# Transcriptome Landscape Variation in the Genus _Thymus_
## 3 Results and Discussion
### Thymol Biosynthetic Pathway  
Triannotated transcriptome contigs were analyzed and mined for putative genes involved in thymol biosynthesis. Thymol formation is proposed to be through p-cymene and \(\gamma\)-terpinene intermediates [80; 81]. Terpenes are derived from two five-carbon precursors, DMAPP (Dimethylallyl diphosphate) and IPP (Isopentenyl diphosphate), generated through the MVA (Mevaluation) and MEP (2-C-methylerythnitro1-4-phosphate) pathways, respectively. IPP and DMAPP are subsequently converted to GPP (Geranyl pyrophosphate), GGPP (Geranyl geranyl pyrophosphate) and FPP (Farnesyl pyrophosphate). GGDS occurs in both homomeric and heteromeric forms. The homomeric form is composed of a large subunit (LSU) and the heterodimer form consists of an LSY and a small subunit (SU). Monoterpenes are produced from GPP. Genes associated with terminal steps leading to thymol biosynthesis are GGDS, \(\gamma\)-terpinene synthase (GTP) and cytochrome P 450 71 family (Figure S2). Based on the integrated annotation process, 81, 91, 80, 97, and 94 transcripts were annotated as 11 genes involved in thymol and other monoterypes biosynthesis (Table S5) in Tdae, Tvul, Tian, Tper, and Tpub, respectively. For _DXS_ (1-deoxy-D-xylulose 6-phosphate (DOXP) synthase), _HDS_ (4-hydroxy-3-methyl-but-2-enyl diphosphate (HMB-PP) synthase), _HDR_ (4-hydroxy-3-methyl-but-2-enyl diphosphate (HMB-PP) reductase), and _TPS_, there were high contig numbers (9 to 25 transcripts). _DXR_ (1-deoxy-D-xylulose 6-phosphate reductoisomerase) and _IDI_ (Isopentenyl diphosphate isomerase) had moderate numbers of contigs (3-10), while _MCT_ (2-C-methyl-D-erythnitro1 cytidyltransferase), _CMK_ (4-diphosphocytidyl-2-C-methyl-D-erythritol kinase), _MDS_ (2-C-methyl-D-erythritol 2,4-cyclodextrin diphosphate synthase), and _GDS_ (Geranyl diphosphate synthase) all had fewer than four contigs. It is reasonable to expect that the differences in contig number  
Figure 4: Distribution of transcripts in different transcription factor (TF) families showing over-representation in bHLH. reflects differences in transcript abundance. Given this, the MEP pathway would seem to be the major contributor of monoterpene thymol production, given the higher numbers of contigs associated with this pathway. Due to the high gene expression level of monoterpene synthase genes in leaves, a query-based search showed the presence of complete coding regions for most of these genes (Table S6). However, IDI, GDS, and some terpene synthases (TPSs) were found as partial sequences in some species. TPSs are highly diversified throughout the plant kingdom and many exhibit tissue-specific expression. Analysis of the several plant genomes indicates TPS genes are a mid-size family, with the gene number ranging from 18 to 152, although not all of them have activity [82]. To date, a number of TPSs including TPS1-TPS9 have been identified and characterized in _T. Vulgaris, T. caespittius_, and _T. albicans_[83; 84; 85; 86]. Nine to 25 _Thymus_ transcripts were identified as putative TPS genes in different species grouped into four TPS classes, including TPS2, TPS3, TPS4, and TPS5. Among the identified TPS genes, the maximum number of unigenes was identified for TPS5, followed by TPS3. TPS5 displays a responsibility for the generation of volatile monoterpenes because of a similar trend with the emission of monoterpenes [87; 88]. Through the homology search in the current assembled transcriptomes, the full-length sequences were identified for TPS5 in all species, TPS2 in only Tdae, TPS3 in Tvul, Tian, and Tper, and finally TPS4 in all species except Tper. Global alignment revealed a >89% similarity between TPS5 and the sequence reported in NCBI (KC461937.1) belonging to _T. vulgaris_ with length of 1795 bp. The sequences of unigene candidates encoding terpenoid biosynthesis enzymes are listed in Additional File 1. All four putative TPS sequences displayed high similarity to known plant TPS genes based on a comparison at the amino acid level.