## 3 Results and Discussion
### Plastome Structure with Codon Usage and Repeat Sequences  
Plastome structure is influenced by multiple factors, such as the loss/inversion of genes, abundant repetitive elements, gene boundary shifts in IR regions, and gene rearrangements [36]. We analyzed the structural composition using codon usage bias and repeat sequences. Codon usage bias is influenced by nucleotide replication, sequence mutation, and the intensity of gene expression [37]. The RSCU index is an actual frequency ratio of each synonymous codon, and this index directly reflects codon usage bias [38]. To analyze codon usage bias, sixty-one synonymous codons and three stop codons (UGA, UAG, and UAA) were detected based on 75 newly annotated protein-coding genes. Twenty amino acids were encoded by the 64 detected codons. Only two amino acids (methionine and tryptophan) contained one codon, and eighteen amino acids contained two or more codons (Figure 2). Notably, three amino acids (leucine, arginine, and serine) showed complex compositions encoded by six codon types. The RSCU values ranged from 0.37 to 1.83 for all \begin{table}
\begin{tabular}{c c c} \hline \hline
**Category** & **Group** & **Gene Name** \\ \hline Photosynthesis & Photosystem I & _psaA_, _psaB_, _psaC_, _psal_, _psal_, _ycf3_ **, _ycf4_ \\ Photosystem II & _psbA_, \(B\), \(C\), \(D\), \(E\), \(F\), \(H\), \(I\), \(J\), \(K\), \(L\), \(M\), \(N\), \(T\), \(Z\) \\ NADH 1 & _ndhA_ *, \(B\) *, \(C\), \(D\), \(E\), \(F\), \(G\), \(H\), \(I\), \(J\), \(K\) \\ ATP synthase & _atpA_, \(B\), \(E\), \(F\) *, \(H\), \(I\) \\ Cytochrome 2 & _petA_, \(B\) *, \(D\) *, \(G\), \(L\), \(N\) \\ Rubisco unit & _rchL_ \\ \hline Self-replication & Small ribosome & _rps2_, \(3\), \(4\), \(7\), \(8\), _11_, _12_ **, _14_, _15_, _16_, _18_, _19_ \\  & Large ribosome & _rpl2_ *, _14_, _16_ *, _20_, _22_, _33_, _36_ \\  & RNA polymerase & _rpoA_, \(B\), \(C\) *, \(C\) \\ Ribosomal RNA & & _rm4.5_, \(5\), _16_, _23_ \\  & & _rmA_-UGC_ *, _rmC-GCA_, _trnD-GUC_, _trnE-LUIC_, _trnM-CAU_, _trnF-GAA_, _trnG-CCC_, _trnG-UCC_ *, _trnH-CUG_, _trnl-CAU_, _trnl-GAU_ *, _trnL-UUU_ *, _trnL-CAA_, _trnl-UAA_ *, _trnl-UAG_, _trnM-CAU_, _trnN-GUIU_, _trnP-LUGG_, _trnQ-LUIG_, _trnR-AGC_, _trnR-UCI_, _trnG-GCU_, _trnG-GCU_, _trnT-GCUU_, _trnT-GCU_, _trnS-GGA_, _trnS-UIGA_, _trnV-CCA_, _trnV-GUA_ \\ \hline Other genes & C-types cytochromes (_ccsA_), envelope membrane proteins (_ccmA_), protease (_clpP_ **), maturase (_matK_), unknown (_yf1_, _yyf2_) \\ \hline \hline \end{tabular}  
* NADH dehydrogenase; 2 chloroplast cytochrome b\({}_{\text{bf}}\) complex. Common names of family genes are omitted.
* and ** indicate that the gene contains one and two introns, respectively.  
\end{table} 
Table 1: Plastome gene composition in classified categories. codons, and synonymous codons were classified into three categories for measuring codon preference. Twenty-six high-frequency codons exhibited a preference region (RSCU > 1), thirty-two low-frequency codons had a frequency bias (RSCU < 1), and three codons (AGU, UGG, and CCU) displayed no frequency bias (RSCU = 1). High-frequency codons (RSCU > 1) play an important role in plastome mutations because they have a more positive bias, whereas low-frequency codons (RSCU < 1) have a negative bias on codon usage [39]. Of these high-frequency codons, 22 codons possess A or U nucleotides at the end point (third position of codons), and these high A/U preference (22/26, 85%) trends have been reported in other Campanulaceae species [40]. Plastome repeats play a major role in structural variations through insertion-deletion (InDel), duplication, and rearrangement [41]. To investigate structural variations, we identified the 145 repeat sequences consisting of 50 SSRs and 95 long tandem repeats. Generally, SSRs are mainly used in evolutionary studies because of their high variability in genetic polymorphism [42]. All SSRs ranged from 10 to 18 bp in length and were classified into three nucleotides types (thirty-eight mono-, four di-, and eight tri-).