## 2 Results  
### RNA-seq-Based Approach for Deciphering Conserved Expressions of Metabolic Enzymes  
To elucidate the transcriptional regulation of both primary and secondary metabolic pathways in the trichomes of the Cannabis plant, an RNA-sequencing (RNA-seq) approach was pursued. Five Cannabis cultivars, Chemdawg (CD), Ghost OG \(\times\) NBK (nothing but Kush) (GT), Westside OG (WS), Tahoe OG \(\times\) NBK (TH), and Headband (HB), were selected for investigation, as they are commonly planted (Figure 1a). CD is a sativa cultivar, while GT, WS, TH, and HB, are indica-dominant cultivars. All of them are known to produce high levels of THC, along with lesser amounts of CBD and other minor cannabinoids. The transcriptome of the trichomes was compared with that of a representative stem of the HB cultivar, whose trichomes were removed entirely. Furthermore, differences in the biosynthetic pathways among the trichomes of the cultivars were picked out to provide the molecular basis for profiling their yield variations. Figure 1: Quality assessment and differential analysis of gene expression data. (**a**) Picture of Cannabis cultivars selected for investigation. (**b**) Score plot of trichome and stem samples according to the first and second components (PC 1 and PC 2) obtained from a principal component analysis. (**c**) Plot of average gene expression values for the trichomes of five cultivars versus the corresponding expression in the stem tissue of HB. (**d**) M-D plot of global gene expression data. (M-value: log\({}_{2}\) FC value; D-value: absolute-difference-in-expression value). Red dots in (**c,d**) denote genes that are differentially expressed. HB: Headband; CD: Chem Dawg; GT: Ghost OG \(\times\) NBK; TH: Tahoe OG \(\times\) NBK; WS: Westside OG. RNA-seq yielded between 248 to 277 million raw reads in both RNA strand directions per sample (Table 1), with 3-8% of the reads subsequently removed by pre-processing and quality-control measures. The resulting reads were quasi-mapped to the latest reference transcriptome of _Cannabis sativa_ (GenBank assembly accession: GCA_900626175.2) for the generation of transcript read counts and further corrected for sequencing biases. In the process, between 200 to 235 million reads were used to tabulate the transcripts in each sample, representing 86-88% of the initial raw reads. The read counts of the transcripts were then added up for each gene. As a result, a total of 22,578 genes had reads mapped to their sequences in at least one sample. After further data normalization to enable sample and gene comparisons, the data quality was assessed using principal component analysis, which revealed a tight clustering of trichome samples away from the stem sample, indicating the absence of any trichome outliers (Figure 1b). For each cultivar, the global gene expression values of the trichomes were then plotted against those of stem tissue using a scatter plot, revealing their broadly similar distribution vis-a-vis the stem tissue: most genes lay near the diagonal through the origin and were thus not differentially expressed, as should be the case after normalization (Figure S1). Their alikeness was also apparent from the plot of absolute-difference-in-expression values (D-values) versus log\({}_{2}\) FC values (M-values), thereby confirming the broad consistency in data quality among the cultivars. A subsequent hierarchical clustering of the M-values of DEGs further revealed regions of similar expression patterns between the GT and TH cultivars, and between WS and TH, with the former being possibly attributed to their common NBK ancestry (Figure S2a). On the other hand, CD and HB showed distinctive profiles. Overall, the five cultivars had about half of their DEGs in common, with 10% (or fewer) unique to each cultivar (Figure S2b, Table S3). Taken together, the observations suggested a moderate degree of diversity among the cultivars.