, Volume 247, Issue 5, pp 1191–1202 | Cite as

Transcriptome-wide identification and functional prediction of novel and flowering-related circular RNAs from trifoliate orange (Poncirus trifoliata L. Raf.)

  • Ren-Fang Zeng
  • Jing-Jing Zhou
  • Chun-Gen Hu
  • Jin-Zhi Zhang
Original Article


Main conclusion

A total of 558 potential circular RNAs (circRNAs) were identified in citrus, and these were analyzed and compared. One hundred seventy-six differentially expressed circRNAs were identified in two genotypes of trifoliate orange.

Circular RNAs (circRNAs) play diverse roles in transcriptional control and microRNA (miRNA) function. However, little information is known about circRNAs in citrus. To identify citrus circRNAs and investigate their functional roles, high-throughput sequencing of precocious trifoliate orange (an early-flowering trifoliate orange mutant, Poncirus trifoliata L. Raf.) and its wild type was performed. A total of 558 potential circRNAs were identified by bioinformatic analysis, and 86.02% of these were sense-overlapping circRNAs. Their sequence features, alternative circularization, and other characteristics were investigated in this study. Compared with the wild type, 176 circRNAs were identified as differentially expressed circRNAs, 61 were significantly up-regulated and 115 were down-regulated in precocious trifoliate orange, indicating that they may play an important role in the early flowering process. Alternative circularization and differential expression of some circRNAs were verified by Sanger sequencing and real-time polymerase chain reaction. The functions of differentially expressed circRNAs and their host genes were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. We found that many differentially expressed circRNAs had abundant miRNA binding sites: 29 circRNAs were found to act as the 16 miRNA targets. Overall, these results will help to reveal the biological functions of circRNAs in growth and development of citrus.


Alternative circularization Back-splicing sites Citrus Host genes microRNAs 



Circular RNAs


Gene Ontology


Kyoto Encyclopedia of Genes and Genomes



This research was supported financially by the National Natural Science Foundation of China (Grant nos. 31772252, 31471863, 31372046, 31521092, and 31672110). Hubei Provincial Natural Science Foundation for Innovative Group (2017CFA018) and the International Foundation for Science No. C/5148-2.

Supplementary material

425_2018_2857_MOESM1_ESM.jpg (3.6 mb)
Suppl. Fig. S1 Sanger sequencing further confirmed head-to-tail back-splicing of 11 selected circRNAs (JPEG 3657 kb)
425_2018_2857_MOESM2_ESM.xlsx (19 kb)
Suppl. Table S1 Primers used in this study (XLSX 19 kb)
425_2018_2857_MOESM3_ESM.xlsx (65 kb)
Suppl. Table S2 Genome-wide identification of circRNAs in two genotypes of the trifoliate orange (XLSX 65 kb)
425_2018_2857_MOESM4_ESM.xlsx (4.4 mb)
Suppl. Table S3 The 558 circRNA sequences from two genotypes of the trifoliate orange (XLSX 4459 kb)
425_2018_2857_MOESM5_ESM.xlsx (30 kb)
Suppl. Table S4 Alternative circularization of trifoliate orange circRNAs (XLSX 29 kb)
425_2018_2857_MOESM6_ESM.xlsx (36 kb)
Suppl. Table S5 Differential expression of circRNAs between precocious trifoliate orange and its wild type (XLSX 36 kb)
425_2018_2857_MOESM7_ESM.xlsx (23 kb)
Suppl. Table S6 The different biological metabolism pathways from up-regulated circRNA host genes were identified by KEGG pathway analysis (XLSX 23 kb)
425_2018_2857_MOESM8_ESM.xlsx (23 kb)
Suppl. Table S7 The different biological metabolism pathways from down-regulated circRNA host genes were identified by KEGG pathway analysis (XLSX 22 kb)
425_2018_2857_MOESM9_ESM.xlsx (20 kb)
Suppl. Table S8 Differential expression of circRNAs predicted as putative miRNA targets (XLSX 19 kb)


