BACKGROUND
with the advent of new genomic techniques such as tiling arrays and next generation sequencing  <cit> , non-coding rnas are increasingly identified and recognized as an integral and functional component of the genome.
while some non-coding rnas perform housekeeping functions, such as trnas, rrnas and small nuclear rnas, others such as micrornas and small interfering rnas  play critical regulatory roles during development or stress responses  <cit> .
non-coding rnas with a length greater than 200bp are defined as long non-coding rnas .
the lncrnas can be grouped into three subclasses: 1) long non-coding natural antisense transcripts , 2) intronic lncrnas, and 3) intergenic lncrnas.
like protein-coding  genes, a majority of lncrnas are transcribed by rna polymerase ii with a 5′ cap and a 3′ poly-a tail in animals  <cit> .
in plants, however, lncrnas can be transcribed by polii, iv, and v, therefore some may lack poly-a tails .
a growing number of reports revealed lncrnas from animals, especially human; they are involved in diverse biological processes, such as development, cellular differentiation, and diseases including cancers.
lncrnas may serve as diagnostic markers or even therapeutic targets .
studies of animal lncrnas showed that lncrnas function through a number of mechanisms.
first, lncrnas act in epigenetic regulation of gene expression.
for instance, the mammalian xist rna initiates x chromosome inactivation in cis to equalize gene expression between males and females  <cit> .
hotair  is able to mediate transcriptional repression of hox loci in trans by modulating histone methylation  <cit> .
second, lncrnas may directly interact with proteins to titrate their functions and are thus called ‘decoys’.
a well-known example is the lncrna named terra, which was demonstrated to be the natural ligand and inhibitor of telomerase  <cit> .
third, lncrnas may act as a scaffold to form a complex with other proteins.
together, lncrnas were demonstrated to play bona fide and essential roles in animals.
in contrast, there are only a handful of reports on the functions of lncrnas in plants.
flowering locus c  is a key flowering repressor in the vernalization pathway.
to ensure epigenetic silencing of flc, a lnc-nat named coolair  and an intronic lncrna called coldair  could be induced after vernalization treatment to gradually repress the expression of flc by promoting methylation  <cit> .
another intergenic lncrna induced by phosphate starvation was found in medicago truncatula , arabidopsis thaliana , tomato , and rice  .
further analysis indicated that ips <dig> acts as a decoy of mirna- <dig> and allows the accumulation of its target gene transcripts  <cit> .
a third intergenic lncrna called ldmar  is required for normal pollen development in rice under long day conditions  <cit> .
a number of lncrnas are differentially expressed under stress stimuli in arabidopsis  <cit> .
despite limited reports on the mechanisms of plant lncrna function, it is evident that plant lncrnas play vital roles in developmental and stress responses.
this realization combined with the advent of next-generation sequencing has prompted tremendous efforts and investments in identifying lncrnas in a wide range of organisms .
f. vesca, the woodland strawberry, is becoming a new model organism for both octoploid cultivated strawberry  and other members of the rosaceae, a family that includes many fruit trees.
f. vesca has a short life cycle, small stature, facile transformation, and small and sequenced genome  .
further, transcriptomic data of various tissues in the cultivated strawberry were available  <cit> .
of significant interest is the strawberry fruit, which is developed from the receptacle   <cit> .
toward the identification of molecular mechanisms of strawberry floral and fruit development, we first generated comprehensive rna-seq datasets from  <dig> distinct f. vesca floral and fruit tissues at different developmental stages to profile genome-wide expression of pc genes  <cit> .
in current work, we seek to identify lncrnas from strawberry floral and fruit transcriptomes with the goal of uncovering lncrnas that function in flower and fruit development.
in total,  <dig>  lncrnas derived from  <dig>  loci were identified from the f. vesca flower and fruit trancriptome dataset.
further analysis indicated that these lncrnas are similar to pc genes in terms of gene structure and transcriptional regulation.
however, lncrnas also show a number of distinctions from pc genes.
for example, a large number of lncrnas are precursors to small rnas, and their sequences are much less conserved than pc genes.
in an effort to identify potential regulatory target genes of lncrnas, we used expression correlation between lncrnas and the pc genes in the f. vesca genome, taking advantage of the rna-seq data from a large number of different floral and fruit tissues.
both positive and negatively correlated lncrna-pc gene pairs are identified.
our analysis provided the first look at the lncrna landscape in a fruit crop and laid the foundation for future studies of lncrna function in strawberry.
to facilitate the study of lncrnas and data sharing, our f. vesca lncrnas can be accessed from the strawberry genome resources  website  <cit>  as a new track in gbrowse  as well as from genome database for rosaceae   <cit> .
RESULTS
identification of f. vesca lncrnas from flower and fruit rna-seq datasets
to globally identify lncrnas in the f.vesca genome, we utilized rna-seq datasets generated from  <dig> distinct floral and fruit tissues plus two vegetative tissues , which were isolated from a 7th generation inbred line of f. vesca    <cit> .
there were two biological replicates for each tissue type, and hence  <dig> rna-seq libraries in total, amounting to about  <dig>  billion total single-end reads of 51bp.
the analysis pipeline is shown in fig.
<dig>  in brief, each library was aligned individually by tophat <dig> in order to preserve junction reads.
known and novel transcripts were assembled by cufflinks based on uniquely mapped reads and, finally, all assemblies were combined by cuffmerge and then compared to the annotations by cuffcompare to characterize each transcript.fig.
<dig> summary of the workflows used for identifying strawberry lncrnas.
a lncrna identification from f. vesca rna-seq datasets.
b lncrna identification from unigenes.
c venn diagram comparing lncrnas identified in  and .
d different classifications of fve-lncrnas based on expression levels, repeats, and precursors of small rnas.
hc-lncrnas: high confidence lncrnas
in total,  <dig>  candidate noncoding transcripts  in class “u” , “o” , “x” , and “i”  were subjected to further filtering .
the transcripts are defined as lncrnas if they are longer than 200bp, are non-coding, and are not a member of known functional rna classes  .
a relatively stringent non-coding standard was used.
first, the coding potential calculator was used to discriminate non-coding from coding transcripts as it takes into account several biologically meaningful sequence features with high accuracy  <cit> ; a non-coding transcript has a coding potential score  lower than “-1”.
second, to reduce false positive, any short peptide coded by “non-coding transcript” should contain no conserved protein domains .
as a result,  <dig>  transcript isoforms in  <dig>  loci were identified as lncrnas .table  <dig> the number of lncrnas in different classes before and after filtering
notes:
athe number of original isoforms and loci predicted by cufflinks
bthe number of lncrnas after filtering
c“total” number is smaller than the sum of the column, because certain loci have multiple isoforms with different class codes
in addition to the rna-seq data, we mined existing ests from fragaria.
a large number of raw est datasets derived from both diploid fragaria vesca and octoploid fragaria × ananassa were found in the ncbi dbest database, but unigenes from gdr  were chosen for lncrna discovery as unigenes do not contain low quality ests or redundant ests  <cit> .
the filtering criteria were similar to the lncrna discovery from rna-seq data  and led to the identification of  <dig> lncrnas out of  <dig>  unigenes .
interestingly, only  <dig> of the  <dig> lncrnas are in common with the rna-seq-derived lncrnas .
we then compared the remaining  <dig> unigene-derived lncrnas with the f. vesca genome annotation and found that a majority of them are from annotated pc loci on either the same or opposite strand sometimes covering the introns.
