Comprehensive comparative analysis of chloroplast genomes from seven Panax species and development of an authentication system based on species-unique single nucleotide polymorphism markers

Abstract

Background: Panax species are important herbal medicinal plants in the Araliaceae family. Recently, we reported the complete chloroplast genomes and 45S nuclear ribosomal DNA sequences from seven Panax species, two (P . quinqu e folius and P . trifolius) from North America and five (P . ginseng, P . notoginseng, P . japonicus, P . vietnamensis, and P . stipuleanatus) from Asia.

Methods: We conducted phylogenetic analysis of these chloroplast sequences with 12 other Araliaceae species and comprehensive comparative analysis among the seven Panax whole chloroplast genomes.

Results: We identified 1,128 single nucleotide polymorphisms (SNP) in coding gene sequences, distributed among 72 of the 79 protein-coding genes in the chloroplast genomes of the seven Panax species. The other seven genes (including psaJ, psbN, rpl23, psbF, psbL, rps18, and rps7) were identical among the Panax species. We also discovered that 12 large chloroplast genome fragments were transferred into the mitochondrial genome based on sharing of more than 90% sequence similarity. The total size of transferred fragments was 60,331 bp, corresponding to approximately 38.6% of chloroplast genome. We developed 18 SNP markers from the chloroplast genic coding sequence regions that were not similar to regions in the mitochondrial genome. These markers included two or three species-specific markers for each species and can be used to authenticate all the seven Panax species from the others.

Conclusion: The comparative analysis of chloroplast genomes from seven Panax species elucidated their genetic diversity and evolutionary relationships, and 18 species-specific markers were able to discriminate among these species, thereby furthering efforts to protect the ginseng industry from economically motivated adulteration.

Keywords: Araliaceae evolution; Chloroplast genome; Ginseng authentication; Panax species; dCAPS markers.

Fig. 1

Complete chloroplast genomes from seven Panax species. Colored boxes represent conserved chloroplast genes that were classified based on product function. Genes shown inside the circle are transcribed clockwise, and those outside the circle are transcribed counterclockwise. Genes belonging to different functional groups are color-coded. The dashed area in the inner circle indicates the GC content.

Fig. 2

ML phylogenetic tree ofsevenPanax species with 12 related species in the Araliaceae family based on entire chloroplast genome sequences. Numbers in the nodes are the bootstrap support values from 1000 replicates. Black triangles indicate tetraploid Panax species. The chloroplast sequence of carrot (Daucus carota) was used as an outgroup. ML, maximum likelihood.

Fig. 3

Single nucleotide polymorphic sites in 79 protein-coding genes fromsevenPanax species. The inner track shows the 79 chloroplast CDS genes. Track A represents the total SNPs in all seven Panax species. Track B–G represents SNPs in P. trifolius, P. stipuleanatus, P. vietnamensis, P. japonicus, P. notoginseng, and P. quinquefolius compared to P. ginseng. The red, green, blue, and black lines on each track indicate the four kinds of SNPs (T, A, C, and G nucleotides), respectively. Yellow lines indicate InDel regions. CDS, coding sequence; InDel, insertions or deletion; SNP, single nucleotide polymorphism.

Fig. 4

Schematic representation of gene transfer between the chloroplast and mitochondrial genomes from Panax species. Each gray line within the circle shows the regions of chloroplast genome that has been inserted into the indicated location in the mitochondrial genome. Colored boxes show conserved chloroplast genes, classified based on product function. Genes shown inside the circle are transcribed clockwise, and those outside the circle are transcribed counterclockwise.

Fig. 5

Validation of 18 dCAPS markers derived from CDS SNP regions of seven Panax chloroplast genomes. The 18 denoted dCAPS markers, Pgdm1–3, Pqdm4–6, Pndm7–9, Pjdm10 and 11, Pvdm12 and 13, Psdm14–16, and Ptdm17 and 18 are unique for P. ginseng, P. quinquefolius, P. notoginseng, P. japonicus, P. vietnamensis, P. stipuleanatus, and P. trifolius, respectively. Abbreviated species names shown on amplicons are as follows: Pg, P. ginseng; Pq, P. quinquefolius; Pn, P. notoginseng; Pj, P. japonicus; Pv, P. vietnamensis; Ps, P. stipuleanatus; Pt, P. trifolius; M, 100-bp DNA ladder. CDS, coding sequence; dCAPS, derived cleaved amplified polymorphic sequence; SNP, single nucleotide polymorphism.