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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. |
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