The knockout of ClaCSLH1 induced dwarfing in watermelon

Abstract In agriculture and horticulture, selecting ideal plant types with desirable traits, such as dwarfing and upright stem structures, significantly enhances crop yield and quality by optimizing light absorption, spatial efficiency, and nutrient utilization. Developing new varieties of dwarf watermelon is a crucial objective in watermelon breeding. In this study, we constructed an F2 generation population by using the wild type V063 as the paternal parent and the dwarf variety dw-n as the maternal parent. The dwarfing trait was found to be recessive, governed by a pair of recessive alleles. Through Bulk Segregant Analysis sequencing (BSA-seq) and RNA sequencing (RNA-seq), we identified the gene Cla97C02G035450, which encodes for cellulose synthase-like H1 (CSLH1), as a candidate gene associated with the dwarfing phenotype. ClaCLSH1 belongs to the ClaCESA/CSLs family, which is involved in the synthesis of cell walls by regulating the synthesis of cellulose and hemicellulose. Microscopic analyses, revealed that dw-n plants exhibited shorter internode cells, thicker cell walls, and elevated hemicellulose content compared to V063. Subcellular localization studies demonstrated that the CLACSLH1 protein is primarily localized in the nucleus and the cell membrane/wall. Notably, the overexpression of CLACSLH1 in the dw-n background was able to rescue its dwarf phenotype. Furthermore, experiments indicated that knockdown of CLACSLH1 resulted in excessive hemicellulose synthesis, inhibited internode cell elongation, and ultimately led to the stunted phenotype observed in dw-n. This research provides innovative insights into the development of superior dwarf watermelon varieties and advances our understanding of the molecular mechanisms underlying watermelon dwarfism.