Epistatic interactions among multiple copies of FLC genes with naturally occurring insertions correlate with flowering time variation in radish

Brassicaceae crops, which underwent whole-genome triplication during their evolution, have multiple copies of flowering-related genes. Interactions among multiple gene copies may be involved in flowering time regulation; however, this mechanism is poorly understood. In this study, we performed comprehensive, high-throughput RNA sequencing analysis to identify candidate genes involved in the extremely late-bolting (LB) trait in radish. Then, we examined the regulatory roles and interactions of radish FLOWERING LOCUS C (RsFLC) paralogs, the main flowering repressor candidates. Seven flowering integrator genes, five vernalization genes, nine photoperiodic/circadian clock genes and eight genes from other flowering pathways were differentially expressed in the early-bolting (EB) cultivar 'Aokubinagafuto' and LB radish cultivar 'Tokinashi' under different vernalization conditions. In the LB cultivar, RsFLC1 and RsFLC2 expression levels were maintained after 40 days of cold exposure. Bolting time was significantly correlated with the expression rates of RsFLC1 and RsFLC2. Using the EB × LB F2 population, we performed association analyses of genotypes with or without 1910- and 1627-bp insertions in the first introns of RsFLC1 and RsFLC2, respectively. The insertion alleles prevented the repression of their respective FLC genes under cold conditions. Interestingly, genotypes homozygous for RsFLC2 insertion alleles maintained high RsFLC1 and RsFLC3 expression levels under cold conditions, and two-way analysis of variance revealed that RsFLC1 and RsFLC3 expression was influenced by the RsFLC2 genotype. Our results indicate that insertions in the first introns of RsFLC1 and RsFLC2 contribute to the late-flowering trait in radish via different mechanisms. The RsFLC2 insertion allele conferred a strong delay in bolting by inhibiting the repression of all three RsFLC genes, suggesting that radish flowering time is determined by epistatic interactions among multiple FLC gene copies.