Identification of MicroRNAs and Their Targets Involved in Paeonia rockii Petal Variegation Using High-throughput Sequencing

in Journal of the American Society for Horticultural Science

Tree peony (Paeonia sp.) is a popular traditional ornamental plant in China. Among the nine wild species, Paeonia rockii displays wide-ranging, deep purple variegation at the base of the petals, whereas Paeonia ostii exhibits purely white petals. Overall, the posttranscriptional regulation involved in tree peony flower opening and pigmentation remains unclear. To identify potential microRNAs (miRNAs) involved in flower variegation, six small RNA libraries of P. ostii and P. rockii petals at three different opening stages were constructed and sequenced. Using Illumina-based sequencing, 22 conserved miRNAs and 27 novel miRNAs were identified in P. rockii and P. ostii petals. Seventeen miRNAs were differentially expressed during flower development, and several putative target genes of these miRNAs belonged to transcription factor families, such as Myb domain (MYB), and basic helix-loop-helix (bHLH) transcription factors. Furthermore, an integrative analysis of the expression profiles of miRNAs and their corresponding target genes revealed that variegation formation might be regulated by miR159c, miR168, miR396a, and novel_miR_05, which target the MYB transcription factors, chalcone synthase (CHS), and ABC transporter. Our preliminary study is the first report of miRNAs involved in Paeonia flower pigmentation. It provides insight regarding the molecular mechanisms underlying the regulation of flower pigmentation in tree peony.

Contributor Notes

This work was supported by the Natural Science Foundation of China (31800599), Natural Science Foundation of Shannxi Province, China (2017JQ3024), General Financial Grant from the China Postdoctoral Science Foundation (2017M623267), the Special Fund for Scientific Research in the Public Welfare (201404701), the Scientific Startup Foundation for Doctors of Northwest A&F University (Z109021611), and Fundamental Research Funds for the Central Universities (Z109021606).

Qianqian Shi and Xiaoxiao Zhang performed the experiments and data analysis and wrote the manuscript. Xiang Li performed some experiments. Xiaoning Luo, Jianrang Luo, and Lixia He helped prepare the plant material. Yanlong Zhang and Long Li designed this research study. All of the authors read and approved the final manuscript.

These authors contributed equally to this work and should be considered co-first authors.

Corresponding authors. Email: or

Article Sections

Article Figures

  • View in gallery

    Photographs of Paeonia ostii and Paeonia rockii during five different flower opening stages. (AE) P. ostii at S1–S5. (FJ) P. rockii at S1–S5 (S1 = unpigmented tight bud; S2 = slightly soft bud without pigmentation; S3 = initially open flower with slight pigmentation; S4 = half-open flower with slight pigmentation; S5 = fully open and pigmented flower with exposed anthers).

  • View in gallery

    A study of the overall expression patterns of all miRNAs in the six miRNA libraries. (A) General distribution of the log10-transformed expression values [transcripts per million (TPM)] of all miRNAs in the six libraries. (B) Correlations among the six miRNA libraries based on the Pearson correlation coefficient. The color scale represents the Pearson correlation coefficients among the different samples. The higher the Pearson correlation coefficient, the closer the relationship between the two libraries. Blue represents a close relationship; pink represents a distant relationship.

  • View in gallery

    A study of Venn diagrams of all miRNAs between Paeonia ostii and Paeonia rockii (A) and differentially expressed miRNAs at different flower opening stages (B). S1, S3, and S5 represent three different developmental stages (stage 1, stage 3, and stage 5, respectively) in P. ostii and P. rockii (S1 = unpigmented tight bud; S2 = slightly soft bud without pigmentation; S3 = initially open flower with slight pigmentation; S4 = half-open flower with slight pigmentation; S5 = fully open and pigmented flower with exposed anthers).

  • View in gallery

    The study of expression patterns of the differentially expressed conserved and novel miRNAs among the three different opening stages of Paeonia rockii (PR) and Paeonia. ostii (PO) based on Illumina (San Diego, CA) sequencing datasets using Heml software (Deng et al., 2014). The bar indicates the log2-transformed expression level scale of the miRNAs. Each column represents a sample, and the colored bar indicates the relative expression level from high (red) to low (black). S1, S3, and S5 represent three different developmental stages (stage 1, stage 3, and stage 5, respectively) in PO and PR (S1 = unpigmented tight bud; S2 = slightly soft bud without pigmentation; S3 = initially open flower with slight pigmentation; S4 = half-open flower with slight pigmentation; S5 = fully open and pigmented flower with exposed anthers).

