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 differentially expressed genes, including two newly annotated genes, co-expressed during early male flower development. Quantitative PCR validation confirmed three SDR genes as candidate sex-determining factors, namely TU_SDR00087 (bZIP domain transcription factor), TU_SDR000168 (RNA-binding splicing factor domain), and TU_SDR000174 (MYB domain transcription factor). Together, these findings provide a foundation for functional characterization of sex determination in Spinacia.