Accepted author version posted online: 29 January 2020
Sequencing and Characterisation of Complete Mitochondrial DNA Genome for
Trigonopoma pauciperforatum (Cypriniformes: Cyprinidae: Danioninae) with
Phylogenetic Consideration
1
Chung Hung Hui*, 1Leonard Lim Whye Kit, 2Liao Yunshi, 2Tommy Lam Tsai Yuk and
1
Chong Yee Ling
1
Faculty of Resource and Technology, Universiti Malaysia Sarawak, Kota Samarahan
94300 Sarawak, Malaysia
2
School of Public Health, The University of Hong Kong, Hong Kong
*Corresponding author: hhchung@unimas.my
Running title: mitochondrial DNA genome of Trigonopoma pauciperforatum
Abstract. The Trigonopoma pauciperforatum or the redstripe rasbora is a cyprinid
commonly found in marshes and swampy areas with slight acidic tannin-stained water
in the tropics. In this study, the complete mitogenome sequence of T. pauciperforatum
was first amplified in two parts using two pairs of overlapping primers and then
sequenced. The size of the mitogenome is 16,707 bp, encompassing 22 transfer RNA
genes, 13 protein-coding genes, two ribosomal RNA genes and a putative control region.
Identical gene organisation was detected between this species and other family
members. The heavy strand accommodates 28 genes while the light strand houses the
remaining nine genes. Most protein-coding genes utilize ATG as start codon except for
COI gene which uses GTG instead. The terminal associated sequence (TAS), central
conserved sequence block (CSB-F, CSB-D and CSB-E) as well as variable sequence
block (CSB-1, CSB-2 and CSB-3) are conserved in the control region. The maximum
likelihood phylogenetic tree revealed the divergence of T. pauciperforatum from the
basal region of the major clade, where its evolutionary relationships with Boraras
maculatus, Rasbora cephalotaenia and R. daniconius are poorly resolved as suggested
by the low bootstrap values. This work contributes towards the genetic resource
enrichment for peat swamp conservation and comprehensive in-depth comparisons
across other phylogenetic researches done on the Rasbora-related genus.
Keywords: Trigonopoma pauciperforatum, mitogenome, gene arrangement, light strand
origin, phylogenetic analysis
INTRODUCTION
The redstripe rasbora (Trigonopoma pauciperforatum) (Weber & de Beaufort, 1916) is
grouped under the subfamiliy Danioninae in the Cyprinidae family. It has the distinctive
thick striking red neon stripe which aligns in parallel to its spine, starting from the side of
its jaw, crossing upper part of the eye and up till before its tail fin (Weber & de Beaufort,
1916). Its greyish brown streamline body are equipped with smoke-grey fins, white belly
as well as a fork-shaped caudal fin tail (Ward, 2003). Females have bigger bellies than
1
males in general because this species is egg-spawning fish. This red-striped rasbora
fish can be found abundantly in school around stagnant fresh waters (rivers, drainages,
lakes and streams) of South East Asia, including Peninsular Malaysia, Sarawak and
Sumatra (Ward, 2003). The type locality of this species is Sumatra. Their natural habitat
has heavily grown and overhanging vegetation with minimal lighting. The diet of this fish
is mainly made up of zooplankton, larvae and insects. Adult fish can grow up to the
length of >6 centimetre (Ward, 2003).
The T. pauciperforatum is a popular ornamental aquarium fish often mistaken for
the Glowlight Tetra (Hemigrammus erythrozonus) (Durbin, 1909) due to their high
morphological similarities but they are distinguishable by the much brighter red stripe
and the absence of adipose fin in the Redline Rasbora (Durbin, 1909; Weber & de
Beaufort, 1916; Ward, 2003). Due to their extremely selective breeding behaviour,
breeding them in aquarium conditions is not easy and the success rate is higher when
they are placed in school of 6 to 10 (Ward, 2003). Adult females scatter their eggs all
over overgrown vegetation before the adult males are stimulated release sperms to
fertilise the eggs during the action of tailing the females. Egg hatching occurs within 1 to
2 days post fertilisation and the fry can swim freely within 3 to 5 days (Ward, 2003). The
lifespan of this fish ranges from 3 to 5 years with good care and maintenance under the
following conditions: pH 6.2 to 7.0, 0 to 6-degree hardness and 22.7 to 26°C (Ward,
2003).
