Access to this full-text is provided by Frontiers.
Content available from Frontiers in Plant Science
This content is subject to copyright.
Comparative analysis of
medicinal plant Isodon
rubescens and its common
adulterants based on
chloroplast genome sequencing
Zhongyu Zhou
1
, Jing Wang
1,2
, Tingting Pu
1
, Jingjing Dong
1
,
Qin Guan
1
, Jun Qian
1
, Linchun Shi
3
*and Baozhong Duan
1
*
1
College of Pharmaceutical Science, Dali University, Dali, China,
2
College of Life Science, Northeast
Forestry University, Harbin, China,
3
Institute of Medicinal Plant Development, Chinese Academy of
Medical Sciences, Peking Union Medical College, Beijing, China
Isodon rubescens (Hemsley) H. Hara is the source of Donglingcao under the
monograph Rabdosiae Rubescentis Herba in Chinese Pharmacopoeia. In the
local marketplace, this medicine can be accidentally contaminated,
deliberately substituted, or mixed with other related species. The
contaminants of herbal products are a threat to consumer safety. Due to the
scarcity of genetic information on Isodon plants, more molecular markers are
needed to avoid misidentification. In the present study, the complete
chloroplast (cp) genome of seven species of Isodon was sequenced, de novo
assembled and characterized. The cp genomes of these species universally
exhibited a conserved quadripartite structure, i.e., two inverted repeats (IRs)
containing most of the ribosomal RNA genes and two unique regions (large
single copy and small single copy). Moreover, the genome structure, codon
usage, and repeat sequences were highly conserved and showed similarities
among the seven species. Five highly variable regions (trnS-GCU-trnT-CGU,
atpH-atpI,trnE-UUC-trnT-GGU, ndhC-trnM-CAU, and rps15-ycf1) might be
potential molecular markers for identifying I. rubescens and its contaminants.
These findings provide valuable information for further species identification,
evolution, and phylogenetic research of Isodon.
KEYWORDS
Isodon rubescens, chloroplast genome, species identification, molecular
marker, phylogenetic
Frontiers in Plant Science frontiersin.org01
OPEN ACCESS
EDITED BY
Zemin Ning,
Wellcome Sanger Institute (WT),
United Kingdom
REVIEWED BY
Qingguo Ma,
Research Institute of Forestry, Chinese
Academy of Forestry, China
Gang Yao,
South China Agricultural University,
China
Cornelius Mulili Kyalo,
University of Chinese Academy of
Sciences, China
*CORRESPONDENCE
Baozhong Duan
bzduan@126.com
Linchun Shi
linchun_shi@163.com
SPECIALTY SECTION
This article was submitted to
Plant Bioinformatics,
a section of the journal
Frontiers in Plant Science
RECEIVED 04 September 2022
ACCEPTED 26 October 2022
PUBLISHED 21 November 2022
CITATION
Zhou Z, Wang J, Pu T, Dong J,
Guan Q, Qian J, Shi L and Duan B
(2022) Comparative analysis of
medicinal plant Isodon rubescens and
its common adulterants based on
chloroplast genome sequencing.
Front. Plant Sci. 13:1036277.
doi: 10.3389/fpls.2022.1036277
COPYRIGHT
© 2022 Zhou, Wang, Pu, Dong, Guan,
Qian, Shi and Duan. This is an open-
access article distributed under the
terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in other
forums is permitted, provided the
original author(s) and the copyright
owner(s) are credited and that the
original publication in this journal is
cited, in accordance with accepted
academic practice. No use,
distribution or reproduction is
permitted which does not comply with
these terms.
TYPE Original Research
PUBLISHED 21 November 2022
DOI 10.3389/fpls.2022.1036277
Introduction
Isodon rubescens (Hemsley) H. Hara belongs to the family
Lamiaceae (Liu et al., 2017), which is listed in the Chinese
Pharmacopoeia, and the Chinese name is “Donglingcao”
(Chinese Pharmacopoeia Commission, 2020). The Rabdosiae
Rubescentis Herba has crucial medicinal value in eliminating
inflammation, reducing sore throats, and treating malignant
tumors (Xue et al., 2020;Guan et al., 2021). The previous
survey has revealed that I. rubescens is generally contaminated
with common adulterants, such as I. inflexus (Thunb.) Kudo,I.
eriocalyx (Dunn) Kudo,I. excisus (Maxim.) Kudo,I.
lophanthoides (Buch.-Ham. ex D.Don) H.Hara, I. coetsa
(Buch.-Ham. ex D.Don) Kudo,andI. japonicus (Burm.f.)
H.Hara (Su et al., 2007;Xia et al., 2013;Ge et al., 2022). These
adulterants are usually of poor quality and some might even be
toxic (Xia et al., 2013;Duan et al., 2018). As the morphology of
these species is similar, interchangeable, and indistinguishable,
the identification of these species remains somewhat
controversial, which may affect their safety and effectiveness in
clinical use (Lazarski et al., 2014;Lin et al., 2019). Therefore, it is
imperative to develop a method for accurately identifying I.
rubescens and its common adulterants.
With the rapid development of molecular technology in
recent years, molecular identification has made significant
progress in Chinese medicine, especially molecular markers,
which involve sequencing specific sections of the genome to
identify differences between individuals of different species or
populations (Roman and Houston, 2020). Recent studies have
shown high levels of genetic variability within species of Isodon
and an associated lack of phylogenetic resolution between
different species (Zhong et al., 2010;Chen et al., 2021).
Universal DNA markers, such as ITS, psbA-trnH, trnD-trnT,
rpl32-trnL, and ETS have been used to identify I. rubescens and
its related taxa (Xia et al., 2013;Yu et al., 2014;Chen et al., 2021).
Moreover, according to Harris and Klooster (2011), they found
that 11 microsatellite loci amplify reliably and are sufficiently
variable for studying population genetics in I. rubescens.
However, some common adulterants were not included in
these studies. Therefore, more scientificandaccurate
identification methods must be developed. The chloroplast
(cp) is an essential organelle that plays a crucial role in plant
photosynthesis and biochemical processes (Bendich, 2004).