  • View in gallery

    The study of expression profiles and the fold changes of six miRNAs and their corresponding target genes in petals at five different flower opening stages Paeonia rockii (PR) (S1 = unpigmented tight bud; S2 = slightly soft bud without pigmentation; S3 = initially open flower with slight pigmentation; S4 = half-open flower with slight pigmentation; S5 = fully open and pigmented flower with exposed anthers) in PR and Paeonia ostii (PO) using qRT-PCR. The expression profiles of six miRNAs and their corresponding target genes and the fold changes (PR/PO) of six miRNAs and their corresponding target genes are shown. U6 snRNA was used as a reference for the miRNA qRT-PCR. TUB was chosen as the internal housekeeping gene control for the target genes. Gene and miRNA expression levels at the S1 stage of PO were selected as the internal control, and the expression level of all genes in the control was set at 1.0. Error bars indicate the sd of three biological replicates and three technical replicates. (A) miR159c and c115438.graph_c0 (Myb DNA-binding protein, MYB1). (B) miR168 and c105628.graph_c0 (Myb DNA-binding protein, MYB2). (C) miR319 and c194399.graph_c0 (Myb DNA-binding protein, MYB3). (D) novel_miR_05 and c121983.graph_c0 (Myb DNA-binding protein, MYB4). (E) novel_miR_05 and c119993.graph_c0 (Myb DNA-binding protein, MYB5). (F) miR396a and c130130.graph_c0 (helix-loop-helix DNA-binding domain, bHLH1). (G) miR396b and c103359.graph_c0 (helix-loop-helix DNA-binding domain, bHLH2). (H) miR168 and c43510.graph_c0 (chalcone synthase, CHS). (I) miR396a and c129851.graph_c0 (ATP-binding glutathione S-conjugate). All miRNAs and their target genes are listed in Supplemental Table 1. The correlations (Cor) between miRNAs and targets were calculated based on fold changes of miRNA and the target gene expression between PR and PO using the Pearson correlation coefficient using SPSS (version 19.0; IBM Corp., Armonk, NY).

  • View in gallery

    The study of the length distribution of miRNAs identified in Paeonia rockii and Paeonia ostii petals. The x-axis represents the length of miRNAs. The y-axis represents the number of miRNAs with a specific length.

  • View in gallery

    Predicted precursor structures of all identified miRNAs. (A) Conserved miRNAs. (B) Novel miRNAs. Red sequences represent the mature sequences. Yellow sequences represent the loop structure. Purple sequences represent the star sequences predicted by miRDeep2 software. Blue sequences represent the star sequences from reads by sRNA sequencing.

  • View in gallery

    The study of correlations between the gene expression results obtained by qRT-PCR analysis and those obtained using Illumina sequencing for the 32 identified miRNAs at three different opening stages (S1, S3, and S5) in Paeonia ostii and P. rockii petals. The correlations were analyzed based on the average log2 values of expression levels of each miRNA during the three opening stages of P. ostii and P. rockii (S1 = unpigmented tight bud; S2 = slightly soft bud without pigmentation; S3 = initially open flower with slight pigmentation; S4 = half-open flower with slight pigmentation; S5 = fully open and pigmented flower with exposed anthers).

Article References

AlbertN.LewisD.ZhangH.SchwinnK.JamesonP.DaviesK.2011Members of an R2R3-MYB transcription factor family in Petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterningPlant J.65771784

AllenE.XieZ.GustafsonA.M.CarringtonJ.C.2005MicroRNA directed phasing during trans-acting siRNA biogenesis in plantsCell121207221

AshburnerM.BallC.A.BlakeJ.A.BotsteinD.ButlerH.CherryJ.M.DavisA.P.DolinskiK.DwightS.S.EppigJ.T.HarrisM.A.HillD.P.Issel-TarverL.KasarskisA.LewisS.MateseJ.C.RichardsonJ.E.RingwaldM.RubinG.M.SherlockG.2000Gene ontology: Tool for the unification of biologyNat. Genet.252529

AxtellM.J.SnyderJ.A.BartelD.P.2007Common functions for diverse small RNAs of land plantsPlant Cell1917501769

BurgeS.W.DaubJ.EberhardtR.TateJ.BarquistL.NawrockiE.P.EddyS.R.GardnerP.P.BatemanA.2013Rfam p 110: 10 Years of RNA familiesNucleic Acids Res.41226232

ChiouC.Y.YehK.W.2008Differential expression of MYB gene (OgMYB1) determines color patterning in floral tissue of Oncidium Gower RamseyPlant Mol. Biol.66379388

