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

Butternut squash (Cucurbita moschata) is an economically important crop; yet its genetic improvement has been hindered by the absence of high-resolution genomic resources and limited germplasm availability. In this study, we assembled a gap-free, telomere-to-telomere genome of Cucurbita moschata PKUMo using high accuracy Oxford Nanopore Technology reads, resulting in a genome assembly of 314.34 Mb, organized into 20 pseudomolecules represented by single contigs. Our analysis revealed that 40.58% of the genome comprises transposable elements, which have undergone significant expansion in the last 0.27 million years. Further, 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. Additionally, our study investigates the evolution of centromeric regions in C. moschata, revealing distinct centromeric structures that differ significantly between PKUMo and HZAU. The centromeric regions of PKUMo demonstrate increased transposon activity, particularly involving LTR retrotransposons. To enhance functional genomics, we optimized a pollen mutagenesis protocol using EMS, generating a mutant library of 60,000 M₁ seeds and 800 M₂ families, demonstrating 15.5% visible phenotypic variation. This library serves as a valuable resource for dissecting agronomic traits and supports forward genetics approaches for identifying critical genes in C. moschata. Utilizing this T2T genome, 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 and extensive mutant library establish a robust foundation for exploring agronomic traits and accelerating genetic improvement in Cucurbita breeding programs.