Introduction {#s1}
============

The genus *Populus* encompasses approximately 100 species divided into 5 sections and many of these species are important components of terrestrial ecosystems or important cultivated trees for wood, pulp, paper, and possibly biofuels uses. As dioecious trees, the genus *Populus* provides opportunities to study perennial woody plant biology and plant sexual dimorphism [@pone.0062681-Tuskan1]. Moreover, the high-quality, annotated genome sequence of *P. trichocarpa* provides a key genomics tool, enabling genome-wide regulatory analyses [@pone.0062681-Tuskan1]. Sexual dimorphism is a widely studied phenomenon in dioecious plants and likely results from different modes of selection operating in males and females [@pone.0062681-Zluvova1]. As dioecious trees, poplar floral development is sexual dimorphism. The poplar flower is composed of two whorls, including a reduced perianth cup surrounding either the stamens or carpels, which begin to develop in spring (early May in Shandong province) of the year before the spring in which they will open [@pone.0062681-Sheppard1], [@pone.0062681-Rottmann1]. Until late May or early June, structural development of the male inflorescence is virtually indistinguishable from development of the female inflorescence. After that, the flower morphology, bract morphology, and cell division patterns differ markedly between the sexes [@pone.0062681-Boes1]. Pioneering experiments found that a series of genes involved in flowering pathways are differently expressed between female and male flowers [@pone.0062681-An1], [@pone.0062681-Song1]. Although the molecular basis of poplar sex-specific flower development remains largely mysterious, increasing evidence suggests that miRNAs may play essential roles in this process.

In plants, there are two classes of endogenous small RNAs: small interfering RNAs (siRNAs) and microRNAs (miRNAs). Both classes, ranging from 20--24 nt, are short non-coding RNA molecules that negatively regulate gene expression at the transcriptional and/or post-transcriptional levels [@pone.0062681-Chen1]. siRNAs are generated from double- stranded RNA. By contrast, miRNAs are transcribed from a long precursor molecule that folds back on itself to form a hairpin. Dicer-Like1 (DCL1) protein cleaves this precursor molecule, resulting in a miRNA:miRNA\* complex, which separates into miRNA and miRNA\* after transport to the cytoplasm [@pone.0062681-Bartel1]. In the cytoplasm, the miRNA is bound by ARGONAUTE proteins to form part of the RNA-induced silencing complex (RISC), which interacts with target mRNAs to cleave the RNA of target genes at the paired region [@pone.0062681-Llave1]. Since the mature miRNA and its complementary target sequence have almost perfect complementarity, identifying a miRNA usually leads to the prediction and/or identification of its target. miRNAs have been shown to target genes during plant organ development, stress tolerance, phytohormone signaling, growth phase change, and disease resistance [@pone.0062681-Navarro1], [@pone.0062681-Wu1].

miRNAs have been identified as regulators of flower development and other pathways. For example, in *Arabidopsis*, the miR164 family targets a subset of NAC transcription factors including *CUP-SHAPED COTYLEDON1* (*CUC1*) and *CUC2*, which mediate organ boundary formation. Among these members, miRNA164c affects petal number in early flowers [@pone.0062681-Baker1]. miRNA172 regulates *APETALA2* (*AP2*) and several of its homologs that share two tandem AP2 DNA-binding domains and play an important role in regulating flower development [@pone.0062681-Aukerman1]--[@pone.0062681-Schmid1]. In maize, *TASSELSEED 4* (*TS4*) and the *AP2* homolog *indeterminate spikelet 1* are confirmed miRNA172 targets. In *ts4* mutants, female organs develop in the male inflorescence. Moreover, branching is increased in *ts4* mutants indicating a link between sex determination and meristem fate [@pone.0062681-Chuck1]. In tree species, the unknown functions of the majority of genes are still the greatest gap. miRNA studies provided a best chance for directing to identify functions for genes [@pone.0062681-Sun1]. Small RNA libraries prepared from poplar leaves and vegetative buds have been analyzed by high-throughput sequencing, finding 48 *Populus*-specific miRNA families [@pone.0062681-Barakat1]. However, sex-specific miRNA differences between female and male flower organs remain unclear, partly because of the effects of different genetic backgrounds of dioecious plants. For this work, we first used andromonoecious poplar, an exceptional model system that has both male and hermaphrodite flowers on a single plant, to eliminate confounding effects of genetic background of dioecious plants ([Figure S1](#pone.0062681.s001){ref-type="supplementary-material"}). In this study, our aim is to screen sex-specific potential functional miRNAs in poplar floral tissue, investigate their defined expression patterns and demonstrate a possible functional network of sex-specific miRNAs in poplar flower organs.

Results {#s2}
=======

Small RNA Sequence Analyses {#s2a}
---------------------------

Two small RNA (sRNA) libraries were constructed from female (F) and male (M) flowers of andromonoecious *P*. *tomentosa*. A total of 58,517,150 (F) and 47,918,351 (M) raw reads were generated from these libraries, respectively, using the Solexa sequencing technology ([Table 1](#pone-0062681-t001){ref-type="table"}). After removing contaminant reads, clean reads were obtained and screened against rRNA, tRNA, snRNA, snoRNA, and mRNA in the Rfam 9.1 and NCBI GenBank databases, resulting in 50,782,479 (F) and 42,837,470 (M) reads remaining for further analyses. Conserved miRNAs were identified by alignment to data in miRBase 19.0 with ±2 nt mismatch; a total of 4,789,550 (F) and 4,306,015 (M) reads that could form characteristic hairpin structures were identified. 4,459,932 (F) and 3,810,615 (M) unannotated unique sRNA sequences, which were further analyzed to predict novel miRNAs. The sequenced sRNAs were then mapped to the *Populus* genome, miRBase19.0, the NCBI GenBank database, and the Rfam database, and classified into six categories: rRNA, known miRNA (miRNAs in miRBase 19.0), exon, intron, repeat associated RNA, and unknown sRNA. More than 60% of the sequences were unknown sRNAs ([Figure 1A](#pone-0062681-g001){ref-type="fig"}). The majority of total sRNA reads were either 21 or 24 nt for both libraries ([Figure 1B](#pone-0062681-g001){ref-type="fig"}). In female flower libraries, the 21-nt sRNAs were the most abundant, making up 41.4% of total sequence reads. The 24-nt sRNAs were the second most abundant class at 17.3% of total sequence reads. In male flower libraries, the 21 and 24 nt sRNAs were not significantly different, making up 29.9% and 29.7%, respectively.

![Small RNAs from female and male flower libraries in andromonoecious poplar.\
(A) Distribution of unique sRNA annotation categories. (B) Size distribution of small RNA reads.](pone.0062681.g001){#pone-0062681-g001}

10.1371/journal.pone.0062681.t001

###### Summary of small RNA sequences from *P*. *tomentosa* female and male flower.

![](pone.0062681.t001){#pone-0062681-t001-1}

                        Female library (F)   Male library (M)
  -------------------- -------------------- ------------------
  Raw reads                 58,517,150          47,918,351
  Clean reads               50,782,479          42,837,470
  Annotated sRNAs           16,625,919          16,030,471
  Un-annotated sRNAs        34,156,560          26,806,999
  Small RNAs                4,789,550           4,306,015
  Annotated sRNAs            329,618             495,400
  Un-annotated sRNAs        4,459,932           3,810,615

Conserved miRNAs in Andromonoecious *P. tomentosa* {#s2b}
--------------------------------------------------

To date, 46 conserved and non-conserved miRNA families have been discovered in the *Populus* genome [@pone.0062681-GriffithsJones1]. In the two libraries sequenced in our study, 134 unique miRNA sequences were identified belonging to 38 conserved miRNA families ([Table 2](#pone-0062681-t002){ref-type="table"}). The expression levels of a few miRNA families, such as miR166 and miR472, were extraordinarily high in both libraries ([Table 2](#pone-0062681-t002){ref-type="table"}). miR166 was the most abundant, with 2,157,665 (F) and 142, 916 (M) reads accounting for 62.5% and 47.1% of all conserved miRNA reads, respectively. Several miRNA families, such as miR156, miR159, miR169, miR319, miR396, and miR1447, had moderate expression levels ([Table 2](#pone-0062681-t002){ref-type="table"}). By contrast, some miRNA families showed very low levels of expression, with fewer than 200 reads. Different members in the same miRNA family displayed drastically different expression levels. For example, miR166 members varied in abundance from 695 to 2,157,665 reads. Also, 38 unique miRNA sequences belonging to 38 conserved miRNA families were produced from 134 loci ([Table S1](#pone.0062681.s003){ref-type="supplementary-material"}). Of these miRNA sequences, 11 were encoded by a single locus in the *Populus* genome, whereas the other 27 sequences had multiple loci. Most of these had 2--6 loci in the genome, and only a few had more than eight loci. For example, Pto-miR166a--l and Pto-miR169a--m had 12 and 13 loci, respectively. Thus, the size of miRNA families varies in andromonoecious *P. tomentosa*. Among these conserved miRNAs, miR6459 was only expressed in the female library. By contrast, two miRNAs, miRNA397 and miRNA6462, were detected as having male-specific expression ([Figure 2](#pone-0062681-g002){ref-type="fig"}).

