BACKGROUND
micrornas  constitute a class of single stranded 19– <dig> nucleotide long non-coding rnas that mediate endogenous gene silencing by binding to their target genes.
mirnas binds either to the open reading frame  or untranslated regions  of target mrnas and silencing occurs either by target cleavage or by inhibiting mrna translation
<cit>  through perfect or partial complementarity respectively
<cit> .
mirna feature to regulate gene expression is exemplified by their indispensable roles in critical biological and cellular events in plants including lateral root formation, leaf morphology and polarity, hormonal control, flowering time
<cit> , cell death and apoptosis
<cit> .
these are also involved in plant adaptation to vast array of biotic and abiotic stresses such as cold, drought, salinity, nutrient deficiency
<cit>  and oxidative stress
<cit> .
majority of the targets of plant mirnas were found to be transcription factors such as squamosa-promoter binding protein , hd-zip transcription factors
<cit>  or regulatory stress response proteins like nac domain protein and myb proteins
<cit> .
mirnas along with other rnas such as small interfering rna , crispr rna , piwi interacting rna and trans-acting sirnas  constitute the complex smrnaome
<cit> .
sirnas and mirnas are near similar in structure and function, however, several characters distinguish mirnas from sirnas which include the nature of precursor, conservation, target gene preference and genetic requirements for biogenesis
<cit> .
mirna biogenesis involves a meshwork of various enzymes.
these are produced from their own transcriptional units
<cit>  generated by stepwise processing of rna polymerase ii dependent primary mirna transcripts  which are subsequently capped, spliced and polyadenylated
<cit> .
in plants, rna binding protein dwaddle  stabilizes pri-mirnas for their conversion into processing centers called nuclear dicing bodies  to stem loop precursor mirnas .
this conversion further requires concerted assistance of serrate , hyponastic leaves <dig> , nuclear cap binding complex  and dicer-like  <dig> .
dcl- <dig> possess both drosha and dicer like activities instigating the initial set of both the cuts responsible for above pri- to pre-mirna conversion and further in mature mirna processing for releasing mirna/mirna* duplex in the nucleus, supposed to be predominant location of dcl- <dig>  hua enhancer  <dig> , a methyl transferase, adds methyl groups to the 3′ ends of duplex for stabilization which is further exported in cytoplasm by hasty  proteins, plant ortholog of exportin-5
<cit> .
finally, depending on the thermodynamic stability of 5’end, mirna strand is preferentially loaded into rna induced silencing complex  whereas mirna* strand is degraded.
the risc complex guides mirna strand to its target mrna which is then cleaved between 10th and 11th bases from 5′ end of mirna match.
the cleaved halves of target mrna are then degraded by exoribonucleases  <dig>  and secondary sirnas
<cit> .
mirnas were initially discovered as developmental timing regulators in caenorhabditis elegans <cit>  and later found to be ubiquitously present in wide range of animals, plants, some viruses and in unicellular organisms such as green algae, chlamydomonas reinhardtii <cit> .
increasing evidences showed that mirna repertoire of plant or animal species includes a set of conserved  and non-conserved or novel  mirnas
<cit> .
a total of  <dig> entries representing hairpin precursor mirnas, expressing  <dig> mature mirna products, across  <dig> species have been deposited in the publicly available online repository for mirna sequences, mirbase release 18
<cit> .
however, the biggest challenge in plants is to identify novel mirnas and to understand their mode of action, role in various metabolic processes.
stevia rebaudiana bertoni  is one of  <dig> members of genus stevia and one of the only two species that produce sweet stevoil glycosides
<cit> .
s. rebaudiana is a perennial herb which accumulates up to 30%  diterpenoid steviol glycosides
<cit> .
sgs are glucosylated derivatives of diterpenoid alcohol steviol and stevioside and rebaudioside a are the major sgs found in stevia.
sweetness indices of these and other related compounds ranges between  <dig> to  <dig> times higher than that of sucrose
<cit>  and are used as non-calorific sweeteners in many countries of the world.
in addition to being a sweetener, stevia has been suggested to exert beneficial effects on human health and is considered to have anti-hyperglycemic, anti-hypersensitive, anti-oxidant, anti-tumour/anti-inflammatory, immunomodulatory, antidiarrheal, antimicrobial and anti-rotavirus activities
<cit> .
stevioside has been reported to lower the postprandial blood glucose level of type ii diabetes patients and blood pressure in mildly hypertensive patients
<cit> .
sgs biosynthesis pathway is divided into two stages; early stage wherein geranylgeranyl diphosphate  is synthesized and late stage, which is involved in the synthesis of sgs from ggdp.
sgs being important compounds of stevia, imparting medicinal properties to the plant, so the molecular studies done on this plant were mainly focussed on understanding the biosynthesis and regulation of the genes involved in sg biosynthesis.
a control over the regulation of these early and late genes can help to manipulate the diterepenoid contents
<cit> .
further, the unraveling of srna guided circuitry in stevia will enhance the value of its gene and est information and improve our ability to devise strategies to enhance certain essential features of stevia that are less amenable to functional genomics analysis leading to its enhanced nutritive value.
the identification of mirna and their targets is important not only to help us learn more about the roles of mirnas in stevia development and physiology but also to provide a framework for further designing rnai based experiments for regulation of gene expression in this species.
genome of s. rebaudiana has not been sequenced yet and further there are only  <dig> stevia expressed sequecend tags  and  <dig> gene sequences available in the ncbi database.
thus, it was not possible to perform an extensive study to discover stevia mirnas using computational analysis with this limited number of available sequences.
experimental cloning combined with computational prediction appears to be the most effective method to predict stevia mirnas.
the availability of high throughput next generation sequencing  technologies such as  <dig> and illumina have further revolutionized srna discovery.
