Figure 1.
Map-based cloning of Crr1a: a genetic map around the Crr1 locus and graphical genotypes of F3 populations in which recombination occurred between AT27 and BZ2–DraI.
The major locus for resistance to Ano-01 was predicted around BSA7, and another locus with minor effect was predicted around BSA2 (Suwabe et al., 2011). We named the former locus Crr1a and the latter Crr1b. Gray bars indicate BAC clones isolated by using BSA7 as a probe. Black bars, homozygous for resistant G004 allele; white bars, homozygous for susceptible A9709 allele. Phenotypes of resistance (R) and susceptibility (S) of F3 populations to Ano-01 are indicated to the right of the graphical genotype, with the number of F3 populations of each in parentheses. The 3 F3 populations in which recombination occurred between B359C3 and B355H7 were susceptible to Ano-01. The position of Crr1a was delimited to an 8-kb region between B355H7 and B359C3. The predicted ORFs of the Crr1a candidate are shown below.
Figure 2.
Complementation test of Crr1a candidate.
A. Resistance of transgenic T2 lines (UpCc_01–24), LsUbi promoter::GUS lines (UGU_07, 08), and wild-type Col-0 to Ano-01. Means (±SD) are based on the average of 2 or 3 clubroot tests (9 plants per test). A lower mean disease index score indicates higher resistance to clubroot. B, C. Root phenotypes of (B) Col-0 inoculated with Ano-01 and (C) transgenic T2 line inoculated with Ano-01. Scale bar indicates 10 mm. D. Resistance of T3 lines to Ano-01 (A) and Wakayama-01 (W).
Figure 3.
Alignment of deduced amino acid sequences of Crr1a between resistant G004 and susceptible A9709 alleles.
Asterisks, identical amino acid residues; dashed lines, gaps for alignment. Blue, TIR domain; green, NB domain; red, LRR domain.
Figure 4.
Sequence polymorphisms of Crr1a between resistant and susceptible alleles.
Crr1aG004: Schematic representation of Crr1a allelic structure in resistant G004. Black boxes, exons; black lines, introns; arrowheads, large insertions. Crr1aA9709: Sequence surrounding the site of the 357-bp insertion in susceptible A9707. White box, retrotransposon-like sequence. Bent arrow, transcription start site of Crr1aA9709 predicted by 2 independent 5′-RACE analyses. Putative target site duplication is underlined. Crr1aChiifu-401: Corresponding sequence from susceptible Chiifu-401. The 357-bp insertion in Crr1aA9709 was almost identical to the 3′-terminal region of a 4546-bp copia-like retrotransposon found in Crr1aChiifu-401.
Figure 5.
A. Semi-quantitative RT-PCR analysis of Crr1 in resistant (R4-8-1) and susceptible (A9709) lines. The B. rapa actin gene (BrACT) was used as a control. PCR cycles are indicated on the right. B-D. Spatial expression of Crr1G004 shown as GUS staining in (B) 12-day-old seedlings of Crr1G004 promoter::GUS plants (arrow indicates extreme end of primary root), (C) stele (arrow) of primary root, and (D) cortex (arrow) of primary root. E. Schematic diagram of gene structure of Crr1aG004 and alternative transcripts obtained by RT-PCR using primers flanking introns 2 (RT-a, -b, and -c) and 3 (RT-d and -e). V-like lines, spliced introns; asterisks, predicted stop codons.
Figure 6.
Expression of the Crr1aG004 transgene in T1 lines inoculated with Ano-01.
Transcript levels of 5 representative T1 plants derived from each of 2 resistant T0 lines and 1 plant derived from each of 2 susceptible T0 lines are shown. Osome is the recipient. The disease index of each plant is indicated in parentheses above each bar. Similar results were obtained in 2 independent tests.
Table 1.
Clubroot resistance of the transgenic Brassica rapa (T1) carrying Crr1a promoter::Crr1a cDNA construct.