BACKGROUND
in recent years, small non coding rnas  have emerged as key regulators of gene expression in both plants and animals  <cit> .
in plants, there are many classes of small ncrnas and the two major classes that have been well-studied and defined are mirnas and sirnas  <cit> .
although both are functionally similar, they have different mode of biogenesis.
sirnas, 20– <dig> nt long, are processed from perfect double stranded rna.
they are mainly involved in heterochromatin modification, maintaining genome integrity by silencing of transgenes and transposons, defence against viruses and regulation of gene expression  <cit> .
there are diverse sirna species depending on the dicer-like  proteins involved in the production, such as natural antisense transcript-derived sirnas , heterochromatic sirnas  and trans-acting sirnas   <cit> .
mirnas are small, endogenous, single-stranded and ~ <dig> nt long rnas that regulate gene expression at the post-transcriptional level by degrading or repressing translation of targeted mrnas, depending on the extent of complementarity between mirna and mrna  <cit> .
mirnas are transcribed by rna polymerase ii into an imperfect long folded back structure, called as primary mirna .
the pri-mirna is cleaved by dicer-like  <dig>  protein , in association with other protein factors , into a precursor sequence with hairpin structure , which is further processed by dcl <dig> into a mirna:mirna* duplex  <cit> .
one of the strands of this duplex  gets incorporated into the silencing complex or effector complex  containing argonaute proteins, while the other strand  gets degraded.
within the complex, mirna guides the target mrna cleavage or inhibits translation with the help of one of the argonaute proteins   <cit> .
there is increasing evidence which indicates that mirnas play vital roles in stress responses and various developmental processes, including shoot apical meristem formation, leaf morphogenesis and polarity, floral organ identity, root development, vegetative phase change and vascular development  <cit> .
a large number of mirnas are evolutionary conserved among diverse species within the plant kingdom while several mirnas, that are considered to be recently evolved show species-specificity and often express at lower levels relative to conserved mirnas  <cit> .
due to their low expression levels, most of the species-specific mirnas remained unidentified in many plant species.
however, in recent years, with the advent of high-throughput sequencing technology, both species-specific and conserved mirnas have been identified in diverse plant species  <cit> .
potato  is the fourth most important crop worldwide, after rice, wheat and barley.
the main reasons for the increasing popularity of potato are high nutritional value combined with the simplicity of its propagation by vegetative reproduction.
potato tubers are derived from the swollen tips of specialized basal lateral juvenile shoots, called stolons.
tuber development is a highly systematic and well-coordinated morpho-physiological process that involves interactions between several environmental, biochemical and genetic factors  <cit> .
since control of tuber initiation impacts many commercial aspects of crop improvement, including scheduling, size grade distribution, yield and expansion of climate zone suitable for propagation, this is a high priority area for potato research.
several transcription factors, hormones, environmental and molecular signals have been studied that regulate tuberization  <cit> .
although, the potato genome sequencing project  <cit>  has revealed that both gene family expansion and recruitment of existing genes for new pathways have contributed to the evolution of tuber development in potato, this phenomena still remains a challenge for plant biologists.
to date, only mir <dig> and mir <dig> have been shown to play a role in tuberization process  <cit>  and only a few mirnas have been shown to regulate defense genes in potato  <cit> .
since the potato genome has been completely sequenced recently  <cit> , it will greatly help in identifying the entire population of small rnas and their target genes that might be involved in various developmental processes in potato.
in previous studies, through an in silico approach, many conserved mirnas were identified in potato based on mirbase repository by using potato est, gss and nr  databases   <cit> .
earlier, large scale sequencing studies were limited to the identification of only conserved mirnas in potato  <cit> .
a recent study reported  <dig> conserved and  <dig> potato-specific mirna families in potato by deep sequencing of srna libraries from leaf and stolon tissues  <cit> .
however, knowledge about the potato-specific mirna population in different vegetative tissues and developmental stages of tuberization remain largely unknown.
in this study, we report a population of potato mirnas from vegetative organs  and four early development stages of tuberization that might be involved in regulating developmental processes, including tuberization.
by taking advantage of the recently sequenced potato genome  <cit> ,  <dig> conserved mirna families and  <dig> potato-specific mirna were identified using high-throughput sequencing and bioinformatics pipeline.
we also report the expression profiles of both conserved and potato-specific mirnas in different tissues  and different developmental stages of tuberization based on digital and qrt-pcr expression analyses.
additionally, the putative target genes for the conserved and potato-specific mirnas were predicted and 5′ rlm-race was carried out to map the cleavage site of four conserved mirnas targets.
go analysis of potato-specific mirnas was also performed to gain a better understanding of their functions in various developmental processes in potato.
RESULTS
analysis of small rna population
to identify mirnas in potato , seven small rna libraries from leaf, stem, root and four early development stages of tuberization  were constructed and sequenced independently.
small rna sequencing results yielded a total of  <dig> million raw reads from these seven libraries.
the raw sequences were processed and filtered through several criteria to identify conserved and potato-specific mirnas.
after removing adaptor sequences, sequences smaller than  <dig> nt and larger than  <dig> nt, poor quality sequences and invalid sequences, a total of approximately  <dig> million raw reads, corresponding to approximately  <dig> million unique sequences, were obtained .
high quality trimmed sequences were further subjected to removal of non coding rnas such as transfer or ribosomal rrnas .
the remaining sequences represented the small rna population, which accounted for 83% of reads , suggesting a useful set of srnas with reasonable sequencing depth.
the size distribution analysis of these small rna sequences showed that they exhibit similar pattern of distribution in length in all the libraries .
the length of the srnas in each library varied from  <dig> nt to  <dig> nt and the majority of reads were  <dig> to  <dig> nt in length, as expected for dcls cleaved products  <cit>  .
in all the small rna libraries the  <dig> nt long sequence size class was the most abundant, followed by  <dig> nt .
this result was consistent with previous studies in other species such as arabidopsis thaliana, tomato, pepper, cucumber, maize, peanut, pepper, citrus trifoliata and rice  <cit>  but in contrast to grapevine, wheat and chinese yew where  <dig> nt or  <dig> nt class predominated the srna transcriptome  <cit> .
analysis of the size distribution of unique sequences revealed that  <dig> nt length read was the most abundant, followed by  <dig> nt class .
thus, for the  <dig> nt size class, which is characteristic of an authentic mirna, the number of total reads was found to be more compared to total number of unique sequences.
the  <dig> nt size class might represent the degraded products of  <dig> nt small rnas.
the high abundance of  <dig> nt long small rnas might indicate the complexity of the potato genome as they are mainly sirnas that are associated with repeats and heterochromatic modifications  <cit> .
these results suggest the existence of complex and diverse srna population in potato.
note: a -  =  <dig>  e -  =  <dig>
identification of conserved and potato-specific mirnas in potato
in order to identify mirnas, all unique reads obtained after filtering were submitted to the uea srna toolkit-plant version mircat pipeline   <cit>  and were mapped onto the potato reference genome   with no mismatch.
the  <dig> nt flanking regions of srna loci were excised from the genome.
the secondary structures of these extracted regions were predicted using rnafold   <cit>  to identify the potential precursor sequences.
the resulting secondary structures were then trimmed and analysed by mircat  <cit> .
to recover the putative mirna candidate, the potential precursors were screened based on the plant mirnas prediction criterion as described by meyers et al.