a lack of over-lap between rna-seq- and unigene-derived lncrnas suggests that the identification of lncrnas is far from saturation, partly due to a lack of strand orientation information from our rna-seq reads.
identification of high confidence -lncrnas
the  <dig>  lncrnas identified from the f. vesca rna-seq data were further characterized based on expression levels and whether or not they contain repeats or generate small rnas .
first,  <dig>  lncrnas are expressed at higher than  <dig> fpkm  in at least one of the tissue types in both replicates; the remaining  <dig>  lncrnas are expressed at lower than  <dig> fpkm.
second, lncrnas that overlapped with transposable elements and/or repeats were identified by repeatmasker and repeatscout, respectively .
in total,  <dig>  transcripts with a percentage of repetitive sequences lower than  <dig> % were classified as “no repeat” .
the filtering pipeline  has removed known pre-mirna transcripts, but a small quantity of the remaining lncrnas may still encode previously unknown mirnas.
moreover, some lncrnas would generate short hairpin rnas and sirnas involved in epigenetic regulation  <cit> .
to distinguish those small rna-generating lncrnas, we used the small rna-seq dataset from nine strawberry tissue types  <cit> .
in total,  <dig> million small rna-seq reads between 18bp and 30bp were mapped against the  <dig>  lncrnas by bowtie  <dig>   <dig>  % of the small rna reads mapped perfectly to the lncrnas; the majority  of these small rnas were 21bp in length.
this is in sharp contrast with the total small rna reads with  <dig> bp small rna as the most abundant species .
we thus identified  <dig>  potentially small rna-generating lncrnas, defined as having higher than  <dig> small rna reads mapped to the lncrna locus .
when all three filtering criteria were applied, only  <dig>  high confidence lncrnas  were obtained .
they are expressed at a relatively high level , contain no repeats, and do not produce small rnas.
characterization of strawberry lncrnas
the  <dig>  predicted pc genes from the f. vesca genome are evenly distributed across chromosomes .
this is in contrast to some other genomes, such as maize and soybean, which have lower gene densities in the pericentromeric regions.
like the pc genes of f. vesca,  <dig>  fve-lncrnas are also distributed evenly across the seven chromosomes .
however, fve-lncrnas also exhibit marked differences from fve-pc genes.
first, lncrnas have fewer exons .
the majority of fve-lncrnas  possess only one or two exons, while only  <dig> % of the pc genes have < =  <dig> exons.
second, lncrnas are generally shorter than pc transcripts .
third, a larger number of pc genes are expressed at a higher level than lncrna-coding loci, according to the fpkm extracted from the output of a single cuffdiff run .
lncrnas are expressed in specific tissues and stages
during the cuffdiff run, both differentially expressed  loci and their isoforms  were examined by pairwise comparisons between successive developmental stages of the same tissue types .
in these comparisons, anthers and embryos have the most de lncrna isoforms and loci; very few de lncrnas were found in receptacle cortex and pith .
in total,  <dig> isoforms from  <dig> loci showed differential expression between at least two different tissue types.
the z-score was obtained for each of these de loci based on averaged fpkm of two biological replicates.
a z-score-based heatmap was made by hierarchical clustering across all tissues .
overall, a large number of lncrnas were specific to one tissue at one specific stage.
the biggest cluster of loci was exclusively expressed in the mature pollen.
relatively more lncrnas were uniquely expressed in anther_ <dig>  anther_ <dig>  and embryo_ <dig>  it may be that anther_ <dig> and mature pollen share some specifically expressed lncrnas since pollen was collected from stage  <dig> anthers.
a similar gene expression trend was also observed when looking at the expression of all isoforms .
hence, lncrna expression is spatially and temporally regulated.
the js  specificity score was used to estimate the degree of tissue specificity.
when a gene is expressed exclusively in a particular tissue, its js score equals to  <dig>  the distribution of the js score shows that more lncrnas have a higher score than pc genes , suggesting that a higher percentage of lncrnas were more exclusively expressed .fig.
<dig> heatmaps showing tissue-specific expression of differentially expressed lncrnas.
a heatmap showing expression patterns of  <dig>  differentially expressed lncrnas.
they are defined as differentially expressed when q-value <  <dig> , fold change >  <dig> in one of the pairwise comparisons.
z-score obtained from averaged fpkm of two replicates was used.
b the distributions of the maximal js  specificity score of coding genes and lncrna-loci, respectively
to validate the rna-seq data, ten lncrnas with anther- or mature pollen-specific expression were selected for verification by rt-pcr .
these lncrnas have a class_code “u” or “x” with fpkm starting from  <dig>  the expected amplicons were observed for all ten lncrnas , but some primers also amplified non-specific bands in the same tissue  or different bands between tissues .
we further tested the expression levels of six lncrnas by quantitative rt-pcr.
xloc_ <dig> is highly expressed in stage 7– <dig> anthers, just before or at the stage of microspore tetrad formation .
xloc_ <dig> and xloc_ <dig> are more abundant in stage  <dig> anthers, the stage at which the tapetum cells start to degenerate .
xloc_ <dig> is highly expressed in stage  <dig> anthers .
both xloc_ <dig> and xloc_ <dig> are predominantly expressed in mature pollen and slightly expressed in stage  <dig> anthers .
in general, the qrt-pcr results  are consistent with the rna-seq results estimated by cufflinks  .fig.
<dig> validation of anther/pollen specific expression of lncrnas by qrt-pcr.
a gel image of rt-pcr products of ten randomly selected anther/pollen specific lncrnas.
b to g the expression of six lncrnas quantified by qrt-pcr .
error bar indicates standard deviation  of two biological replicates with three technical replicates each.
the relative fpkm of the same six lncrnas based on rna-seq data was also shown .
gene <dig> was used as the internal control for both rna-seq  and qrt-pcr .
rnas were from anthers at stage7/ <dig>  stage <dig>  stage <dig>  stage <dig>  and stage <dig> as well as mature pollen
expression correlation of lncrnas with neighboring pc genes
lncrnas regulate gene expression via a number of mechanisms including the regulation of neighboring loci in cis  <cit> .
we thus examined the correlation in expression between lncrnas and their respective neighboring pc genes.
since the location of lncrnas in the unanchored pseudo-molecule of the f. vesca genome is ambiguous, those lncrnas were not included in the analysis.
<dig>  out of  <dig>  lncrna loci have pc neighbors either upstream or downstream within a  <dig> kb distance.
<dig>  gene pairs showed absolute value of correlation coefficient >  <dig>   and are thus promising candidates for cis-regulation.
although lncrnas were slightly more positively correlated with neighboring coding genes than pc genes with their neighbors in statistics  test, p <  <dig> ; additional file 1: figure s4), the distributions of correlation coefficients for lncrna-to-neighbor and pc-to-neighbor are quite similar, in agreement with findings in human  <cit> .
further, many more lncrna-to-neighbor gene pairs showed a positive correlation than those showing a negative correlation .
however, an in depth examination of negatively correlated lncrna-neighbor pairs revealed some intriguing findings.
as shown in fig.
