Watermelon, Citrullus lanatus, is the world’s third largest fruit crop. Reference genomes with gaps and a narrow genetic base hinder functional genomics and genetic improvement of watermelon. Here, we report the assembly of a telomere-to-telomere gap-free genome of the elite watermelon inbred line G42 by incorporating high-coverage and accurate long-read sequencing data with multiple assembly strategies. All 11 chromosomes have been assembled into single-contig pseudomolecules without gaps, representing the highest completeness and assembly quality to date. The G42 reference genome is 369 321 829 bp in length and contains 24 205 predicted protein-coding genes, with all 22 telomeres and 11 centromeres characterized. Furthermore, we established a pollen-EMS mutagenesis protocol and obtained over 200 000 M1 seeds from G42 . In a sampling pool, 48 monogenic phenotypic mutations, selected from 223 M1 and 78 M2 mutants with morphological changes, were confirmed. The average mutation density was 1 SNP/1.69 Mb and 1 indel/4.55 Mb per M1 plant and 1 SNP/1.08 Mb and 1 indel/6.25 Mb per M2 plant. Taking advantage of the gap-free G42 genome, 8039 mutations from 32 plants sampled from M1 and M2 families were identified with 100% accuracy, whereas only 25% of the randomly selected mutations identified using the 97103v2 reference genome could be confirmed. Using this library and the gap-free genome, two genes responsible for elongated fruit shape and male sterility (ClMS1) were identified, both caused by a single base change from G to A. The validated gap-free genome and its EMS mutation library provide invaluable resources for functional genomics and genetic improvement of watermelon.

西瓜、西瓜，是世界第三大水果作物。具有空白和狭窄遗传基础的参考基因组阻碍了西瓜的功能基因组学和遗传改良。在这里，我们通过将高覆盖率和准确的长读长测序数据与多种组装策略相结合，报告了优良西瓜近交系 G42 的端粒到端粒无间隙基因组的组装。11条染色体全部组装成单重叠群假分子，没有间隙，代表了迄今为止最高的完整性和组装质量。G42 参考基因组长度为 369 321 829 bp，包含 24 205 个预测的蛋白质编码基因，具有所有 22 个端粒和 11 个着丝粒的特征。此外，我们建立了花粉-EMS 诱变方案，从 G42 获得了超过 200 000 M1 种子。在采样池中，48 个单基因表型突变，从 223 个 M1 和 78 个具有形态变化的 M2 突变体中选出。平均突变密度为每 M1 植物 1 SNP/1.69 Mb 和 1 indel/4.55 Mb，每 M2 植物 1 SNP/1.08 Mb 和 1 indel/6.25 Mb。利用无间隙的 G42 基因组，来自 M1 和 M2 家族的 32 株植物的 8039 个突变被鉴定为 100% 准确率，而使用 97103v2 参考基因组鉴定的随机选择突变中只有 25% 可以被确认。使用这个库和无间隙基因组，两个负责拉长果实形状和雄性不育的基因（来自 M1 和 M2 家族的 32 株植物的 8039 个突变以 100% 的准确率被鉴定，而使用 97103v2 参考基因组鉴定的随机选择的突变中只有 25% 可以被确认。使用这个库和无间隙基因组，两个负责拉长果实形状和雄性不育的基因（来自 M1 和 M2 家族的 32 株植物的 8039 个突变以 100% 的准确率被鉴定，而使用 97103v2 参考基因组鉴定的随机选择的突变中只有 25% 可以被确认。使用这个库和无间隙基因组，两个负责拉长果实形状和雄性不育的基因（ClMS1 ) 被鉴定，两者都是由从 G 到 A 的单个碱基变化引起的。经过验证的无间隙基因组及其 EMS 突变库为西瓜的功能基因组学和遗传改良提供了宝贵的资源。