Establishing a 2-gene MiMe to produce clonal gametes in allotetraploid Brassica napus

Apomixis, or clonal reproduction through seeds, bypasses meiosis and fertilization to produce progeny that are genetically identical to the mother plant, thereby allowing fixation of hybrid vigor (heterosis) across generations. Synthetic apomixis has been established in Arabidopsis thaliana and rice (Oryza sativa) by combining 3 mutations into a Mitosis instead of Meiosis (MiMe) system where meiosis is transformed into a single mitosis-like division that produces clonal unreduced gametes, together with factors inducing parthenogenesis or haploidy. MiMe requires the simultaneous inactivation of 3 genes involved in different meiotic events, which poses challenges for its application in polyploids, which harbor multiple copies of each target gene. Here, we demonstrate that mutating 2 genes in the allotetraploid oilseed crop Brassica napus effectively converts meiosis into a mitosis-like division, producing over 95% viable clonal gametes, and fixing the genetic heterozygosity of F1 hybrids. This simplified MiMe system provides a practical framework for the production of clonal gametes, paving the way for engineering synthetic apomixis in Brassica napus and potentially other polyploid crops.