ChuZ.L.ChenJ.Y.XuH.X.DongZ.D.ChenF.CuiD.Q.2016Identification and comparative analysis of microRNA in wheat (Triticum aestivum L.) callus derived from mature and immature embryos during in vitro cultureFront. Plant Sci.71302

ConesaA.GötzS.García-GómezJ.M.TerolJ.TalónM.RoblesM.2005Blast2GO: A universal tool for annotation, visualization and analysis in functional genomics researchBioinforma2136743676

De PaolaD.CattonaroF.PignoneD.SonnanteG.2012The miRNAome of globe artichoke: Conserved and novel microRNAs and target analysisBMC Genomics1341

DengW.K.WangY.B.LiuZ.X.ChengH.XueY.2014Heml: A toolkit for illustrating heatmapsPLoS One9e111988

DongM.YangD.LangQ.ZhouW.XuS.XuT.2013Microarray and degradome sequencing reveal microRNA differential expression profiles and their target in Pinellia pedatisectaPLoS One8e75978

EspleyR.V.HellensR.P.PutterillJ.StevensonD.E.Kutty-AmmaS.AllanA.C.2007Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10Plant J.49414427

GardnerP.P.DaubJ.TateJ.MooreB.L.OsuchI.H.Griffiths-JonesS.FinnR.D.NawrockiE.P.KolbeD.L.EddyS.R.BatemanA.2011Rfam: Wikipedia, clans and the “decimal” releaseNucleic Acids Res.39D141D145

Griffiths-JonesS.SainiH.K.van DongenS.EnrightA.J.2008miRBase: Tools for microRNA genomicsNucleic Acids Res.36D154D158

GonzalezA.ZhaoM.LeavittJ.M.LloydA.M.2008Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/ Myb transcriptional complex in Arabidopsis seedlingsPlant J.53814827

GouJ.Y.FelippesF.F.LiuC.J.WeigelD.WangJ.W.2011Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factorPlant Cell2315121522

GuZ.Y.ZhuJ.HaoQ.YuanY.W.DuanY.W.MenS.Q.WangQ.Y.HouQ.Z.LiuZ.A.ShuQ.Y.WangL.S.2018A novel R2R3-MYB transcription factor contributes to petal blotch formation by regulating organ-specific expression of PsCHS in tree peony (Paeonia suffruticosa)Plant Cell Physiol.doi: 10.1093/pcp/pcy232

HongY.JacksonS.2015Floral induction and flower formation-the role and potential applications of miRNAsPlant Biotechnol. J.13282292

HsuC.ChenY.TsaiW.ChenW.ChenH.2015Three R2R3-MYB transcription factors regulate distinct floral pigmentation patterning in Phalaenopsis sppPlant Physiol.168175191

JiaX.LiuH.ShenJ.LiF.DingN.SunY.GaoC.LiR.2015Negative regulation of anthocyanin biosynthesis in tomato by microRNA828 under phosphate deficiencyScientia Agrcultura Sinica4829112924

KapitonovV.V.JurkaJ.2008A universal classification of eukaryotic transposable elements implemented in RepbaseNat. Rev. Genet.9411412

KoesR.VerweijW.QuattrocchioF.2005Flavonoids: A colorful model for the regulation and evolution of biochemical pathwaysTrends Plant Sci.10236242

LangmeadB.TrapnellC.PopM.SalzbergS.L.2009Ultrafast and memory-efficient alignment of short DNA sequences to the human genomeGenome Biol.10R25

LiC.DuH.WangL.S.ShuQ.Y.ZhengY.R.XuY.J.2009Flavonoid composition and antioxidant activity of tree peony (Paeonia section Moutan) yellow flowersJ. Agr. Food Chem.5784968503

LiJ.J.1999Chinese tree peony and herbaceous peony. For. Publ. House Beijing China

LiL.ChengZ.C.MaY.J.BaiQ.S.LiX.Y.CaoZ.H.WuZ.N.GaoJ.2018The association of hormone signaling genes, transcription and changes in shoot anatomy during moso bamboo growthPlant Biotechnol. J.167285

LiuR.LaiB.HuB.QinY.HuG.ZhaoJ.2017Identification of microRNAs and their target genes related to the accumulation of anthocyanins in Litchi chinensis by high-throughput sequencing and degradome analysisFront. Plant Sci.720592070

LuY.P.LiZ.S.ReaP.A.1997AtMRP1 gene of Arabidopsis encodes a glutathione S-conjugate pump: Isolation and functional definition of a plant ATP-binding cassette transporter geneProc. Natl. Acad. Sci. USA9482438248

