Development and application of a 40K liquid chip enriched with structural variation markers for watermelon breeding

Watermelon (Citrullus lanatus) has undergone severe loss of genetic diversity due to domestication, necessitating the exploitation of wild relatives for trait improvement. While structural variations (SVs) represent a major source of genetic diversity, high-throughput SV genotyping remains technically challenging, and existing single-nucleotide polymorphism (SNP) arrays fail to capture presence/absence variations (PAVs) that differentiate wild from cultivated germplasm. Here, we developed the 'Watermelon 40K SNP-SV Chip,' the first liquid-phase genotyping chip enriched with SV markers, leveraging a telomere-to-telomere super-pangenome of seven Citrullus species. The chip comprises 7087 SV markers (enriched for PAVs) and 11 383 SNP/InDel markers. Technical validation in 12 accessions and large-scale genotyping of 228 diverse accessions demonstrated high reproducibility (≥98.5% concordance) and robust call rates (>97%), though performance declined in deeply divergent wild species. Genome-wide association studies revealed SV-specific signals for flesh color and rind pattern that co-localized with known quantitative trait loci (QTL), including a strong candidate regulatory PAV upstream of LCYB. The predominance of rare variants (74.3% SVs with minor allele frequency < 0.05) limits statistical power for detecting QTL underlying polygenic domestication traits such as yield but confers exceptional utility for tracking wild introgressions in early-generation breeding populations. The 40K SNP-SV chip bridges the gap between fundamental SV research and practical breeding, enabling SV-assisted genomic selection for watermelon improvement.