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Deep sequencing discovery of novel and conserved microRNAs in wild type and a white-flesh mutant strawberry

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MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by base pairing to mRNA target sequences, and play crucial roles in plant development and stress responses. The knowledge on post-transcriptional regulation by miRNAs in strawberry is rather limited so far. In order to understand the role of miRNA in the molecular control during strawberry fruit development, small RNA libraries were constructed from fruits at the turning stage of strawberry cultivar ‘Sachinoka’ and its white-flesh mutant by using the Solexa platform. One hundred and twenty conserved miRNAs belonging to 27 miRNA families and 33 putative novel strawberry miRNAs were identified in both libraries. Their target genes were predicted using the Fragaria vesca genome. Nine of all miRNAs showed significant expression differences between two types of samples. Four miRNAs were up-regulated and five were down-regulated in white-flesh mutant. The sequencing results were partially validated by quantitative RT-PCR. Among them, the expression of miR399a shows the biggest change between the two samples. The prediction of its target gene showed that miR399 may play an important role in phosphate homeostasis of strawberry fruits. Furthermore, we deduce that the expression of miR399 has negative correlation with the content of sugars.

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Deoxynucleotide triphosphate


Minimal folding free energy




Mutant type


National Center for Biotechnology Information


miRNA precursor


Primary miRNA


Quantitative reverse transcription-polymerase chain reaction


RNA-induced silencing complex


Wild type


  1. Achard P, Herr A, Baulcombe DC, Harberd NP (2004) Modulation of floral development by a gibberellin-regulated microRNA. Development 131:3357–3365

  2. Adai A, Johnson C, Mlotshwa S, Archer-Evans S, Manocha V, Vance V, Sundaresan V (2005) Computational prediction of miRNAs in Arabidopsis thaliana. Genome Res 15:78–91

  3. Allen E, Xie Z, Gustafson AM, Carrington JC (2005) microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121:207–221

  4. Ambros V, Bartel B, Bartel DP, Burge CB, Carrington JC, Chen X, Dreyfuss G, Eddy SR, Griffiths-Jones S, Marshall M, Matzke M, Ruvkun G, Tuschl T (2003) A uniform system for microRNA annotation. RNA 9:277–279

  5. Axtell MJ, Bartel DP (2005) Antiquity of microRNAs and their targets in land plants. Plant Cell 17:1658–1673

  6. Bari R, Datt Pant B, Stitt M, Scheible WR (2006) PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants. Plant Physiol 141:988–999

  7. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297

  8. Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW (2011) GenBank. Nucleic Acids Res 39:D32–D37

  9. Bonnet E, Wuyts J, Rouzé P, de Peer VY (2004) Evidence that microRNA precursors, unlike other non-coding RNAs, have lower folding free energies than random sequences. Bioinformatics 20:2911–2917

  10. Carra A, Mica E, Gambino G, Pindo M, Moser C, Enrico MP, Schubert A (2009) Cloning and characterization of small non-coding RNAs from grape. Plant J 59:750–763

  11. Chang L, Zhang Z, Yang H, Li H, Dai H (2007) Detection of strawberry RNA and DNA viruses by RT-PCR using total nucleic acid as a template. J Phytopathol 155:431–436

  12. Chen X (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303:2022–2025

  13. Chen X (2008) MicoRNA metabolism in plants. Curr Top Microbiol Immunol 320:117–136

  14. Chen CF, Ridzon DA, Broomer AJ, Zhou ZH, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33:e179

  15. Chen R, Hu Z, Zhang H (2009) Identification of microRNAs in wild soybean (Glycine soja). J Integr Plant Biol 51:1071–1079

  16. Chen L, Ren Y, Zhang Y, Xu J, Zhang Z, Wang Y (2011) Genome-wide profiling of novel and conserved Populus microRNAs involved in pathogen stress response by deep sequencing. Planta 235:873–883

  17. Csukasi F, Donaire L, Casañal A, Martínez-Priego L, Botella MA, Medina-Escobar N, Llave C, Valpuesta V (2012) Two strawberry miR159 family members display developmental-specific expression patterns in the fruit receptacle and cooperatively regulate Fa-GAMYB. New Phytol 195:47–57

  18. Dolgosheina EV, Morin RD, Aksay G, Sahinalp SC, Magrini V, Mardis ER, Mattsson J, Unrau PJ (2008) Conifers have a unique small RNA silencing signature. RNA 14:1508–1515