Compared with the gene fragments, the cp genome is
relatively conserved and slightly varied (Drouin et al., 2008;
Ferrarini et al., 2013), which has been widely used for identifying
Paris,Polygonatum, and its contaminants (Kawabe and
Miyashita, 2003;Jiang et al., 2022;Wang et al., 2022).
Recently, although the complete plastid genomes of I.
rubescens has reported by Lian et al. (2022) and Yue et al.,
(2021), the focus of these papers was to compare the intraspecific
variation or characterize one genome information. However, the
use of cp genomes for comparing Isodon species with their
common adulterants has not been reported.
Our study aims to: (i) contribute new fully-sequenced cp
genomes in Isodon and improve the understanding of the overall
structure of these genomes, (ii) perform comparative analyses
and elucidate the phylogenetic evolution of Isodon cp genomes,
and (iii) screen molecular markers to differentiate I. rubescens
and its adulterants. In the current work, the complete cp
genomes of seven Isodon species were sequenced, de novo
assembled, and annotated. These genomes were then used in a
comparative analysis of genome structure and evolution
relationships. The data acquired in this study increase the
genomic resources available for the Isodon genus and provide
valuable information support for the phylogenetic analysis and
identification of the Isodon genus, as well as safe medical
applications of I. rubescens. This study is the first
comprehensive research on identifying I. rubescens and its
adulterants based on the cp genomes.
Materials and methods
Plant and DNA resources
The fresh, healthy leaves for seven species of I. rubescens
and its common adulterants, including I. inflexus,I. eriocalyx,
I. excisus,I. lophanthoides,I. coetsa,andI. japonicus,were
collected from the Germplasm Resource Garden (Yunnan,
China, 24°49′55″N, 102°48′58″E), Kunming Zhifen
Biotechnology Co., Ltd. One individual sample of
approximately 1.0 g of fresh leaves per plant species was
gathered and stored in an ice-filled cooler or refrigerator (4°
C) until DNA extractions could be performed. Professor
Baozhong Duan authenticated the specimens, and the
detailed sample information is available in Supplementary
Table S1 and Figure S1. The voucher specimens were
deposited in the Dali University herbarium. Genomic DNA
was extracted from tissue samples using the Plant Genomic
DNA kit (Tiangen, Beijing, China) following the
manufacturer’s protocol. The extracted DNA was quantified
on high-sensitivity Qubit 4.0 fluorometry (Life Technologies,
Inc.),andallPCRproductswereexaminedforthepresenceof
amplified products in agarose gels.
DNA sequencing, assembly,
and annotation
For sequencing library preparation, we used thirty
microlitres of high-quality (>100 ng/mL) DNA per individual.
All libraries were sequenced on the Illumina NovaSeq system
(Illumina, San Diego, CA). Paired-end sequence reads were
Zhou et al. 10.3389/fpls.2022.1036277
Frontiers in Plant Science frontiersin.org02
trimmed to remove low-quality bases and adapter sequences in
the Toolkit_v2.3.3 software. The cp genomes were assembled by
GetOrganelle v.1.6.4, exploiting Bowtie2 v.2.4.4, SPAdes v.3.13.0,
and Blast v.2.5.0 as dependencies (Jin et al., 2019). After
assembly, two online annotation tools, CpGAVAS2 and GeSeq
were used to annotate the circular cp genomes (Wick et al.,
2015), and the annotated cp genome sequences were submitted
to the GenBank database of the National Center for
Biotechnology Information (NCBI) (Table 1). Gene maps of
the cp genomes were produced with the online IRscope (https://
irscope.shinyapps.io/Chloroplot/).
Repeat analysis
The GC content was analyzed using the Geneious 9.0.2
software (Kearse et al., 2012). Four kinds of the dispersed
repeat sequence, including Forward (F), Reverse (R),
Palindromic (P), and Complementary (C), were detected using
the REPuter program (https://bibiserv.cebitec.uni-bielefeld.de/
reputer/)(Kurtz, 2001). The criteria for repeat determination
include a minimum repeat size of 20 bp with a similarity
between repeat pairs of 90% by putting edit value 3.
Furthermore, MISA software (http://pgrc.ipk-gatersleben.de/
misa/) was used to evaluate the simple sequence repeats (SSRs)
with the parameters of ‘10’for mono, ‘5’for di-, ‘4’for tri-, and
‘3’for tetra-, penta-, and hexanucleotide motifs (Beier
et al., 2017).
Comparative and phylogenetic analyses
Relative synonymous codon usage (RSCU) and codon usage
values were analyzed by CodonW v.1.4.2. Moreover, the RSCU
values were shown in a heatmap by Tbtools (Chen et al., 2020).
The contraction and expansion of IR regions at the junctions
were visualized using the online IRscope (https://irscope.
shinyapps.io/irapp/)(Amiryousefiet al., 2018). The mVISTA
program in Shuffle LAGAN mode was used to compare the cp
genomes of seven species of Isodon (Frazer et al., 2004), using the
annotation information of I. serra (GenBank NC064127) as a
reference. Additionally, the nucleotide variability (Pi) across the
cp genome sequences was assessed using DnaSP v.6.12.03, with a
window length of 600 sites and a step size of 200 sites (Rozas
et al., 2017). A value of Pi higher than 0.014 was recommended
as mutational hotspots (Cui et al., 2019a). In phylogenetic
analyses, 36 species, including 29 species downloaded from
NCBI (Table S2), were used to infer the phylogeny.
Simultaneously, two species, Scrophularia dentata (GenBank
NC036942) and S. henryi (GenBank NC036943) were used as
outgroups (Chase et al., 2016). All these sequences were aligned
using MAFFT, and alignments were checked manually. The
Maximum-likelihood (ML) tree was reconstructed with an
IQtree using default parameters of 1000 iterations, 1000
replications, and best-fit model selection (Nguyen et al., 2015).
