Article | . 2019 Vol. 37, Issue. 1
Development of a Molecular Marker Using GWAS to Select the Resistance Resource for the Yeoncheon Strain Causing Kimchi Cabbage Clubroot Disease

Vegetable Division, National Institute of Horticultural & Herbal Science, Rural Development Administration1
Postharvest Research Division, National Institute of Horticultural & Herbal Science, Rural Development Administration2
Department of Crop Science, College of College of Agriculture and Life Sciences, Chungnam National University3

2019.. 92:107


Resistant cultivars have been developed as a response to clubroot disease, which poses a challenge to Kimchi cabbage cultivation in Korea. However, the recent problem of disease vulnerability in resistant varieties has led to collective efforts to promote genetically diverse resistant sources. The National Institute of Horticultural & Herbal Science (NIHHS, Vegetable Research Division) promotes and conserves resistant resources, and despite using the molecular markers reported thus far in analyzing the resources, a need for novel molecular markers has become apparent owing to the large number of resources that could not be amplified. To address this problem, 96 resources for resistance and vulnerability were selected among the ones conserved at the NIHHS, from which 20,540 SNPs were detected using genotype-by-sequencing (GBS) analysis. Subsequently, a molecular marker associated with resistance to the Yeoncheon strain that causes clubroot disease was developed using a genome-wide association study (GWAS). The corresponding gene had an SNP (G↔T) at the 23098380 position of A07, which was used to develop the CAPS marker that can differentiate resistance and susceptibility to the Yeoncheon strain causing clubroot disease. The gene at the position of the CAPS marker was found to be an associated gene within 21 kb, encoding a glucose-methanol-choline (GMC) oxidoreductase family protein with four V-type H+-transporting ATPase subunit Gs. In this study, a molecular marker specific for the Yeoncheon strain was developed. Its use, alongside other previously developed markers in selecting resistance resources, is expected to prove useful in the selection of a wide array of disease-resistant breeding sources. Furthermore, the resulting GBS data could be used for future analysis of the resistance genes associated with various strains causing clubroot disease.

1. Butcher DN, Searle LM, Mousdale DMA (1976) The role of glucosinolates in the clubroot disease of the cruciferae. Med Fac Landbouw Rijksuniv Gent 41:525-532  

2. Cheah LH, Veerakone S, Kent G (2000) Biological control of clubroot on cauliflower with Trichoderma and Streptomyces spp. Org Biocontrol 53:18-21  

3. Cox MP, Peterson DA, Biggs PJ (2010) SolexaQA: At-a-glance quality assessment of Illumina second-generation sequencing data. BMC Bioinformatics 11:485. doi:10.1186/1471-2105-11-485  

4. Datnoff LE, Kroll TK, Lacy GH (1987) Efficacy of chlorine for decontaminating water infested with resting spores of Plasmodiophora brassicae. Plant Dis 71:734-736. doi:10.1094/PD-71-0734  

5. Dekhuizen HM, Overeem JC (1971) The role of cytokinins in clubroot formation. Physiol Plant Pathol 1:151-161. doi:10.1016/0048- 4059(71)90024-5  

6. Dietz KJ, Tavakoli N, Kluge C, Mimura T, Sharma SS, Harris GC, Chardonnens AN, Golldack D (2001) Significance of the V-type ATPase for the adaptation to stressful growth conditions and its regulation on the molecular and biochemical level. J Exp Bot 52:1969-1980. doi:10.1093/jexbot/52.363.1969  

7. Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, Mitchell SE (2011) A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS ONE 6:e19379. doi:10.1371/journal.pone.0019379  

8. Grsic-Rausch S, Kirchheim B, Pieper K, Fritsch M, Hilgenberg W, Ludwig-Müller J (1999) Induction of auxin biosynthetic enzymes by jasmonic acid and in clubroot diseased Chinese cabbage plants. Physiol Plant 105:521-531. doi:10.1034/j.1399-3054.1999.105318.x  

9. Grsic-Rausch S, Kobelt P, Siemens JM, Bischoff M, Ludwig-Müller J (2000) Expression and localization of nitrilase during symptom development of the clubroot disease in Arabidopsis. Plant Physiol 122:369-378. doi:10.1104/pp.122.2.369  

10. 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 8:1. doi:10.1371/ journal.pone.0054745  

11. Hirai M, Harada T, Kubo N, Tsukada M, Suwabe K, Matsumoto S (2004) A novel locus for clubroot resistance in and its linkage markers. Theor Appl Genet 108:639-643. doi:10.1007/s00122-003-1475-x  

12. Huang Z, Peng G, Liu X, Deora A, Falk KC, Gossen BD, McDonald MR, Yu F (2017) Fine mapping of a clubroot resistance gene in Chinese cabbage using SNP markers identified from bulked segregant RNA sequencing. Front Plant Sci 8:1448. doi:10.3389/fpls.2017.01448  

13. Jo SJ, Shim SA, Jang KS, Choi YH, Kim JC, Choi GJ (2011) Resistance of cultivars of Chinese cabbage to Plasmodiophora brassicae isolates of several races collected in Korea. Korean J Hortic Sci Technol 29:610-616  

14. Kato T, Hatakeyama K, Fukino N, Matsumoto S (2012) Identificaiton 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. doi:10.1270/jsbbs.62.282  

15. Kawabe M, Onokubo AO, Arimoto Y, Yoshida T, Azegami K, Teraoka T, Arie T (2011) GMC oxidoreductase, a highly expressed protein in a potent biocontrol agent Fusarium oxysporum Cong:1-2, is dispensable for biocontrol activity. J Gen Appl Microbiol 57:207-217. doi:10.2323/jgam.57.207  

