Tropical Plant Biology

, Volume 12, Issue 1, pp 44–54 | Cite as

Analysis of Small RNAs from Solanum torvum Swartz by Deep Sequencing

  • Xu Yang
  • Yu Zhang
  • Jinyan Xue
  • Fei Liu
  • Yufu ChengEmail author


Verticillium wilt causes substantial economic losses in eggplant by reducing its yield and quality. However, the relationship between the host and fungal pathogen is poorly understood. Large amounts of high quality clean reads were obtained from our samples. We used bioinformatics analysis to detect 373 differentially expressed miRNAs and 18 differentially expressed novel miRNAs. Eleven highly enriched MIRNA families were found to be involved in the resistance to Verticillium Wilt in eggplant. We identified the differentially expressed miRNAs among samples and analyzed their 78 target genes. These target genes were annotated with GO function enrichment and KEGG pathway association. The target genes included transcription factors such as TCP, E3 and FEX6 etc. Functional categorization of those target genes reflected a number of important pathways, which are involved in the plant’s resistance to various pathogens. These differentially expressed miRNAs and their target genes will provide a theoretical basis for the further study of Verticillium wilt-related miRNAs and the regulation of disease resistance mechanisms of eggplant at the miRNA level.


Verticillium wilt Solanum torvum Swartz Verticillium dahlia Klep Small RNA 



Clusters of Orthologous Groups


False Discovery Rate


Gene Ontology


Kyoto Encyclopedia of Genes and Genomes




next-generation sequencing


RNA-induced silencing complex


small interfering RNAs


small RNAs


tags per Million reads





The work presented here was supported by the National Natural Science Foundation of China (Grant No.NSFC31171954), the Natural Science Foundation of Jiangsu Province (Grant No. BK2010320), China Postdoctoral Science Foundation (Grant No. 20110491463) and Jiangsu Postdoctoral Science Foundation (Grant No. 1002001C).

Author’s Contributions

All authors contributed experimental design oversight. XY and Y-f C designed the experiments. YZ performed the experiments. YZ, FL and J-y X analyzed the data. YZ and FL wrote the paper. All authors read and approved the final manuscript.

Compliance with Ethical Standards

Ethics Approval and Consent to Participate

Before the sampling phase of the study, the approval for handling and sampling of eggplant samples were obtained from school of Horticulture and Plant Protection, yangzhou university.

Competing Interests

The authors declare that they have no competing interests.

Supplementary material

12042_2018_9216_MOESM1_ESM.xls (414 kb)
ESM 1 (XLS 414 kb)
12042_2018_9216_MOESM2_ESM.doc (284 kb)
ESM 2 (DOC 284 kb)


