Assembly and analysis of stephania japonica mitochondrial genome provides new insights into its identification and energy metabolism

Abstract

Stephania japonica, a popular indoor ornamental and medicinal plant widely found in southern China, contains many natural compounds with potential medicinal value. S. japonica is also favored by researchers for its ability to produce catharanthine. Energy metabolism functions in plant development, and the composition of mitochondrial genome is regarded as the foundation for understanding energy metabolism and getting insights into plant environmental adaptation. In present investigation, the whole mitochondrial genome of S. japonica was assembled from both second- and third-generation sequencing data. The mitochondrial genome size of S. japonica is 555,117 bp. It is depicted as a complex polycyclic structure. In addition, we conducted an in-depth study of the cytochrome c oxidase (cox) gene, of which expression levels in different tissues of S. japonica were measured by real-time quantification PCR. Two phylogenetic trees were established in the light of sequences concerning 19 conserved mitochondrial protein-coding genes and cox gene, respectively. Both phylogenetic trees show that S. japonica is more closely related to Aconitum kusnezoffii. The result showed that the cox genes were the most highly expressed in the roots. A high-quality mitochondrial genome exhibits potential application value for the progress of molecular markers, identification of species as super DNA barcoding, and resolve mitochondrial energy metabolism mechanisms in response to the environment using genomic information. With the recognition of the medicinal value of Stephania plants, the genomic information of S. japonica has been thoroughly studied and the comprehensive analysis of its mitochondrial genome in this investigation can offer valuable insights for the breeding of new plant varieties.

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Wu et al. BMC Genomics (2025) 26:185
https://doi.org/10.1186/s12864-025-11359-6 BMC Genomics
†Ya Wu and Zhihao Sun contributed equally to this work.
*Correspondence:
Liang Leng
lling@cdutcm.edu.cn
Shilin Chen
slchen@cdutcm.edu.cn
1School of Pharmacy, School of Modern Chinese Medicine Industry,
Chengdu University of Traditional Chinese Medicine, Chengdu
611137, China
2Institute of Herbgenomics, Chengdu University of Traditional Chinese
Medicine, Chengdu 611137, China
3College of Pharmacy, Hubei University of Chinese Medicine,
Wuhan 430065, China
4School of Chinese Materia Medica, Tianjin University of Traditional
Chinese Medicine, Tianjin 300193, China
5Wuhan Benagen Technology Co., Ltd., Wuhan, Hubei 430000, China
Abstract
Stephania japonica, a popular indoor ornamental and medicinal plant widely found in southern China, contains
many natural compounds with potential medicinal value. S. japonica is also favored by researchers for its ability to
produce catharanthine. Energy metabolism functions in plant development, and the composition of mitochondrial
genome is regarded as the foundation for understanding energy metabolism and getting insights into plant
environmental adaptation. In present investigation, the whole mitochondrial genome of S. japonica was assembled
from both second- and third-generation sequencing data. The mitochondrial genome size of S. japonica is
555,117bp. It is depicted as a complex polycyclic structure. In addition, we conducted an in-depth study of the
cytochrome c oxidase (cox) gene, of which expression levels in dierent tissues of S. japonica were measured by
real-time quantication PCR. Two phylogenetic trees were established in the light of sequences concerning 19
conserved mitochondrial protein-coding genes and cox gene, respectively. Both phylogenetic trees show that
S. japonica is more closely related to Aconitum kusnezoi. The result showed that the cox genes were the most
highly expressed in the roots. A high-quality mitochondrial genome exhibits potential application value for the
progress of molecular markers, identication of species as super DNA barcoding, and resolve mitochondrial energy
metabolism mechanisms in response to the environment using genomic information. With the recognition of
the medicinal value of Stephania plants, the genomic information of S. japonica has been thoroughly studied and
the comprehensive analysis of its mitochondrial genome in this investigation can oer valuable insights for the
breeding of new plant varieties.
Keywords Stephania Japonica, Mitochondrial genome, Cytochrome c oxidase, Evolution, Polymerase chain reaction
Assembly and analysis of stephania japonica
mitochondrial genome provides new insights
into its identication and energy metabolism
YaWu1,2†, ZhihaoSun2,3†, ZhaoyuLiu2,4, TingQiu5, XiaojingLi5, LiangLeng2* and ShilinChen2*
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

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Wu et al. BMC Genomics (2025) 26:185
Introduction
During the last several years, Coronavirus Disease 2019
(COVID-19) was deemed a signicant issue in people’s
lives. Research on the repurposing of clinically autho-
rized medications for treating COVID-19 has shown that
cepharanthine has signicant therapeutic promise [1].
