Application of chloroplast genome in the identification of Traditional Chinese Medicine Viola philippica

Abstract

Background: Viola philippica Cav. is the only source plant of "Zi Hua Di Ding", which is a Traditional Chinese Medicine (TCM) that is utilized as an antifebrile and detoxicant agent for the treatment of acute pyogenic infections. Historically, many Viola species with violet flowers have been misused in "Zi Hua Di Ding". Viola have been recognized as a taxonomically difficult genera due to their highly similar morphological characteristics. Here, all common V. philippica adulterants were sampled. A total of 24 complete chloroplast (cp) genomes were analyzed, among these 5 cp genome sequences were downloaded from GenBank and 19 cp genomes, including 2 "Zi Hua Di Ding" purchased from a local TCM pharmacy, were newly sequenced.

Results: The Viola cp genomes ranged from 156,483 bp to 158,940 bp in length. A total of 110 unique genes were annotated, including 76 protein-coding genes, 30 tRNAs, and four rRNAs. Sequence divergence analysis screening identified 16 highly diverged sequences; these could be used as markers for the identification of Viola species. The morphological, maximum likelihood and Bayesian inference trees of whole cp genome sequences and highly diverged sequences were divided into five monophyletic clades. The species in each of the five clades were identical in their positions within the morphological and cp genome tree. The shared morphological characters belonging to each clade was summarized. Interestingly, unique variable sites were found in ndhF, rpl22, and ycf1 of V. philippica, and these sites can be selected to distinguish V. philippica from samples all other Viola species, including its most closely related species. In addition, important morphological characteristics were proposed to assist the identification of V. philippica. We applied these methods to examine 2 "Zi Hua Di Ding" randomly purchased from the local TCM pharmacy, and this analysis revealed that the morphological and molecular characteristics were valid for the identification of V. philippica.

Conclusions: This study provides invaluable data for the improvement of species identification and germplasm of V. philippica that may facilitate the application of a super-barcode in TCM identification and enable future studies on phylogenetic evolution and safe medical applications.

Keywords: Chloroplast genome; Identification; Morphological characteristics; Phylogeny; Unique variable site; Viola philippica.

Fig. 1

Morphology and cp genome map of Viola species. Photo credits: Dong-Ling Cao. A The morphological character of V. philippica. B a. root; b. flower; c. upper leaf blade (ventral); d. upper leaf blade (dorsal); e. lower leaf blade (ventral); f. lower leaf blade (dorsal); g. sepal; h. nectar glands; i. petal; j. style; k. capsule; l. capsule longitudinal section; m. capsule transverse section; n. capsule dehiscence. Scale bar =1cm in a; 50mm in b, c, d, e, f, g, h, i, and n; 5mm in k, l, m; 1mm in j. C Chloroplast genome map of V. philippica with annotated genes. The genes transcribed clockwise are shown inside of the circle whereas genes transcribed anti-clockwise are shown outside of the circle. The colored bars indicate different functional groups. The darker gray area in the inner circle denotes GC content while the lighter gray corresponds to the AT content of the genome. LSC: large single copy, SSC: small single copy, IR: inverted repeat

Fig. 2

Sequence polymorphism among 17 Viola cp genomes. A Sequence alignment of Viola cp genomes, with Viola acuminata as the reference using mVISTA. The position and transcriptional direction of each gene are indicated by gray arrows. The vertical scale indicates the percentage of identity, ranging from 50 to 100%. The horizontal axis indicates the coordinates within the chloroplast genome. Genome regions are color-coded as exons, introns, and intergenic spacer (IGS). B The nucleotide diversity (Pi) values of the 17 Viola cp genomes; Pi values of coding genes and Pi values of IGS

Fig. 3

Unique variation sites in ndhF, rpl22, and ycf1 of Viola philippica. Alignment of four fragment sequences of 17 Viola species. AndhF, (B) rpl22, (C) ycf1. The black arrow at the bottom indicated four species-specific variation sites in Viola philippica

Fig. 4

The primer sequences and electrophoretogram for four sequence fragments with unique variation sites in 14 newly sequenced Viola species. AndhF, (B) rpl22, (C) ycf1 primer pairs. Left and right arrows indicate forward and reverse primers, respectively. M: Marker. 1–14: V. acuminata; V. chaerophylloides; V. collina; V. dissecta; V.inconspicua; V. monbeigii; V. mongolica; V. patrinii; V. phalacrocarpa; V. philippica; V. prionantha; V. variegate; V. yezoensis; V. yunnanfuensis

Fig. 5

Morphological clustering of 17 Viola species. Top left inset shows cods of five morphological characters in 17 Viola species. Lobe: 0 = entire leaf, 1 = lobed leaf; Length of stipules adnate to petioles: 0 = stipule adnate with petioles shorter than one-half, 1 = stipule adnate with petioles longer than one-half; Leaf blade baes: 0 = explanate, 1 = reflexed; Stigma type: 0 = immarginate, 1 = margined; Fruit shape: 0 = capsule ellipsoid, 1 = capsule globose. Each trait was represented by a different shape on the cluster analysis tree. Use solid or hollow to indicate different types

Fig. 6

Maximum Likelihood (ML) and Bayesian Inference (BI) phylogenetic trees based on whole cp genomes of 24 samples. Paspalum paniculatum was set as the outgroup. The numbers above the branches represent ML bootstrap values/BI posterior probabilities. The shared morphological characteristics for each of the five clades were shown next to each species in the way of hand-drawn illustrations, and the dry leaves of “ZHDD” bought from TCM pharmacy were shown next to “Zi Hua Di Ding1, 2”. The hand-drawn illustrations credits: Dong-Ling Cao. In the mature period with dimorphic leaf blades, i.e., smaller triangular-ovate for the lower leaf blades and longer oblong-ovate for the upper leaf blades was shown next to V. philippica. For section Viola, the fruit is the globose capsule. For section Trigonocarpae, the stigmas are clavate, slightly curved forward at the base, and the apex is uncinate or papillose; For section Bilobatae, the stigmas are 2-lobed, thick, with a conspicuous stigma hole; For section Pinnatae, the leaf blades are lobe; For section Adnatae, the length of stipules adnate with petioles is longer than one-half length of stipules