<cit> .
to identify the conserved mirnas in potato, all the predicted mirna sequences were mapped against the known plant mirnas, deposited in mirbase v <dig>  <cit> , allowing up to  <dig> mismatches during the alignment.
only those sequences that matched to known plant mirnas with 0– <dig> mismatches were considered as conserved mirnas.
the remaining sequences that had  <dig> or more than  <dig> mismatches or showed no homology to any previously known plant mirnas were regarded as novel mirnas or potato-specific mirnas.
the predicted pre-mirnas loci were explored further to find out their distribution among the intergenic and genic regions of the genome.
it was found that  <dig> pre-mirnas were encoded by intergenic regions and only  <dig> were encoded by genic regions .
in the genic regions,  <dig> of the pre-mirnas were located in introns,  <dig> in exon regions and  <dig> in both intron and exon regions .
these results suggests that majority of the pre-mirnas in potato are transcribed from non-coding part of the genome.
digital expression profiling of predicted mirnas
the read abundance of predicted mirnas in different libraries was normalized and expressed as transcripts per million  to measure the expression levels of these mirnas in different samples .
conserved mirnas
a total of  <dig> mirnas, belonging to  <dig> families, were identified as conserved mirnas in potato since these were also detected in other plant species.
star sequences were detected for  <dig> conserved mirnas .
the star sequences for the remaining conserved mirnas were not detected which could be either due to their low expression count in the sequencing dataset or poor stability.
a significant difference in the number of members was detected for each conserved mirna family .
the largest families identified were mir <dig> and mir <dig> with  <dig> members each.
the second largest families, with  <dig> members each, were mir <dig>  mir <dig> and mir <dig>  of the remaining families,  <dig> comprised of 2– <dig> members while  <dig> were represented by a single member .
a considerable variation in expression levels was found among mirna families.
of the  <dig> conserved mirna families, mir <dig>  mir <dig>  mir <dig> and mir <dig> showed high abundance, similar to those observed in other species  <cit> , each with total tpm > <dig> , <dig> .
among these, mir <dig> was the most abundantly expressed mirna family .
while mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir <dig> were found to be least abundant, each with total tpm less than  <dig> and amongst these poorly expressed mirnas, mir <dig> had lowest read number .
it was also observed that different members of a given family showed variations in their expression levels which suggest functional divergence within the family .
for example, the sequencing frequency of the mir <dig> family varied from  <dig> – <dig> tpm.
similar variations in read abundance were also observed among members of the mirna families such as mir <dig> , mir <dig> , mir <dig>  and mir <dig> .
most of the mirnas identified in potato were also found across various plant species, which suggests their highly conserved function.
potato-specific mirnas
a total of  <dig> novel mirna candidates were identified in potato that met our criteria and had no matches to any previously known plant mirnas.
these were therefore termed as potato-specific mirnas and further categorised into  high-confidence potato-specific mirnas - that had their star sequences detected in our sequencing dataset  and  candidate potato-specific mirnas - for which star sequences were not detected .
of these mirnas,  <dig> belonged to category i while the remaining  <dig> sequences belonged to category ii.
out of the  <dig> category ii sequences,  <dig> candidate mirnas were sequenced with less than  <dig> reads and thus excluded from the analysis resulting in  <dig> candidate potato-specific mirnas.
as the detection of star sequence is considered to increase the authenticity of predicted mirnas  <cit> , potato-specific mirnas with star sequences  were selected for further analysis and termed as potato-specific mirnas in this study.
the stem loop structure of precursors of all potato-specific mirnas were predicted using rnafold  <cit> , and are provided in additional file  <dig>  figure  <dig> shows the secondary structure of precursors of a few potato-specific mirnas, predicted using mfold web server  <cit> .
the predicted hairpin length of these precursors were found to be in the range of 64– <dig> nt, similar to that observed in other plant species  <cit>  .
the adjusted minimum free energy  of mirna precursors ranged from - <dig>  to - <dig>  kcal mol- <dig>  with an average of about -54 kcal mol- <dig>  which was in accordance with the free energy values reported for mirna precursors in other plant species   <cit>  .
this indicates a high stability of the hairpin structures.
thus, the pre-mirnas had mfe much lower than the free folding energies of transfer rna  and ribosomal rna   <cit> .
the sequencing frequencies of a majority of potato-specific mirnas were found to be low, consistent with the hypothesis that species-specific mirnas are often expressed at a lower level as compared to conserved mirnas  <cit> .
most of these mirnas  were detected with total tpm less than  <dig> .
however, a few mirnas  were found to have total tpm more than  <dig>  among all the potato-specific mirnas, mirna <dig> and mirna <dig> were the most abundant, with total tpm more than  <dig> .
some predicted conserved and potato-specific mirnas displayed significant variation in expression level among the different tissues .
for example, conserved mirna - mir <dig> exhibited high expression during tuberization stages, with highest level at pt <dig> .
among potato-specific mirnas, mirna <dig> had highest sequencing frequencies in stem and displayed significant change in expression level during tuberization stages  .
these findings suggest the probable role of these regulatory mirnas in specific tissues or during specific developmental stages in potato.
in this study, biological replicates were not used for sequencing.
hence, the digital expression data only gives an indication of variation in expression level between different tissues.
this could be further confirmed by qrt-pcr as has been done for some of the mirnas in this study .
expression profile of conserved and potato-specific mirnas in different tissues by qrt-pcr
quantitative real time pcr was performed to determine the expression levels of conserved and potato-specific mirnas in different vegetative tissues  and four early developmental stages of tuberization  to validate results obtained through digital expression profiling of potato mirnas .
in this study, based on the tpm values, we selected  <dig> conserved mirnas and  <dig> potato-specific mirnas .
the expression level of each mirna in pt <dig> was set as control, taken as  <dig> and expression level in all other tissues was quantified relative to it.
the expression profiles of conserved and potato-specific mirnas have been shown in figure 6a and figure 6b, respectively.
expression profile of mirnas in vegetative tissues
among conserved mirnas, the expression of mir164_ <dig> and mirna171_ <dig> appeared to be restricted to stem and root, respectively while mir8044_ <dig> showed abundant expression in all the examined vegetative tissues.
mir394_ <dig> exhibited the highest expression in stem and moderate expression in leaf.
mir171_ <dig> and mir157_ <dig> showed low expression in root whereas relatively high expression was observed in leaf and stem.
the expression of mir396_ <dig> and mir172_ <dig> was found to be most abundant in stem and moderate in root whereas the expression of mir399_ <dig> was relatively high in root.
mir172_ <dig> had high expression level in root, low expression in stem and could rarely be detected in leaf while mir159_ <dig> demonstrated highest expression in stem but low expression in leaf and root.