The T. pauciperforatum was previously classified under the genus Rasbora. The
Rasbora genus encompasses a large group of diversified freshwater fishes, making it
the most species-enriched genus (87 species as of 2015) in the Cyprinidae family (Fricke
et al., 2018). The classification of the Rasbora genus possesses complications as it is
known as the catch-all group lacking synapomorphies or shared derived characters
(Brittan, 1954; Kottelat & Vidthayanon, 1993; Liao et al., 2010; Tang et al, 2010). The
eight Rasbora species complexes defined by Brittan (1954) had been revised recurrently
over the years by various researchers (Kottelat & Vidthayanon, 1993; Siebert & Guiry,
1996; Kottelat, 2005; Liao et al., 2010) with some new genera being introduced and till
now majority of them still hold firm on the Rasbora sensu lato concept by Brittan (1954)
which encompasses all the new genera created. Yet, most of the Rasbora species lack
the distinctive characters to form a monophyletic clade of its own both morphologically
(Liao et al., 2010) and molecularly (mitochondrial COI, Cytb and nuclear RAG1)
(Kusuma et al., 2016).
The use of Rasbora species in genetic research is picking up its pace recently with
the discovery of their potential as ecotoxicology models (Lim et al., 2018; Wijeyaratne &
Pathiratne, 2006). To date, only nine Rasbora species (namely R. argyrotaenia, R.
sumatrana, R. trilineata, R. aprotaenia, R. steineri, R. lateristriata, R. daniconius, R.
borapetensis and R. cephalotaenia) and four other species previously classified under
the Rasbora genus (Rasboroides vaterifloris, Trigonostigma heteromorpha, T. espei and
Boraras maculatus) (Miya, 2009; Tang et al., 2010; Chang et al., 2013; Ho et al., 2014;
Zhang et al., 2014; Kusuma & Kumazawa, 2015; Kusuma et al., 2017) had their
mitochondrial genomic sequences published out of the total 87 species discovered thus
far (Fricke et al., 2018), a mere 14.94%. The genus T. pauciperforatum resides in
(Trigonopoma) contains only two species thus far, where its sole genus counterpart is T.
gracile. To the best of our knowledge, T. pauciperforatum is the only species from this
genus that have had its mitogenome sequenced and this accounts for the urgency to
unravel more about the mitogenomes of its genus as well as natural habitat counterparts
2
in order to obtain a bigger picture of the genetic biodiversity in the peat swamp for
conservation purposes (Chen et al., 2016; Sule et al., 2018). On the other hand, the
phylogenetic data based on whole mitogenome sequences of this species provides
opportunities for comprehensive comparison of the phylogenetic tree constructed based
on morphologies (Liao et al., 2010).
Thus, this study had shed light on the landscape of the complete mitochondrial
genome of T. pauciperforatum beside further dissecting on the genetic contents and
revealing the molecular phylogenetic relationship across 13 other closely related
members of the Danioninae subfamily (from Rasbora genus and other species
previously classified under Rasbora genus). This study also contributes towards the
genetic resource enrichment for peat swamp conservation (Sule et al., 2018) and
comprehensive in-depth comparisons across other phylogenetic researches (Liao et al.,
2010; Kusuma et al., 2016) done on the Rasbora-related genus.
MATERIALS AND METHODS
Sampling and Genomic DNA Extraction
The T. pauciperforatum specimen was collected from Matang River, Sarawak, Malaysia
(1.5755° N, 110.2990° E) with the permit issued by Sarawak Forestry Department
(permit number: NCCD.94047(Jld13)-178). Adult fish was sacrificed humanely using
TricaneTM as anaesthetics with permission from Universiti Malaysia Sarawak Animal
Ethics Committee (reference number: UNIMAS/TNC(PI)-04.01/06-09(17)). The muscle
tissues were harvested from the fish body before subjecting to storage in 95% ethanol.
The genomic DNA was extracted using CTAB method (Thomas et al., 2010).
Primers Design, Long-PCR Amplification and DNA Sequencing
A total of two pairs of primers were designed based on the multiple alignment outcomes
from the complete mitochondrial genome of four closely related Rasbora species
including R. argyrotaenia, R. sumatrana, R. trilineata and R. aprotaenia. The primer pairs
(Table 1) were designed to amplify two large fragments from the mitochondrial genome
with overlapping of at least 2 kb at both ends of fragments to ensure good sequencing
reads. The complete mitochondrial genome of T. pauciperforatum was assembled by
joining the two large amplicon fragments and trimming overlapping sequences.