Results and discussion
Genome structure
The cp genome size and gene content of seven Isodon species
are listed in Table 1. All the genome sizes were similar to that of
the reference genome, i.e., around 150 kb. I. inflexu had the
largest genome, with a size of 152,701 bp, and I. japonicus had
the smallest genome, with a size of 152,208 bp. Moreover, the
length of large single copy (LSC) regions ranged from 83,079 bp
(I. lophanthoides) to 83,577 bp (I. rubescens), small single copy
(SSC) regions ranged from 17,656 bp (I. excisus) to 17,729 bp (I.
lophanthoides), and IRa and IRb regions ranged from 25,700 bp
(I. lophanthoides and I. japonicus) to 25,728 bp (I. excisus). In
addition, the cp genomes of Isodon contained 132 genes,
including 88 protein-coding genes, 8 rRNA genes, and 36
tRNA genes, 18 of which are repeated as members of IR
regions (Figure 1), which were congruent and largely
concordant with recent studies of Isodon (Yue et al., 2021;
Lian et al., 2022). It is worth noting that the chIB,chIL, and
ycf68 were lost during evolution, typically in most angiosperms
(Millen et al., 2001). Meanwhile, the number and types of
introns were similar among the seven Isodon species (Table
TABLE 1 Information of Isodon cp genome features.
Genome
characterristics
Total length
(bp)
GC content
(%)
AT content
(%)
LSC length
(bp)
SSC length
(bp)
IR length
(bp)
GenBank
accession
I. inflexus 152695 37.63 62.37 83558 17663 25722 OM808733
I. eriocalyx 152657 37.63 62.37 83546 17657 25727 OM808731
I. excisus 152643 37.64 62.36 83531 17656 25728 OM808732
I. lophanthoides 152208 37.61 62.39 83079 17729 25700 OM808735
I. japonicus 152238 37.60 62.40 83139 17699 25700 OM808734
I. coetsa 152441 37.63 62.37 83289 17670 25726 OM808730
I. rubescens 152690 37.62 62.38 83577 17661 25726 OM808736
Zhou et al. 10.3389/fpls.2022.1036277
Frontiers in Plant Science frontiersin.org03
S3). Each of the 18 genes contained one intron, including trnA-
UGC (×2), trnI-GAU (×2), rpl2 (×2), and ndhB (×2) were located
in the IR, and the genes (trnK-UUU,rps16,trnT-CGU,atpF,
rpoC1,trnL-UAA,petB,petD, and rpl16) were located in the LSC,
and the ndhA was the only present in the SSC region.
Furthermore, ycf3 and clpP have two introns, respectively,
consistent with previous genetic studies (Jiang et al., 2022).
The GC content ranged from 37.60% to 37.64% and varied
among the different regions. The findings were identical to those
of other Isodon species (Yue et al., 2021), which is not
unexpected, given that the angiosperms possess the highly
conserved cp character at the genus level (Meng et al., 2018;
Cui et al., 2019b;Shahzadi et al., 2020).
Codon usage bias of cp genomes
The analyses of relative synonymous codon usage (RSCU)
provide information about the encoding frequency of codons for
an amino acid. As shown in Table S4, the results of RSCU
revealed that the cp protein sequences encoded 21 amino acids,
and 30 codons were used frequently in Isodon species, consistent
with recent codon usage studies (Lian et al., 2022). Moreover,
amino acid frequency analyses confirmed the highest frequency
of leucine and isoleucine, whereas cysteine was a rare amino acid
(Table S4), which was supported by other researchers based on
codon usage bias (Lian et al., 2022). Notably, the use of start
codon AUG for methionine and UGG for tryptophan in the
Isodon genus showed no codon usage bias, consistent with
previous reports of Isodon (Lian et al., 2022). In general, we
found high similarities in codon usage and amino acid frequency
among the seven species of Isodon. Furthermore, as illustrated in
Figure 2, higher RSCU values (≥1) were found for codons with A
or T at the 3’position, which showed high encoding efficacy.
Similar findings were reported for codon usage and amino acid
frequency in the cp genomes of other angiosperms, which may
be attributable to the high overall AT content in the cp genome
(Kawabe and Miyashita, 2003;Jiang et al., 2022;Wang et al.,
2022). In addition, the codon usage was similar in I. rubescens,I.
lophanthoides, and I. japonicaus, whereas the codon usage of I.
eriocalyx and I. excisus was relatively close to that of I. inflexus
and I. coetsa (Figure 2).
It was shown that the GC content of synonymous third
codons positions (GC3s) was closely related to codon bias, which
provided the foundation for assessing the codon usage pattern
(Shang et al., 2011). In our study, the values of GC3s ranged
from 29.4% to 29.5%, demonstrating that the genus Isodon had a
greater preference for the A/U ending codons, which, along with
the highly conserved GC content in seven Isodon cp genomes,
suggest that natural selection had a profound impact on codon
usage patterns (Zhang et al., 2018). In addition, the values for the
effective number of codons ranged from 51.73 to 51.81, and both
FIGURE 1
Cp genome map of Isodon.
Zhou et al. 10.3389/fpls.2022.1036277
Frontiers in Plant Science frontiersin.org04
the codon adaptation index and frequency of optimal were less
than 0.5. These findings indicated a slight bias of codon usage in
the seven Isodon species.
Repeat analysis
Repetitive sequences play a crucial role in the rearrangement
and stability of cp genomes (Rahemi et al., 2012;Kumar et al.,
2015). A total of 51, 50, 50, 51, 50, 46, and 53 SSRs were
identified in I. inflexus,I. eriocalyx,I. excisus,I. lophanthoides,I.
japonicus,I. coetsa, and I. rubescens, respectively (Figure 3).
More than half of SSRs (62.00% –69.60%) were mononucleotide
A/T motifs, which is consistent with the previous works in the cp
genomes of angiosperms (Qian et al., 2013;Liang et al., 2019).
The second was dinucleotide (11.65% –18.00%) with a
predominant motif of AT/TA, followed by tetranucleotide
repeats (14.00% –19.61%) with a predominant motif of
AAAT/ATTT and AATC/ATTG, trinucleotide (1.89% –
2.17%), pentanucleotide (1.89% –2.00%) with a predominant
motif of AATAT/ATATT, AATAG/ATTCT. Hexanucleotides
(1.96% –4.00%) were absent in the cp genomes of I. eriocalyx,I.
coetsa, and I. rubescens. This result was consistent with previous
findings that most SSRs include mono- and dinucleotide repeats,
while tri-, tetra-, penta-, and hexanucleotide repeat sequences
exhibit lower frequencies (Dong et al., 2018).