  19. Floyd SK, Bowman JL (2004) Gene regulation: ancient microRNA target sequences in plants. Nature 428:485–486

  20. Frazier TP, Xie F, Freistaedter A, Burklew CE, Zhang B (2010) Identification and characterization of microRNAs and their target genes in tobacco (Nicotiana tabacum). Planta 232:1289–1308

  21. Fujii H, Chiou TJ, Lin SI, Aung K, Zhu JK (2005) A miRNA involved in phosphate-starvation response in Arabidopsis. Curr Biol 15:2038–2043

  22. Ge AJ, Shangguan LF, Zhang X, Dong QH, Han J, Liu H, Wang XC, Fang JG (2013) Deep sequencing discovery of novel and conserved microRNAs in strawberry (Fragaria × ananassa). Physiol Plantarum 148:387–396

  23. Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36:D154–D158

  24. 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

  25. Henderson IR, Zhang XY, Lu C, Johnson L, Meyers BC, Green PJ, Jacobsen SE (2006) Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat Genet 38:721–725

  26. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAs and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53

  27. Juarez MT, Kui JS, Thomas J, Heller BA, Timmermans MC (2004) MicroRNA-mediated repression of rolled leaf specifies maize leaf polarity. Nature 428:84–88

  28. Karthikeyan AS, Varadarajan DK, Jain A, Held MA, Carpita NC, Raghothama KG (2007) Phosphate starvation responses are mediated by sugar signaling in Arabidopsis. Planta 225:907–918

  29. Li R, Li Y, Kristiansen K, Wang J (2008) SOAP: short oligonucleotide alignment program. Bioinformatics 24:713–714

  30. Li H, Zhang Z, Huang F, Chang L, Ma Y (2009) MicroRNA expression profiles in conventional and micropropagated strawberry (Fragaria × annanassa Duch.) plants. Plant Cell Rep 28:891–902

  31. Li B, Qin Y, Duan H, Yin W, Xia X (2011) Genome-wide characterization of new and drought stress responsive microRNAs in Populus euphratica. J Exp Bot 62:3765–3779

  32. Liang Y, Li H, Ma Y, Cao F, Dou Y, Zhang Z (2012) Comparative analysis of biological characteristics and quality in a white-flesh strawberry mutant ‘Sachinoka’ and its wild type. Scientia Agricultura Sinica 45:3115–3123

  33. Lin SI, Chiang SF, Lin WY, Chen JW, Tseng CY, Wu PC, Chiou TJ (2008) Regulatory network of microRNA399 and PHO2 by systemic signaling. Plant Physiol 147:732–746

  34. Liu J, Samac DA, Bucciarelll B, Allan DL, Vance CP (2005) Signaling of phosphorus deficiency-induced gene expression in white lupin requires sugar and phloem transport. Plant J 41:257–268

  35. Liu TY, Chang CY, Chiou TJ (2009) The long-distance signaling of mineral macronutrients. Curr Opin Plant Biol 12:312–319

  36. Liu JQ, Allan DL, Vance CP (2010) Systemic signaling and local sensing of phosphate in common bean: cross-talk between photosynthate and microRNA399. Mol Plant 3:428–437

  37. Llave C, Xie Z, Kasschau KD, Carrington JC (2002) Cleavage of scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297:2053–2056

  38. Lu S, Sun YH, Shi R, Clark C, Li L, Chiang VL (2005) Novel and mechanical stress-responsive microRNAs in Populus trichocarpa that are absent from Arabidopsis. Plant Cell 17:2186–2203

  39. Lu S, Sun YH, Chiang VL (2008) Stress-responsive microRNAs in Populus. Plant J 55:131–151

  40. 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

  41. Mohorianu I, Schwach F, Jing R, Lopez-Gomollon S, Moxon S, Szittya G, Sorefan K, Moulton V, Dalmay T (2011) Profiling of short RNAs during fleshy fruit development reveals stage-specific sRNAome expression patterns. Plant J 67:232–246

  42. Morin RD, Aksay G, Dolgosheina E, Ebhardt HA, Magrini V, Mardis ER, Sahinalp SC, Unrau PJ (2008) Comparative analysis of the small RNA transcriptomes of Pinus contorta and Oryza sativa. Genome Res 18:571–584

  43. Moxon S, Jing R, Szittya G, Schwach F, Rusholme Pilcher RL, Moulton V, Dalmay T (2008) Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening. Genome Res 18:1602–1609

  44. Pantaleo V, Szittya G, Moxon S, Miozzi L, Moulton V, Dalmay T, Burgyan J (2010) Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis. Plant J 62:960–976