![Summary of common and specific sequences between female and male libraries. (A) Conserved sRNAs and (B) Novel sRNAs.](pone.0062681.g002){#pone-0062681-g002}

10.1371/journal.pone.0062681.t002

###### Summary of conserved miRNAs found in andromonoecious *P. tomentosa*.

![](pone.0062681.t002){#pone-0062681-t002-2}

  miRNAfamily    Members    Reads    Ratios (female/male)  
  ------------- --------- --------- ---------------------- -------
  miR156            6       3629             8330           0.44
  miR159            3      808474           20798           38.87
  miR160            4       2695             523            5.15
  miR162            2       5094             342            14.89
  miR166           12      2157665          142916          15.10
  miR167            8       1995            15568           0.13
  miR168            2       38333            5129           7.47
  miR169           13       41555            3514           11.83
  miR171            5        820             298            2.75
  miR172            2        58              105            0.55
  miR319            6      115137            2239           51.42
  miR393            5       2498             1911           1.31
  miR394            2       4814             1143           4.21
  miR396            9       30598           30786           0.99
  miR397            1         0               8             0.00
  miR398            3       1783             752            2.37
  miR399           10       11180            116            96.38
  miR403            3       19815            5087           3.90
  miR408            1        157             161            0.98
  miR472            2      126848           48300           2.63
  miR475            7       1524             394            3.87
  miR476            3       1101             637            1.73
  miR482            2       1973             312            6.32
  miR530            1        337              11            30.64
  miR1444           1        29               36            0.81
  miR1446           4        22               51            0.43
  miR1447           1       22381            2700           8.29
  miR1448           1       16637            4935           3.37
  miR1450           1       16340            826            19.78
  miR6421           1        641             711            0.90
  miR6425           4        219             245            0.89
  miR6427           1        345             112            3.08
  miR6433           2       1026             374            2.74
  miR6445           2       9334             3382           2.76
  miR6447           1         2               10            0.20
  miR6459           1        603              0             0.00
  miR6462           1         0               5             0.00
  miR6478           1       1846             1028           1.80
  Total            134     3447508          303795          11.35

Identification of Novel miRNAs in Andromonoecious *P. tomentosa* {#s2c}
----------------------------------------------------------------

The characteristic hairpin structure of miRNA precursors was used to predict novel miRNAs. In total, 78 unique sRNA sequences were identified including 8 sRNAs with complementary miRNA\*s ([Table 3](#pone-0062681-t003){ref-type="table"}, [4](#pone-0062681-t004){ref-type="table"} and [5](#pone-0062681-t005){ref-type="table"}). The majority of these novel miRNA candidates had lengths of 21 and 22 nt, and started with a 5′ U ([Figure 3](#pone-0062681-g003){ref-type="fig"}). The 78 sRNA sequences were transcribed from 85 loci; most were only produced from one locus, but Pto-F2, Pto-F13, Pto-F31 and Pto-F31 were each generated from 3 loci. The length of the predicted novel miRNA precursors ranged from 76 to 303 nt, with an average of 140 nt. The average minimum free energy (MFE) value was −55.58 kcal/mol, with a range of −21.2 to −177.4 kcal/mol. The structures of the 78 novel miRNA precursors are shown in [Figure S2](#pone.0062681.s002){ref-type="supplementary-material"}. Most of them showed differential expression in the two libraries. For instance, the mature miRNA counts ranged from 63 to 8,480,860. Among those 78 miRNAs, only 6 sRNAs were found both in female and male libraries. 61 female-specific sRNAs and 11 male-specific sRNAs were detected, suggesting that more kinds of sRNA are involved in female floral development than in male development. To investigate whether these novel miRNA sequences were conserved across plant species, we used them as query sequences to perform BLASTN searches against the *Arabidopsis thaliana*, *Brassica rapa*, *Citrus sinensis*, *Medicago truncatula*, *Oryza sativa*, *Ricinus communis*, *Prunus persica*, *Vitis vinifera* and *Zea mays* genome databases in Phytozome v7.0. A large proportion of the novel sRNAs identified in andromonoecious *P. tomentosa* did not have perfect matches in different genome databases. Only 6 male-specific sRNA and 5 female-specific sRNAs were detected in other species ([Table S2](#pone.0062681.s004){ref-type="supplementary-material"}).

![First nucleotide bias of novel candidate miRNAs in andromonoecious poplar female and male flower libraries.](pone.0062681.g003){#pone-0062681-g003}

10.1371/journal.pone.0062681.t003

###### Summary of common novel miRNAs in female and male flower libraries.

![](pone.0062681.t003){#pone-0062681-t003-3}

  Name          Sequence (5′-3′)      LP     MFE         miRNA location        miRNA reads   Ratio(Female/Male)   miR\*reads  
  --------- ------------------------ ----- ------- -------------------------- ------------- -------------------- ------------ -----
  Pto-F1     ATAGGTTTCCGATCATTCCTCC   169   −81.7     chr13∶103779--103800        8990              391             22.99      NO
  Pto-F2a    TGAAATGCCATGAACAACGATC   125   −53.4   chr13∶11993451--11993472       433              8111             0.05      NO
  Pto-F2b    TGAAATGCCATGAACAACGATC   115    −47     chr16∶4142967--4142987        377              942              0.40      NO
  Pto-F3     ATGTAATCAAGGCTGCTAGTC    141   −72.3    chr17∶8682859--8682879        71               434              0.16      Yes
  Pto-F4     TCTAAAGATCCGGCGGTCGGC    124    −44     chr18∶2804698--2804718        63                90              0.70      NO
  Pto-F5     TATGTTTGACAGAAACCCCCT    171   −89.2    chr18∶8350113--8350132        729              1991             0.37      NO
  Pto-F6     TGTGGTAGATATGTGAGGATT    148   −65.2   chr19∶10983127--10983147       472              233              2.03      NO

10.1371/journal.pone.0062681.t004

###### Summary of male-specific novel miRNAs.

![](pone.0062681.t004){#pone-0062681-t004-4}

  Name            Sequence (5-3)       LP     MFE          miRNA location        miRNA reads   miR\*reads
  ---------- ------------------------ ----- -------- -------------------------- ------------- ------------
  Pto-F7      CTCAAACTTTTCTCTTACTTC    283   −168.1    chr2∶1282971--1282991         147           NO
  Pto-F8      TACCGTTAGAAAGATCTCAAC    179    −62      chr2∶8266846--8266866         126           NO
  Pto-F9      ATAGGTTTCCGATCATTCCTCC   199   −85.04     chr5∶119707--119728          819          YES
  Pto-F10     TTTTGCACGTAGAATTCAGGA    139    −38     chr5∶18398105--18398125        147           NO
  Pto-F11     ACGAGCCATCATAACTGTAGG    125   −81.92    chr6∶6767480--6767500         714          YES
  Pto-F12     TAGTTCGAGGGACCAAAATCA    219   −111.4    chr6∶8063061--8063081         357          YES
  Pto-F13     ATCTGCTTTCTGCGACTCCTC    183   −68.8     chr8∶8028138--8028158         126           NO
  Pto-F14     GCATTTGGACGTCGGGGAACT    90    −23.7     chr9∶7157296--7157316         357           NO
  Pto-F15a    AGGATTGGAGGGAATTAAACA    135   −40.55   chr10∶20020250--20020270       105           NO
  Pto-F15b    AGGATTGGAGGGAATTAAACA    129   −41.7    chr12∶11845073--11845093       105           NO
  Pto-F16     TAATTCCATGACTGTGTACAG    156   −37.82   chr15∶10265155--10265175       189           NO
  Pto-F17a    TTTAATTTCCTCCAATATCTTA   133   −45.54   chr18∶14578403--14578424       210           NO
  Pto-F17b    TTTAATTTCCTCCAATATCTTA   136   −45.54   scaffold_269∶8974--8995        210           NO
  Pto-F17c    TTTAGTTTCCTCCAATATCTTA   136   −41.2    scaffold_269∶9454--9475        189           NO

