## 5 Conclusions  
In this study, stable reference genes were identified in fruits at various developmental stages and in different tissues of _E. rutaocarpa_ var. _officialis_. The most stable reference genes in the fruits during the different developmental periods were _Act1_ and _UBQ_, whereas in the different tissues, _Act1_ and _His3_ exhibited the highest levels of stability. The potential pathway genes involved in the limonin biosynthesis of _E. rutaocarpa_ var. _officialis_ were determined through \"Full length transcriptome + expression profile + metabolome\" correlation analysis. The expression levels of these pathway genes were relatively quantified using the two most stable reference genes that were identified. A majority of the pathway genes displayed initial increases followed by decreases in expression levels. Interestingly, the genes _HMGCR_, _SQE_, and _CYP450_ exhibited consistent and continuous decreases in expression levels, aligning perfectly with the trends observed in the limonin content changes. This suggested a potential role for these genes in the biosynthesis of limonin in _E. rutaocarpa_ var. _officialis_. This research is the first to investigate the molecular mechanism of effective component metabolism in _E. rutaocarpa_ var. _officialis_. Reliable reference genes were screened in _E. rutaocarpa_ var. _officialis_, and the pathway genes were quantified using the reference genes to verify the expression trends of the pathway genes. This study lays the foundation for further research on the metabolic pathway of limonin in _E. rutaocarpa_ var. _officialis_, and it provides a reliable reference gene for other researchers to use for conducting expression analyses.  
**Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/plants12183197/s1](https://www.mdpi.com/article/10.3390/plants12183197/s1)]([https://www.mdpi.com/article/10.3390/plants12183197/s1](https://www.mdpi.com/article/10.3390/plants12183197/s1)), Text S1: The sequences of the ten candidate reference genes (_Actin-1_, _Histone H3_, _UBQ_, _GAPDH_, _UBQ 10_, _CHS 1_, _B-TUB6_, _a-TUB_, _Actin-100_, _B-TUB2_). Text S2: The sequences of the pathway genes (_DXS_, _DXR_, _ISPD_, _ISPE_, _ISPP_, _ISPG_, _ISPH_, _AGCT_, _HMGCC_, _HMGCR_, _MVK_, _MVD_, _GPP/FPP_, _IDI_, _SQS_, _SQE_, _CYP_), Figures S1-S28: Dissociation curves of the ten candidate reference genes and pathway genes.  
**Author Contributions:** Conceptualization, Q.T. and Y.Z. ([PERSON]); methodology, Y.Z. ([PERSON]) and Y.Z. ([PERSON]); software, Y.Z. ([PERSON]) and Y.Z. ([PERSON]); validation, Q.T., Y.Z. ([PERSON]) and D.M.; formal analysis, Y.Z. ([PERSON]); investigation, Q.T.; resources, Q.T., J.Y. and Y.L.; data curation, Y.Z. ([PERSON]) and Y.Q.; writing--original draft preparation, Y.Z. ([PERSON]) and D.M.; writing--review and editing, Q.T., D.M. and W.C.; visualization, Y.Z. ([PERSON]) and R.Z.; supervision, Q.T. and L.P.; project administration, Q.T. and L.P.; funding acquisition, Q.T. and L.P. All authors have read and agreed to the published version of the manuscript.  
**Funding:** This research was funded by the Hunan Provincial Science and Technology Plan Project, grant number (2017 ZK3133), Chinese herbal medicine support project of the Ministry of Industry and Information Technology (2013082830), and Research project of Changsha Science and Technology Bureau (kh2005099).  
**Data Availability Statement:** Not applicable.  
**Conflicts of Interest:** The authors declare no conflict of interest.