Moreover, oligonucleotide repeats analysis of four types of
repeats in the cp genome, including F, R, P, and C, was
performed by REPuter. As illustrated in Figure 4, the number
of repeat types varied and presented random permutations
among the cp genomes of seven species. The number of
repeats varied among these species, but most repeat sequences
existed in 20 –29 bp, which was supported by recent literature
(Lian et al., 2022). Meanwhile, the abundance of F and P repeats
was higher than that of R and C repeats. A total of 36 F repeats
and 47 P repeats were observed in I. rubescens, 38 and 48 in I.
inflexus, 34 and 39 in I. eriocalyx, 39 and 48 in I. excisus, 38 and
43 in I. lophanthoides, 39 and 45 in I. japonicus, 42 and 47 in I.
coetsa, respectively. These repeats play a crucial role in the
generation of substitutions and indels, which makes them
important for detecting mutational hotspots (McDonald et al.,
2011;Ahmed et al., 2012)
FIGURE 2
Heat map of the RSCU values among Isodon cp genome.
Zhou et al. 10.3389/fpls.2022.1036277
Frontiers in Plant Science frontiersin.org05
Inverted repeats
The contraction and expansion of IRs are regarded as crucial
evolutionary phenomena that result in the pseudogenes, gene
duplication, or the reduction of duplicate genes to a single copy
(Abdullah et al., 2020). As illustrated in Figure 5, the rpl22 gene
was present in the LSC region, and rpl2 was entirely in the IRb
region, consistent with a previous study (Lian et al., 2022). A
truncated copy of rps19 gene was observed in all species at the
IRb/LSC junction except for I. ternifolious, which starts in LSC
and integrates into the IRb regions, while rp12 is exclusively
located in the IRb region. In monocotyledons, the rps19 gene is
in the IR region (Ahmed et al., 2012;Henriquez et al., 2020), but
our findings show that things are different in Lamiaceae.
Additionally, the ndhF gene was also found at the junction of
IRb/SSC and integrated into the IRb with a size ranging from 42
to 46 kb. At the IRb/LSC junction, another truncated copy of
ycf1 gene was observed in all species except for I. ternifolious and
I. serra. Contrary to the findings of Henriquez et al. (2020), the
de novo assembled genomes of seven Isodon species did not
include a ycf pseudogene at the IRa/SSC junction. Neither an
internal stop codon nor double peaks were observed in the
electropherograms, indicating that the seven Isodon species lack
amplified pseudogenes. In addition, psbA and trnH were in the
LSC, and rpl2 in the IRa.
Genome comparison and
nucleotide diversity
A comparison of overall sequence variation showed that the
cp genome of Isodon is highly conserved, and the coding region
is more conserved than non-coding regions. Except for ndhF,
ycf1, and ycf2 genes, all protein-coding genes showed a highly
conserved character (Figure 6); the intergenic spacers (IGS) with
the highest divergence were trnH-GUG-psbA,trnQ-UUG-psbK,
FIGURE 3
The number and type of SSRs in the cp genome of Isodon.
FIGURE 4
Repeat sequences detected in Isodon cp genome. P, F, C, and R indicate the repeat types: R (Reverse repeats), P (Palindromic repeats), F
(Forward repeats), C (Complement repeats).
Zhou et al. 10.3389/fpls.2022.1036277
Frontiers in Plant Science frontiersin.org06
trnS-GCU-trnT-CGU,atpH-atpI,trnE-UUC-trnT-GGU, psaA-
ycf3, ndhC-trnM-CAU, psbH-petB, and rps15-ycf1,as
predicted. In addition, the sliding window analysis revealed
that seven regions, including rps16-trnQ-UUG,ndhF,ndhB,
ccsA-ndhD,ndhA,ndhH,andycf1 genes, exhibited higher
nucleotide diversity values (> 0.014, Figure 7); the IR regions
exhibited lower sequence divergence than LSC and SSC regions,
consistent with the previous comparisons of cp genomes (Jiang
et al., 2022;Wang et al., 2022). Among these 16 high
polymorphic regions, 11 intergenic spacers were found in the
trnH-GUG-psbA,trnQ-UUG-psbK,trnS-GCU-trnT-CGU,atpH-
atpI,trnE-UUC-trnT-GGU, psaA-ycf3, ndhC-trnM-CAU, psbH-
petB, rps15-ycf1,rps16-trnQ-UUG,andccsA-ndhD.Itis
noteworthy that the atpH-atpI,rps16-trnQ-UUG, and ndhC-
trnM-CAU were also identified as mutational hotspots in a
previous study (Lian et al., 2022). IGS was considered one of
the evolutionary hotspots that exhibited more significant rates of
nucleotide substitutions and indel mutations (Drummond,
2008). Therefore, these IGS might be undergoing more rapid
nucleotide substitution at the species level, which could be
served as a potential molecular marker for application in
phylogenetic analyses of the Isodon genus.
Species authentication analysis based
on IGS
Intergenic spacer regions are the most frequently used cp
markers for phylogenetic studies at lower taxonomic levels in
plants (Shaw et al., 2005), as they are regarded as more variable
and could provide more phylogenetically informative characters.
To find candidate markers for identifying I. rubescens and its
adulterants, the 11 IGSs were extracted using the PhyloSuite
v1.2.2 from 14 Isodon species (Zhang et al., 2019). Each of the 11
IGSs was subject to maximum likelihood analyses in IQtree
(Nguyen et al., 2015). As illustrated in Figure S2.1-S2.11,five
IGSs, including trnS-GCU-trnT-CGU,atpH-atpI,trnE-UUC-
trnT-GGU,ndhC-trnM-CAU,andrps15-ycf1 could be
FIGURE 5
Comparisons of the borders of LSC, SSC, and IRa/b regions among the 11 Isodon plastid genomes. The numbers represent the distance
between the gene ends and the border sites, and the numbers below represent the length of the LSC, SSC, and IRa/b regions. This Figure is not
to scale.
