CRISPR-Cas9-mediated mutagenesis of the flowering repressor gene VcCENTRORADIALIS (VcCEN) induces early flowering in tetraploid highbush blueberry

Flowering marks the vegetative-to-reproductive growth transition and is the most important event in the plant life cycle. Unlike annual plants, perennial fruit trees flower and set fruits only after an extended juvenile phase (i.e., several years), which is an impediment to efficient breeding and gene function analyses. In this study, we generated an early flowering blueberry line via the CRISPR-Cas9-mediated mutagenesis of VcCENTRORADIALIS ( VcCEN ). The expression of VcCEN in the apical bud was negatively correlated with flower bud formation. Moreover, in the cultivar that flowers in both autumn and spring, the VcCEN expression level was lower and decreased earlier than in the normal cultivar that flowers in only spring. The expression data suggested that VcCEN functions as a flowering repressor. The CRISPR-Cas9 vector harboring a gRNA targeting VcCEN was introduced into the blueberry genome via Agrobacterium-mediated transformation. Mutations (e.g., 1–10 bp indels) were detected in the stable transformants, with all VcCEN alleles of the tetraploid genome mutated in some lines. Compared with the wild-type (WT), the cen mutants exhibited repressed vegetative growth. Additionally, in the mutants, first flowering occurred within 1 year after the Agrobacterium infection, which was approximately 1–2 years earlier than in WT. The mutants set a single terminal flower without entering dormancy, whereas WT produced an apical flower and multiple axillary flowers that bloomed after an exposure to chilling conditions and then warm temperatures. This early flowering trait is conducive to efficient breeding and gene functional analyses, especially in fruit crops with a long juvenile phase.