whereas, transcribed sequences such as ests has led to the identification of only conserved  mirnas or mirnas previously identified in other species, on the other hand, ngs provides high throughput tools to make new discoveries of additional species specific or lowly expressed mirnas in plants irrespective of whether their genome is sequenced or not, e.g.
arabidopsis <cit> , oryza sativa <cit> , populus trichocarpa <cit> , triticum aestivum <cit> , brachypodium distachyon <cit> , vitis vinifera <cit> , arachis hypogaea <cit> , citrus trifoliate <cit>  , carthamus tinctorius <cit>  and many more.
this huge list signifies that mirnas have been extensively studied since years and the digging of smrnaome of any organism is of utmost importance to understand the srna mediated gene regulations and the diversity of srnas.
our research aimed to examine srna population in leaves of s. rebaudiana using illumina platform to identify conserved and novel mirnas.
the downstream analysis of srna data generated by genome analyzer ii led to the identification of  <dig> conserved mirna sequences belonging to  <dig> mirna families sharing very high homology with that already known in other species.
in addition,  <dig> putative novel mirna families were also identified with their precursors.
a scan of the s. rebaudiana est and nucleotide databases with mirna sequences revealed their putative targets.
putative targets have been predicted for  <dig> out of above  <dig> conserved mirna families and  <dig> novel mirnas using computational methods.
RESULTS
deep sequencing of srnas
using  <dig> cycled single end sequencing by genome analyzer ii
<cit> , a total of  <dig> , <dig> sequences were obtained from smrnaome of s. rebaudiana.
after removing the low quality and adaptor sequences,  <dig> , <dig> sequences were obtained.
among these sequences  <dig> , <dig> sequences ranged from 16– <dig> nt in length.
further removing t/rrnas, a total of  <dig> , <dig> srna sequences were obtained with a size range of 19– <dig> nt .
although some srnas were high in abundance and present thousands of times in our dataset, majority of srnas were sequenced only once in our experiment e.g.
<dig> , <dig> sequences out of  <dig> , <dig> sequences were sequenced only once .
this suggested that  there is a significant variation in expression of various srnas in stevia;  srna survey in stevia is far from being exhausted.
this ultimately directs that stevia contains a large and diverse srna population as was the case seen in c. trifoliate <cit> .
in stevia, the size of srnas was not evenly distributed .
among these sequences, the number of 23nt long sequences was unusually greater than other longer and shorter sequences and accounted for  <dig> % of the total sequence number.
class of 23– <dig> nt has been recently reported and named as long mirnas discovered in arabidopsis <cit> .
this result was unlike as seen in peanut
<cit> , cotton
<cit> , medicago
<cit>  and early phase maize seedlings
<cit>  where fraction of  <dig> nt was very small as compared to others.
in total,  <dig> , <dig> sequences of  <dig> nt length  represented the second highest followed by 22nt sequences  and finally  <dig> nt sequences  which is the typical length of plant mature mirnas .
the latter trend of abundances of 24nt sequences followed by 22nt and 21nt was same as observed during seed germination in maize .
additionally,  <dig>  sequences with a length of 27nt were present, covering  <dig> % in the stevia library.
they may represent the degradation products during srna library construction.
identification of conserved stevia mirnas
the filtered srna dataset were compared with the currently known mature plant mirnas in mirbase v18
<cit>  using mirprof.
after multiple blastn searches and further sequence analysis, a total of  <dig> conserved mirnas, belonging to  <dig> mirna families  were identified.
most of the mirna families were found to be conserved in a variety of plant species e.g.
using a comparative genomics based strategy homologs of mir <dig>  mir156/ <dig>  mir <dig>  mir165/ <dig>  mir <dig> and mir <dig> were found in  <dig> , <dig> , <dig> and  <dig> diverse plant species respectively
<cit> .
deep sequencing aids by providing an alternative way to estimate the expression profiles of mirna genes
<cit> .
it not only determines the abundance of various mirna families but simultaneously distinguishes between different members of a given family.
on the basis of similarity in mature mirna sequence, these mirna were grouped into families, with members often varying upto  <dig> or 2nt.
diversity of stevia mirnas could also be found in the aspect of number of members each family contained .
further, mirna families displayed significant varied abundance from each other.
for e.g.
majority of stevia mirnas were sequenced less than  <dig> times whereas some mirnas were detected in low counts i.e.
less than  <dig> times .
among the  <dig> mirna families, mir <dig> proved to be largest one with highest number of sequences.
in addition, mir <dig> and mir <dig> were found to have some thousands to tens of thousands of redundancies while mir <dig>  mir <dig> and mir <dig> had more than one hundred redundancies.
the remaining families were infrequently sequenced .
a vast diversity was further seen in abundance of different members of a single family.
for e.g.
- as mir159a showed  <dig>  sequenced clones i.e.
more than  <dig>  times.
on the other hand, mir159e was sequenced just  <dig> times.
as the samples were collected in the pre-flowering phase the existence of a dominant member in a mirna family may suggest that the regulatory role of this family was performed by the dominant member in that particular developmental phase.
identification of novel stevia mirnas
in this study, due to unavailability of whole genome sequence of s. rebaudiana, we predicted potential novel mirnas based on bioinformatics analysis using the stevia est and nt sequences available in ncbi database.
first, we removed the sequencing tags that can be annotated as non- coding rnas including t or rrnas and then filtered the known mirna sequences  from further analysis.
a total of  <dig> , <dig> redundant sequences survived the above filtration steps and  <dig>  unique sequences were analyzed for novel mirna prediction.
a distinguishing feature of mirnas is the ability of their pre mirna sequences to adopt canonical stem loop hairpin structure.
these unique srna sequences were mapped to est and nt sequences by searching all s. rebaudiana srnas and predicting the secondary structure of sequences surrounding them  .
we identified  <dig> sequences that satisfied the secondary structure and other criterions .
corresponding hairpin loop structures are provided in supplementary information.
a srna is considered as a potential mirna candidate only if it meets the following strict criteria as defined by zhang et al.