the expression of mir6149_ <dig> and mir482_ <dig> was higher in leaf, stem and lower in root.
mir171_ <dig> showed strong expression in stem and root but weak expression in leaf and mir398_ <dig> was found to be expressed predominantly in stem, followed by leaf and root.
among potato-specific mirnas, mirna <dig> had higher level of expression in leaf and stem, and moderate level in root.
mirna <dig> appeared to be highly expressed in leaf and weakly in stem and root.
the expression pattern of mirna <dig>  mirna <dig>  mirna <dig> were found to be very similar - high expression in stem, followed by root and leaf while mirna <dig> exhibited moderate level of expression in root and low expression in leaf, stem.
expression profile of mirnas during tuber developmental stages
the expression of mir164_ <dig>  mir399_ <dig>  mir157_ <dig>  mir398_ <dig>  mir171_ <dig>  mir171_ <dig>  and mirna <dig>  could barely be detected during tuberization stages.
among potato-specific mirnas, mirna <dig>  mirna <dig> and among conserved mirnas, mir171_ <dig>  mir396_ <dig>  mir159_ <dig>  mir6149_ <dig>  mir8044_ <dig> and mir482_ <dig> had very similar pattern of expression during different tuberization stages.
they showed a slight increase in expression at pt <dig> stage and then a gradual decrease during later stages whereas mirna <dig> and mir394_ <dig> was found to be expressed at low level in all the stages with no variation in expression levels.
mir172_ <dig> showed moderate level of expression during tuberization - similar expression level at pt <dig> and pt <dig> stage, then a slight decrease in abundance at pt <dig> stage.
the expression of mirna <dig>  mirna <dig>  and mir172_ <dig> was relatively high during tuberization.
mirna <dig> and mir172_ <dig> showed a gradual increase in abundance from pt1–pt <dig>  reaching the maximum expression at pt <dig>  then a decrease thereafter whereas mirna <dig> showed similar level of expression during all the stages.
it was found that digital expression data and qrt-pcr expression profiles of mirnas in different tissues and developmental stages of tuberization do not correlate with each other.
the probable reason for this variation could be the biasness of rna ligase activity during small rna library preparation  <cit> .
target prediction for conserved and potato-specific mirnas
in order to explore the functional role of all the identified mirnas in diverse biological processes, their putative target genes were predicted using the open source web server psrnatarget  <cit>  with default parameters .
the transcript sequences of the potato genome  were used as a reference set.
additional file  <dig> and additional file  <dig> provide detailed information regarding the predicted targets for conserved and potato-specific mirnas respectively.
a majority of the mirnas were found to target more than one transcript.
among conserved mirna targets, most of them were found to be transcripts coding for transcription factors , such as squamosa promoter-binding protein , gras family transcription factors , gamyb-like <dig> , apetala <dig> , nac domain containing protein , auxin response factors , phavoluta-like hd-zipiii protein  and nuclear transcription factors - ya <dig>  ya <dig>  ya <dig> .
in addition to transcription factors, other targets included mrna coding for f-box family protein , disease resistance protein and fiber expressed protein , laccase , salt tolerance protein , udp-glucoronate decarboxylase  <dig> , dna binding protein , nl <dig> disease resistance protein , protein phosphatase and kinase , ago1- <dig> , galactose oxidase  and proteins with unknown functions .
most of the conserved mirna targeted plant transcription factors  were found to be similar to the conserved mirna targets predicted in arabidopsis and other plants  <cit> , underlining the role of conserved mirnas in essential biological processes.
for potato-specific mirnas, targets were predicted successfully for  <dig> out of  <dig> mirnas.
target genes were not found for the remaining five mirnas which could be due to stringent cut-off used in our prediction analysis to reduce the number of false positives and to obtain reliable results.
their targets included mrna coding for tropinone reductase i, cdk10/ <dig>  endo- <dig> -beta-d-glucanase, xh/xs domain containing protein, ankyrin repeat containing protein, cytochrome p <dig>  alpha amylase, glucosyltransferase and others .
a few potato-specific mirnas showed lipoxygenase and protein phosphatase type 2a, that are known to be involved in potato tuberization  <cit> , as their predictive targets .
in addition, some potato-specific mirnas were also found to target transcription factors including nac domain protein, gras family transcription factors, mads-box family protein and arf domain class transcription factor .
these transcription factors have been known to be involved in regulating developmental processes in plants  <cit> .
these results imply that these potato-specific mirnas might be involved in some specific developmental as well as essential biological processes in potato.
many of the potato-specific mirna target transcripts that code for proteins of unknown function.
further, all the predicted targets of potato-specific mirnas, identified in this study, were subjected to go analysis using blast2go  <cit>  to gain a better understanding of their functions.
go terms were categorized into biological processes, molecular functions and cellular component.
the go results revealed that within the cellular component category, most of the genes are localized in membrane and nucleus .
in the category of molecular functions, majority of targets are involved in binding activities .
with respect to biological processes, most of the genes participate in oxidation-reduction processes, metabolic processes, regulation of transcription, transport, signal transduction and defense responses .
detailed information of go annotation is provided in additional file  <dig>
experimental validation of conserved mirna targets
cleavage site of predicted targets of four conserved mirnas in potato were further verified by 5′ rlm-race.
the predicted targets of mir <dig>  mir <dig>  mir <dig> and mir <dig> were arf <dig>  nam, rap <dig> and ham, respectively.
the cleavage site was found to be between 10– <dig> bases, from the 5′ end of the mirna, for arf <dig> , nam  and rap <dig>  .
however, ham  was found to be cleaved between 10– <dig> and 13– <dig> bases from the 5′ end of the mirna binding site .
discussion
since the discovery of srnas  as regulators of gene expression in c. elegans <cit> , there has been intense interest in the srna biology in eukaryotic organisms.
in plants, extensive studies in the past several years have clearly shown that mirnas regulate gene expression at the post-transcriptional level by degrading or repressing translation of targeted mrnas and play important roles during plant development and growth  <cit> .
here, we report identification and characterization of a population of both conserved and potato-specific mirnas from different vegetative tissues and various well-defined stages of tuberization.
also, we discuss the potential role of these validated mirnas in various developmental processes in potato.
in this study, with the application of high-throughput sequencing technology, we identified  <dig> conserved mirna ,  <dig> potato-specific mirnas and  <dig> candidate potato-specific mirnas, based on the sequenced potato genome dataset  <cit> .
consistent with previous reports  <cit> , a majority of the potato-specific mirnas predicted in our analysis, showed low sequencing frequencies as compared to conserved mirnas in the dataset.
the majority of the pre-mirnas were found to be encoded by intergenic regions which is also consistent with earlier findings  <cit> .
the analysis of srna population in potato revealed that  <dig> nt long srnas  dominated the srna transcriptome, in both total reads and unique sequences, as observed in many other plant species  <cit> .
the high percentage of  <dig> nt srnas suggests an important role of this class in maintaining genome integrity by heterochromatic-histone modification  <cit> .
