Plant Molecular Biology Reporter

, Volume 36, Issue 5–6, pp 725–737 | Cite as

Intronic Sequence Variations in a Gene with Peroxidase Domain Alter Bolting Time in Cabbage (Brassica oleracea var. capitata)

  • Md. Abuyusuf
  • Ujjal Kumar Nath
  • Hoy-Taek Kim
  • Manosh Kumar Biswas
  • Jong-In ParkEmail author
  • Ill-Sup NouEmail author
Original Paper


Cabbage (Brassica oleracea var. capitata) is the most popular leafy vegetable; however, its quality as a vegetable depends on its growth stage. Premature bolting triggered by low temperatures leads to a reduction of yield and quality of cabbage. Late bolting is preferred by growers to increase market value, whereas early bolting plants are ideal for quality seed production. Herein, we reported a gene BolPrx.2 annotated as Q9FLC0 in the SwissPort, involved in bolting time variation in cabbage and designed molecular markers to characterize early- and late-bolting cabbage populations and lines. The BolPrx.2 gene encodes a peroxidase domain and has been identified as a candidate showed almost similar effect as the previously reported MADS-box domain-containing FLC genes for controlling bolting time. An insertion/deletion (InDel) variation in intron1 has been identified as a causal factor for variation between late- and early-bolting inbred lines. By using this InDel, we designed molecular markers for characterizing the bolting time variation and validated them with 141 F2 generation plants. These markers predicted about 84% of the variation within the population and commercial lines. Therefore, it could be a potential genetic tool to predict bolting time variation and support marker-assisted back crossing (MABC) programs for developing desired bolting types of cabbage cultivars.


InDel markers Peroxidase domain Intron sequence Early- and late-bolting cabbages 


Author Contributions

The work presented here was carried out in collaboration among all authors. MA carried out the experiments and prepared the tables, figures, and manuscript draft. UKN collected primary data regarding genes and wrote, edited, and finalized the manuscript. JIP formulated the experimental concept and provided the plant materials. HTK conducted greenhouse experiments and took care of the plant populations. MKB performed in silico analyses and helped with cloning. ISN designed and participated in all the experiments and assisted in improving the technical sites for the project. All authors have read and approved the final manuscript.


This research was supported by the Golden Seed Project (Center for Horticultural Seed Development, No. 213007-05-2-SB110), the Ministry of Agriculture, the Food and Rural Affairs (MAFRA), the Ministry of Oceans and Fisheries (MOF), the Rural Development Administration (RDA), and Korea Forest Service (KFS), Korea.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

11105_2018_1113_MOESM1_ESM.doc (90 kb)
ESM 1 Supplementary Table S1. Primer list of 8 genes of BolFLC, BolPrxs, BolATL73 and BolCYP. Supplementary Table S2. Nucleotide and amino acid similarities of BolPrx.2 gene with reported FLC gene and other flowering pathway genes in Brassica oleracea, respectively. (DOC 89 kb)
11105_2018_1113_MOESM2_ESM.ppt (493 kb)
Supplementary Fig. S1 Validation of 20 commercial inbred early- and late-bolting cabbage lines with InDel marker of BolPrx.2 gene matched 85%, the heterozygous lines (lanes: 10, 13, and 16) are not matched with the phenotypes. Here, P1= late-bolting parent (BN4730), P2= early-bolting parent (BN7115), F1= (BN4730 × BN7115), M=100 bp DNA marker, red color numbered (10 early-bolting lines): lane 1, 2, 4, 7, 9, 11, 12, 15,17 are 17FLE1, 17FLE2, 17FLE3, 17FLE4, 17FLE5, 17FLE6, 17FLE7, 17FLE8, 17FLE9, and 17FLE10, respectively and black color numbered (10 late-bolting lines): lanes 3, 5, 6, 8, 10, 13, 14, 16 ,19 and 20 are 17FLL1, 17FLL2, 17FLL3, 17FLL4, 17FLL5, 17FLL6, 17FLL7, 17FLL8, 17FLL9, and 17FLL10, respectively. (PPT 493 kb)
11105_2018_1113_MOESM3_ESM.doc (46 kb)
Additional file 1. List of the genes and their sequences used for constructing of phylogenetic tree. (DOC 46 kb)
11105_2018_1113_MOESM4_ESM.doc (41 kb)
Additional file 2. Sequence alignment of the total sequences of the gene BolPrx.2 cloned from late bolting line (BN4730) and early bolting line (BN7115) aligned with reference sequence of BolPrx.2 gene collected from Brassica database ( Blue and black colour regions are the introns and exons of the gene, respectively. Gray highlighted codons indicate the start and stop codon of the gene; yellow, pink, and green highlighted portion indicate the deletion, insertion and SNPs variations in the late bolting line. (DOC 41 kb)