Stephania japonica was of interest to researchers because
of its ability to produce catharanthine [1–3]. S. japonica
is a widely distributed medicinal plant and widespread
indoor decorative crop in southern China [4]. Medicinal
plants contain numerous natural compounds with poten-
tial medicinal value. Taking inspiration from natural
products is easier than designing specic small molecules
based on the structure of proteins [5]. In recent stud-
ies, it has been found that S. japonica contains a variety
of valuable alkaloid components, which can treat many
types of diseases, including cancer [6] and leukopenia [3],
which has garnered the attention of researchers in recent
years, particularly in the development of drugs targeting
receptors [7]. erefore, S. japonica possesses extensive
medicinal properties, and the recent high-quality nuclear
genome sequencing of the Stephania genus has enhanced
our understanding of the biosynthetic mechanisms of
cepharanthine within Stephania species [8–10].
Chloroplast genomes exhibit simpler structural features
compared to mitochondrial genomes, and consequently,
chloroplast genome research in Stephania plants has also
been extensively conducted [11, 12]. Despite the valu-
able data provided by chloroplast and nuclear genomes
for various research purposes, the analysis of mitochon-
drial genome also oers indispensable and eective data
for delving deeper into the genetic basis of its agronomic
traits. Because of its maternal inheritance qualities,
the mitochondrial genome is a valuable addition to the
nuclear genome in many species’ evolutionary studies
[13, 14]. Mitochondria produce energy for each cell as
their main function. Furthermore, they are vital for the
multiplication, dierentiation, and death of cells [15, 16].
Maguire’s endosymbiosis theory holds that mitochondria
develop into unique organelles during long-term plant
symbiosis after emerging from the nucleogenetic archaea
[17, 18]. For mitochondrial structure, early research sug-
gested that the mitochondrial genome had a closed-loop
structure and that all of the genetic information about
mitochondria was included in the circular shape [19, 20].
However, recent research found that the mitochondrial
genome may be more than a simple closed-loop structure
[19]. Plant mitochondrial genomes dier in structure and
sequence due to rapid invasion by short textual insertions
and fragment migration of the chloroplast genome [14].
e complex structures of mitochondria have been found
in many plants, such as Abelmoschus esculentus [19] and
Rhopalocnemis phalloides [21].
Mitochondria are the primary sites of oxidative phos-
phorylation and ATP production in plant cells, inuenc-
ing intracellular energy demand. ey also act as pivotal
organelles in the response of plants to environmental sig-
nals and play a vital role in energy balance [22, 23]. When
faced with stress, mitochondria have the function of con-
verting stress perception into signals of energy deciency,
which subsequently aid in restoring metabolic balance
[24]. e mitochondrial genome also has potential for
plant molecular identication; although the evolutionary
rate is slower, it provides polymorphism and stability and
can be used as a candidate sequence for multi-genome,
multi-fragment barcoding [25].
rough this investigation, we achieved the assembly
and annotation of mitochondrial genome of S. japonica
for the rst time. We also examined migration between
mitochondria and chloroplast genomes, repetitive
regions, as well as codon use bias. Clarifying the roles
of plant mitochondria as well as their evolutionary and
genetic links requires thorough investigations of plant
mitochondrial genomes. We further investigated the
evolutionary link between S. japonica and the degree of
cox gene expression. e mitochondria are essential to
plant growth and energy metabolism, and a comprehen-
sive mitochondrial genome analysis of S. japonica will
contribute to acknowledge the genetic information of S.
japonica growth and development as well as evolution in
the Ranunculales.
Materials and methods
DNA extraction and sequencing
ree healthy S. japonica plants were chosen from the
Wuhan, China. Using the CTAB, DNA was extracted
from roots, stems, leaves, and shoots. Wuhan Benagen
Tech Solutions Company conducted both Oxford and
Illumina sequencing (http://en.benagen.com/). HiSeq
Xten PE150 Illumina, San Diego, CA, USA was deployed
to obtain the Illumina sequencing data, and Oxford
Nanopore GridION × 5 Oxford Nanopore Technologies,
Oxford, UK, handled the Nanopore sequencing.
Acquirement of S. Japonica mitochondrial genome
e mitochondrial genome of S. japonica was acquired
by a hybrid assembly strategy with second- combined
with third-generation sequencing data. GetOrganelle (v
1.7.5) was utilized to assign the mitochondrial genome
from about 10 G of sequenced second-generation data
[26], and the graphical S. japonica mitochondrial genome
was obtained. e long-read sequencing data was aligned
to the assembled contigs by BWA software and the cover-
age depth was then determined via Samtools (v 0.9) [27].
