Local Duplication of TIR-NBS-LRR Gene Marks Clubroot Resistance in Brassica napus cv. Tosca

Abstract

Clubroot, caused by Plasmodiophora brassicae infection, is a disease of growing importance in cruciferous crops, including oilseed rape (Brassica napus). The affected plants exhibit prominent galling of the roots that impairs their capacity for water and nutrient uptake, which leads to growth retardation, wilting, premature ripening, or death. Due to the scarcity of effective means of protection against the pathogen, breeding of resistant varieties remains a crucial component of disease control measures. The key aspect of the breeding process is the identification of genetic factors associated with variable response to the pathogen exposure. Although numerous clubroot resistance loci have been described in Brassica crops, continuous updates on the sources of resistance are necessary. Many of the resistance genes are pathotype-specific, moreover, resistance breakdowns have been reported. In this study, we characterize the clubroot resistance locus in the winter oilseed rape cultivar "Tosca." In a series of greenhouse experiments, we evaluate the disease severity of P. brassicae-challenged "Tosca"-derived population of doubled haploids, which we genotype with Brassica 60 K array and a selection of SSR/SCAR markers. We then construct a genetic map and narrow down the resistance locus to the 0.4 cM fragment on the A03 chromosome, corresponding to the region previously described as Crr3. Using Oxford Nanopore long-read genome resequencing and RNA-seq we review the composition of the locus and describe a duplication of TIR-NBS-LRR gene. Further, we explore the transcriptomic differences of the local genes between the clubroot resistant and susceptible, inoculated and control DH lines. We conclude that the duplicated TNL gene is a promising candidate for the resistance factor. This study provides valuable resources for clubroot resistance breeding programs and lays a foundation for further functional studies on clubroot resistance.

Keywords: Brassica napus; Oxford Nanopore; Plasmodiophora brassicae; QTL; RNA-Seq; TNL; duplication; resistance.

FIGURE 1

Distribution of the Disease Index (DI) in (A) a series of phenotyping experiments, (B) joint phenotyping of 240 lines in conditions promoting pathogenesis, and (C) a cumulative estimation of the DI-BLUP. The color lines connect DI’s of 8 checks tested in each experiment.

FIGURE 2

Results of genetic mapping. (A) LOD score for QTL presence along the A03 chromosome genetic map. Red lines span Bayes Credible Intervals. (B) Effect of “BRH-1” (yellow) and “Tosca” (green) alleles along the A03 chromosome genetic map. (C) Phenotype (BLUP-DI) distribution of DH lines carrying “BRH-1” yellow and “Tosca” green alleles at the peak marker. (D) Analysis of recombinants. The plot shows a genotype (white: “BRH-1”-inherited, gray: “Tosca”-inherited) at markers surrounding the mapped locus. The phenotype for 18 DH lines recombining in the proximity of the locus is shown as a DI from one of the 1–6 batches, and, if available, 7th, common batch, as well as BLUP-DI. A marker solely explaining the phenotype is highlighted in teal.

FIGURE 3

Physical localization of the resistance locus (black line) in different assemblies of the Brassica napus genome defined by the bin of cosegregating markers at 11.98 cM. Colored bars represent Darmor-bzh 4.1 contigs. Gray color depicts sequences in the pangenome and Express 617 not covered by the Darmor-bzh 4.1 contigs.

FIGURE 4

Schematic location of the Crr3^Tsc region in the context of other resistance loci and markers located on Express 617 chromosome A03. (A) A general overview of the location of Rcr1/Cra/Crb/Crb^Kato and Crr3/CRd/Crk regions on the A03 chromosome. (B) Zoomed region from the Crr3/CRd/Crk fragment (marked with a black box on A). Markers labeled with the same color cosegregate in the mapping DH population. No genetic data were obtained for markers indicated with black. The precise start of the CRd locus could not be physically mapped onto Express 617 assembly.

FIGURE 5

(A) Simplified plot showing resistance locus coverage of ONT long reads aligned to the Express 617 reference genome. A sharp, flat peak (indicated by an arrow) in “Tosca” genomic data marks the location of the fragment containing the TNL gene (yellow). Darker colors on the coverage plot represent insertions larger than 10 bp. (B) Schematic overview of (i) depth of coverage of ON reads (DoC) and (ii) within the duplicated “Tosca” region. Yellow – a fragment corresponding to the BnaA03g29300D gene; green – homologous fragments of STP6 gene. (C) Pairwise alignment-based comparison of CDS sequence variability between TNL homologs in “Tosca” (T1,T2) and “BRH-1.” Synonymous substitutions are marked as empty circles below the axis, non-synonymous are indicated by filled circles above.

FIGURE 6

Allele-discriminating PCR. The gel electropherogram (B) shows a result of PCR reaction designed as depicted on the schematic (A). B, T1, and T2 stand for BnaA03g29300D copies from “BRH-1” and “Tosca,” respectively. C03 represents a homoeologous region on the C03 chromosome, identical between the parental lines. Expected products are color-coded according to the schematic, and juxtaposed with the gel. Gel lanes: M – size marker, T – “Tosca,” B – “BRH-1,” W – water-containing control reaction, 266-56 – selected lines, recombining in the proximity of the resistance locus. Susceptible lines are underlined with red, resistant with teal. Both “Tosca” alleles (fragments 224 bp and 577 bp) segregated with the resistance.