<dig>  xloc_ <dig>  an intergenic lncrna locus with three isoforms and two exons, has a correlation coefficient of − <dig>  with the neighboring gene xloc_ <dig> , which codes for a pentatricopeptide repeat-containing protein.
the opposite expression pattern is evident in all  <dig> tissues  and is illustrated in detail for the fruit cortex tissues .
the lncrna gene xloc_ <dig> is highly expressed in the stage  <dig> cortex, while xloc_ <dig> is lowly expressed in the stage  <dig> cortex.
in contrast, xloc_ <dig> is lowly expressed in the stage  <dig> cortex, while xloc_ <dig> is highly expressed in the stage  <dig> cortex.
while the observed expression correlations between fve-lncrnas and their co-expressed neighbors are highly intriguing, whether they reflect true regulatory relationships or not require further testing.fig.
<dig> an example of negative correlation in expression between an lncrna and its neighboring pc gene.
a the expression of lncrna xloc_ <dig>  and neighboring coding gene xloc_ <dig>  is negatively correlated.
y-axis shows the expression level by z-score obtained from averaged fpkm of two replicates.
b igv view of aligned rna-seq read counts for xloc_ <dig> and xloc_ <dig> based on two fruit tissue stages: cortex- <dig>  and cortex- <dig> .
the panel of “reference” shows the gene structure based on genome annotation version <dig> .
thin line indicates intron and thick line denotes exon.
the panel of “isoform” shows transcript variants predicted by cufflinks.
the bottom four panels illustrate the rna-seq read counts in respective tissues.
the two replicates are shown with identical color
expression correlation of lncrnas with pc genes in trans
prior reports in animal systems also suggest trans-acting modes of some lncrnas  <cit> .
to identify lncrnas that may act in trans, the expression correlations between lncrnas and all pc genes in the f. vesca genome were calculated.
<dig>  out of  <dig>  lncrnas were negatively correlated with pc genes .
<dig> lncrnas each showed negative coefficients with more than  <dig> genes; the expression patterns of these  <dig> lncrnas are shown in the heatmap .
while a majority of these lncrnas showed high levels of expression in the pollen, two clusters of lncrnas  showed complementary expression patterns.
one cluster  was specifically and more abundantly expressed in the receptacle fruit  at post fertilization stages .
the complementary cluster  was expressed in other tissues excluding the receptacle  at stages 2– <dig>  successful fertilization of ovules has been shown to induce receptacle fruit initiation  <cit> , therefore the c <dig> cluster of lncrna genes may be induced by fertilization with potential roles in promoting fleshy fruit initiation.
in contrast, the c <dig> cluster of lncrnas may possess opposite roles and may be involved in repressing receptacle fruit development.fig.
<dig> negative correlation in expression between lncrnas and pc genes across the genome.
a heatmap showing the expression of  <dig> lncrnas, which were identified as having  <dig> or more negatively correlated pc genes across the genome at a cut-off of r < − <dig> .
clusters c <dig> and c <dig> were highlighted and are specifically repressed or activated in the receptacle.
b top five lncrnas with the highest number of negatively correlated protein coding genes are shown.
each of the five lncrnas is connected to its negatively correlated protein–coding genes with lines.
the expression pattern of each of the five lncrnas is marked in  by arrows with the same colors.
c the opposite expression trend between lncrna xloc_ <dig>  and its  <dig> negatively correlated pc genes .
z-score used in  and  was obtained from averaged fpkm from two replicates
the five lncrnas with the highest number of negatively co-regulated genes are shown in a graph .
each of the five lncrnas is connected to the co-regulated genes in the genome with colored lines.
these  <dig> lncrnas are all expressed at a low level in the receptacle fruit, with four belonging to the c <dig> cluster .
in contrast, their negatively correlated  <dig> pc genes are more highly expressed in the receptacle .
fig.
5c illustrates the expression of lncrna xloc_ <dig> and its corresponding  <dig> negatively co-regulated pc genes.
given that these  <dig> lncrnas could potentially contribute to the regulation of such a large number of negatively correlated pc genes, their impact on the development of fruit could be significant.
enriched go terms for these  <dig> pc genes include metabolic processes and intracellular transport processes, consistent with functions in fruit tissue development .
these analyses not only identified fve-lncrnas with potential regulatory roles in trans, but also highlighted specific lncrnas that could potentially serve as hubs in a coordinated gene expression networks underlying receptacle fruit development.
examination of evolutionary conservation of lncrnas
if lncrnas perform evolutionarily conserved functions, they could be conserved across species even though a lack of coding constraints may enable rapid changes in dna sequences.
thus, we investigated if any of the fve-lncrnas identified in this study are conserved across different plant species.
first, we used the fve-lncrnas to blast against the genomes of arabidopsis thaliana, maize , rice , apple , and peach ;  <dig>   <dig>   <dig>   <dig> and  <dig> fve-lncrnas were found to share similarities with certain sequences in the respective plant genomes .
since apple, peach, and strawberry belong to the rosaceae family, they are more closely related to one another.
the numbers above suggest that about  <dig> % of the fve-lncrnas have potential conserved counterparts in the non-roseceae species and about  <dig> % fve-lncrnas have potential homologs in the roseceae species.
hence, the evolutionary conservation of lncrna is rather limited.
the above analysis could not determine if the homologous sequence in the other species encode lncrnas.
therefore, we investigated if the fve-lncrnas are homologous to lncrnas already identified in these species.
currently,  <dig>  lncrnas were identified in arabidopsis  <cit> ,  <dig>  lncnras were identified in rice  <cit> , and several thousand lncrnas were reported from two studies in zea mays.
boerner and mcginnis first identified  <dig>  maize lncrnas  <cit> ; li reported  <dig> high confidence lncrnas and  <dig>  pre-lncrnas in maize  <cit> .
<dig>  fve-lncrnas were blasted against the lncrnas from arabidopsis, rice and maize, only one fve-lncrna  was found to share similarity to the pre-lncrna transcript tcons_ <dig> in maize .table  <dig> identification of conserved lncrnas in related species
arabidopsis
fragaria vesca
notes:
column  <dig> shows the number of fve-lncrnas with homologs in other  species listed in column  <dig>  the number before ‘/’ refers to lncrna number in fragaria and the number after ‘/’ refers to the number of lncrna in the target  species.
the numbers in column  <dig> were derived from blast searches using strawberry lncrnas as queries against the target species.
the blast e-value cutoff is <  <dig> .
the second column shows results using arabidopsis lncrnas as queries against corresponding target species.
no blast was performed against the query itself and hence was marked as ‘-/-’
to test conservation of fve-lncrnas in the rosaceae species, malus and prunus, we took advantage of available ests for these two species.
we first identified lncrnas from each species applying the same filtering criteria as for f. vesca.
specifically, we analyzed  <dig>  assembled unigenes in the malus unigenes v. <dig> downloaded from gdr; these unigenes were built previously based on ests from mainly malus domestica.
among them,  <dig>  unigenes were found to possess lncrnas .
<dig> of these m. domestica unigenes exhibited sequence similarities to  <dig> f. vesca lncrnas  .
similarly, analysis of  <dig>  assembled unigenes in the prunus unigenes v. <dig>  yielded  <dig>  unigenes that likely produce lncrnas .
