Molecular Biology Reports

, Volume 45, Issue 5, pp 773–785 | Cite as

Identification of NBS-encoding genes linked to black rot resistance in cabbage (Brassica oleracea var. capitata)

  • Khandker Shazia Afrin
  • Md Abdur Rahim
  • Jong-In Park
  • Sathishkumar Natarajan
  • Hoy-Taek Kim
  • Ill-Sup NouEmail author
Original Article


Heading cabbage is a nutritionally rich and economically important cruciferous vegetable. Black rot disease, caused by the bacterium Xanthomonas campestris pv. campestris, reduces both the yield and quality of the cabbage head. Nucleotide binding site (NBS)-encoding resistance (R) genes play a vital role in the plant immune response to various pathogens. In this study, we analyzed the expression and DNA sequence variation of 31 NBS-encoding genes in cabbage (Brassica oleracea var. capitata). These genes encoded TIR, NBS, LRR and RPW8 protein domains, all of which are known to be involved in disease resistance. RNA-seq revealed that these 31 genes were differentially expressed in leaf, root, silique, and stem tissues. Furthermore, qPCR analyses revealed that several of these genes were more highly expressed in resistant compared to susceptible cabbage lines, including Bol003711, Bol010135, Bol010559, Bol022784, Bol029866, Bol042121, Bol031422, Bol040045 and Bol042095. Further analysis of these genes promises to yield both practical benefits, such as molecular markers for marker-assisted breeding, and fundamental insights to the mechanisms of resistance to black rot in cabbage.


Black rot Cabbage Resistance NBS-encoding genes Expression 



Analysis of variance

R gene

Resistance gene




Conserved domain database


Fragments per kilobase of transcript per million mapped reads


Gene expression omnibus




Leucine-rich repeats


Nucleotide binding site


Quantitative real time polymerase chain reaction


Single nucleotide polymorphism


Toll interleukin-1 receptor



This research work was financially supported by the Golden Seed Project (Center for Horticultural Seed Development, Grant No. 213007-05-2-CG100) of the Ministry of Agriculture, Food and Rural affairs in the Republic of Korea (MAFRA).

Author contributions

I-SN, J-IP and H-TK designed the research. KSA conducted the experiments. KSA and MAR analyzed the data and created figures and tables. SN contributed to the in silco identification of genetic variants. KSA and MAR wrote the manuscript. All the authors contributed to, read and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no competing interests.

Ethical approval

This article does not contain any studies with human participants and/or animals performed by any of the authors.

Supplementary material

11033_2018_4217_MOESM1_ESM.docx (460 kb)
Supplementary material Figure S1 (DOCX 460 KB)
11033_2018_4217_MOESM2_ESM.docx (20 kb)
Supplementary material Table S1 (DOCX 20 KB)
11033_2018_4217_MOESM3_ESM.xlsx (16 kb)
Supplementary material Table S2 (XLSX 16 KB)
11033_2018_4217_MOESM4_ESM.xlsx (15 kb)
Supplementary material Table S3 (XLSX 14 KB)
11033_2018_4217_MOESM5_ESM.xlsx (19 kb)
Supplementary material Table S4 (XLSX 18 KB)
11033_2018_4217_MOESM6_ESM.xlsx (10 kb)
Supplementary material Table S5 (XLSX 9 KB)


