Screening Key Genes for Salt Tolerance in Maize Inbred Lines via Time-Series Transcriptomics and Machine Learning

A systematic evaluation of salt tolerance at the seedling stage was conducted using 143 maize inbred lines under a 150 mM mixed-salt solution (NaCl:Na₂SO₄ = 9:1, EC = 16.78 dS/m) that mirrors the ionic composition of saline groundwater in the Yellow River Delta. The comprehensive salt tolerance index (D value) ranged from 0.15 to 0.85 across the population, with the elite line B114 exhibiting the highest D value (0.835) and the sensitive line PHT55 ranking near the bottom. Under salt stress, B114 displayed remarkable growth stability, with plant height decreasing by only 25.9%, fresh weight by 13.3%, and dry weight remaining unchanged, whereas PHT55 suffered severe growth inhibition (plant height: 61.5% decrease; fresh weight: 63.2% decrease; dry weight: 33.3% decrease). Time-series RNA-seq of root tissues across four time points (5, 8, 11, and 14 days) revealed markedly distinct transcriptional dynamics: B114 exhibited relatively stable temporal regulation (2261-9124 DEGs), whereas PHT55 showed a pronounced early transcriptional burst that progressively intensified (3728-10,108 DEGs). Using random forest-based machine learning, 50 core salt tolerance-related genes were unbiasedly identified from 16,194 significantly differentially expressed genes. Functional enrichment analysis revealed that these genes were primarily involved in redox regulation, ion homeostasis maintenance, and stress signal transduction pathways. qRT-PCR validation confirmed biphasic expression patterns, with Zm00001d024160 showing the strongest early induction (48-fold at 5 h). This study established a maize salt tolerance evaluation system closely aligned with field conditions and demonstrated that coordinated temporal transcriptional regulation represents a core molecular mechanism underlying high salt tolerance in maize. The elite salt-tolerant germplasm and key candidate genes identified here provide valuable genetic resources and a theoretical foundation for molecular breeding of salt-tolerant maize adapted to saline-alkaline soils.