*3.1. Verification of Amplicon Size, Primers Specificity and PCR Amplification Efficiency*

A total of 20 candidate genes from *L. megaphylla* were selected as potential reference genes for the normalization of target gene transcript levels using RT-qPCR. Standard PCR amplification with primers (Table 2, Supplementary Table S1) targeting the candidate genes was performed with reverse-transcribed cDNA from each sample as templates. Agarose gel electrophoresis indicated that all PCR products were single bands of the expected sizes, indicating that the primers were specific (Supplementary Figure S1). The melting curves, obtained after 40 cycles of amplification by RT-qPCR, showed single peaks, which also verified that the primers for the 20 candidate reference genes had strong specificity (Supplementary Figure S2). Standard curves, generated using a five-fold serial dilution for each candidate gene, had linear correlation coefficients (R2) greater than 0.99 for each specific primer pair, and the amplification efficiencies of the RT-qPCR reactions were 64.42–107.17% (Table 2). Because the amplification efficiencies of *ICIn* (64.42%), *UBQ* (85.95%), *SDE2* (86.75%), and *EF1α* (83.49%) were less than 90%, and the R2 values of *CYP95* and *RHA2A* were <0.99, these six candidate genes, based on the designed primers, were not appropriate for validating expression. Therefore, the remaining 14 candidate genes were used for subsequent experiments.