<cit> ; 1) the sequence could fold into an appropriate stem loop hairpin secondary structure; 2) the small rna sits in one arm of hairpin structure; 3) no more than  <dig> mismatches between mature mirna sequence and its opposite mirna * sequence in the secondary structure; 4) no loop or break in the mirna or mirna * sequences; 5) predicted secondary structure has negative minimal folding free energy; 6) minimum a+u%  as mature mirnas contain more a+u nucleotides than g+c
<cit>  and 7) usually begins with 5' u which is one of the characteristic feature of mirnas.
target prediction of mirnas
mirna target prediction in plants is easier due to high and significant complementarities of mirna- mrna targets
<cit>  and targets of most plant mirnas possesses a single perfect or near perfect complementary site in coding region
<cit> .
assuming this to be generally the case, the stevia transcript library  was searched for complementarity with the sequences of identified conserved and novel mirnas using the two target prediction web servers psrnatarget and tapir .
almost all the targets predicted through psrna target server were further authenticated by tapir which showed the same targets.
further, tapir predicted additional putative targets.
a deep insight into mirna targets helps us in understanding the range of srna expression, regulation and their functional importance.
to analyze probable srna targets is significant in plants because the complimentary sites of potential srna can exist anywhere along the target mrna rather than at 3’utr in case of animals
<cit> .
we predicted target genes and putative targets were identified for  <dig> out of  <dig> conserved families  and for  <dig> out of  <dig> stevia specific or novel mirnas .
further, mode of action of mirnas on targets was found to be predominantly through cleavage which is usually the case seen in plants
<cit> .
mostly multiple regulatory targets were predicted e.g.
mir  <dig> has been predicted to have almost  <dig> targets in stevia est and nucleotide transcript library.
the predicted targets are mainly mrna encoding enzymes regulating essential plant metabolic and signaling pathways which include transcription factors, elongation factors, protein kinases, heat shock proteins, f-box family proteins, catalases, peroxidases, aminopeptases, dehydrogenases, transferases, synthases, esterases, oxidoreductases etc.
mfe: free energy ratio; start: start position of the duplex on the mrna; seed gap: no.of gaps in seed region; seed mismatch: no.of mismatches in seed region; seed gu: number of g–u pairs in seed region; mismatches: number of mismatches, gu: number of g–u pairs; gap: number of gaps introduced by bulges and loop structures; score: score calculated for each mirna–mrna duplex taking into account all the above described parameters.
e : scores <  <dig>  considered as a potential target of mirna; upe :target accessibility - allowed maximum energy to unpair the target site.
discussion
a sudden inclination of extensive research towards investigation of plant mirnas is due to their indispensable roles in plant development and adaption.
at present more than thousand mirna genes among diverse plant species have been identified, annotated and some of them have been well characterised
<cit> .
but still a large number of plant species are unexplored and stevia is one of them.
till date several agrotechniques, bioproduct extraction, phytochemical, biological and toxicological studies have been carried out on s. rebaudiana <cit> .
a large flux of research activities have diverted to understand the biosynthesis and possible manipulation of diterpenoids in stevia, particularly sgs which are mainly responsible for its ecological and commercial importance.
but till date no research activity is reported for exploring the mirna repertoire in stevia.
a vast survey of mirnas in stevia will provide useful information to elucidate their physiological functions, gene regulation, biogenesis and evolutionary roles in plants.
traditional strategies like genetic screening, microarray and bioinformatics prediction have miserably failed in species where genome sequence is not available such as stevia.
the availability of high throughput ngs technologies has overshadowed this weakness by generating an accurate and comprehensive picture of smrnaome and identification of known mirnas but also for successful discovery of novel mirnas with high precision.
it is also used as mirna expression profiling tool to understand mirna function in various molecular and physiological pathways.
in this report, we describe the first screen for stevia mirnas by deep sequencing with an aim of gaining insights into various roles of mirnas.
in total,  <dig> , <dig> high quality sequences were generated from  <dig> , <dig> raw sequences representing 57% of the total raw reads.
rna species  <dig>   <dig>   <dig> and  <dig> nt in length dominated the small rna transcriptome with the  <dig> nt class being the most abundant in our library.
this class of 23– <dig> nt has been named as long mirnas.
this finding is consistent with the working hypothesis that evolutionary change in structure of hairpin can change their affinity for dcl’s and hence the size of mirnas.
so, it is actually dcl <dig> which is responsible for pri-mirna processing to generate  <dig> nt long mirnas in contrast to the highly stringent dcl  <dig> for 20– <dig> nt canonical mirnas.
further at times dcl <dig> can also process certain pri-mirnas and generate  <dig> nt long mirnas
<cit>  justifying their presence.
further a total of  <dig>   <dig>   <dig> raw sequences generated a profile of  <dig> conserved mirna families.
our data were in good harmony with previous studies of mirna profiling based on exhaustive sequencing of srna populations  .
this probably reflects the generally accepted high level of expression reported for conserved mirnas.
stevia mirna families showed varied abundance from each other.
among the  <dig> mirna families mir <dig> showed the largest number of sequenced clones which are in agreement as mir <dig> is also the most abundant family in arabidopsis <cit> .
further mir <dig> and mir <dig> with highest abundance in stevia were among the moderately expressed mirnas in arachis hypogea <cit>  and among the lowly expressed mirnas in c. trifoliate <cit> .
in stevia mir <dig> was among the lowly expressed mirnas but usually mir <dig> represents one of the highly abundant mirna families in diverse plant species e.g.