10.1371/journal.pone.0062681.t005

###### Summary of female-specific novel miRNAs.

![](pone.0062681.t005){#pone-0062681-t005-5}

  Name            Sequence (5-3)       LP     MFE          miRNA location         miRNA reads   miR\*reads
  ---------- ------------------------ ----- -------- --------------------------- ------------- ------------
  Pto-F18     TTGGTTAAACTCCCATCAGGA    133   −65.6      chr1∶7117690--7117710         126           NO
  Pto-F19      CTCAGATTAGCCAGGTGCCT    117   −44.3     chr1∶10983482--10983501        105           NO
  Pto-F20     TTATTAAACCCGGACCGGCCT    122   −54.62    chr1∶18312617--18312637        126           NO
  Pto-F21     TACCCGGTTACTGTGCATGTGC   76    −33.3     chr1∶38996098--38996119        126           NO
  Pto-F22     TATCCTTGGATCTGACGACCA    220   −123.4    chr1∶39819960--39819980        294           NO
  Pto-F23     TTTTCGATCCAAGCTTCGGGT    80    −27.1     chr1∶42987729--42987749        105           NO
  Pto-F24     TTACTTCCTTTTGTCCCTCTC    303   −177.4     chr2∶1282957--1282977         357           NO
  Pto-F25      TATAGATTGCAGAGGGAACC    203   −71.04    chr2∶12233873--12233892        126           NO
  Pto-F26     TTATTAAACCCGGACCGGCCT    78    −42.8     chr2∶19952785--19952805        126           NO
  Pto-F27     TCCGAGCTCTAATTATGTGGG    182    −92      chr2∶21648214--21648234        798           NO
  Pto-F28     TTGGGCTGGCAGTTGTGATGAC   161   −45.87    chr3∶19076220--19076241        231           NO
  Pto-F29     TTCCGGTTTTTGGGACTCCGAT   103   −35.1     chr4∶20319258--20319279        105           NO
  Pto-F30     TTCTTTCCTATCAAACTTCAAC   191   −92.4     chr4∶22210258--22210279        105           NO
  Pto-F31     TGAAGATAAGAGCTTGTTTGG    169   −75.86     chr5∶6548077--6548097         651           NO
  Pto-F32     TTTGCACGTAGAATTCAGGATT   139    −38      chr5∶18398103--18398124        588           NO
  Pto-F33     ATGGATTGATGAAGATGAGGA    92    −26.3     chr5∶21111319--21111339        105           NO
  Pto-F34     TTATTTCCTCAAACTTTCCTC    256   −159.8    chr5∶24262419--24262439        273           NO
  Pto-F35     TAGAACCTGACCAGTTGAGCT    116    −30       chr6∶2313141--2313161         105           NO
  Pto-F36     TGGCCCATGATCTTCATTGTG    115   −78.02     chr6∶6767559--6767579         357          YES
  Pto-F37     TTTTTGTTGTTTAAGAACCCT    301   −128.6    chr6∶21307737--21307757        315           NO
  Pto-F38     TTGGGCTGGCAGTTGTGATGAC   161   −46.2       chr7∶301518--301539          231           NO
  Pto-F39     TGTGGGACTCGAATTAGTGAT    96    −26.43    chr7∶13307862--13307882        147           NO
  Pto-F40a     GAGGAGGGAGAGAGATCTGT    136   −38.3      chr8∶5498368--5498387         126           NO
  Pto-F40b     GAGGAGGGAGAGAGATCTGT    114   −34.3      chr8∶5522852--5522871         126           NO
  Pto-F41     ATCCAACGGTTAGATCTCCCT    296   −162.7     chr8∶8443078--8443098         105           NO
  Pto-F42     CTTGAAACAATGTTTGGTGCAG   121   −31.9     chr8∶10699331--10699352        105           NO
  Pto-F43     TAGAGTATAAGAGGATCGACT    124   −32.4     chr8∶15869891--15869911        147           NO
  Pto-F44      ACTTTGACCTGAACTTGCCC    79     −40       chr9∶8255922--8255941         105           NO
  Pto-F45     TGTCCTGACTCGAACTCGAGA    82    −21.2     chr10∶7102937--7102957         189           NO
  Pto-F46     TGAAGATAAGAGCTTGTTTGG    169   −77.06    chr10∶7227906--7227926         651           NO
  Pto-F47     GGGACTGCTGTAGATGCTTGG    90    −34.7    chr10∶13462743--13462763        105           NO
  Pto-F48     TTGATGACTGATCTTGAGCAT    99    −62.8    chr10∶16376128--16376148        189          YES
  Pto-F49     TTGTAAGAGCCTGAGACAGCC    143   −51.8    chr10∶18334850--18334870        714           NO
  Pto-F50     TTTAATTTCCTCCAATATCTCA   169   −45.91   chr10∶20020296--20020317        399           NO
  Pto-F51     TTGGAATCCTCTCTGATAATGC   105   −30.5    chr10∶20322598--20322619        105           NO
  Pto-F52     TTTAGGACCCGTGGGATTAGTC   102    −27      chr11∶8039096--8039117         189           NO
  Pto-F53     TCAATGGGTAAAAATTCTGACC   96    −24.4     chr12∶3834076--3834097         126           NO
  Pto-F54     TTGGAGCTCGAGACTTGGCAC    90    −25.74    chr13∶1347925--1347945         105           NO
  Pto-F55     ATCACTGTGTCTATTAGGATGG   82    −28.1     chr13∶6333415--6333436         105           NO
  Pto-F56     TGCTAGGACCAAGTTTTCTGG    103   −53.34    chr13∶9970875--9970895         609          YES
  Pto-F57     TTCAGGACCTGTAGAATTAGTC   92    −24.2    chr13∶12124499--12124520        105           NO
  Pto-F58a    TGAATGGCTGTTGAACTTGGC    156   −49.5      chr14∶293794--293814          168           NO
  Pto-F58b    TGAATGGCTGTTGAACTTGGC    158   −45.51     chr14∶726835--726855          168           NO
  Pto-F58c    TGAATGGCTGTTGAACTTGGC    158   −52.6     chr14∶1080042--1080062         168           NO
  Pto-F59     TGATGGGTCTCATTTAGTAGA    215   −86.36    chr14∶5877228--5877248         441           NO
  Pto-F60     AACCGAACCGAACTGAACCGA    98    −33.4    chr14∶16267732--16267752        105           NO
  Pto-F61     TTATAGTTTTGAAATCCGGCC    95    −47.2     chr15∶5715046--5715066         147           NO
  Pto-F62     AACCCAAGAACTCAACTCTGT    171   −68.37    chr15∶6997081--6997101         126           NO
  Pto-F63     GTACGATCCTATAGAACAGAT    116   −34.5    chr15∶15015652--15015672        189          YES
  Pto-F64     CAACACCAGACCCAAGAGCTT    117   −44.51    chr16∶5544896--5544916         210           NO
  Pto-F65      TTAGGGTTTAGGGTTTAGAA    157   −41.6      chr17∶210550--210569          126           NO
  Pto-F66      TCGAGATTGTACTGTTCATA    77    −23.6     chr17∶4120360--4120379         105           NO
  Pto-F67     TGGCAAATGATCTGCATCTGC    90    −24.52   chr17∶13666346--13666366        105           NO
  Pto-F68      GTGGGTGGGTCTGGGTGGCA    118   −47.7    chr18∶10507789--10507808        147           NO
  Pto-F69     TTTGCTCTTCGTTTTCTCATG    170   −77.8    chr18∶14336738--14336758        294           NO
  Pto-F70     TTTTATTCCTGACTCTGGCATC   166   −43.31     chr19∶907650--907671          126           NO
  Pto-F71     TTGGGAATTATTACAATGGCA    106   −31.8     chr19∶1460855--1460875         147           NO
  Pto-F72     TTATTAAACCCGGACCGGCCT    87    −39.6     chr19∶2013039--2013059         126           NO
  Pto-F73     TGTTTTCCGGAAAGTAGTTTC    99    −27.1     chr19∶8890465--8890485         378           NO
  Pto-F74     TAGGGCAGAGAATTGAATGACT   119   −45.4    chr19∶10646670--10646691        252           NO
  Pto-F75     TCGAGATTTCTTTGTTGGAGCT   125   −42.72   scaffold_1233∶2309--2330        126           NO
  Pto-F76     TCGGCGTTGATGTAGAATGGC    153   −53.1    scaffold_401∶10122--10142       126           NO
  Pto-F77     TTTTGAGACAATGCTGAAAAT    99    −28.4    scaffold_65∶42511--42531       1953           NO
  Pto-F78     ACATGAAACGGCGTCGTTTTG    91     −26     scaffold_76∶39681--39701        126           NO

