, Volume 237, Issue 4, pp 1149–1161 | Cite as

Genome-wide identification and characterization of microRNA genes and their targets in flax (Linum usitatissimum)

Characterization of flax miRNA genes
  • Vitthal T. Barvkar
  • Varsha C. Pardeshi
  • Sandip M. Kale
  • Shuqing Qiu
  • Meaghen Rollins
  • Raju Datla
  • Vidya S. Gupta
  • Narendra Y. Kadoo
Original Article


MicroRNAs (miRNAs) are small (20–24 nucleotide long) endogenous regulatory RNAs that play important roles in plant growth and development. They regulate gene expression at the post-transcriptional level by translational repression or target degradation and gene silencing. In this study, we identified 116 conserved miRNAs belonging to 23 families from the flax (Linum usitatissimum L.) genome using a computational approach. The precursor miRNAs varied in length; while most of the mature miRNAs were 21 nucleotide long, intergenic and showed conserved signatures of RNA polymerase II transcripts in their upstream regions. Promoter region analysis of the flax miRNA genes indicated prevalence of MYB transcription factor binding sites. Four miRNA gene clusters containing members of three phylogenetic groups were identified. Further, 142 target genes were predicted for these miRNAs and most of these represent transcriptional regulators. The miRNA encoding genes were expressed in diverse tissues as determined by digital expression analysis as well as real-time PCR. The expression of fourteen miRNAs and nine target genes was independently validated using the quantitative reverse transcription PCR (qRT-PCR). This study suggests that a large number of conserved plant miRNAs are also found in flax and these may play important roles in growth and development of flax.


Linseed MiRNA Promoter analysis Digital expression analysis Gene cluster MiRNA target transcript 

Supplementary material

425_2012_1833_MOESM1_ESM.xlsx (68 kb)
Supplementary material 1 (XLSX 67 kb)
425_2012_1833_MOESM2_ESM.pdf (3.7 mb)
Supplementary material 2 (PDF 3781 kb)
425_2012_1833_MOESM3_ESM.xlsx (288 kb)
Supplementary material 3 (XLSX 287 kb)
425_2012_1833_MOESM4_ESM.tif (3.2 mb)
Hierarchical representation of GO terms overrepresented in biological process (a), cellular component (b) and molecular function (c) categories generated by agriGO analysis. The boxes represent GO terms, GO id, term definition and statistical information and are color coded as per the level of significance (0.05). Non-significant terms are shown as white boxes. The degree of color saturation of a box is positively correlated to the enrichment level of the term. Solid, dashed, and dotted lines represent two, one and zero enriched terms at both ends connected by the line, respectively. The rank direction of the graph is set to from left to right (TIFF 3284 kb)


