*3.2. Transcript Abundance of Candidate Reference Genes*

The transcript abundance of 14 candidate reference genes was estimated using the average cycle threshold (Ct) values for RNA extracted from different tissues or plants grown under different experimental conditions. All candidate reference genes were expressed at a wide range of transcript levels under the different experimental conditions. The gene expression level is negatively correlated with the Ct value, which means that a gene with a higher transcript level has a smaller Ct value. As shown in Figure 1, the minimum Ct value was 17.94, and the maximum was 27.51. Among the genes, *UBC36* exhibited the highest transcript abundance, with the minimum, median and maximum Ct values of 20.91, 22.90, and 27.51, respectively. *PAB2* showed the lowest expression abundance, with minimum, median and maximum Ct values being 17.94, 20.20, and 22.65, respectively. Compared with other genes, *GAPDH*, *UBC28,* and *TCTP* had obviously narrow range of Ct values (19.14–22.69, 20.74–23.95, and 21.10–24.98, respectively), indicating that they have a relatively stable expression level. While all the candidate reference genes exhibited significant expression abundance, none of the candidate genes were expressed stably in all samples. Thus, it is necessary to screen the appropriate internal reference genes for *L. megaphylla* under different experimental conditions or tissue types.

**Figure 1.** Distribution of average cycle threshold (Ct) values of the 14 candidate reference genes across all 38 samples from *L. megaphylla*. Boxes represent the interquartile range, with the middle solid lines indicating the median Ct values. The upper and lower boundaries of each box represent the 75th and 25th percentiles, respectively. The upper and lower bars represent the maximum and minimum Ct values, respectively, and the small squares represent the average values.
