Abstract
To facilitate prevention of clubroot disease, a major threat to the successful cultivation of Chinese cabbage (Brassica rapa L.), we bred clubroot-resistant (CR) cultivars by introducing resistance genes from CR turnips via conventional breeding. Among 11 CR loci found in B. rapa, we identified CRb in Chinese cabbage cultivar ‘CR Shinki’ as a single dominant gene for resistance against Plasmodiophora brassicae pathotype group 3, against which the stacking of Crr1 and Crr2 loci was not effective. However, the precise location and pathotype specificity of CRb have been controversial, because CRa and Rcr1 also map near this locus. Previously, our fine-mapping study revealed that CRb is located in a 140-kb genomic region on chromosome A03. Here, we determined the nucleotide sequence of an approximately 64-kb candidate region in the resistant line; this region contains six open reading frames (ORFs) similar to NB-LRR encoding genes that are predicted to occur in tandem with the same orientation. Among the six ORFs present, only four on the genome of the resistant line showed a strong DNA sequence identity with each other, and only one of those four could confer resistance to P. brassicae isolate No. 14 of the pathotype group 3. These results suggest that these genes evolved through recent gene duplication and uneven crossover events that could lead to the acquisition of clubroot resistance. The DNA sequence of the functional ORF was identical to that of the previously cloned CRa gene; thus, we showed that the independently identified CRb and CRa are one and the same clubroot-resistance gene.
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Aruga D, Ueno H, Matsumura H, Matsumoto E, Hayashida N (2013) Distribution of CRa in clubroot resistance (CR) cultivars of Chinese cabbage. Plant Biotechnol 30:393–397
Bulgarelli D, Biselli C, Collins NC, Consonni G, Stanca AM, Schulze-Lefert P, Vale G (2010) The CC-NB-LRR-type Rdg2a resistance gene confers immunity to the seed-borne barley leaf stripe pathogen in the absence of hypersensitive cell death. PLoS One 5(9):e12599. doi:10.1371/journal.pone.0012599
Chen J, Jing J, Zhan Z, Zhang T, Zhang C, Piao Z (2013) Identification of novel QTLs for isolate-specific partial resistance to Plasmodiophora brassicae in Brassica rapa. PLoS One 8:e85307
Chu M, Song T, Falk KC, Zhang X, Liu X, Chang A, Lahlali R, McGregor L, Gossen BD, Peng G, Yu F (2014) Fine mapping of Rcr1 and analyses of its effect on transcriptome patterns during infection by Plasmodiophora brassicae. BMC Genom 15:1166
Dangl JL, Jones JD (2001) Plant pathogens and integrated defence responses to infection. Nature 411:826–833
Deslandes L, Olivier J, Theulieres F, Hirsch J, Feng DX, Bittner-Eddy P, Beynon J, Marco Y (2002) Resistance to Ralstonia solanacearum in Arabidopsis thaliana is conferred by the recessive RRS1-R gene, a member of a novel family of resistance genes. Proc Natl Acad Sci USA 99:2404–2409
Diederichsen E, Frauen M, Linders EGA, Hatakeyama K, Hirai M (2009) Status and perspectives of clubroot resistance breeding in Crucifer Crops. J Plant Growth Regul 28:265–281
Dixon G (2009) The Occurrence and Economic Impact of Plasmodiophora brassicae and Clubroot Disease. J Plant Growth Regul 28:194–202
Eitas TK, Dangl JL (2010) NB-LRR proteins: pairs, pieces, perception, partners, and pathways. Curr Opin Plant Biol 13:472–477
Ellis JG, Dodds PN, Lawrence GJ (2007) Flax rust resistance gene specificity is based on direct resistance-avirulence protein interactions. Annu Rev Phytopathol 45:289–306
Fredua-Agyeman R, Rahman H (2016) Mapping of the clubroot disease resistance in spring Brassica napus canola introgressed from European winter canola cv. ‘Mendel’. Euphytica 211:201–213
