LAUSR

Abstract Fruit color is an important horticultural trait, which greatly affects consumer preferences. In tomato, fruit color is determined by the accumulation of different pigments, such as carotenoids in the pericarp and flavonoids in the peel, along with the degradation of chlorophyll during fruit ripening. Since fruit color is a multigenic trait, it takes years to introgress all color-related genes in a single genetic background via traditional crossbreeding, and the avoidance of linkage drag during this process is difficult. Here, we proposed a rapid breeding strategy to generate tomato lines with different colored fruits from red-fruited materials by CRISPR/Cas9-mediated multiplex gene editing of three fruit color-related genes (PSY1, MYB12, and SGR1). Using this strategy, the red-fruited cultivar ‘Ailsa Craig’ has been engineered to a series of tomato genotypes with different fruit colors, including yellow, brown, pink, light-yellow, pink-brown, yellow-green, and light green. Compared with traditional crossbreeding, this strategy requires less time and can obtain transgene-free plants with different colored fruits in less than 1 year. Most importantly, it does not alter other important agronomic traits, like yield and fruit quality. Our strategy has great practical potential for tomato breeding and serves as a reference for improving multigene-controlled traits of horticultural crops.