  1. 1.
    Singh B, Sharma S, Singh B (2009) Heterosis for mineral elements in single cross-hybrids of cabbage (Brassica oleracea var. capitata L.). Sci Hortic 122:32–36CrossRefGoogle Scholar
  2. 2.
    Lema M, Cartea ME, Sotelo T, Velasco P, Soengas P (2012) Discrimination of Xanthomonas campestris pv. campestris races among strains from northwestern Spain by Brassica spp. genotypes and rep-PCR. Eur J Plant Pathol 133:159–169CrossRefGoogle Scholar
  3. 3.
    Vicente JG, Holub EB (2013) Xanthomonas campestris pv. campestris (cause of black rot of crucifers) in the genomic era is still a worldwide threat to brassica crops. Mol Plant Pathol 14:2–18CrossRefPubMedGoogle Scholar
  4. 4.
    Williams PH (1980) Black rot: a continuing threat to world crucifers. Plant Dis 64:736–742CrossRefGoogle Scholar
  5. 5.
    Cook A, Walker J, Larson R (1952) Studies on the disease cycle of black rot of crucifers. Phytopathology 42:162–167Google Scholar
  6. 6.
    Sharma BB, Kalia P, Singh D, Sharma TR (2017) Introgression of black rot resistance from Brassica carinata to cauliflower (Brassica oleracea botrytis group) through embryo rescue. Front Plant Sci 8:1255CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Leyns F, De Cleene M, Swings J, De Ley J (1984) The host range of the genus Xanthomonas. Bot Rev 50:308–356CrossRefGoogle Scholar
  8. 8.
    Bradbury JF (1986) Guide to plant pathogenic bacteria. CAB International, Farnham RoyalGoogle Scholar
  9. 9.
    Tonu NN, Doullah MA, Shimizu M, Karim MM, Kawanabe T, Fujimoto R, Okazaki K (2013) Comparison of positions of QTLs conferring resistance to Xanthomonas campestris pv. campestris in Brassica oleracea. Am J Plant Sci 4:11–20CrossRefGoogle Scholar
  10. 10.
    Kifuji Y, Hanzawa H, Terasawa Y, Nishio T (2013) QTL analysis of black rot resistance in cabbage using newly developed EST-SNP markers. Euphytica 190:289–295CrossRefGoogle Scholar
  11. 11.
    Massomo SM, Nielsen H, Mabagala RB, Mansfeld-Giese K, Hockenhull J, Mortensen CN (2003) Identification and characterisation of Xanthomonas campestris pv. campestris strains from Tanzania by pathogenicity tests, biolog, rep-PCR and fatty acid methyl ester analysis. Eur J Plant Pathol 109:775–789CrossRefGoogle Scholar
  12. 12.
    Fargier E, Manceau C (2007) Pathogenicity assays restrict the species Xanthomonas campestris into three pathovars and reveal nine races within X. campestris pv. campestris. Plant Pathol 56:805–818CrossRefGoogle Scholar
  13. 13.
    Kamoun S, Kamdar HV, Tola E, Kado CI (1992) A vascular hypersensitive response: role of the hrpK locus. Mol Plant-Microbe Interact 5:22–33CrossRefGoogle Scholar
  14. 14.
    Vicente JG, Conway J, Roberts S, Taylor J (2001) Identification and origin of Xanthomonas campestris pv. campestris races and related pathovars. Phytopathology 91:492–499CrossRefPubMedGoogle Scholar
  15. 15.
    Cruz J, Tenreiro R, Cruz L (2017) Assessment of diversity of Xanthomonas campestris pathovars affecting cruciferous plants in Portugal and disclosure of two novel X. campestris pv. campestris races. J Plant Pathol 99:403–414Google Scholar
  16. 16.
    Jensen BD, Massomo SM, Swai IS, Hockenhull J, Andersen SB (2005) Field evaluation for resistance to the black rot pathogen Xanthomonas campestris pv. campestris in cabbage (Brassica oleracea). Eur J Plant Pathol 113:297–308CrossRefGoogle Scholar
  17. 17.
    Guo H, Dickson M, Hunter J (1991) Brassica napus sources of resistance to black rot in crucifers and inheritance of resistance. HortScience 26:1545–1547Google Scholar