the high ratio of total reads/unique sequences for  <dig> nt length class indicated the high abundance of this size class.
the majority of the identified mirnas were  <dig> nt in length, which is the canonical size for mirnas generated from dcl <dig> processing.
only a few mirnas showed minor variations in size  which could be due to inaccuracy of dcl <dig> processing  <cit> .
previously, many conserved mirnas were reported in potato by a computational approach using available est, gss and nr databases  <cit> .
a recent study by zhang et al.
has led to the identification of  <dig> potato-specific mirna families and  <dig> conserved mirna families in leaf and stolon tissues by high-throughput sequencing  <cit> .
however, majority of potato-specific mirnas predicted by zhang et al.
were also detected in our sequencing dataset but could not meet the criteria for mirna annotation employed in our analysis and thus excluded from further analysis.
to observe the expression profile of identified mirnas, qrt-pcr was performed for  <dig> conserved and  <dig> potato-specific mirnas.
our qrt-pcr results showed that majority of potato-specific  and conserved mirnas  expressed at relatively high level in the stem tissues.
this indicates that either these mirnas express highly in the stem tissues or most of these mirnas are being continuously transported through the conductive vascular tissues.
in case of their high expression in stem it is possible that they might play an important role in stem development and differentiation.
on the other hand, potato mir <dig> and other mirnas  have been shown to be involved in graft transmissible movement through the conductive vascular tissues  <cit> .
interestingly, potato-specific mirnas mirna <dig>  mirna <dig> and the conserved mir172_ <dig>  mir172_ <dig> showed significant expression during developmental stages of tuberization.
this indicates a probable role for these mirnas in potato tuberization.
in our analysis, the expression profile of mir <dig>  during developmental stages of tuberization was consistent with the previous report  <cit> .
also, we found that mir172_ <dig> expressed at a high level in potato roots, which was not reported previously.
to investigate the functional importance of the identified mirnas, targets were predicted using psrna target finder server  <cit>  and the annotated potato genome transcript data  <cit> .
among conserved mirnas, mir <dig> was predicted to target mrna encoding for nac domain containing proteins.
it has been also reported that mir <dig> is a key regulator in diverse developmental processes including floral, embryonic, vegetative organs and lateral root development  <cit> .
phavoulta like hd-zip iii transcription factor was one of the predicted targets of mir <dig> in our study.
previous studies showed that mir166/mir <dig> targets hd-zip iii transcription factor genes and regulate a wide range of developmental processes including meristem formation, vascular development, lateral organ polarity, root and nodule development  <cit> .
in our analysis, mir <dig> and mir <dig> were predicted to target mrna coding for gamyb like transcription factor.
gamyb transcription factors are involved in ga signalling and it has been proven that mir159-mediated regulation of gamyb affects plant anther development and flowering time in arabidopsis  <cit> .
auxin response factor was found as a predicted target of mir <dig>  it has been observed that mir <dig> targets arf <dig>  arf <dig> and arf <dig> and regulate various aspects of plant development in arabidopsis  <cit> .
squamosa-promoter binding protein was predicted as the target of mir <dig> and mir <dig>  previous studies have demonstrated that mir <dig> plays a vital role in controlling leaf development, apical dominance, floral transition and development  <cit>  by targeting several members of the squamosa promoter binding protein like  family of plant-specific transcription factors.
interestingly, a recent study has shown that overexpression of mir <dig> in potato leads to development of stolon-borne aerial tubers from distal nodes  <cit> .
the digital expression profile reported in this study also showed high abundance of mir <dig> in tuber developmental stages with highest level at pt <dig> , suggesting a role of mir <dig> in the phase transition from basal lateral juvenile shoots to a storage organ in the process of tuber development.
in our analysis, mir <dig> was found to target gras family transcription factors and hairy meristem .
they have been shown to be involved in regulating various aspects of plant growth and development  <cit> .
in potato, our qrt-pcr data showed that all the three members of mir <dig> expressed highly in vegetative tissues , indicating a role of mir <dig> in regulating potato developmental process.
previous studies have indicated that mir <dig> largely targets gene encoding members of apetala <dig>  and ap2-like transcription factor family and plays a major role in the regulation of flowering time, vegetative phase change and floral organ identity, by cleaving and repressing the translation of its target mrnas  <cit> .
recently, mir <dig> has been shown to induce tuberization by regulating long distance signals and targeting rap <dig> mrna   <cit> .
our prediction analysis showed rap <dig>  which is a known target of mir <dig> in potato  <cit> , as a predicted target of mir <dig>  this data supports the efficacy of our target prediction analysis.
further, mirna-mediated cleavage of target of conserved mirnas was confirmed by 5′ rlm-race.
several targets of conserved mirnas have been validated using race analysis in tomato, pepper and rice  <cit> .
however, this is the first study in potato where 5′ rlm-race was performed to authenticate predicted targets of conserved mirnas.
our race results confirmed that transcripts of afr <dig>  nam, rap <dig> and ham were cleaved in vivo by mir <dig>  mir <dig>  mir <dig> and mir <dig>  respectively.
for potato-specific mirnas, degradome sequencing approach is being used for mapping of the cleavage site.
our target prediction analysis in potato and other studies on plant species, especially rice and arabidopsis, strongly suggest that most of the conserved mirnas target plant transcription factors   <cit> , thereby emphasizing the role of conserved mirna in essential biological processes in plants, including potato tuberization.
go was performed on potato-specific mirnas targets to gain a better understanding of their possible function  <cit> .
go results revealed that majority of the targets are involved in oxidation-reduction processes, metabolic processes, regulation of transcription, transport, signal transduction and defense responses.
these results suggest that they covered a broad range of physiological and metabolic processes.
thus, in contrast to conserved mirna targets, most of the putative targets of potato-specific mirnas were not transcription factors.
a few potato-specific mirnas were predicted to target transcripts encoding transcription factors, including nac domain protein, gras family transcription factors, mads-box family protein and arf domain class transcription factor.
these transcription factors are known to be targeted by conserved mirnas and play a major role in various aspects of plant growth and development  <cit> .
thus, it appears that the potato-specific mirnas might play important roles in essential biological processes as well.
future studies are essential to explain the functional significance of the inverse relationship between mirna and their target which will improve our knowledge about the regulatory role of mirnas in potato.
with respect to potato tuberization, expression profiling by qrt-pcr and target prediction studies indicated that a few mirnas might play a role during transition from stolon to tuber development.
qrt-pcr results indicated that mir172_ <dig>  mir172_ <dig>  and mirna <dig>  mirna <dig>  showed significant expression during developmental stages of tuberization.
lipoxygenase  was predicted as one of the targets of mirna <dig>  mirna <dig>  mirna <dig>  mirna <dig> and mirna <dig>  protein phosphatase type 2a  was found to be a predicted target of mirna <dig>  previous studies have suggested the role of mir <dig>  lox and pp2a in potato tuber developmental process  <cit> .
therefore, these potential mirnas and their target genes are being further investigated to determine their roles in potato tuber development.