arachis hypogea <cit> , brachypodium <cit>  and early maize seedlings
<cit> .
the actual reason why these mirna families showed varied abundances in different plant species in unknown.
thus this varied abundance of mirna families suggested that mirna genes would be differentially transcribed at this young leaves stage.
certain mirna families are monocot or dicot specific which is further justified by the presence of mir <dig>  in stevia.
the abundance of mir <dig> was  <dig> times low as compared to mir <dig> in our dataset which is consistent with the previous finding that these two mirnas are conversely regulated
<cit> .
further, the largest mirnas family size identified was mir <dig> that consisted of  <dig> members.
mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig> and mir <dig> possessed  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> members respectively whereas other mirna families such as mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig>  mir <dig> etc.
had only one member detected in this library.
the size of mirnas families may be indicative of their function.
based on the results from the deep sequencing, different family members displayed drastically different expression levels.
for example, the abundance of mir <dig> family varied from  <dig> reads to  <dig>  reads in the deep sequencing.
this was also the case for some other mirna families, such as mir <dig>  mir <dig>  and mir <dig> .
abundance comparisons of different members in one mirna family may provide useful information on the role that mirnas play in the pre-flowering stage and even importance of the dominant member at that particular stage of plant growth.
on the contrary  <dig> novel mirnas were predicted according the criterion mentioned before.
all the  <dig> followed secondary structure criteria forming a stem loop structure with mirna sequence sitting at one arm of the hairpin and negative minimal folding energy.
the a+u contents of predicted mirnas were found in the range of  <dig> -  <dig> %.
out of these  <dig> srnas, sequences of stv_ <dig>   <dig>   <dig>   <dig> and  <dig> start with 5’u.
though stv_ <dig> and stv_ <dig> contained less than 50% a+u content but they start with u. keeping this in view, these two srnas could be putative stevia mirnas.
earlier accepted findings state that non-conserved mirnas are usually expressed at lower levels showing a tissue or development specific pattern
<cit>  as was seen in case of stevia where the read number of novel mirnas was much lower than that for conserved mirnas.
majority of them had only 1– <dig> sequenced clones .
this suggests mirnas identified in stevia might represent only a meager portion of novel mirnas due to the fact that srna library was constructed from young plant leaves under normal conditions.
it has been shown that plant mirnas exhibit high degree of sequence complementarity to their targets which facilitates effective target prediction.
target genes identified seem to be associated not only with development but also with diverse physiological processes.
it was found that target sites for conserved mirnas in this plant were similar or functionally related to validated plant mirna targets e.g.
most members of the squamosa promoter binding protein like  transcription factor family are targeted by mir <dig> in plants
<cit> .
for instance, mir <dig> targets  <dig> of the  <dig> spl genes in arabidopsis.
spls affect diverse developmental processes such as leaf development, shoot maturation, phase change and flowering in plants.
similarly, in stevia mir <dig> has been found to target spl.
similarly, mir <dig> targets auxin response factor  <dig>  and mir <dig> targets zinc finger related protein .
mir <dig> is predicted to regulate multiple targets including ring/u-box domain-containing protein, phytochrome e, wd-repeat protein, vacuolar atp synthase subunit e, sin <dig> component, translation elongation factor, molecular chaperone hsp <dig>  alf domain class transcription factor, programmed cell death  <dig> c-terminal domain-containing protein, resistance protein, nac, dna-damage repair protein and several proteins involved in synthesis i.e.
60s acidic ribosomal protein etc.
further, mir <dig> is predicted to have interesting stevia genes as targets like kaurene synthase , calmodulin and udp-glycosyltransferase 73e <dig>  kaurene synthase is a vital late gene involved in steviol glycoside biosynthesis pathway.
further targets were predicted for certain more conserved mirnas including mir <dig>  mir <dig>  mir <dig>  mir  <dig> and mir <dig>  mir <dig> and mir <dig> .
mirnas regulate gene expression predominantly by cleavage  due to high complementarity of mirna and targets.
putative targets were also predicted for some newly identified mirnas.
stv_ <dig> targets one of the chaperonin_like superfamily protein whose common function is to sequester non-native proteins inside their central cavity and promote folding by using energy derived from atp hydrolysis.
on the other hand, stv_ <dig> targets phosphoglycerate kinase  which is a monomeric enzyme that catalyzes the transfer of the high-energy phosphate group of  <dig> -bisphosphoglycerate to adp, forming atp and 3-phosphoglycerate.
this reaction represents the first of the two substrate-level phosphorylation events in the glycolytic pathway.
stv_ <dig> targets pectinacetylesterase family protein which is located in cell wall or membrane and exhibits carboxylesterase activity.
further, stv_ <dig> is predicted to target ribulose- <dig> -bisphosphate carboxylase activase involved in diverse cellular functions as well as lupus la ribonucleoprotein or rrm  found in proteins involved in post-transcriptional gene expression processes including mrna and rrna processing, rna export, and rna stability.
additionally, we predicted a few genes with unknown function and hypothetical genes for mirna targeting e.g.
stv_ <dig> .
target of only  <dig> novel mirnas were predicted speculating that rest of the mirna may be typically present in stevia and needs further experimental work to locate their gene targets.
the above described information regarding predicted targets could be utilized efficiently to assess the regulatory roles of these novel mirnas in stevia.
further if any of these putative novel mirna is found to have control over the regulation of the early and late genes in steviol glycosides biosynthetic pathway as seen in case of conserved mirna  <dig> can help to manipulate the diterepenoid contents.