<dig> of these prunus unigenes showed sequence similarity to  <dig> f. vesca lncrnas  .
the conservation of these lncrnas within the same plant family suggests that these lncrnas may arise in the common ancestor of these species and may confer biological functions unique to this family of plants.
to investigate possible conservation of lncrnas across any plant species described above, the  <dig>  lncrnas from arabidopsis  <cit>  were blasted against the lncrnas from maize, rice, malus and prunus, respectively.
five of these arabidopsis lncrnas showed similarity to  <dig> of  <dig>  pre-lncrnas from maize   <cit> .
five arabidopsis lncrnas are similar to  <dig> of the  <dig>  lncrnas from rice   <cit> .
two arabidopsis lncrnas are similar to  <dig> of the  <dig>  lncrnas from malus .
no homolog was identified in other blasts.
therefore, lncrnas were not well conserved in higher plants, perhaps reflecting the fast evolving nature of lncrnas due to the lack of constraint normally imposed upon pc genes  <cit> .
discussion
lncrnas are being increasingly recognized as an important class of regulatory molecules in both animals and plants.
while lncrnas have been widely studied in human and animals, they are still poorly studied in plants with the exception of a limited number of model plant species.
to date, no lncrna has been described in strawberry nor in any other rosaceae species.
in this study, we performed genome-wide identification of lncrnas from diploid strawberry fragaria vesca as well as several other rosaceae species including malus and prunus, thereby providing a first look at the landscape of noncoding genes in the rosaceae genomes.
since f. vesca possesses a full complement of molecular genetic tools, the discovery of fve-lncrnas laid the groundwork for future functional studies of lncrnas.
since rosaceae is an important family for fruit crops, the regulation of their flower and fruit development is of considerable interests.
previously we developed extensive floral and fruit tissue transcriptomes for f. vesca via rna-seq and small rna-seq  <cit> .
in this study, we mined these transcriptome datasets for lncrnas using a set of stringent filtering criteria.
we identified  <dig>  f. vesca lncrnas coded by  <dig>  loci.
we showed that these f. vesca noncoding loci possess features similar to pc loci; they contain promoters, exons, and introns, and are alternatively spliced.
on the other hand, we showed that the lncrnas are not well conserved when compared with pc genes, have fewer exons, are expressed at lower levels, and are shorter in transcript length .
these findings are consistent with other studies on plant lncrnas .
one most striking feature of these lncrnas is their tissue-specific expression indicating possible function in specific flower or fruit tissues or at specific stages of development.
the lncrnas identified from our study may represent only a fraction of lncrnas coded by the f. vesca genome.
this is because these lncrnas were only identified from libraries derived from flower and fruit tissues grown under normal conditions.
further, since the rna-seq libraries were made from polya-selected mrnas, nonpolyadenylated lncrnas were missed from these libraries.
finally, natural antisense transcripts  transcribed from the presumptive non-coding dna strand could be missed due to a lack of strand-specific information in our libraries.
therefore, our work only represents the initial genome scale identification of lncrnas.
nevertheless, the large number of lncrnas identified in this study suggests that lncrnas may contribute significantly to strawberry flower and fruit development.
plant lncrnas are not well conserved during evolution
blast searches of fve-lncrnas against the lncrnas identified in several non rosaceae plant species yielded none or very few homologs , indicating limited evolutionary conservation.
though non-coding rnas evolve faster than pc genes  <cit> , thousands of conserved lncrnas have been found among primates and several tetrapod species  <cit> , possibly owing to more ancient origin of these lncrnas found in animals and more time to be stabilized in function.
in contrast, flowering plants only arose  <dig> million years ago and their lncrnas may be of recent origin and maybe relatively young and transient.
it is not surprising why lncrnas are not well conserved.
first, lncrnas are not constrained by codon usage.
second, lncrnas may possess short conserved motifs, but these short motifs are not easily identifiable by blast.
for example, an intergenic lncrna family mt4/ips1/at4/tpsi1/ospi <dig> found in multiple plant species  contains a ~ <dig> bp short and conserved motif; this motif binds mir- <dig> via sequence complementarity, the basis of “target mimicry”  <cit> .
target mimicry is emerging as a prevalent mode of action by lncrnas since more lncrnas are found to act through target mimicry  <cit> .
third,  <dig> % lncrnas are probably associated with small rnas in this study, most of which should be sirnas .
the sirnas could be generated from a short pairing fragment of two rnas, such as lnc-nats, or the stem-loop structure of a single rna, which are less constrained in other parts of the transcripts  <cit> .
fourth, some lncrnas may directly interact with rna-binding proteins through conserved secondary structures  <cit> .
the expression of lncrnas is highly tissue-specific
a major challenge in deducing lncrna function resides in that lncrnas do not encode proteins.
gene expression, specifically tissue-specific expression, may help shed light on the potential function of these lncrnas.
pairwise comparisons between tissues or stages revealed that  <dig>  % of lncrna isoforms and  <dig>  % of lncrna loci are differentially expressed and show high tissue specificity , suggesting that lncrnas are subject to active transcriptional regulation.
most notably, a large number of lncrnas are highly and specifically expressed in the mature pollen .
however, pc genes are also highly and specifically expressed in the mature pollen  <cit> .
this similarity may simply reflect that mature pollen is a very unique tissue.
nevertheless, the action of pollen-specific lncrnas and their interaction with pollen-specific pc genes may underlie the unique characteristics of mature pollen.
the precise regulation of lncrnas in specific tissues support that lncrnas may play important functions during reproductive development.
expression correlations of lncrnas with pc genes in cis and in trans
in animals and plants, lncrnas have been shown to act either in cis or in trans to regulate pc gene expression  <cit> .
based on the idea that lncrnas and their regulatory targets may exhibit highly correlative expression either positively or negatively, we sought to identify potential regulatory targets of the fve-lncrnas by taking advantages of the available rna-seq data for a large number  of f. vesca tissues.
a similar strategy was successfully employed in finding candidate regulatory targets of lncrnas in mammals and model plants  <cit> .
through this strategy, we identified  <dig>  positively correlated and  <dig> negatively correlated pairs of lncrnas and neighboring genes .
the positive expression correlations may simply result from their subjecting to common regulations of local chromatin.
however, some correlations could reflect authentic regulatory relationships between an lncrna and its neighboring gene.
an encouraging example is the positive expression correlation between lncrna apolo and its regulatory target pid, which codes for a key regulator of polar auxin transport  <cit> .
in our study, we showed an example of an lncrna xloc_ <dig> and its negatively correlated pc gene xloc_ <dig> .
xloc_ <dig> encodes a protein belonging to the ppr protein family that has rna binding capacity  <cit> .
the opposite expression pattern and the immediate upstream location of the lncrna xloc_ <dig> with respect to the ppr gene warrants further investigation into a possible direct regulatory relationship between the lncrna and its neighboring ppr gene.
to identify lncrnas acting in trans, we performed a correlation analysis between  <dig>  lncrnas and all pc genes in the f. vesca genome .
one third of the lncrnas have negatively correlated pc genes at a cut-off of − <dig> ; this is not surprising given the pervasive in trans regulations reported in animals  <cit> .
in examining the expression profiles of these lncrnas, we identified clusters of lncrnas exhibiting receptacle fruit-specific repression or activation .
it will be interesting to determine if c <dig> and c <dig> clusters play opposite roles in the development of this unique strawberry fruit type.
among the lncrnas that show receptacle-specific repression, we selected  <dig> that have expression patterns correlated with the highest number of pc genes .
the  <dig> coding genes targeted by these five lncrnas are enriched in various metabolic processes associated with active cell proliferation and growth .
these results suggest that the receptacle-repressive lncrnas could serve as the hubs of a gene regulatory network, the repression of which may lead to positive receptacle fruit growth.