  1. Achard P, Herr A, Baulcombe DC, Harberd NP (2004) Modulation of floral development by a gibberellin-regulated microrna. Development 131(14):3357–3365Google Scholar
  2. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402Google Scholar
  3. Apweiler R, Bairoch A, Wu CH, Barker WC, Boeckmann B, Ferro S, Gasteiger E, Huang H, Lopez R, Magrane M, Martin MJ, Natale DA, O'Donovan C, Redaschi N, Yeh LSL (2004) UniProt: the universal protein knowledgebase. Nucleic Acids Res 32:115–119Google Scholar
  4. Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, Gomez-Gomez L, Boller T, Ausubel FM, Sheen J (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415(6875):977–983Google Scholar
  5. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G (2000) Gene ontology: tool for the unification of biology. The gene ontology consortium. Nat Genet 25(1):25–29Google Scholar
  6. Baker CC, Sieber P, Wellmer F, Meyerowitz EM (2005) The early extra petals1 mutant uncovers a role for microRNA miR164c in regulating petal number in Arabidopsis. Curr Biol 15(4):303–315Google Scholar
  7. Belkhadir Y, Jaillais Y, Epple P, Balsemão-Pires E, Dangl JL, Chory J (2012) Brassinosteroids modulate the efficiency of plant immune responses to microbe-associated molecular patterns. Proc Natl Acad Sci U S A 109(1):297–302Google Scholar
  8. Bhat RG, Subbarao KV (1999) Host range specificity in Verticillium dahliae. Phytopathology 89(12):1218–1225Google Scholar
  9. Brodersen P, Voinnet O (2006) The diversity of RNA silencing pathways in plants. Trends Genet 22(5):268–280Google Scholar
  10. Burge SW, Daub J, Eberhardt R, Tate J, Barquist L, Nawrocki EP, Eddy SR, Gardner PP, Bateman A (2013) Rfam 11.0: 10 years of RNA families. Nucleic Acids Res 41:226–232Google Scholar
  11. Chen X (2009) Small RNAs and their roles in plant development. Annu Rev Cell Dev Biol 25(1):21–44Google Scholar
  12. Covarrubias AA, Reyes JL (2010) Post-transcriptional gene regulation of salinity and drought responses by plant microRNAs. Plant Cell Environ 33(4):481–489Google Scholar
  13. Czech B, Hannon GJ (2010) Small RNA sorting: matchmaking for Argonautes. Nat Rev Genet 12(1):19–31Google Scholar
  14. Deng YY, Li JQ, Wu SF, Chen YW, He FC (2006) Integrated nr database in protein annotation system and its localization. Comput Eng 32(5):71–74Google Scholar
  15. Ellendorff U, Fradin EF, Jonge RD, Thomma BPHJ (2009) RNA silencing is required for Arabidopsis defence against Verticillium wilt disease. J Exp Bot 60(2):591–602Google Scholar
  16. Eynck C, Koopmann B, Grunewaldt-Stoecker G, Karlovsky P, Von Tiedemann A (2007) Differential interactions of Verticillium longisporum and V. dahliae with Brassica napus detected with molecular and histological techniques. Eur J Plant Pathol 118(3):259–274Google Scholar
  17. George N (2005) Agrios: plant pathology, 5th edn. Elsevier Academic Press, CAGoogle Scholar
  18. Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36:154–158Google Scholar
  19. Hao J, Tu LL, Hu HY, Tan JF, Deng FL, Tang WX, Nie YC, Zhang XL (2012) GbTCP, a cotton TCP transcription factor, confers fibre elongation and root hair development by a complex regulating system. J Exp Bot 63(17):6267–6281Google Scholar
  20. Jacob A, Lancaster J, Buhler J, Harris B, Chamberlain RD (2008) Mercury BLASTP: accelerating protein sequence alignment. ACM Trans Reconfigurable Technol Syst 1(2):9Google Scholar
  21. Jain M, Nijhawan A, Arora R, Agarwal P, Ray S, Sharma P, Kapoor S, Tyagi AK, Khurana JP (2007) F-box proteins in rice. Genome-wide analysis, classification, temporal and spatial gene expression during panicle and seed development, and regulation by light and abiotic stress. Plant Physiol 143(4):1467–1483Google Scholar
  22. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol 57(1):19–53Google Scholar
  23. Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori M (2004) The KEGG resource for deciphering the genome. Nucleic Acids Res 32:277–280Google Scholar
  24. Kapitonov VV, Jurka J (2008) A universal classification of eukaryotic transposable elements implemented in Repbase. Nat Rev Genet 9:411–412Google Scholar
  25. Katiyar-Agarwal S, Jin H (2010) Role of small RNAs in host-microbe interactions. Annu Rev Phytopathol 48(1):225–246Google Scholar
  26. Kiełbasa SM, Blüthgen N, Fähling M, Mrowka R (2010) a resource for systematic discovery of transcription factor target genes. Nucleic Acids Res 38:233–238Google Scholar
  27. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3):R25Google Scholar
  28. Li Y, Zhang Z, Liu F, Vongsangnak W, Jing Q, Shen BR (2012) Performance comparison and evaluation of software tools for microRNA deep-sequencing data analysis. Nucleic Acids Res 40:4298–4305Google Scholar
  29. Liu SP, Zhu YP, Xie C, Jue DW, Hong YB, Chen M, Hubdar AK, Yang Q (2012) Transgenic potato plants expressing StoVe1 exhibit enhanced resistance to Verticillium dahliae. Plant Mol Biol Rep 30(4):1032–1039Google Scholar
  30. Liu YL, Chen YH, Liu FZ, Zhang Y, Lian Y (2015) Cloning of E2 gene StUBCc and analysis of induced expression of Verticillium wilt. J Hortic 42(6):1185–1194Google Scholar
  31. Lu D, Lin W, Gao X, Wu S, Cheng C, Avila J, Heese A, Devarenne TP, He P, Shan L (2011) Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity. Science 332(6036):1439–1442Google Scholar
  32. Mallory AC, Vaucheret H (2006) Functions of microRNAs and related small RNAs in plants. Nat Genet 38:S31–S36Google Scholar
