# Unraveling the Biosynthesis of Carvacrol in Different Tissues of _Origanum vulgarae_
## 2 Results
### SMRT Sequencing Achieves Full-Length Transcripts of O. vulgare 'HS'  
To obtain the precise and comprehensive full-length sequences of _O. vulgare_ 'HS', we used the combination of SMRT and short-read sequencing technology via PacBio RS and Illumina platforms. First of all, we constructed two SMRT cells to obtain 21.84 Gb of clean data and acquired 308,935 circular consensus (CCS) reads (with the filter criteria of full passes \(\geq\) 3 and sequence accuracy \(>\) 90%; Figure S1), and 270,324 full-length non  
Figure 1: Photographs of _Origanum vulgare_ ‘Hot and Spicy’ plants at the full bloom stage. The aerial part of _O. vulgare_ ‘HS’ (**A**). The infloresence of _O. vulgare_ ‘HS’ (**B**). Different tissues of petals (PE; **(C)**), sepsis (SE; **(D)**), bracts (BR; **(E)**), leaves (LE; **(F)**), and stems (ST; **(G)**). Pellate glandular trichomes of _O. vulgare_ ‘HS’ (**H**).  
Figure 2: Analysis of volatile organic compounds from different tissues of _Origanum vulgare_ ‘Hot and Spicy’. Score plot (**A**), loading plot (**B**), and variable importance in projection values (**C**) from PLS-DA model based on the chemical profiles of all samples. UpSet plot (**D**) and shared component Heatmap (**E**) based on the volatile organic compounds from different tissues of _Origanum vulgare_ ‘Hot and Spicy’.  
chimeric (FLNC) reads were identified (FLNC % = 87.50%; Figure S2). Accordingly, we used SMRTLink software to cluster these FLNC reads and achieved 101,447 consensus isoforms (Figure S3A). After polishing, we obtained 97,949 high-quality isoforms (sequence accuracy > 99%) and revised the low-quality isoforms using Proofread software compared to relevant Illumina RNA sequence data. We merged all the high-quality and revised consensus isoforms, removed sequence redundancy through cDNA_Cupcake, and ultimately obtained 53,802 non-redundant sequences with an N50 of 2232 bp. Next, we used the MIcroSAtellite identification tool to analyze non-redundant sequences and certified 37,217 SSRs (Figure S4), and applied TransDecoder software to predict complete 40,365 CDSs and relevant protein sequences (Figure S3B; Table S2).  
A total of 49,175 isoforms were annotated after analyzing the annotation, route, and functional categorization of SMRT data. Among them, 37,697, 49,049, 35,702, 21,634, 31,164, 40,288, and 21,462 isoforms were respectively annotated according to the GO (Gene Ontology; Figure S3C), NR (Non-Redundant; Figure S3D), Swissprot, COG (Clusters of Orthologous Groups; Figure S5), KOG (euKaryotic Ortholog Groups; Figure S6), Pfam (Protein family), and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases. In detail, 37,697 isoforms annotated according to the GO database were classified into 3 main categories and distributed across 50 subcategories (Figure S3C). For the biological process (82,110 isoforms), the greatest number of isoforms were enriched in'metabolic process' (20,210 isoforms; 24.61%), 'cellular process' (18,902 isoforms; 23.02%), and'single-organism process' (14,270 isoforms; 17.38%). For the molecular function (45,571 isoforms), the greatest number of isoforms were enriched in 'catalytic activity' (20,561 isoforms; 45.12%) and 'binding' (18221 isoforms; 39.98%). As shown in Figure S3D, there were three major species (_Sesamum indicum_, 30,719 isoforms, 62.64%; _Erythranthe guttata_, 8640 isoforms, 17.62%; _Salvia miliorrhiza_, 895 isoforms, 1.83%) with high similarity to _O. vulgare_ 'HS'. In this setting, SMRT technology was an efficient method for further biological study of plants and yielded a comprehensive transcriptome resource of _O. vulgare_ 'HS'.