Figure 4. Comparison of the Repeats and SSRs in Cymbaria chloroplast genomes. (A) Repeats and SSRs. (B) Repeat types. (C) Occurrences of SSRs. (D) SSR types. (E) SSR motif types. Bar colors of green and orange correspond to C. mongolica (left) and C. daurica (right), respectively.
3.4. | Development and validation of DNA barcodes
High conservation with some degree of divergence was observed in the two chloroplast genomes (Figure 5). Most of the sequence differences were observed in the non-coding regions. Nucleotide diversity (Pi) was 0.02099, and higher divergence was observed in the LSC and SSC regions (Fig. S4). We also detected several divergence hotspot regions (Pi > 0.05), including trn M-CAU-ndh C,psa A, mat K,acc D-psa I,ycf 4-cem A, rpl 32-trn L-UAG,ndh D-ndh G,rps 15-ycf 1, rrn 23S, andtrn A-UGC-trn E-UUC. We designed four specific DNA markers (CymN1, CymN2, CymY, and CymR), and they were validated using sequences from different regions from seven individuals of each species (Table S3; Table S4). DNA fragments varying in length were obtained from C. mongolica and C. daurica using each primer, and both species could be distinguished via agarose gel electrophoresis (Fig. 6). Likewise, the same results were observed from the sanger sequencing alignment and chromatogram of the amplification of DNA barcodes. Overall, these findings suggest that the four pairs of DNA barcodes could be used to distinguish among Cymbaria species.