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Genome-Wide Identification of Embryogenesis-Associated microRNAs in Radish (Raphanus sativus L.) by High-Throughput Sequencing

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Abstract

microRNAs (miRNAs), endogenous non-coding RNAs of approximately 21–24 nucleotides, are important regulators of transcriptional and post-transcriptional gene expression. These regulators play a key role during plant growth and development, including embryogenesis, which is crucial to the life cycle of most plant species. However, although embryogenesis-associated miRNAs have been isolated in a few species, the diversity of these regulatory miRNAs remains largely unexplored, especially in radish. In this study, two small RNA libraries were constructed from radish ovules before and after fertilization. Both libraries were sequenced by next generation sequencing (NGS) technology. This analysis identified 144 conserved and 34 non-conserved miRNAs (representing 43 known miRNA families) and 38 novel miRNAs (representing 28 miRNA families). Comparative analysis revealed that 29 known and 10 novel miRNA families were differentially expressed during embryogenesis. QRT-PCR analysis confirmed miRNA expression patterns and revealed tissue-specific and/or developmental stage-dependent expression for some miRNAs. Moreover, potential target predictions indicated that most of these targets were transcription factors involved in regulating plant growth, development and metabolism. Notably, target transcripts such as squamosa promoter-binding protein, auxin response factor and agamous-like MADS-box protein participated in radish embryogenesis.

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References

  • Ágyi Á, Havelda Z (2013) Analysis of gradient-like expression of miR167 in Arabidopsis thaliana embryonic tissue. J Plant Biology 56:336–344

    Article  Google Scholar 

  • Barvkar VT, Pardeshi VC, Kale SM, Qiu S, Rollins M, Datla R, Gupta VS, Kadoo NY (2013) Genome-wide identification and characterization of microRNA genes and their targets in flax (Linum usitatissimum): Characterization of flax miRNA genes. Planta 237:1149–1161

    Article  CAS  PubMed  Google Scholar 

  • Braybrook SA, Harada JJ (2008) LECs go crazy in embryo development. Trends Plant Sci 13:624–630

    Article  CAS  PubMed  Google Scholar 

  • Buxdorf K, Hendelman A, Stav R, Lapidot M, Ori N, Arazi T (2010) Identification and characterization of a novel miR159 target not related to MYB in tomato. Planta 232:1009–1022

    Article  CAS  PubMed  Google Scholar 

  • Carrington JC, Ambros V (2003) Role of microRNAs in plant and animal development. Science 301:336–338

    Article  CAS  PubMed  Google Scholar 

  • Chen L, Ren Y, Zhang Y, Xu J, Sun F, Zhang Z, Wang Y (2012) Genome-wide identification and expression analysis of heat-responsive and novel microRNAs in Populus tomentosa. Gene 504:160–165

    Article  CAS  PubMed  Google Scholar 

  • Chorostecki U, Crosa VA, Lodeyro AF, Bologna NG, Martin AP, Carrillo N, Schommer C, Palatnik JF (2012) Identification of new microRNA-regulated genes by conserved targeting in plant species. Nucleic Acids Res 40:8893–8904

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Coudert Y, Perin C, Courtois B, Khong NG, Gantet P (2010) Genetic control of root development in rice, the model cereal. Trends Plant Sci 15:219–226

    Article  CAS  PubMed  Google Scholar 

  • Dai X, Zhao PX (2011) psRNATarget: a plant small RNA target analysis server. Nucleic Acids Res 39:W155–W159

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Eyles RP, Williams PH, Ohms SJ, Weiller GF, Ogilvie HA, Djordjevic MA, Imin N (2013) microRNA profiling of root tissues and root forming explant cultures in Medicago truncatula. Planta 238:91–105

    Article  CAS  PubMed  Google Scholar 

  • Galla G, Barcaccia G, Ramina A, Collani S, Alagna F, Baldoni L, Cultrera NG, Martinelli F, Sebastiani L, Tonutti P (2009) Computational annotation of genes differentially expressed along olive fruit development. BMC Plant Biology 9:128

