**5. Conclusions**

We verified the pepper varieties 17-03 and H1023 as being heat-resistant and heatsensitive varieties and used RNA-seq to elucidate the effects of HS and the subsequent recovery on the expression of genes regulating the HS response and thermotolerance mechanisms. A total of 11,633 DEGs were identified in the four groups, with 1229 common DEGs among all four groups. Functional enrichment analysis showed that in 17-03 and H1023, DEGs were most enriched in metabolic processes under stress and photosynthesis and light harvesting during HS and after recovery from HS. The most significantly enriched pathways in 17-03 and H1023 were the same under HS, but differed during recovery. Furthermore, 38 Hsps, 17 Hsfs, 38 NAC TFs, and 35 WRKY TFs were identified as participating in the HS or recovery responses. These findings facilitate a better understanding of the molecular mechanisms underlying HS and recovery after HS in different pepper genotypes.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/horticulturae7100339/s1, Table S1: Statistical analysis of pepper clean reads in 18 libraries for RNA-seq. Table S2: Gene read count, FPKM value, annotation, and functional enrichment. Table S3: Detailed list of DEGs in 17-03 and H1023 under HS (42 ◦C) for 3 d and recovery (25 ◦C) for 1 d relative to the control. Table S4: Significantly enriched GO terms of DEGs in 17-03 and H1023 in the four groups (FDR ≤ 0.05). Table S5: Significantly enriched KEGG pathway in 17-03 and H1023 under HS (42 ◦C) for 3 d and recovery (25 ◦C) for 1 d (FDR ≤ 0.05). Table S6: DEGs encoding Hsps in 17-03 and H1023 at the heat treatment and recovery stages. Table S7: Differentially expressed transcription factors in 17-03 and H1023 at the heat treatment and recovery stages. Table S8: qRT-PCR primers used for validation of RNA-Seq data.

**Author Contributions:** Conceptualization, M.Y. and C.J.; methodology, M.Y. and C.J.; validation, F.W., Y.Y., C.Y., N.L., S.S., Y.L., S.G., C.J. and M.Y.; formal analysis, F.W., Y.Y., C.Y., N.L. and S.G.; investigation, S.S. and Y.L.; resources, F.W. and M.Y.; data curation, C.J. and M.Y.; writing—original draft preparation, F.W. and Y.Y.; writing—review and editing, C.J. and M.Y.; visualization, C.J. and M.Y.; supervision, C.J. and M.Y.; project administration, C.J. and M.Y.; funding acquisition, M.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grants from the Natural Science Foundation of Hubei Province, China (2020CFA010), the earmarked fund for the China Agriculture Research System (CARS-23-G28), Key R & D Program Projects in Hubei Province (2020BBA037), China Postdoctoral Science Foundation (2017M620305), and Youth Fund of Hubei Academy of Agricultural Sciences (2021NKYJJ04).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data used for the analysis in this study are available in the article and Supplementary Materials.

**Acknowledgments:** We thank Personal Biotechnology Co., Ltd., Shanghai, China for RNA-Sequencing.

**Conflicts of Interest:** The authors declare no conflict of interest.
