## 4 Materials and Methods
### RNA Isolation and Quantitative Real-Time PCR (qRT-PCR)  
For expression analysis of genes involved in flower color and scent, approximately 200 mg of petal sample was used. The samples were ground into a powder in liquid nitrogen, and total RNA was extracted using a modified CTAB method [114; 115]. Genomic DNA contamination was eliminated using _DNase I_, RNase-Free DNase Set (Fermentas, Waltham, MA, USA). The quality and quantity of RNA samples were assessed using agarose gel electrophoresis and Nanodrop ND-1000, respectively [116; 117]. To synthesize the first strand complementary DNA (cDNA), 1 \(\upmu\)g of total RNA was converted into cDNA using the cDNA synthesis kit (A101161, Reverse Transcription Kit made by Parstous Iran) following the manufacturer's instructions. The main genes identified by GC-MS data analysis and literature review were selected (Figures 8 and 9).  
The online version of primer3 software [118] was used to design the corresponding primers for _PAL_, _FLS_, _MYB1_, _PAR_, _GPS_, _GGPS_, _ANS_, _CCD1_, _LIS1_, _AAT1_, _DXR_, _DXS_, and _CER1_ genes. The primer efficiency was checked using the Oli-go-analyzer tool and NCBI/Primer-BLAST software ([[https://www.ncbi.nlm.nih.gov/tools/primer-blast/](https://www.ncbi.nlm.nih.gov/tools/primer-blast/)]([https://www.ncbi.nlm.nih.gov/tools/primer-blast/](https://www.ncbi.nlm.nih.gov/tools/primer-blast/))). Table 4 lists the sequences of the forward and reverse primers used for qRT-PCR. The beta-actin gene served as a housekeeping gene for normalizing the expression of target genes. QIAGEN's real-time PCR system was used to perform qRT-PCR following the manufacturer's instructions [119; 120]. Each reaction consisted of a 15 \(\upmu\)L mixture containing 7.5 \(\upmu\)L of SYBR green Master Mix 2X (ROX), 4.7 \(\upmu\)L of RNase-free H\({}_{2}\)O, 2 \(\upmu\)L of cDNA, 0.4 \(\upmu\)L of forward primer, and 0.4 \(\upmu\)L of reverse primer. The qRT-PCR thermal and temporal profiles were set to 15 min at 95 \({}^{\circ}\)C for initial denaturation, followed by 40 three-step cycles of amplification: 15 s of denaturation at 95 \({}^{\circ}\)C, 20 s of annealing at 56-59 \({}^{\circ}\)C (depending on the specific annealing temperature for each primer pair according to the gradient PCR results), and 20 s of extension at 72 \({}^{\circ}\)C. The 2\({}^{-\Delta\Delta\text{ACT}}\) method [121] was used to calculate the relative expression levels of mRNAs. SAS 9.4 software was used to analyze the gene ex pression results based on a Completely Random Design (CRD). [PERSON]'s test was applied to examine the mean values of significance at the 5% (\(p\leq 0.05\)) and 1% (\(p\leq 0.01\)) levels.  
Figure 8: Schematic representation of the MEP pathway for terpenoids biosynthesis in roses. G3P, glyceraldehyde-3-phosphate; _DXS_, 1-deoxy-d-xylulose-5-phosphate synthase; _DXR_, 1-Deoxy-D-xylulose 5-phosphate reductoisomerase; _LIS_, linalool synthase; _AAT1_, aromatic amino acid aminotransferase; _CCD1_, carotenoid cleavage dioxygenase 1.  
Figure 9: Schematic illustration of phenylpropanoids/benzenoids biosynthesis in rose flowers. _PLAL_-phenylalanine ammonia lyase; _PAR_-phenyl acetaldehyde reductase; _AAT_-alcohol acetyltransferase.  
To examine the patterns of similarity among the gene expression results, cluster analysis was performed using the R package. A heatmap was generated to visualize the clustering results based on the Ward distance method. The heatmap indicates the degree of association between the flower color and scent biosynthesis pathway by using a color scale [122; 123].