## 4 Materials and Methods
### qRT-PCR Analysis  
The qRT-PCR reactions were conducted using a Thermal Cycler Dice(tm) Real Time System III TP970 (TAKARA, Dalian, China), which is a real-time fluorescence quantitative PCR instrument. The total reaction volume was 25 \(\mu\)L, which included TB Green(r) Premix Ex Taq(tm) II (Tli RNaseH Plus) (TAKARA, Dalian, China) at 12.5 \(\mu\)L, cDNA at 2 \(\mu\)L, primer at 1 \(\mu\)L, and RNase-free water at 8.5 \(\mu\)L. The preparation process was performed on ice. The cDNA template was diluted 10 times as the template before use, and the two-step PCR amplification protocol was as follows: 95 \({}^{\circ}\)C for 30 s (1 cycle), followed by 95 \({}^{\circ}\)C for 5 s and then 60 \({}^{\circ}\)C for 30 s (40 cycles), ending with dissociation. Each reaction was repeated three times biologically and three times technically. After the reaction, the primer's amplification specificity was determined based on the dissociation curve analysis.  
To conduct the qRT-PCR pre-experiments, cDNA from the different tissues and fruits at the various developmental stages (reverse transcribed from 1 \(\mu\)g RNA) were mixed. The mixed template was then diluted to concentrations of 5\({}^{-1}\), 5\({}^{-2}\), 5\({}^{-3}\), 5\({}^{-4}\), and 5\({}^{-5}\) times the original concentration. These pre-experiments were carried out on the ten candidate reference genes. The standard curves were generated, yielding the slopes (\(k\)) and linear correlation coefficients (\(R^{2}\)). The amplification efficiency of the reference gene primers was calculated using the formula E = (10\({}^{-1/k}\) - 1) \(\times\) 100%.  
\begin{table}
\begin{tabular}{c c c} \hline
**Gene** & **Full Name and the E.C. Number** & **Primer Sequence (5\({}^{\prime}\)\(\rightarrow\) 3\({}^{\prime}\))** \\ \hline _ISPE_ & 4-diphosphocytidyl-2-C-methyl-D-erythitol kinase (EC: 2.7.1.148) & F:CCACCAACTCCATCTACCT \\  & R:CCTGTCTACTCTTCATCTGCC \\ \hline _ISPF_ & 2-C-methyl-D-erythitol 2,4-cyclodiphosphate synthase (EC: 4.6.1.12) & F:GCTCCAAAATCAAAAATACAT \\  & R:AATCGCATCCACAACACAG \\ \hline _ISPG_ & (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase (EC: 1.17.7.1) & F:CAGAAGCACCAGAGAGAGGAGA \\  & R:CCAGAACGACGATGAAAAT \\ \hline _ISPH_ & 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (EC: 1.17.7.4) & F:AAATCATTCACAACCCGC \\  & R:CCAGACCTTAGACACCCA \\ \hline _FPS/GPS_ & Geranyl pyrophosphate synthetase/Farnesyl pyrophosphate synthetase (EC: 2.5.1.1 2.5.1.10) & F:GGAAAAATTACAGGCCA \\  & (EC: 2.5.1.1 2.5.1.10) & R:AGACAGGCCAAGTAGAGCA \\ \hline _ACAT_ & acetyl-CoA C-acetyltransferase (EC: 2.3.1.9) & F:CCTGTGAAAGGGGAAAAC \\  & R:GAGCCGCCATCAGCAAATC \\ \hline _HMGCS_ & hydroxymethylglutaryl-CoA synthase (EC: 2.3.3.10) & F:TGGCACATCATCATCTCCCTCG \\  & R:CACAGTTTCGCCTTCTCT \\ \hline _HMGCR_ & hydroxymethylglutaryl-CoA reductase (EC: 1.1.1.34) & F:CGACGAACAAAGAG \\  & R:GTAAAACCCAACGGAAAAG \\ \hline _MVK_ & mevalonate kinase (EC: 2.7.1.36) & F:ACACAGAATTGGGAGA \\  & R:TATGAGGAATGGCTGC \\ \hline _MVAK_ & phosphomevalonate kinase (EC: 2.7.4.2) & F:TCAGAAGAGCCAT \\  & R:GATCAAGAACACAGAGCAT \\ \hline _MVD_ & diphosphomevalonate decarboxylase (EC:4.1.1.33) & F:TCGTAGTCTTTTTGGGTGA \\  & R:TGTATTACGCTTTTCGG \\ \hline _IDI_ & isopentenyl-diphosphate Delta-isomerase (EC: 5.3.3.2) & F:CAGCCATCCTCTATTCCGC \\  & R:ATCAAGTTCATGTCCTCCC \\ \hline _SQS_ & squalene synthase (EC: 2.5.1.21) & F:TGACACACACACAC \\  & R:AATGCCTCTCTCTGATAACC \\ \hline _SQE_ & squalene epoxidase (EC: 1.14.14.17) & F:GGTTCGGTGTGTGTGA \\  & R:GCTTCTATTTGCCATGTTT \\ \hline _CYP450_ & cytochromeP450 (1.14.x.x) & F:GGTCAGGTAGCCGTT \\  & R:TCTCATCCGCGGTAGCC \\ \hline \end{tabular}
\end{table}
Table 3: _Cont._