Lonicera japonica is a well-known medicinal plant, and the Damaohua cultivar is one of the oldest known honeysuckle cultivars in China. The 155,151 bp chloroplast genome of this cultivar was obtained through Illumina sequencing. The genome includes a pair of inverted repeats (IRa and IRb; 23,789 bp each), a large single-copy region (88,924 bp), and a small single-copy (SSC) region (18,649 bp). In total, 127 unique genes were identified: 80 protein-coding, 39 tRNA, and 8 rRNA genes. Only ycf3 contained two introns. Eighty-nine large repetitive sequences and 54 simple sequence repeats were detected. Fifty potential RNA editing sites were predicted. Adaptive evolution analysis revealed that infA, matK, petB, petD, rbcL, rpl16, rpl2, rps3, ycf1, and ycf2 were positively selected, possibly reflecting the specific environmental adaptations of this cultivar. Sequence alignment and analysis revealed several candidate fragments for Lonicera species identification, such as the intergenic regions rpoB-petN, rbcL-accD, and psaA-ycf3. The IR region boundary and phylogenetic analysis revealed that the L. japonica cv. Damaohua chloroplast genome was most closely related to the L. japonica genome, but there were five distinct differences between the two. There are four sites with high variability between L. japonica and L. japonica cv. Damaohua with nucleotide variability (Pi) greater than 0.002, including rps2-rpoC2, atpB-rbcL, ycf1, and ycf1-trnN GUU. The differences between L. japonica and L. japonica cv. Damaohua were further confirmed by the single nucleotide polymorphism sites between these two species. Therefore, this study revealed that the chloroplast genome can serve as a universal super barcode for plant identification, which can identify differences and help distinguish Lonicera japonica from related species. An understanding of Lonicera japonica cv. Damaohua chloroplast genomics and a comparative analysis of Lonicera species will provide a scientific basis for breeding, species identification, systematic evolution analysis, and chloroplast genetic engineering research on medicinal honeysuckle plants.
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