The complete chloroplast genome of Artemisia annua (Asteraceae), the primary source of artemisinin, was sequenced and analyzed. The A. annua cp genome is 150,995 bp, and harbors a pair of inverted repeat regions (IRa and IRb), of 24,850 bp each that separate large (LSC, 82,988 bp) and small (SSC, 18,267 bp) single-copy regions. Our annotation revealed that the A. annua cp genome contains 113 genes and 18 duplicated genes. The gene order in the SSC region of A. annua is inverted; this fact is consistent with the sequences of chloroplast genomes from three other Artemisia species. Fifteen (15) forward and seventeen (17) inverted repeats were detected in the genome. The existence of rich SSR loci in the genome suggests opportunities for future population genetics work on this anti-malarial medicinal plant. In A. annua cpDNA, the rps19 gene was found in the LSC region rather than the IR region, and the rps19 pseudogene was absent in the IR region. Sequence divergence analysis of five Asteraceae species indicated that the most highly divergent regions were found in the intergenic spacers, and that the differences between A. annua and A. fukudo were very slight. A phylogenetic analysis revealed a sister relationship between A. annua and A. fukudo. This study identified the unique characteristics of the A. annua cp genome. These results offer valuable information for future research on Artemisia species identification and for the selective breeding of A. annua with high pharmaceutical efficacy.

DNA barcoding has been used for decades, although it has mostly been applied to some single-species. Traditional Chinese medicine (TCM), which is mainly used in the form of combination-one type of the multi-species, identification is crucial for clinical usage. Next-generation Sequencing (NGS) has been used to address this authentication issue for the past few years, but conventional NGS technology is hampered in application due to its short sequencing reads and systematic errors. Here, a novel method, Full-length multi-barcoding (FLMB) via long-read sequencing, is employed for the identification of biological compositions in herbal compound formulas in adequate and well controlled studies. By directly sequencing the full-length amplicons of ITS2 and psbA-trnH through single-molecule real-time (SMRT) technology, the biological composition of a classical prescription Sheng-Mai-San (SMS) was analyzed. At the same time, clone-dependent Sanger sequencing was carried out as a parallel control. Further, another formula—Sanwei-Jili-San (SJS)—was analyzed with genes of ITS2 and CO1. All the ingredients in the samples of SMS and SJS were successfully authenticated at the species level, and 11 exogenous species were also checked, some of which were considered as common contaminations in these products. Methodology analysis demonstrated that this method was sensitive, accurate and reliable. FLMB, a superior but feasible approach for the identification of biological complex mixture, was established and elucidated, which shows perfect interpretation for DNA barcoding that could lead its application in multi-species mixtures.