  18. 18.
    Ignatov A, Kuginuki Y, Hida K (1998) Race-specific reaction of resistance to black rot in Brassica oleracea. Eur J Plant Pathol 104:821–827CrossRefGoogle Scholar
  19. 19.
    Williams P, Staub T, Sutton J (1972) Inheritance of resistance in cabbage to black rot. Phytopathology 62:247–252CrossRefGoogle Scholar
  20. 20.
    Hunter J, Dickson M, Ludwig J (1987) Source of resistance to black rot of cabbage expressed in seedlings and adult plants. Plant Dis 71:263–266CrossRefGoogle Scholar
  21. 21.
    Bain D (1952) Reaction of brassica seedlings to black rot. Phytopathology 42:316–319Google Scholar
  22. 22.
    Ignatov A, Kuginuki Y, Hidam K (2000) Distribution and inheritance of race-specific resistance to Xanthomonas campestris pv. campestris in Brassica rapa and B. napus. J Russ Phytopathol Soc 1:89–94Google Scholar
  23. 23.
    Vicente JG, Taylor J, Sharpe A, Parkin I, Lydiate D, King G (2002) Inheritance of race-specific resistance to Xanthomonas campestris pv. campestris in Brassica genomes. Phytopathology 92:1134–1141CrossRefPubMedGoogle Scholar
  24. 24.
    Westman AL, Kresovich S, Dickson MH (1999) Regional variation in Brassica nigra and other weedy crucifers for disease reaction to Alternaria brassicicola and Xanthomonas campestris. pv. campestris. Euphytica 106:253–259CrossRefGoogle Scholar
  25. 25.
    Taylor J, Conway J, Roberts S, Astley D, Vicente JG (2002) Sources and origin of resistance to Xanthomonas campestris pv. campestris in Brassica genomes. Phytopathology 92:105–111CrossRefPubMedGoogle Scholar
  26. 26.
    Lema M, Cartea ME, Francisco M, Velasco P, Soengas P (2015) Screening for resistance to black rot in a Spanish collection of Brassica rapa. Plant Breed 134:551–556CrossRefGoogle Scholar
  27. 27.
    Afrin KS, Rahim MA, Park J, Natarajan S, Rubel MH, Kim H, Nou I (2018) Screening of cabbage (Brassica oleracea L.) germplasm for resistance to black rot. Plant Breed Biotech 6:30–43CrossRefGoogle Scholar
  28. 28.
    Dangl JL, Jones JD (2001) Plant pathogens and integrated defense responses to infection. Nature 411:826–833CrossRefPubMedGoogle Scholar
  29. 29.
    Flor HH (1971) Current status of the gene-for-gene concept. Annu Rev Phytopathol 9:275–296CrossRefGoogle Scholar
  30. 30.
    Yu J, Tehrim S, Zhang F, Tong C, Huang J, Cheng X, Dong C, Zhou Y, Qin R, Hua W (2014) Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana. BMC Genomics 15:3CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Van der Biezen EA, Jones JD (1998) Plant disease-resistance proteins and the gene-for-gene concept. Trends Biochem Sci 23:454–456CrossRefPubMedGoogle Scholar
  32. 32.
    Wan H, Yuan W, Ye Q, Wang R, Ruan M, Li Z, Zhou G, Yao Z, Zhao J, Liu S (2012) Analysis of TIR-and non-TIR-NBS-LRR disease resistance gene analogous in pepper: characterization, genetic variation, functional divergence and expression patterns. BMC Genomics 13:502CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Torii KU (2004) Leucine-rich repeat receptor kinases in plants: structure, function, and signal transduction pathways. Int Rev Cytol 234:1–46CrossRefPubMedGoogle Scholar
  34. 34.