  1. Anderson JT, Lee CR, Mitchell-Olds T (2011) Life-history QTLS and natural selection on flowering time in Boechera stricta, a perennial relative of Arabidopsis. Evolution 65:771–787CrossRefGoogle Scholar
  2. Andrés F, Coupland G (2012) The genetic basis of flowering responses to seasonal cues. Nat Rev Genet 13:627–639CrossRefGoogle Scholar
  3. Badiani M, Paolacci A, Miglietta F, Kimball B, Pinter P, Garcia R, Hunsaker D, LaMorte R, Wall G (1996) Seasonal variations of antioxidants in wheat (Triticum aestivum) leaves grown under field conditions. Funct Plant Biol 23:687–698CrossRefGoogle Scholar
  4. Blázquez MA, Green R, Nilsson O, Sussman MR, Weigel D (1998) Gibberellins promote flowering of Arabidopsis by activating the LEAFY promoter. Plant Cell 10:791–800CrossRefGoogle Scholar
  5. Camargo L, Osborn T (1996) Mapping loci controlling flowering time in Brassica oleracea. Theor Appl Genet 92:610–616CrossRefGoogle Scholar
  6. Cao J, Schneeberger K, Ossowski S, Günther T, Bender S, Fitz J, Koenig D, Lanz C, Stegle O, Lippert C (2011) Whole-genome sequencing of multiple Arabidopsis thaliana populations. Nat Genet 43:956–963CrossRefGoogle Scholar
  7. Chai L, Wang J, Fan Z, Liu Z, Wen G, Li X, Yang Y (2012) Regulation of the flowering time of Arabidopsis thaliana by thylakoid ascorbate peroxidase. Afr J Biotechnol 11:7151–7157Google Scholar
  8. Consortium EUAGS (2000) Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana. Nature 408:823CrossRefGoogle Scholar
  9. Fornara F, de Montaigu A, Coupland G (2010) SnapShot: control of flowering in Arabidopsis. Cell 141:550–550. e552CrossRefGoogle Scholar
  10. Franks SJ, Perez-Sweeney B, Strahl M, Nowogrodzki A, Weber JJ, Lalchan R, Jordan KP, Litt A (2015) Variation in the flowering time orthologs BrFLC and BrSOC1 in a natural population of Brassica rapa. PeerJ 3:e1339CrossRefGoogle Scholar
  11. Gazzani S, Gendall AR, Lister C, Dean C (2003) Analysis of the molecular basis of flowering time variation in Arabidopsis accessions. Plant Physiol 132:1107–1114CrossRefGoogle Scholar
  12. Gielis J, Goetghebeur P, Debergh P (1999) Physiological aspects and experimental reversion of flowering in Fargesia murieliae (Poaceae, Bambusoideae). Syst Geogr Plants 68:147–158CrossRefGoogle Scholar
  13. Gómez-Mena C, Piñeiro M, Franco-Zorrilla JM, Salinas J, Coupland G, Martínez-Zapater JM (2001) Early bolting in short days: an Arabidopsis mutation that causes early flowering and partially suppresses the floral phenotype of leafy. Plant Cell 13:1011–1024CrossRefGoogle Scholar
  14. Gotô N, Hamada M (1988) Promotion of flowering by DNA base analogues and changes in acid phosphatase and peroxidase isozyme composition in dark-grown Arabidopsis thaliana. Plant Cell Physiol 29:683–688Google Scholar
  15. Grillo MA, Li C, Hammond M, Wang L, Schemske DW (2013) Genetic architecture of flowering time differentiation between locally adapted populations of Arabidopsis thaliana. New Phytol 197:1321–1331CrossRefGoogle Scholar
  16. Guo P, Baum M, Grando S, Ceccarelli S, Bai G, Li R, Von Korff M, Varshney RK, Graner A, Valkoun J (2009) Differentially expressed genes between drought-tolerant and drought-sensitive barley genotypes in response to drought stress during the reproductive stage. J Exp Bot 60:3531–3544CrossRefGoogle Scholar
  17. Hepworth SR, Valverde F, Ravenscroft D, Mouradov A, Coupland G (2002) Antagonistic regulation of flowering-time gene SOC1 by CONSTANS and FLC via separate promoter motifs. EMBO J 21:4327–4337CrossRefGoogle Scholar
