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Comparative Study
. 2018 Jul 5;19(7):1962.
doi: 10.3390/ijms19071962.

Comparative Chloroplast Genome Analyses of Species in Gentiana section Cruciata (Gentianaceae) and the Development of Authentication Markers

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Comparative Study

Comparative Chloroplast Genome Analyses of Species in Gentiana section Cruciata (Gentianaceae) and the Development of Authentication Markers

Tao Zhou et al. Int J Mol Sci. .

Abstract

Gentiana section Cruciata is widely distributed across Eurasia at high altitudes, and some species in this section are used as traditional Chinese medicine. Accurate identification of these species is important for their utilization and conservation. Due to similar morphological and chemical characteristics, correct discrimination of these species still remains problematic. Here, we sequenced three complete chloroplast (cp) genomes (G. dahurica, G. siphonantha and G. officinalis). We further compared them with the previously published plastomes from sect. Cruciata and developed highly polymorphic molecular markers for species authentication. The eight cp genomes shared the highly conserved structure and contained 112 unique genes arranged in the same order, including 78 protein-coding genes, 30 tRNAs, and 4 rRNAs. We analyzed the repeats and nucleotide substitutions in these plastomes and detected several highly variable regions. We found that four genes (accD, clpP, matK and ycf1) were subject to positive selection, and sixteen InDel-variable loci with high discriminatory powers were selected as candidate barcodes. Our phylogenetic analyses based on plastomes further confirmed the monophyly of sect. Cruciata and primarily elucidated the phylogeny of Gentianales. This study indicated that cp genomes can provide more integrated information for better elucidating the phylogenetic pattern and improving discriminatory power during species authentication.

Keywords: Gentiana section Cruciata; chloroplast genome; molecular markers; species authentication.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Merged gene map of the complete chloroplast genomes of three Gentiana species. Genes belonging to different functional groups are classified by different colors. The genes drawn outside of the circle are transcribed counterclockwise, while those inside are clockwise. Dashed area in the inner circle represent GC content of chloroplast genome.
Figure 2
Figure 2
Comparison of chloroplast genome borders of LSC, SSC, and IRs among eight species in Gentiana sect. Cruciata. Ψ indicates a pseudogene.
Figure 3
Figure 3
Analysis of different repeats in eight chloroplast genomes of Gentiana sect. Cruciata. (A) Histogram showing the number of palindromic repeats and dispersed repeats; (B) histogram showing the number of tandem repeats; (C) number of different simple sequence repeat (SSR) types detected in eight chloroplast genomes; (D) total numbers of different SSR motifs in eight chloroplast genomes.
Figure 4
Figure 4
mVISTA percent identity plot comparing the eight chloroplast genomes of Gentiana sect. Cruciata with G. crassicaulis as a reference. The y-axis represents the percent identity within 50–100%. Genome regions are color-coded as protein coding (purple), rRNA, or tRNA coding genes (blue), and noncoding sequences (pink).
Figure 5
Figure 5
Percentage of variable characters in eight aligned chloroplast genomes of Gentiana sect. Cruciata. (A) Coding region; (B) Noncoding region.
Figure 6
Figure 6
Validation of 16 molecular markers derived from InDel regions of eight chloroplast genomes of Gentiana sect. Cruciata. Inserted sequences and tandem repeats are designated by diamonds and triangle, respectively. Solid and dotted lines indicate conserved and deleted sequences, respectively. Left and right black arrows indicate forward and reverse primers, respectively. Abbreviated species names were shown on schematic diagrams: Gd, G. dahurica; Go, G. officinalis; Gm, G. macrophylla; Gsi, G. siphonantha; Gst, G. straminea; Gr, G. robusta; Gc, G. crassicaulis; M, D600 DNA ladder.
Figure 7
Figure 7
Phylogenetic relationships of species belong to Gentiana sect. Cruciata inferred from MP/ML/BI analysis based on complete chloroplast genome sequences. The numbers associated with each node are bootstrap support and posterior probability values, and the symbol formula image in the phylogenetic tree indicated that the support value of branch is 100/100/1.0.

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