Integrative k-mer and Transcriptomic Analysis Reveals Putative Sex-Determining Genes in Spinacia turkestanica

Deciphering the regulation of sex-determining gene/s in dioecious crops is crucial for molecular breeding. However, the identification of sex-determining gene/s in Spinacia is challenging due to incomplete genome assemblies, high genomic similarity between males and females, and the limitations of transcriptome-only analyses, which may miss unannotated or novel genes in poorly assembled or absent genomic regions. To overcome these limitations, we employed a reference-genome-free k-mer approach to identify candidate sex-determining genes in Spinacia turkestanica , the closest evolutionary relative of cultivated spinach ( S. oleracea ). Male-specific reads were de novo assembled into contigs, revealing 21.5 Mb of the sex-determining region (SDR). Using the MAKER pipeline, which integrates transcriptomic and proteomic evidence, we predicted 226 protein-coding genes within the SDR, including nine previously unannotated. Transcriptomic profiling combined with weighted gene co-expression network analysis identified eight SDR DEGs, including two newly annotated genes, co-expressing during early male flower development. qPCR validation confirmed three SDR genes as candidate sex-determining factors, including TU_SDR00087 (bZIP domain), TU_SDR000168 (RNA-binding splicing factor domain), and TU_SDR000174 (MYB domain). Together, these findings provide a foundation for functional characterization of sex determination in Spinacia .