Bandage [28] was applied for visualizing the mitochon-
drial genome. BWA (v 0.7.17) [29] was deployed to depict
the Nanopore data to the base sequences and manually
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Wu et al. BMC Genomics (2025) 26:185
remove the redundant segments of the chloroplast and
nuclear. e mitochondrial genome of S. japonica with
seven ring structures was obtained.
e Geseq software was conducted for the annotation
of protein-coding genes (PCGs) from S. japonica mito-
chondrial genome [30], and Aconitum kusnezoi, Hepat-
ica maxima, and A. thaliana was selected as a reference.
Annotation of mitochondrial genomic tRNAs utilizing
tRNAscan-SE (v 2.0.2) [31] and the blastn of BLAST (v
2.12.0+) was used to annotate the genomic rRNA based
on nucleic acid sequence similarity. Apollo (v. 6. 0) was
also used for manually adjusting the position of genes
[32].
Relative synonymous codon usage and Ka/Ks analyses
Phy-losuite was used for obtaining the PCGs from S.
japonica’s mitochondrial genome [4]. e relative synon-
ymous codon usage (RSCU)was calculated and analyzed
with MEGA 11 [33]. e synonymous (Ks) and nonsyn-
onymous (Ka) substitution rates of the PCGs in the S.
japonica mitochondrial genome were examined by three
species (A. kusnezoi, H. maxima, and A. thaliana).
TBtools [34] was utilized in this investigation to compute
Ka/Ks.
Analysis of repeated sequences
Detection of simple sequence repeats (SSRs) using the
Microsatellite Identication tool named as MISA ( h t t p s :
/ / w e b b l a s t . i p k - g a t e r s l e b e n . d e / m i s a /) [35]. e repetition
counts of ten, ve, four, three, three, and three repeats for
mono-, di-, tri-, tetra-, penta-, and hexameric bases were
found. Tandem Repeats Finder (v 4.09) ( h t t p : / / t a n d e m . b u
. e d u / t r f / t r f . s u b m i t . o p t i o n s . h t m l) [36] was used with the
default parameter to identify tandem repeats with > six
bp repeat. e REPuter webserver ( h t t p s : / / b i b i s e r v . c e b
i t e c . u n i - b i e l e f e l d . d e / r e p u t e r /) [37] was utilized to d e t e r
m i n e the forward and reverse repeats, with the minimal
repeat size set to 30bp.
Migration of the mitochondrial genome to the chloroplast
genome and RNA editing analysis
Based on the chloroplast genome of S. japonica already
held by our research group, BLASTN software [38]
was used for homologous fragment comparison with S.
japonica mitochondrial genome to explore the evolu-
tion of mitochondrial fragments and the migration of the
chloroplast fragments. Circos was used to visualize the
comparison results [39]. Under a threshold of 0.2, RNA
editing events were predicted depending on the PREP
suit online website (http://prep.unl.edu/) [37].
Construction of phylogenetic tree and collinearity analysis
To establish the phylogenetic tree, the shared mitochon-
drial PCGs between S. japonica and 26 other species
were utilized. (Table S5 provides full species informa-
tion). e mitochondrial genome information was down-
loaded from NCBI. PhyloSuite was utilized to extract the
conserved PCGs (atp1, atp4, atp6, atp8, matR, rps3, cox
2 ~ 3, ccmB, ccmC, ccmFC, ccmFN, nad 1 ~ 3, nad 5 ~ 7,
and nad9) [4]. We aligned sequences with the MAFFT
algorithm [40]. e evolutionary relationship was ana-
lyzed via MRBAYES (v. 3.2.2) [41]. e results of the tree
analysis were displayed by ITOL (https://itol.embl.de/)
[42].
MCscanX was utilized for creating a multiple synteny
plot of S. japonica with H. maxima and A. kusnezof-
i [43]. e BLASTN results of pairwise comparisons
of each mitochondrial genome were obtained via the
BLAST program; homologous sequences larger than
500bp were kept as conserved collinearity blocks. e
Maximum-Likelihood technique was used in MEGA 11
(v 11.0.13) to create a phylogenetic tree of the cox gene
as follows: General Time Reversible Model as adopted
model; Gamma Distributed With Invariant Sites (G + I)
as Rates among Sites. Table S5 provides full species
information.