  18. Hirai N, Kojima Y, Shinozaki M, Koshimizu K, Murofushi N, Takimoto A (1995) Accumulation of ascorbic acid in the cotyledons of morning glory (Pharbitis nil) seedlings during the induction of flowering by low-temperature treatment and the effect of prior exposure to high-intensity light. Plant Cell Physiol 36:1265–1271CrossRefGoogle Scholar
  19. Hu TT, Pattyn P, Bakker EG, Cao J, Cheng J-F, Clark RM, Fahlgren N, Fawcett JA, Grimwood J, Gundlach H (2011) The Arabidopsis lyrata genome sequence and the basis of rapid genome size change. Nat Genet 43:476–481CrossRefGoogle Scholar
  20. Ishizawa M, Kobayashi Y, Miyamura T, Matsuura S (1991) Simple procedure of DNA isolation from human serum. Nucleic Acids Res 19:5792CrossRefGoogle Scholar
  21. Johanson U, West J, Lister C, Michaels S, Amasino R, Dean C (2000) Molecular analysis of FRIGIDA, a major determinant of natural variation in Arabidopsis flowering time. Science 290:344–347CrossRefGoogle Scholar
  22. Jung C, Müller AE (2009) Flowering time control and applications in plant breeding. Trends Plant Sci 14:563–573CrossRefGoogle Scholar
  23. Komeda Y (2004) Genetic regulation of time to flower in Arabidopsis thaliana. Annu Rev Plant Biol 55:521–535CrossRefGoogle Scholar
  24. Koornneef M, Alonso-Blanco C, Blankestijn-de Vries H, Hanhart C, Peeters A (1998a) Genetic interactions among late-flowering mutants of Arabidopsis. Genetics 148:885–892PubMedPubMedCentralGoogle Scholar
  25. Koornneef M, Alonso-Blanco C, Peeters AJ, Soppe W (1998b) Genetic control of flowering time in Arabidopsis. Annu Rev Plant Biol 49:345–370CrossRefGoogle Scholar
  26. Krekule J, Machackova I (1986) possible role of auxin and of its metabolic changes in the photoperiodic control of flowering. Molecular and physiological aspects of plant peroxidases/edited by H Greppin, C Penel, Th Gaspar. Information Systems Division, National Agricultural Library, Beltsville, Maryland and Washington, D.C., USAGoogle Scholar
  27. Levy YY, Dean C (1998) The transition to flowering. Plant Cell 10:1973–1989CrossRefGoogle Scholar
  28. Li Y, Roycewicz P, Smith E, Borevitz JO (2006) Genetics of local adaptation in the laboratory: flowering time quantitative trait loci under geographic and seasonal conditions in Arabidopsis. PLoS One 1:e105CrossRefGoogle Scholar
  29. Li P, Filiault D, Box MS, Kerdaffrec E, van Oosterhout C, Wilczek AM, Schmitt J, McMullan M, Bergelson J, Nordborg M (2014) Multiple FLC haplotypes defined by independent cis-regulatory variation underpin life history diversity in Arabidopsis thaliana. Genes Dev 28:1635–1640CrossRefGoogle Scholar
  30. Lokhande SD, Ogawa KI, Tanaka A, Hara T (2003) Effect of temperature on ascorbate peroxidase activity and flowering of Arabidopsis thaliana ecotypes under different light conditions. J Plant Physiol 160:57–64CrossRefGoogle Scholar
  31. Mao Y, Wu F, Yu X, Bai J, Zhong W, He Y (2014) MicroRNA319a-targeted Brassica rapa ssp. pekinensis TCP genes modulate head shape in Chinese cabbage by differential cell division arrest in leaf regions. Plant Physiol 164:710–720CrossRefGoogle Scholar
  32. Michaels SD, Amasino RM (1999) FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11:949–956CrossRefGoogle Scholar
  33. Michaels SD, Amasino RM (2000) Memories of winter: vernalization and the competence to flower. Plant Cell Environ 23:1145–1153CrossRefGoogle Scholar
  34. Michaels SD, He Y, Scortecci KC, Amasino RM (2003) Attenuation of FLOWERING LOCUS C activity as a mechanism for the evolution of summer-annual flowering behavior in Arabidopsis. Proc Natl Acad Sci U S A 100:10102–10107CrossRefGoogle Scholar
  35. Michaels SD, Himelblau E, Kim SY, Schomburg FM, Amasino RM (2005) Integration of flowering signals in winter-annual Arabidopsis. Plant Physiol 137:149–156CrossRefGoogle Scholar
