Characterization of the CHTBD1 gene family in Spinacia oleracea  reveals candidate genes for downy mildew resistance

Spinach (Spinacia oleracea) is a globally cultivated leafy vegetable, and its yield is severely threatened by downy mildew (Peronospora effusa), yet the molecular basis of resistance remains poorly understood. Chitin-binding type 1 (CHTBD1) proteins play critical roles in pathogen recognition and immune activation across plant species. However, their genomic organization and potential involvement in disease resistance have not been explored in spinach. In this study, we identified 27 CHTBD1 homologs (SoCHIBI) in the spinach genome and performed a comprehensive analysis of their physicochemical properties, predicted subcellular localization, chromosomal distribution, gene structure, conserved motifs, phylogenetic relationships, and macrosynteny with four representative angiosperms. Promoter analysis revealed an enrichment of stress-responsive and pathogen-associated cis-acting elements. To evaluate transcriptional responses, we profiled the expression of 14 SoCHIBI genes using quantitative real-time PCR across six tissues in three spinach cultivars under downy mildew infection. Eight genes, including SOV2g016080.1, SOV2g016120.1, and SOV3g012940.1, were significantly upregulated following infection, with stronger induction observed in the resistant cultivar U9 compared to the susceptible S2. This study presents the first systematic characterization of the CHTBD1 gene family in spinach, identifies defense-associated candidate genes, and provides valuable genetic resources for future functional validation and resistance breeding.