  1. Alexandrov NN, Troukhan ME, Brover VV, Tatarinova T, Flavell RB, Feldmann KA (2006) Features of Arabidopsis genes and genome discovered using full-length cDNAs. Plant Mol Biol 60:69–85PubMedCrossRefGoogle Scholar
  2. Altuvia Y, Landgraf P, Lithwick G, Elefant N, Pfeffer S, Aravin A, Brownstein MJ, Tuschl T, Margalit H (2005) Clustering and conservation patterns of human microRNAs. Nucleic Acids Res 33:2697–2706PubMedCrossRefGoogle Scholar
  3. Ambros V, Bartel B, Bartel DP, Burge CB, Carrington JC, Chen XM, Dreyfuss G, Eddy SR, Griffiths-Jones S, Marshall M, Matzke M, Ruvkun G, Tuschl T (2003) A uniform system for microRNA annotation. RNA 9:277–279PubMedCrossRefGoogle Scholar
  4. Arnaud D, Dejardin A, Leple JC, Lesage-Descauses MC, Boizot N, Villar M, Benedetti H, Pilate G (2012) Expression analysis of LIM gene family in poplar, toward an updated phylogenetic classification. BMC Res Notes 5:102PubMedCrossRefGoogle Scholar
  5. Aukerman MJ, Sakai H (2003) Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes. Plant Cell 15:2730–2741PubMedCrossRefGoogle Scholar
  6. Axtell MJ, Bartel DP (2005) Antiquity of microRNAs and their targets in land plants. Plant Cell 17:1658–1673PubMedCrossRefGoogle Scholar
  7. Barakat A, Wall PK, Diloreto S, Depamphilis CW, Carlson JE (2007) Conservation and divergence of microRNAs in Populus. BMC Genomics 8:481PubMedCrossRefGoogle Scholar
  8. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297PubMedCrossRefGoogle Scholar
  9. Bentwich I (2005) Prediction and validation of microRNAs and their targets. FEBS Lett 579:5904–5910PubMedCrossRefGoogle Scholar
  10. Bonnet E, Wuyts J, Rouze P, Van de Peer Y (2004) Evidence that microRNA precursors, unlike other non-coding RNAs, have lower folding free energies than random sequences. Bioinformatics 20:2911–2917PubMedCrossRefGoogle Scholar
  11. Brennecke J, Stark A, Russell RB, Cohen SM (2005) Principles of MicroRNA-target recognition. PLoS Biol 3:404–418CrossRefGoogle Scholar
  12. Calvino M, Bruggmann R, Messing J (2011) Characterization of the small RNA component of the transcriptome from grain and sweet sorghum stems. BMC Genomics 12:356PubMedCrossRefGoogle Scholar
  13. Cui X, Xu SM, Mu DS, Yang ZM (2009) Genomic analysis of rice microRNA promoters and clusters. Gene 431:61–66PubMedCrossRefGoogle Scholar
  14. Dai XB, Zhao PX (2011) psRNATarget: a plant small RNA target analysis server. Nucleic Acids Res 39:155–159CrossRefGoogle Scholar
  15. Deng W, Nickle DC, Learn GH, Maust B, Mullins JI (2007) ViroBLAST: a stand-alone BLAST web server for flexible queries of multiple databases and user’s datasets. Bioinformatics 23:2334–2336PubMedCrossRefGoogle Scholar
  16. Du Z, Zhou X, Ling Y, Zhang ZH, Su Z (2010) AgriGO: a GO analysis toolkit for the agricultural community. Nucleic Acids Res 38:W64–W70PubMedCrossRefGoogle Scholar
  17. Fenart S, Ndong YPA, Duarte J, Riviere N, Wilmer J, van Wuytswinkel O, Lucau A, Cariou E, Neutelings G, Gutierrez L, Chabbert B, Guillot X, Tavernier R, Hawkins S, Thomasset B (2010) Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray. BMC Genomics 11:592PubMedCrossRefGoogle Scholar
  18. Frazier TP, Xie FL, Freistaedter A, Burklew CE, Zhang BH (2010) Identification and characterization of microRNAs and their target genes in tobacco (Nicotiana tabacum). Planta 232:1289–1308PubMedCrossRefGoogle Scholar
  19. Gou JY, Felippes FF, Liu CJ, Weigel D, Wang JW (2011) Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. Plant Cell 23:1512–1522PubMedCrossRefGoogle Scholar
  20. Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36:D154–D158PubMedCrossRefGoogle Scholar
  21. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Res 27:297–300PubMedCrossRefGoogle Scholar
  22. Hofacker IL (2003) Vienna RNA secondary structure server. Nucleic Acids Res 31:3429–3431PubMedCrossRefGoogle Scholar
  23. Huang Y, Niu BF, Gao Y, Fu LM, Li WZ (2010) CD-HIT Suite: a web server for clustering and comparing biological sequences. Bioinformatics 26:680–682PubMedCrossRefGoogle Scholar
  24. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAs and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53PubMedCrossRefGoogle Scholar
