Abstract
Spinach is a beloved vegetable crop and widely cultivated worldwide. It is dioecious with male and female plants, although monoecious mutations exist. Female spinach exhibits two distinct sepal morphologies-thorn-shaped and round-shaped determine seed shape and strongly influence seed handling, mechanized sowing, and cultivar classification. To dissect the genetic basis of this trait, we developed an F2 population from contrasting parental lines and constructed a high-density linkage map with ~1615 bin markers spanning ~994.04 cm. A major 4.31 Mb genomic interval on chromosome 4, flanked by tightly linked markers, was consistently associated with sepal morphology. Transcriptome profiling across early and mature sepal developmental stages revealed significant enrichment of cell cycle-related pathways, including DNA replication, repair, mitosis, and cytokinesis. By integrating differential expression analysis with weighted gene co-expression network analysis, we identified 25 DEGs within the mapped interval, 11 of which showed strong co-expression with hub genes in trait-associated modules. Structural variation analysis further uncovered promoter and coding sequence polymorphisms in a subset of candidate genes. This study highlights 11 promising candidate genes potentially regulating sepal-derived seed morphology in spinach, rather than confirming definitive causal genes, providing valuable targets for functional validation and new insights into the genetic regulation of sepal development.