CONCLUSIONS
<dig>  fve-lncrnas derived from  <dig>  loci were identified from diploid strawberry fragaria vesca using the flower and fruit rna-seq datasets, thereby providing a first look at the landscape of noncoding genes in one fruit crop of the rosaceae family.
the tissue-specific lncrna expression patterns and the gene expression correlation analysis between lncrnas and pc genes identified a set of lncrnas with potential roles in flower and fruit development.
the discovery of fve-lncrnas laid the groundwork for future functional studies of lncrnas in strawberry.

 with the advent of new genomic techniques such as tiling arrays and next generation sequencing, non-coding rnas are increasingly identified and recognized as an integral and functional component of the genome. while some non-coding rnas perform housekeeping functions, such as trnas, rrnas and small nuclear rnas, others such as micrornas and small interfering rnas play critical regulatory roles during development or stress responses. non-coding rnas with a length greater than 200bp are defined as long non-coding rnas. the lncrnas can be grouped into three subclasses: 1) long non-coding natural antisense transcripts, 2) intronic lncrnas, and 3) intergenic lncrnas. like protein-coding genes, a majority of lncrnas are transcribed by rna polymerase ii with a 5′ cap and a 3′ poly-a tail in animals. in plants, however, lncrnas can be transcribed by polii, iv, and v, therefore some may lack poly-a tails. a growing number of reports revealed lncrnas from animals, especially human; they are involved in diverse biological processes, such as development, cellular differentiation, and diseases including cancers. lncrnas may serve as diagnostic markers or even therapeutic targets. studies of animal lncrnas showed that lncrnas function through a number of mechanisms. first, lncrnas act in epigenetic regulation of gene expression. for instance, the mammalian xist rna initiates x chromosome inactivation in cis to equalize gene expression between males and females. hotair is able to mediate transcriptional repression of hox loci in trans by modulating histone methylation. second, lncrnas may directly interact with proteins to titrate their functions and are thus called ‘decoys’. a well-known example is the lncrna named terra, which was demonstrated to be the natural ligand and inhibitor of telomerase. third, lncrnas may act as a scaffold to form a complex with other proteins. together, lncrnas were demonstrated to play bona fide and essential roles in animals. in contrast, there are only a handful of reports on the functions of lncrnas in plants. flowering locus c is a key flowering repressor in the vernalization pathway. to ensure epigenetic silencing of flc, a lnc-nat named coolair and an intronic lncrna called coldair could be induced after vernalization treatment to gradually repress the expression of flc by promoting methylation. another intergenic lncrna induced by phosphate starvation was found in medicago truncatula, arabidopsis thaliana, tomato, and rice. further analysis indicated that ips acts as a decoy of mirna- and allows the accumulation of its target gene transcripts. a third intergenic lncrna called ldmar is required for normal pollen development in rice under long day conditions. a number of lncrnas are differentially expressed under stress stimuli in arabidopsis. despite limited reports on the mechanisms of plant lncrna function, it is evident that plant lncrnas play vital roles in developmental and stress responses. this realization combined with the advent of next-generation sequencing has prompted tremendous efforts and investments in identifying lncrnas in a wide range of organisms. f. vesca, the woodland strawberry, is becoming a new model organism for both octoploid cultivated strawberry and other members of the rosaceae, a family that includes many fruit trees. f. vesca has a short life cycle, small stature, facile transformation, and small and sequenced genome. further, transcriptomic data of various tissues in the cultivated strawberry were available. of significant interest is the strawberry fruit, which is developed from the receptacle. toward the identification of molecular mechanisms of strawberry floral and fruit development, we first generated comprehensive rna-seq datasets from distinct f. vesca floral and fruit tissues at different developmental stages to profile genome-wide expression of pc genes. in current work, we seek to identify lncrnas from strawberry floral and fruit transcriptomes with the goal of uncovering lncrnas that function in flower and fruit development. in total, lncrnas derived from loci were identified from the f. vesca flower and fruit trancriptome dataset. further analysis indicated that these lncrnas are similar to pc genes in terms of gene structure and transcriptional regulation. however, lncrnas also show a number of distinctions from pc genes. for example, a large number of lncrnas are precursors to small rnas, and their sequences are much less conserved than pc genes. in an effort to identify potential regulatory target genes of lncrnas, we used expression correlation between lncrnas and the pc genes in the f. vesca genome, taking advantage of the rna-seq data from a large number of different floral and fruit tissues. both positive and negatively correlated lncrna-pc gene pairs are identified. our analysis provided the first look at the lncrna landscape in a fruit crop and laid the foundation for future studies of lncrna function in strawberry. to facilitate the study of lncrnas and data sharing, our f. vesca lncrnas can be accessed from the strawberry genome resources website as a new track in gbrowse as well as from genome database for rosaceae.  identification of f. vesca lncrnas from flower and fruit rna-seq datasets to globally identify lncrnas in the f.vesca genome, we utilized rna-seq datasets generated from distinct floral and fruit tissues plus two vegetative tissues, which were isolated from a 7th generation inbred line of f. vesca. there were two biological replicates for each tissue type, and hence rna-seq libraries in total, amounting to about billion total single-end reads of 51bp. the analysis pipeline is shown in fig.  in brief, each library was aligned individually by tophat in order to preserve junction reads. known and novel transcripts were assembled by cufflinks based on uniquely mapped reads and, finally, all assemblies were combined by cuffmerge and then compared to the annotations by cuffcompare to characterize each transcript.fig. summary of the workflows used for identifying strawberry lncrnas. a lncrna identification from f. vesca rna-seq datasets. b lncrna identification from unigenes. c venn diagram comparing lncrnas identified in and. d different classifications of fve-lncrnas based on expression levels, repeats, and precursors of small rnas. hc-lncrnas: high confidence lncrnas in total, candidate noncoding transcripts in class “u”, “o”, “x”, and “i” were subjected to further filtering. the transcripts are defined as lncrnas if they are longer than 200bp, are non-coding, and are not a member of known functional rna classes. a relatively stringent non-coding standard was used. first, the coding potential calculator was used to discriminate non-coding from coding transcripts as it takes into account several biologically meaningful sequence features with high accuracy; a non-coding transcript has a coding potential score lower than “-1”. second, to reduce false positive, any short peptide coded by “non-coding transcript” should contain no conserved protein domains. as a result, transcript isoforms in loci were identified as lncrnas.table the number of lncrnas in different classes before and after filtering notes: athe number of original isoforms and loci predicted by cufflinks bthe number of lncrnas after filtering c“total” number is smaller than the sum of the column, because certain loci have multiple isoforms with different class codes in addition to the rna-seq data, we mined existing ests from fragaria. a large number of raw est datasets derived from both diploid fragaria vesca and octoploid fragaria × ananassa were found in the ncbi dbest database, but unigenes from gdr were chosen for lncrna discovery as unigenes do not contain low quality ests or redundant ests. the filtering criteria were similar to the lncrna discovery from rna-seq data and led to the identification of lncrnas out of unigenes. interestingly, only of the lncrnas are in common with the rna-seq-derived lncrnas. we then compared the remaining unigene-derived lncrnas with the f. vesca genome annotation and found that a majority of them are from annotated pc loci on either the same or opposite strand sometimes covering the introns. a lack of over-lap between rna-seq- and unigene-derived lncrnas suggests that the identification of lncrnas is far from saturation, partly due to a lack of strand orientation information from our rna-seq reads. identification of high