Telomere-to-telomere genome assembly and a mutant library empower functional genomics and genetic improvement in Cucurbita moschata

Butternut squash (Cucurbita moschata) is an economically important crop; however, its genetic improvement has been hindered by the lack of high-resolution genomic resources and limited germplasm availability. In this study, we present a gap-free, telomere-to-telomere (T2T) genome assembly of C. moschata PKUMo, generated using high-accuracy Oxford Nanopore reads. The final assembly spans 314.34 Mb and is organized into 20 pseudomolecules, each represented by a single contig. Our analysis revealed that 40.58% of the genome consists of transposable elements, which have undergone significant expansion over the past 0.27 million years. Comparative genomic analysis with Cucurbita maxima (HZAU) identified substantial structural differences, including 27.20 Mb of inversions and 9.50 Mb of translocations, mainly affecting pericentromeric regions. We further investigated the evolution of centromeric regions in C. moschata and revealed distinct centromeric structures between PKUMo and HZAU. Notably, PKUMo centromeres exhibit increased transposon activity, particularly involving LTR retrotransposons. To facilitate functional genomics, we optimized an EMS-based pollen mutagenesis protocol, generating a mutant library comprising 60,000 M₁ seeds and 800 M₂ families, with 15.5% showing visible phenotypic variation. This library provides a valuable resource for dissecting agronomic traits and supports forward genetic approaches for identifying key genes in C. moschata. Using this T2T genome assembly, we successfully identified the causal genes Cmos16G0077000 linked to a yellow-leaf phenotype and Cmos14G0126400 associated with a miniature squash (mSq) phenotype. Overall, the PKUMo T2T genome assembly, together with the extensive mutant library, provides a robust foundation for exploring agronomic traits and accelerating genetic improvement in Cucurbita breeding programs.