Comparative Chloroplast Genome Analysis of Anchusa and the Adulterants of HERBA ANCHUSAE

Background: The genus Anchusa L. includes plants used in Uyghur medicine for their anti-inflammatory and analgesic effects. However, in China, the botanical origin of HERBA ANCHUSAE (Niushecao, a Uyghur medicinal herb) is severely confused. Traditional identification methods and standard DNA barcodes do not work well for these close relatives. Chloroplast genomes are known to contain variable regions that can help distinguish species, yet no such study has been done for Anchusa. Therefore, We compared the complete chloroplast genomes of six Anchusa species and the main adulterants of Niushecao. Methods: We analyzed genome structure, repeat sequences, codon usage bias, and nucleotide diversity (Pi), as well as conducted comparative and phylogenetic analyses. Results: All genomes shared a typical ring-shaped quadripartite structure, ranged from 150,178 to 150,844 bp in size, and contained the same set of genes. Despite this overall conservation, we identified several highly variable spots, mostly located in non-coding intergenic spacer regions. Using two complementary approaches—sliding window analysis and mVISTA-based sequence visualization—we identified three overlapping regions (rbcL-psaI, petA-psbJ, and trnC-GCA-petN) as candidate DNA barcodes for species identification. Our phylogenetic tree showed that Anchusa strigosa Banks & Sol. is most closely related to the true medicinal species Anchusa azurea Mill. (Bootstrap support (BS) = 100%), while other look-alikes formed separate branches. Conclusions: These findings provide the first chloroplast genomic resources for this genus and offer potential molecular markers for authenticating Anchusa medicinal materials, laying a foundation for future development of molecular authentication methods.