confidence -lncrnas the lncrnas identified from the f. vesca rna-seq data were further characterized based on expression levels and whether or not they contain repeats or generate small rnas. first, lncrnas are expressed at higher than fpkm in at least one of the tissue types in both replicates; the remaining lncrnas are expressed at lower than fpkm. second, lncrnas that overlapped with transposable elements and/or repeats were identified by repeatmasker and repeatscout, respectively. in total, transcripts with a percentage of repetitive sequences lower than % were classified as “no repeat”. the filtering pipeline has removed known pre-mirna transcripts, but a small quantity of the remaining lncrnas may still encode previously unknown mirnas. moreover, some lncrnas would generate short hairpin rnas and sirnas involved in epigenetic regulation. to distinguish those small rna-generating lncrnas, we used the small rna-seq dataset from nine strawberry tissue types. in total, million small rna-seq reads between 18bp and 30bp were mapped against the lncrnas by bowtie % of the small rna reads mapped perfectly to the lncrnas; the majority of these small rnas were 21bp in length. this is in sharp contrast with the total small rna reads with bp small rna as the most abundant species. we thus identified potentially small rna-generating lncrnas, defined as having higher than small rna reads mapped to the lncrna locus. when all three filtering criteria were applied, only high confidence lncrnas were obtained. they are expressed at a relatively high level, contain no repeats, and do not produce small rnas. characterization of strawberry lncrnas the predicted pc genes from the f. vesca genome are evenly distributed across chromosomes. this is in contrast to some other genomes, such as maize and soybean, which have lower gene densities in the pericentromeric regions. like the pc genes of f. vesca, fve-lncrnas are also distributed evenly across the seven chromosomes. however, fve-lncrnas also exhibit marked differences from fve-pc genes. first, lncrnas have fewer exons. the majority of fve-lncrnas possess only one or two exons, while only % of the pc genes have < = exons. second, lncrnas are generally shorter than pc transcripts. third, a larger number of pc genes are expressed at a higher level than lncrna-coding loci, according to the fpkm extracted from the output of a single cuffdiff run. lncrnas are expressed in specific tissues and stages during the cuffdiff run, both differentially expressed loci and their isoforms were examined by pairwise comparisons between successive developmental stages of the same tissue types. in these comparisons, anthers and embryos have the most de lncrna isoforms and loci; very few de lncrnas were found in receptacle cortex and pith. in total, isoforms from loci showed differential expression between at least two different tissue types. the z-score was obtained for each of these de loci based on averaged fpkm of two biological replicates. a z-score-based heatmap was made by hierarchical clustering across all tissues. overall, a large number of lncrnas were specific to one tissue at one specific stage. the biggest cluster of loci was exclusively expressed in the mature pollen. relatively more lncrnas were uniquely expressed in anther_ anther_ and embryo_ it may be that anther_ and mature pollen share some specifically expressed lncrnas since pollen was collected from stage anthers. a similar gene expression trend was also observed when looking at the expression of all isoforms. hence, lncrna expression is spatially and temporally regulated. the js specificity score was used to estimate the degree of tissue specificity. when a gene is expressed exclusively in a particular tissue, its js score equals to the distribution of the js score shows that more lncrnas have a higher score than pc genes, suggesting that a higher percentage of lncrnas were more exclusively expressed.fig. heatmaps showing tissue-specific expression of differentially expressed lncrnas. a heatmap showing expression patterns of differentially expressed lncrnas. they are defined as differentially expressed when q-value < , fold change >  in one of the pairwise comparisons. z-score obtained from averaged fpkm of two replicates was used. b the distributions of the maximal js specificity score of coding genes and lncrna-loci, respectively to validate the rna-seq data, ten lncrnas with anther- or mature pollen-specific expression were selected for verification by rt-pcr. these lncrnas have a class_code “u” or “x” with fpkm starting from the expected amplicons were observed for all ten lncrnas, but some primers also amplified non-specific bands in the same tissue or different bands between tissues. we further tested the expression levels of six lncrnas by quantitative rt-pcr. xloc_ is highly expressed in stage 7– anthers, just before or at the stage of microspore tetrad formation. xloc_ and xloc_ are more abundant in stage anthers, the stage at which the tapetum cells start to degenerate. xloc_ is highly expressed in stage anthers. both xloc_ and xloc_ are predominantly expressed in mature pollen and slightly expressed in stage anthers. in general, the qrt-pcr results are consistent with the rna-seq results estimated by cufflinks.fig. validation of anther/pollen specific expression of lncrnas by qrt-pcr. a gel image of rt-pcr products of ten randomly selected anther/pollen specific lncrnas. b to g the expression of six lncrnas quantified by qrt-pcr. error bar indicates standard deviation of two biological replicates with three technical replicates each. the relative fpkm of the same six lncrnas based on rna-seq data was also shown. gene was used as the internal control for both rna-seq and qrt-pcr. rnas were from anthers at stage7/ stage stage stage and stage as well as mature pollen expression correlation of lncrnas with neighboring pc genes lncrnas regulate gene expression via a number of mechanisms including the regulation of neighboring loci in cis. we thus examined the correlation in expression between lncrnas and their respective neighboring pc genes. since the location of lncrnas in the unanchored pseudo-molecule of the f. vesca genome is ambiguous, those lncrnas were not included in the analysis. out of lncrna loci have pc neighbors either upstream or downstream within a kb distance. gene pairs showed absolute value of correlation coefficient >  and are thus promising candidates for cis-regulation. although lncrnas were slightly more positively correlated with neighboring coding genes than pc genes with their neighbors in statistics test, p < ; additional file 1: figure s4), the distributions of correlation coefficients for lncrna-to-neighbor and pc-to-neighbor are quite similar, in agreement with findings in human. further, many more lncrna-to-neighbor gene pairs showed a positive correlation than those showing a negative correlation. however, an in depth examination of negatively correlated lncrna-neighbor pairs revealed some intriguing findings. as shown in fig.  xloc_ an intergenic lncrna locus with three isoforms and two exons, has a correlation coefficient of − with the neighboring gene xloc_, which codes for a pentatricopeptide repeat-containing protein. the opposite expression pattern is evident in all tissues and is illustrated in detail for the fruit cortex tissues. the lncrna gene xloc_ is highly expressed in the stage cortex, while xloc_ is lowly expressed in the stage cortex. in contrast, xloc_ is lowly expressed in the stage cortex, while xloc_ is highly expressed in the stage cortex. while the observed expression correlations between fve-lncrnas and their co-expressed neighbors are highly intriguing, whether they reflect true regulatory relationships or not require further testing.fig. an example of negative correlation in expression between an lncrna and its neighboring pc gene. a the expression of lncrna xloc_ and neighboring coding gene xloc_ is negatively correlated. y-axis shows the expression level by z-score obtained from averaged fpkm of two replicates. b igv view of aligned rna-seq read counts for xloc_ and xloc_ based on two fruit tissue stages: cortex- and cortex-. the panel of “reference” shows the gene structure based on genome annotation version. thin line indicates intron and thick line denotes exon. the panel of “isoform” shows transcript variants predicted by cufflinks. the bottom four panels illustrate the rna-seq read counts in respective tissues. the two replicates are shown with identical color expression correlation of lncrnas with pc genes in trans prior reports in animal systems also suggest trans-acting modes of some lncrnas. to identify lncrnas that may act in trans, the expression correlations between lncrnas and all pc genes in the f. vesca genome were calculated. out of lncrnas were negatively correlated with pc genes. lncrnas each showed negative coefficients with more than genes; the expression patterns of these lncrnas are shown in the heatmap. while