CONCLUSIONS
in summary, we report a comprehensive study of potato mirnas at genome-wide level using three different vegetative tissues and four early developmental stages of tuberization by high-throughput sequencing and high-end bioinformatics methods.
we have identified a population of potato-specific mirnas that could fill the gap in understanding the regulatory role of mirnas in potato.
the digital expression data was further quantified by qrt-pcr to determine up- and down-regulations of potato mirnas in various vegetative tissues and developmental stages of tuberization.
the putative target genes were predicted for conserved and potato-specific mirnas to gain a better understanding of their function.
further, a few conserved mirna targets were also validated by 5′ rlm-race and the predicted target genes for potato-specific mirnas were subjected to go annotation to infer the functional role of target genes.

 in recent years, small non coding rnas have emerged as key regulators of gene expression in both plants and animals. in plants, there are many classes of small ncrnas and the two major classes that have been well-studied and defined are mirnas and sirnas. although both are functionally similar, they have different mode of biogenesis. sirnas, 20– nt long, are processed from perfect double stranded rna. they are mainly involved in heterochromatin modification, maintaining genome integrity by silencing of transgenes and transposons, defence against viruses and regulation of gene expression. there are diverse sirna species depending on the dicer-like proteins involved in the production, such as natural antisense transcript-derived sirnas, heterochromatic sirnas and trans-acting sirnas. mirnas are small, endogenous, single-stranded and ~ nt long rnas that regulate gene expression at the post-transcriptional level by degrading or repressing translation of targeted mrnas, depending on the extent of complementarity between mirna and mrna. mirnas are transcribed by rna polymerase ii into an imperfect long folded back structure, called as primary mirna. the pri-mirna is cleaved by dicer-like protein, in association with other protein factors, into a precursor sequence with hairpin structure, which is further processed by dcl into a mirna:mirna* duplex. one of the strands of this duplex gets incorporated into the silencing complex or effector complex containing argonaute proteins, while the other strand gets degraded. within the complex, mirna guides the target mrna cleavage or inhibits translation with the help of one of the argonaute proteins. there is increasing evidence which indicates that mirnas play vital roles in stress responses and various developmental processes, including shoot apical meristem formation, leaf morphogenesis and polarity, floral organ identity, root development, vegetative phase change and vascular development. a large number of mirnas are evolutionary conserved among diverse species within the plant kingdom while several mirnas, that are considered to be recently evolved show species-specificity and often express at lower levels relative to conserved mirnas. due to their low expression levels, most of the species-specific mirnas remained unidentified in many plant species. however, in recent years, with the advent of high-throughput sequencing technology, both species-specific and conserved mirnas have been identified in diverse plant species. potato is the fourth most important crop worldwide, after rice, wheat and barley. the main reasons for the increasing popularity of potato are high nutritional value combined with the simplicity of its propagation by vegetative reproduction. potato tubers are derived from the swollen tips of specialized basal lateral juvenile shoots, called stolons. tuber development is a highly systematic and well-coordinated morpho-physiological process that involves interactions between several environmental, biochemical and genetic factors. since control of tuber initiation impacts many commercial aspects of crop improvement, including scheduling, size grade distribution, yield and expansion of climate zone suitable for propagation, this is a high priority area for potato research. several transcription factors, hormones, environmental and molecular signals have been studied that regulate tuberization. although, the potato genome sequencing project has revealed that both gene family expansion and recruitment of existing genes for new pathways have contributed to the evolution of tuber development in potato, this phenomena still remains a challenge for plant biologists. to date, only mir and mir have been shown to play a role in tuberization process and only a few mirnas have been shown to regulate defense genes in potato. since the potato genome has been completely sequenced recently, it will greatly help in identifying the entire population of small rnas and their target genes that might be involved in various developmental processes in potato. in previous studies, through an in silico approach, many conserved mirnas were identified in potato based on mirbase repository by using potato est, gss and nr databases. earlier, large scale sequencing studies were limited to the identification of only conserved mirnas in potato. a recent study reported conserved and potato-specific mirna families in potato by deep sequencing of srna libraries from leaf and stolon tissues. however, knowledge about the potato-specific mirna population in different vegetative tissues and developmental stages of tuberization remain largely unknown. in this study, we report a population of potato mirnas from vegetative organs and four early development stages of tuberization that might be involved in regulating developmental processes, including tuberization. by taking advantage of the recently sequenced potato genome, conserved mirna families and potato-specific mirna were identified using high-throughput sequencing and bioinformatics pipeline. we also report the expression profiles of both conserved and potato-specific mirnas in different tissues and different developmental stages of tuberization based on digital and qrt-pcr expression analyses. additionally, the putative target genes for the conserved and potato-specific mirnas were predicted and 5′ rlm-race was carried out to map the cleavage site of four conserved mirnas targets. go analysis of potato-specific mirnas was also performed to gain a better understanding of their functions in various developmental processes in potato.  analysis of small rna population to identify mirnas in potato, seven small rna libraries from leaf, stem, root and four early development stages of tuberization were constructed and sequenced independently. small rna sequencing results yielded a total of million raw reads from these seven libraries. the raw sequences were processed and filtered through several criteria to identify conserved and potato-specific mirnas. after removing adaptor sequences, sequences smaller than nt and larger than nt, poor quality sequences and invalid sequences, a total of approximately million raw reads, corresponding to approximately million unique sequences, were obtained. high quality trimmed sequences were further subjected to removal of non coding rnas such as transfer or ribosomal rrnas. the remaining sequences represented the small rna population, which accounted for 83% of reads, suggesting a useful set of srnas with reasonable sequencing depth. the size distribution analysis of these small rna sequences showed that they exhibit similar pattern of distribution in length in all the libraries. the length of the srnas in each library varied from nt to nt and the majority of reads were to nt in length, as expected for dcls cleaved products. in all the small rna libraries the nt long sequence size class was the most abundant, followed by nt. this result was consistent with previous studies in other species such as arabidopsis thaliana, tomato, pepper, cucumber, maize, peanut, pepper, citrus trifoliata and rice but in contrast to grapevine, wheat and chinese yew where nt or nt class predominated the srna transcriptome. analysis of the size distribution of unique sequences revealed that nt length read was the most abundant, followed by nt class. thus, for the nt size class, which is characteristic of an authentic mirna, the number of total reads was found to be more compared to total number of unique sequences. the nt size class might represent the degraded products of nt small rnas. the high abundance of nt long small rnas might indicate the complexity of the potato genome as they are mainly sirnas that are associated with repeats and heterochromatic modifications. these results suggest the existence of complex and diverse srna population in potato. note: a -  =  e -  =  identification of conserved and potato-specific mirnas in potato in order to identify mirnas, all unique reads obtained after filtering were submitted to the uea srna toolkit-plant version mircat pipeline and were mapped onto the potato reference genome with no mismatch. the nt flanking regions of srna loci were excised from the genome. the secondary structures of these extracted regions were predicted using rnafold to identify the potential precursor sequences. the resulting secondary structures