CONCLUSIONS
the present study provides an important glimpse of srna abundance and diversity in stevia.
we discovered, for the first time,  <dig> highly conserved mirna families and  <dig> novel potential mirnas from s. rebaudiana using high throughput solexa sequencing of srnas, which indicated that species specific mirnas exist in stevia.
the isolated srnas may be considered as stevia putative mirnas based on their characteristic features.
these results show that regulatory mirnas exist in nutritionally important stevia and play a role in stevia growth, development and response to stress.
further prediction of target genes adds to our understanding of mechanisms regulating their cellular function and evolution.
most importantly, this study will serve as a foundation of future research into functional roles of mirnas and will prove to be a major breakthrough if any of these mirna will be targeting the genes involved in steviol glycosides pathway.

 micrornas constitute a class of single stranded 19– nucleotide long non-coding rnas that mediate endogenous gene silencing by binding to their target genes. mirnas binds either to the open reading frame or untranslated regions of target mrnas and silencing occurs either by target cleavage or by inhibiting mrna translation through perfect or partial complementarity respectively. mirna feature to regulate gene expression is exemplified by their indispensable roles in critical biological and cellular events in plants including lateral root formation, leaf morphology and polarity, hormonal control, flowering time, cell death and apoptosis. these are also involved in plant adaptation to vast array of biotic and abiotic stresses such as cold, drought, salinity, nutrient deficiency and oxidative stress. majority of the targets of plant mirnas were found to be transcription factors such as squamosa-promoter binding protein, hd-zip transcription factors or regulatory stress response proteins like nac domain protein and myb proteins. mirnas along with other rnas such as small interfering rna, crispr rna, piwi interacting rna and trans-acting sirnas constitute the complex smrnaome. sirnas and mirnas are near similar in structure and function, however, several characters distinguish mirnas from sirnas which include the nature of precursor, conservation, target gene preference and genetic requirements for biogenesis. mirna biogenesis involves a meshwork of various enzymes. these are produced from their own transcriptional units generated by stepwise processing of rna polymerase ii dependent primary mirna transcripts which are subsequently capped, spliced and polyadenylated. in plants, rna binding protein dwaddle stabilizes pri-mirnas for their conversion into processing centers called nuclear dicing bodies to stem loop precursor mirnas. this conversion further requires concerted assistance of serrate, hyponastic leaves, nuclear cap binding complex and dicer-like. dcl- possess both drosha and dicer like activities instigating the initial set of both the cuts responsible for above pri- to pre-mirna conversion and further in mature mirna processing for releasing mirna/mirna* duplex in the nucleus, supposed to be predominant location of dcl- hua enhancer, a methyl transferase, adds methyl groups to the 3′ ends of duplex for stabilization which is further exported in cytoplasm by hasty proteins, plant ortholog of exportin-5. finally, depending on the thermodynamic stability of 5’end, mirna strand is preferentially loaded into rna induced silencing complex whereas mirna* strand is degraded. the risc complex guides mirna strand to its target mrna which is then cleaved between 10th and 11th bases from 5′ end of mirna match. the cleaved halves of target mrna are then degraded by exoribonucleases and secondary sirnas. mirnas were initially discovered as developmental timing regulators in caenorhabditis elegans and later found to be ubiquitously present in wide range of animals, plants, some viruses and in unicellular organisms such as green algae, chlamydomonas reinhardtii. increasing evidences showed that mirna repertoire of plant or animal species includes a set of conserved and non-conserved or novel mirnas. a total of entries representing hairpin precursor mirnas, expressing mature mirna products, across species have been deposited in the publicly available online repository for mirna sequences, mirbase release 18. however, the biggest challenge in plants is to identify novel mirnas and to understand their mode of action, role in various metabolic processes. stevia rebaudiana bertoni is one of members of genus stevia and one of the only two species that produce sweet stevoil glycosides. s. rebaudiana is a perennial herb which accumulates up to 30% diterpenoid steviol glycosides. sgs are glucosylated derivatives of diterpenoid alcohol steviol and stevioside and rebaudioside a are the major sgs found in stevia. sweetness indices of these and other related compounds ranges between to times higher than that of sucrose and are used as non-calorific sweeteners in many countries of the world. in addition to being a sweetener, stevia has been suggested to exert beneficial effects on human health and is considered to have anti-hyperglycemic, anti-hypersensitive, anti-oxidant, anti-tumour/anti-inflammatory, immunomodulatory, antidiarrheal, antimicrobial and anti-rotavirus activities. stevioside has been reported to lower the postprandial blood glucose level of type ii diabetes patients and blood pressure in mildly hypertensive patients. sgs biosynthesis pathway is divided into two stages; early stage wherein geranylgeranyl diphosphate is synthesized and late stage, which is involved in the synthesis of sgs from ggdp. sgs being important compounds of stevia, imparting medicinal properties to the plant, so the molecular studies done on this plant were mainly focussed on understanding the biosynthesis and regulation of the genes involved in sg biosynthesis. a control over the regulation of these early and late genes can help to manipulate the diterepenoid contents. further, the unraveling of srna guided circuitry in stevia will enhance the value of its gene and est information and improve our ability to devise strategies to enhance certain essential features of stevia that are less amenable to functional genomics analysis leading to its enhanced nutritive value. the identification of mirna and their targets is important not only to help us learn more about the roles of mirnas in stevia development and physiology but also to provide a framework for further designing rnai based experiments for regulation of gene expression in this species. genome of s. rebaudiana has not been sequenced yet and further there are only stevia expressed sequecend tags and gene sequences available in the ncbi database. thus, it was not possible to perform an extensive study to discover stevia mirnas using computational analysis with this limited number of available sequences. experimental cloning combined with computational prediction appears to be the most effective method to predict stevia mirnas. the availability of high throughput next generation sequencing technologies such as and illumina have further revolutionized srna discovery. whereas, transcribed sequences such as ests has led to the identification of only conserved mirnas or mirnas previously identified in other species, on the other hand, ngs provides