  25. Jung JH, Seo PJ, Kang SK, Park CM (2011) miR172 signals are incorporated into the miR156 signaling pathway at the SPL3/4/5 genes in Arabidopsis developmental transitions. Plant Mol Biol 76:35–45PubMedCrossRefGoogle Scholar
  26. Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843–854PubMedCrossRefGoogle Scholar
  27. Lu C, Meyers BC, Green PJ (2007) Construction of small RNA cDNA libraries for deep sequencing. Methods 43:110–117PubMedCrossRefGoogle Scholar
  28. Masoudi-Nejad A, Tonomura K, Kawashima S, Moriya Y, Suzuki M, Itoh M, Kanehisa M, Endo T, Goto S (2006) EGassembler: online bioinformatics service for large-scale processing, clustering and assembling ESTs and genomic DNA fragments. Nucleic Acids Res 34:W459–W462PubMedCrossRefGoogle Scholar
  29. Mathews DH, Sabina J, Zuker M, Turner DH (1999) Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol 288:911–940PubMedCrossRefGoogle Scholar
  30. McCarthy FM, Wang N, Magee GB, Nanduri B, Lawrence ML, Camon EB, Barrell DG, Hill DP, Dolan ME, Williams WP, Luthe DS, Bridges SM, Burgess SC (2006) AgBase: a functional genomics resource for agriculture. BMC Genomics 7:229PubMedCrossRefGoogle Scholar
  31. Megraw M, Baev V, Rusinov V, Jensen ST, Kalantidis K, Hatzigeorgiou AG (2006) MicroRNA promoter element discovery in Arabidopsis. RNA 12:1612–1619PubMedCrossRefGoogle Scholar
  32. Meyers BC, Axtell MJ, Bartel B, Bartel DP, Baulcombe D, Bowman JL, Cao X, Carrington JC, Chen XM, Green PJ, Griffiths-Jones S, Jacobsen SE, Mallory AC, Martienssen RA, Poethig RS, Qi YJ, Vaucheret H, Voinnet O, Watanabe Y, Weigel D, Zhui JK (2008) Criteria for annotation of plant microRNAs. Plant Cell 20:3186–3190PubMedCrossRefGoogle Scholar
  33. Moss TY, Cullis CA (2012) Computational prediction of candidate microRNAs and their targets from the completed Linum usitatissimum genome and EST database. J Nucleic Acids Investig 3:e2Google Scholar
  34. Moxon S, Schwach F, Dalmay T, MacLean D, Studholme DJ, Moulton V (2008) A toolkit for analysing large-scale plant small RNA datasets. Bioinformatics 24:2252–2253PubMedCrossRefGoogle Scholar
  35. Neutelings G, Fenart S, Lucau-Danila A, Hawkins S (2012) Identification and characterization of miRNAs and their potential targets in flax. J Plant Physiol 169:1754–1766PubMedCrossRefGoogle Scholar
  36. Prufer K, Stenzel U, Dannemann M, Green RE, Lachmann M, Kelso J (2008) PatMaN: rapid alignment of short sequences to large databases. Bioinformatics 24:1530–1531PubMedCrossRefGoogle Scholar
  37. Rajagopalan R, Vaucheret H, Trejo J, Bartel DP (2006) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev 20:3407–3425PubMedCrossRefGoogle Scholar
  38. Ruan MB, Zhao YT, Meng ZH, Wang XJ, Yang WC (2009) Conserved miRNA analysis in Gossypium hirsutum through small RNA sequencing. Genomics 94:263–268PubMedCrossRefGoogle Scholar
  39. Smale ST (2001) Core promoters: active contributors to combinatorial gene regulation. Genes Dev 15:2503–2508PubMedCrossRefGoogle Scholar
  40. Solovyev V, Salamov A (1997) The gene-finder computer tools for analysis of human and model organisms genome sequences. In: Rawling C, Clark D, Altman R, Hunter L, Lengauer T, Wodak S (eds) fifth international conference on intelligent systems for molecular biology, proceedings. AAAI Press, Halkidiki, Greece, pp 294–302Google Scholar
  41. Song C, Jia Q, Fang J, Li F, Wang C, Zhang Z (2010) Computational identification of citrus microRNAs and target analysis in citrus expressed sequence tags. Plant Biology 12:927–934PubMedCrossRefGoogle Scholar
  42. Sun G (2012) MicroRNAs and their diverse functions in plants. Plant Mol Biol 80:17–36PubMedCrossRefGoogle Scholar
  43. Tang GL (2010) Plant microRNAs: an insight into their gene structures and evolution. Semin Cell Dev Biol 21:782–789PubMedCrossRefGoogle Scholar
  44. Thakur V, Wanchana S, Xu M, Bruskiewich R, Quick WP, Mosig A, Zhu XG (2011) Characterization of statistical features for plant microRNA prediction. BMC Genomics 12:108PubMedCrossRefGoogle Scholar
  45. Turner M, Yu O, Subramanian S (2012) Genome organization and characteristics of soybean microRNAs. BMC Genomics 13:169PubMedCrossRefGoogle Scholar
  46. Unver T, Budak H (2009) Conserved microRNAs and their targets in model grass species Brachypodium distachyon. Planta 230:659–669PubMedCrossRefGoogle Scholar
