# Transcriptome Landscape Variation in the Genus _Thymus_
## 3 Results and Discussion
### Database Searches and Gene Ontology  
We used the BinPacker assemblies for each species as queries and ran BLASTX to search against the UniProt database with no e-value cut-off, but an e-value of \(1\times 10^{-5}\) as the threshold to report the results. A total of 39,968 (percentage of all unigenes in a species: 60.02%), 54,555 (61.33%), 53,922 (56.79%), 41,372 (61.58%), and 44,347 (65.39) unigenes of Tdae, Tvul, Tian, Tper. and Tpub, respectively, were annotated. Overall, Tpub had a higher percentage of unigenes annotated compared to the other species. The number of predicted proteins ranged from 13,571 to 11,549. Tvul and Tian had the highest numbers of predicted proteins, possibly because a greater size of sequencing data can provide a better assembly. Furthermore, predicted protein lists with significant sequence similarity were further used to compare the number of shared proteins among species (Figure 1). Five assemblies shared 6158 proteins. The highest number of protein sequence homology (21.5-24%) was shared between Tian and Tvul (9447 proteins), while Tian and Tpub shared the lowest number of proteins (8596 proteins), followed closely by Tdae and Tper (8597 proteins). To categorize the biological functions of transcripts, the contigs having sequence homology with UniProt annotations were given GO (gene ontology) assignments under biological processes, cellular components, and molecular function domains. The distribution of functional categories for all five transcriptomes was visualized using the Web Gene Ontology Annotation Plotting (WEGO) application [32]. The derived graph (Figure 2) indicates that enriched gene ontology term annotations were broadly distributed across the three main domains, and the percentages of sequences mapped to  
Figure 1: Venn diagram showing the number of overlapping proteins between the five _Thymus_ transcriptome assemblies. a given sub-ontology were highly similar for all species. Based on the obtained results, most of the GO terms were significantly overrepresented in cellular component domain. Within this category, cell class, followed by cell part and intracellular classes, were predominant. In the biological process domain, the two most common categories were cellular process and metabolic process. As far as molecular function, the most abundant classes were binding and catalytic activity. Overall, the GO term abundance results revealed that the main GO classifications were metabolism and fundamental biological regulation, and a majority of unigenes involved in terpenoid biosynthesis were found in the categories of metabolic processes. These results are similar to other terpene-producing plant species such as _Salvia miltiorrhiza_[61] and _Melaleuca alternifolia_[62]. According to functional annotations based on the COG databases, unigenes were clustered into 24 COG categories (Figure 3). The largest category was function unknown, followed by posttranslational modification, transcription, and signal transduction mechanisms. Nuclear structures and extracellular structures were the smallest groups. The number of assigned proteins to secondary metabolite biosynthesis ranged from 350 to 409. The presence of genes with unknown functions in these species suggests that the regulation of biosynthesis of secondary metabolites may involve novel factors that require further study. Figure 2: Gene ontology (GO) classification map derived from _Thymus_ transcriptomes. The results are summarized in three GO categories: biological process, molecular function, and cellular component. The x-axis indicates the percentage of sequences in each category. Based on the KEGG assignment, the majority of predicted protein-coding genes were distributed under the \"metabolism\" classification (Figure S1). The genes in this category were further classified into 12 subcategories, of which \"global and overview maps\" were the most prevalent, followed by \"carbohydrate metabolism\", while the metabolism of terpenoids and polyketides categories contained a few gene members.