Lettuce (Lactuca sativa L.) is among the most widely cultivated and consumed vegetables globally, valued for its phytonutrients beneficial for human health. Here, we report the high-quality reference super-pangenome for the Lactuca genus by integrating 12 chromosome-scale genomes from representative cultivated lettuce morphotypes, landrace, and wild relatives, and investigate lettuce genome evolution and domestication. These assemblies exhibit diverse genome sizes ranging from 2.1 Gb to 5.5 Gb with abundant repetitive sequences, and expansion of repetitive sequences, associated with low DNA methylation levels, likely contributes to the genome size variations. Furthermore, by constructing a graph-based lettuce pangenome reference, we explore the landscape, genomic, and epigenetic features of structural variations, and reveal their contributions to gene expression and domestication. We also identify copy number variations in FLOWERING LOCUS C in association with delayed flowering in cultivated lettuce. Overall, this comprehensive Lactuca super-pangenome assembly will expedite functional genomics studies and breeding efforts of this globally important crop.
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