Integrated GWAS and Transcriptome Analysis Reveal the Genetic and Molecular Basis of Low Nitrogen Tolerance in Maize Seedlings

Nitrogen is an essential nutrient for the growth and development of maize (<i>Zea mays</i> L.), and soil nitrogen deficiency is an important factor limiting maize yield. Although excessive application of nitrogen fertilizer can increase yield, it can also cause environmental problems. Therefore, screening low-nitrogen-tolerant (LNT) germplasm resources and analyzing their genetic mechanisms are of great significance for the development of efficient and environmentally friendly agriculture. In this study, 201 maize inbred lines were used as materials. Two levels of low nitrogen (LN) (0.05 mmol/L, N1) and normal nitrogen (4 mmol/L, N2) were set up. Phenotypic indicators such as seedling length, root length and biomass were measured, and they were classified into LNT type (18 samples), nitrogen-sensitive (NS) type (27 samples) and intermediate type (156 samples). A total of 47 significant SNP loci were detected through a genome-wide association study (GWAS), and 36 candidate genes were predicted. Transcriptome sequencing (RNA-seq) analysis revealed that the differentially expressed genes (753 upregulated and 620 downregulated) in LNT materials under low nitrogen stress (LNS) were significantly fewer than those in NS materials (2436 upregulated and 2228 downregulated). Further analysis using WGCNA identified a total of eight co-expression modules. Among them, the red module was significantly correlated with root length and underground fresh weight under LN conditions (r = 0.75), and three key genes for stress response (<i>Zm00001d005264</i>, <i>Zm00001d053931</i>, <i>Zm00001d044292</i>) were screened out. Combined with GWAS, RNA-seq and qRT-PCR verification, eight candidate genes closely related to LNT at the seedling stage of maize were finally determined, involving biological processes such as stress response, nitrogen metabolism and substance formation. This study initially revealed the molecular mechanism of maize tolerance to LN through multi-omics analysis, providing a theoretical basis and genetic resources for breeding new nitrogen-efficient maize varieties.