LuoQ.MittalA.JiaF.RockC.2012An autoregulatory feedback loop involving PAP1 and TAS4 in response to sugars in ArabidopsisPlant Mol. Biol.80117129

MartinsT.JiangP.RausherM.2016How petals change their spots: Cis-regulatory re-wiring in Clarkia (Onagraceae)New Phytol.216510

MeyersB.C.AxtellM.J.BartelB.BartelD.P.BaulcombeD.BowmanJ.L.CaoX.F.CarringtonJ.C.ChenX.M.GreenP.J.Griffiths-JonesS.JacobsenS.E.MalloryA.C.MartienssenR.A.PoethigR.S.QiY.J.VaucheretH.VoinnetO.WatanabeY.WeigelD.ZhuJ.K.2008Criteria for annotation of plant microRNAsPlant Cell2031863190

MishraA.K.DuraisamyG.S.MatoušekJ.RadisekS.JavornikB.JakseJ.2016Identification and characterization of microRNAs in Humulus lupulus using highthroughput sequencing and their response to citrus bark cracking viroid (CBCVd) infectionBMC Genomics17919

NawrockiE.P.BurgeS.W.BatemanA.DaubJ.EberhardtR.Y.EddyS.R.FlodenE.W.GardnerP.P.JonesT.A.TateJ.FinnR.D.2015Rfam: Updates to the RNA families databaseNucleic Acids Res.43D130D137

NishiharaM.NakatsukaT.2011Genetic engineering of flavonoid pigments to modify flower color in floricultural plantsBiotechnol. Lett.33433441

QuD.YanF.MengR.JiangX.YangH.GaoZ.DongY.YangY.ZhaoZ.2016Identification of microRNAs and their targets associated with fruit-bagging and subsequent sunlight re-exposure in the ‘Granny Smith’ apple exocarp using high-throughput sequencingFront. Plant Sci.72740

RomualdiC.BortoluzziS.D’AlessiF.DanieliG.A.2003IDEG6: A web tool for detection of differentially expressed genes in multiple tag sampling experimentsPhysiol. Genomics12159162

RoyS.TripathiA.M.YadavA.MishraP.NautiyalC.S.2016Identification and expression analyses of miRNAs from two contrasting flower color cultivars of canna by deep sequencingPLoS One11e0147499

SchwabR.PalatnikJ.F.RiesterM.SchommerC.SchmidM.WeigelD.2005Specific effects of microRNAs on the plant transcriptomeDev. Cell8517527

SchwinnK.VenailJ.ShangY.MackayS.AlmV.ButelliE.OyamaR.BaileyP.DaviesK.MartinC.2006A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus AntirrhinumPlant Cell18831851

ShangY.VenailJ.MackayS.BaileyP.SchwinnK.JamesonP.MartinC.DaviesK.2011The molecular basis for venation patterning of pigmentation and its effect on pollinator attraction in flowers of AntirrhinumNew Phytol.189602615

ShiQ.LiL.ZhangX.LuoJ.LiX.ZhaiL.HeL.ZhangY.2017Biochemical and comparative transcriptomic analyses identify candidate genes related to variegation formation in Paeonia rockiiMolecules221364

SunY.QiuY.DuanM.WangJ.ZhangX.WangH.SongJ.LiX.2017Identification of anthocyanin biosynthesis related microRNAs in a distinctive Chinese radish (Raphanus sativus L.) by high-throughput sequencingMol. Genet. Genomics292215229

SunkarR.ChinnusamyV.ZhuJ.ZhuJ.2007Small RNAs as big players in plant abiotic stress responses and nutrient deprivationTrends Plant Sci.12301309

SuzukiK.SuzukiT.NakatsukaT.DohraH.YamagishiM.MatsuyamaK.MatsuuraH.2016RNA-seq-based evaluation of bicolor tepal pigmentation in Asiatic hybrid lilies (Lilium spp.)BMC Genomics17611

TanakaY.SasakiN.OhmiyaA.2008Biosynthesis of plant pigments: Anthocyanins, betalains and carotenoidsPlant J.54733749

TatusovR.L.GalperinM.Y.NataleN.A.KooninE.V.2000The COG database: A tool for genome-scale analysis of protein functions and evolutionNucleic Acids Res.283336

UnverT.BudakH.2009Conserved microRNAs and their targets in model grass species Brachypodium distachyonPlanta230659669

WangL.HashimotoF.ShiraishiA.ShimizuK.AokiN.SakataY.2000Petal coloration and pigmentation of tree peony cultivars of Xibei (the northwest of China)J. Jpn. Soc. Hort. Sci.69233

