# Transcriptome Landscape Variation in the Genus _Thymus_
## 4 Conclusions  
In this study, we presented annotated transcriptomes for five different _Thymus_ species using mRNA sequencing (RNA-seq), providing valuable resources for studying molecular mechanisms of terpenoid biosynthesis, the development of molecular markers, and evaluating phylogenetic relationships among species. This also led to the identification of putative genes involved in thymol biosynthesis. Most notably, three unigenes (germacrene D synthase, \(\gamma\)-terpinene synthase, and a member of the cytochrome P450 71 family) were identified as probable terminal steps of thymol biosynthesis. GO, COG, and KEGG enrichment analysis revealed that numerous contigs encode enzymes involved in secondary metabolic pathways. The functional characterization identified key TPS genes that were clustered into the TPS-b subfamily. The phylogenetic tree based on single-copy orthogroups showed a close genetic relationship between _T. daenensis_ and _T. vulgaris_. Between 50 and 60 genes showed evidence of positive selection in the _Thymus_ lineages. The present analysis based on L-shaped Ks distribution indicated that WGD is probably absent in _Thymus_ species, or that WGD has probably not contributed substantially to duplicated genes in these species, and that polyploidy does not appear to be responsible for the large genome size in _T. lancifolius_, _T. persicus_, or _T. pubescens_. The study also provides worthy resources for a bioengineering study of terpenoids in _Thymus_ species with pharmacologically increased metabolite content.  
**Supplementary Materials:** The following are available online at [[http://www.mdpi.com/2073-4425/10/8/620/s1](http://www.mdpi.com/2073-4425/10/8/620/s1)]([http://www.mdpi.com/2073-4425/10/8/620/s1](http://www.mdpi.com/2073-4425/10/8/620/s1)), Figure S1: KEGG classification of unigenes for _Thymus_ transcriptomes, Figure S2: Schematic representation of thymol and carvoxel biosynthetic pathway, Figure S3: GO enrichment of PSGs related to ecological adaptation in _Thymus_ species, Table S1: Assembly statistics results of _Thymus_ species, Table S2: Velvet/Oases assembly statistics results, Table S3: The number of full-length transcripts for three assemblers and five species, Table S4: Number of transcripts for each transcription factor genes families of _Thymus_ species, Table S5: The contigs number of 11 genes involved in thymol and other monoterrene biosynthesis identified in _Thymus_ transcriptomes, Table S6: Lengths of genes involved in thymol and other monoterrene biosynthesis identified in _Thymus_ transcriptomes, Table S7: The unigenes subjected to positive selection.  
**Author Contributions:** [PERSON] and [PERSON] designed the experiment. [PERSON], [PERSON] and [PERSON] collected the samples. [PERSON], [PERSON], and [PERSON] performed RNA extraction and prepared the libraries. [PERSON] carried out bioinformatics and comparative analyses. [PERSON] and [PERSON] wrote the manuscript. All authors reviewed and approved the final manuscript for publication.  
**Funding:** This research was funded by the Iran National Science Foundation (INSF, No. 96014503) for the financial support of part of this work.  
**Conflicts of Interest:** The authors declare no conflict of interest.