Gassmann W, Hinsch ME, Staskawicz BJ (1999) The Arabidopsis RPS4 bacterial-resistance gene is a member of the TIR-NBS-LRR family of disease-resistance genes. Plant J 20:265–277
Hatakeyama K, Fujimura M, Ishida M, Suzuki T (2004) New classification method for Plasmodiophora brassicae field isolates in Japan based on resistance of F1 cultivars of Chinese cabbage (Brassica rapa L.) to clubroot. Breed Sci 54:197–201
Hatakeyama K, Suwabe K, Tomita RN, Kato T, Nunome T, Fukuoka H, Matsumoto S (2013) Identification and characterization of Crr1a, a gene for resistance to clubroot disease (Plasmodiophora brassicae Woronin) in Brassica rapa L. PLoS One 8:e54745
Hirai M (2006) Genetic analysis of clubroot resistance in Brassica crops. Breed Sci 56:223–229
Hirai M, Harada T, Kubo N, Tsukada M, Suwabe K, Matsumoto S (2004) A novel locus for clubroot resistance in Brassica rapa and its linkage markers. Theor Appl Genet 108:639–643
Jones DA, Jones JDG (1997) The role of leucine-rich repeat proteins in plant defences. In: Andrews JH, Tommerup IC, Callow JA (eds) Advances in botanical research, vol 24. Academic Press, pp 89–167. doi:10.1016/S0065-2296(08)60072-5
Kato T, Hatakeyama K, Fukino N, Matsumoto S (2012) Identification of a clubroot resistance locus conferring resistance to a Plasmodiophora brassicae classified into pathotype group 3 in Chinese cabbage (Brassica rapa L.). Breed Sci 62:282–287
Kato T, Hatakeyama K, Fukino N, Matsumoto S (2013) Fine mapping of the clubroot resistance gene CRb and development of a useful selectable marker in Brassica rapa. Breed Sci 63:116–124
Kuginuki Y, Yoshikawa H, Hirai M (1999) Variation in virulence of Plasmodiophora brassicae in Japan tested with clubroot-resistant cultivars of Chinese cabbage (Brassica rapa L. ssp pekinensis). Eur J Plant Pathol 105:327–332
Leister D (2004) Tandem and segmental gene duplication and recombination in the evolution of plant disease resistance gene. Trends Genet 20:116–122
Manzanares-Dauleux MJ, Divaret I, Baron F, Thomas G (2001) Assessment of biological and molecular variability between and within field isolates of Plasmodiophora brassicae. Plant Pathol 50:165–173
Marone D, Russo M, Laidò G, De Leonardis A, Mastrangelo A (2013) Plant Nucleotide Binding Site-Leucine-Rich Repeat (NBS-LRR) genes: active guardians in host defense responses. Int J Mol Sci 14:7302
Matsumoto E, Yasui C, Ohi M, Tsukada M (1998) Linkage analysis of RFLP markers for clubroot resistance and pigmentation in Chinese cabbage (Brassica rapa ssp. pekinensis). Euphytica 104:79–86
Narusaka M, Shirasu K, Noutoshi Y, Kubo Y, Shiraishi T, Iwabuchi M, Narusaka Y (2009) RRS1 and RPS4 provide a dual Resistance-gene system against fungal and bacterial pathogens. Plant J 60:218–226
Piao ZY, Deng YQ, Choi SR, Park YJ, Lim YP (2004) SCAR and CAPS mapping of CRb, a gene conferring resistance to Plasmodiophora brassicae in Chinese cabbage (Brassica rapa ssp pekinensis). Theor Appl Genet 108:1458–1465
Piao ZY, Ramchiary N, Lim YP (2009) Genetics of clubroot resistance in Brassica species. J Plant Growth Regul 28:252–264
Sakamoto K, Saito A, Hayashida N, Taguchi G, Matsumoto E (2008) Mapping of isolate-specific QTLs for clubroot resistance in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Theor Appl Genet 117:759–767
Stirnweis D, Milani SD, Brunner S, Herren G, Buchmann G, Peditto D, Jordan T, Keller B (2014) Suppression among alleles encoding nucleotide-binding-leucine-rich repeat resistance proteins interferes with resistance in F1 hybrid and allele-pyramided wheat plants. Plant J 79:893–903
Suwabe K, Tsukazaki H, Iketani H, Hatakeyama K, Fujimura M, Nunome T, Fukuoka H, Matsumoto S, Hirai M (2003) Identification of two loci for resistance to clubroot (Plasmodiophora brassicae Woronin) in Brassica rapa L. Theor Appl Genet 107:997–1002