16. Kim H, Jo EJ, Choi YH, Jang KS, Choi GJ (2016) Pathotype classification of Plasmodiophora brassicae isolates using clubroot resistant cultivars of Chinese cabbage. Plant Pathol J 32:423-430. doi:10.5423/PPJ.OA.04.2016.0081  

17. Kim JE, Oh SK, Lee JH, Lee BM, Jo SH (2014) Genome-wide SNP calling using next generation sequencing data in tomato. Mol Cells 37:36-42. doi:10.14348/molcells.2014.2241  

18. Kuginuki Y, Ajisaka H, Yui M, Yoshikawa H, Hida KI, Hirai M (1997) RAPD markers linked to a clubroot-resistance locus in Brassica rapa L. Euphytica 98:149-154. doi:10.1023/A:1003147815692  

19. 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. doi:10.1023/A:1008705413127  

20. Lee JG, Kim JS (2010) Variation of glucosinolate content in the root of susceptibel and resistant Chinese cabbage cultivars during development of clubroot disease. Korean J Hortic Sci Technol 28:200-208  

21. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754-1760. doi:10.1093/bioinformatics/btp324  

22. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R; 1000 Genome Project Data Processing Subgroup (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078-2079. doi:10.1093/bioinformatics/ btp352  

23. Lipka AE, Tian F, Wang Q, Peiffer J, Li M, Bradbury PJ, Gore MA, Buckler ES, Zhang Z (2012) GAPIT: genome association and prediction integrated tool. Bioinformatics 28:2397–2399. doi:10.1093/bioinformatics/bts444  

24. Ludwig-Müller J, Schubert B, Pieper K, Ihmig S, Hilgenberg W (1997) Glucosinolate content in susceptible and resistant Chinese cabbage varieties during development of clubroot disease. Phytochemistry 44:407-414. doi:10.1016/S0031-9422(96)00498-0  

25. Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17:10-12. doi:10.14806/ej.17.1.200  

26. Matsumoto E, Hayashida N, Sakamoto K, Ohi M (2005) Behavior of DNA markers linked to a clubroot resistance gene in segregating populations of Chinese cabbage (Brassica rapa L. ssp. pekinensis). J Jpn Soc Hortic Sci 74:367-373. doi:10.2503/jjshs.74.367  

27. Matsumoto E, Yasui C, Ohi M, Tsukada M (1998) Linkage analysis of RFLP markers for clubroot resistance and pigmentation in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Euphytica 104:79-86. doi:10.1023/A:1018370418201  

28. Park SH, Yoon MK, Lim YP (2011) Development of clubroot race4 resistant inbreds using conventional breeding and microspore culture method in Chinese cabbage. CNU J Agric Sci 38:613-618  

29. Piao ZY, Deng YQ, Park YJ, Choi YS, Lim YP (2004) SCAR and CAPS mapping of a resistance gene, CRb, that confers resistance to Plasmodiophora brassicae in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Theor Appl Genet 108:1458-1465. doi:10.1007/ s00122-003-1577-5  

30. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959  

31. Ratajczak R (2000) Structure, function and regulation of the plant vacuolar H-translocating ATPase. Biochim Biophys Acat 1465:17-36. doi:10.1016/S0005-2736(00)00129-2  

32. 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. doi:10.1007/s00122-008-0817-0  

33. 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. doi:10.1007/s00122-003-1309-x  

34. Suwabe K, Tsukazaki H, Iketani H, Hatakeyama K, Kondo M, Fujimura M, Nunome T, Fukuoka H, Hirai M, et al (2006) Simple sequence repeat-based comparative genomics between Brassica rapa and Arabidopsis thaliana: The genetic origin of clubroot resistance. Genetics 173:309-319. doi:10.1534/genetics.104.038968  

35. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol 30:2725-2729. doi:10.1093/molbev/mst197  

36. Tanaka S, Fujiyama S, Shigemori A, Nakayama S, Ito M, Kamaya I (1998) Pathogenesis of isolates of Plasmodiophora brassicae from Japan (1). Race and pathogenicities in clubroot resistant cultivars (in Japanese). Kyushu Plant Prot Res 44 :15-19. doi:10.4241/kyubyochu.44.15  

37. Yeoung YR, Kim JH, Kim BS, Jeon JY, Yoon CS (2003) Effects of beneficial antagonists (Bacillus sp., Pseudomonas sp., and Trichoderma sp.) on control of club root of Chinese cabbage. Korean J Hortic Sci Technol 21:194-198  

38. Yoshida T, Nomura Y, Akimoto T (1998) Symptomless infection with pathogenicity-deficient isolates of Fusarium oxysporum f. sp. conglutinans on cabbage plants. Ann Phytophathol Soc Jpn 64:611  

39. Yoshikawa H (1981) Breeding for clubroot resistance in Chinese cabbage. In NS Taleker, TD Griggs, eds, Chinese Cabbage. AVRDC, Shanhua, Taiwan, pp 405-413  

40. Yu F, Zhang X, Peng G, Falk KC, Strelkov SE, Gossen BD (2017) Genotyping-by-sequencing reveals three QTL for clubroot resistance to six pathotypes of Plasmodiophora brassicae in Brassica rapa. Sci Rep 7:4516. doi:10.1038/s41598-017-04903-2  

41. Zheng X, Levine D, Shen J, Gogarten S, Laurie C and Weir B (2012) A high-performance computing toolset for relatedness and principal component analysis of SNP data. Bioinformatics 28:3326-3328. doi:10.1093/bioinformatics/bts606