    Article  PubMed Central  PubMed  Google Scholar 

  • Gao Z, Shi T, Luo X, Zhang Z, Zhuang W, Wang L (2012) High-throughput sequencing of small RNAs and analysis of differentially expressed microRNAs associated with pistil development in Japanese apricot. BMC Genomics 13:371

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Goldberg RB, Gd P, Yadegari R (1994) Plant embryogenesis: zygote to seed science. Science 266:605–614

    Article  CAS  PubMed  Google Scholar 

  • Hafner M, Landgraf P, Ludwig J, Rice A, Ojo T, Lin C, Holoch D, Lim C, Tuschl T (2008) Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods 44:3–12

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Huang D, Koh C, Feurtado JA, Tsang EW, Cutler AJ (2013) MicroRNAs and their putative targets in Brassica napus seed maturation. BMC Genomics 14:140

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Jung JH, Park CM (2007) MIR166/165 genes exhibit dynamic expression patterns in regulating shoot apical meristem and floral development in Arabidopsis. Planta 225:1327–1338

    Article  CAS  PubMed  Google Scholar 

  • Lan Y, Su N, Shen Y, Zhang R, Wu F, Cheng Z, Wang J, Zhang X, Guo X, Lei C, Wang J, Jiang L, Mao L, Wan J (2012) Identification of novel miRNAs and miRNA expression profiling during grain development in indica rice. BMC Genomics 13:264

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Li H, Dong Y, Sun Y, Zhu E, Yang J, Liu X, Xue P, Xiao Y, Yang S, Wu J, Li X (2011) Investigation of the microRNAs in safflower seed, leaf, and petal by high-throughput sequencing. Planta 233:611–619

    Article  CAS  PubMed  Google Scholar 

  • Li RQ, Yu C, Li YR, Lam TW, Yiu SM, Kristiansen K, Wang J (2009) SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics 25:1966–1967

    Article  CAS  PubMed  Google Scholar 

  • Li S, Yang X, Wu F, He Y (2012) HYL1 controls the miR156-mediated juvenile phase of vegetative growth. J Exp Bot 63:2787–2798

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Liang C, Zhang X, Zou J, Xu D, Su F, Ye N (2010) Identification of miRNA from Porphyra yezoensis by high-throughput sequencing and bioinformatics analysis. PloS One 5:e10698

    Article  PubMed Central  PubMed  Google Scholar 

  • Lin Y, Lai Z (2013) Comparative analysis reveals dynamic changes in miRNAs and their targets and expression during somatic embryogenesis in Longan (Dimocarpus longan Lour.). PloS One 8:e60337

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Liu PP, Montgomery TA, Fahlgren N, Kasschau KD, Nonogaki H, Carrington JC (2007) Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post-germination stages. Plant J 52:133–146

    Article  CAS  PubMed  Google Scholar 

  • Lopez-Gomollon S, Mohorianu I, Szittya G, Moulton V, Dalmay T (2012) Diverse correlation patterns between microRNAs and their targets during tomato fruit development indicates different modes of microRNA actions. Planta 236:1875–1887

    Article  CAS  PubMed  Google Scholar 

  • Ma X, Shao C, Wang H, Jin Y, Meng Y (2013) Construction of small RNA-mediated gene regulatory networks in the roots of rice (Oryza sativa). BMC Genomics 14:510

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mao W, Li Z, Xia X, Li Y, Yu J (2012) A combined approach of high-throughput sequencing and degradome analysis reveals tissue specific expression of microRNAs and their targets in cucumber. PloS One 7:e33040

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Marin E, Jouannet V, Herz A, Lokerse AS, Weijers D, Vaucheret H, Nussaume L, Crespi MD, Maizel A (2010) miR390, Arabidopsis TAS3 tasiRNAs, and their AUXIN RESPONSE FACTOR targets define an autoregulatory network quantitatively regulating lateral root growth. Plant Cell 22:1104–1117

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Martin RC, Liu PP, Goloviznina NA, Nonogaki H (2010) microRNA, seeds, and darwin?: diverse function of miRNA in seed biology and plant responses to stress. J Exp Bot 61:2229–2234

