Stay-green trait is an important agronomic characteristic closely related to high yield, good quality, and stress resistance of maize. This study explored the differences in nitrogen uptake and translocation of different stay-green maize hybrids, aiming to provide a theoretical basis for the physiological mechanism of high nitrogen efficiency in maize.

The tested materials were the stay-green hybrid Shandan 650 and the non-stay-green hybrid Zhengdan 958. In 2023, 6 N treatments were applied: N1 (0 kg·hm^-2), N2 (60 kg·hm^-2), N3 (120 kg·hm^-2), N4 (180 kg·hm^-2), N5 (240 kg·hm^-2), and N6 (300 kg·hm^-2). In 2024, a nitrogen×density interaction experiment was conducted with three N levels—low N (LN, 0 kg·hm^-2), medium N (MN, 180 kg·hm^-2), and high N (HN, 240 kg·hm^-2)—and two planting densities—low density (LD, 60000 plants·hm^-2) and high density (HD, 75000 plants·hm^-2). After the silking stage of maize, indicators were determined for each treatment, such as SPAD value of ear leaves, total number of green leaves per plant, dry matter, and nitrogen accumulation in vegetative organs and grains. Meanwhile, nitrogen absorption and translocation rates as well as nitrogen use efficiency-related indicators were analyzed.

Grain yield of both hybrids initially increased and then stabilized with rising N rates, with Shandan 650 consistently outperforming Zhengdan 958 across all N and density treatments. Post-silking, Shandan 650 exhibited faster chlorophyll degradation (SPAD decline: 65.1% vs. 49.9%) and greater green leaf loss than Zhengdan 958, particularly under low N. Shandan 650 demonstrated superior N remobilization efficiency, especially under low N and high density, with significantly higher N translocation from leaves to grains. Overall, Shandan 650 achieved significantly higher N remobilization efficiency, nitrogen use efficiency, nitrogen agronomic efficiency, and nitrogen harvest index than Zhengdan 958. Furthermore, under high-density planting conditions, reasonable nitrogen reduction further enhanced its nitrogen efficiency performance.

The functional stay-green maize variety Shandan 650 maintains consistent greenness and photosynthetic capacity until a certain period before physiological maturity, at which point a rapid decline occurs along with nitrogen remobilization. Its strong nitrogen translocation capacity in vegetative organs enhances nitrogen translocation rate and nitrogen use efficiency, and higher nitrogen efficiency could be achieved under reasonable nitrogen reduction and density increase.

Functional stay-green is generally considered a desirable trait in major crop varieties including maize. Finding new loci and candidate genes related to stay-green, and providing new theoretical basis for the genetic research on stay-green.

Using 150 recombinant inbred lines (RIL) populations derived from the cross between Xu 178 and K12, QTL mapping of three stay-green related traits (visual stay green (VSG), green leaf number at silking stage (GLNS) and green leaf number at mature stage (GLNM) were performed by the composite interval mapping (CIM) method of Windows QTL Cartographer V2.5. Besides, an association population, which composed of 139 natural materials genotyped with 50790 high-quality SNP markers, was used to dissect genetic locus of three traits by genome-wide association study (GWAS) based on the mixed linear model MLM).

Based on CIM, three traits (GLNM, GLNS and VSG) were mapped using phenotypic values in a single environment and best linear unbiased prediction (BLUP) value. A total of 37 QTLs were detected on all chromosomes except Chromosome 10, and the LOD score ranged from 2.58-11.36, with a phenotypic variation contribution rate of 4.34%-22.40%. Among them, 14, 12 and 11 loci were detected for GLNM, GLNS and VSG traits, respectively. Four of the QTLs, qGLNS2-1, qVSG1-1, qVSG1-2 and qVSG7-1, were genetically stable and were detected simultaneously in three or more different single environments. GWAS was performed on three stay-green related traits using MLM, and a total of 44 significant SNPs above the threshold line were detected. According to the physical position of SNP markers in the B73 reference genome, a total of 15 SNP were found to fall into the QTL interval mapped by linkage analysis.

Combined with the results of QTL mapping and genome-wide association study, a total of 4 genetically stable colocalization genetic regions were detected (the corresponding physical position intervals on the B73 reference genome version 4 are 6.2-8.2 Mb on chromosome 1, 209.1-221.4 Mb on chromosome 2, 96.8-102.1 Mb on chromosome 6, and 4.9-11.4 Mb on chromosome 7), and four important candidate genes (Zm00001d006119, Zm00001d018975, Zm00001d006535 and Zm00001d036763) related to photosynthesis and stress response were mined.

Genetic improvement for efficient utilization of maize nutrients represents a crucial method to ensure national food security. Exploring quantitative trait locus (QTL) and related candidate genes of nitrogen use efficiency can provide a theoretical basis for improving the efficiency of nitrogen fertilizer in maize and cultivating high-yield and high-efficiency maize varieties.

In this study, QTL mapping analysis in one recombinant inbred line (RIL) population constructed by KA105 and KB024 was performed for grain yield under two different nitrogen treatments, including the derived traits partial factor productivity from applied nitrogen (PFPN), low nitrogen tolerance coefficient (LNTC) and nitrogen agronomic efficiency (NAE). Concurrently, integrating the seedling transcriptome data of the parent KA105 under nitrogen treatment, differentially expressed genes were identified, and candidate genes associated with maize nitrogen use efficiency were mined through co-expression analysis. Subsequently, the selected candidate genes were validated using qRT-PCR.

Through mapping analysis, a total of 36 QTLs distributed across different chromosomes were detected, explaining 1.63% to 17.26% of the phenotypic variation. Among these, eight major QTLs with a phenotypic variation explanation rate exceeding 10% were identified, along with seven genetically stable QTLs commonly identified across different traits or environments. Notably, qNNGYP1 located on chromosome 1 has been repeatedly detected in previous studies, with a phenotypic explanation rate of up to 11.73%. Additionally, other QTLs (qNNGYP1, qPFPN1) co-located in this interval across different environments, suggesting it as a focal region for further investigation. Combining transcriptome data of seedlings under low nitrogen stress, 39 differentially expressed genes within these QTL intervals were identified, and 6 key genes were identified through co-expression network prediction. The result of qRT-PCR indicated that the expression trends of the candidate genes under both nitrogen treatments were consistent with the transcriptome data. Specifically, GRMZM2G366873 was involved in the regulation of auxin homeostasis and may participate in maize responses to low nitrogen stress, drought stress, and boron stress through auxin signal transduction, also regulating ear length. GRMZM2G414192 was involved in the response of the photosynthetic system to low nitrogen stress and was regulated by brassinosteroids. GRMZM2G414043 was associated with maize grain length and biomass, while GRMZM2G040642 may be involved in the long-distance signal transduction of nitrogen.

In summary, a total of 36 QTLs were identified, distributed across chromosomes 1, 4, 5, 7, 8, and 9, including eight major QTLs (PVE>10%). The candidate genes GRMZM2G366873, GRMZM2G414192, GRMZM2G414043, and GRMZM2G040642 were identified as potential genes for maize nitrogen efficiency.