Integrative transcriptomic and haplotype-based analyses identified candidate genes potentially associated with nitrogen use efficiency in cucumber (Cucumis sativus L.)

Nitrogen use efficiency (NUE) is a key determinant of crop productivity and agricultural sustainability; however, the molecular mechanisms underlying NUE in cucumber remain largely unclear. In this study, we performed a comprehensive multi-level analysis integrating phenotypic evaluation, root transcriptome profiling under contrasting nitrogen conditions (high, normal, and low nitrogen at 3 h and 48 h), weighted gene co-expression network analysis (WGCNA), gene set enrichment analysis (GSEA), and haplotype-based association analysis in a natural cucumber population. Significant phenotypic variation was observed among nitrogen treatments. Nitrogen deficiency reduced total biomass by more than 57% and markedly decreased chlorophyll content, whereas high nitrogen promoted shoot growth and decreased the root-to-shoot ratio. Transcriptomic analyses revealed a clear temporal shift from early transcriptional regulation to late-stage metabolic reprogramming. Protein interaction dynamics and redox regulation emerged as core nitrogen-responsive modules shared across treatments, while low nitrogen conditions triggered broader activation of carbon and nitrogen metabolism as well as oxidative stress-related pathways. By integrating WGCNA and GSEA results, six high-confidence candidate genes associated with NUE were identified: CsaV4_3G004580 (CLV1-like receptor kinase), CsaV4_6G000207 (DREB2A-like), CsaV4_6G003173 (WRKY15-like), CsaV4_3G003483 (LRK81-like L-type lectin receptor kinase), CsaV4_3G000093 (MED10B-like Mediator subunit), and CsaV4_3G000718 (AIL6-like AP2 transcription factor). Promoter analysis showed that these genes were enriched in hormone- and stress-responsive cis-elements, particularly ABRE and methyl jasmonate (MeJA)-responsive motifs, and several genes exhibited preferential expression in roots. Multi-genotype qPCR validation further demonstrated that CsaV4_3G004580 (CLV1-like) was induced more than 10-fold under nitrogen treatments specifically in the high-NUE genotype, whereas the low-NUE genotype failed to maintain this induction, revealing a genotype-dependent molecular response associated with NUE. Moreover, multilocus haplotype analysis demonstrated that superior haplotypes across the six loci significantly increased the low-nitrogen dry weight ratio (Cohen’s d = 0.65, p < 0.05), indicating that NUE is regulated by multiple loci with moderate additive effects. Overall, this study establishes a comprehensive framework linking nitrogen availability with transcriptional regulation and phenotypic variation in cucumber. The identified genotype-dependent expression patterns and superior haplotypes provide valuable genetic resources for marker-assisted breeding of nitrogen-efficient cucumber cultivars.