high throughput tools to make new discoveries of additional species specific or lowly expressed mirnas in plants irrespective of whether their genome is sequenced or not, e.g. arabidopsis, oryza sativa, populus trichocarpa, triticum aestivum, brachypodium distachyon, vitis vinifera, arachis hypogaea, citrus trifoliate, carthamus tinctorius and many more. this huge list signifies that mirnas have been extensively studied since years and the digging of smrnaome of any organism is of utmost importance to understand the srna mediated gene regulations and the diversity of srnas. our research aimed to examine srna population in leaves of s. rebaudiana using illumina platform to identify conserved and novel mirnas. the downstream analysis of srna data generated by genome analyzer ii led to the identification of conserved mirna sequences belonging to mirna families sharing very high homology with that already known in other species. in addition, putative novel mirna families were also identified with their precursors. a scan of the s. rebaudiana est and nucleotide databases with mirna sequences revealed their putative targets. putative targets have been predicted for out of above conserved mirna families and novel mirnas using computational methods.  deep sequencing of srnas using cycled single end sequencing by genome analyzer ii, a total of, sequences were obtained from smrnaome of s. rebaudiana. after removing the low quality and adaptor sequences,, sequences were obtained. among these sequences, sequences ranged from 16– nt in length. further removing t/rrnas, a total of, srna sequences were obtained with a size range of 19– nt. although some srnas were high in abundance and present thousands of times in our dataset, majority of srnas were sequenced only once in our experiment e.g., sequences out of, sequences were sequenced only once. this suggested that there is a significant variation in expression of various srnas in stevia; srna survey in stevia is far from being exhausted. this ultimately directs that stevia contains a large and diverse srna population as was the case seen in c. trifoliate. in stevia, the size of srnas was not evenly distributed. among these sequences, the number of 23nt long sequences was unusually greater than other longer and shorter sequences and accounted for % of the total sequence number. class of 23– nt has been recently reported and named as long mirnas discovered in arabidopsis. this result was unlike as seen in peanut, cotton, medicago and early phase maize seedlings where fraction of nt was very small as compared to others. in total,, sequences of nt length represented the second highest followed by 22nt sequences and finally nt sequences which is the typical length of plant mature mirnas. the latter trend of abundances of 24nt sequences followed by 22nt and 21nt was same as observed during seed germination in maize. additionally, sequences with a length of 27nt were present, covering % in the stevia library. they may represent the degradation products during srna library construction. identification of conserved stevia mirnas the filtered srna dataset were compared with the currently known mature plant mirnas in mirbase v18 using mirprof. after multiple blastn searches and further sequence analysis, a total of conserved mirnas, belonging to mirna families were identified. most of the mirna families were found to be conserved in a variety of plant species e.g. using a comparative genomics based strategy homologs of mir mir156/ mir mir165/ mir and mir were found in,, and diverse plant species respectively. deep sequencing aids by providing an alternative way to estimate the expression profiles of mirna genes. it not only determines the abundance of various mirna families but simultaneously distinguishes between different members of a given family. on the basis of similarity in mature mirna sequence, these mirna were grouped into families, with members often varying upto or 2nt. diversity of stevia mirnas could also be found in the aspect of number of members each family contained. further, mirna families displayed significant varied abundance from each other. for e.g. majority of stevia mirnas were sequenced less than times whereas some mirnas were detected in low counts i.e. less than times. among the mirna families, mir proved to be largest one with highest number of sequences. in addition, mir and mir were found to have some thousands to tens of thousands of redundancies while mir mir and mir had more than one hundred redundancies. the remaining families were infrequently sequenced. a vast diversity was further seen in abundance of different members of a single family. for e.g. - as mir159a showed sequenced clones i.e. more than times. on the other hand, mir159e was sequenced just times. as the samples were collected in the pre-flowering phase the existence of a dominant member in a mirna family may suggest that the regulatory role of this family was performed by the dominant member in that particular developmental phase. identification of novel stevia mirnas in this study, due to unavailability of whole genome sequence of s. rebaudiana, we predicted potential novel mirnas based on bioinformatics analysis using the stevia est and nt sequences available in ncbi database. first, we removed the sequencing tags that can be annotated as non- coding rnas including t or rrnas and then filtered the known mirna sequences from further analysis. a total of, redundant sequences survived the above filtration steps and unique sequences were analyzed for novel mirna prediction. a distinguishing feature of mirnas is the ability of their pre mirna sequences to adopt canonical stem loop hairpin structure. these unique srna sequences were mapped to est and nt sequences by searching all s. rebaudiana srnas and predicting the secondary structure of sequences surrounding them. we identified sequences that satisfied the secondary structure and other criterions. corresponding hairpin loop structures are provided in supplementary information. a srna is considered as a potential mirna candidate only if it meets the following strict criteria as defined by zhang et al.; 1) the sequence could fold into an appropriate stem loop hairpin secondary structure; 2) the small rna sits in one arm of hairpin structure; 3) no more than mismatches between mature mirna sequence and its opposite mirna * sequence in the secondary structure; 4) no loop or break in the mirna or mirna * sequences; 5) predicted secondary structure has negative minimal folding free energy; 6) minimum a+u% as mature mirnas contain more a+u nucleotides than g+c and 7) usually begins with 5' u which is one of the characteristic feature of mirnas. target prediction of mirnas mirna target prediction in plants is easier due to high and significant complementarities of mirna- mrna targets and targets of most plant mirnas possesses a single perfect or near perfect complementary site in coding region. assuming this to be generally the case, the stevia transcript library was searched for complementarity with the sequences of identified conserved and novel mirnas using the two target prediction web servers psrnatarget and tapir. almost all the targets predicted through psrna target server were further authenticated by tapir which showed the same targets. further, tapir predicted additional putative targets. a deep insight into mirna targets helps us in