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

Bitter gourd (Momordica charantia L.), an economically vital vegetable crop with significant medicinal attributes, faces bottlenecks in genetic improvement due to scarce genomic resources and limited functional genomics tools. Here, we present a comprehensive genomic and functional resource platform to overcome these limitations. Utilizing PacBio HiFi and ONT ultra-long sequencing technologies combined with Hi-C, we achieved 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 accurate annotation of complex genomic regions, including centromeres characterized using chromatin immunoprecipitation (ChIP-seq). We constructed a large-scale EMS-induced mutant library consisting of 3,223 M₁ plants and performed whole-genome resequencing on 320 mutants, identifying approximately 76,002 induced mutations affecting 13,984 genes. Leveraging this mutant population, we rapidly isolated the causal gene McEGY1 responsible for chlorotic leaf and fruit phenotypes using a MutMap-based method. Furthermore, we applied Bulked Segregant Analysis (BSA) and RNA-seq (BSR) to identify genomic regions and candidate genes underlying critical agronomic traits, including fruit length, fruit tubercle formation and powdery mildew resistance. Additionally, integrated transcriptomic and metabolomic analyses across multiple tissues and developmental stages generated comprehensive gene expression and metabolite accumulation profiles, elucidating tissue-specific regulatory networks. Finally, we integrated the genomic, mutant, transcriptomic, and metabolomic resources generated in this study to build Bittergourd DB (http://bittergourddb.omicsplant.cn/), a comprehensive platform to support bitter gourd functional genomics and molecular breeding. Collectively, our study provides an unprecedented genomic and functional resource, including a complete T2T reference genome, extensive EMS mutant library, trait-associated loci, and multi-omics maps, significantly accelerating bitter gourd functional genomics research and molecular breeding efforts.