# Transcriptomic Analysis of the Candidate Genes Related to Aroma Formation in _Osmanthus fragrans_
## 2 Results
### Annotation and Functional Classification  
To determine the putative functions of unigenes, the transcriptome was annotated using protein functions, pathways, KOG (euKaryotic Ortholog Groups) functions, and GO (Gene Ontology) annotations. The unigenes were aligned using the BLASTx program with an E-value threshold of \(10^{-5}\) to the Nr (NCBI non-redundant protein), the Swiss-Prot protein, and the KEGG (Kyoto Encyclopedia of Genes and Genomes) and KOG databases for which the percentages of the annotated unigenes were 42.86, 34.62, 28.58, and 17.44%, respectively (Table 2). Approximately 47.43% of the unigenes were aligned. According to the best alignment results to the species of homologous sequences, the number of homologous sequences of the top ten species is shown in Figure S2.  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Transcript** & **Unigene** \\ \hline Total assembled bases & 108,311,010 \\ Total number of genes & 136,611 \\ Max length of unigenes (bp) & 16,876 \\ Min length of unigenes (bp) & 201 \\ Average length ofunigenes (bp) & 792 \\ N50 (bp) & 1424 \\ GC percentage (\%) & 38.9215 \\ \hline \hline \end{tabular}
\end{table}
Table 1: The Summary of the _O. fragrans_ transcriptome.  
Figure 1: Photographs of flowers at different flowering stages: (**S1**): Initial flowering stage, (**S2**): Full flowering stage, and (**S3**): Final flowering stage.  
The GO offered a strictly defined concept to comprehensively describe the properties of genes and their products in any organism. The GO functional annotations of the \"RiXiangGui\" unigenes were classified into three categories (Molecular Function, Cellular Component, and Biological Process) and 50 subcategories (Figure 2). There were 77,416 (56.67%) unigenes assigned to 20 subcategories in the Biological Process for which \"metabolic process\", \"cellular process\", and \"single-organism process\" were the dominant subcategories. The molecular function categories with the most abundant members were \"catalytic activity\" and \"binding\"; in the Cellular category, \"cell\", \"cell part\", and \"organelle\" were the abundant classes.  
In total, 136,611 out of the 39,043 unigenes showing Nr hits were assigned to COG classifications and grouped into 25 categories (Figure S3). The cluster of \"general function prediction\" was the largest group, representing 35.72%, followed by \"post-translational modification, protein turnover, chaperones\" and \"signal transduction mechanisms\". There were 1537 unigenes in the \"secondary metabolites biosynthesis, transport, and catabolism\" group.  
A total of 23,825 unigenes were annotated and 23,166 sequences were assigned to 132 KEGG pathways (Figure S4). Metabolism (~61.21%) had the largest number of sequences in the primary pathway hierarchy and the unigenes of Genetic Information Processing followed (~27.15%). The flower scents and colors were the main ornamental traits of _O. frigrans_, so we focused on the secondary metabolic pathways, especially those of phenylpropanoids, carotenoids, flavonoids, flavones and flavonoid, anthocyanins, and terpenoids. These data provided a valuable resource and narrowed down the special pathways and candidate genes of interest in _O. frigrans_.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Database** & **Number of Annotated Unigenes** & **Percentage of Annotated Unigenes (\%)** \\ \hline Nr & 58,556 & 42.86 \\ Swiss-prot & 47,294 & 34.62 \\ KOG & 39,043 & 28.58 \\ KO (KEGG Orthology) & 23,825 & 17.44 \\ Total & 64,795 & 47.43 \\ \hline \hline \end{tabular}
\end{table}
Table 2: The statistics of annotation on unigenes against public databases.  
Figure 2: The GO functional classification of differentially expressed genes (DEGs) among different samples.