Cloning clubroot resistance genes from B. nigra and transferring the genes into canola through a CRISPR/Cas9 based technology

Term: 4 years, ending May 2025
Status: Completed
Researcher(s): Fengqun Yu, AAFC
SaskCanola Investment: $90,475
Total Project Cost: $361,900
Funding Partners: Agriculture Development Fund, Western Grains Research Foundation

Objectives

1. Identify the most probable candidates for the CR genes identified in B. nigra.

2. Clone and verify the function of CR genes.

3. Transfer the validated CR genes into canola using CRISPR/Cas9.

Project Description

Clubroot, caused by Plasmodiophora brassicae, is a severe soil-borne disease threatening canola (Brassica napus) production in Western Canada. With the emergence of new virulent races capable of overcoming existing resistance sources, there is a critical need to identify novel clubroot resistance (CR) genes for integration into elite cultivars. Initially, this project aimed to clone the previously identified CR gene, Rcr6, from Brassica nigra (black mustard), a relative known for its robust clubroot resistance, and to transfer it into canola using a CRISPR/Cas9-based intragenic breeding approach. Remarkably, the project surpassed its original objectives by identifying and characterizing an additional distinct CR gene, Rcr12, closely located near Rcr6. Bulked Segregant RNA sequencing (BSR-Seq) initially located the region of Rcr12 on chromosome B3. Subsequent fine-mapping efforts narrowed the candidate region down to a 0.33 Mb interval, partially overlapping the previously characterized Rcr6 locus. Comprehensive sequence analyses within the mapped region identified nine nucleotide-binding leucine-rich-repeat (NLR)-type candidate resistance genes clustered at the Rcr6-Rcr12 loci. Two extensive rounds of cloning efforts were conducted, involving three resistant B. nigra lines—PI (Rcr6 donor), BRA (Rcr12 donor), and CR2716 (presumed Rcr12 donor)—and one susceptible control line (CR2748). This extensive effort resulted in the cloning of nineteen unique alleles from resistant donors, all distinct from susceptible counterparts, thereby significantly exceeding the original goal of cloning a single gene. These nineteen alleles were transferred into canola breeding line DH12075 through Agrobacterium-mediated transformation. Rigorous testing of the transgenic plants against P. brassicae strains successfully identified five functionally validated CR alleles: B33 and C638 at the NLR1 locus, B71 at the NLR5 locus, and 15510C6 and 15510BP at the NLR3 locus. Each allele displayed distinct sequence variations and provided robust resistance against specific pathogen strains, clearly demonstrating multiple independent resistance mechanisms within the Rcr6-Rcr12 clusters. Leveraging the established CRISPR/Cas9 intragenic breeding system, intragenic canola lines carrying the two most potent alleles (15510BP and 15510C6) were also successfully developed. This precise and efficient technology significantly accelerated breeding timelines, delivering selection-marker-free, genetically improved lines within two years—a transformative improvement over traditional introgression methods. Moreover, this approach effectively minimized linkage drag and enhanced breeding precision. This project's achievements have significantly advanced CR research by providing validated genetic resources and molecular markers essential for marker-assisted selection and gene stacking strategies. The discovery of the novel NLR cluster-singleton organization of Rcr12 and Rcr6 on chromosome B3 is unprecedented in Brassica crops, underscoring the rich genetic diversity within the B genome. By introducing these five functionally validated CR alleles into elite canola lines, the study provides the canola breeding community with critical genetic tools necessary for developing durable, broad-spectrum resistance strategies. Future research efforts will focus on the development of intragenic canola lines with additional CR alleles, the characterization and nomenclature of CR genes in the Rcr6–Rcr12 clusters, the exploration of additional CR candidates, the investigation of CR gene synergism, the optimization of gene stacking protocols, and proactive regulatory engagement.

Grower Benefits

• Genetic resistance is the most effective and efficient way to manage clubroot disease in canola. Black mustard (Brassica nigra, B genome) is highly resistant to clubroot and offers abundant genetic resources for clubroot resistance (CR).

• A novel CR gene, Rcr12, which confers strong resistance to clubroot, was genetically mapped to chromosome B3, near our previously identified CR gene, Rcr6.

• Nineteen alleles encoding disease resistance proteins were cloned from three B. nigra resistant lines, and five of them responsible for CR were functionally validated in the Rcr12 and Rcr6 clusters.

• Canola breeding lines with CR derived from black mustard were developed using CRISPR/Cas9 gene-editing technology.

• The molecular markers and canola breeding lines developed in the project can be used for canola breeding programs.