were expressed at relatively low levels during seed development and maturation. for example, mir expression increased substantially but spls were expressed at low levels from to daf. in arabidopsis, the expression of spls significantly decreased from the globular to heart stage of seed development and remained low throughout the rest of seed development. thus, in b. napus, it is likely that expression of spls was already suppressed by mir before 10daf. we also observed that typically, the range of expression changes exhibited by the mirna targets during seed development were smaller than the overall range of gene expression changes. this observation is consistent with previous reports that mirna-mediated post-transcriptional regulation might contribute to buffering changes in target gene expression by establishing a threshold for target-triggered regulatory effects within a complex regulatory network. the buffering function of mirna might be critical to prevent the fluctuation of target gene expression levels and therefore prevent aberrant development events. the occurrence of mirna multiple targeting and co-targeting also complicates the relationship between mirna and their targets. given the complexity of gene regulatory networks, an individual mirna target might also be regulated by various factors in addition to the cognate mirnas and one of the other factors may be predominant in a given tissue or stage. in addition, recent studies have shown that plant mirnas can repress target genes at the level of translation with little or no influence on the mrna abundance. the roles of mirnas in seed development and maturation plant mirnas are important for fine-tuning plant development including organ identity, patterning, polarity, developmental phase transitions and development of cellular organelles. previous reports suggested the potential involvement of mir mir mir and mir in seed development, dormancy, and germination. however, the specific functions of mirnas in seed maturation are largely unknown. mir mir and their targets are key players in coordinating plant phase transitions, from juvenile to adult and from the vegetative to the reproductive phase, during post-embryonic development. in arabidopsis and maize, mir maintains the juvenile phase and prevents precocious flowering while mir acts downstream of mir and promotes flowering by repressing apetala 2-like repressors of flowering locust. mir targets and represses the expression of spl genes that are positive regulators of mir the levels of mir and mir exhibit contrasting age/development-specific expression patterns: mir levels decline during vegetative development whereas mir levels correspondingly increase. as noted earlier, the mir family is the most abundant mirna in seed development. ath-mir156a, the most abundant variant of the mir family, is preferentially expressed in embryo and increased 113-fold from 15daf to 50daf. ath-mir172c, the most abundant variant of the mir family, was preferentially expressed in endosperm in early seed development and decreased substantially during subsequent maturation, showing the opposite expression pattern to mir the complementary tissue- and stage-specific expression patterns of mir and mir are analogous to the contrasting age/development-specific expression patterns of mir and mir during the transition from the vegetative phase to flowering. this suggests that indirect repression of mir by the highly expressed mir is important for embryo maturation and that the mir156-spls-mir regulatory cascade also plays an important role during seed development and germination. nodine and bartel proposed that mirnas enable proper embryonic patterning by preventing precocious expression of genes normally expressed during seed maturation, partially through mir156-mediated repression of spl transcripts. however, the substantial increase of mir during seed maturation and very low expression of the target spl genes in late seed development indicate that the mir156-spl regulatory cascade is not likely to suppress the expression of maturation genes. indeed it seems intuitively more likely that mir represses floral identity in order to allow vegetative identity to become established during maturation. the plant life-cycle is characterized by two major developmental phase transitions: germination and flowering. recent studies indicate that genes involved in flowering and other phase transitions also regulate the transition from dormancy to germination, suggesting that conserved mechanisms control all plant phase transitions. a study of global transcriptional interactions revealed coordinated regulation of plant cellular phase transitions in seed germination. an intermediate transition phase between dormancy and germination is enriched with genes involved in cellular phase transitions such as early bolting in short days, flowering locus c, flowering locus t and abscisic acid insensitive. the hyper-abundance of mir in mature seeds and the presence of common regulators of phase changes in seeds and flowering suggest that the mir156-spl regulatory cascade might control the developmental phase transition from embryonic to seedling stage, analogous to shoot maturation and the transition from the vegetative state to flowering. high levels of mir and reduced spls and mir in the mature embryo may repress the developmental transition and keep seeds in the maturation/dormant state. ath-mir is the second most abundant mirna during seed development and maturation. the expression pattern of mir is very similar to that of ath-mir in a study by reyes and chua, mir was induced by aba via the transcription factor abi and cleaves myb domain protein and myb domain protein transcripts, which were described as positive regulators of aba signaling during germination. however, this study is inconsistent with other papers that provide evidence that gamb-like genes myb myb and myb domain protein mediate ga effects. for example, mir was implicated in floral and anther development by targeting the expression of myb and myb which were shown to be involved in ga-promoted activation of leafy. recently, alonso-peral et al. demonstrated that in mir mutants, deregulation of myb and myb in vegetative tissues resulted in up-regulation of genes that are highly expressed in the aleurone and induced by ga during germination, suggesting that mir acts as a molecular switch confining the expression of myb and myb to anthers and seeds. our data shows that mir is preferentially expressed in the embryo, presumably allowing expression of myb and myb in the endosperm to facilitate storage product hydrolysis and later programmed cell death. furthermore, within the embryo, mir is preferentially expressed in the cotyledons and hypocotyl, perhaps repressing specific ga processes to allow aba-associated maturation to be maximized. aba regulates many aspects of plant seed development such as seed desiccation tolerance, dormancy, and the inhibition of the phase transitions from embryonic to germinative growth. gas are antagonists of aba and release seed dormancy, promote germination and induce flowering. taken together, we speculate that mir restricts specific ga effects in the embryo, primarily affecting cotyledon and hypocotyl tissues. several mirnas that target components of the auxin response were identified during seed development. mir and mir are predicted to target auxin response factors, specifically