Multi-omics analyses unveil dual genetic loci governing four distinct watermelon flesh color phenotypes

Watermelon fruit flesh displays various colors. Although genetic loci underlying these variations are identified, the molecular mechanism remains elusive. Here, we assembled a chromosome-scale reference genome of an elite watermelon and developed integrated genetic maps using single nucleotide polymorphism (SNP) and structural variation markers. Several key genetic varients for fruit shape and flesh color were identified. Two variants associated with flesh color were further studied, including one copy number variant (CNV, a triplicate of 1.2 kb DNA) in the promoter region of REDUCED CHLOROPLAST COVERAGE 2 (ClREC2) and one SNP in Lycopene β-Cyclase (ClLCYB) coding region. These two variants together explained 99.7% of the flesh color variations in 314 watermelon accessions. The SNP in ClLCYB was the same as previously reported, disrupting ClLCYB function. The CNV could strongly enhance ClREC2 expression, consequently increasing the expression of carotenoid biosynthesis genes, the number of plastoglobules within chromoplasts, and carotenoid level in mature fruit flesh. Finally, we proposed a "two-switch" genetic model by integrating two major causative loci, which can explain the formation of the four main flesh colors in different watermelon accessions. These results provide new insights into the regulation of carotenoid biosynthesis and color formation in plants.