Real-time quantitative PCR
Using the total RNA extraction kit of plant (Foregene
Biotech, Chengdu, China, CodeNo. RE-05011), the
experiment was conducted on three more S. japonica
that are presently being grown in our laboratory from
the same source (2.1) (30°68’N,103°81’E). en the total
RNA was transferred to cDNA utilizing the reagent kit
with gDNA Eraser (Foregene Biotech, Chengdu, China,
Code No. RT-01032). A quantitative real-time poly-
merase chain reaction (qRT-PCR) was deployed using the
QuantStudio5 real-time PCR system. TB Green® Premix
ExTaq™ II (Vazyme Biotech, Beijing, China, Code No.
Q711-02) was used as the PCR reagent, during which
three technical replications were carried out. e ACT2
gene sequence (AT3G18780.2) [44] and the GAPDH
gene sequence (AT3G04120.1) of A. thaliana was used
as housekeeping genes [45]. rough the Blast result, the
SjapChr2G00058590.1 corresponds to the ACT2 gene
and SjapChr10G00234090.1 corresponds to the GAPDH
gene of S. japonica. ese gene were used as internal ref-
erence for qPCR analysis [8]. e 2−△△CT calculations
were utilized to quantied gene expression. Table S6 con-
tains a list of the primer sequences that were employed in
this investigation.
Verifying recombination events mediated by repetitions
To verify the homologous recombination products sup-
ported by Nanopore long reads, we extracted sequences
of 300–400bp anking the predicted repeat sequences
to form a reference sequence and designed specic prim-
ers [46]. PCR amplication was performed on a 25 µL
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Wu et al. BMC Genomics (2025) 26:185
sample, which contained 12.5 µL 2 × Taq Master Mix
(Dye Plus) (Vazyme, Nanjing, China), 9.5 µL ddH2O, 1
µL of each primer (10 µmol/L), and 1 µL DNA. e PCR
reaction was carried out under the following conditions:
denaturation at 95°C for 3min; followed by 35 cycles of
95°C for 15s, 58°C for 15s, and 72°C for 2min; and
extension at 72°C for 5min. We observed the PCR ampli-
cons by 1.0% agarose gel electrophoresis and sequenced
the PCR amplicons using Sanger sequencing technology
from Beijing Tsingke Biotech Co., Ltd. (Beijing, China).
Results
Characteristics of the mitochondrial genome of S. Japonica
In this investigation, the complete mitochondrial genome
of S. japonica was obtained. e seven circular structures
that make up the mitochondrial genome of S. japonica
have a total length of 555,117 bp, 46.56% GC content
and average depth over 90X (Table S1). e complete
mitochondrial genome of S. japonica consists of seven
ring structures. In order to verify the accuracy of the
assembled structure of S. japonica, we performed PCR
amplication and Sanger sequencing on 300–400bp spe-
cic primers designed for PCR amplication at both ends
of the predicted repeat sequences in the assembled seven
ring structures (Figure S1 A). PCR amplication showed
that the band length was as expected (Figure S1C, D and
E), and Sanger sequencing conrmed the existence of
this complex conformation (Figure S2-10). e S. japon-
ica contains three rRNA genes, 18 tRNA genes, and 40
PCGs (Fig. 1c). e annotated PCGs in the S. japonica
mitochondrial genome are grouped into ten categories,
as shown in Table1.
A detailed analysis of the genes of the species can pro-
vide the necessary help for the identication of species
and can also facilitate the in-depth study of species [47].
For the purpose of better analysis, the mitochondrial
Fig. 1 S. japonica’s mitochondrial genome structure and annotation. (a) GetOrganelle predicted seven circular contigs in S. japonica’s mitochondrial
genome. (b) The 2D structure of S. japonica’s mitochondrial genome following the removal of nuclear and articial chloroplast gene segments. (c) The
mitochondrial genome annotations for S. japonica. The sequence information in (b) is accordingly marked on each ring. Dierent colors correspond to
dierent roles of genes
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Wu et al. BMC Genomics (2025) 26:185
genes of S. japonica, H. maxima, and A. kusnezoi were
compared, and duplication and loss of some genes were
found. In H. maxima, the mitochondrial genome was
found for atp1 replication and deletion of rps14 and
rps10 in contrast to the other. In S. japonica, the mito-
chondrial genome was relatively complete with the dele-
tion of only one gene (sdh4) compared with the other two
species, while four genes were found absent in A. kusne-
zoi (rpl2, rps2, rps11, and rps19).
