*2.1. Plant Materials and Heat Treatments*

Two pepper varieties, heat-tolerant 17-03 and heat-sensitive H1023, were obtained from the Hubei Key Laboratory of Vegetable Germplasm Enhancement and Genetic Improvement, Hubei Academy of Agricultural Sciences, for transcriptome analysis. Seeds were sown in 50-hole trays and grown in a growth chamber under cool white fluorescent lights (approximately 200 <sup>μ</sup>mol/m2/s) at 25 ± <sup>2</sup> ◦C with a photoperiod of 16 h light/8 h dark and 70–80% relative humidity. At two weeks, seedlings with uniform growth were transplanted to plastic pots (10 × 10 × 10 cm), containing peat, vermiculite, and soil (v/v/v = 1:1:1), until they reached the stage of 6–8 true leaves. For heat treatment, seedlings were well watered and cultivated at 42 ◦C for 3 d before recovering at 25 ◦C for 1 d. The control seedlings were placed at 25 ◦C for 4 d. The lighting conditions and humidity were not changed. The first fully expanded leaf from the top of each plant was sampled from eight seedlings (three replicates) in the control and treatment groups. All samples were immediately frozen in liquid nitrogen and stored at −80 ◦C for RNA sequencing. The samples were named CK1 (HT1\_1-HT1\_3) (control group of 17-03), T1 (HT2\_1-HT2\_3) (heat treatment group of 17-03), M1 (HT3\_1-HT3\_3) (recovered group of 17-03), CK2 (HS1\_1-HS1\_3) (control group of H1023), T2 (HS2\_1-HS2\_3) (heat treatment group of H1023), and M2 (HS3\_1-HS3\_3) (recovered group of H1023).
