Phenotypic Evaluation and Genome-Wide Association Analysis of Cold Tolerance at Seedling Stage in Maize

Low temperature exerts severe adverse effects on maize growth, particularly during the seedling stage. Screening for cold-tolerant maize genotypes is highly significant for identifying genes associated with cold tolerance and enhancing maize performance under low, suboptimal temperature conditions. The identification of representative cold tolerance-related genes is of great significance for the breeding of cold-resistant maize varieties. In this study, a diversity panel of 205 materials was evaluated and classified for cold tolerance at the seedling stage. The coefficients of variation of all materials ranged from 14.53% to 35.71%, reflecting considerable genetic diversity within the panel. The correlation coefficients for each phenotypic trait between the cold-treated (CT) and control (CK) maize materials ranged from 0.60 to 0.90, further indicating that all traits displayed varying degrees of sensitivity to cold stress. A comprehensive evaluation of cold tolerance using the D value was conducted. The D values of all materials ranged from 0.355 to 0.863, with a mean value of 0.64. A hierarchical clustering analysis was performed to classify all materials into five categories based on their cold tolerance. Further, 17 SNPs were identified using GWAS analysis, and 12 candidate genes were located within the regions related to the SNPs. Some candidate genes were closely associated with cold tolerance, such as genes encoding MYB and GRAS transcription factors, leucine-rich repeat (LRR) proteins, and protein kinases. Validation by qRT-PCR confirmed that the expression of some genes was induced under cold stress conditions. These findings lay a crucial foundation for breeding cold-tolerant maize varieties and for further exploration of genes associated with cold tolerance.