A single nucleotide mutation in ClphyB gene is associated with a short lateral branch phenotype in watermelon

The plant branching habit is a unique morphological trait of watermelon that regulates the plant architecture as well as crop yield, but the in-depth understanding of molecular regulation of the branching habit in watermelon is less studied. In this experiment, two contrasted watermelon lines with different phenotypes “a normal branching line (GWAS-38) and a mutant short lateral branched (slb) line” were crossed to derive the bi-parental F2 mapping populations. Genetic segregation analysis of collected phenotypic data depicted a good-fit 3:1 segregating ratio, which indicated that the slb locus in the mapping population was regulated by one recessive gene. A bulk segregant sequencing analysis (BSA-seq) method and genetic linkage mapping with F2 mapping populations (336 plants) preliminary identified the 1.63 Mbp region on chromosome 5. Fine genetic mapping with an expanded F2 mapping population (1020 plants) narrowed down the candidate slb locus to a 25.355 kb interval with two predicted genes. The sequence variations indicated a 1 bp deletion (A→-) causing a truncated protein lacking the conserved domains for the phytochrome PAS2 structural domain and HATPase structural domain in the mutant slb line, which was also co-segregated with the short lateral branched phenotype. The haplotype analysis and gene expression analysis suggested that the Cla97C05G088180.1 gene encoding phytochrome B, is the major gene regulating the phenotype of short lateral branched trait in mutant slb line. Further, CRISPR/Cas9-mediated mutation of CsphyB led to hypocotyl elongation, reduced leaf angle, and inhibited branch elongation, suggesting that CsphyB is mainly responsible for regulation of branch elongation in cucumber plants. To the best of our knowledge, we first time reported the vital molecular characterization and cloning of the ClphyB gene regulating branch elongation in watermelon. We believe that our results would provide beneficial genetic insights for understanding the possible roles of ClphyB in the architecture watermelon plant.