4.3. Combined use of multiple barcodes could get more accurate and complete analysis results
This study selected multiple barcodes for species identification. Among them, the nuclear genome ITS2 region showed higher authentication efficiency at the species level (Yao et al., 2020). Chloroplast gene sequences including psbA-trnH , matK , and rbcL have been widely used in the field of plant identification (Group et al., 2009). However, there were certain limitations in the identification of some chloroplast sequences at the species level (Chen et al., 2013; Liu et al., 2014). For example, the psbA-trnH , matK , andrbcL sequences of P. ginsen and P. quinquefoliushad no mutation sites, so the BLAST results matched to the two species simultaneously. Therefore, it is necessary to combine the ITS2 region to accurately distinguish the two species. In this study, the ITS2 assembly sequence of A. macrocephalala , one of the labeled ingredients, could be obtained in all samples, but its chloroplast psbA-trnH ,matK , and rbcL sequences were not obtained. To analyze the reasons for this phenomenon, we downloaded the chloroplast genome sequence of A. macrocephala from NCBI as Query sequence and found that some samples can obtain incomplete matK sequence after assembly (Supplementary Figure 6) . It might be the reason that traditional herbal patent medicine has been concocted and the DNA is degraded seriously. It may also be due to uneven sequencing of the genome, resulting in low coverage of some regions. At the same time, the nuclear genome exists in all parts of the plant. But the underground parts of some plants such as Atractylodis Macrocephalae Rhizomamay contain fewer chloroplasts (Xiong & Yan, 2016). However, for D. oppositifolia , its ITS2 sequence was not obtained in any of the four pharmaceutical samples. Subsequently, the four pharmaceutical samples were checked by blast to observe if they contain the reads of theD. oppositifolia ITS2 sequence. The results showed that there were zero, six, one, and nine reads in A03, HSZY152, HSZY191, and HSZY192, but the sequencing depth was relatively low, and ITS2 sequences could not be obtained directly. However, in combination with chloroplast regions, D. oppositifolia was further be detected in all pharmaceutical samples. Therefore, the combined use of multiple barcodes could guarantee the high accuracy analytical results (Arulandhu et al., 2017; Group et al., 2009)