Codon usage analysis among protein-coding genes
e ratio of synonymous codon usage frequency to
expected frequency is known as RSCU. e predicted
frequency represents the average usage frequency of all
codons that encode a specic amino acid. A codon’s rela-
tive high use bias is shown when its RSCU value is more
than one. As shown in Fig.2, the codon in each amino
acid was shown in dierent colors. For mitochondrial
PCGs, there is a general bias in the codon usage, except
for the initiation codons AUG and UGG, both of which
have RSCU values of 1. For example, tyrosine (Tyr) has
the greatest RSCU value of 1.52 and is highly biased for
UAU, whereas histidine (His) is biased for CAU and has
an RSCU value of 1.5. Additionally, all three show a high
codon use bias, with the maximum RSCU of arginine
(Arg), glutamine (Gln), and glycine (Gly) values being
larger than 1.4.
Repeat sequence analysis
Based on the MISA online service platform, 187 SSRs
were found in S. japonica, and they were distributed
among seven mitochondrial circular molecules: circu-
lar molecule 1 (63 SSRs), circular molecule 2 (23 SSRs),
circular molecule 3 (29 SSRs), circular molecule 4 (24
SSRs), circular molecule 5 (19 SSRs), circular molecule 6
(13 SSRs), and circular molecule 7 (16 SSRs). e over-
all distribution detail is shown in Figure S11. Adenine
monomeric repeat sequences (27) accounted for 48.2% of
Table 1 The encoding genes of S. Japonica mitochondrial
genome
Groups Gene Names
ATP synthase atp1, atp4, atp6, atp8, atp9
NADH dehydrogenase nad1, nad2, nad3, nad4, nad4L, nad5,
nad6,
nad7, nad9
Cytochrome c biogenesis cob
Ubiquinol cytochrome c
reductase
ccmB, ccmC, ccmFC, ccmFN
Cytochrome c oxidase cox1, cox2, cox3
Maturases matR
Transport membrane protein mttB
Large subunit of ribosome rpl2, rpl5, rpl10, rpl16
Small subunit of ribosome rps1, rps2, rps3, rps4, rps7, rps10, rps11,
rps12, rps13, rps14, rps19
Succinate dehydrogenase sdh3
Ribosome RNA rrn5, rrn18, rrn26
Transfer RNA trnC-GCA, trnD-GUC, trnE-UUC (×2)
trnF-GAA, trnG-GCC, trnH-GUG, trnK-
UUU, trnM-CAU (×3), trnN-GUU (×2),
trnP-CGG, trnP-UGG (×2), trnQ-UUG,
trnS-GCU, trnS-UGA, trnT-GGU, trnV-
GAC, trnW-CCA, trnY-GUA
Fig. 2 RSCU in PCGs of S. japonica. The height of the bar means the total number of amino acid subtypes, and the fraction of each codon is shown by
the dierent colors
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Wu et al. BMC Genomics (2025) 26:185
the 59 monomeric SSRs, whereas 49.2% of all SSRs were
composed of monomeric and dimeric repeats. Addition-
ally, the proportion of tetrameric repeat sequences (61)
was high and accounted for 32.6% of the total SSRs. How-
ever, no SSR sequences containing hexameric repeats
were found within the assembled mitochondrial genome.
Tandem repeats are widely observed in prokaryotic and
eukaryotic genomes [48–50]. Totally 19 tandem repeats
were determined (Table S3), 7 of which had a 100%
match rate. Additionally, dispersed repeats in the mito-
chondrial genome were examined. Consequently, 257
duplicates with lengths higher than 30 were noted in total
(shown in Fig.3); the most scattered repeats were found
in circular molecule 1 (163bp), while the least scattered
repeats (four bp) were found in circular molecule 5.
DNA migration from chloroplast to mitochondria
A total of 16 homologous fragments totaling 4,831 bp
in length were discovered to migrate from chloroplast
to mitochondria in accordance with sequence similarity
and the chloroplast genome. ese fragments account for
0.87% of the complete mitochondrial genome. In partic-
ular, the two ultralong fragments were found in circular
molecule 4, each 916bp in length, and both contain parts
of the tRNA gene (tRNA-UGG). A total of ve intact
tRNA genes (trnM-CAU, trnN-GUU, trnI-CAU, trnD-
GUC, and trnT-GGU) were found in 16 homologous
fragments. It may be evident from the data that two chlo-
roplast PCGs migrated to the mitochondrial genome but
lost their integrity during the migration (Table S4, Fig.4).
