Genome-wide identification and expression analysis of nucleotide-binding site leucine-rich repeat genes associated...

Downy mildew is a destructive disease that causes severe losses in spinach (Spinacia oleracea L.) yield and quality. Nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes are core regulators of plant innate immunity and play critical roles in defense against pathogen infection. However, the characteristics of NBS-LRR family genes in spinach and their specific functions in downy mildew resistance remain unclear, limiting the development of resistant spinach cultivars.

Using available spinach genome data, we identified NBS-LRR family members via bioinformatics approaches. We classified these members into subclasses, analyzed their chromosomal distribution, conserved motifs, and within-clade functional similarity. Combining genome-wide association study (GWAS) results, we selected candidate genes associated with downy mildew resistance. We detected the expression patterns of these candidate genes in different tissues and between resistant and susceptible spinach genotypes. We also analyzed the expression dynamics of candidate genes in resistant spinach (Whale) under downy mildew stress and performed protein-protein interaction (PPI) analysis to identify core proteins involved in the resistance response and their potential disease-related associations.

We identified 114 NBS-LRR family members in spinach, which were categorized into coiled-coil (CC) domain-NBS (40 members), CC-NBS-LRR (27 members), NBS (19 members), NBS-LRR (12 members), and other subclasses. These genes were asymmetrically distributed across chromosomes, with enrichment on chromosomes 1 and 3 (accounting for 26.3% and 28.1% of the total, respectively). NBS-LRR members shared conserved motifs, with motifs 2 and 12 being the most highly conserved; genes within the same phylogenetic clade showed functional similarity. Ten candidate genes were selected through integration with GWAS results. These candidates exhibited tissue-specific expression (e.g., SoarcTNL1 was highly expressed in petioles and roots) and differential expression between resistant and susceptible materials (SoarcRN1 showed the highest expression in resistant US78). Under downy mildew stress, candidate genes were more strongly upregulated in resistant Whale, with expression inflection points or peaks at 6 and 8 days post-inoculation (dpi) with expression inflection points or peaks at 6 and 8 dpi. PPI analysis identified core proteins SoarcRN1 and SoarcNL8 in high-confidence networks. This study clarifies the characteristics of spinach NBS-LRR genes and their associations with downy mildew resistance, laying a critical foundation for subsequent gene function research and resistant spinach breeding.