  1. Abe N, Matsumoto K, Nishihara M, Nakano Y, Shibata A, Maruyama H, Shuto S, Matsuda A, Yoshida M, Ito Y, Abe H (2015) Rolling circle translation of circular RNA in living human cells. Sci Rep 5:16435CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ashwal-Fluss R, Meyer M, Pamudurti NR, Ivanov A, Bartok O, Hanan M, Evantal N, Memczak S, Rajewsky N, Kadener S (2014) circRNA biogenesis competes with pre-mRNA splicing. Mol Cell 56:55–66CrossRefPubMedGoogle Scholar
  3. Blankenberg D, Gordon A, Von Kuster G, Coraor N, Taylor J, Nekrutenko A (2010) Manipulation of FASTQ data with Galaxy. Bioinformatics 26:1783–1785CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chen LL (2016) The biogenesis and emerging roles of circular RNAs. Nat Rev 17:205–211CrossRefGoogle Scholar
  5. Chen L, Yu Y, Zhang X, Liu C, Ye C, Fan L (2016) PcircRNA_finder: a software for circRNA prediction in plants. Bioinformatics 32:3528–3529PubMedPubMedCentralGoogle Scholar
  6. Chen L, Zhang P, Fan Y, Lu Q, Li Q, Yan JB, Muehlbauer GJ, Schnable PS, Dai MQ, Li L (2018) Circular RNAs mediated by transposons are associated with transcriptomic and phenotypic variation in maize. New Phytol 217(3):1292–1306CrossRefPubMedGoogle Scholar
  7. Cheng Y-J, Guo W-W, Yi H-L, Pang X-M, Deng X (2003) An efficient protocol for genomic DNA extraction from Citrus species. Plant Mol Biol Rep 21:177–178CrossRefGoogle Scholar
  8. Chu Q, Zhang X, Zhu X, Liu C, Mao L, Ye C, Zhu Q-H, Fan L (2017) PlantcircBase: a database for plant circular RNAs. Mol Plant 10:1126–1128CrossRefPubMedGoogle Scholar
  9. Dai X, Zhao PX (2011) psRNATarget: a plant small RNA target analysis server. Nucleic Acids Res 39:155–159CrossRefGoogle Scholar
  10. Danan M, Schwartz S, Edelheit S, Sorek R (2012) Transcriptome-wide discovery of circular RNAs in Archaea. Nucleic Acids Res 40:3131–3142CrossRefPubMedGoogle Scholar
  11. Darbani B, Noeparvar S, Borg S (2016) Identification of circular RNAs from the parental genes involved in multiple aspects of cellular metabolism in barley. Front Plant Sci 7:776CrossRefPubMedPubMedCentralGoogle Scholar
  12. Dong WW, Li HM, Qing XR, Huang DH, Li HG (2016) Identification and characterization of human testis derived circular RNAs and their existence in seminal plasma. Sci Rep 6:39080CrossRefPubMedPubMedCentralGoogle Scholar
  13. Gao Y, Wang JF, Zhao FQ (2015) CIRI: an efficient and unbiased algorithm for de novo circular RNA identification. Genome Biol 16:4CrossRefPubMedPubMedCentralGoogle Scholar
  14. Hansen TB, Venø MT, Damgaard CK, Kjems J (2016) Comparison of circular RNA prediction tools. Nucleic Acids Res 44:e58CrossRefPubMedGoogle Scholar
  15. Hua W, Zhang L, Liang S, Jones RL, Lu YT (2004) A tobacco calcium/calmodulin-binding protein kinase functions as a negative regulator of flowering. J Biol Chem 279:31483–31494CrossRefPubMedGoogle Scholar
  16. Kanehisa M, Goto S (2000) KEGG: Kyoto encyclopaedia of genes and genomes. Nucleic Acids Res 28:27–30CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kozomara A, Griffithsjones S (2014) miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 42:68–73CrossRefGoogle Scholar
  18. Kramer MC, Liang D, Tatomer DC, Gold B, March ZM, Cherry S, Wilusz JE (2015) Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins. Gene Dev 29:2168–2182CrossRefPubMedPubMedCentralGoogle Scholar
