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Lettuce (Lactuca sativaL.) is a leafy vegetable crop with ongoing breeding efforts related to quality, resilience, and innovative production systems. Genetic variation of important traits in close relatives is necessary to meet lettuce breeding goals.Lactuca virosa(2x=2n=18), a wild relative assigned to the tertiary lettuce gene pool, has a much larger genome (3.7 Gbp) thanLactuca sativa(2.5 Gbp). It has been used in interspecific crosses and is a donor to modern crisphead lettuce cultivars. Here, we present ade novoreference assembly ofL. virosawith high continuity and complete gene space. This assembly facilitated comparisons to the genome ofL. sativaand to that of the wild speciesL. saligna, a representative of the secondary lettuce gene pool. To assess the diversity in gene content, we classified the genes of the threeLactucaspecies as core, accessory and unique. In addition, we identified three interspecific chromosomal inversions compared toL. sativa, which each may cause recombination suppression and thus hamper future introgression breeding. Using three-way comparisons in both reference-based and reference-free manners, we show that the proliferation of long-terminal repeat elements has driven the genome expansion ofL. virosa. Further, we performed a genome-wide comparison of immune genes, nucleotide-binding leucine-rich repeat, and receptor-like kinases amongLactucaspp. and indicate the evolutionary patterns and mechanisms behind their expansions. These genome analyses greatly facilitate the understanding of genetic variation inL. virosa, which is beneficial for the breeding of improved lettuce varieties. |