The dwarf architecture is an important and valuable agronomic trait in watermelon breeding and has the potential to increase fruit yield and reduce labor cost in crop cultivation. However, the molecular basis for dwarfism in watermelon remains largely unknown. In this study, a recessive dwarf allele (designated as Cldf (Citrullus lanatus dwarfism)) was fine mapped in a 32.88 kb region on chromosome 09 using F₂ segregation populations derived from reciprocal crossing of a normal line M08 and a dwarf line N21. Gene annotation of the corresponding region revealed that the Cla015407 gene encoding a gibberellin 3β-hydroxylase functions as the best possible candidate gene for Cldf. Sequence analysis showed that the fourth polymorphism site (a G to A point mutation) at the 3′ AG splice receptor site of the intron leads to a 13 bp deletion in the coding sequence of Cldf in dwarf line N21 and thus results in a truncated protein lacking the conserved domain for binding 2-oxoglutarate. In addition, the dwarf phenotype of Cldf could be rescued by exogenous GA₃ application. Phylogenetic analysis suggested that the small multigene family GA3ox (GA3 oxidase) in cucurbit species may originate from three ancient lineages in Cucurbitaceae. All these data support the conclusion that Cldf is a GA-deficient mutant, which together with the cosegregated marker can be used for breeding new dwarf cultivars.

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