WangY.Q.HouX.J.ZhangB.ChenW.J.LiuD.C.LiuB.L.ZhangH.G.2016Identification of a candidate gene for Rc-D1, a Locus controlling red coleoptile colour in wheatCereal Res. Commun.443546

XiaR.ZhuH.AnY.Q.BeersE.P.LiuZ.2012Apple miRNAs and tasiRNAs with novel regulatory networksGenome Biol.1347

XieF.FrazierT.P.ZhangB.2010Identification and characterization of microRNAs and their targets in the bioenergy plant switchgrass (Panicum virgatum)Planta232417434

XuW.DubosC.LepiniecL.2015Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexesTrends Plant Sci.20176185

YamagishiM.2016A novel R2R3-MYB transcription factor regulates light mediated floral and vegetative anthocyanin pigmentation patterns in Lilium regaleMol. Breed.363

YamagishiM.2018Involvement of a LhMYB18 transcription factor in large anthocyanin spot formation on the flower tepals of the Asiatic hybrid lily (Lilium spp.) cultivar ‘Grand Cru’Mol. Breed.3860

YamagishiM.ShimoyamadaY.NakatsukaT.MasudaK.2010Two R2R3-MYB genes, homologs of Petunia AN2, regulate anthocyanin biosyntheses in flower tepals, tepal spots and leaves of asiatic hybrid lilyPlant Cell Physiol.51463474

YamagishiM.TodaS.TasakiK.2014The novel allele of the LhMYB12 gene is involved in splatter-type spot formation on the flower tepals of Asiatic hybrid lilies (Lilium spp.)New Phytol.20110091020

YangF.LiangG.LiuD.YuD.2009Arabidopsis mir396 mediates the development of leaves and flowers in transgenic tobaccoJ. Plant Biol.52475481

YinD.LiS.ShuQ.Y.GuZ.Y.WuQ.FengC.Y.XuW.Z.WangL.S.2018Identification of microRNAs and long non-coding RNAs involved in fatty acid biosynthesis in tree peony seedsGene6667282

YinH.F.FanZ.Q.LiX.L.WangJ.Y.LiuW.X.WuB.YingZ.LiuL.P.LiuZ.C.LiJ.Y.2016Phylogenetic tree-informed microRNAome analysis uncovers conserved and lineage-specific miRNAs in Camelia during floral organ developmentJ. Expt. Bot.6726412653

YoshidaK.IwasakaR.ShimadaN.AyabeS.AokiT.SakutaM.2010Transcriptional control of the dihydroflavonol 4-reductase multigene family in Lotus japonicasJ. Plant Res.123801805

YuanY.W.SagawaJ.M.FrostL.VelaJ.P.BradshawH.D.2014Transcriptional control of floral anthocyanin pigmentation in monkeyflowers (Mimulus)New Phytol.20410131027

ZhangB.WangQ.2015MicroRNA-based biotechnology for plant improvementJ. Cell. Physiol.230115

ZhangY.ChengY.YaH.XuS.HanJ.2015Transcriptome sequencing of purple spot region in tree peony reveals differentially expressed anthocyanin structural genesFront. Plant Sci.6964972

ZhangY.WangY.GaoX.LiuC.GaiS.2018Identification and characterization of microRNAs in tree peony during chilling induced dormancy release by high-throughput sequencingScientific Rpt.84537

ZhaoD.WeiM.ShiM.HaoZ.TaoJ.2017Identification and comparative profiling of miRNAs in herbaceous peony (Paeonia lactiflara Pall.) with red/yellow bicoloured flowersScientific Rpt.744926

ZhaoD.GongS.J.HaoZ.J.TaoJ.2015Identification of miRNAs responsive to Botrytis cinerea in herbaceous peony (Paeonia lactiflora Pall.) by high-throughput sequencingGenes (Basel)6918934

ZhouS.L.ZouX.H.ZhouZ.Q.LiuJ.XuC.YuJ.WangQ.ZhangD.M.WangX.Q.GeS.SangT.PanK.Y.HongD.Y.2014Multiple species of wild tree peonies gave rise to the ‘king of flowers’, Paeonia suffruticosa AndrewsProc. Biol. Sci.2811687

ZongY.XiX.Y.LiS.M.ChenW.J.ZhangB.LiuD.C.LiuB.L.WangD.W.ZhangH.G.2017Allelic variation and transcriptional isoforms of wheat TaMYC1 gene regulating anthocyanin synthesis in pericarpFront. Plant Sci.81645

Article Information

Google Scholar

Related Content

Article Metrics

All Time Past Year Past 30 Days
Abstract Views 87 87 41
Full Text Views 15 15 9
PDF Downloads 10 10 7