An intronic SNP in candidate gene ClALB3 is associated with light green coloration in watermelon

• ClALB3 is a newly identified candidate gene in watermelon that regulates leaf coloration, serving as a valuable genetic resource for studying chloroplast development and photosynthesis. • Although ClALB3 is essential for chloroplast protein transport and photosystem assembly, a C-terminal mutation located outside the conserved domain results in only a mild light-green phenotype. This suggests that this mutation may not severely compromise the protein's core function. • The enrichment of light- and stress-responsive elements in the CLALB3 promoter indicates that its transcription may be environmentally regulated, which could facilitate plant adaptation by modulating chloroplast protein assembly efficiency. Ethyl methane sulfonate (EMS) mutagenesis generates genetic variation that broadens germplasm resources for watermelon breeding. In this study, a pollen EMS-induced watermelon mutant, g42lg, exhibited a consistently light-green phenotype in leaves and fruit skin, accompanied by significantly reduced in chlorophyll a and b contents. Starch granules and thylakoid structures were also degraded. Genetic analysis indicated that this mutation is conferred by a recessive nuclear gene. A bulk segregant sequencing analysis (BSA-seq) identified ClALB3 as the candidate gene underlying the target trait. A G-to-A mutation at the first base of the ninth intron in Clalb3 was associated with a 7-bp deletion at the boundary between exons 9 and 10 in the CDS. Evolutionary conservation analyses suggests that the 60kDIMP domain of ClALB3 plays a critical role in SRP (signal recognition particle) -mediated light-harvesting protein insertion. Expression analysis revealed significant suppression of ClALB3 but concurrent up-regulation of SRP components (cpSRP54/43/FtsY) and the PSII subunit CP24 in the mutant. Collectively, our work identifies a previously unreported gene associated with the light-green phenotype. The mutant potentially serves as a valuable genetic material for studying chloroplast protein transport and assembly. Additionally, the light-green mutant provides a practical morphological marker for hybrid purity assessment of watermelon seed.