  19. Li ZM, Zhang JZ, Mei L, Deng XX, Hu CG, Yao JL (2010) PtSVP, an SVP homolog from trifoliate orange (Poncirus trifoliata L. Raf.), shows seasonal periodicity of meristem determination and affects flower development in transgenic Arabidopsis and tobacco plants. Plant Mol Biol 74:129–142CrossRefPubMedGoogle Scholar
  20. Li Z, Huang C, Bao C, Chen L, Lin M, Wang X, Zhong G, Yu B, Hu W, Dai L, Zhu P, Chang Z, Wu Q, Zhao Y, Jia Y, Xu P, Liu H, Shan G (2015) Exon-intron circular RNAs regulate transcription in the nucleus. Nat Struct Mol Biol 22:256–264CrossRefPubMedGoogle Scholar
  21. Liang S, Zhu W, Xiang W (1999) Precocious trifoliate orange (Poncirus trifoliata L. Raf.) biology characteristic and its stock experiment. ZheJiang Citrus 16:2–4Google Scholar
  22. Lu TT, Cui LL, Zhou Y, Zhu CR, Fan DL, Gong H, Zhao Q, Zhou CC, Zhao Y, Lu DF, Wo PH, Wang YC, Tian QL, Feng Q, Huang T, Han B (2015) Transcriptome-wide investigation of circular RNAs in rice. RNA 21:2076–2087CrossRefPubMedPubMedCentralGoogle Scholar
  23. Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A, Maier L, Mackowiak SD, Gregersen LH, Munschauer M, Loewer A, Ziebold U, Landthaler M, Kocks C, le Noble F, Rajewsky N (2013) Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495:333–338CrossRefPubMedGoogle Scholar
  24. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628CrossRefPubMedGoogle Scholar
  25. Noh B, Lee SH, Kim HJ, Yi G, Shin EA, Lee M, Jung KJ, Doyle MR (2004) Divergent roles of a pair of homologous jumonji/zinc-finger-class transcription factor proteins in the regulation of Arabidopsis flowering time. Plant Cell 16:2601–2613CrossRefPubMedPubMedCentralGoogle Scholar
  26. Park DH, Somers DE, Yang SK, Choy YH, Lim HK, Soh MS, Kim HJ, Kay SA, Hong GN (1999) Control of circadian rhythms and photoperiodic flowering by the Arabidopsis GIGANTEA gene. Science 285:1579–1582CrossRefPubMedGoogle Scholar
  27. Rivals I, Personnaz L, Taing L, Potier M-C (2007) Enrichment or depletion of a GO category within a class of genes: which test? Bioinformatics 23:401–407CrossRefPubMedGoogle Scholar
  28. Roe JL, Rivin CJ, Sessions RA, Feldmann KA, Zambryski PC (1993) The Tousled gene in A. thaliana encodes a protein kinase homolog that is required for leaf and flower development. Cell 75:939–950CrossRefPubMedGoogle Scholar
  29. Salzman J, Gawad C, Wang PL, Lacayo N, Brown PO (2012) Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLoS ONE 7:e30733CrossRefPubMedPubMedCentralGoogle Scholar
  30. Shen Y, Guo X, Wang W (2017) Identification and characterization of circular RNAs in zebrafish. FEBS Lett 591:213–220CrossRefPubMedGoogle Scholar
  31. Shiu SH, Bleecker AB (2001) Plant receptor-like kinase gene family: diversity, function, and signaling. Sci STKE 2001:re22PubMedGoogle Scholar
  32. Stein JC, Howlett B, Boyes DC, Nasrallah ME, Nasrallah JB (1991) Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proc Natl Acad Sci USA 88:8816–8820CrossRefPubMedPubMedCentralGoogle Scholar
  33. Sun LM, Ai XY, Li WY, Guo WW, Deng XX, Hu CG, Zhang JZ (2012) Identification and comparative profiling of miRNAs in an early flowering mutant of trifoliate orange and its wild type by genome-wide deep sequencing. PLoS ONE 7:e43760CrossRefPubMedPubMedCentralGoogle Scholar
  34. Takatsuji H (1998) Zinc-finger transcription factors in plants. Cell Mol Life Sci 54:582–596CrossRefPubMedGoogle Scholar
  35. Tarazona S, Garcia-Alcalde F, Dopazo J, Ferrer A, Conesa A (2011) Differential expression in RNA-seq: a matter of depth. Genome Res 21:2213–2223CrossRefPubMedPubMedCentralGoogle Scholar
