# Unraveling the Biosynthesis of Carvacrol in Different Tissues of _Origanum vulgarae_
## 2 Results
### RNA-Seq Data Reveal Carvacrol Biosynthesis in Different Tissues of O. vulgare 'HS'  
To identify the tissue-specific expression of key genes involved in carvacrol production in _O. vulgaris_ 'HS' aerial parts, fifteen RNA samples from five different tissues with three biological replications of STs (ST1-3), LEs (LE1-3), BRs (BR1-3), SEs (SE1-3), and PEs (PE1-3) at the full bloom stage were subjected to short-read sequencing. Totally, we obtained 100.18 G clean data with a Q30 \(>\) 85% and an approximate GC content of 48.9% (Table S3). Moreover, we conducted the sequence alignment of short-read sequencing-based clean reads with the reference SMRT-based non-redundant transcripts, enabling us to achieve the expression patterns of SMRT transcripts (Table S4). To test the repeatability and reliability between sample biological replications, we observed the transcriptional abundance of each sample based on FPKM density and values (Figure S7A, Figure S7C), and conducted the Pearson Correlation Coefficient analysis of expression levels among samples (Figure S7B). Figure S7 indicates that the SE1 sample was extraordinary compared to SE2 and SE3. Biological replications of other PE, BR, LE, and ST samples clustered together. As a whole, we removed the SE1 sample and used the other 14 samples to carry out further gene differential expression analysis to obtain tissue-specific DEGs (FC > 2; FDR < 0.01; Table S5). In this study, we predicated the biosynthesis pathways of monoterpenes and sesquiterpenpenes in _O. vulgare_ 'HS', especially for carvacrol. As a result, we investigated the expression of DEGs encoding enzymes in different tissues. The DEGs were subjected to the filters of FC > 2 or < 0.5 and FDR < 0.01. Analysis of our transcriptome dataset revealed 24 (across 7 gene families) and 37 (across 6 gene families) candidate DEGs of _O. vulgare_ 'HS' related to MVA and MEP pathways, respectively. As for MVA genes, 19 DEGs (79.17%) exhibited high expression levels in STs, 16 DEGs (66.67%) in SEs and PEs, 13 DEGs (54.17%) in BRs, and 10 DEGs (41.67%) in LEs (FPKM > 10), while STs presented more highly expressed DEGs than other tissues (FC > 2) (Figure 3A). Notably, five DEGs were most highly expressed in STs, comprising three IDIs (A_transcript_50897, A_transcript_19352, A_transcript_82985), one _AACT_ (A_transcript_14352), and one _HMGR_ (A_transcript_9446) (FPKM > 100). Two _IDIs_ (A_transcript_19352, A_transcript_82985) were most highly expressed in PEs (FPKM > 100). As for MEP genes, 24 DEGs (64.86%) exhibited high expression levels in BRs, 23 DEGs (62.16%) in SEs, 21 DEGs (56.76%) in LEs, 16 DEGs (43.24%) in PEs, and 14 DEGs (37.84%) in STs (FPKM > 10) (Figure 3B). Compared to STs, 21, 23, 19, and 15 DEGs in SEs, BRs, LEs, and PEs presented upregulated expression (FC > 2). Significantly, three _HDR_s (A_transcript_14338, A_transcript_97750, A_transcript_35759) were most highly expressed in SEs, BRs, PEs, and one _HDS_ (A_transcript_81125) in SEs (FPKM > 10; FC > 100). The gene expression patterns of five _GPPS_s and six _FPPS_s (Figure 3C) revealed that three of five _GPPS_s exhibited upregulated expression in SEs, LEs, and BRs (FC > 2) compared with STs, especially for _GPPS_ (A_transcript_46736) highly expressed in SEs (FPKM = 38.02; FC > 100). Observably, _FPPS_s, contributing to sesquiterpene biosynthesis, presented more downregulations in PEs (6/6), SEs (4/6), BRs (4/6), and LEs (4/6) than in STs (FC < 0.5). In general, tissue-specific expression of MEP genes revealed maximum expression in SEs, followed by BRs, LEs, PEs, and STs. Conversely, MVA genes were highly expressed even in STs, followed by SEs, PEs, BRs, and LEs. Transcriptomic Analysis of Tissue-Specific Expression Patterns of TPS/CYP/SDR Genes in O. vulgare 'HS'  
In _O. vulgare_ 'HS', various distinct TPSs can be predicted for final products of monoterpenes and sesquiterpenes, especially for carvacrol content in SEs, PEs, LEs, and BRs, but not in STs.