Suwabe K, Tsukazaki H, Iketani H, Hatakeyama K, Kondo M, Fujimura M, Nunome T, Fukuoka H, Hirai M, Matsumoto S (2006) Simple sequence repeat-based comparative genomics between Brassica rapa and Arabidopsis thaliana: the genetic origin of clubroot resistance. Genetics 173:309–319
Suwabe K, Suzuki G, Kondo M, Tomita RN, Mukai Y, Fukuoka H, Hirai M, Matsumoto S (2012) Microstructure of the Brassica rapa genome segment that are homeologous to resistance gene cluster in Arabidopsis chromosome 4. Breed Sci 62:170–177
Ueno H, Matsumoto E, Aruga D, Kitagawa S, Matsumura H, Hayashida N (2012) Molecular characterization of the CRa gene conferring clubroot resistance in Brassica rapa. Plant Mol Biol 80:621–629
Wang XW, Wang HZ, Wang J, Sun RF, Wu J, Liu SY, Bai YQ, Mun JH, Bancroft I, Cheng F, Huang SW, Li XX, Hua W, Wang JY, Wang XY, Freeling M, Pires JC, Paterson AH, Chalhoub B, Wang B, Hayward A, Sharpe AG, Park BS, Weisshaar B, Liu BH, Li B, Liu B, Tong CB, Song C, Duran C, Peng CF, Geng CY, Koh CS, Lin CY, Edwards D, Mu DS, Shen D, Soumpourou E, Li F, Fraser F, Conant G, Lassalle G, King GJ, Bonnema G, Tang HB, Wang HP, Belcram H, Zhou HL, Hirakawa H, Abe H, Guo H, Wang H, Jin HZ, Parkin IAP, Batley J, Kim JS, Just J, Li JW, Xu JH, Deng J, Kim JA, Li JP, Yu JY, Meng JL, Wang JP, Min JM, Poulain J, Hatakeyama K, Wu K, Wang L, Fang L, Trick M, Links MG, Zhao MX, Jin MN, Ramchiary N, Drou N, Berkman PJ, Cai QL, Huang QF, Li RQ, Tabata S, Cheng SF, Zhang S, Zhang SJ, Huang SM, Sato S, Sun SL, Kwon SJ, Choi SR, Lee TH, Fan W, Zhao X, Tan X, Xu X, Wang Y, Qiu Y, Yin Y, Li YR, Du YC, Liao YC, Lim Y, Narusaka Y, Wang YP, Wang ZY, Li ZY, Wang ZW, Xiong ZY, Zhang ZH (2011) The genome of the mesopolyploid crop species Brassica rapa. Nat Genet 43:U1035–U1157
Williams PH (1966) A system for determination of races of Plasmodiophora brassicae that infect cabbage and rutabaga. Phytopathology 56:624–626
Zhang T, Zhao Z, Zhang C, Pang W, Choi SR, Lim YP, Piao Z (2014) Fine genetic and physical mapping of the CRb gene conferring resistance to clubroot disease in Brassica rapa. Mol Breed 34:1173–1183
Zhang H, Feng J, Hwang S-H, Strelkov SE, Falak I, Huang X, Sun R (2016) Mapping of clubroot (Plasmodiophora brassicae) resistance in canola (Brassica napus). Plant Pathol 65:435–440
Acknowledgements
The authors thank S. Negoro, T. Yamakawa, and Y. Higashi, Institute of Vegetable and Floriculture Science, National Agriculture and Food Research Organization, for their technical assistance.
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This work was supported by the Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics for Agricultural Innovation, Grant number HOR1002).
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438_2016_1281_MOESM1_ESM.pptx
Fig. S1 Root phenotype of the T1 plants derived from the three representative T0 lines of the transgenic B. rapa carrying the CRb_α transgene. The presence (+)/absence (—) of the transgene and disease index (DI) of each plant are indicated under the picture. (PPTX 3339 kb)
438_2016_1281_MOESM2_ESM.pptx
Fig. S2 Alignment of the sequences of the fosmid clone P005H06 and CRa genomic DNA (accession no. AB751516). Identical nucleotide sequences are shown in gray. The start and stop codons are boxed. Exon–intron junctions are indicated under the sequences. (PPTX 21183 kb)
438_2016_1281_MOESM3_ESM.pptx
Fig. S3 Alignment of TIR (a), NB (b), and LRR (c) domains between the predicted Crr1a and CRa proteins. The identical amino acid residues are shown in gray. (PPTX 311 kb)
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Hatakeyama, K., Niwa, T., Kato, T. et al. The tandem repeated organization of NB-LRR genes in the clubroot-resistant CRb locus in Brassica rapa L.. Mol Genet Genomics 292, 397–405 (2017). https://doi.org/10.1007/s00438-016-1281-1
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DOI: https://doi.org/10.1007/s00438-016-1281-1