Novel miRNA and Targets were Located in Gender Determination Regions {#s2d}
--------------------------------------------------------------------

It was reported that Chromosome XIX is an incipient sex chromosome in *Populus* [@pone.0062681-Yin1], [@pone.0062681-Tuskan2]. In the present study, 5 novel miRNAs located in chromosome XIX were identified, including Pto-F6, Pto-F70, Pto-F71, Pto-F72 and Pto-F73 ([Table 6](#pone-0062681-t006){ref-type="table"}; [Figure 4A](#pone-0062681-g004){ref-type="fig"}). Pto-F6 was expressed both in female and male flower libraries, but expressed at higher levels in female flowers than in male ([Table 3](#pone-0062681-t003){ref-type="table"}). The Pto-70, Pto-71, Pto-72 and Pto-73 female-specific miRNAs were weakly expressed in female flowers ([Table 5](#pone-0062681-t005){ref-type="table"}). A total of 33 targets located on chromosome XIX were identified for these novel miRNAs. Among these, Pto-F6 and Pto-73 were predicted to match 14 targets respectively that are located on chromosome XIX ([Table 6](#pone-0062681-t006){ref-type="table"}). By contrast, no targets were identified for Pto-F72. Putative functions of these targets genes were annotated by BLAST searches against the TAIR, NCBI and JGI protein datasets. The results revealed that target genes are involved in disease resistance, response to auxin stimulus, decreased DNA methylation and membrane transport. Yin et al (2008) indicated that a 2.45 Mb segment in the peritelomeric region of chromosome XIX and is associated with gender determination [@pone.0062681-Yin1]. In our study, Pto-70 and 4 targets genes were found in sex-determination regions ([Figure 4B](#pone-0062681-g004){ref-type="fig"}). Among the 4 target genes, 3 targets were predicted to match Pto-73 miRNAs and one was predicted to match Pto-F6 miRNA.

![Distribution of novel miRNAs on chromosome XIX.\
A. 6 novel miRNAs and their targets located on chromosome XIX. B. Schematic diagram of the order of 6 novel miRNAs and their targets on chromosome XIX. Each miRNA and corresponding targets are marked the same color. Putative sex determination region in a 2.45-Mb region at the peritelomeric end of chromosome XIX, based on Yin et al (2008).](pone.0062681.g004){#pone-0062681-g004}

10.1371/journal.pone.0062681.t006

###### Targets of novel *P. tomentosa* miRNAs verified in XIX chromosome of poplar.

![](pone.0062681.t006){#pone-0062681-t006-6}

  miRNA      Target Gene Model^a^                        Putative function^b^                                Location            TAIR Gene Model^c^
  --------- ---------------------- ---------------------------------------------------------------- --------------------------- --------------------
  Pto-F6       POPTR_0019s00970        negative regulation of catalytic activity, proteolysis,         Chr19∶737356--739497          AT1G04110
               POPTR_0019s01610                       response to auxin stimulus                      Chr19∶1429616--1430026         AT5G53590
               POPTR_0019s02760                        protein phosphorylation                        Chr19∶2561092--2568350         AT1G53430
               POPTR_0019s02880                        Pentatricopeptide repeat                       Chr19∶2750320--2751951         AT2G34400
               POPTR_0019s03110     Encodes a lipid acyl hydrolase with wide substrate specificity    Chr19∶3122643--3124999         AT2G26560
               POPTR_0019s03700          NB-ARC domain-containing disease resistance protein          Chr19∶3775544--3779105         AT4G27220
               POPTR_0019s03970          NB-ARC domain-containing disease resistance protein          Chr19∶4034540--4038218         AT4G27220
               POPTR_0019s06080           KAR-UP F-box 1 (KUF1); CONTAINS InterPro DOMAIN/s           Chr19∶6589704--6590843         AT1G31350
               POPTR_0019s10290      LRR and NB-ARC domains-containing disease resistance protein     Chr19∶11867256--1870843        AT3G14460
               POPTR_0019s10300      LRR and NB-ARC domains-containing disease resistance protein    Chr19∶11884201--11888700        AT3G14460
               POPTR_0019s10890                similar to expression supported by MPSS               Chr19∶12374345--12375027        AT1G35430
               POPTR_0019s12450               similar to chloroplast precursor (TRX-M1)              Chr19∶13509839--13510150        AT4G03520
               POPTR_0019s13380                                                                      Chr19∶14290118--14294196        AT5G08415
               POPTR_0019s15030                 similar to decreased DNA methylation 1               Chr19∶15655586--15661763        AT5G66750
  Pto-F70      POPTR_0019s02280                     member of Myosin-like proteins                    Chr19∶2112309--2122774         AT1G54560
               POPTR_0019s10800                           Encodes a cyclase                          Chr19∶12293003--122983961       AT1G78955
               POPTR_0019s15290                            unknown protein                           Chr19∶15825895--15830273        AT5G01970
  Pto-F71      POPTR_0019s03120     Encodes a lipid acyl hydrolase with wide substrate specificity    Chr19∶3134481--3137841         AT2G26560
               POPTR_0019s08530                     ATP phosphoribosyl transferase                   Chr19∶10043845--10045971        AT1G09795
  Pto-F73      POPTR_0019s00820                      ENTH/VHS/GAT family protein                       Chr19∶545449--549077          AT5G63640
               POPTR_0019s01010          NB-ARC domain-containing disease resistance protein           Chr19∶774829--779781          AT4G27220
               POPTR_0019s01080          NB-ARC domain-containing disease resistance protein           Chr19∶820958--823839          AT4G27220
               POPTR_0019s02030                                                                       Chr19∶1889627--1891189    
               POPTR_0019s02060      LRR and NB-ARC domains-containing disease resistance protein     Chr19∶1922498--1924769         AT4G10780
               POPTR_0019s02180          NB-ARC domain-containing disease resistance protein          Chr19∶2029692--2032970         AT4G27220
               POPTR_0019s02200          Disease resistance protein (CC-NBS-LRR class) family         Chr19∶2058206--2062218         AT1G12290
               POPTR_0019s02410                                                                       Chr19∶2215592--2216219    
               POPTR_0019s03080          NB-ARC domain-containing disease resistance protein          Chr19∶3094719--3100126         AT4G27220
               POPTR_0019s03480           similar to phosphoenolpyruvate-carboxylase kinase           Chr19∶3561751--3563659         AT3G04530
               POPTR_0019s03800               similar to SNF2 domain-containing protein               Chr19∶3871132--3874229         AT1G08600
               POPTR_0019s05300      similar to putative disease resistance gene analog NBS-LRR.      Chr19∶5594198--5607481         AT4G27190
               POPTR_0019s05990              integral membrane transporter family protein             Chr19∶6422952--6425657         AT1G64890
               POPTR_0019s12230                     similar to GTP-binding protein                   Chr19∶13325801--13327862        AT5G60860

Target Gene Model^a^ and Putative function^b^ were derived from Joint Genome Institute (JGI: <http://www.jgi.doe.gov/>) and (TAIR : <http://www.arabidopsis.org>).