understanding the range of srna expression, regulation and their functional importance. to analyze probable srna targets is significant in plants because the complimentary sites of potential srna can exist anywhere along the target mrna rather than at 3’utr in case of animals. we predicted target genes and putative targets were identified for out of conserved families and for out of stevia specific or novel mirnas. further, mode of action of mirnas on targets was found to be predominantly through cleavage which is usually the case seen in plants. mostly multiple regulatory targets were predicted e.g. mir has been predicted to have almost targets in stevia est and nucleotide transcript library. the predicted targets are mainly mrna encoding enzymes regulating essential plant metabolic and signaling pathways which include transcription factors, elongation factors, protein kinases, heat shock proteins, f-box family proteins, catalases, peroxidases, aminopeptases, dehydrogenases, transferases, synthases, esterases, oxidoreductases etc. mfe: free energy ratio; start: start position of the duplex on the mrna; seed gap: no.of gaps in seed region; seed mismatch: no.of mismatches in seed region; seed gu: number of g–u pairs in seed region; mismatches: number of mismatches, gu: number of g–u pairs; gap: number of gaps introduced by bulges and loop structures; score: score calculated for each mirna–mrna duplex taking into account all the above described parameters. e: scores < considered as a potential target of mirna; upe:target accessibility - allowed maximum energy to unpair the target site. discussion a sudden inclination of extensive research towards investigation of plant mirnas is due to their indispensable roles in plant development and adaption. at present more than thousand mirna genes among diverse plant species have been identified, annotated and some of them have been well characterised. but still a large number of plant species are unexplored and stevia is one of them. till date several agrotechniques, bioproduct extraction, phytochemical, biological and toxicological studies have been carried out on s. rebaudiana. a large flux of research activities have diverted to understand the biosynthesis and possible manipulation of diterpenoids in stevia, particularly sgs which are mainly responsible for its ecological and commercial importance. but till date no research activity is reported for exploring the mirna repertoire in stevia. a vast survey of mirnas in stevia will provide useful information to elucidate their physiological functions, gene regulation, biogenesis and evolutionary roles in plants. traditional strategies like genetic screening, microarray and bioinformatics prediction have miserably failed in species where genome sequence is not available such as stevia. the availability of high throughput ngs technologies has overshadowed this weakness by generating an accurate and comprehensive picture of smrnaome and identification of known mirnas but also for successful discovery of novel mirnas with high precision. it is also used as mirna expression profiling tool to understand mirna function in various molecular and physiological pathways. in this report, we describe the first screen for stevia mirnas by deep sequencing with an aim of gaining insights into various roles of mirnas. in total,, high quality sequences were generated from, raw sequences representing 57% of the total raw reads. rna species and nt in length dominated the small rna transcriptome with the nt class being the most abundant in our library. this class of 23– nt has been named as long mirnas. this finding is consistent with the working hypothesis that evolutionary change in structure of hairpin can change their affinity for dcl’s and hence the size of mirnas. so, it is actually dcl which is responsible for pri-mirna processing to generate nt long mirnas in contrast to the highly stringent dcl for 20– nt canonical mirnas. further at times dcl can also process certain pri-mirnas and generate nt long mirnas justifying their presence. further a total of raw sequences generated a profile of conserved mirna families. our data were in good harmony with previous studies of mirna profiling based on exhaustive sequencing of srna populations. this probably reflects the generally accepted high level of expression reported for conserved mirnas. stevia mirna families showed varied abundance from each other. among the mirna families mir showed the largest number of sequenced clones which are in agreement as mir is also the most abundant family in arabidopsis. further mir and mir with highest abundance in stevia were among the moderately expressed mirnas in arachis hypogea and among the lowly expressed mirnas in c. trifoliate. in stevia mir was among the lowly expressed mirnas but usually mir represents one of the highly abundant mirna families in diverse plant species e.g. arachis hypogea, brachypodium and early maize seedlings. the actual reason why these mirna families showed varied abundances in different plant species in unknown. thus this varied abundance of mirna families suggested that mirna genes would be differentially transcribed at this young leaves stage. certain mirna families are monocot or dicot specific which is further justified by the presence of mir in stevia. the abundance of mir was times low as compared to mir in our dataset which is consistent with the previous finding that these two mirnas are conversely regulated. further, the largest mirnas family size identified was mir that consisted of members. mir mir mir mir mir and mir possessed and members respectively whereas other mirna families such as mir mir mir mir mir mir etc. had only one member detected in this library. the size of mirnas families may be indicative of their function. based on the results from the deep sequencing, different family members displayed drastically different expression levels. for example, the abundance of mir family varied from reads to reads in the deep sequencing. this was also the case for some other mirna families, such as mir mir and mir. abundance comparisons of different members in one mirna family may provide useful information on the role that mirnas play in the pre-flowering stage and even importance of the dominant member at that particular stage of plant growth. on the contrary novel mirnas were predicted according the criterion mentioned before. all the followed secondary structure criteria forming a stem loop structure with mirna sequence sitting at one arm of the hairpin and negative minimal folding energy. the a+u contents of predicted mirnas were found in the range of - %. out of these srnas, sequences of stv_ and start with 5’u. though stv_ and stv_ contained less than 50% a+u content but they start with u. keeping this in view, these two srnas could be putative stevia mirnas. earlier accepted findings state that non-conserved mirnas are usually expressed at lower levels showing a tissue or development specific pattern as was seen in case of stevia where the read number of novel mirnas was much lower than that for conserved mirnas. majority of them had only 1– sequenced clones. this suggests mirnas identified in stevia might represent only a meager portion of novel mirnas due to the fact that srna library was constructed from young plant leaves under normal conditions. it has been shown that plant mirnas exhibit high degree