Efficient CRISPR/Cas9-Mediated Knockout of an Endogenous PHYTOENE DESATURASE Gene in T1 Progeny of Apomictic Hieracium Enables New Strategies for Apomixis Gene Identification

Most subgenus species are self-incompatible. Some undergo facultative apomixis where most seeds form asexually with a maternal genotype. Most embryo sacs develop by mitosis, without meiosis and seeds form without fertilization. Apomixis is controlled by dominant loci where recombination is suppressed. Loci deletion by γ-irradiation results in reversion to sexual reproduction. Targeted mutagenesis of genes at identified loci would facilitate causal gene identification. In this study, the efficacy of CRISPR/Cas9 editing was examined in apomictic by targeting mutations in the endogenous ( ) gene using -mediated leaf disk transformation. In three experiments, the expected albino dwarf-lethal phenotype, characteristic of knockout, was evident in 11% of T0 plants, 31.4% were sectorial albino chimeras, and the remainder were green. The chimeric plants flowered. Germinated T1 seeds derived from apomictic reproduction in two chimeric plants were phenotyped and sequenced to identify gene edits. Up to 86% of seeds produced albino seedlings with complete knockout. This was attributed to continuing Cas9-mediated editing in chimeric plants during apomictic seed formation preventing segregation from the target. This successful demonstration of efficient CRISPR/Cas9 gene editing in apomictic , enabled development of the discussed strategies for future identification of causal apomixis genes.