  47. Venglat P, Xiang D, Qiu S, Stone SL, Tibiche C, Cram D, Alting-Mees M, Nowak J, Cloutier S, Deyholos M, Bekkaoui F, Sharpe A, Wang E, Rowland G, Selvaraj G, Datla R (2011) Gene expression analysis of flax seed development. BMC Plant Biol 11:74PubMedCrossRefGoogle Scholar
  48. Wang YP, Li KB (2009) Correlation of expression profiles between microRNAs and mRNA targets using NCI-60 data. BMC Genomics 10:218Google Scholar
  49. Wang JW, Czech B, Weigel D (2009) miR156-Regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana. Cell 138:738–749PubMedCrossRefGoogle Scholar
  50. Wang Z, Hobson N, Galindo L, Zhu S, Shi D, McDill J, Yang L, Hawkins S, Neutelings G, Datla R, Lambert G, Galbraith DW, Grassa CJ, Geraldes A, Cronk QC, Cullis C, Dash PK, Kumar PA, Cloutier S, Sharpe A, Wong GK, Wang J, Deyholos MK (2012) The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads. Plant J 72:461–473PubMedCrossRefGoogle Scholar
  51. Weber MJ (2005) New human and mouse microRNA genes found by homology search. FEBS J 272:59–73PubMedCrossRefGoogle Scholar
  52. Wightman B, Ha I, Ruvkun G (1993) Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75:855–862PubMedCrossRefGoogle Scholar
  53. Wu MF, Tian Q, Reed JW (2006) Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression, and regulates both female and male reproduction. Development 133:4211–4218PubMedCrossRefGoogle Scholar
  54. Xie ZX, Allen E, Fahlgren N, Calamar A, Givan SA, Carrington JC (2005) Expression of Arabidopsis MIRNA genes. Plant Physiol 138:2145–2154PubMedCrossRefGoogle Scholar
  55. Xie FL, Xiao P, Zhang BH (2011) Target-align: a tool for plant MicroRNA target identification. In Vitro Cell Dev Biol-Animal 47:S71–S72Google Scholar
  56. Yang TW, Xue LG, An LZ (2007) Functional diversity of miRNA in plants. Plant Sci 172:423–432CrossRefGoogle Scholar
  57. Zeng CY, Wang WQ, Zheng Y, Chen X, Bo WP, Song S, Zhang WX, Peng M (2010) Conservation and divergence of microRNAs and their functions in Euphorbiaceous plants. Nucleic Acids Res 38:981–995PubMedCrossRefGoogle Scholar
  58. Zhang BH, Pan XP, Anderson TA (2006a) Identification of 188 conserved maize microRNAs and their targets. FEBS Lett 580:3753–3762PubMedCrossRefGoogle Scholar
  59. Zhang BH, Pan XP, Cannon CH, Cobb GP, Anderson TA (2006b) Conservation and divergence of plant microRNA genes. Plant J 46:243–259PubMedCrossRefGoogle Scholar
  60. Zhang BH, Pan XP, Cox SB, Cobb GP, Anderson TA (2006c) Evidence that miRNAs are different from other RNAs. Cell Mol Life Sci 63:246–254PubMedCrossRefGoogle Scholar
  61. Zhang BH, Wang QL, Wang KB, Pan XP, Liu F, Guo TL, Cobb GP, Anderson TA (2007) Identification of cotton microRNAs and their targets. Gene 397:26–37PubMedCrossRefGoogle Scholar
  62. Zhang BH, Pan XP, Stellwag EJ (2008) Identification of soybean microRNAs and their targets. Planta 229:161–182PubMedCrossRefGoogle Scholar
  63. Zhang LF, Chia JM, Kumari S, Stein JC, Liu ZJ, Narechania A, Maher CA, Guill K, McMullen MD, Ware D (2009) A genome-wide characterization of microRNA genes in Maize. PLoS Genet 5:11Google Scholar
  64. Zhao BT, Ge LF, Liang RQ, Li W, Ruan KC, Lin HX, Jin YX (2009) Members of miR-169 family are induced by high salinity and transiently inhibit the NF-YA transcription factor. BMC Mol Biol 10:29Google Scholar
  65. Zhong R, Ye ZH (2007) Regulation of HD-ZIP III Genes by MicroRNA 165. Plant Signal Behav 2:351–353PubMedCrossRefGoogle Scholar
  66. Zhou M, Sun J, Wang QH, Song LQ, Zhao GA, Wang HZ, Yang HX, Li X (2011) Genome-wide analysis of clustering patterns and flanking characteristics for plant microRNA genes. FEBS J 278:929–940PubMedCrossRefGoogle Scholar
  67. Zhu RS, Li X, Chen QS (2011) Discovering numerical laws of plant microRNA by evolution. Biochem Biophys Res Commun 415:313–318PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Vitthal T. Barvkar
    • 1
  • Varsha C. Pardeshi
    • 1
  • Sandip M. Kale
    • 1
  • Shuqing Qiu
    • 2
  • Meaghen Rollins
    • 2
  • Raju Datla
    • 2
  • Vidya S. Gupta
    • 1
  • Narendra Y. Kadoo
    • 1
  1. 1.Biochemical Sciences DivisionCSIR-National Chemical LaboratoryPuneIndia
  2. 2.Plant Biotechnology Institute, NRCSaskatoonCanada

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