  36. Moharekar S, Tanaka R, Ogawa K, Tanaka A, Hara T (2007) Great promoting effect of high irradiance from germination on flowering in Arabidopsis thaliana—a process of photo-acclimation. Photosynthetica 45:259–265CrossRefGoogle Scholar
  37. Ner-Gaon H, Halachmi R, Savaldi-Goldstein S, Rubin E, Ophir R, Fluhr R (2004) Intron retention is a major phenomenon in alternative splicing in Arabidopsis. Plant J 39:877–885CrossRefGoogle Scholar
  38. Ogawa KI, Tasaka Y, Mino M, Tanaka Y, Iwabuchi M (2001) Association of glutathione with flowering in Arabidopsis thaliana. Plant Cell Physiol 42:524–530CrossRefGoogle Scholar
  39. Österberg MK, Shavorskaya O, Lascoux M, Lagercrantz U (2002) Naturally occurring indel variation in the Brassica nigra COL1 gene is associated with variation in flowering time. Genetics 161:299–306PubMedPubMedCentralGoogle Scholar
  40. Ratcliffe OJ, Nadzan GC, Reuber TL, Riechmann JL (2001) Regulation of flowering in Arabidopsis by an FLC homologue. Plant Physiol 126:122–132CrossRefGoogle Scholar
  41. Ratcliffe OJ, Kumimoto RW, Wong BJ, Riechmann JL (2003) Analysis of the Arabidopsis MADS AFFECTING FLOWERING gene family: MAF2 prevents vernalization by short periods of cold. Plant Cell 15:1159–1169CrossRefGoogle Scholar
  42. Razi H, Howell E, Newbury H, Kearsey M (2008) Does sequence polymorphism of FLC paralogues underlie flowering time QTL in Brassica oleracea? Theor Appl Genet 116:179–192CrossRefGoogle Scholar
  43. Ridge S, Brown PH, Hecht V, Driessen RG, Weller JL (2014) The role of BoFLC2 in cauliflower (Brassica oleracea var. botrytis L.) reproductive development. J Exp Bot 66:125–135CrossRefGoogle Scholar
  44. Ross J, O'Neill D (2001) New interactions between classical plant hormones. Trends Plant Sci 6:2–4CrossRefGoogle Scholar
  45. Saha G, Park J-I, Jung H-J, Ahmed NU, Kayum MA, Chung M-Y, Hur Y, Cho Y-G, Watanabe M, Nou I-S (2015) Genome-wide identification and characterization of MADS-box family genes related to organ development and stress resistance in Brassica rapa. BMC Genomics 16:178CrossRefGoogle Scholar
  46. Salomé PA, Bomblies K, Laitinen RA, Yant L, Mott R, Weigel D (2011) Genetic architecture of flowering-time variation in Arabidopsis thaliana. Genetics 188:421–433CrossRefGoogle Scholar
  47. Sánchez-Bermejo E, Méndez-Vigo B, Pico FX, Martínez-Zapater JM, Alonso-Blanco C (2012) Novel natural alleles at FLC and LVR loci account for enhanced vernalization responses in Arabidopsis thaliana. Plant Cell Environ 35:1672–1684CrossRefGoogle Scholar
  48. Schiessl SV, Huettel B, Kuehn D, Reinhardt R, Snowdon RJ (2017) Flowering time gene variation in Brassica species shows evolutionary principles. Front Plant Sci 8:1742CrossRefGoogle Scholar
  49. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108CrossRefGoogle Scholar
  50. Schranz ME, Quijada P, Sung S-B, Lukens L, Amasino R, Osborn TC (2002) Characterization and effects of the replicated flowering time gene FLC in Brassica rapa. Genetics 162:1457–1468PubMedPubMedCentralGoogle Scholar
  51. Scortecci KC, Michaels SD, Amasino RM (2001) Identification of a MADS-box gene, FLOWERING LOCUS M that represses flowering. Plant J 26:229–236CrossRefGoogle Scholar
  52. Searle I, He Y, Turck F, Vincent C, Fornara F, Kröber S, Amasino RA, Coupland G (2006) The transcription factor FLC confers a flowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis. Genes Dev 20:898–912CrossRefGoogle Scholar
  53. Sheldon CC, Burn JE, Perez PP, Metzger J, Edwards JA, Peacock WJ, Dennis ES (1999) The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation. Plant Cell 11:445–458CrossRefGoogle Scholar