    Tameling WI, Elzinga SD, Darmin PS, Vossen JH, Takken FL, Haring MA, Cornelissen BJ (2002) The tomato R gene products I-2 and MI-1 are functional ATP binding proteins with ATPase activity. Plant Cell 14:2929–2939CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Meyers BC, Kozik A, Griego A, Kuang H, Michelmore RW (2003) Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis. Plant Cell 15:809–834CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Mun J, Yu H, Park S, Park B (2009) Genome-wide identification of NBS-encoding resistance genes in Brassica rapa. Mol Gen Genomics 282:617–631CrossRefGoogle Scholar
  37. 37.
    Zhou T, Wang Y, Chen J, Araki H, Jing Z, Jiang K, Shen J, Tian D (2004) Genome-wide identification of NBS genes in japonica rice reveals significant expansion of divergent non-TIR NBS-LRR genes. Mol Genet Genomics 271:402–415CrossRefPubMedGoogle Scholar
  38. 38.
    Monosi B, Wisser R, Pennill L, Hulbert S (2004) Full-genome analysis of resistance gene homologues in rice. Theor Appl Genet 109:1434–1447CrossRefPubMedGoogle Scholar
  39. 39.
    Lv S, Changwei Z, Tang J, Li Y, Wang Z, Jiang D, Hou X (2015) Genome-wide analysis and identification of TIR-NBS-LRR genes in Chinese cabbage (Brassica rapa ssp. pekinensis) reveal expression patterns to TuMV infection. Physiol Mol Plant Pathol 90:89–97CrossRefGoogle Scholar
  40. 40.
    Wan H, Yuan W, Bo K, Shen J, Pang X, Chen J (2013) Genome-wide analysis of NBS-encoding disease resistance genes in Cucumis sativus and phylogenetic study of NBS-encoding genes in Cucurbitaceae crops. BMC Genom 14:109CrossRefGoogle Scholar
  41. 41.
    Lozano R, Ponce O, Ramirez M, Mostajo N, Orjeda G (2012) Genome-wide identification and mapping of NBS-encoding resistance genes in Solanum tuberosum group phureja. PLoS ONE 7:e34775CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Porter BW, Paidi M, Ming R, Alam M, Nishijima WT, Zhu YJ (2009) Genome-wide analysis of Carica papaya reveals a small NBS resistance gene family. Mol Gen Genomics 281:609–626CrossRefGoogle Scholar
  43. 43.
    Yang S, Zhang X, Yue J, Tian D, Chen J (2008) Recent duplications dominate NBS-encoding gene expansion in two woody species. Mol Gen Genomics 280:187–198CrossRefGoogle Scholar
  44. 44.
    Lee S, Seo J, Rodriguez-Lanetty M, Lee D (2003) Comparative analysis of super families of NBS-encoding disease resistance gene analogs in cultivated and wild apple species. Mol Gen Genomics 269:101–108Google Scholar
  45. 45.
    Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95:14863–14868CrossRefPubMedGoogle Scholar
  46. 46.
    Saldanha AJ (2004) Java Treeview–extensible visualization of microarray data. Bioinformatics 20:3246–3248CrossRefPubMedGoogle Scholar
  47. 47.
    Cheng F, Sun R, Hou X, Zheng H, Zhang F, Zhang Y, Liu B, Liang J, Zhuang M, Liu Y (2016) Subgenome parallel selection is associated with morphotype diversification and convergent crop domestication in Brassica rapa and Brassica oleracea. Nat Genet 48:1218–1224CrossRefPubMedGoogle Scholar
  48. 48.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–∆∆CT method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  49. 49.
    Xiao S, Ellwood S, Calis O, Patrick E, Li T, Coleman M, Turner JG (2001) Broad-spectrum mildew resistance in Arabidopsis thaliana mediated by RPW8. Science 291:118–120CrossRefPubMedGoogle Scholar
  50. 50.
    van der Biezen EA, Jones JD (1998) The NB-ARC domain: a novel signalling motif shared by plant resistance gene products and regulators of cell death in animals. Curr Biol 8:R226–R228CrossRefPubMedGoogle Scholar
  51. 51.
