The fast breeding of selection-marker-free canola with Rcr1-rendered clubroot resistance by a CRISPR/Cas9-based vector system.

Disease resistance breeding for major crops is of crucial importance for global food security and societal sustainability. However, common biotechnologies like traditional transgenesis or genome editing are not ideal for this type of breeding need, while selection-marker-free transgenic crops like cisgenic/intragenic crops could fit into this niche. In this study, after cloning and functional verification of the clubroot resistance gene Rcr1, we first confirmed that clubroot resistance genes Rcr1, Rcr2, Rcr4, and CRa from Brassica rapa vegetables and the resistance gene from B. napus oilseed rape cv. "Mendel" on chromosome A03 were identical in their coding regions and that Rcr1 has a wide distribution in Brassica breeding materials and renders potent resistance against multiple representative clubroot strains in Canada. With this valuable genetic resource, we modified a CRISPR/Cas9-based cisgenic vector system to demonstrate the fast breeding of selection-marker-free transgenic crops with add-on traits, and selection-marker-free canola (B. napus L.) germplasms with Rcr1-rendered stable resistance to clubroot disease were successfully developed within 2 years. In the B. napus background, the intragenic vector system can remove unwanted residue sequences from the final product with high editing efficiency, and off-target mutations were not detected. Our study demonstrated the potential of applying this breeding strategy to other crops that can be transformed by Agrobacterium. Following the streamlined working procedure, intragenic germplasms could be developed within 2 generations, which could significantly reduce the breeding time and labor but achieve comparable or better breeding results compared to traditional introgression.