CRISPR editing of TERMINAL FLOWER 1 recapitulates perpetual flowering in strawberry, mimicking repression by the PERPETUAL FLOWERING locus.

The development of perpetual flowering (PF) cultivars revolutionized octoploid strawberry (Fragaria  ×  ananassa) cultivation by enabling nearly year-round fruit production and doubling fruit yields compared to historic seasonal flowering (SF) cultivars, which flower in spring. Most commercial PF cultivars result from the introgression of a dominant PERPETUAL FLOWERING (PF) allele at the PERPETUAL FLOWERING RUNNERING (PFRU) locus. PFRU has been genetically and physically mapped to chromosome 4B, but the causal gene(s) remain unknown. Here we used the haplotype-phased "UCD Royal Royce" (PFpf) genome, next-generation sequencing-based k-mer genome-wide association study (k-mer GWAS), and RNAseq to investigate PFRU. We narrowed the locus down to a precise physical address and revealed abundant genetic and transcriptomic differences in the locus and associated with flowering. Outside the PFRU locus, flowering regulation diverges at expression of TERMINAL FLOWER 1 (TFL1) in all 4 subgenomes, analogous to diploid Fragaria vesca. This critical step of the flowering pathway is altered by PF in diverse cultivars, and gene editing knockout of TFL1 is sufficient to induce flowering under long days in a seasonal flowering cultivar. Together, these results shed light on the mechanism of flowering regulation by the PF locus and expand biotechnological approaches to achieve extended flowering in any strawberry cultivar.