*3.7. BestKeeper Algorithm*

The BestKeeper algorithm evaluates the most stable candidate reference genes based on the standard deviation (SD) and coefficient of variance (CV) of the average cycle threshold (Ct) values [16,19,32]. In general, the smaller the SD value is, the more stable the gene is. As shown in Table 3, the genes *UBC7* (0.06 ± 0.24) and *helicase-15* (0.14 ± 0.57) had the lowest SD values in seedlings, while *GAPDH* (0.09 ± 0.48) and *TCTP* (0.18 ± 0.83) were the most stable genes in adult trees. The genes with the most stable expression across different tissues were *ACT7* (0.29 ± 1.36) and *UBC28* (0.31 ± 1.40), while the expression of *UBC28* (0.60 ± 2.76) and *GAPDH* (0.66 ± 3.24) was the most stable across different leaf developmental stages. The genes with the most stable expression across the entire growth cycle were *UBC28* (0.58 ± 2.64) and *TCTP* (0.59 ± 2.73). Under cold treatment, *PPR* (0.18 ± 0.82) and *EIF4A-3* (0.19 ± 0.91) were the most stable genes, while under heat treatment, *UBC28* (0.22 ± 1.03) and *ubiquinone* (0.54 ± 2.35) were the most stable genes. Under both types of temperature stress, the expression of *UBC28* (0.37 ± 1.70) and *PPR* (0.42 ± 1.86) were the most stable. For all samples, the top four most stable reference genes were *UBC28* (0.51 ± 2.32), *TCTP* (0.56 ± 2.57), *GAPDH* (0.60 ± 2.98) and *ubiquinone* (0.64 ± 2.76), while *TUA* showed the highest value and thus the lowest expression stability.

**Figure 5.** Ranking of expression stability of the 14 candidate reference genes in *L. megaphylla* using NormFinder analysis. Results from (**A**) different seedling tissues; (**B**) different adult tree tissues; (**C**) all seedling and adult tree tissues; (**D**) 16 leaf developmental stages; (**E**) entire growth cycle including all different seedling and adult tree tissues as well as 16 leaf developmental stages; (**F**) cold treatment for 7 days; (**G**) cold treatment for 24 h; (**H**) cold treatments including 7 days and 24 h; (**I**) heat treatment for 24 h; (**J**) different temperature treatments including cold and heat; (**K**) total samples.


**Table 3.** Ranking of expression stability of the 14 candidate reference genes in *L. megaphylla* using BestKeeper

 analysis.