Zhou et al. 10.3389/fpls.2022.1036277
Frontiers in Plant Science frontiersin.org07
distinguished I. rubescens from I. japonicus and I. lophanthoides.
Whereas the rest of the Isodon species cannot be distinguished
based on these IGSs, bootstrap values for the relationship among
these major clades were weak (<70%). These results are partially
in line with the previous study, which found that the atpH-atpI
and ndhC-trnM-CAU could be potential molecular markers for
distinguishing Isodon species (Lian et al., 2022). Moreover, the
remaining fragment, including trnS-GCU-trnT-CGU, trnE-
UUC-trnT-GGU,andrps15-ycf1 also reported as potential
markers for other species identification (Kim et al., 2020;Li
et al., 2020;Alzahrani, 2021). Although the previous study has
revealed that universal DNA barcodes (e.g., psbA-trnH) could
differentiate I. rubescens from their related species (Xia et al.,
2013), some common adulterants were not included in this
FIGURE 6
Global comparison of complete genomes of Isodon.
FIGURE 7
Sliding window analysis of Isodon cp genome. The X-axis represents the midpoint of the window; The Y-axis represents nucleotide diversity
values. Window length: 600 bp; step size: 200 bp.
Zhou et al. 10.3389/fpls.2022.1036277
Frontiers in Plant Science frontiersin.org08
study. Furthermore, the comparative analysis showed that the
screened IGSs exhibit higher variability than psbA-trnH. These
IGSs could theoretically distinguish the selected 7 species,
whereas a much more detailed investigation of identification
accuracy and amplification efficiency needs to be accomplished,
and more experimental evidence is needed. Moreover, the ML
phylogenetic tree was also inferred using a combination of these
five IGSs. The results (Figure S3) showed that I. rubescens
(GenBank NW018469) and I. rubescens (GenBank NC053708)
clustered together as sister group groups and closely related to I.
excisus. Simultaneously, I. rubescens (GenBank OM808736) was
located in independent branches in the phylogeny, and well-
supported sister relationship between I. rubescens and I.
japonicus +I. lophanthoides (100% B/S). These results
indicated that the combination of five IGSs could effectively
discriminate I. rubescens from its common adulterants.
Species identification and
phylogenetic analysis
The ML phylogenetic tree was inferred using 36 species,
with Scrophularia as the outgroup. As illustrated in Figure 8,on
the consensus trees, most nodes were supported with
maximum support (100% bootstrap support). Ocimoideae
and Lamioideae were sister taxa within the three subfamilies,
and Ajugoideae was sister to the clade containing Ocimoideae
+ Lamioideae. The phylogenetic tree’s crown was occupied by
the subfamily Ocimoideae, which included the genera Isodon
and Ocimum.Thesefindings confirm the position of Isodon
within the Lamiaceae and are consistent with previous
phylogenomic studies (Chen et al., 2021;Yue et al., 2021).
The genus Isodon is further divided into three clades: (i) clade
A, including I. rubescens (GenBank NC053708, GenBank
NW018469), I. excise,I. serra,I. nervosus,I. amethystoides,I.
coetsa,I. inflexus,I. eriocalyx,andI. rubescens (GenBank
NW376483); (ii) clade B, only I. ternifolious; (iii) clade C,
including I. japonicas,I. lophanthoides,andI. rubescens
(GenBank OM808736). It is worth noting that the I.
rubescens (GenBank OM808736) clustered differently from
the other three samples (GenBank NC053708, GenBank
NW018469, GenBank NW376483) of the same species in the
phylogeny, which is consistent with the finding of other
researcher based on rps16,trnL-trnF, and ITS sequence
(Harris et al., 2012). Moreover, Lian et al. (2022) also found
that the samples of I. rubescens from different geographical
FIGURE 8
ML phylogenetic tree reconstruction containing the cp genomes of 36 plants. The Sceophularia species were set as the outgroup.
Zhou et al. 10.3389/fpls.2022.1036277
Frontiers in Plant Science frontiersin.org09
areas were not recovered as monophyletic and were placed in
different branches in the previous report, which is well
distinguished by sampling locations, suggesting that the
intraspecificdiversitywaspresentinI. rubescens. This
phenomenon might be explained by the fact that the
geographical area of origin might influence the variation of I.
rubescens. Another previous study supported the same
conclusion; the Artemisia argyi collected from different
geographical areas display high intraspecific diversity in the
cp genome (Chen et al., 2022). In addition, clade B only
includes a species of I. ternifolious, which was supported by
the findings of other researchers based on trnD-trnT,psbA-
trnH,rpl32-trnL,trnL-trnF,rps16,andnrITS (Zhong et al.,
2010;Yu et al., 2014;Chen et al., 2021). Moreover, maximum
likelihood analysis demonstrated that I. rubescens (GenBank
OM808736) was located in independent branches in the
phylogeny and deeply nested within clade C, and the sister
relationship between I. rubescens and I. japonicus +I.
lophanthoides was highly supported (100% B/S), indicating
that the cp genome could discriminate I. rubescens from its
common adulterants. The findings supported the results of
morphological classification reported by Ge et al. (2022) and
Xia et al. (2013).
Conclusion
In the current study, the complete cp genome of seven
species of Isodon was de novo assembled from Illumina high
throughput sequencing reads, and cp genome sequences of I.
inflexus,I. eriocalyx,I. excisus, and I. coetsa were reported for the
first time. These cp genomes were generally conserved and
exhibited similar gene content and genomic structure. Five
highly variable cp loci, including trnS-GCU-trnT-CGU,atpH-
atpI,trnE-UUC-trnT-GGU, ndhC-trnM-CAU, and rps15-ycf1,
were identified, which could serve as potential markers for
identifying I. rubescens and its common adulterants. In
conclusion, our study provides a powerful tool and valuable
scientific reference for the safety and effectiveness of clinical drug
use, and it also contributes to the bioprospecting and
conservation of Isodon species.
Data availability statement
Thedatapresentedinthestudyaredepositedinthe
GenBank repository, accession numbers were from OM808730
to OM808736.
Author contributions
ZZ, JW, TP, and BD participated in the conception and
design of the research. JW, QG, and BD collected the species. JQ,
LS, and JD are responsible for analyzing and processing data. ZZ
wrote the manuscript. The paper was revised by JQ, LS, and BD.