Predicting RNA editing
RNA editing, the post-transcriptional bioprocess, rou-
tinely improves protein folding [17]. It is widespread and
impressive for regulating mitochondrial gene expression
among advanced plants. PREP was utilized to predict
RNA editing loci in mitochondrial genes of S. japonica.
e outcome revealed 684 RNA editing loci in total. e
nad4 gene, which includes 55 editing sites, has the most
in S. japonica’s mitochondrial genome, as seen in Fig.5.
e rps1 and rps7 genes had the fewest editing sites in S.
japonica mitochondrial genes, with only two RNA edit-
ing sites predicted, respectively. Among the 684 edited
sites, 330 were found at the triplet code’s rst position,
while 331 were found at the triplet code’s second base.
And 20 edited sites showed special cases where both the
rst position and second base were changed by editing,
such as the substitution of phenylalanine (TTT / TTC)
for the original proline (CCT / CCC). e properties of
the original amino acids can also be changed after RNA
editing, with 8.5% of amino acids performing hydropho-
bic to hydrophilic conversion, whereas 47.22% perform
hydrophilic to hydrophobic conversion. Additionally,
our ndings demonstrated a leucine propensity in amino
acids of projected editing codons following RNA edit-
ing, which is corroborated by the nding that 44.59% of
amino acids underwent leucine conversion following
RNA editing (Table2). Additionally, we found that three
genes (ccmFC, atp6, and rps11) have altered open reading
frames as a result of RNA editing that produces a termi-
nation codon.
Phylogenetic analysis and collinearity analysis
Phylogenetic trees were constructed for 26 species under
six orders of angiosperms (shown in Fig.6), as well as S.
japonica, according to the DNA sequences of 19 of the
shared PCGs (ccmB, ccmC, ccmFC, atp1, atp4, atp6, atp8,
Fig. 4 Mitochondrial genome migration events in S. japonica. The ge-
nomes of the mitochondria and chloroplasts are represented by the blue
and yellow arcs. The green lines between the arcs signify homologous ge-
nomic segments; the deeper color denotes a more similar section
Fig. 3 The number of repetitive sequences in S. japonica. Blue denotes
forward repetitions, red denotes reverse repeats, purple suggests palin-
dromic repeats, and green represents tandem repeats
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Wu et al. BMC Genomics (2025) 26:185
ccmFN, cox 2 ~ 3, matR, nad 1 ~ 3, nad 5 ~ 7, nad9, and
rps3). As shown in Figs.6a and 20 of the 24 nodes exhib-
ited bootstrap support of at least 80%, while 15 nodes had
100% bootstrap support. e evolutionary closeness of S.
japonica to the branches created by the two Ranunculales
and the closer anity of A. kusnezoi are supported by
the generated phylogenetic tree and align with the latest
Angiosperm Phylogeny Group IV classication.
Between S. japonica species and two closely related
species of the Ranunculales family, H. maxima and A.
kusnezoi, several homologous collinearity blocks were
found; however, these collinearity blocks were short
(Fig.6B). ere were some unmatched areas discovered,
and these sequences are exclusive to the species and are
not linked to any genes with the rest of the species. As
shown in Fig.6B, the collinearity blocks between these
three mitochondrial genomes are not aligned in the same
precedence. Signicant genomic rearrangements of the
mitochondrial genome of S. japonica occurred in com-
parison with H. maxima and A. kusnezoi. e collin-
earity blocks are short, and the mitochondrial genomic
sequences are nonconservative alignments and undergo
frequent genomic recombination.
The substitution rates of protein-coding genes
We performed Ka/Ks calculations based on these shared
PCGs with A. kusnezoi, H. maxima, and A. thaliana
(Fig. 7). For phylogenetic reconstruction and compre-
hending the evolutionary processes of PCG sequences
in H. maxima and A. kusnezoi, Ka and Ks are crucial.
e pressure of positive and negative selection can be
inferred from the ratio of Ka/Ks. e ccmB genes in A.
kusnezoi, H. maxima, A. thaliana, and S. japonica
have Ka/Ks values greater than 1, revealing that positive
choice of this gene occurred during evolution (Fig. 7).
