Telomere-to-telomere genome assembly and comprehensive mutation library facilitate bitter gourd breeding and functional genomics research

plants and performed whole-genome resequencing of 320 mutants, identifying approximately 76 002 induced mutations affecting 13 984 genes. Using this mutant population, we rapidly identified the causal gene McEGY1, responsible for chlorotic leaf and fruit phenotypes, through a MutMap-based approach. In addition, we conducted bulked segregant analysis and bulked segregant RNA sequencing to identify genomic regions and candidate genes associated with key agronomic traits, including fruit length, fruit tubercle formation, and powdery mildew resistance. Moreover, integrated transcriptomic and metabolomic analyses across multiple tissues and developmental stages generated comprehensive profiles of gene expression and metabolite accumulation, revealing tissue-specific regulatory networks. Finally, we integrated the genomic, mutant, transcriptomic, and metabolomic resources generated in this study to establish Bittergourd DB, a comprehensive platform supporting bitter gourd functional genomics and molecular breeding. Collectively, this study provides an unprecedented genomic and functional resource for bitter gourd, including a complete T2T reference genome, an extensive EMS mutant library, trait-associated loci, and multi-omics maps, which will substantially accelerate functional genomics research and molecular breeding in this species.