  45. Qiu D, Pan X, Wilson IW, Ketchum REB, Li F, Liu M, Teng W, Zhang BH (2009) High throughput sequencing technology reveals that the taxoid elicitor methyl jasmonate regulates microRNA expression in Chinese yew (Taxus chinensis). Gene 436:37–44

  46. Rajagopalan R, Vaucheret H, Trejo J, Bartel DP (2006) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev 20:3407–3425

  47. Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev 16:1616–1626

  48. Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP (2002) Prediction of plant microRNA targets. Cell 110:513–520

  49. Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, Weigel D (2005) Specific effects of microRNAs on the plant transcriptome. Dev Cell 8:517–527

  50. Shulaev V, Sargent DJ, Crowhurst RN, Mockler TC, Folkerts O, Delcher AL, Jaiswal P, Mockaitis K, Liston A, Mane SP, Burns P, Davis TM, Slovin JP, Bassil N, Hellens RP, Evans C, Harkins T, Kodira C, Desany B, Crasta OR, Jensen RV, Allan AC, Michael TP, Setubal JC, Celton JM, Rees DJG, Williams KP, Holt SH, Rojas JJR, Chatterjee M, Liu B, Silva H, Meisel L, Adato A, Filichkin SA, Troggio M, Viola R, Ashman TL, Wang H (2011) The genome of woodland strawberry (Fragaria vesca). Nat Genet 43:109–116

  51. Sunkar R, Jagadeeswaran G (2008) In silico identifcation of conserved microRNAs in large number of diverse plant species. BMC Plant Biol 8:37

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

  53. Sunkar R, Girke T, Jain PK, Zhu JK (2005) Cloning and characterization of microRNAs from rice. Plant Cell 17:1397–1411

  54. Sunkar R, Zhou X, Zheng Y, Zhang W, Zhu JK (2008) Identification of novel and candidate miRNAs in rice by high throughput sequencing. BMC Plant Biol 8:25

  55. Xie Z, Khanna K, Ruan S (2010) Expression of microRNAs and its regulation in plants. Semin Cell Dev Biol 21:790–797

  56. Xu Q, Liu Y, Zhu A, Wu X, Ye J, Yu K, Guo W, Deng X (2010) Discovery and comparative profiling of microRNAs in a sweet orange red-flesh mutant and its wild type. BMC Genomics 11:246

  57. Yang JH, Liu XY, Xu BC, Zhao N, Yang XD, Zhang MF (2013) Identification of miRNAs and their targets using high-throughput sequencing and degradome ananlysis in cytoplasmic male-sterile and its maintainer fertile lines of Brassica juncea. BMC Genomics 14:9

  58. Yao YY, Guo GG, Ni ZF, Sunkar R, Du JK, Zhu JK, Sun QX (2007) Cloning and characterization of microRNAs from wheat (Triticum aestivum L.). Genome Biol 8:R96

  59. Zhang BH, Pan XP, Cox SB, Cobb GP, Anderson TA (2006) Evidence that miRNAs are different from other RNAs. Cell Mol Life Sci 63:246–254

  60. Zhang J, Xu Y, Huan Q, Chong K (2009) Deep sequencing of Brachypodium small RNAs at the global genome level identifies microRNAs involved in cold stress response. BMC Genomics 10:449

  61. Zhao CZ, Xia H, Frazier TP, Yao YY, Bi YP, Li AQ, Li MJ, Li CS, Zhang BH, Wang XJ (2010) Deep sequencing identifies novel and conserved microRNAs in peanuts (Arachis hypogaea L.). BMC Plant Biol 10:3

  62. Zorrilla-Fontanesi Y, Cabeza A, Domínguez P, Medina JJ, Valpuesta V, Denoyes-Rothan B, Sánchez-Sevilla JF, Amaya I (2011) Quantitative trait loci and underlying candidate genes controlling agronomical and fruit quality traits in octoploid strawberry (Fragaria × ananassa). Theor Appl Genet 123:755–778

  63. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415

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This work was financially supported by National Natural Science Foundation of China (31101524), Special Fund for Agro-scientific Research in the Public Interest (201003064), Postdoctoral Foundation of China (20100481212 and 2012T50270) and Program for Innovation Team in University of Liaoning Province (LT2010094).

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Correspondence to Zhihong Zhang.

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Li, H., Mao, W., Liu, W. et al. Deep sequencing discovery of novel and conserved microRNAs in wild type and a white-flesh mutant strawberry. Planta 238, 695–713 (2013). https://doi.org/10.1007/s00425-013-1917-x

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  • Fruit development
  • Fragaria × ananassa
  • MicroRNA
  • Solexa sequencing
  • qRT-PCR