Table 2 RNA editing sites in S. Japonica
Type RNA editing Number Percentage
hydrophobic CTT (L) = > TTT (F) 18 30.83%
CCC (P) = > C TC (L) 13
CCG (P) = > C TG (L) 42
CCA (P) = > C TA (L) 64
GCG (A) = > GTG (V ) 7
CCC (P) = > TTC (F) 8
CCT (P) = > CTT (L) 31
GCA (A) = > GTA ( V ) 1
CTC (L) = > TTC (F) 10
CCT (P) = > TTT (F) 12
GCT (A) = > GTT (V ) 3
GCC (A) = > GTC (V ) 2
hydrophilic CGC (R) = > TGC (C) 13 13.01%
CGT (R) = > TGT (C) 39
CAT (H) = > TAT (Y ) 25
CAC (H) = > TAC ( Y) 12
hydrophobic-hydrophilic CCC (P) = > TCC (S) 16 8.5%
CCA (P) = > TCA (S) 14
CCT (P) = > TCT (S) 20
CCG (P) = > TCG (S) 8
hydrophilic-hydrophobic TCG (S) = > TTG (L) 58 47.22%
TCA (S) = > TTA (L) 97
TCC (S) = > TTC (F) 52
TCT (S) = > TTT (F) 55
ACT (T ) = > ATT (I) 6
ACA ( T ) = > ATA (I) 7
CGG (R) = > TGG (W ) 39
ACG ( T) = > ATG (M) 8
ACC ( T) = > ATC (I) 1
hydrophilic-stop CAA (Q) = > TAA (*) 2 0.4%
CGA (R) = > TGA (*) 1
Note: “*” rep resents the terminatio n codon
Fig. 5 RNA editing events of dierent genes in S. japonica
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Page 8 of 12
Wu et al. BMC Genomics (2025) 26:185
Among the 39 PCGs of S. japonica, six genes have Ka/Ks
values greater than one compared to H. maxima and less
than one (Fig.7).
Evolution and expression analysis of the cox genes
e phylogenetic tree of the cox genes was consistent
with that built by 19 conserved genes of the entire spe-
cies (Figs.6A and 8A). e cox gene expression levels in
dierent tissues of S. japonica were also analyzed (root,
stem, leaf, and bud). e relative expression levels of
the cox genes veried tissue specicity. at is, they had
superior expressions in the root than in other tissues,
which may be connected to how the plant is growing
[51]. e root of S. japonica also had higher metabolic
activity during growth and required more energy, which
could explain the higher cox expression in these tissues
[52].
Discussion
Decoding complete genome sequence information
oers insights into genetic diversity, enabling the pre-
cise identication of gene variations that are crucial for
understanding the genetic basis of traits, diseases, and
evolutionary process. Plant cells possess three distinct
genomes, containing the nuclear, chloroplast, as well as
mitochondrial genomes, oers valuable resources for
investigating plant evolution [53]. Plant mitochondrial
genomes exhibit greater complexity compared to animal
mitochondrial genomes due to many factors, includ-
ing genome dierences in size and repetitive sequences
Fig. 6 The evolutionary analysis of S. japonica. (A) Phylogenetic tree analysis based on 19 conserved proteins encoded by S. japonica. (B) Analysis of col-
linearity between S. japonica and other two Ranunculales species
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Page 9 of 12
Wu et al. BMC Genomics (2025) 26:185
[54]. In early research on plant mitochondrial genomes,
they were reported to exhibit a single circular structure
analogous to that of animal mitochondrial genomes
[55]. However, numerous studies have shown that their
actual structures can also possess a variety of branched,
linear, or mixed forms of genomic organization [56–58].
A high-quality S. japonica genome has been assembled
with a size of 643.4 Mb [8]. rough this investigation,
we investigated the mitochondrial genome of S. japonica.
S. japonica shows marked structural specicity com-
pared to its relatives, being a complex structure with
multiple branches, whereas the mitochondrial structure
of two Ranunculales species presents a classical one-loop
structure [59, 60]. Multiring structures have also been
discovered in the mitochondrial genomes of various spe-
cies, including ferns, basal angiosperms, monocots, and
dicots. For instance, the mitochondrial genome of Fritil-
laria ussuriensis Maxim consists of 12 circular structures,
among which ve rings contain only a single functional
gene [56]. Similarly, the mitochondrial genome of Angel-
ica dahurica also consists of 12 circular structures [61].
ese results indicate that plant mitochondrial genomes
are diverse and complex in terms of structure, size, and
gene content.
Fig. 8 Evolution and expression analysis of cox genes. A. Phylogenetic tree analysis based on cox genes with 24 species. The color of the box indicates
categories of species: Caryophyllales in blue, Santalaes in yellow, Proteales in perilla, Ranunculales in red, and Alismatales in green. B. Real-time quantica-
tion of cox genes expression in dierent tissues of S. japonica
Fig. 7 The Ka/Ks values of 39 PCGs of S. japonica versus three species. Among them, H. maxima and A. kusnezoi belong to Ranunculaes
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 10 of 12
Wu et al. BMC Genomics (2025) 26:185
RNA editing is critical for most events, such as gener-
ating initiation codons, termination codons, or chang-
ing codons to specify conserved amino acid positions. In
plants, RNA editing of plant organ genomes frequently
results in C-to-U/U-to-C transitions [62], and in the
present study, It was all C to U editing in the mitochon-
drial genome of S. japonica, and there were 687 editing
loci in the mitochondrial genes of H. maxima with the
same orders as S. japonica, which were also for C to U
editing [60]. Fewer editing sites (441) were revealed in A.
thaliana compared to S. japonica, but the changes were
consistent with the overall increase in overall hydropho-
bicity [62]. RNA editing is indispensable for regulating
various physiological processes, such as plasmid prog-
ress and the response to stress [63]. Alterations in RNA
editing are common in stress responses [64–66]. Fur-
thermore, we found that three genes (ccmFC, atp6, and
rps11) had altered open reading frames due to RNA edit-
ing to generate termination codons.