of sequence complementarity to their targets which facilitates effective target prediction. target genes identified seem to be associated not only with development but also with diverse physiological processes. it was found that target sites for conserved mirnas in this plant were similar or functionally related to validated plant mirna targets e.g. most members of the squamosa promoter binding protein like transcription factor family are targeted by mir in plants. for instance, mir targets of the spl genes in arabidopsis. spls affect diverse developmental processes such as leaf development, shoot maturation, phase change and flowering in plants. similarly, in stevia mir has been found to target spl. similarly, mir targets auxin response factor and mir targets zinc finger related protein. mir is predicted to regulate multiple targets including ring/u-box domain-containing protein, phytochrome e, wd-repeat protein, vacuolar atp synthase subunit e, sin component, translation elongation factor, molecular chaperone hsp alf domain class transcription factor, programmed cell death c-terminal domain-containing protein, resistance protein, nac, dna-damage repair protein and several proteins involved in synthesis i.e. 60s acidic ribosomal protein etc. further, mir is predicted to have interesting stevia genes as targets like kaurene synthase, calmodulin and udp-glycosyltransferase 73e kaurene synthase is a vital late gene involved in steviol glycoside biosynthesis pathway. further targets were predicted for certain more conserved mirnas including mir mir mir mir and mir mir and mir. mirnas regulate gene expression predominantly by cleavage due to high complementarity of mirna and targets. putative targets were also predicted for some newly identified mirnas. stv_ targets one of the chaperonin_like superfamily protein whose common function is to sequester non-native proteins inside their central cavity and promote folding by using energy derived from atp hydrolysis. on the other hand, stv_ targets phosphoglycerate kinase which is a monomeric enzyme that catalyzes the transfer of the high-energy phosphate group of -bisphosphoglycerate to adp, forming atp and 3-phosphoglycerate. this reaction represents the first of the two substrate-level phosphorylation events in the glycolytic pathway. stv_ targets pectinacetylesterase family protein which is located in cell wall or membrane and exhibits carboxylesterase activity. further, stv_ is predicted to target ribulose- -bisphosphate carboxylase activase involved in diverse cellular functions as well as lupus la ribonucleoprotein or rrm found in proteins involved in post-transcriptional gene expression processes including mrna and rrna processing, rna export, and rna stability. additionally, we predicted a few genes with unknown function and hypothetical genes for mirna targeting e.g. stv_. target of only novel mirnas were predicted speculating that rest of the mirna may be typically present in stevia and needs further experimental work to locate their gene targets. the above described information regarding predicted targets could be utilized efficiently to assess the regulatory roles of these novel mirnas in stevia. further if any of these putative novel mirna is found to have control over the regulation of the early and late genes in steviol glycosides biosynthetic pathway as seen in case of conserved mirna can help to manipulate the diterepenoid contents.  the present study provides an important glimpse of srna abundance and diversity in stevia. we discovered, for the first time, highly conserved mirna families and novel potential mirnas from s. rebaudiana using high throughput solexa sequencing of srnas, which indicated that species specific mirnas exist in stevia. the isolated srnas may be considered as stevia putative mirnas based on their characteristic features. these results show that regulatory mirnas exist in nutritionally important stevia and play a role in stevia growth, development and response to stress. further prediction of target genes adds to our understanding of mechanisms regulating their cellular function and evolution. most importantly, this study will serve as a foundation of future research into functional roles of mirnas and will prove to be a major breakthrough if any of these mirna will be targeting the genes involved in steviol glycosides pathway. 
deep sequencing of small rnas combined with transcriptomic data is a powerful tool for identifying conserved and novel mirnas irrespective of availability of genome sequence data. also, we identified novel mirnas whose precursors were potentially generated from stevia est and nucleotide sequences. putative target genes were predicted for most conserved and novel mirnas. this study led to the identification of highly conserved mirna families and novel potential mirnas indicating that specific mirnas exist in stevia species. stevia rebaudiana bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. micrornas constitute a family of small rna population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. our results provided information on stevia mirnas and their targets building a foundation for future studies to understand their roles in key stevia traits. to identify mirnas in s. rebaudiana, srna library was constructed and sequenced using illumina genome analyzer ii. a total of, reads representing, distinct sequences were obtained from srna library. several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about mirnas has been lacking in s. rebaudiana. the predicted targets are mainly mrna encoding enzymes regulating essential plant metabolic and signaling pathways. all novel sequences have not been earlier described in other plant species.
 micrornas constitute a family of small rna population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. extensive studies on mirnas have been performed in different plants such as arabidopsis thaliana, oryza sativa etc. and volume of the mirna database, mirbase, has been increasing on day to day basis. stevia rebaudiana bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about mirnas has been lacking in s. rebaudiana. deep sequencing of small rnas combined with transcriptomic data is a powerful tool for identifying conserved and novel mirnas irrespective of availability of genome sequence data.  to identify mirnas in s. rebaudiana, srna library was constructed and sequenced using illumina genome analyzer ii. a total of, reads representing, distinct sequences were obtained from srna library. based on sequence similarity, we identified mirnas belonging to highly conserved families. also, we identified novel mirnas whose precursors were potentially generated from stevia est and nucleotide sequences. all novel sequences have not been earlier described in other plant species. putative target genes were predicted for most conserved and novel mirnas. the predicted targets are mainly mrna encoding enzymes regulating essential plant metabolic and signaling pathways.  this study led to the identification of highly conserved mirna families and novel potential mirnas indicating that specific mirnas exist in stevia species. our results provided information on stevia mirnas and their targets building a foundation for future studies to understand their roles in key stevia traits. stevia rebaudiananext generation sequencingsmall rnasconserved and novel mirnamirna targets 