## 4 Discussion  
_Fusarium_ will disease is one of the biggest threats endangering lilies. It can cause huge economic losses through its adverse impacts on lily yield and quality [24; 25; 26]. Currently, chemical control is the primary method to control or prevent this disease. However, chemical pesticides have shown diminishing effects due to the soil-borne nature of _F. oxysporum_ and the development of pathogenic resistance [27]. Moreover, chemicals are expensive and harm the environment [24]. Therefore, breeding novel resistant varieties is the most effective approach for managing _Fusarium_ wilt diseases in lilies [28; 40]. Some Chinese endemic wild lily species, including _L. sargentiae_ [PERSON], _L. regale_ [PERSON], and _L. pumilum_ [PERSON], have shown higher resistance to _F. oxysporum_ and have been integrated into disease resistance breeding programs [30; 33]. With the rapid developments of genomics-assisted breeding, it is essential to integrate quantitative genetics and functional genomics to decipher the regulation network of _Fusarium_ wilt resistance in lilies. These studies require qRT-PCR analysis for precise and efficient quantification of expression levels of  
Figure 4: The relative expression levels of four resistance-related genes at different time points of _L. sargentiae_. The relative expression levels of _POD_ (**a**), _PAL_ (**b**), _CHI_ (**c**), and _DIR_ (**d**). F and W represent the different treatments with _F. oxysporum_ and sterile water, respectively. The 0 h time point with sterile water treatment (0 hW) is the control. Each time period was compared with the control. *, **, and *** represent \(p\) < 0.05, \(p\) < 0.01, and \(p\) < 0.001, respectively. The expression of the four target genes was normalized according to the most suitable reference gene _HIS_ using the \(2^{-\Delta\Delta\text{Ct}}\) method. One-way ANOVA with [PERSON]’s HSD post-hoc test was used to compare the means of multiple samples at the 5% significance level. target genes. Accordingly, the present study identified reliable reference genes for precise qRT-PCR normalization of candidate _Fusarium_ wilt-related resistant genes' expressions to facilitate further molecular studies in _L. sargentiae_. Diverse molecular studies have been conducted in _L. regale_ [PERSON] [25; 28; 29; 41; 42; 43] and _L. pumilum_ [PERSON] [30], but rarely in _L. sargentiae_ [PERSON]. Transcriptome sequencing technology was applied to uncover the resistance-related genes of _F. oxysporum_ and gain insight into the molecular mechanisms that govern disease resistance in _L. sargentiae._ Based on the transcriptome data, we selected nine candidate reference genes for qRT-PCR normalization of _F. oxysporum_-resistant candidate genes' expressions. The expression stabilities of these genes were assessed via three public programs (GeNorm, BestKeeper, and NormFinder). To our knowledge, this study represents the first comprehensive identification of reliable reference genes for molecular dissection of _F. oxysporum_-resistance mechanisms in _L. sargentiae._ Previous related studies were focused on internal reference genes for other phenotypic traits of lily and other close species. For example, the genes _TIP41_ and _ACT_ were identified as the best combination of reference genes for analyzing candidate genes associated with shading in the lily cultivar 'Tiny Padhye' [2]. _EF-1a_ and 18S rRNA were unveiled as the most suitable reference genes for investigating the expression levels of abiotic stresses related candidate genes, including heat, cold, heavy metals, and drought, in _L. browniti_ var. _Viridulum_[44]. In _L. davidiu_ var. _Unticolor_, _FP_, _ACT_, _GAPDH_, and _AP4_ were detected as reference genes for studying abiotic stress and developmental process-related genes [45]. These results indicate that reference genes are specific to experimental conditions, and for the same trait, they may differ according to the species and studied organs. In _L.