| Summary: | <i>Ampelopsis humulifolia</i> (<i>A. humulifolia</i>) and <i>Ampelopsis japonica</i> (<i>A. japonica</i>), which belong to the family Vitaceae, are valuably used as medicinal plants. The chloroplast (cp) genomes have been recognized as a convincing data for marker selection and phylogenetic studies. Therefore, in this study we reported the complete cp genome sequences of two <i>Ampelopsis</i> species. Results showed that the cp genomes of <i>A. humulifolia</i> and <i>A. japonica</i> were 161,724 and 161,430 bp in length, respectively, with 37.3% guanine-cytosine (GC) content. A total of 114 unique genes were identified in each cp genome, comprising 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. We determined 95 and 99 small sequence repeats (SSRs) in <i>A. humulifolia</i> and <i>A. japonica</i>, respectively. The location and distribution of long repeats in the two cp genomes were identified. A highly divergent region of <i>psbZ</i> (Photosystem II reaction center protein Z) -<i>trnG</i> (tRNA-Glycine) was found and could be treated as a potential marker for Vitaceae, and then the corresponding primers were designed. Additionally, phylogenetic analysis showed that <i>Vitis</i> was closer to <i>Tetrastigma</i> than <i>Ampelopsis</i>. In general, this study provides valuable genetic resources for DNA barcoding marker identification and phylogenetic analyses of <i>Ampelopsis</i>.
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