Blueberry (Vaccinium spp.) is one of the most economically important woody plant species because its fruit is rich in antioxidants such as anthocyanin, which offer beneficial effects on human health. Thus, the demand for blueberry production and for the development of novel elite cultivars has been continuously increasing. Although new blueberry cultivars have been developed by cross breeding for more than a century, the conventional breeding approaches are time-consuming and labor-intensive due to the associated characteristics that hinder efficient breeding such as long juvenile phase, polyploidy, and heterozygosity. Genetic engineering offers a promising approach to confer a desirable trait to elite cultivars. In particular, the recently developed genome editing technology enables precise modifications of plant genomes. We aim to apply genome editing to improve agronomically important traits in blueberry, specifically anthocyanin and sugar content. Towards the development of high anthocyanin blueberry, we targeted an anthocyanin repressor gene, VcMYBC2. So far, we successfully obtained 3 lines with all-alleles of VcMYBC2 were mutated. An increase in anthocyanin content in fruit is expected in the mybc2 mutants. Regarding sugar content, we targeted invertase inhibitor (INVINH) gene that is involved in sugar metabolism. Invertase promotes sucrose unloading in the fruit by maintaining a gradient of sucrose concentration between source leaves and fruits, while INVINH represses this process. Thus, higher sugar accumulation is expected in invinh mutants. Three transgenic lines harboring CRISPR-Cas9 vectors targeting VcINVINH genes that are highly expressed in fruit tissue, were obtained. The mutated allele frequencies of the mutants ranged from 21-67%. Additionally, we generated transgenic blueberries overexpressing FLOWERING LOCUS T (FT), a mobile florigen signal gene that induces flowering. A previous study demonstrated that blueberry scions grafted to FT-overexpressing lines could show early flowering. We thus assume that the mybc2 and invinh mutants may flower earlier when they are grafted onto the FT overexpressing rootstock, which will accelerate our fruit phenotype evaluations. The FT-overexpressing rootstock may also be utilized to facilitate the production of null-segregant mutants.
