Lettuce, an important member of the Asteraceae family, is a globally cultivated cash vegetable crop. With a highly complex genome (~2.5 Gb; 2n = 18) rich in repeat sequences, current lettuce reference genomes exhibit thousands of gaps, impeding a comprehensive understanding of the lettuce genome. Here, we present a near-complete gapless reference genome for cutting lettuce with high transformability, comprising 7 telomere-to-telomere and 2 near-complete pseudo-chromosomes, using high-coverage and long-read PacBio HiFi and Nanopore sequencing data. By further using the RNA-seq-based transcriptomics data, whole-genome DNA methylation data, and Nanopore long-read direct RNA sequencing data, we construct genome annotations, detect structural variations (SVs) between cutting and stem lettuce, and explore the genomic and epigenetic features of SVs and WGT genes after diploidization. Interestingly, we identify 127,681 hitherto unknown SVs related to transposons and DNA methylation states, revealing for the first time the major divergence of leafy and stem lettuce. We also find that 4,706 retained whole-genome triplication genes exhibit high expression levels, a characteristic associated with low DNA methylation levels and high N6-methyladenosine (m6A) RNA modifications. DNA methylation changes are also associated with activation of genes involved in callus formation. Overall, our study reports the first near-complete gapless genome of lettuce, representing the highest completeness and assembly quality for a plant species in the Asteraceae family to date. This gapless lettuce reference genome will serve as a cornerstone in functional genomics and breeding, and the revealed epigenetic features of SVs and retained WGT genes signify a major step forward in understanding the complexity of transcriptional and post-transcriptional regulations associated with the dynamics of DNA and RNA epigenetics during genome evolution. Importantly, the cutting lettuce is easily transformed, providing a solid foundation for functional genomics study and breeding.
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