arf arf and arf mir and mir165/ are predicted to target transcription factors nac and hd-zip respectively, which also play an important role in early auxin response. our data showed very specific expression patterns of these auxin responsive mirnas. mir is one of the most abundant mirnas during seed development and is preferentially expressed in the seed coat and endosperm. there are two peaks of mir reads at and daf and expression of mir was negatively correlated with that of its targets arf and arf mir was preferentially expressed in the embryo cotyledons and mir165/ was preferentially expressed in the hypocotyl. mir was preferentially expressed in early embryo development. the expression of the mirna targets arf phv and nac were negatively correlated with expression of the corresponding mirnas. the mir166/ group and its target genes have been reported to regulate apical and lateral meristem formation, leaf polarity, and vascular development. the key role of auxin in plant embryogenesis has been well described and a proper distribution and activity of auxin are crucial for embryo development. the high abundance and specific expression of mir165/ mir mir and mir indicate that these mirnas play important roles in maintaining proper auxin signaling homeostasis in seed development. one of the predicted novel mirna, mir also the most abundant putative mirna sequence with total reads, was predicted to target homologs of arabidopsis demeter. dme and dem-like proteins are required for appropriate distribution of dna methylation marks, endosperm gene imprinting and seed viability in arabidopsis. recently, kim et al. reported that mir affects seed germination of arabidopsis thaliana under stress conditions via targeting demeter-like protein mrna. it is hypothesized that the induction of mir by stress guides cleavage of dml which in turn maintains dna methylation in genes that play a negative role in seed germination. lu et al. found both methylation and demethylation events are detected during seed germination in b. napus. our data showed high level of mir and low level of dme in developing and mature seeds, suggesting that mirnas contribute to the epigenetic regulation of genes during seed development, maturation and germination through regulation of dme or dme-like genes.  large numbers of mirnas with diverse expression patterns, multiple-targeting and co-targeting of many mirnas, and complex relationships between expression of mirnas and targets were identified in this study. development is regulated by complex networks consisting mainly of interactions between tfs, micrornas and hormones. the relative abundance as well as specific temporal and spatial expression patterns of these mirnas and their targets suggested that mir mir mir mir mir and mir are the major contributors to the network controlling seed development and maturation through their pivotal roles in plant development. our data suggests a possible role for mirna in regulating the phase transition from seed to vegetative state that is analogous to its role in the vegetative to floral transition. altogether, the large diversity of conserved and novel mirnas identified in b. napus seed development, provide new perspectives on the regulation of gene expression networks and on developmental timing by mirnas during seed development and maturation. 
large numbers of mirnas with diverse expression patterns, multiple-targeting and co-targeting of many mirnas, and complex relationships between expression of mirnas and targets were identified in this study. numerous mirnas were expressed in a stage- or tissue-specific manner suggesting that they have specific functions related to the fine tuning of transcript abundance during seed development. global correlation analysis of the expression patterns of mirnas and their targets revealed complex mirna-target gene regulatory networks during seed development. several key mirna-target expression patterns were identified and new roles of mirnas in regulating seed development are suggested. mir mir mir mir mir and mir are the major contributors to the network controlling seed development and maturation through their pivotal roles in plant development. using next-generation sequencing technologies, we performed a comprehensive analysis of mirnas during seed maturation at time points from 10 days after flowering to daf using whole seeds and included separate analyses of radicle, hypocotyl, cotyledon, embryo, endosperm and seed coat tissues at selected time points. despite the thousands of mirnas identified and validated in numerous plant species, only small numbers have been identified from the oilseed crop plant brassica napus – especially in seeds. only of the conserved mirna sequences had been previously identified in b. napus. mirna targets in b. napus were predicted and their expression patterns profiled using microarray analyses. mir may regulate the developmental transition to germination. micrornas are 20– nucleotide rna molecules that suppress the transcription of target genes and may also inhibit translation. more than mirna loci were identified/annotated using the b. rapa genome as a surrogate for the b.napus a genome. we identified more than conserved mirna or variant unique sequences with > sequence reads and also found novel mirnas.
 micrornas are 20– nucleotide rna molecules that suppress the transcription of target genes and may also inhibit translation. despite the thousands of mirnas identified and validated in numerous plant species, only small numbers have been identified from the oilseed crop plant brassica napus – especially in seeds.  using next-generation sequencing technologies, we performed a comprehensive analysis of mirnas during seed maturation at time points from 10 days after flowering to daf using whole seeds and included separate analyses of radicle, hypocotyl, cotyledon, embryo, endosperm and seed coat tissues at selected time points. we identified more than conserved mirna or variant unique sequences with > sequence reads and also found novel mirnas. only of the conserved mirna sequences had been previously identified in b. napus. more than mirna loci were identified/annotated using the b. rapa genome as a surrogate for the b.napus a genome. numerous mirnas were expressed in a stage- or tissue-specific manner suggesting that they have specific functions related to the fine tuning of transcript abundance during seed development. mirna targets in b. napus were predicted and their expression patterns profiled using microarray analyses. global correlation analysis of the expression patterns of mirnas and their targets revealed complex mirna-target gene regulatory networks during seed development. the mir family was the most abundant and the majority of the family members were primarily expressed in the embryo.  large numbers of mirnas with diverse expression patterns, multiple-targeting and co-targeting of many mirnas, and complex relationships between expression of mirnas and targets were identified in this study. several key mirna-target expression patterns were identified and new roles of mirnas in regulating seed development are suggested. mir mir mir mir mir and mir are the major contributors to the network controlling seed development and maturation through their pivotal roles in plant development. mir may regulate the developmental transition to germination. seed developmentembryonext generation sequencing 