were then trimmed and analysed by mircat. to recover the putative mirna candidate, the potential precursors were screened based on the plant mirnas prediction criterion as described by meyers et al.. to identify the conserved mirnas in potato, all the predicted mirna sequences were mapped against the known plant mirnas, deposited in mirbase v, allowing up to mismatches during the alignment. only those sequences that matched to known plant mirnas with 0– mismatches were considered as conserved mirnas. the remaining sequences that had or more than mismatches or showed no homology to any previously known plant mirnas were regarded as novel mirnas or potato-specific mirnas. the predicted pre-mirnas loci were explored further to find out their distribution among the intergenic and genic regions of the genome. it was found that pre-mirnas were encoded by intergenic regions and only were encoded by genic regions. in the genic regions, of the pre-mirnas were located in introns, in exon regions and in both intron and exon regions. these results suggests that majority of the pre-mirnas in potato are transcribed from non-coding part of the genome. digital expression profiling of predicted mirnas the read abundance of predicted mirnas in different libraries was normalized and expressed as transcripts per million to measure the expression levels of these mirnas in different samples. conserved mirnas a total of mirnas, belonging to families, were identified as conserved mirnas in potato since these were also detected in other plant species. star sequences were detected for conserved mirnas. the star sequences for the remaining conserved mirnas were not detected which could be either due to their low expression count in the sequencing dataset or poor stability. a significant difference in the number of members was detected for each conserved mirna family. the largest families identified were mir and mir with members each. the second largest families, with members each, were mir mir and mir of the remaining families, comprised of 2– members while were represented by a single member. a considerable variation in expression levels was found among mirna families. of the conserved mirna families, mir mir mir and mir showed high abundance, similar to those observed in other species, each with total tpm >,. among these, mir was the most abundantly expressed mirna family. while mir mir mir mir and mir were found to be least abundant, each with total tpm less than and amongst these poorly expressed mirnas, mir had lowest read number. it was also observed that different members of a given family showed variations in their expression levels which suggest functional divergence within the family. for example, the sequencing frequency of the mir family varied from – tpm. similar variations in read abundance were also observed among members of the mirna families such as mir, mir, mir and mir. most of the mirnas identified in potato were also found across various plant species, which suggests their highly conserved function. potato-specific mirnas a total of novel mirna candidates were identified in potato that met our criteria and had no matches to any previously known plant mirnas. these were therefore termed as potato-specific mirnas and further categorised into high-confidence potato-specific mirnas - that had their star sequences detected in our sequencing dataset and candidate potato-specific mirnas - for which star sequences were not detected. of these mirnas, belonged to category i while the remaining sequences belonged to category ii. out of the category ii sequences, candidate mirnas were sequenced with less than reads and thus excluded from the analysis resulting in candidate potato-specific mirnas. as the detection of star sequence is considered to increase the authenticity of predicted mirnas, potato-specific mirnas with star sequences were selected for further analysis and termed as potato-specific mirnas in this study. the stem loop structure of precursors of all potato-specific mirnas were predicted using rnafold, and are provided in additional file figure  shows the secondary structure of precursors of a few potato-specific mirnas, predicted using mfold web server. the predicted hairpin length of these precursors were found to be in the range of 64– nt, similar to that observed in other plant species. the adjusted minimum free energy of mirna precursors ranged from - to -  kcal mol- with an average of about -54 kcal mol- which was in accordance with the free energy values reported for mirna precursors in other plant species. this indicates a high stability of the hairpin structures. thus, the pre-mirnas had mfe much lower than the free folding energies of transfer rna and ribosomal rna. the sequencing frequencies of a majority of potato-specific mirnas were found to be low, consistent with the hypothesis that species-specific mirnas are often expressed at a lower level as compared to conserved mirnas. most of these mirnas were detected with total tpm less than. however, a few mirnas were found to have total tpm more than among all the potato-specific mirnas, mirna and mirna were the most abundant, with total tpm more than. some predicted conserved and potato-specific mirnas displayed significant variation in expression level among the different tissues. for example, conserved mirna - mir exhibited high expression during tuberization stages, with highest level at pt. among potato-specific mirnas, mirna had highest sequencing frequencies in stem and displayed significant change in expression level during tuberization stages. these findings suggest the probable role of these regulatory mirnas in specific tissues or during specific developmental stages in potato. in this study, biological replicates were not used for sequencing. hence, the digital expression data only gives an indication of variation in expression level between different tissues. this could be further confirmed by qrt-pcr as has been done for some of the mirnas in this study. expression profile of conserved and potato-specific mirnas in different tissues by qrt-pcr quantitative real time pcr was performed to determine the expression levels of conserved and potato-specific mirnas in different vegetative tissues and four early developmental stages of tuberization to validate results obtained through digital expression profiling of potato mirnas. in this study, based on the tpm values, we selected conserved mirnas and potato-specific mirnas. the expression level of each mirna in pt was set as control, taken as and expression level in all other tissues was quantified relative to it. the expression profiles of conserved and potato-specific mirnas have been shown in figure 6a and figure 6b, respectively. expression profile of mirnas in vegetative tissues among conserved mirnas, the expression of mir164_ and mirna171_ appeared to be restricted to stem and root, respectively while mir8044_ showed abundant expression in all the examined vegetative tissues. mir394_ exhibited the highest expression in stem and moderate expression in leaf. mir171_ and mir157_ showed low expression in root whereas relatively high expression was observed in leaf and stem. the expression of mir396_ and mir172_ was found to be most abundant in stem and moderate in root whereas the expression of mir399_ was relatively high in root. mir172_ had high expression level in root, low expression in stem and could rarely be detected in leaf while mir159_ demonstrated highest expression in stem but low expression in leaf and root. the expression of mir6149_ and mir482_ was higher in leaf, stem and lower in root. mir171_ showed strong expression in stem and root but weak expression in leaf and mir398_ was found to be expressed predominantly in stem, followed by leaf and root. among potato-specific mirnas, mirna had higher level of expression in leaf and stem, and moderate level in root. mirna appeared to be highly expressed in leaf and weakly in stem and root. the expression pattern of mirna mirna mirna were found to be very similar - high expression in stem, followed by root and leaf while mirna exhibited moderate level of expression in root and low expression in leaf, stem. expression profile of mirnas during tuber developmental stages the expression of mir164_ mir399_ mir157_ mir398_ mir171_ mir171_ and mirna could barely be detected during tuberization stages. among potato-specific mirnas, mirna mirna and among conserved mirnas, mir171_ mir396_ mir159_ mir6149_ mir8044_ and mir482_ had very similar pattern of expression during different tuberization stages. they showed a slight increase in expression at pt stage and then a gradual decrease during later stages whereas mirna and mir394_ was found to be expressed at low level in all the stages with no variation in expression levels. mir172_ showed moderate level of expression during tuberization - similar expression level at pt and pt stage, then a slight decrease in abundance at pt stage. the expression of mirna mirna and mir172_ was relatively high during tuberization. mirna and mir172_ showed a gradual increase in abundance from pt1–pt reaching the maximum expression at pt then a decrease thereafter whereas mirna showed similar level of expression during all the stages. it was found that