## 3 Results
### Expression Stability Analysis of the Candidate Reference Genes  
Three programs (GeNorm, BestKeeper, and NormFinde) were used to determine the rankings of the nine potential reference genes separately. Thereafter, the GM method was applied to compute the comprehensive stability ranking. By computing the gene expression stability measure (M) based on the average pairwise expression ratio, GeNorm was used to determine the most stable reference gene. The gene with the lowest M value is considered the most stable. A gene can be considered a reference gene if its M value is less than 1.5; lower M value indicates a more stable expression of the gene [3]. Eight of these reference genes had M values lower than 1.0, with the exception of _GAPDH_, which recorded an M value of 1.466. _HIS_ and _EF-1a_ were certified as the two most stable reference genes, with M values of 0.533 and 0.535, respectively (Table 2). In some gene expression analyses, a single reference gene may not be able to satisfy the experiment requirements if the precision requirements are high or the quantity change of gene expression has to be expressed quantitatively. It is therefore necessary to use two or more reference genes in order to obtain accurate and reliable normalization results. It is possible to determine the optimal number of reference genes for normalization based on the pairwise variation value (Vn/n + 1, V value). When Vn/n +1 is greater than or equal to 0.15, the optimal number of reference genes for accurate normalization should be \(n+1\). On  
Figure 2: qRT-PCR Ct values for all candidate reference genes in all samples. The box graph represents the 75 th to 25 th percentiles and the line across the box represents the median. The whiskers on each box represent the minimum and maximum values and the circles represent outliers. the contrary, if \(\rm{Vn/n}+1\) is less than 0.15, n is the optimal number of reference genes [3; 5]. GeNorm V value analysis showed that when the V value of the two to eight reference genes (V2/3 to V7/8) was less than 0.15 (Figure 3), the optimal number of reference genes is two. Based on the V values, the most appropriate combination of reference genes for qRT-PCR normalization of resistance-related genes' expression levels in \(L\). _sargentiae_ was _HIS_ and _EF-1a_. Unlike GeNorm analysis, the NormFinder is commonly used to evaluate the stability value (SV) of reference genes and to detect, via intra- and inter-group variations, the optimal number of reference genes for precise normalization. A gene is considered more stable if its expression level is lower than the average expression stability. The results of NormFinder analyses were the same as the GeNorm results in stability rankings of five reference genes including _HIS_, _CYP_, _TUB_, _RPL13_, and _GAPDH_ (Table 2). Similarly, _HIS_ (SV = 0.086) and _GAPDH_ (SV = 0.994) were confirmed as the most stable and unstable genes, respectively. BestKeeper is another tool to explore the stability index of potential reference genes through the evaluation of SD (standard deviation), CV (coefficient of variation), and \(R\) (correlation coefficient) [36]. If SD \(<1.0\), the gene is considered stable [1; 4]. According to the BestKeeper results, all nine genes are suitable for use as reference genes (SD \(<1.0\)). _CYP_