The complete chloroplast genomes of Tetrastigma hemsleyanum (Vitaceae) from different regions of China: molecular structure, comparative analysis and development of DNA barcodes for its geographical origin discrimination.

Background: Tetrastigma hemsleyanum is a valuable traditional Chinese medicinal plant widely distributed in the subtropical areas of China. It belongs to the Cayratieae tribe, family Vitaceae, and exhibited significant anti-tumor and anti-inflammatory activities. However, obvious differences were observed on the quality of T. hemsleyanum root from different regions, requiring the discrimination strategy for the geographical origins.

Result: This study characterized five complete chloroplast (cp) genomes of T. hemsleynum samples from different regions, and conducted a comparative analysis with other representing species from family Vitaceae to reveal the structural variations, informative markers and phylogenetic relationships. The sequenced cp genomes of T. hemsleyanum exhibited a conserved quadripartite structure with full length ranging from 160,124 bp of Jiangxi Province to 160,618 bp of Zhejiang Province. We identified 112 unique genes (80 protein-coding, 28 tRNA and 4 rRNA genes) in the cp genomes of T. hemsleyanum with highly similar gene order, content and structure. The IR contraction/expansion events occurred on the junctions of ycf1, rps19 and rpl2 genes with different degrees, causing the differences of genome sizes in T. hemsleyanum and Vitaceae plants. The number of SSR markers discovered in T. hemsleyanum was 56-57, exhibiting multiple differences among the five geographic groups. Phylogenetic analysis based on conserved cp genome proteins strongly grouped the five T. hemsleyanum species into one clade, showing a sister relationship with T. planicaule. Comparative analysis of the cp genomes from T. hemsleyanum and Vitaceae revealed five highly variable spacers, including 4 intergenic regions and one protein-coding gene (ycf1). Furthermore, five mutational hotspots were observed among T. hemsleyanum cp genomes from different regions, providing data for designing DNA barcodes trnL and trnN. The combination of molecular markers of trnL and trnN clustered the T. hemsleyanum samples from different regions into four groups, thus successfully separating specimens of Sichuan and Zhejiang from other areas.

Conclusion: Our study obtained the chloroplast genomes of T. hemsleyanum from different regions, and provided a potential molecular tracing tool for determining the geographical origins of T. hemsleyanum, as well as important insights into the molecular identification approach and and phylogeny in Tetrastigma genus and Vitaceae family.