Tests for CR and CR marker analysis of nabana cultivars

Inoculation tests were performed according to Kuginuki et al. (1999). P. brassicae isolates Ng2 and Ng9, whose pathotypes are groups 4 and 2, respectively (Kubo et al. 2017), were inoculated to 10 CR and two non-CR nabana cultivars (Table 2), in which approximately 20 individuals were tested for each cultivar. A Chinese cabbage cultivar set with differential pathogenicity (Hatakeyama et al. 2004) was also inoculated as a control.

Test for clubroot resistance (CR) of nabana cultivars

Six markers linked to four known CR loci (CRb and Crr1-3) were tested by PCR amplification using 10 CR and one non-CR nabana cultivars (four individuals per cultivar) (Supplemental Table 4) according to previous reports (Hirai et al. 2004, Kato et al. 2013, Matsumoto et al. 2017, Suwabe et al. 2006). DNAs of three CR lines (‘CR Shinki’, G004, and N-WMR-3) (Hirai et al. 2004, Suwabe et al. 2003) were used for positive controls of CRb, Crr1/Crr2, and Crr3 resistance alleles, respectively. Alleles were detected using a DNA sequencer with a post-labeling method (Shimizu and Yano 2011) or by agarose gel electrophoresis.