Moreover, only sdh3 was found in the mitochondria of
S. japonica. e sdh4 gene was not found in S. japonica,
in contrast to H. maxima and A. kusnezoi. It has been
demonstrated that sdh contributed to the production of
ROS in mitochondria, regulating plant development and
stress response in A. thaliana and Rice [67]. Whether it
is related to the change in the response of S. japonica to
its growth environment is worthy of further study. As
the respiratory chain’s nal electron acceptor. cox is vital
for oxidative phosphorylation and the conversion of O2
to H2O [68]. Inhibition of cox17 gene expression in A.
thaliana can reduce the response to salt stress [61]. e
cox genes are usually thought to function in the evolu-
tion of species and the growth and progress of plants.
For example, the cox decient mutant of A. thaliana has
problems in seed germination and root growth retarda-
tion [69]. And cox expression is specic and preferentially
expressed in tissues taking high energy requirements,
such as the root meristem [51]. e ndings verify that
the cox gene was found to be highly expressed in roots
in S. japonica. To make species identication easier, some
researchers have even suggested utilizing the organelle
genome as a super DNA barcode [70]. e cox genes are
relatively conserved in evolution and widely considered
to be important enzymes involved in respiration and bio-
logical processes. It has served as a species identication
DNA barcode and has been well applied for animal spe-
cies identication [71]. In this investigation, the cox genes
of S. japonica were utilized to build the phylogenetic tree,
in agreement with one with 19 conserved genes. is
result further shows the reliability of the evolutionary
relationship and the conservation of cox genes, proving
that the cox gene is also relatively conservative among
plant species, although the mitochondrial genome is
more complex. Although the application of the cox gene
for the determination of closely related plants has certain
limitations, it is undeniable that the cox gene is still used
to identify plant species. is study looked more closely
at the cox gene expression pattern in S. japonica. e cox
genes in S. japonica were expressed in the roots, stem,
leaf, and bud. Among these tissues, cox gene expression
was highest in roots. is might be related to the growth
stage of the plant or the oxygen content in the air [72, 73].
Conclusions
is investigation successfully obtained the whole mito-
chondrial genome of S. japonica, providing a valuable
resource for a better understanding of Stephania species.
We conducted an interspecies study on the cox gene and
believe that it can still be used as an alternative solution
for species identication. We also further investigated the
cox gene expression in dierent tissues, which may assist
in research related to plant energy metabolism. is lays
the foundation for mitochondrial-based species identi-
cation techniques, such as DNA barcoding, as well as
research related to energy metabolism.
Supplementary Information
The online version contains supplementary material available at h t t p s : / / d o i . o r
g / 1 0 . 1 1 8 6 / s 1 2 8 6 4 - 0 2 5 - 1 1 3 5 9 - 6 .
Supplementary Material 1
Supplementary Material 2
Author contributions
CSL and LL designed and oversaw the study. LL oered both experimental
supplies and nancial support. QT, LXJ, LZY, SZH, and WY will engage in data
analysis. WY to experiment. WY, SZH, and LZY char ts to create a rst draft.
Funding
This work is supported by the talented person scientic research start funds
subsidization project of Chengdu University of Traditional Chinese Medicine
(030040016/030).
Data availability
All the mitochondrial genomes mentioned in this study can be available
in NCBI ( h t t p s : / / w w w . n c b i . n l m . n i h . g o v /). And the accession numbers can
be found in the supplementary material. The mitochondrial genome of S.
japonica can be available at Genbank under accession number OR500009.
All raw sequencing data were deposited under the National Center for
Biotechnology Information (NCBI) GenBank accession number PRJNA888087.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Collection of plant material
The collection of plant material complies with relevant institutional, national,
and international guidelines and legislation.
Competing interests
The authors declare no competing interests.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 11 of 12
Wu et al. BMC Genomics (2025) 26:185
Received: 15 November 2024 / Accepted: 11 February 2025
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