Cucumis sativus L., commonly known as cucumber, is an important vegetable crop worldwide, with China as the largest producer, particularly of the North and South China types. While extensive genomic research has focused on the North China type, especially the Chinese Long 9930, studies on the South China type remain limited. In this study, we assembled high-quality genomes of two widely cultivated and representative parent varieties: S36 (North China type) and H19 (South China type), and conducted mutagenesis analyses. Comparative genome analysis revealed a large number of structural variants between two North China types and two South China types, with many of the affected genes showing strong homology to known functional loci, potentially contributing to phenotypic divergence. We also constructed an EMS mutant library through the mutagenesis of S36 and identified a gene that encodes chlorophyll oxidase, demonstrating the method’s effectiveness for rapid gene discovery. In conclusion, this study provides valuable insights into the classification and evolution of cucumber, highlighting the promising potential of forward genetic approaches in cucumber breeding.

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