## 2 Results and Discussion
### Gene Function Annotation and Classification  
All the 76,717 assembled putative unigenes were aligned using the BLAST program against the NR, NT, Swiss-Prot and COG databases with the E-value cutoff of 10\({}^{-5}\). A total of 33,855 unigenes were annotated, accounting for 44.13% (Table 3). Among them, 26,686 unigenes (34.78%) showed high homology, with sequences in the NR database, 24,371 unigenes (31.77%) matched to protein sequences in TAIR10, and 18,627 unigenes (24.28%) showed homology with known genes in SwissProt. The detailed results are shown in Tables 3 and S1-S3. Based on the top-hit species distribution of the homology result against NR databases, 26,361 unigenes (92.08%) showed high homology with sequences from land plants, among which the highest matches were to genes from _Coffea canephora_ (36.08%), followed by _Vitis vinifera_ (8.57%), and _Sesamum indicum_ (7.36%) (Figure 2). Putative protein sequences were obtained by translating using a standard codon table. The CDSs of unigenes that did not match the above databases were predicted with the ESTSCAN software. The gene length distribution is shown in Figure 3. The length of peptides predicted by BLASTp ranges from 60-810, while that of ESTSCAN are 30-240. In this study, all unigenes were searched against the GO database. Out of 76,717 unigenes, 26,494 were successfully annotated and classified into three GO categories: biological process, cellular component, and molecular function, and assigned to 56 functional groups (Figure 4). As shown in Figure 4, assignments which fell under cellular component ranked the highest, followed by biological process, and molecular function. In the biological process category, \"cellular process\" (16,075, 60.67%) and \"metabolic process\" (15,223, 57.46%) were the two most representative subcategories. In the cellular component category, unigenes related to \"cell\" (10,308, 38.91%) and \"cell part\" (10,282, 38.81%) were dominant, while in the molecular function category, the majority of unigenes were involved in \"binding\" (14,903, 56.25%) and \"catalytic activity\" (12,326, 46.52%). These results suggested that many kinds of enzyme pathways were active in gentian. A total of 10,524 sequences were classified into 26 KOG/COG (Clusters of Orthologous Groups of proteins) groups (Figure 5), where \"General function prediction only\" category accounted for the most frequent group (1948, 18.51%), with the second largest group being \"Post-translational modification, protein turnover, chaperon\" (1319, 12.53%), followed by \"Signal transduction\" (932, 8.86%) and \"Translation\" (654, 6.21%). These results showed that in the flower stage of gentian, the protein translation and signal transduction are active. Figure 3: Length distributions of predicated peptides. (**A**) Predicated by BLAST; (**B**) Predicated by ESTScan program (Available online: [[http://www.ch.embnet.org/software/ESTScan.html](http://www.ch.embnet.org/software/ESTScan.html)]([http://www.ch.embnet.org/software/ESTScan.html](http://www.ch.embnet.org/software/ESTScan.html))). The abscissa represents the peptide length, while the ordinate represents thee number of the corresponding number. The KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic system is a group of metabolic maps which represents current understanding of biomolecular interaction networks. In order to determine the active pathways in flowering gentian, KEGG assignments of all unigenes were performed. Figure 4: GO classification map. The abscissa represents the next level GO term of the three GO categories, while the ordinate represents the number of genes annotated into the corresponding term, and its proportion of the total number of annotated genes. Figure 5: KOG/COG classification map. The abscissa represents 26 group names of KOG/COG, while the vertical axis represents the number of genes annotated into the group and its proportion of total number of annotated genes. Referencing the 7998 unigenes of _G.