Title: Functional Analysis of TM6 MADS-box gene in the Octoploid Strawberry by CRISPR/Cas9 directed mutagenesis.

The B-class of MADS-box transcription factors has been studied in many plant species, but remains functionally uncharacterized in Rosaceae. APETALA3 (AP3), a member of this class, controls petal and stamen identities in Arabidopsis. In this study, we identified two members of the AP3 lineage in cultivated strawberry, Fragaria x ananassa, namely FaAP3 and FaTM6. FaTM6, and not FaAP3, showed an expression pattern equivalent to that of AP3 in Arabidopsis. We used the CRISPR/Cas9 genome editing system for the first time in an octoploid species to characterize the function of TM6 in strawberry flower development. An analysis by high-throughput sequencing of the FaTM6 locus spanning the target sites showed highly efficient genome editing already present in the T0 generation. Phenotypic characterization of the mutant lines indicated that FaTM6 plays a key role in anther development in strawberry. Our results validate the use of the CRISPR/Cas9 system for gene functional analysis in F. x ananassa as an octoploid species, and offer new opportunities for engineering strawberry to improve traits of interest in breeding programs.