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)