*2.5. RNA-Seq Analysis*

The samples used for RNA-seq were seed embryos soaked in 0 mM and 10 mM of betaine, heat-stressed at 38 ◦C for 24 h. We labeled the treatments BT and HT+BT, respectively, and we performed three biological replicates of each treatment. We extracted the total RNA from young ear tissue with Trizol (Invitrogen, Waltham, MA, USA) reagent. Wuhan Huada Gene Technology Co., Ltd. completed the RNA-seq-library construction and sequencing. The screening conditions of differential genes were a Q value ≤ 0.05 and a fold change (|log2 ratio|) >1.5. We used the FPKM values to analyze the differential gene expression levels, and we used gene ontology (GO) enrichment analysis (agriGO v2.0: a GO analysis toolkit for the agricultural community, 2017 update) to analyze the biological processes of the differential gene participation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional enrichment analyses were performed via the KEGG Database (https://www.genome.jp/kegg (accessed on 14 August 2022)) [38]. We used the phyper function in the R software for enrichment analysis and to calculate the *p*-value, and then FDR corrected the *p*-value, considering a Q value ≤ 0.05 as significant enrichment.