    Article  CAS  PubMed  Google Scholar 

  • Meng Y, Huang F, Shi Q, Cao J, Chen D, Zhang J, Ni J, Wu P, Chen M (2009) Genome-wide survey of rice microRNAs and microRNA-target pairs in the root of a novel auxin-resistant mutant. Planta 230:883–898

    Article  CAS  PubMed  Google Scholar 

  • Meyers BC, Axtell MJ, Bartel B, Bartel DP, Baulcombe D, Bowman JL, Cao X, Carrington JC, Chen X, Green PJ, Griffiths-Jones S, Jacobsen SE, Mallory AC, Martienssen RA, Poethig RS, Qi Y, Vaucheret H, Voinnet O, Watanabe Y, Weigel D, Zhu JK (2008) Criteria for annotation of plant MicroRNAs. Plant Cell 20:3186–3190

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Muvva C, Tewari L, Aruna K, Ranjit P, MD ZS, MD KAM, Veeramachaneni H (2012) In silico identification of miRNAs and their targets from the expressed sequence tags of Raphanus sativus. Bioinformation 8:98–103

    Article  PubMed Central  PubMed  Google Scholar 

  • Nodine MD, Bartel DP (2010) MicroRNAs prevent precocious gene expression and enable pattern formation during plant embryogenesis. Genes Dev 24:2678–2692

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Nonogaki H (2010) MicroRNA gene regulation cascades during early stages of plant development. Plant Cell Physiol 51:1840–1846

    Article  CAS  PubMed  Google Scholar 

  • Oh TJ, Wartell RM, Cairney J, Pullman GS (2008) Evidence for stage-specific modulation of specific microRNAs (miRNAs) and miRNA processing components in zygotic embryo and female gametophyte of loblolly pine (Pinus taeda). New Phytol 179:67–80

    Article  CAS  PubMed  Google Scholar 

  • Palovaara J, Saiga S, Weijers D (2013) Transcriptomics approaches in the early Arabidopsis embryo. Trends Plant Sci 18:514–521

  • Rademacher EH, Lokerse AS, Schlereth A, Llavata-Peris CI, Bayer M, Kientz M, Freire Rios A, Borst JW, Lukowitz W, Jurgens G, Weijers D (2012) Different auxin response machineries control distinct cell fates in the early plant embryo. Dev Cell 22:211–222

    Article  CAS  PubMed  Google Scholar 

  • Reyes JL, Chua NH (2007) ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination. Plant J 49:592–606

    Article  CAS  PubMed  Google Scholar 

  • Rodriguez RE, Mecchia MA, Debernardi JM, Schommer C, Weigel D, Palatnik JF (2010) Control of cell proliferation in Arabidopsis thaliana by microRNA miR396. Development 137:103–112

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Rubio-Somoza I, Cuperus JT, Weigel D, Carrington JC (2009) Regulation and functional specialization of small RNA-target nodes during plant development. Curr Opin Plant Biol 12:622–627

    Article  CAS  PubMed  Google Scholar 

  • Song QX, Liu YF, Hu XY, Zhang WK, Ma B, Chen SY, Zhang JS (2011) Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing. BMC Plant Biology 11:5

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sunkar R (2010) MicroRNAs with macro-effects on plant stress responses. Semin Cell Dev Biol 21:805–811

    Article  CAS  PubMed  Google Scholar 

  • Sunkar R, Kapoor A, Zhu JK (2006) Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance. Plant Cell 18:2051–2065

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Voinnet O (2009) Origin, biogenesis, and activity of plant microRNAs. Cell 136:669–687

    Article  CAS  PubMed  Google Scholar 

  • Wan LC, Wang F, Guo X, Lu S, Qiu Z, Zhao Y, Zhang H, Lin J (2012) Identification and characterization of small non-coding RNAs from Chinese fir by high throughput sequencing. BMC Plant Biology 12:146

