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Abstract Background Breeding of lettuce (Lactuca sativa L.), the most important leafy vegetable worldwide, for enhanced disease resistance and resilience relies on multiple wild relatives to provide the necessary genetic diversity. In this study, we constructed a super-pangenome based on four Lactuca species (representing the primary, secondary and tertiary gene pools) and comprising 474 accessions. We include 68 newly sequenced accessions to improve cultivar coverage and add important foundational breeding lines. Results With the super-pangenome we find substantial presence/absence variation (PAV) and copy-number variation (CNV). Functional enrichment analyses of core and variable genes show that transcriptional regulators are conserved whereas disease resistance genes are variable. PAV-genome-wide association studies (GWAS) and CNV-GWAS are largely congruent with single-nucleotide polymorphism (SNP)-GWAS. Importantly, they also identify several major novel quantitative trait loci (QTL) for resistance against Bremia lactucae in variable regions not present in the reference lettuce genome. The usability of the super-pangenome is demonstrated by identifying the likely origin of non-reference resistance loci from the wild relatives Lactuca serriola, Lactuca saligna and Lactuca virosa. Conclusions The super-pangenome offers a broader view on the gene repertoire of lettuce, revealing relevant loci that are not in the reference genome(s). The provided methodology and data provide a strong basis for research into PAVs, CNVs and other variation underlying important biological traits of lettuce and other crops.

Abstract Background Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a congenital malformation of multifactorial etiology. Research has identified >40 genome‐wide significant risk loci, which explain less than 40% of nsCL/P heritability. Studies show that some of the hidden heritability is explained by rare penetrant variants. Methods To identify new candidate genes, we searched for highly penetrant de novo variants (DNVs) in 50 nsCL/P patient/parent‐trios with a low polygenic risk for the phenotype (discovery). We prioritized DNV‐carrying candidate genes from the discovery for resequencing in independent cohorts of 1010 nsCL/P patients of diverse ethnicities and 1574 population‐matched controls (replication). Segregation analyses and rare variant association in the replication cohort, in combination with additional data (genome‐wide association data, expression, protein–protein‐interactions), were used for final prioritization. Conclusion In the discovery step, 60 DNVs were identified in 60 genes, including a variant in the established nsCL/P risk gene CDH1. Re‐sequencing of 32 prioritized genes led to the identification of 373 rare, likely pathogenic variants. Finally, MDN1 and PAXIP1 were prioritized as top candidates. Our findings demonstrate that DNV detection, including polygenic risk score analysis, is a powerful tool for identifying nsCL/P candidate genes, which can also be applied to other multifactorial congenital malformations.