Development of Single-Nucleotide Polymorphism Markers for Seed Coat Color in Watermelon

Seed coat color affects water absorption, gas diffusion, seed dormancy, seed quality, germination, and seedling appearance. These attributes of watermelon seeds thus necessitate the establishment of watermelon breeding programs that produce desired seed coat colors. However, the hereditary and molecular mechanisms underlying this feature remain unknown. Here, we aimed to identify the genomic region responsible for seed coat color in watermelon and to design single-nucleotide polymorphism (SNP) markers that are closely associated with this characteristic. We crossed two inbred lines, RG-21 (white seed coat, WSC) and Wr-609 (red seed coat, RSC), to generate F₁ hybrids (tan seed coat) and F₂ populations for locus mapping and a SNP marker analysis. Our findings indicated that the F₂ seed coat color is controlled by negative epistasis-expressing WSC. A bulked segregant analysis combined with Illumina whole-genome resequencing revealed a putative causative genomic area for WSC at 5.1–9.7 Mb on chromosome (Chr.) 6 and RSC at 3.6–7.7 Mb on Chr. 5. Based on the four-gene hypothesis, the genotypes of the WSC, RSC, and F₁ hybrids were Rtwd, rtWd, and RtWd, respectively. We then applied 19 high-resolution melting markers to 106 F₂ progeny to identify the causative genomic regions. We created a strongly co-segregated SNP marker for WSC at 6.9–7.1 Mb on Chr. 6 and RSC at 4.2–4.8 Mb on Chr. 5. Our findings serve as a basis for utilizing seed coat color in watermelon breeding programs and for expediting F₁ cultivar generation.