All authors contributed to the article and approved the
submitted version.
Funding
This work was supported by the Yunnan academician expert
workstation (202205AF150026, 202105AF150053), the key
technology projects in the Yunnan province of China
(202002AA100007), and the Yunnan Xingdian talent support
plan (YNWR-QNBJ-2020251).
Acknowledgments
We would like to thank Ms. Qingshu Yang for her assistance
in obtaining specimens for this study. We also thank Northeast
Forestry University and the China Academy of Chinese Medical
Sciences for technical assistance.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed
or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/
fpls.2022.1036277/full#supplementary-material
Zhou et al. 10.3389/fpls.2022.1036277
Frontiers in Plant Science frontiersin.org10
References
Abdullah,, Mehmood, F., Shahzadi, I., Waseem, S., Mirza, B., Ahmed, I., et al.
(2020). Chloroplast genome of Hibiscus rosa-sinensis (Malvaceae): comparative
analyses and identification of mutational hotspots. Genomics (San Diego Calif.)
112, 581–591. doi: 10.1016/j.ygeno.2019.04.010
Ahmed, I., Biggs, P. J., Matthews, P. J., Collins, L. J., Hendy, M. D., and Lockhart,
P. J. (2012). Mutational dynamics of aroid chloroplast genomes. Genome Biol. Evol.
4, 1316–1323. doi: 10.1093/gbe/evs110
Alzahrani, D. A. (2021). Complete chloroplast genome of Abutilon fruticosum:
genome structure, comparative and phylogenetic analysis. Plants 10, 270. doi:
10.3390/plants10020270
Amiryousefi, A., Hyvönen, J., and Poczai, P. (2018). IRscope: an online program
to visualize the junction sites of chloroplast genomes. Bioinformatics 34, 3030–
3031. doi: 10.1093/bioinformatics/bty220
Beier, S., Thiel, T., Münch, T., Scholz, U., and Mascher, M. (2017). MISA-web: a
web server for microsatellite prediction. Bioinformatics 33, 2583–2585.
doi: 10.1093/bioinformatics/btx198
Bendich, A. J. (2004). Circular chloroplast chromosomes: the grand illusion.
Plant Cell 16, 1661–1666. doi: 10.1105/tpc.160771
Chase, M. W., Christenhusz, M. J. M., Fay, M. F., Byng, J. W., Judd, W. S., Soltis,
D. E., et al. (2016). An update of the angiosperm phylogeny group classification for
the orders and families of flowering plants: APG IV. Bot. J. Linn. Soc 181, 1–20.
doi: 10.1111/boj.12385
Chen, C., Chen, H., Zhang, Y., Thomas, H. R., Frank, M. H., He, Y., et al. (2020).
TBtools: An integrative toolkit developed for interactive analyses of big biological
data. Mol. Plant 13, 1194–1202. doi: 10.1016/j.molp.2020.06.009
Chen, Y. P., Huang, C. Z., Zhao, Y., and Xiang, C. L. (2021). Molecular and
morphological evidence for a new species of Isodon (Lamiaceae) from southern
China. Plant Diversity 43, 54–62. doi: 10.1016/j.pld.2020.06.004
Chen, C. J., Miao, Y. H., Luo, D. D., Li, J. X., Wang, Z. X., Luo, M., et al. (2022).
Sequence characteristics and phylogenetic analysis of the Artemisia argyi
chloroplast genome. Front. Plant Sci. 13. doi: 10.3389/fpls.2022.906725
Chinese Pharmacopoeia Commission (2020). Pharmacopoeia of the peoples
republic of China (Beijing: China Med. Sci. Press).
Cui, Y. X., Chen, X. L., Nie, L. P., Sun, W., Hu, H. Y., Lin, Y. L., et al. (2019a).
Comparison and phylogenetic analysis of chloroplast genomes of three medicinal
and edible Amomum species. Int. J. Mol. Sci. 20, 4040. doi: 10.3390/ijms20164040
Cui, Y. X., Nie, L. P., Sun, W., Xu, Z. C., Wang, Y., Yu, J., et al. (2019b).
Comparative and phylogenetic analyses of ginger (Zingiber officinale) in the family
zingiberaceae based on the complete chloroplast genome. Plants (Basel) 8, 283–295.
doi: 10.3390/plants8080283
Dong, W. L., Wang, R. N., Zhang, N. Y., Fan, W. B., Fang, M. F., and Li, Z. H.
(2018). Molecular evolution of chloroplast genomes of orchid species: insights into
phylogenetic relationship and adaptive evolution. Int. J. Mol. Sci. 19, 716–736.
doi: 10.3390/ijms19030716
Drouin, G., Daoud, H., and Xia, J. N. (2008). Relative rates of synonymous
substitutions in the mitochondrial, chloroplast and nuclear genomes of seed plants.
Mol. Phylogenet. Evol. 49, 827–831. doi: 10.1016/j.ympev.2008.09.009
Drummond, C. S. (2008). Diversification of Lupinus (Leguminosae) in the
western new world: derived evolution of perennial life history and colonization of
montane habitats. Mol. Phylogenet. Evol. 48, 408–421. doi: 10.1016/
j.ympev.2008.03.009
Duan, B. Z., Wang, Y. P., Fang, H. L., Xiong, C., Li, X. W., Wang, P., et al. (2018).
Authenticity analyses of rhizoma par idis using barcoding coupled with high
resolution melting (Bar-HRM) analysis to control its quality for medicinal plant
product. Chin. Med-Uk 13, 8–18. doi: 10.1186/s13020-018-0162-4
Ferrarini, M., Moretto, M., Ward, J. A., S
urbanovski, N., Stevanovic, V., Giongo,
L., et al. (2013). An evaluation of the PacBio RS platform for sequencing and de
novo assembly of a chloroplast genome. BMC Genomics 14, 670. doi: 10.1186/1471-
2164-14-670
Frazer, K. A., Pachter, L., Poliakov, A., Rubin, E. M., and Dubchak, I. (2004).
