A CRISPR/Cas9-based vector system enables the fast breeding of selection-marker-free canola with Rcr1-rendered clubroot resistance

Breeding for disease resistance in major crops is of crucial importance for global food security and sustainability. However, common biotechnologies such as traditional transgenesis or genome editing do not provide an ideal solution, whereas transgenic crops free of selection markers such as cisgenic/intragenic crops might be suitable. In this study, after cloning and functional verification of the Rcr1 gene for resistance to clubroot (Plasmodiophora brassicae), we confirmed that the genes Rcr1, Rcr2, Rcr4, and CRa from Brassica rapa crops and the resistance gene from B. napus oilseed rape cv. 'Mendel' on chromosome A03 were identical in their coding regions. We also determined that Rcr1 has a wide distribution in Brassica breeding materials and renders potent resistance against multiple representative clubroot strains in Canada. We then modified a CRISPR/Cas9-based cisgenic vector system and found that it enabled the fast breeding of selection-marker-free transgenic crops with add-on traits, with selection-marker-free canola (B. napus) germplasms with Rcr1-rendered stable resistance to clubroot disease being successfully developed within 2 years. In the B. napus background, the intragenic vector system was able to remove unwanted residue sequences from the final product with high editing efficiency, and off-target mutations were not detected. Our study demonstrates the potential of applying this breeding strategy to other crops that can be transformed by Agrobacterium. Following the streamlined working procedure, intragenic germplasms can be developed within two generations, which could significantly reduce the breeding time and labor compared to traditional introgression whilst still achieving comparable or even better breeding results. A modified CRISPR/Cas9-based vector system enables the fast breeding of selection-marker-free canola with Rcr1 -rendered clubroot resistance, and has potential for developing other transgenic crops free of markers.