TAIR Gene Model^c^ were derived from TAIR (TAIR : <http://www.arabidopsis.org>).

Target Prediction for Pto-miR156, Pto-miR159, Pto-miR172 and Pto-miR319 and Novel miRNAs in Andromonoecious *P. tomentosa* {#s2e}
--------------------------------------------------------------------------------------------------------------------------

To understand the functions of sex-specific flower development related miRNAs, the first step is to predict and experimentally validate their targets. Consequently, we predicted 25 target genes for Pto-miR156, Pto-miR159, Pto-miR172 and Pto-miR319 that were different from the previously defined *Populus* miRNA targets (miRBase 19.0), and a total of 464 targets were identified for the 78 novel miRNAs ([Table S3](#pone.0062681.s005){ref-type="supplementary-material"}). The number of predicted targets varied from 1 to 15 per miRNA. The categorization of female and male-specific miRNA target genes according to biological process is shown in [Figure 5](#pone-0062681-g005){ref-type="fig"}. After putative functional annotation by BLAST searches against the TAIR, NCBI and JGI protein datasets, all targets of female and male-specific miRNAs that had a greater than twofold difference in expression between the sexes (Ratio≧2or≦0.5) were identified as candidate genes for gene functional enrichment analysis. All the target genes between the sexes were functionally characterized using enrichment analysis of gene sets, mostly as described by Gene Ontology terms ([Figure 5](#pone-0062681-g005){ref-type="fig"}). GO analysis showed that response to hormone stimulus, negative regulation of gene expression, regulation of flower development, and organ formation were significantly enriched terms for biological processes.

![AgriGO analysis of target genes for statistically enriched GO terms in the 'Biological process' ontology.\
Coloring of GO term nodes is proportional to their significance as indicated by the scale.](pone.0062681.g005){#pone-0062681-g005}

Expression Profiling of miRNAs and Targets in Andromonoecious *P. tomentosa* {#s2f}
----------------------------------------------------------------------------

Andromonoecious poplar, an exceptional model system, eliminates confounding effects of genetic background of dioecious plants and thus is a powerful system in which to identify accurate sex-specific factors related to flower development. Therefore, the comparison of the small RNA profiles of female and male flowers of andromonoecious poplar was informative for characterization of functional miRNAs involved in sex-specific flower development. We sequenced 134 conserved miRNA sequences and 78 predicted novel miRNA sequences. 70.1% (94/134) of conserved miRNA sequences were up-regulated in female flower, and only 40 conserved miRNAs were up-regulated in male flower. A total of 104 conserved and 2 novel miRNAs were verified as differentially expressed by greater than two-fold between the two libraries (ratio \>2 or \<0.5 and p\<0.01), including 94 conserved and 2 novel miRNAs that were up-regulated in female flower and 37 conserved and 4 novel miRNAs that were up-regulated in male flower ([Table 2](#pone-0062681-t002){ref-type="table"} and [3](#pone-0062681-t003){ref-type="table"}). Moreover, 3 conserved miRNAs (miRNA 397, miRNA 6459 and miRNA6462) and 72 novel miRNAs, including 11 male-specific and 61 female-specific, were expressed in a sex-specific manner ([Table 4](#pone-0062681-t004){ref-type="table"} and [5](#pone-0062681-t005){ref-type="table"}). To examine the detailed expression pattern of these potential miRNAs in andromonoecious poplar flowers, the 27 differentially expressed miRNAs, including four conserved and 23 novel miRNAs, were verified by real-time quantitative PCR ([Figure 6A](#pone-0062681-g006){ref-type="fig"}). Overall, comparisons of these data correspond to the deep sequencing results, indicating that the miRNA expression levels determined by high-throughput sequencing are reliable.

![Pearson correlation Coefficient (PCC) heat map representing expression of miRNAs and their targets in andromonoecious poplar flower organs.\
Red and yellow indicate higher levels of transcriot and green and blue indicate lower levels of transcript. The names of samples are shown at the top. The gene model and gene annotation are shown on the right side. A: Heatmap of conserved and novel miRNAs. B: Heatmap of target genes.](pone.0062681.g006){#pone-0062681-g006}

Base on the results of gene enrichment analysis, 31 putative target genes were selected as candidate genes for gene expression analysis ([Table 7](#pone-0062681-t007){ref-type="table"} and [Figure 6B](#pone-0062681-g006){ref-type="fig"}). The expression of miRNA target genes was detected using real-time quantitative PCR. For most target genes, expression was negatively correlated with the levels of a given miRNA, which is in accordance with the gene silencing function of miRNAs. It should be noted that expression of *CONSTANS-LIKE 6* (*COL6*), the target of PtoF25, was not correlated with the abundance of PtoF25 ([Figure 6B](#pone-0062681-g006){ref-type="fig"}), indicating that *COL6* expression might be regulated by other mechanisms.

10.1371/journal.pone.0062681.t007

###### Candidate targets of novel and conserved miRNAs in andromonoecious *P. tomentosa*.

![](pone.0062681.t007){#pone-0062681-t007-7}

  miRNA       Cleave sites   Target Gene Model^a^                      Putative function^b^                      Accession number   TAIR Gene Model
  ---------- -------------- ---------------------- ------------------------------------------------------------ ------------------ -----------------
  miRNA167        392          POPTR_0013s00750               similar to MAINTENANCE OF METHYLATION                  KC477290          AT5G66750
  Pto-F6          235          POPTR_0005s14640            similar to gibberellin-responsive protein 5               KC477281          AT3G02380
  Pto-F6          305          POPTR_0004s18020         similar to myb family transcription factor (MYB4);           KC477284          AT1G74670
  Pto-F6          394          POPTR_0019s15030               similar to DECREASED DNA METHYLATION 1                 KC297686          AT1G08060
  Pto-F7          193          POPTR_0004s10800               similar to CONSTANS-like protein CO2.                  KC477292          AT4G38620
  Pto-F11         228          POPTR_0012s03530             similar to EARLY-PHYTOCHROME-RESPONSIVE1;                KC477285          AT1G18330
  Pto-F14         336          POPTR_0010s13870            similar to PHYTOCHROME AND FLOWERING TIME 1               KC477286          AT3G60460
  Pto-F16         310          POPTR_0015s07740          calcium-dependent protein kinase family protein             KC477266          AT1G68520
  Pto-F19         548          POPTR_0002s02970           similar to Zinc finger protein CONSTANS-LIKE 6             KC477293          AT1G25540
  Pto-F19         270          POPTR_0014s05370             similar to myb family transcription factor               KC297692          AT1G75540
  Pto-F25         139          POPTR_0010s13490           similar to Zinc finger protein CONSTANS-LIKE 6             KC477294          AT1G19870
  Pto-F28         293          POPTR_0002s02680                 calmodulin-binding family protein                    KC477283          AT4G36920
  Pto-F36         283          POPTR_0005s18400                  similar to APETAL2-like protein                     KC477268          AT3G10300
  Pto-F45         481          POPTR_0006s04590               calcium-binding EF hand family protein                 KC477282          AT3G15210
  Pto-F47         125          POPTR_0001s40770      similar to Ethylene responsive element binding factor 4         KC477274          AT4G34760
  Pto-F47          99          POPTR_0004s17150                similar to auxin-responsive protein                   KC477279          AT4G36920
  Pto-F51         815          POPTR_0007s10780                  similar to APETAL2-like protein                     KC477295          AT3G16990
  Pto-F54         224          POPTR_0008s10610              similar to seed maturation protein PM36                 KC477289          AT1G74740
  Pto-F54         180          POPTR_0001s16100              similar to UNUSUAL FLORAL ORGANS protein                KC477287          AT1G30950
  Pto-F55         321          POPTR_0007s08330                     similar to ABA DEFICIENT 3                       KC477272          AT5G66750
  Pto-F56         513          POPTR_0010s11350                  similar to CYTOKININ RESPONSE 1                     KC477269          AT2G01830
  Pto-F56         167          POPTR_0007s12710               similar to DECREASED DNA METHYLATION 1                 KC477291          AT1G16540
  Pto-F58a        311          POPTR_0008s13720                  similar to CYTOKININ RESPONSE 1                     KC477270          AT2G01830
  Pto-F64         168          POPTR_0018s06080     similar to ethylene-responsive transcriptional coactivator       KC477275          AT3G24500
  Pto-F65         152          POPTR_0002s25330                    similar to ABA insensitive 3                      KC477273          AT3G02380
  Pto-F66          71          POPTR_0004s10800                 similar to CONSTANS-like protein 1                   KC477292          AT3G24650
  Pto-F66         187          POPTR_0017s14410                 similar to CONSTANS-like protein 1                   KC477267          AT5G43700
  Pto-F68         253          POPTR_0012s04470            similar to Auxin efflux carrier component 1               KC477277          AT3G02380
  Pto-F68         166          POPTR_0005s05550               similar to Auxin-induced protein IAA4                  KC477278          AT1G73590
  Pto-F68          89          POPTR_0008s10100                  similar to gibberellin 2-oxidase                    KC477280          AT1G47990
  Pto-F73         421          POPTR_0013s01640              dormancy/auxin associated family protein                KC477276          AT5G21482
  Pto-F76         221          POPTR_0006s23760      similar to cytokinin oxidase; similar to putative (CKX5)        KC477271          AT1G56220