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wang C, Han J, Liu C, Kibet KN, Kayesh E, Shangguan L, Li X, Fang J (2012a) Identification of microRNAs from Amur grape (Vitis amurensis Rupr.) by deep sequencing and analysis of microRNA variations with bioinformatics. BMC Genomics 13:122

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wang F, Li L, Liu L, Li H, Zhang Y, Yao Y, Ni Z, Gao J (2012b) High-throughput sequencing discovery of conserved and novel microRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Mol Genet Genomics 287:555–563

    Google Scholar 

  • Wang J, Czech B, Weigel D (2009) miR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana. Cell 138:738–749

    Article  CAS  PubMed  Google Scholar 

  • Wang ZJ, Huang JQ, Huang YJ, Li Z, Zheng BS (2012c) Discovery and profiling of novel and conserved microRNAs during flower development in Carya cathayensis via deep sequencing. Planta 236:613–621

    Article  CAS  PubMed  Google Scholar 

  • Wang ZM, Xue W, Dong CJ, Jin LG, Bian SM, Wang C, Wu XY, Liu JY (2012d) A comparative miRNAome analysis reveals seven fiber initiation-related and 36 novel miRNAs in developing cotton ovules. Mol Plant 5:889–900

    Article  CAS  PubMed  Google Scholar 

  • Wei LQ, Yan LF, Wang T (2011) Deep sequencing on genome-wide scale reveals the unique composition and expression patterns of microRNAs in developing pollen of Oryza sativa. Genome Biol 12:R53

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wei M, Wei H, Wu M, Song M, Zhang J, Yu J, Fan S, Yu S (2013) Comparative expression profiling of miRNA during anther development in genetic male sterile and wild type cotton. BMC Plant Biology 13:66

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Williams L, Grigg SP, Xie M, Christensen S, Fletcher JC (2005) Regulation of Arabidopsis shoot apical meristem and lateral organ formation by microRNA miR166g and its AtHD-ZIP target genes. Development 132:3657–3668

    Article  CAS  PubMed  Google Scholar 

  • Willmann MR, Mehalick AJ, Packer RL, Jenik PD (2011) MicroRNAs regulate the timing of embryo maturation in Arabidopsis. Plant Physiol 155:1871–1884

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wu G, Park MY, Conway SR, Wang JW, Weigel D, Poethig RS (2009) The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell 138:750–759

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wu XM, Liu MY, Ge XX, Xu Q, Guo WW (2011) Stage and tissue-specific modulation of ten conserved miRNAs and their targets during somatic embryogenesis of Valencia sweet orange. Planta 233:495–505

    Article  CAS  PubMed  Google Scholar 

  • Xiang D, Venglat P, Tibiche C, Yang H, Risseeuw E, Cao Y, Babic V, Cloutier M, Keller W, Wang E, Selvaraj G, Datla R (2011) Genome-wide analysis reveals gene expression and metabolic network dynamics during embryo development in Arabidopsis. Plant Physiol 156:346–356

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Xie C, Mao X, Huang J, Ding Y, Wu J, Dong S, Kong L, Gao G, Li CY, Wei L (2011) KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res 39:W316–W322

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Xing S, Salinas M, Hohmann S, Berndtgen R, Huijser P (2010) miR156-targeted and nontargeted SBP-box transcription factors act in concert to secure male fertility in Arabidopsis. Plant Cell 22:3935–3950

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Xu L, Wang Y, Xu Y, Wang L, Zhai L, Zhu X, Gong Y, Ye S, Liu L (2013a) Identification and characterization of novel and conserved microRNAs in radish (Raphanus sativus L.) using high-throughput sequencing. Plant Sci 201–202:108–114

    Article  PubMed  Google Scholar 

  • Xu L, Wang Y, Zhai L, Xu Y, Wang L, Zhu X, Gong Y, Yu R, Limera C, Liu L (2013b) Genome-wide identification and characterization of cadmium-responsive microRNAs and their target genes in radish (Raphanus sativus L.) roots. J Exp Bot 64:4271–4287

