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Key message: The mutated LsTT2 and Ls2OGD genes are responsible for white seeds and yellow seeds in lettuce, respectively. Three LsCHS genes are involved in the biosynthesis of flavonoid in seed coats. Lettuce seeds have several different colors, including black, yellow, and white. The genetic mechanisms underlying color variations of lettuce seeds remain unknown. We used genome-wide association studies (GWAS) and map-based cloning approaches to clone genes controlling the color of lettuce seeds. LsTT2, which encodes an R2R3-MYB transcription factor and is homologous to the TT2 gene in Arabidopsis, was shown to be the causal gene for the variation of black and white seeds in lettuce. A point mutation leads to the lack of stop codon in the LsTT2 transcript, resulting in white seeds. Knockout of the LsTT2 gene converted black seeds to white seeds. The locus controlling yellow seeds was mapped to Chromosome 2. Knockout of two 2-oxoglutarate-dependent dioxygenases (2OGD) genes from the candidate region converted black seeds to yellow seeds, suggesting that these two 2OGD proteins catalyze the conversion of yellow metabolites to black metabolites. We also showed that three LsCHS genes from the candidate region are associated with flavonoid biosynthesis in seeds. Knockout mutants of the three LsCHS genes decreased color intensity. This study provides new insights into the regulation of flavonoid biosynthesis in plants. [ABSTRACT FROM AUTHOR]

Summary: Anthocyanins protect plants from biotic and abiotic stressors and provide great health benefits to consumers. In this study, we cloned four genes (Red Lettuce Leaves 1 to 4: RLL1 to RLL4) that contribute to colour variations in lettuce. The RLL1 gene encodes a bHLH transcription factor, and a 5‐bp deletion in some cultivars abolishes its function to activate the anthocyanin biosynthesis pathway. The RLL2 gene encodes an R2R3‐MYB transcription factor, which was derived from a duplication followed by mutations in its promoter region. The RLL3 gene encodes an R2‐MYB transcription factor, which down‐regulates anthocyanin biosynthesis through competing with RLL2 for interaction with RLL1; a mis‐sense mutation compromises the capacity of RLL3 to bind RLL1. The RLL4 gene encodes a WD‐40 transcription factor, homologous to the RUP genes suppressing the UV‐B signal transduction pathway in Arabidopsis; a mis‐sense mutation in rll4 attenuates its suppressing function, leading to a high concentration of anthocyanins. Sequence analysis of the RLL1‐RLL4 genes from wild and cultivated lettuce showed that their function‐changing mutations occurred after domestication. The mutations in rll1 disrupt anthocyanin biosynthesis, while the mutations in RLL2, rll3 and rll4 activate anthocyanin biosynthesis, showing disruptive selection for leaf colour during domestication of lettuce. The characterization of multiple polymorphic genes in this study provides the necessary molecular resources for the rational breeding of lettuce cultivars with distinct levels of red pigments and green cultivars with high levels of health‐promoting flavonoids. [ABSTRACT FROM AUTHOR]