VISTA: computational tools for comparative genomics. Nucleic Acids Res. 32,
W273–W279. doi: 10.1093/nar/gkh458
Ge, J. W., Li, P., Zhang, T. T., Gao, F., Wang, W. Y., Wang, J. N., et al. (2022).
Molecular identification of a newly discovered medicinal plant, Isodon rubescens,in
Shandong using DNA barcoding. Shandong Sci. 35, 15–20. doi: 10.3976/j.issn.1002-
4026.2022.04.003
Guan, Y., Liu, X., Pang, X., Liu, W., Yu, G. X., Li, Y. R., et al. (2021). Recent
progress of oridonin and its derivatives for cancer therapy and drug resistance.
Med. Chem. Res. 30, 1795–1821. doi: 10.1007/s00044-021-02779-6
Harris, E. S. J., Cao, S. G., Schoville, S. D., Dong, C. M., Wang, W. Q., Jian, Z. Y.,
et al. (2012). Selection for high oridonin yield in the chinese medicinal plant Isodon
(Lamiaceae) using a combined phylogenetics and population genetics approach.
PLoS One 7, e50753. doi: 10.1371/journal.pone.0050753
Harris, E. S. J., and Klooster, M. R. (2011). Development of microsatellite
markers for the medicinal plant Isodon rubescens (Lamiaceae) and related species.
Am. J. Bot. 98, e293–e295. doi: 10.3732/ajb.1100190
Henriquez, C. L., Abdullah Ahmed, I., Carlsen, M. M., Zuluaga, A., Croat, T. B.,
et al. (2020). Evolutionary dynamics of chloroplast genomes in subfamily Aroideae
(Araceae). Genomics 112, 2349–2360. doi: 10.1016/j.ygeno.2020.01.006
Jiang, Y., Miao, Y. J., Qian, J., Zheng, Y., Xia, C. L., Yang, Q. S., et al. (2022).
Comparative analysis of complete chloroplast genome sequences of five
endangered species and new insights into phylogenetic relationships of Paris.
Gene 833, 146572. doi: 10.1016/j.gene.2022.146572
Jin, J. J., Yu, W. B., Yang, J. B., Song, Y., DePamphilis, C. W., Yi, T. S., et al.
(2019). GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of
organelle genomes. Genome Biol. 21, 241–272. doi: 10.1101/256479
Kawabe, A., and Miyashita, N. T. (2003). Patterns of codon usage bias in three
dicot and four monocot plant species. Genes Genet. Syst. 78, 343–352. doi: 10.1266/
ggs.78.343
Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., et al.
(2012). Geneious basic: an integrated and extendable desktop software platform for
the organization and analysis of sequence data. Bioinformatics 28, 1647–1649.
doi: 10.1093/bioinformatics/bts199
Kim, G., Lim, C. E., Kim, J., Kim, K., Lee, J. H., Yu, H., et al. (2020). Comparative
chloroplast genome analysis of Artemisia (Asteraceae) in East Asia: Insights into
evolutionary divergence and phylogenomic implications. BMC Genomics 21, 415–
432. doi: 10.1186/s12864-020-06812-7
Kumar, M., Choi, J., Kumari, N., Pareek, A., and Kim, S. (2015). Molecular
breeding in Brassica for salt tolerance: importance of microsatellite (SSR) markers
for molecular breeding in Brassica.Front. Plant Sci. 6. doi: 10.3389/fpls.2015.00688
Kurtz, S. (2001). REPuter: the manifold applications of repeat analysis on a
genomic scale. Nucleic Acids Res. 29, 4633–4642. doi: 10.1093/nar/29.22.4633
Lazarski, K. E., Moritz, B. J., and Thomson, R. J. (2014). The total synthesis of
Isodon diterpenes. Angewandte Chemie Int. Edition 53, 10588–10599. doi: 10.1002/
anie.201404482
Liang, C. L., Wang, L., Lei, J., Duan, B. Z., Ma, W. S., Xiao, S. M., et al. (2019). A
comparative analysis of the chloroplast genomes of four Salvia medicinal plants.
Engineering-Prc 5, 907–915. doi: 10.1016/j.eng.2019.01.017
Lian, C. L., Yang, H., Lan, J. X., Zhang, X. Y., Zhang, F., Yang, J. F., et al. (2022).
Comparative analysis of chloroplast genomes reveals phylogenetic relationships
and intraspecific variation in the medicinal plant Isodon rubescens.PLoS One 17,
e266546. doi: 10.1371/journal.pone.0266546
Lin, C. Z., Liu, F. L., Zhang, R. J., Liu, M. T., Zhu, C. C., Zhao, J., et al. (2019).
High-performance thin-layer chromatographic fingerprints of triterpenoids for
distinguishing between Isodon lophanthoides and Isodon lophanthoides var.
gerardianus.J. Aoac Int. 102, 714–719. doi: 10.5740/jaoacint.18-0305
Liu, M., Wang, W. G., Sun, H. D., and Pu, J. X. (2017). Diterpenoids from Isodon
species: an update. Nat. Prod. Rep. 34, 1090–1140. doi: 10.1039/C7NP00027H
Li, D. M., Ye, Y. J., Xu, Y. C., Liu, J. M., and Zhu, G. F. (2020). Complete
chloroplast genomes of Zingiber montanum and Zingiber zerumbet: Genome
structure, comparative and phylogenetic analyses. PLoS One 15, e236590.
doi: 10.1371/journal.pone.0236590
McDonald, M. J., Wang, W., Huang, H., and Leu, J. (2011). Clusters of
nucleotide substitutions and insertion/deletion mutations are associated with
repeat sequences. PLoS Biol. 9, e1000622. doi: 10.1371/journal.pbio.1000622
Meng, J., Li, X. P., Li, H. T., Yang, J. B., Wang, H. D., and He, J. (2018).
