## 2 Results
### Stability Analysis of the Candidate Reference Genes
#### 2.4.5 RefFinder Comprehensive Stability Analysis  
The RefFinder website provides a comprehensive ranking by integrating the analysis results from various software tools to identify the most stable reference gene [28; 29]. The results indicated that in the fruits at the different developmental stages, the stability ranking was as follows: _Act1_ (1.19) > _UBQ_ (1.86) > _GAPDH_ (2.45) > _g-TUB6_ (3.72) > _His3_ = _UBQ10_ (5.48) > _g-TUB2_ (7.24) > _Act100_ (8.24) > _CHS1_ (8.45) > _\(\alpha\)-TUB_ (10.00). Among these, _Act1_ and _UBQ_ emerged as the two most stable genes, while _\(\alpha\)-TUB_ was identified as the least stable. In the distinct tissue types, the stability ranking was as follows: _Act1_ (1.19) > _His3_ (1.86) > _GAPDH_ (2.28) > _\(\alpha\)-TUB_ (4.43) > _CHS1_ (5.44) > _UBQ10_ (5.83) > _g-TUB2_ (6.85) > _UBQ_ (7.97) > _g-TUB6_ (8.35) > _Act100_ (9.46). Among these, _Act1_ and _His3_ stood out as the two most stable genes, while _Act100_ was found to be the least stable.  
Gene Expression Analysis of the Related Enzymes in the Biosynthesis Pathway of Limonin in E. rutacacarpa var. officinalis  
The candidate genes involved in the biosynthesis pathway of limonin were identified through transcriptome sequencing. When the number of candidate pathway genes exceeded two, the gene most closely associated with the limonin content was selected for investigation. In cases where there was only one candidate pathway gene, the co-correlation analysis method was not required. Limonin was not detected in the roots, leaves, or buds, and so the co-correlation analysis method could only be applied to the fruits for the different developmental stages. Taking _CYP450_ as an illustrative example (Figure 8), a total of 32 _CYP450_ genes were identified within the transcriptome. Subsequently, by performing co-expression cluster analyses of both the transcriptome and the metabolome data, the _CYP450_ gene most likely to be involved in the limonin biosynthesis pathway was identified as _PB.10523.1_. The relative expression levels of the pathway genes in the fruits for 14 August, 1 September, and 24 October were determined, with the fruits from 17 July serving as the control. As shown in Figure 9a, the genes exhibiting decreasing expression trends encompassed _HMGCR_, _SQE_, and _CYP_, while the expression levels of the other genes displayed patterns of initial increases followed by decreases. The changing trends in the relative gene expression levels closely corresponded with the fragments per kilobase of the exon model per million mapped fragments (FPKM) values from the transcriptome data (Figure 9b).  
Figure 8: Screening of cytochrome P450 associated with limonin biosynthesis. (WZY1\(-\)1, WZY1\(-\)2, and WZY1\(-\)3 represent the three repeats of limonin content in the _E. rutacarpa_ var. _officialis_ fruit on17 July. WZY3\(-\)1, WZY3\(-\)2, and WZY3\(-\)3 represent the three repeats of limonin content in the _E. rutaocarpa_ var. _officialis_ fruit on 14 August. WZY4\(-\)1, WZY4\(-\)2, and WZY4\(-\)3 represent the three repeats of limonin content in the _E. rutaocarpa_ var. _officialis_ fruit on 1 September. WZY6\(-\)1, WZY6\(-\)2, and WZY6\(-\)3 represent the three repeats of limonin content in the _E. rutaocarpa_ var. _officialis_ fruit on 24 October.  
Figure 9: Analysis of the expression patterns of the genes related to the limonin synthesis pathway. (**a**) The expression pattern calculated by the qRT-PCR. (**b**) The expression pattern calculated by the FPKM values in the transcriptome (** indicates \(p<0.01\) and ** indicates \(p<0.05\)).