Target Gene Model^a^ and Putative function^b^ were derived from Joint Genome Institute (JGI: <http://www.jgi.doe.gov/>) and (TAIR : <http://www.arabidopsis.org>).

A Possible Functional Network of Sex-specific Flower Development Related miRNAs in Andromonoecious *P. tomentosa* {#s2g}
-----------------------------------------------------------------------------------------------------------------

The idea that miRNAs could play important roles in flower development has been suggested in previous studies. However, no systematic investigation has been conducted into how flower development may be regulated via miRNA-mediated processes. In the present study, a possible functional network, which connects flower development related miRNAs with their targets were first reported ([Table 7](#pone-0062681-t007){ref-type="table"} and [Figure 7](#pone-0062681-g007){ref-type="fig"}). Base on the gene functional annotation, all target genes were divided into four subgroups as shown in [Figure 7](#pone-0062681-g007){ref-type="fig"}.

![A possible functional network of flower development related miRNAs in andromonoecious *P. tomentosa*.\
Black line represent repression of target gene expression.](pone.0062681.g007){#pone-0062681-g007}

The first subgroup related to floral development was regulated by 9 miRNAs including Pto-F6, Pto-F11, Pto-F14, Pto-F19, Pto-25, Pto-36, Pto-F51, Pto-F54 and Pto-F66. Four members of the *CONSTANS-LIKE* gene family were negatively regulated by three female-specific miRNAs including Pto-F19, Pto-F25 and Pto-F66. Of these, Pto-F66 was found to target 2 members of the *CONSTANS-LIKE2* gene family. Also, *EARLY-PHYTOCHROME-RESPONSIVE1* (*EPR1*) and *PHYTOCHROME AND FLOWERING TIME 1* (*PFT1*) were negatively regulated by two male-specific miRNAs, Pto-F11 and Pto-F14, respectively. In our study, we detected two novel miRNAs, Pto-F36 and Pto-51, targeting *APETALA 2* (*AP2*). Female flower specific Pto-F54 was found to target two genes, *UNUSUAL FLORAL ORGANS* (*UFO*) and *SEED MATURATION PROTEIN* (*PM36*).

Three miRNAs are involved in regulation of the second subgroup, genes that relate to Ca^2+^ transport. Two female flower specific miRNAs, Pto-F28 and Pto-F45, were found to be negative factors for the expression of *CALMODULIN-BINDING FAMILY PROTEIN* and *CALCIUM-BINDING EF HAND FAMILY PROTEIN*. By contrast, *CALCIUM-DEPENDENT PROTEIN KINASE FAMILY PROTEIN* (*CDPK*) gene expression was repressed by Pto-F16, a male-specific miRNA.

In the third subgroup, there were 10 miRNAs involved in hormone related gene regulation. All of these miRNAs were female flower specific, except Pto-F6, which had expression significantly higher in female flower than in male. Pto-F68 has three target genes, including *AUXIN EFFLUX CARRIER COMPONENT 1*, *AUXIN-INDUCED PROTEIN* (*IAA4*) and *GIBBERELLIN 2-OXIDASE*. By contrast, *ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR 4* (*EREBF4*) and *AUXIN-RESPONSIVE PROTEIN* (*SAUR29*) were targeted by the same novel miRNA, Pto-F47. Two genes related to ABA, *ABA DEFICIENT 3* (*ABA3*) and *ABA INSENSITIVE 3* (*ABI3*), were targeted by female flower specific miRNAs Pto-F55 and Pto-F65, respectively.

In the last subgroup, genes that relate to DNA methylation were regulated by 3 miRNAs. *MAINTENANCE OF METHYLATION1* (*MET1*) was negatively regulated by miRNA167, which had expression significant higher in male flower than in female. By contrast, two members of *DECREASED DNA METHYLATION 1* (*DDM1*) were negatively regulated by Pto-F6, which had higher expression in female flower and by female flower specific Pto-F56, respectively.

Discussion {#s3}
==========

Overview of the Deep Sequencing Datasets {#s3a}
----------------------------------------

Although an increasing number of studies about miRNAs and their expression patterns from different tissues have been reported, there are still few studies on sex-specific miRNAs between female and male flowers of poplar. In this study, andromonoecious poplar, an exceptional model system, was used to eliminate confounding effects of genetic background of dioecious plants. We generated sRNA libraries from female and male flower of andromonoecious poplar and obtained over 40 million reads of 18--30 nt per library, substantially increasing the available data on poplar flower sRNAs [@pone.0062681-Pantaleo1], [@pone.0062681-Zhao1]. Mapping analysis of unique sRNAs revealed that over 60% of the identified sRNAs were previously unknown, suggesting that even more sRNAs remain to be identified. The size distribution of small RNAs indicated that the numbers of 21-nt sRNAs were significantly higher than 24-nt sRNAs in female flower, suggesting that female flower development may require more sRNAs in this size class. By contrast, the numbers of 21-nt sRNAs and 24-nt sRNAs were not significantly different in male flower libraries. The 24-nt sRNAs mainly comprised siRNAs associated with repeats and transposons [@pone.0062681-Lippman1], [@pone.0062681-Lu1]. 24-nt sRNA numbers in male flower were significantly higher than in female, suggesting that a number of siRNAs related to repeats and transposons are involved in male flower development.

Conserved miRNAs Expressed in Flower {#s3b}
------------------------------------

We detected dramatically different expression levels among different members of the same miRNA family, suggesting that expression of conserved miRNAs varies in andromonoecious *P. tomentosa*. These conserved miRNAs have been identified in multiple flowering pathways. For example, three miRNA families (miR172, miR159/miR319 and miR156) are involved in flowering-time regulation [@pone.0062681-JonesRhoades1]. In this study, miRNA159 and miRNA319 expression in female flower was 38.8-fold and 51.42-fold higher, respectively, than in male flower. miRNA172 was weakly expressed in female and male flower, with no significant difference. By contrast, miRNA156 was expressed in male flower at higher levels than in female flower. This result suggested that different miRNAs are involved in female and male flowering time regulation. miRNA159 and miRNA319 also have been reported to act with miRNA164 and miRNA167 in specifying particular cell types during the later stages of flower development [@pone.0062681-Nag1]. The present study revealed that miRNA164 was not expressed in female or male flower, indicating miRNA164 might show stage-specific expression in flower development. miRNA167 was up-regulated dramatically in male flower, indicating that it may play an important role in specifying male flower cell types. miRNA156, miRNA157 and miRNA172 may be components of a regulatory pathway mediating the transition between the vegetative and reproductive phases in plants [@pone.0062681-Nag1]. miRNA166, targeting *APETALA2* and type III homeodomain-leucine zipper (HD-Zip) family genes, was dramatically up-regulated in female flower, suggesting *AP2* and HD-ZiP genes might be negatively regulated in female flower [@pone.0062681-Ji1]. miR167 and miR160 are thought to control transcription in response to the phytohormone auxin by targeting mRNAs coding for ARF DNA-binding proteins [@pone.0062681-Rajagopalan1], [@pone.0062681-Reinhart1]. These miRNAs were detected in this study as having opposite expression patterns between female and male flower, suggesting that two miRNAs involved in regulation of auxin responses in female flower are different compare with in male flower.