    Article  CAS  Google Scholar 

  • Xu MY, Dong Y, Zhang QX, Zhang L, Luo YZ, Sun J, Fan YL, Wang L (2012) Identification of miRNAs and their targets from Brassica napus by high-throughput sequencing and degradome analysis. BMC Genomics 13:421

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Xue LJ, Zhang JJ, Xue HW (2009) Characterization and expression profiles of miRNAs in rice seeds. Nucleic Acids Res 37:916–930

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yan K, Liu P, Wu CA, Yang GD, Xu R, Guo QH, Huang JG, Zheng CC (2012) Stress-induced alternative splicing provides a mechanism for the regulation of microRNA processing in Arabidopsis thaliana. Mol Cell 48:521–531

    Article  CAS  PubMed  Google Scholar 

  • Yang J, Liu X, Xu B, Zhao N, Yang X, Zhang M (2013a) Identification of miRNAs and their targets using high-throughput sequencing and degradome analysis in cytoplasmic male-sterile and its maintainer fertile lines of Brassica juncea. BMC Genomics 14:9

    Article  PubMed Central  PubMed  Google Scholar 

  • Yang X, Wang L, Yuan D, Lindsey K, Zhang X (2013b) Small RNA and degradome sequencing reveal complex miRNA regulation during cotton somatic embryogenesis. J Exp Bot 64:1521–1536

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yang X, Zhang X, Yuan D, Jin F, Zhang Y, Xu J (2012) Transcript profiling reveals complex auxin signalling pathway and transcription regulation involved in dedifferentiation and redifferentiation during somatic embryogenesis in cotton. BMC Plant Biology 12:110

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yazawa K, Takahata K, Kamada H (2004) Isolation of the gene encoding Carrot leafy cotyledon1 and expression analysis during somatic and zygotic embryogenesis. Plant Physiol Biochem 42:215–223

    Article  CAS  PubMed  Google Scholar 

  • Yu S, Galvao VC, Zhang YC, Horrer D, Zhang TQ, Hao YH, Feng YQ, Wang S, Schmid M, Wang JW (2012) Gibberellin regulates the Arabidopsis floral transition through miR156-targeted SQUAMOSA promoter binding-like transcription factors. Plant Cell 24:3320–3332

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zhai L, Xu L, Wang Y, Cheng H, Chen Y, Gong Y, Liu L (2013) Novel and useful genic-SSR markers from de novo transcriptome sequencing of radish (Raphanus sativus L.). Mol Breed. doi:10.1007/s11032-013-9978-x

  • Zhang H, Ogas J (2009) An epigenetic perspective on developmental regulation of seed genes. Mol Plant 2:610–627

    Article  CAS  PubMed  Google Scholar 

  • Zhang J, Zhang S, Han S, Wu T, Li X, Li W, Qi L (2012) Genome-wide identification of microRNAs in larch and stage-specific modulation of 11 conserved microRNAs and their targets during somatic embryogenesis. Planta 236:647–657

    Article  CAS  PubMed  Google Scholar 

  • Zhang XN, Li X, Liu JH (2013) Identification of conserved and novel cold-responsive microRNA in trifoliate orange (Poncirus trifoliata (L.) Raf.) using high-throughput sequencing. Plant Mol Biol Rep. doi:10.1007/s11105-013-0649-1

    PubMed Central  PubMed  Google Scholar 

  • Zhu QH, Helliwell CA (2011) Regulation of flowering time and floral patterning by miR172. J Exp Bot 62:487–495

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported in part by grants from the Program for the National Natural Science Foundation of China (31171956, 31372064), the Key Technologies R & D Program of Jiangsu Province, China (BE2010328,BE2013429), the PAPD and JASTIF [CX(12)2006]. We thank Dr. Oliver R. E. from North Dakota State University, ND, USA for her critical review and helpful comments during the preparation of this manuscript.

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Zhai, L., Xu, L., Wang, Y. et al. Genome-Wide Identification of Embryogenesis-Associated microRNAs in Radish (Raphanus sativus L.) by High-Throughput Sequencing. Plant Mol Biol Rep 32, 900–915 (2014). https://doi.org/10.1007/s11105-014-0700-x

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