a majority of these lncrnas showed high levels of expression in the pollen, two clusters of lncrnas showed complementary expression patterns. one cluster was specifically and more abundantly expressed in the receptacle fruit at post fertilization stages. the complementary cluster was expressed in other tissues excluding the receptacle at stages 2– successful fertilization of ovules has been shown to induce receptacle fruit initiation, therefore the c cluster of lncrna genes may be induced by fertilization with potential roles in promoting fleshy fruit initiation. in contrast, the c cluster of lncrnas may possess opposite roles and may be involved in repressing receptacle fruit development.fig. negative correlation in expression between lncrnas and pc genes across the genome. a heatmap showing the expression of lncrnas, which were identified as having or more negatively correlated pc genes across the genome at a cut-off of r < −. clusters c and c were highlighted and are specifically repressed or activated in the receptacle. b top five lncrnas with the highest number of negatively correlated protein coding genes are shown. each of the five lncrnas is connected to its negatively correlated protein–coding genes with lines. the expression pattern of each of the five lncrnas is marked in by arrows with the same colors. c the opposite expression trend between lncrna xloc_ and its negatively correlated pc genes. z-score used in and was obtained from averaged fpkm from two replicates the five lncrnas with the highest number of negatively co-regulated genes are shown in a graph. each of the five lncrnas is connected to the co-regulated genes in the genome with colored lines. these lncrnas are all expressed at a low level in the receptacle fruit, with four belonging to the c cluster. in contrast, their negatively correlated pc genes are more highly expressed in the receptacle. fig. 5c illustrates the expression of lncrna xloc_ and its corresponding negatively co-regulated pc genes. given that these lncrnas could potentially contribute to the regulation of such a large number of negatively correlated pc genes, their impact on the development of fruit could be significant. enriched go terms for these pc genes include metabolic processes and intracellular transport processes, consistent with functions in fruit tissue development. these analyses not only identified fve-lncrnas with potential regulatory roles in trans, but also highlighted specific lncrnas that could potentially serve as hubs in a coordinated gene expression networks underlying receptacle fruit development. examination of evolutionary conservation of lncrnas if lncrnas perform evolutionarily conserved functions, they could be conserved across species even though a lack of coding constraints may enable rapid changes in dna sequences. thus, we investigated if any of the fve-lncrnas identified in this study are conserved across different plant species. first, we used the fve-lncrnas to blast against the genomes of arabidopsis thaliana, maize, rice, apple, and peach; and fve-lncrnas were found to share similarities with certain sequences in the respective plant genomes. since apple, peach, and strawberry belong to the rosaceae family, they are more closely related to one another. the numbers above suggest that about % of the fve-lncrnas have potential conserved counterparts in the non-roseceae species and about % fve-lncrnas have potential homologs in the roseceae species. hence, the evolutionary conservation of lncrna is rather limited. the above analysis could not determine if the homologous sequence in the other species encode lncrnas. therefore, we investigated if the fve-lncrnas are homologous to lncrnas already identified in these species. currently, lncrnas were identified in arabidopsis, lncnras were identified in rice, and several thousand lncrnas were reported from two studies in zea mays. boerner and mcginnis first identified maize lncrnas; li reported high confidence lncrnas and pre-lncrnas in maize. fve-lncrnas were blasted against the lncrnas from arabidopsis, rice and maize, only one fve-lncrna was found to share similarity to the pre-lncrna transcript tcons_ in maize.table identification of conserved lncrnas in related species arabidopsis fragaria vesca notes: column shows the number of fve-lncrnas with homologs in other species listed in column the number before ‘/’ refers to lncrna number in fragaria and the number after ‘/’ refers to the number of lncrna in the target species. the numbers in column were derived from blast searches using strawberry lncrnas as queries against the target species. the blast e-value cutoff is <. the second column shows results using arabidopsis lncrnas as queries against corresponding target species. no blast was performed against the query itself and hence was marked as ‘-/-’ to test conservation of fve-lncrnas in the rosaceae species, malus and prunus, we took advantage of available ests for these two species. we first identified lncrnas from each species applying the same filtering criteria as for f. vesca. specifically, we analyzed assembled unigenes in the malus unigenes v. downloaded from gdr; these unigenes were built previously based on ests from mainly malus domestica. among them, unigenes were found to possess lncrnas. of these m. domestica unigenes exhibited sequence similarities to f. vesca lncrnas. similarly, analysis of assembled unigenes in the prunus unigenes v. yielded unigenes that likely produce lncrnas. of these prunus unigenes showed sequence similarity to f. vesca lncrnas. the conservation of these lncrnas within the same plant family suggests that these lncrnas may arise in the common ancestor of these species and may confer biological functions unique to this family of plants. to investigate possible conservation of lncrnas across any plant species described above, the lncrnas from arabidopsis were blasted against the lncrnas from maize, rice, malus and prunus, respectively. five of these arabidopsis lncrnas showed similarity to of pre-lncrnas from maize. five arabidopsis lncrnas are similar to of the lncrnas from rice. two arabidopsis lncrnas are similar to of the lncrnas from malus. no homolog was identified in other blasts. therefore, lncrnas were not well conserved in higher plants, perhaps reflecting the fast evolving nature of lncrnas due to the lack of constraint normally imposed upon pc genes. discussion lncrnas are being increasingly recognized as an important class of regulatory molecules in both animals and plants. while lncrnas have been widely studied in human and animals, they are still poorly studied in plants with the exception of a limited number of model plant species. to date, no lncrna has been described in strawberry nor in any other rosaceae species. in this study, we performed genome-wide identification of lncrnas from diploid strawberry fragaria vesca as well as several other rosaceae species including malus and prunus, thereby providing a first look at the landscape of noncoding genes in the rosaceae genomes. since f. vesca possesses a full complement of molecular genetic tools, the discovery of fve-lncrnas laid the groundwork for future functional studies of lncrnas. since rosaceae is an important family for fruit crops, the regulation of their flower and fruit development is of considerable interests. previously we developed extensive floral and fruit tissue transcriptomes for f. vesca via rna-seq and small rna-seq. in this study, we mined these transcriptome datasets for lncrnas using a set of stringent filtering criteria. we identified f. vesca lncrnas coded by loci. we showed that these f. vesca noncoding loci possess features similar to pc loci; they contain promoters, exons, and introns, and are alternatively spliced. on the other hand, we showed that the lncrnas are not well conserved when compared with pc genes, have fewer exons, are expressed at lower levels, and are shorter in transcript length. these findings are consistent with other studies on plant lncrnas. one most striking feature of these lncrnas is their tissue-specific expression indicating possible function in specific flower or fruit tissues or at specific stages of development. the lncrnas identified from our study may represent only a fraction of lncrnas coded by the f. vesca genome. this is because these lncrnas were only identified from libraries derived from flower and fruit tissues grown under normal conditions. further, since the rna-seq libraries were made from polya-selected mrnas, nonpolyadenylated lncrnas were missed from these libraries. finally, natural antisense transcripts transcribed from the presumptive non-coding dna strand could be missed due to a lack of strand-specific information in our libraries. therefore, our work only represents the initial genome scale identification of lncrnas. nevertheless, the large number of lncrnas identified in this study suggests that lncrnas may contribute significantly to strawberry flower and fruit