digital expression data and qrt-pcr expression profiles of mirnas in different tissues and developmental stages of tuberization do not correlate with each other. the probable reason for this variation could be the biasness of rna ligase activity during small rna library preparation. target prediction for conserved and potato-specific mirnas in order to explore the functional role of all the identified mirnas in diverse biological processes, their putative target genes were predicted using the open source web server psrnatarget with default parameters. the transcript sequences of the potato genome were used as a reference set. additional file and additional file provide detailed information regarding the predicted targets for conserved and potato-specific mirnas respectively. a majority of the mirnas were found to target more than one transcript. among conserved mirna targets, most of them were found to be transcripts coding for transcription factors, such as squamosa promoter-binding protein, gras family transcription factors, gamyb-like, apetala, nac domain containing protein, auxin response factors, phavoluta-like hd-zipiii protein and nuclear transcription factors - ya ya ya. in addition to transcription factors, other targets included mrna coding for f-box family protein, disease resistance protein and fiber expressed protein, laccase, salt tolerance protein, udp-glucoronate decarboxylase, dna binding protein, nl disease resistance protein, protein phosphatase and kinase, ago1-, galactose oxidase and proteins with unknown functions. most of the conserved mirna targeted plant transcription factors were found to be similar to the conserved mirna targets predicted in arabidopsis and other plants, underlining the role of conserved mirnas in essential biological processes. for potato-specific mirnas, targets were predicted successfully for out of mirnas. target genes were not found for the remaining five mirnas which could be due to stringent cut-off used in our prediction analysis to reduce the number of false positives and to obtain reliable results. their targets included mrna coding for tropinone reductase i, cdk10/ endo- -beta-d-glucanase, xh/xs domain containing protein, ankyrin repeat containing protein, cytochrome p alpha amylase, glucosyltransferase and others. a few potato-specific mirnas showed lipoxygenase and protein phosphatase type 2a, that are known to be involved in potato tuberization, as their predictive targets. in addition, some potato-specific mirnas were also found to target transcription factors including nac domain protein, gras family transcription factors, mads-box family protein and arf domain class transcription factor. these transcription factors have been known to be involved in regulating developmental processes in plants. these results imply that these potato-specific mirnas might be involved in some specific developmental as well as essential biological processes in potato. many of the potato-specific mirna target transcripts that code for proteins of unknown function. further, all the predicted targets of potato-specific mirnas, identified in this study, were subjected to go analysis using blast2go to gain a better understanding of their functions. go terms were categorized into biological processes, molecular functions and cellular component. the go results revealed that within the cellular component category, most of the genes are localized in membrane and nucleus. in the category of molecular functions, majority of targets are involved in binding activities. with respect to biological processes, most of the genes participate in oxidation-reduction processes, metabolic processes, regulation of transcription, transport, signal transduction and defense responses. detailed information of go annotation is provided in additional file experimental validation of conserved mirna targets cleavage site of predicted targets of four conserved mirnas in potato were further verified by 5′ rlm-race. the predicted targets of mir mir mir and mir were arf nam, rap and ham, respectively. the cleavage site was found to be between 10– bases, from the 5′ end of the mirna, for arf, nam and rap. however, ham was found to be cleaved between 10– and 13– bases from the 5′ end of the mirna binding site. discussion since the discovery of srnas as regulators of gene expression in c. elegans, there has been intense interest in the srna biology in eukaryotic organisms. in plants, extensive studies in the past several years have clearly shown that mirnas regulate gene expression at the post-transcriptional level by degrading or repressing translation of targeted mrnas and play important roles during plant development and growth. here, we report identification and characterization of a population of both conserved and potato-specific mirnas from different vegetative tissues and various well-defined stages of tuberization. also, we discuss the potential role of these validated mirnas in various developmental processes in potato. in this study, with the application of high-throughput sequencing technology, we identified conserved mirna, potato-specific mirnas and candidate potato-specific mirnas, based on the sequenced potato genome dataset. consistent with previous reports, a majority of the potato-specific mirnas predicted in our analysis, showed low sequencing frequencies as compared to conserved mirnas in the dataset. the majority of the pre-mirnas were found to be encoded by intergenic regions which is also consistent with earlier findings. the analysis of srna population in potato revealed that nt long srnas dominated the srna transcriptome, in both total reads and unique sequences, as observed in many other plant species. the high percentage of nt srnas suggests an important role of this class in maintaining genome integrity by heterochromatic-histone modification. the high ratio of total reads/unique sequences for nt length class indicated the high abundance of this size class. the majority of the identified mirnas were nt in length, which is the canonical size for mirnas generated from dcl processing. only a few mirnas showed minor variations in size which could be due to inaccuracy of dcl processing. previously, many conserved mirnas were reported in potato by a computational approach using available est, gss and nr databases. a recent study by zhang et al. has led to the identification of potato-specific mirna families and conserved mirna families in leaf and stolon tissues by high-throughput sequencing. however, majority of potato-specific mirnas predicted by zhang et al. were also detected in our sequencing dataset but could not meet the criteria for mirna annotation employed in our analysis and thus excluded from further analysis. to observe the expression profile of identified mirnas, qrt-pcr was performed for conserved and potato-specific mirnas. our qrt-pcr results showed that majority of potato-specific and conserved mirnas expressed at relatively high level in the stem tissues. this indicates that either these mirnas express highly in the stem tissues or most of these mirnas are being continuously transported through the conductive vascular tissues. in case of their high expression in stem it is possible that they might play an important role in stem development and differentiation. on the other hand, potato mir and other mirnas have been shown to be involved in graft transmissible movement through the conductive vascular tissues. interestingly, potato-specific mirnas mirna mirna and the conserved mir172_ mir172_ showed significant expression during developmental stages of tuberization. this indicates a probable role for these mirnas in potato tuberization. in our analysis, the expression profile of mir during developmental stages of tuberization was consistent with the previous report. also, we found that mir172_ expressed at a high level in potato roots, which was not reported previously. to investigate the functional importance of the identified mirnas, targets were predicted using psrna target finder server and the annotated potato genome transcript data. among conserved mirnas, mir was predicted to target mrna encoding for nac domain containing proteins. it has been also reported that mir is a key regulator in diverse developmental processes including floral, embryonic, vegetative organs and lateral root development. phavoulta like hd-zip iii transcription factor was one of the predicted targets of mir in our study. previous studies showed that mir166/mir targets hd-zip iii transcription factor genes and regulate a wide range of developmental processes including meristem formation, vascular development, lateral organ polarity, root and nodule development. in our analysis, mir and mir were predicted to target mrna coding for gamyb like transcription factor. gamyb transcription factors are involved in ga signalling and it has been proven that mir159-mediated regulation of gamyb affects plant anther development and flowering time in arabidopsis. auxin response factor was found as a predicted target of mir it has been observed that mir targets arf arf and arf and regulate various aspects of plant development in arabidopsis. squamosa-promoter binding protein was predicted as the target of mir and mir previous studies have demonstrated