Root architecture is intricately linked to the acquisition of water and nutrients in maize seedlings. Despite its functional importance, few genes controlling root development have been targeted for drought resistance in breeding. Here, we performed a genome-wide association analysis to detect genetic variants linked to primary root length (PRL) across 307 inbred lines grown under hydroponic conditions. We identified 28 SNPs significantly associated with 25 candidate genes, accounting for 6.09%–11.07% of the phenotypic variation. Among them, ZmHSP20-5, encoding a cytoplasm-localized small heat shock protein (sHSP) with preferential expression particularly in lateral root primordia emerged as a promising candidate. Functional validation using knockout mutants revealed that disruption of ZmHSP20-5 impaired root architecture, causing reduced primary root elongation, shorter lateral roots, decreased lateral root density, and compromised drought tolerance. Further analysis revealed that InDel-1224 in the ZmHSP20-5 promoter likely contributed to differential gene expression and variation in root development among inbred lines. Evolutionary evidence suggested that the ZmHSP20-5 locus may have undergone selection during domestication, with the favorable ZmHSP20-5^In-1224 allele increasing in frequency over time. Overall, these findings establish that natural variation in ZmHSP20-5, particularly the ZmHSP20-5^In-1224 allele, contributes to root growth and drought resistance, providing a valuable genetic resource for the breeding of drought-resistant maize varieties with optimized root systems.

Stalk strength increases resistance to stalk lodging, which causes maize (Zea mays L.) production losses worldwide. The genetic mechanisms regulating stalk strength remain unclear. In this study, three stalk strength-related traits (rind penetrometer resistance, stalk crushing strength, and stalk bending strength) and four plant architecture traits (plant height, ear height, stem diameter, stem length) were measured in three field trials. Substantial phenotypic variation was detected for these traits. A genome-wide association study (GWAS) was conducted using general and mixed linear models and 372,331 single-nucleotide polymorphisms (SNPs). A total of 94 quantitative trait loci including 241 SNPs were detected. By combining the GWAS data with public gene expression data, 56 candidate genes within 50 kb of the significant SNPs were identified, including genes encoding flavonol synthase (GRMZM2G069298, ZmFLS2), nitrate reductase (GRMZM5G878558, ZmNR2), glucose-1-phosphate adenylyltransferase (GRMZM2G027955), and laccase (GRMZM2G447271). Resequencing GRMZM2G069298 and GRMZM5G878558 in all tested lines revealed respectively 47 and 2 variants associated with RPR. Comparison of the RPR of the zmnr2 EMS mutant and the wild-type plant under high- and low-nitrogen conditions verified the GRMZM5G878558 function. These findings may be useful for clarifying the genetic basis of stalk strength. The identified candidate genes and variants may be useful for the genetic improvement of maize lodging resistance.