Novel miRNAs Displayed Different Expression Patterns between Female and Male Flower {#s3c}
-----------------------------------------------------------------------------------

The 78 novel miRNAs identified here spanned a size range of 20--22 nt, and the majority started with a 5′ U, consistent with results in Japanese apricot [@pone.0062681-Gao1]. The majority of novel miRNAs showed relatively low expression levels, which was consistent with previous studies predicting that novel miRNAs are often expressed at lower levels than conserved miRNAs [@pone.0062681-Fahlgren1]. The miRNA\*s were generally less abundant than the corresponding mature miRNAs. It has been reported that miRNA\*s have important functions through regulation of the miRNA precursors themselves when miRNA\*s were more abundant than their corresponding miRNAs [@pone.0062681-German1]. miRNA\*s identified were used as extremely strict criteria to identify novel miRNAs [@pone.0062681-Meyers1]. In our study, only 8 unique miRNAs were detected with complementary miRNA\*s. Most novel miRNAs do not have identified miRNAs\*, likely because of the depth of sequencing, which results in a low probability of identifying low-frequency miRNA\*s. The numbers of female-specific novel miRNAs was 6-fold higher than male-specific novel miRNAs, suggesting that abundant novel miRNAs are involved in female flower development. A large proportion of the novel sRNAs identified in andromonoecious *P. tomentosa* did not have perfect matches in different genome databases, providing more evidence to support our conclusion that these novel miRNAs might be poplar-specific and were first reported in present study. Among all of the novel miRNAs, 11 miRNAs were found with perfect matches in other species, suggesting that some of these newly identified miRNAs may be broadly conserved within angiosperms.

Novel miRNAs Located in Sex Determination Regions {#s3d}
-------------------------------------------------

Although studies on determination of gender indicate that dioecy in *Populus* is genetically controlled, the precise gender-determining systems remain unclear. Based on genetic mapping results, a gender determination locus mapped to different positions on chromosome XIX, depending upon the *Populus* species [@pone.0062681-Gaudet1]--[@pone.0062681-Paolucci1]. Klevebring et al. (2009) found that the proposed sex-determining peritelomeric region of chromosome XIX showed a distinctive pattern of sRNA occurrence that differed significantly from the rest of the genome [@pone.0062681-Klevebring1]. In our study, 5 novel miRNAs and 33 targets genes were located on chromosome XIX, indicating that these miRNAs might be involved in sex determination of poplar. The annotated results revealed that over one-third of targets were annotated as disease resistance genes. It has been reported that a NBS--LRR gene supercluster was located on *Populus* chromosome XIX. These results are consistent with the hypothesis that resistance to a floral pathogen and regulation of gender determination coevolved in a region of the genome that experiences reduced recombination [@pone.0062681-Tuskan2]. Interestingly, Pto-F6 was predicted to match a target that encodes DNA (cytosine-5) -methyltransferase implying that DNA methylation might act in flower development.

miRNA Targets and Sub-network Analysis {#s3e}
--------------------------------------

To better understand the functions of identified miRNAs, we sought to identify potential targets in andromonoecious *P. tomentosa* using computational analyses. Many targets of conserved miRNAs have been predicted and their functions annotated [@pone.0062681-Lu2], [@pone.0062681-Lu3]. Integrating target genes and miRNA expression data identified sub-networks for further investigation. *COL1* encodes a zinc finger protein belonging to the first groupings of the flowering-time gene *CONSTANS* (*CO*) [@pone.0062681-Griffiths1]. Over-expression of *COL1* can shorten the period of two distinct circadian rhythms by affecting a light input pathway [@pone.0062681-Ledger1]. In our study, *COL1* was targeted by female flower specific Pto-F66, which was expressed in female flowers suggesting that the novel miRNA Pto-F66 likely plays a negative role in the light input pathway in female flower. By contrast, *COL6* is part of the third groupings of *CO*, and is a target of Pto-F19 and Pto-F25. However, these two miRNAs were not correlated with *COL6* transcript abundance, indicating that *COL6* expression might be regulated by other mechanisms. The target of male specific miRNA Pto-F11, *EPR1* encodes a nuclear-localized MYB protein showing rapid induction in response to red light irradiation. *EPR1* was significantly down-regulated in male flower, suggesting that this primary phytochrome-responsive factor is likely repressed by the male flower specific novel miRNA Pto-F11 [@pone.0062681-Kuno1]. Another gene involved in the photoperiodic pathway, *PFT1* transcript abundances were significantly decreased in male flower. *PFT1* is a target of PtoF14 and functions downstream of phyB to regulate the expression of *FLOWERING LOCUS T* (*FT*), providing evidence that Pto-F14 might be involved in regulation of flowering time by affecting the light quality pathway [@pone.0062681-Cerdn1].

The *AP2* gene family contains 144 members encoding at least one AP2 DNA-binding domain and the varied biological functions of AP2 family members range from development to stress and defense responses [@pone.0062681-Weigel1]--[@pone.0062681-Riechmann1]. Conserved miRNA172 has been identified as an important factor regulating AP2 transcription factors [@pone.0062681-Chuck1]. In our study, miRNA172 was equally expressed in female and male flower, but two novel miRNAs, Pto-F36 and Pto-F51, were detected with female-specific expression. *AP2* transcript abundance decreased in female flower, providing evidence that these two miRNAs act in regulation of *AP2*. *UFO* encodes an F-box protein required for the determination of floral-organ and floral-meristem identity [@pone.0062681-Hepworth1]. *PM36* is involved in reproductive system development. These genes were targeted by the female-specific novel miRNA Pto-F54 and *UFO* and *PM36* expression were down-regulated in female flower, indicating the broad biological functions of Pto-F54. Calcium is a ubiquitous second messenger in plant signal transduction cascades [@pone.0062681-Cheng1]. Three novel miRNAs were identified as related to Ca^2+^ transport, including one male flower specific and two female flower specific miRNAs, suggesting that Pto-F16, Pto-28 and Pto-45 are likely play different roles in regulating calcium transport in female and male flower development.

Hormones play a central role in the coordination of internal developmental processes with external environmental signals [@pone.0062681-Stepanova1]. A series of genes related to phytohormone synthesis and metabolism are differentially expressed between female and male during flower development (unpublished data). In our study, 13 genes related to five important phytohormones were found to be targeted by 12 novel miRNAs, suggesting that these miRNAs act broadly in regulation of phytohormone synthesis and metabolism. Previous work reported that auxin is necessary for GA-mediated control of root growth, and that attenuation of auxin transport or signaling delays the GA-induced disappearance of RGA (repressor of gal-3) [@pone.0062681-Fu1]. Among these miRNAs, Pto-F68 targets three genes involved in auxin transport and gibberellin synthesis suggesting that Pto-F68 may regulate the interaction of auxin and gibberellin. Also, a series of physiological studies indicate that ethylene and auxin interact in a number of different ways depending on the cell type, developmental stage, and environmental conditions [@pone.0062681-Stepanova1]. *EREBF4* and *SAUR2* expression were downregulated in female flower and targeted by Pto-F47 miRNA. This result may provide a clue that deepens our understanding of the important regulatory functions of miRNA Pto-F47 in phytohormone interactions. Poplar male flowers abscise earlier than female flowers, suggesting the ABA accumulation and regulation of related genes might be different between female and male flowers. *ABA3* encodes an enzyme that catalyzes the generation of the sulfurylated form of MoCo, a cofactor required by aldehyde oxidase, which functions in the last step of ABA biosynthesis in plants [@pone.0062681-Xiong1]. *ABI3* encodes a DNA binding factor targeted to promoters responsive to ABA and auxin [@pone.0062681-Nag2]. These two genes related to ABA biosynthesis and responses are targeted by novel miRNAs Pto-F55 and Pto-F65, which have female flower specific expression, suggesting these miRNAs have negative roles in flower abscission through regulation of ABA metabolism.

