The lemon (Citrus limon; family Rutaceae) is one of the most important and popular fruits worldwide. Lemon also tolerates huanglongbing (HLB) disease, which is a devastating citrus disease. Here we produced a gap-free and haplotype-resolved chromosome-scale genome assembly of the lemon by combining Pacific Biosciences circular consensus sequencing, Oxford Nanopore 50-kb ultra-long, and high-throughput chromatin conformation capture technologies. The assembly contained nine-pair chromosomes with a contig N50 of 35.6 Mb and zero gaps, while a total of 633.0 Mb genomic sequences were generated. The origination analysis identified 338.5 Mb genomic sequences originating from citron (53.5%), 147.4 Mb from mandarin (23.3%), and 147.1 Mb from pummelo (23.2%). The genome included 30 528 protein-coding genes, and most of the assembled sequences were found to be repetitive sequences. Several significantly expanded gene families were associated with plant–pathogen interactions, plant hormone signal transduction, and the biosynthesis of major active components, such as terpenoids and flavor compounds. Most HLB-tolerant genes were expanded in the lemon genome, such as 2-oxoglutarate (2OG)/Fe(II)-dependent oxygenase and constitutive disease resistance 1, cell wall-related genes, and lignin synthesis genes. Comparative transcriptomic analysis showed that phloem regeneration and lower levels of phloem plugging are the elements that contribute to HLB tolerance in lemon. Our results provide insight into lemon genome evolution, active component biosynthesis, and genes associated with HLB tolerance.

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