*Article* **Selection and Validation of Optimal RT-qPCR Reference Genes for the Normalization of Gene Expression under Different Experimental Conditions in** *Lindera megaphylla*

**Hongli Liu 1,†, Jing Liu 1,†, Peng Chen 1, Xin Zhang 1, Ke Wang 2, Jiuxing Lu 1,\* and Yonghua Li 1,\***


**Abstract:** *Lindera megaphylla*, a broad-leaved evergreen that is used as a landscape ornamental plant and medicinal plant, is an ecologically important and dominant tree species. However, little is known about the molecular mechanisms of its growth, development, and metabolism. The selection of suitable reference genes is critical for molecular biological analyses. To date, no research on reference genes as a foundation for gene expression analysis has been undertaken in *L. megaphylla*. In this study, 14 candidate genes were selected from the transcriptome database of *L. megaphylla* for RT-qPCR assay under different conditions. Results showed that *helicase-15* and *UBC28* were most stable in different tissues of seedlings and adult trees. For different leaf developmental stages, the best combination of reference genes was *ACT7* and *UBC36*. *UBC36* and *TCTP* were the best under cold treatment, while *PAB2* and *CYP20-2* were the best under heat treatment. Finally, a RT-qPCR assay of *LmNAC83* and *LmERF60* genes were used to further verify the reliability of selected reference genes above. This work is the first to select and evaluate the stability of reference genes for the normalization of gene expression analysis in *L. megaphylla* and will provide an important foundation for future genetic studies of this species.

**Keywords:** *Lindera megaphylla*; normalization genes; RT-qPCR; tissue specificity; temperature; transcript analysis