  36. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7:562–578CrossRefPubMedPubMedCentralGoogle Scholar
  37. Wang PL, Bao Y, Yee MC, Barrett SP, Hogan GJ, Olsen MN, Dinneny JR, Brown PO, Salzman J (2014) Circular RNA is expressed across the eukaryotic tree of life. PLoS ONE 9:e95116CrossRefGoogle Scholar
  38. Wang CY, Liu SR, Zhang XY, Ma YJ, Hu CG, Zhang JZ (2017) Genome-wide screening and characterization of long non-coding RNAs involved in flowering development of trifoliate orange (Poncirus trifoliata L. Raf.). Sci Rep 7:43226CrossRefPubMedPubMedCentralGoogle Scholar
  39. Wu C, You C, Li C, Long T, Chen G, Byrne ME, Zhang Q (2008) RID1, encoding a Cys2/His2-type zinc finger transcription factor, acts as a master switch from vegetative to floral development in rice. Proc Natl Acad Sci USA 105:12915–12920CrossRefPubMedPubMedCentralGoogle Scholar
  40. Wu HJ, Ma YK, Chen T, Wang M, Wang XJ (2012) PsRobot: a web-based plant small RNA meta-analysis toolbox. Nucleic Acids Res 40:W22–W28CrossRefPubMedPubMedCentralGoogle Scholar
  41. Wu GA, Prochnik S, Jenkins J et al (2014) Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication. Nat Biotechnol 32:656–662CrossRefPubMedPubMedCentralGoogle Scholar
  42. Xia S, Feng J, Lei L, Hu J, Xia L, Wang J, Xiang Y, Liu L, Zhong S, Han L, He C (2017) Comprehensive characterization of tissue-specific circular RNAs in the human and mouse genomes. Brief Bioinform 18(6):984–992PubMedGoogle Scholar
  43. Ye CY, Chen L, Liu C, Zhu QH, Fan LJ (2015) Widespread noncoding circular RNAs in plants. New Phytol 208:88–95CrossRefPubMedGoogle Scholar
  44. Ye C-Y, Zhang X, Chu Q, Liu C, Yu Y, Jiang W, Zhu Q-H, Fan L, Guo L (2017) Full-length sequence assembly reveals circular RNAs with diverse non-GT/AG splicing signals in rice. RNA Biol 14:1055–1063CrossRefPubMedGoogle Scholar
  45. Zhang JZ, Ai XY, Sun LM, Zhang DL, Guo WW, Deng XX, Hu CG (2011) Transcriptome profile analysis of flowering molecular processes of early flowering trifoliate orange mutant and the wild-type [Poncirus trifoliata (L.) Raf.] by massively parallel signature sequencing. BMC Genom 12:63CrossRefGoogle Scholar
  46. Zhang JZ, Ai XY, Guo WW, Peng SA, Deng XX, Hu CG (2012) Identification of miRNAs and their target genes using deep sequencing and degradome analysis in trifoliate orange [Poncirus trifoliata L. Raf]. Mol Biotechnol 51:44–57CrossRefPubMedGoogle Scholar
  47. Zhang XO, Wang HB, Zhang Y, Lu XH, Chen LL (2014) Complementary sequence-mediated exon circularization. Cell 159:134–147CrossRefPubMedGoogle Scholar
  48. Zheng Q, Bao C, Guo W, Li S, Chen J, Chen B, Luo Y, Lyu D, Li Y, Shi G, Liang L, Gu J, He X, Huang S (2016) Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Nat Commun 7:11215CrossRefPubMedPubMedCentralGoogle Scholar
  49. Zhu Q-H, Helliwell CA (2011) Regulation of flowering time and floral patterning by miR172. J Exp Bot 62:487–495CrossRefPubMedGoogle Scholar
  50. Zuo JH, Wang Q, Zhu BZ, Luo YB, Gao LP (2016) Deciphering the roles of circRNAs on chilling injury in tomato. Biochem Biophys Res Commun 479:132–138CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture and Forestry ScienceHuazhong Agricultural UniversityWuhanChina
  2. 2.College of Horticulture and Forestry ScienceHuazhong Agricultural UniversityWuhanChina

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