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

Bitter gourd (Momordica charantia) is an economically important vegetable crop with notable medicinal properties, yet its genetic improvement has been hindered by limited genomic resources and insufficient functional genomics tools. Here, we present a comprehensive genomic and functional resource platform to address these limitations. Using PacBio HiFi and Oxford Nanopore ultra-long sequencing technologies combined with Hi-C, we generated a telomere-to-telomere (T2T), gap-free genome assembly of the elite bitter gourd cultivar Y52, comprising 11 chromosomes with a final assembly size of 298.0 Mb. This high-quality assembly enabled precise annotation of complex genomic regions, including centromeres characterized through chromatin immunoprecipitation sequencing. We further constructed a large-scale ethyl methanesulfonate (EMS)-induced mutant library consisting of 3223 M₁ 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.