Comparative analysis of the complete chloroplast genomes of four Aconitum
medicinal species. Molecules 23, 1015. doi: 10.3390/molecules23051015
Millen, R. S., Olmstead, R. G., Adams, K. L., Palmer, J. D., Lao, N. T., Heggie, L.,
et al. (2001). Many parallel losses of infA from chloroplast DNA during angiosperm
evolution with multiple independent transfers to the nucleus. Plant Cell 13, 645–
658. doi: 10.1105/tpc.13.3.645
Nguyen, L. T., Schmidt, H. A., Haeseler, A. V., and Minh, B. Q. (2015). IQ-
TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood
phylogenies. Mol. Biol. Evol. 32, 268–274. doi: 10.1093/molbev/msu300
Qian, J., Song, J. Y., Gao, H. H., Zhu, Y. J., Xu, J., Pang, X. H., et al. (2013). The
complete chloroplast genome sequence of the medicinal plant Salvia miltiorrhiza.
PLoS One 8, e57607. doi: 10.1371/journal.pone.0057607
Rahemi, A., Fatahi, R., Ebadi, A., Taghavi, T., Hassani, D., Gradziel, T., et al.
(2012). Genetic diversity of some wild almonds and related Prunus species revealed
by SSR and EST-SSR molecular markers. Plant Syst. Evol. 298, 173–192.
doi: 10.1007/s00606-011-0536-x
Zhou et al. 10.3389/fpls.2022.1036277
Frontiers in Plant Science frontiersin.org11
Roman, M. G., and Houston, R. (2020). Investigation of chloroplast regions
rps16 and clpP for determination of Cannabis sativa crop type and biogeographical
origin. Legal Med-Tokyo 47, 101759. doi: 10.1016/j.legalmed.2020.101759
Rozas, J., Ferrer-Mata, A., Sanchez-DelBarrio, J. C., Guirao-Rico, S., Librado, P.,
Ramos-Onsins, S. E., et al. (2017). DnaSP 6: DNA sequence polymorphism analysis
of large data sets. Mol. Biol. Evol. 34, 3299–3302. doi: 10.1093/molbev/msx248
Shahzadi, I., Abdullah,, Mehmood, F., Ali, Z., Ahmed, I., and Mirza, B. (2020).
Chloroplast genome sequences of Artemisia maritima and Artemisia absinthium:
Comparative analyses, mutational hotspots in genus Artemisia and phylogeny in
family asteraceae. Genomics 112, 1454–1463. doi: 10.1016/j.ygeno.2019.08.016
Shang, M. Z., Liu, F., Hua, J. P., and Wang, K. B. (2011). Analysis on codon usage
of chloroplast genome of Gossypium hirsutum.Sci. Agr. Sin. 44, 245–253.
doi: 10.3864/j.issn.0578-1752.2011.02.003
Shaw, J., Lickey, E. B., Beck, J. T., Farmer, S. B., Liu, W., Miller, J., et al. (2005). The
tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences
for phylogenetic analysis. Am. J. Bot. 92, 142–166. doi: 10.3732/ajb.92.1.142
Su, X. H., Dong, C. M., Feng, W. S., Chen, S. Q., Wang, L., Wang, W. L., et al.
(2007). Study on the diversity of morphological characteristics of leaves of Rabdosia
rubescens(Hemls.) hara. Lishizhen Med. Mat. Med. 18, 2351–2353. doi: 10.3969/
j.issn.1008-0805.2007.10.010
Wang, J., Qian, J., Jiang, Y., Chen, X. C., Zheng, B. J., Chen, S. L., et al. (2022).
Comparative analysis of chloroplast genome and new insights into phylogenetic
relationships of Polygonatum and tribe polygonatea. Front. Plant Sci. 13.
doi: 10.3389/fpls.2022.882189
Wick, R. R., Schultz, M. B., Zobel, J., and Holt, K. E. (2015). Bandage: interactive
visualization ofde novo genome assemblies. Bioinformatics 31, 3350–3352.
doi: 10.1093/bioinformatics/btv383
Xia,Z.,Zhang,H.R.,Li,H.M.,andGao,Z.M.(2013).Molecular
authentication and phylogenetic relationship of Isodon rubescens and its
relatives. Chin. Trad. Herb. Drugs 44, 2904–2910. doi: 10.7501/j.issn.0253-
2670.2013.20.021
Xue, J. T., Yu, S. S., Wang, J. C., Li, C. Y., Yang, M. Y., Shi, Y. L., et al. (2020).
Network pharmacological screening of the active ingredients and hypoglycemic
effect of Isodon rubescens in the treatment of diabetes. Planta Med. 86, 556–564.
doi: 10.1055/a-1147-9196
Yue, Z. Y., Wang, Y., Zhou, B. Z., and Wang, H. P. (2021). The complete
chloroplast genome of Isodon rubescens, a traditional Chinese herb. Mitochondrial.
DNA B Resour. 6, 337–338. doi: 10.1080/23802359.2020.1860704
Yu, X., Maki, M., Drew, B. T., Paton, A. J., Li, H., Zhao, J., et al. (2014).
Phylogeny and historical biogeography of Isodon (Lamiaceae): rapid radiation in
south-west China and Miocene overland dispersal into Africa. Mol. Phylogenet.
Evol. 77, 183–194. doi: 10.1016/j.ympev.2014.04.017
Zhang, Z., Chuang, Y., Huang, N., and Mitch, W. A. (2019). Predicting the
contribution of chloramines to contaminant decay during Ultraviolet/Hydrogen
peroxide advanced oxidation process treatment for potable reuse. Environ. Sci.
Technol. 53, 4416–4425. doi: 10.1021/acs.est.8b06894
Zhang,D.S.,Hu,P.,Liu,T.G.,Wang,J.,Jiang,S.W.,Xu,Q.H.,etal.(2018).
GC bias lead to increased small amino acids and random coils of proteins in
cold-water fishes. BMC Genomics 19, 315–325. doi: 10.1186/s12864-018-
4684-z
Zhong, J. S., Li, J., Li, L., Conran, J. G., and Li, H. W. (2010). Phylogeny of Isodon
(Schrad. ex benth.) spach (Lamiaceae) and related genera inferred from nuclear
ribosomal ITS, trnL–trnF region, and rps16 intron sequences and morphology. Syst.
Bot. 35, 207–219. doi: 10.1600/036364410790862614
Zhou et al. 10.3389/fpls.2022.1036277
Frontiers in Plant Science frontiersin.org12
Available via license: CC BY
Content may be subject to copyright.