that mir plays a vital role in controlling leaf development, apical dominance, floral transition and development by targeting several members of the squamosa promoter binding protein like family of plant-specific transcription factors. interestingly, a recent study has shown that overexpression of mir in potato leads to development of stolon-borne aerial tubers from distal nodes. the digital expression profile reported in this study also showed high abundance of mir in tuber developmental stages with highest level at pt, suggesting a role of mir in the phase transition from basal lateral juvenile shoots to a storage organ in the process of tuber development. in our analysis, mir was found to target gras family transcription factors and hairy meristem. they have been shown to be involved in regulating various aspects of plant growth and development. in potato, our qrt-pcr data showed that all the three members of mir expressed highly in vegetative tissues, indicating a role of mir in regulating potato developmental process. previous studies have indicated that mir largely targets gene encoding members of apetala and ap2-like transcription factor family and plays a major role in the regulation of flowering time, vegetative phase change and floral organ identity, by cleaving and repressing the translation of its target mrnas. recently, mir has been shown to induce tuberization by regulating long distance signals and targeting rap mrna. our prediction analysis showed rap which is a known target of mir in potato, as a predicted target of mir this data supports the efficacy of our target prediction analysis. further, mirna-mediated cleavage of target of conserved mirnas was confirmed by 5′ rlm-race. several targets of conserved mirnas have been validated using race analysis in tomato, pepper and rice. however, this is the first study in potato where 5′ rlm-race was performed to authenticate predicted targets of conserved mirnas. our race results confirmed that transcripts of afr nam, rap and ham were cleaved in vivo by mir mir mir and mir respectively. for potato-specific mirnas, degradome sequencing approach is being used for mapping of the cleavage site. our target prediction analysis in potato and other studies on plant species, especially rice and arabidopsis, strongly suggest that most of the conserved mirnas target plant transcription factors, thereby emphasizing the role of conserved mirna in essential biological processes in plants, including potato tuberization. go was performed on potato-specific mirnas targets to gain a better understanding of their possible function. go results revealed that majority of the targets are involved in oxidation-reduction processes, metabolic processes, regulation of transcription, transport, signal transduction and defense responses. these results suggest that they covered a broad range of physiological and metabolic processes. thus, in contrast to conserved mirna targets, most of the putative targets of potato-specific mirnas were not transcription factors. a few potato-specific mirnas were predicted to target transcripts encoding transcription factors, including nac domain protein, gras family transcription factors, mads-box family protein and arf domain class transcription factor. these transcription factors are known to be targeted by conserved mirnas and play a major role in various aspects of plant growth and development. thus, it appears that the potato-specific mirnas might play important roles in essential biological processes as well. future studies are essential to explain the functional significance of the inverse relationship between mirna and their target which will improve our knowledge about the regulatory role of mirnas in potato. with respect to potato tuberization, expression profiling by qrt-pcr and target prediction studies indicated that a few mirnas might play a role during transition from stolon to tuber development. qrt-pcr results indicated that mir172_ mir172_ and mirna mirna showed significant expression during developmental stages of tuberization. lipoxygenase was predicted as one of the targets of mirna mirna mirna mirna and mirna protein phosphatase type 2a was found to be a predicted target of mirna previous studies have suggested the role of mir lox and pp2a in potato tuber developmental process. therefore, these potential mirnas and their target genes are being further investigated to determine their roles in potato tuber development.  in summary, we report a comprehensive study of potato mirnas at genome-wide level using three different vegetative tissues and four early developmental stages of tuberization by high-throughput sequencing and high-end bioinformatics methods. we have identified a population of potato-specific mirnas that could fill the gap in understanding the regulatory role of mirnas in potato. the digital expression data was further quantified by qrt-pcr to determine up- and down-regulations of potato mirnas in various vegetative tissues and developmental stages of tuberization. the putative target genes were predicted for conserved and potato-specific mirnas to gain a better understanding of their function. further, a few conserved mirna targets were also validated by 5′ rlm-race and the predicted target genes for potato-specific mirnas were subjected to go annotation to infer the functional role of target genes. 
this study reports the identification and characterization of potato mirnas in three different vegetative tissues and four stages of tuber development by high throughput sequencing. we report a comprehensive study of potato mirnas at genome-wide level by high-throughput sequencing and demonstrate that these mirnas have tissue and/or developmental stage-specific expression profile. our findings indicate the existence of extensive and complex small rna population in this crop and suggest their important role in pathways involved in diverse biological processes, including tuber development. small rna libraries were constructed from leaf, stem, root and four early developmental stages of tuberization and subjected to deep sequencing, followed by bioinformatics analysis. mirnas are small, single-stranded and ~ nt long rnas which regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of plant development and growth. the digital expression profiling based on tpm and qrt-pcr analysis of conserved and potato-specific mirnas revealed that some of the mirnas showed tissue specific expression while a few demonstrated tuberization stage-specific expressions. a total of conserved mirnas, potato-specific mirnas and candidate potato-specific mirnas were identified. targets were predicted for identified conserved and potato-specific mirnas, and predicted targets of four conserved mirnas, mir mir mir and mir which are arf, nam, rap and ham respectively, were experimentally validated using 5′ rlm-race. gene ontology analysis for potato-specific mirnas was also performed to predict their potential biological functions. however, identification of mirnas through deep sequencing approach was limited to a few tissue types and developmental stages.
 micrornas are ubiquitous components of endogenous plant transcriptome. mirnas are small, single-stranded and ~ nt long rnas which regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of plant development and growth. previously, a number of mirnas have been identified in potato through in silico analysis and deep sequencing approach. however, identification of mirnas through deep sequencing approach was limited to a few tissue types and developmental stages. this study reports the identification and characterization of potato mirnas in three different vegetative tissues and four stages of tuber development by high throughput sequencing.  small rna libraries were constructed from leaf, stem, root and four early developmental stages of tuberization and subjected to deep sequencing, followed by bioinformatics analysis. a total of conserved mirnas, potato-specific mirnas and candidate potato-specific mirnas were identified. the digital expression profiling based on tpm and qrt-pcr analysis of conserved and potato-specific mirnas revealed that some of the mirnas showed tissue specific expression while a few demonstrated tuberization stage-specific expressions. targets were predicted for identified conserved and potato-specific mirnas, and predicted targets of four conserved mirnas, mir mir mir and mir which are arf, nam, rap and ham respectively, were experimentally validated using 5′ rlm-race. gene ontology analysis for potato-specific mirnas was also performed to predict their potential biological functions.  we report a comprehensive study of potato mirnas at genome-wide level by high-throughput sequencing and demonstrate that these mirnas have tissue and/or developmental stage-specific expression profile. also, predicted targets of conserved mirnas were experimentally confirmed for the first time in potato. our findings indicate the existence of extensive and complex small rna population in this crop and suggest their important role in pathways involved in diverse biological processes, including tuber development. potatotuberizationmicrornahigh-throughput sequencingdigital expression profilingqrt-pcrtargetgene ontologyrlm-race 