Spinach (Spinacia oleracea L.) is a dioecious, diploid, wind-pollinated crop cultivated worldwide. Sex determination plays an important role in spinach breeding. Hence, this study aimed to understand the differences in sexual differentiation and floral organ development of dioecious flowers, as well as the differences in the regulatory mechanisms of floral organ development of dioecious and monoecious flowers. We compared transcriptional-level differences between different genders and identified differentially expressed genes (DEGs) related to spinach floral development, as well as sex-biased genes to investigate the flower development mechanisms in spinach. In this study, 9189 DEGs were identified among the different genders. DEG analysis showed the participation of four main transcription factor families, MIKC_MADS, MYB, NAC, and bHLH, in spinach flower development. In our key findings, abscisic acid (ABA) and gibberellic acid (GA) signal transduction pathways play major roles in male flower development, while auxin regulates both male and female flower development. By constructing a gene regulatory network (GRN) for floral organ development, core transcription factors (TFs) controlling organ initiation and growth were discovered. This analysis of the development of female, male, and monoecious flowers in spinach provides new insights into the molecular mechanisms of floral organ development and sexual differentiation in dioecious and monoecious plants in spinach.

Vegetative to reproductive phase transition is an important developmental process in flowering plants, regulated in part by microRNAs that repress target genes post-transcriptionally. However, the role of miR156 and its target Squamosa Promoter Binding Protein-Like (SPL) transcription factors remains poorly understood. In this study, we identified 14 SPL gene members in spinach and analyzed their expression during phase transition. Genome-wide identification and transcriptome-based analysis revealed that 11 of these genes are likely direct targets of SpmiR156a/b/c/d, with binding sites confirmed by sequence-based interaction prediction. Expression profiling showed that SpSPL3 and SpSPL8, which are strongly repressed during vegetative growth, were significantly regulated during the transition phase. Gene Ontology (GO) and promoter cis-element analyses support that SPL genes are involved in hormonal pathways and floral development Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) further validated the transcriptome expression patterns of key SPL genes. Together, these findings outline a regulatory framework in which SpmiR156 modulates SpSPL gene activity to control developmental phase change in spinach, highlighting both the expansion and functional diversification of the SPL gene and the central role of SpmiR156 in vegetative to reproductive transition.

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.