DNA Barcoding Identification of Angelicae Sinensis Radix and Its Adulterants Based on Internal Transcribed Spacer 2 Region and Secondary Structure Prediction

<b>Background</b>: Morphological similarities among <i>Angelicae Sinensis</i> Radix and related species often lead to market substitution. This study develops a DNA barcoding method to authenticate these herbs and identify adulterants derived from <i>Angelica</i>, <i>Heracleum</i>, and <i>Peucedanum</i> genera. <b>Methods</b>: Phylogenetic analysis was conducted using MEGA 11.0 software with ITS2 sequences from <i>Angelicae Sinensis</i> Radix, <i>Ligusticopsis Pubescens</i> Radix, <i>Angelicae Pubescens</i> Radix, and their related species within the genera <i>Angelica</i>, <i>Peucedanum</i>, and <i>Heracleum</i>. Additionally, ITS2 secondary structures were predicted for the three herbs to provide supplementary evidence for identification. <b>Results:</b> The amplification success rate was 86.67%, and the interspecific genetic distance, ranging from 0.009~0.220, was significantly greater than the intraspecific genetic distance range from 0.000~0.062, indicating that the ITS2 sequence is suitable for differentiating three herbs and their related species. Additionally, their ITS2 secondary structures exhibited significant differences, which can also serve as a reliable criterion for their identification. <b>Conclusions:</b> This study not only validated the identification efficacy of ITS2 sequences and their secondary structures for the three herbs, but more importantly, enabled precise traceability of adulterants through the construction of a comprehensive phylogenetic framework.