Targeted Mutagenesis in Natural Apomicts

Apomixis-clonal reproduction through seeds-is an alternative reproductive strategy that takes place in less than 0.1% of plant species and has evolved independently in diverse plant lineages. To date, the genetic basis of apomixis has been unraveled genetically in only a few genera. The identification of causal apomixis genes is technically challenging, as apomictic species are typically polyploid and the genetic loci associated with apomixis are often in low-recombination regions limiting conventional fine-mapping. In triploid apomictic dandelion (Taraxacum officinale), after conventional genetic mapping, deletion mapping, and complete apomixis loci haplotype assembly, we made use of targeted mutagenesis using CRISPR/Cas9 technology to identify the Taraxacum officinale PARTHENOGENESIS (ToPAR) gene that is responsible for embryogenesis in the absence of fertilization. Here, we report the methods used to clone the ToPAR gene by targeted mutagenesis and we expect that the general principles could be applied in other systems to identify novel apomixis genes.