A variety of changes, including CRISPR/Cas9-mediated deletions, in CENH3 lead to haploid induction on outcrossing

Creating true-breeding lines is a critical step in plant breeding. Novel, completely homozygous true-breeding lines can be generated by doubled haploid technology in single generation. Haploid induction through modification of the centromere-specific histone 3 variant (CENH3), including chimeric proteins, expression of non-native CENH3 and single amino acid substitutions, has been shown to induce, on outcrossing to wild type, haploid progeny possessing only the genome of the wild-type parent, inArabidopsis thaliana. Here, we report the characterization of 31 additional EMS-inducible amino acid substitutions inCENH3for their ability to complement a knockout in the endogenousCENH3gene and induce haploid progeny when pollinated by the wild type. We also tested the effect of double amino acid changes, which might be generated through a second round of EMS mutagenesis. Finally, we report on the effects of CRISPR/Cas9-mediated in-frame deletions in the alpha N helix of the CENH3 histone fold domain. Remarkably, we found that complete deletion of the alpha N helix, which is conserved throughout angiosperms, results in plants which exhibit normal growth and fertility while acting as excellent haploid inducers when pollinated by wild-type pollen. Both of these technologies, CRISPR mutagenesis and EMS mutagenesis, represent non-transgenic approaches to the generation of haploid inducers.