development. plant lncrnas are not well conserved during evolution blast searches of fve-lncrnas against the lncrnas identified in several non rosaceae plant species yielded none or very few homologs, indicating limited evolutionary conservation. though non-coding rnas evolve faster than pc genes, thousands of conserved lncrnas have been found among primates and several tetrapod species, possibly owing to more ancient origin of these lncrnas found in animals and more time to be stabilized in function. in contrast, flowering plants only arose million years ago and their lncrnas may be of recent origin and maybe relatively young and transient. it is not surprising why lncrnas are not well conserved. first, lncrnas are not constrained by codon usage. second, lncrnas may possess short conserved motifs, but these short motifs are not easily identifiable by blast. for example, an intergenic lncrna family mt4/ips1/at4/tpsi1/ospi found in multiple plant species contains a ~ bp short and conserved motif; this motif binds mir- via sequence complementarity, the basis of “target mimicry”. target mimicry is emerging as a prevalent mode of action by lncrnas since more lncrnas are found to act through target mimicry. third, % lncrnas are probably associated with small rnas in this study, most of which should be sirnas. the sirnas could be generated from a short pairing fragment of two rnas, such as lnc-nats, or the stem-loop structure of a single rna, which are less constrained in other parts of the transcripts. fourth, some lncrnas may directly interact with rna-binding proteins through conserved secondary structures. the expression of lncrnas is highly tissue-specific a major challenge in deducing lncrna function resides in that lncrnas do not encode proteins. gene expression, specifically tissue-specific expression, may help shed light on the potential function of these lncrnas. pairwise comparisons between tissues or stages revealed that % of lncrna isoforms and % of lncrna loci are differentially expressed and show high tissue specificity, suggesting that lncrnas are subject to active transcriptional regulation. most notably, a large number of lncrnas are highly and specifically expressed in the mature pollen. however, pc genes are also highly and specifically expressed in the mature pollen. this similarity may simply reflect that mature pollen is a very unique tissue. nevertheless, the action of pollen-specific lncrnas and their interaction with pollen-specific pc genes may underlie the unique characteristics of mature pollen. the precise regulation of lncrnas in specific tissues support that lncrnas may play important functions during reproductive development. expression correlations of lncrnas with pc genes in cis and in trans in animals and plants, lncrnas have been shown to act either in cis or in trans to regulate pc gene expression. based on the idea that lncrnas and their regulatory targets may exhibit highly correlative expression either positively or negatively, we sought to identify potential regulatory targets of the fve-lncrnas by taking advantages of the available rna-seq data for a large number of f. vesca tissues. a similar strategy was successfully employed in finding candidate regulatory targets of lncrnas in mammals and model plants. through this strategy, we identified positively correlated and negatively correlated pairs of lncrnas and neighboring genes. the positive expression correlations may simply result from their subjecting to common regulations of local chromatin. however, some correlations could reflect authentic regulatory relationships between an lncrna and its neighboring gene. an encouraging example is the positive expression correlation between lncrna apolo and its regulatory target pid, which codes for a key regulator of polar auxin transport. in our study, we showed an example of an lncrna xloc_ and its negatively correlated pc gene xloc_. xloc_ encodes a protein belonging to the ppr protein family that has rna binding capacity. the opposite expression pattern and the immediate upstream location of the lncrna xloc_ with respect to the ppr gene warrants further investigation into a possible direct regulatory relationship between the lncrna and its neighboring ppr gene. to identify lncrnas acting in trans, we performed a correlation analysis between lncrnas and all pc genes in the f. vesca genome. one third of the lncrnas have negatively correlated pc genes at a cut-off of −; this is not surprising given the pervasive in trans regulations reported in animals. in examining the expression profiles of these lncrnas, we identified clusters of lncrnas exhibiting receptacle fruit-specific repression or activation. it will be interesting to determine if c and c clusters play opposite roles in the development of this unique strawberry fruit type. among the lncrnas that show receptacle-specific repression, we selected that have expression patterns correlated with the highest number of pc genes. the coding genes targeted by these five lncrnas are enriched in various metabolic processes associated with active cell proliferation and growth. these results suggest that the receptacle-repressive lncrnas could serve as the hubs of a gene regulatory network, the repression of which may lead to positive receptacle fruit growth.  fve-lncrnas derived from loci were identified from diploid strawberry fragaria vesca using the flower and fruit rna-seq datasets, thereby providing a first look at the landscape of noncoding genes in one fruit crop of the rosaceae family. the tissue-specific lncrna expression patterns and the gene expression correlation analysis between lncrnas and pc genes identified a set of lncrnas with potential roles in flower and fruit development. the discovery of fve-lncrnas laid the groundwork for future functional studies of lncrnas in strawberry. 
while much effort has been invested in the analysis of lncrnas from established plant models arabidopsis, maize, and rice, almost nothing is known about lncrnas from fruit crops, including those in the rosaceae family. our study identifies a large number of tissue-specifically expressed lncrnas in f. vesca, thereby highlighting their potential contributions to strawberry flower and fruit development and paving the way for future functional studies. finally, blast searches within plant species indicate that lncrnas are not well conserved. to identify regulatory interactions between lncrnas and their potential protein-coding gene targets, pairs of lncrnas and pc genes with positively or negatively correlated expression trends were identified based on their expression; these pairs may be candidates of cis- or trans-acting lncrnas and their targets. long non-coding rnas are a new class of regulatory molecules with roles in diverse biological processes. these lncrnas were carefully cataloged based on expression level and whether or not they contain repetitive sequences or generate small rnas. about one fourth of them are termed high-confidence lncrnas because they are expressed at a level of fpkm higher than and produce neither small rnas nor contain repetitive sequence. here, we present a genome-scale identification and characterization of lncrnas from a diploid strawberry, fragaria vesca, based on rich rna-seq datasets from different flower and fruit tissues.
 long non-coding rnas are a new class of regulatory molecules with roles in diverse biological processes. while much effort has been invested in the analysis of lncrnas from established plant models arabidopsis, maize, and rice, almost nothing is known about lncrnas from fruit crops, including those in the rosaceae family.  here, we present a genome-scale identification and characterization of lncrnas from a diploid strawberry, fragaria vesca, based on rich rna-seq datasets from different flower and fruit tissues. fve-lncrnas derived from loci were identified. these lncrnas were carefully cataloged based on expression level and whether or not they contain repetitive sequences or generate small rnas. about one fourth of them are termed high-confidence lncrnas because they are expressed at a level of fpkm higher than and produce neither small rnas nor contain repetitive sequence. to identify regulatory interactions between lncrnas and their potential protein-coding gene targets, pairs of lncrnas and pc genes with positively or negatively correlated expression trends were identified based on their expression; these pairs may be candidates of cis- or trans-acting lncrnas and their targets. finally, blast searches within plant species indicate that lncrnas are not well conserved.  our study identifies a large number of tissue-specifically expressed lncrnas in f. vesca, thereby highlighting their potential contributions to strawberry flower and fruit development and paving the way for future functional studies. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords lncrnastrawberryflowerfruitrna-seqfragaria vescarosaceaeissue-copyright-statement© the author 2015 