The B-class of MADS-box transcription factors has been studied in many plant species, but remain functionally uncharacterized in the Rosaceae family. APETALA3 (AP3), a member of this class, controls the identity of petals and stamens in Arabidopsis thaliana. In this work, we identified two members of the AP3 lineage in the cultivated strawberry (Fragaria x ananassa): FaAP3 and FaTM6. Interestingly, FaTM6, and not FaAP3, shows an expression pattern equivalent to that of AP3 in Arabidopsis. Genome editing using Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is becoming a robust tool for targeted and stable mutagenesis of DNA. However, whether it can be efficiently used in an octoploid species such as F. ananassa is not known. Here we report for the first time the application of CRISPR/Cas9 in F. x ananassa to characterize the function of FaTM6 in flower development. An exhaustive analysis by high-throughput sequencing of the FaTM6 locus spanning the target sites showed a high efficiency genome editing already in the T0 generation. The phenotypic characterization of the mutant lines indicates that FaTM6 plays a key role in petal and especially in anther development in strawberry. Our results validate the CRISPR/Cas9 strategy for gene functional analysis in an octoploid species such as F. x ananassa, and offer new opportunities for engineering strawberry to improve traits of interest in breeding programs