    Chandra S, Kazmi AZ, Ahmed Z, Roychowdhury G, Kumari V, Kumar M, Mukhopadhyay K (2017) Genome-wide identification and characterization of NB-ARC resistant genes in wheat (Triticum aestivum L.) and their expression during leaf rust infection. Plant Cell Rep 36:1097–1112CrossRefPubMedGoogle Scholar
  52. 52.
    Ng A, Xavier RJ (2011) Leucine-rich repeat (LRR) proteins: integrators of pattern recognition and signaling in immunity. Autophagy 7:1082–1084CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Parry G (2012) Assessing the function of the plant nuclear pore complex and the search for specificity. J Exp Bot 64:833–845CrossRefPubMedGoogle Scholar
  54. 54.
    Govindaraj M, Vetriventhan M, Srinivasan M (2015) Importance of genetic diversity assessment in crop plants and its recent advances: an overview of its analytical perspectives. Genet Res Int 2015:431–448Google Scholar
  55. 55.
    Bent AF, Mackey D (2007) Elicitors, effectors, and R genes: the new paradigm and a lifetime supply of questions. Annu Rev Phytopathol 45:399–436CrossRefPubMedGoogle Scholar
  56. 56.
    Lv H, Fang Z, Yang L, Zhang Y, Wang Q, Liu Y, Zhuang M, Yang Y, Xie B, Liu B (2014) Mapping and analysis of a novel candidate Fusarium wilt resistance gene FOC1 in Brassica oleracea. BMC Genom 15:1094CrossRefGoogle Scholar
  57. 57.
    Kim SH, Kwon SI, Saha D, Anyanwu NC, Gassmann W (2009) Resistance to the Pseudomonas syringae effector HopA1 is governed by the TIR-NBS-LRR protein RPS6 and is enhanced by mutations in SRFR1. Plant Physiol 150:1723–1732CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Izzah NK, Lee J, Jayakodi M, Perumal S, Jin M, Park B, Ahn K, Yang T (2014) Transcriptome sequencing of two parental lines of cabbage (Brassica oleracea L. var. capitata L.) and construction of an EST-based genetic map. BMC Genom 15:149CrossRefGoogle Scholar
  59. 59.
    Voorrips R (2002) MapChart: software for the graphical presentation of linkage maps and QTLs. J Hered 93:77–78CrossRefPubMedGoogle Scholar
  60. 60.
    Lee J, Izzah NK, Jayakodi M, Perumal S, Joh HJ, Lee HJ, Lee S, Park JY, Yang K, Nou I (2015) Genome-wide SNP identification and QTL mapping for black rot resistance in cabbage. BMC Plant Biol 15:32CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Saha P, Kalia P, Sonah H, Sharma TR (2014) Molecular mapping of black rot resistance locus Xca1bo on chromosome 3 in Indian cauliflower (Brassica oleracea var. botrytis L.). Plant Breed 133:268–274CrossRefGoogle Scholar
  62. 62.
    Doullah M, Mohsin G, Ishikawa K, Hori H, Okazaki K (2011) Construction of a linkage map and QTL analysis for black rot resistance in Brassica oleracea L. Int J Nat Sci 1:1–6CrossRefGoogle Scholar
  63. 63.
    Camargo L, Williams P, Osborn T (1995) Mapping of quantitative trait loci controlling resistance of Brassica oleracea to Xanthomonas campestris pv. campestris in the field greenhouse. Phytopathology 85:1296–1300CrossRefGoogle Scholar
  64. 64.
    Wang C, Yang Y, Yuan X, Xu Q, Feng Y, Yu H, Wang Y (2014) Genome-wide association study of blast resistance in indica rice. BMC Plant Biol 14:311CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Khandker Shazia Afrin
    • 1
  • Md Abdur Rahim
    • 1
    • 2
  • Jong-In Park
    • 1
  • Sathishkumar Natarajan
    • 1
  • Hoy-Taek Kim
    • 1
  • Ill-Sup Nou
    • 1
    Email author
  1. 1.Department of HorticultureSunchon National UniversitySuncheonRepublic of Korea
  2. 2.Department of Genetics and Plant BreedingSher-e-Bangla Agricultural UniversityDhakaBangladesh

Personalised recommendations