  54. Sheldon CC, Rouse DT, Finnegan EJ, Peacock WJ, Dennis ES (2000) The molecular basis of vernalization: the central role of FLOWERING LOCUS C (FLC). Proc Natl Acad Sci U S A 97:3753–3758CrossRefGoogle Scholar
  55. Sheldon CC, Conn AB, Dennis ES, Peacock WJ (2002) Different regulatory regions are required for the vernalization-induced repression of FLOWERING LOCUS C and for the epigenetic maintenance of repression. Plant Cell 14:2527–2537CrossRefGoogle Scholar
  56. Shigeto J, Tsutsumi Y (2016) Diverse functions and reactions of class III peroxidases. New Phytol 209:1395–1402CrossRefGoogle Scholar
  57. Shindo C, Aranzana MJ, Lister C, Baxter C, Nicholls C, Nordborg M, Dean C (2005) Role of FRIGIDA and FLOWERING LOCUS C in determining variation in flowering time of Arabidopsis. Plant Physiol 138:1163–1173CrossRefGoogle Scholar
  58. Song X, Duan W, Huang Z, Liu G, Wu P, Liu T, Li Y, Hou X (2015) Comprehensive analysis of the flowering genes in Chinese cabbage and examination of evolutionary pattern of CO-like genes in plant kingdom. Sci Rep 5:14631CrossRefGoogle Scholar
  59. Strange A, Li P, Lister C, Anderson J, Warthmann N, Shindo C, Irwin J, Nordborg M, Dean C (2011) Major-effect alleles at relatively few loci underlie distinct vernalization and flowering variation in Arabidopsis accessions. PLoS One 6:e19949CrossRefGoogle Scholar
  60. Sung S, He Y, Eshoo TW, Tamada Y, Johnson L, Nakahigashi K, Goto K, Jacobsen SE, Amasino RM (2006) Epigenetic maintenance of the vernalized state in Arabidopsis thaliana requires LIKE HETEROCHROMATIN PROTEIN 1. Nat Genet 38:706–710CrossRefGoogle Scholar
  61. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefGoogle Scholar
  62. Wang X, Wang H, Wang J, Sun R, Wu J, Liu S, Bai Y, Mun J-H, Bancroft I, Cheng F (2011) The genome of the mesopolyploid crop species Brassica rapa. Nat Genet 43:1035–1039CrossRefGoogle Scholar
  63. Wang Y, Wu F, Bai J, He Y (2014) BrpSPL9 (Brassica rapa ssp. pekinensis SPL9) controls the earliness of heading time in Chinese cabbage. Plant Biotechnol J 12:312–321CrossRefGoogle Scholar
  64. Weigel D, Meyerowitz EM (1993) Activation of floral homeotic genes in Arabidopsis. Science 261:1723–1726CrossRefGoogle Scholar
  65. Werner JD, Borevitz JO, Uhlenhaut NH, Ecker JR, Chory J, Weigel D (2005) FRIGIDA-independent variation in flowering time of natural Arabidopsis thaliana accessions. Genetics 170:1197–1207CrossRefGoogle Scholar
  66. Xia G, He Q, Zhao S (2015) Physiological and biochemical properties analysis of late-bolting transgenic Chinese cabbage (Brassica rapa L. ssp. pekinensis). J Anim Plant Sci 25:152–157Google Scholar
  67. Zang YX, Kim HU, Kim JA, Lim MH, Jin M, Lee SC, Kwon SJ, Lee SI, Hong JK, Park TH (2009) Genome-wide identification of glucosinolate synthesis genes in Brassica rapa. FEBS J 276:3559–3574CrossRefGoogle Scholar
  68. Zou X, Suppanz I, Raman H, Hou J, Wang J, Long Y, Jung C, Meng J (2012) Comparative analysis of FLC homologues in Brassicaceae provides insight into their role in the evolution of oilseed rape. PLoS One 7:e45751CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Md. Abuyusuf
    • 1
  • Ujjal Kumar Nath
    • 1
    • 2
  • Hoy-Taek Kim
    • 1
    • 3
  • Manosh Kumar Biswas
    • 1
  • Jong-In Park
    • 1
    Email author
  • Ill-Sup Nou
    • 1
    Email author
  1. 1.Department of HorticultureSunchon National UniversitySuncheonRepublic of Korea
  2. 2.Department of Genetics and Plant BreedingBangladesh Agricultural UniversityMymensinghBangladesh
  3. 3.University-Industry Cooperation FoundationSunchon National UniversitySuncheonRepublic of Korea

Personalised recommendations