Agriculture and Agri-Food Canada, Saskatoon Research and Development Centre, 107 Science Place, Saskatoon, SK, S7N 0X2, Canada.
J Exp Bot. 2024 Feb 28;75(5):1347-1363. doi: 10.1093/jxb/erad471.
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.
在主要作物中培育抗病性对于全球粮食安全和可持续性至关重要。然而，传统的转基因技术或基因组编辑等常见生物技术并不能提供理想的解决方案，而不含选择标记的转基因作物，如 cisgenic/intragenic 作物，可能是合适的。在这项研究中，我们克隆并验证了 clubroot（Plasmodiophora brassicae）抗性基因 Rcr1 的功能后，证实了来自 Brassica rapa 作物的基因 Rcr1、Rcr2、Rcr4 和 CRa 以及来自 B. napus 油菜品种 'Mendel' 的 A03 染色体上的抗性基因在编码区是相同的。我们还确定 Rcr1 在 Brassica 育种材料中广泛分布，并对加拿大的多个代表性 clubroot 菌株具有强大的抗性。然后，我们修改了基于 CRISPR/Cas9 的 cisgenic 载体系统，发现它能够快速培育具有附加性状的无选择标记的转基因作物，成功地在 2 年内培育出具有 Rcr1 抗性的无选择标记的油菜（B. napus）种质，对 clubroot 病具有稳定的抗性。在 B. napus 背景下，该基因内载体系统能够以高编辑效率从最终产物中去除不需要的残留序列，并且没有检测到脱靶突变。我们的研究表明，这种育种策略有可能应用于其他可以通过农杆菌转化的作物。按照简化的工作流程，基因内种质可以在两代内得到开发，与传统的基因渗入相比，这可以显著减少育种时间和劳动力，同时仍能达到可比甚至更好的育种效果。
