## 3 Results
### DEGs Related to terpenoids Biosynthesis  
In the present study, there were a total of 34 DEGs between high and low oleoresin-yielding slash pine identified to function in the MEP and MVA pathways that are associated with the biosynthesis of terpenoids (Figure 2A). Annotated DEGs associated with 1-deoxy-d-xylulose 5-phosphate synthases (_dxs_), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), 2-C-methyl-d-erythitol 4-phosphate cytidyltransferase (_ispD_), 4-diphosphocytidyl-2-c-methyl-d-erythnitol kinase, 2-c-methyl-d-erythnitol 2,4-cyclodiphosphate synthases (_ispF_), and (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase (_ispG_) in the MEP pathway, and mevalonate kinase, phosphomevalonate kinase, as well as diphosphomevalonate decarboxylase in the MVA pathway encode enzymes that are involved in the biosynthesis of IPP and its isomer DMAPP. In addition, there were also DEGs encoding (2Z,6Z)-farnesyl diphosphate synthase (_ZFPS_) (_c213774.graph_c0_), geranylgeranyl diphosphate synthases (_idsA_) (_c228743.graph_c0_ and _c216266.graph_c1_), and all-trans-nonaprenyl-diphosphate synthase (_SPS_) (_c206960.graph_c0_) that catalyze the condensation reactions to form GPP, FPP, and GGPP. There were also nine DEGs associated with TPS, including six isoprene synthases (_ispS_), two (+)-\(\alpha\)-pinene synthases (_PT30_), and one (-)-\(\beta\)-pinene synthase(_QH6_) encoding genes(Figure 2A). Intriguingly, not all these abovementioned DEGs were upregulated in high-yield slash pine. For instance, _QH6_ (_C204351.graph_c1_), _idsA_ (_c216266.graph_c1_), and _ZFPS_ (_c213774.graph_c0_) were significantly downregulated in high-yielding slash pine at different stages. Moreover, the DEGs in relation to MEP pathway had dramatically higher expression levels in high-yielding slash pine than in low-yielding slash pine, whereas this pentomenon was not observed in the expression profile of DEGs related to the MVP pathway. These results indicate that the high oleoresin yield of high-yielding slash pine is attributed to the enhanced terpenoid biosynthesis mediated by the MEP pathway.  
To confirm the reliability of the results of DEGs from RNA-seq, seven representative DEGs were selected for qRT-PCR analysis (Figure 2B). The relative expression levels of _c204351.graph_c1_ and _c223139.graph_c0_ in the low-yielding slash pine were lower than high-yielding slash pine, while the other five genes, _c214654.graph_c1_, _c193143.graph_c1_, _c220531.graph_c1_, _c206960.graph_c0_, and _c213744.graph_c0_, were highly expressed in the high-yielding slash pine. Moreover, the percentage proportion of \(\alpha\)-pinene in the oleoresin produced by the high-yielding slash pine is remarkably lower than low-yielding slash pine, while the \(\beta\)-pinene found in the high-yielding slash pine is rather notably higher than in low-yielding slash pines (Figure 2C). Collectively, the data of the qRT-PCR and pineine content measuring results were consistent with the RNA-seq results, indicating the high level of reliability of the RNA-seq data used in this study.  
Figure 2: DEGs associated with the biosynthesis of terpenoids and steroids. **(A)** Typical DEGs in the biosynthesis of terpenoids and steroids pathway. The boxes colored red and green represent upregulated and downregulated genes, respectively. H or L refer to high or low oleoresin-yielding slash pine. **(B)** Results of RT-PCR of 7 selected DEGs between high- and low-yielding slash. All exhibitions were standardized by the expression level of _Actin_ (_c2171751.graph_c0_). **(C)** The content of \(\alpha\)- and \(\beta\)-pinees in the vascular cambium of high- and low-yielding slash pines at P4, P7, and P10. All bars represent means \(\pm\) s.d and those columns in histograms labeled with different letters are significantly different with \(p\) value < 0.05 by Duncan’s test. Four biological replications in the experiment were carried out.