  33. Mallory AC, Bartel DP, Bartel B (2005) MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell 17:1360–1375Google Scholar
  34. 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(5):911–940Google Scholar
  35. Meyers BC et al (2008) Criteria for annotation of plant microRNAs. Plant Cell 20:3186–3190Google Scholar
  36. Mi S, Cai T, Hu Y, Chen Y, Hodges E, Ni F, Wu L, Li S, Zhou H, Long C, Chen S, Hannon GJ, Qi Y (2008) Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5′ terminal nucleotide. Cell 18(2):185–199Google Scholar
  37. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-seq. Nat Methods 5(7):621–628Google Scholar
  38. Ntoukakis V, Mucyn TS, Gimenez-Ibanez S, Chapman HC, Gutierrez JR, Balmuth AL, Jones AM, Rathjen JP (2009) Host inhibition of a bacterial virulence effector triggers immunity to infection. Science 324(5928):784–787Google Scholar
  39. Poethig RS (2009) Small RNAs and developmental timing in plants. Curr Opin Genet Dev 19(4):374–378Google Scholar
  40. Prieto P, Navarro-Raya C, Valverde-Corredor A, Amyotte SG, Dobinson KF, Mercado-Blanco J (2009) Colonization process of olive tissues by Verticillium dahliae and its in planta interaction with the biocontrol root endophyte Pseudomonas fluorescens PICF7. Microb Biotechnol 2(4):499–511Google Scholar
  41. Reyes JL, Chua NH (2007) ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination. Plant J 49(4):592–606Google Scholar
  42. Romualdi C, Bortoluzzi S, D'Alessi F, Danieli GA (2003) IDEG6: a web tool for detection of differentially expressed genes in multiple tag sampling experiments. Physiol Genomics 12(2):159–162Google Scholar
  43. 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(5):622–627Google Scholar
  44. Ruiz-Ferrer V, Voinnet O (2009) Roles of plant small RNAs in biotic stress responses. Annu Rev Plant Biol 60(1):485–510Google Scholar
  45. Sunkar R, Chinnusamy V, Zhu J, Zhu JK (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci 12(7):301–309Google Scholar
  46. 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(1):25Google Scholar
  47. Tatusov RL, Galperin MY, Natale DA, Koonin EV (2000) The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res 28(1):33–36Google Scholar
  48. Vallad GE, Subbarao KV (2008) Colonization of resistant and susceptible lettuce cultivars by a green fluorescent protein-tagged isolate of Verticillium dahlia. Phytopathology 98(8):871–885Google Scholar
  49. Voinnet O (2009) Origin, biogenesis, and activity of plant microRNAs. Cell 136(4):669–687Google Scholar
  50. Wang JW, Wang LJ, Mao YB, Cai WJ, Xue HW, Chen XY (2005) Control of root cap formation by MicroRNA-targeted auxin response factors in Arabidopsis. Plant Cell 17(8):2204–2216Google Scholar
  51. Wang D, Pajerowska-Mukhtar K, Culler AH, Dong X (2007) Salicylic acid inhibits pathogen growth in plants through repression of the auxin signaling pathway. Curr Biol 17(20):1784–1790Google Scholar
  52. Wang W, Barnaby JY, Tada Y, Li H, Tör M, Caldelari D, Lee DU, Fu XD, Dong X (2011) Timing of plant immune responses by a central circadian regulator. Nature 470(7332):110–114Google Scholar
  53. Wei XS, Cao BH, Lei JJ, Chen GJ, Chen QH (2010) Research Progress on disease resistance breeding of eggplant. China Vegetables 10:1–8Google Scholar
  54. Wollmann H, Weigel D (2010) Small RNAs in flower development. Eur J Cell Biol 89(2):250–257Google Scholar
  55. Yan YS, Chen XY, Yang K, Sun ZX, Fu YP, Zhang YM, Fang RX (2011) Overexpression of an f-box protein gene reduces abiotic stress tolerance and promotes root growth in rice. Mol Plant 4(1):190–197Google Scholar
  56. Yang L, Jue DW, Li W, Zhang RJ, Chen M, Yang Q (2013) Identification of MiRNA from eggplant (Solanum melongena L.) by small RNA deep sequencing and their response to Verticillium dahliae infection. PLoS One 8(8):e72840Google Scholar
  57. Yin ZJ, Li Y, Han XL, Shen FF (2012) Genome-wide profiling of miRNAs and other small non-coding RNAs in the Verticillium dahliae-inoculated cotton roots. PLoS One 7(4):e35765Google Scholar
  58. Zhang H (2003) Biological characteristics of pathogen of eggplant verticillium wilt and its control. Master's thesis. In: Yangzhou UniversityGoogle Scholar
  59. Zhao YT, Wang M, Fu SX, Yang WC, Qi CK, Wang XJ (2012) Small RNA profiling in two Brassica napus cultivars identifies microRNAs with oil production- and development-correlated expression and new small RNA classes. Plant Physiol 158(2):813–823Google Scholar
  60. Zhao P, Zhao YL, Jin Y, Zhang T, Guo HS (2014) Colonization process of Arabidopsis thaliana roots by a green fluorescent protein-tagged isolate of Verticillium dahliae. Protein Cell 5(2):94–98Google Scholar
  61. Zhu QH, Spriggs A, Matthew L, Fan L, Kennedy G, Gubler F, Helliwell C (2008) A diverse set of microRNAs and microRNA-like small RNAs in developing rice grains. Genome Res 18(9):1456–1465Google Scholar
  62. Zhuang Y, Wang SB (2009) Identification and inheritance of Verticillium wilt resistance in eggplant related species. Jiangsu J Agric Sci 4:847–850Google Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.College of Horticulture and Plant ProtectionYangzhou UniversityYangzhouChina
  2. 2.Jiangsu Key Laboratory for Horticultural Crop Genetic ImprovementJiangsu Academy of Agricultural SciencesNanjingChina

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