*MET1*, encoding DNA (cytosine-5) --methyltransferase, which maintains CG methylation, was down-regulated in male flower [@pone.0062681-Finnegan1]. *DDM1*, encoding a chromatin remodeling complex with ATPase activity that can remodel nucleosomes, was up-regulated in male flower [@pone.0062681-Brzeski1]. *MET1* and *DDM1* play opposite roles in the regulation of DNA methylation levels; therefore, the different expression of these genes in female and male flowers might alter DNA methylation levels, which are higher in female flower than male (unpublished). Our study indicated that miRNA167, Pto-F6 and Pto-F56 might play a positive regulatory role in maintaining DNA methylation levels of female flower through regulation of *MET 1* and *DDM1*.

Materials and Methods {#s4}
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Plant Materials {#s4a}
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Four-hundred and sixty unrelated individuals, representing almost the whole geographic distribution of *P. tomentosa*, were collected in the national nursery of Guan Xian County (Shandong Province, China). After observation of flower morphology, three 29-year-old unrelated andromonoecious clones ('2--14', '3605', and '5103') were found.

All three individuals were used in our study. Sampling that female and male flowers were dissected from the andromonoecious flowers was performed at the last phase of flower development, before pollination. All materials were collected from poplar individuals and immediately frozen in liquid nitrogen, and stored at −80°C for isolation of RNA. To eliminate genetic backgroud, the two bulks were made by mixing equal amounts of RNA extracted from three andromonoecious poplar female and male flower, respectively.

This study was carried out in strict accordance with the recommendations in the Guide for Observational and field studies (<http://www.plosone.org/static/publication> **)**. All necessary permits were obtained for the described field studies. The sampling of all individuals of *P*. *tomentosa* was approved by Youhui Zhang, director of National Garden of *P*. *tomentosa* in Guan Xian County, Shandong Province.

Total RNA Isolation, sRNA Library Construction, and Solexa Sequencing {#s4b}
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Total RNA was isolated from female and male floral tissue by a modified CTAB method [@pone.0062681-Chang1] with isopropanol instead of lithium chloride for RNA precipitation. Extracted RNA was used for sRNA library construction. Small RNAs were sequenced using an Illumina HiSeq 2000 at the Shanghai Bio Institute.

Sequence data from this article have been deposited in the GenBank Data Library under the accession Nos: KC477266--KC477295.

Bioinformatic Analyses of Sequencing Data and Target Prediction of miRNAs {#s4c}
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Clean reads were screened from raw reads by removing contaminating reads, including sequences with adapters, without the insert tag, with poly-A tails, shorter than 18 nt, or longer than 30 nt. Clean reads were then aligned with the *Populus* genome (<http://www.phytozome.net/poplar>) using SOAP [@pone.0062681-Li1], with no mismatches allowed. Small RNA tags matching exons and introns of mRNAs were excluded from further analysis. Tags matching non-coding RNAs, including rRNAs, scRNA, tRNAs, snRNAs, and snoRNAs, deposited at the Rfam (<http://www.sanger.ac.uk/Software/Rfam>) and NCBI GenBank databases (<http://www.ncbi.nlm.nih.gov/blast/Blast.cgi>) [@pone.0062681-Benson1] were also excluded from further analysis. The remaining unannotated sRNAs were searched against miRBase 19.0 with a maximum of two mismatches allowed [@pone.0062681-GriffithsJones1] to identify conserved miRNAs in *P*. *beijingensis* and then the resulting sequences were screened for the presence of the characteristic hairpin structures using the program RNAfold (<http://www.tbi.univie.ac.at/ivo/RNA/ViennaRNA-1.8.1.tar.gz>) [@pone.0062681-Mathews1]. We used the prediction software Mireap (<https://sourceforge.net/projects/mireap/>) to predict novel miRNAs by exploring the secondary structures, DL1 cleavage sites, and minimum free energies of the unannotated sRNA tags that could be mapped to the *Populus* genome. Basic criteria [@pone.0062681-Meyers1] were used for selecting the potential novel miRNAs. Target genes were predicted in accordance with previous research [@pone.0062681-Allen1], [@pone.0062681-Schwab1] but with higher stringency; only three mismatches were allowed in the miRNA/target duplex. Target searches were performed using *Populus* transcripts deposited in the Phytozome v7.0 (<http://www.phytozome>. net/) and Pfam databases (<http://pfam.sanger.ac.uk/>), and the results were used to annotate the functions of potential targets. We compared miRNA expression between the two libraries (control and treatment) to determine which miRNAs were differentially expressed. We normalized the expression levels of miRNAs within each library to get the expression of transcripts per million.

5′-RACE {#s4d}
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RNA Ligase-Mediated 5′-RACE (RLM-RACE) was performed with the First Choice RLM-RACE Kit (Ambion) according to the manufacturer's instructions, with slight modifications. Briefly, 10µg of total RNA was directly ligated to the 5′ adaptor followed by reverse transcription with an oligo (dT) primer. PCR was performed with 5′ adaptor primers and 3′ gene-specific primers ([Table S4](#pone.0062681.s006){ref-type="supplementary-material"}) using cDNA as the template. The RACE products were gel-purified, cloned, and sequenced.

qRT-PCR {#s4e}
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Small RNAs (\<200 nt) were isolated using the mirVana miRNA Isolation Kit (Ambion, USA) following the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was carried out as previously described [@pone.0062681-Shi1] in an ABI PRISM 7500 Fast Real-time PCR System (Ambion, USA) using the SYBR Premix Ex TaqTM Kit (TaKaRa, Japan). The reactions were carried out in a 20 µl volume containing 2 µl of diluted cDNA, 200 nM of each primer, and 19 PCR Master Mix with the following conditions: 95°C for 30 s, and 45 cycles of 95°C for 5 s, 58°C for 15 s, and 72°C for 20 s. Then, a thermal denaturing cycle of 95°C for 15 s and 60°C for 1 min was applied to determine the dissociation curves, which were used to verify the specificity of PCR amplifications. All reactions were run in triplicate for each sample. Twelve miRNAs, including six conserved and six novel miRNAs were validated, and 5.8S rRNA was selected as a reference gene for normalization [@pone.0062681-Lu3] ([Table S5](#pone.0062681.s007){ref-type="supplementary-material"} and [Table S6](#pone.0062681.s008){ref-type="supplementary-material"}).

Network Analysis {#s4f}
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The details of sex-specific flower development related miRNAs functional network methods were done according to previously described [@pone.0062681-Wang1]. Comparative miRNAome analysis combined with realtime quantitative PCR and RACE--PCR revealed 23 miRNAs expressed in andromonoecious poplar flower and each of them has functional targets. Negative correlation between the miRNA and target expression profiles was validated using real-time quantitative PCR. These results indicated that miRNA-mediated regulation does occur in flower development and that these miRNAs with dynamic changes ultimately down-regulate their targets. All miRNA and functional targets were connected for network analysis.

Supporting Information {#s5}
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**Morphological and microscopic observation of andromonoecious poplar.** A:Morphological characteristics of andromonoecious poplar flowers. B Pollen tubes stained with aniline blue, showing that pollen viability is high and that pollen grows well during selfing. Pt pollen tube, Sti stigma, Pg pollen grain.

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**Mature and precursor sequences and the predicted stem-loop structures of novel miRNAs from andromonoecious poplar flower tissue.** The mature miRNAs are in green, and the miRNA\*s are in red.

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134 loci of 38 unique miRNAs.

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11 poplar novel miRNAs were detected in other species.

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miRNAs and their targets.

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Primers for 5′ RACE mapping of miRNA cleavage sites.

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Real-time PCR primer sequences for candidate miRNAs.

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Real-time PCR primer sequences for candidate target genes.

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[^1]: **Competing Interests:**The authors have declared that no competing interests exist.

[^2]: Conceived and designed the experiments: DZ. Performed the experiments: YS KM DC ZZ DZ. Analyzed the data: YS DC DZ. Wrote the paper: YS DC DZ.
