From haploid inducer to CMS donor: repurposing of CENH3 to create a CMS line in a single step in Brassica napus

Mutation in centromere histone H3 (CENH3) protein could induce a paternal haploid with maternal cytoplasm in rapeseed. By paternal haploid induction, a cytoplasmic male sterile line can be created in any genetic background within one breeding cycle. Hybrid development in rapeseed relies primarily on the three-line system, which includes a cytoplasmic male sterile (CMS) line. Conventionally, these CMS lines are developed through backcrossing, a process that requires several breeding cycles to complete. More recently, the doubled haploid technique has been employed in various crops to generate homozygous lines within a single breeding cycle. In the present study, we utilized a haploid induction (HI) strategy to produce fertile homozygous lines and CMS lines via paternal haploid induction. We have created single homozygous and double heterozygous mutants of the BnaCENH3 gene in the rapeseed cultivar ganA (hau-CMS) using CRISPR/Cas9 technique. Upon hybridization of CMS-HI line with wild type can successfully induced paternal haploids with maternal sterile cytoplasm. This system offers the ability to introduce sterile cytoplasm into any genetic background within a single generation.