Long-Polymerase Chain Reaction (Long-PCR) was conducted using Bio-Rad T-100
Thermal Cycler in 20 μL total reaction volume encompassing 0.4 μL 10 μM forward and
reverse primer each, 1.6 μL 2.5mM dNTP, 2.0 μL 10X PCR buffer (with Mg2+), 2.5 U
high-fidelity Taq polymerase, 14.6 μL nuclease-free water and 0.8 μL genomic DNA
extract orchestrated under conditions: one cycle of pre-denaturation at 94°C for 2 min,
followed by 35 cycles of denaturation, annealing and extension at 94°C (30 s), primerspecific temperature (30 s) and 72°C (5 min) respectively and a final extension cycle at
72°C for 5 min. Agarose gel electrophoresis was performed to size separate the
amplicons on 1% agarose gel for visualization under UV light. PCR purification was done
prior to pair-ended short-read DNA sequencing on Illumina HiSeq 4000 System.
Sequencing reads are quality-checked, adaptor-trimmed using cutadapt (Martin, 2011)
3
and assembled into the complete genome sequences using de novo assembler SPAdes
(Bankevich et al., 2012).
Mitochondrial Genome Characterisation and Gene Analysis
The mitochondrial genome map was constructed using MitoFish (Iwasaki et al., 2013)
(http://mitofish.aori.u-tokyo.ac.jp/annotation/input.html). Using MEGA 7.0 (Kumar et al.,
2016), the protein-coding genes were subjected to translation into amino acid sequences
to amend truncated or premature stop codons to ensure their functionalities. The codon
usage was determined using MEGA 7.0 (Kumar et al., 2016) whereas the nucleotide
composition was calculated using DNA nucleotide counter (Heracle BioSoft, 2014). All
anti-codons of tRNA genes were identified using default search mode of the tRNA-scan
SE v. 2.0 software (Lowe & Chan, 2016) (http://lowelab.ucsc.edu/cgi-bin/tRNAscanSE2.cgi). The L-strand origin (OL) determined thru sequence homology was then
subjected to secondary structure visualisation using RNA structure 6.0 (Reuter &
Mathews, 2010). All DNA sequences forming the complete mitochondrial genome was
deposited
into
the
GenBank
database
via
the
Sequin
software
(http://www.ncbi.nlm.nih.gov/Sequin/).
Phylogenetic Tree Construction
The raw data for phylogenetic analysis was collected from GenBank database which
includes 13 other closely related members of the Danioninae subfamily (from Rasbora
genus and other species previously classified under Rasbora genus) with complete
mitochondrial genomic DNA available publicly; Acheilognathus typus and Danio rerio
were selected as the outgroup. A total of 12 protein-coding genes (except for ND6 due
to its high heterogeneity (Miya & Nishida, 2000) were concatenated to one single fasta
format entry for each species to be analysed by first conducting multiple sequence
alignment using clustal w in MEGA 7.0. A model test was performed using MEGA 7.0
prior to phylogenetic tree construction and the best suited model determined, the GTR+G
(General Time Reversible model with Gamma distributed rates among sites) was
employed via Maximum Likelihood (ML) analysis with bootstrap of 1000 replicates. The
resultant phylogenetic tree was viewed using FigTree v1.4.2.
RESULTS AND DISCUSSION
Mitochondrial DNA Genome Structure
The size of the complete mitochondrial genome of T. pauciperforatum is 16,707 bp with
the inclusion of 22 tRNA genes, 13 protein-coding genes, two rRNA genes and a control
region (Figure 1; Table 2). The complete mitochondrial genome sequence was deposited
in the GenBank database with the assigned accession number MK034301. The heavy
strand (H-strand) of the mitochondrion carries a total of 28 genes whereas the remaining
are housed on the light strand (L-strand). All 4 overlaps detected from the entire
mitochondrial genome are found on the H-strand. The greatest overlap (7 bp) was
observed in both between genes ATP8 and ATP6 as well as between genes ND4L and
4
ND4. The lengthiest intergenic spacer (34 bp) was detected between genes tRNAAsn and
tRNACys.
The overall A+T content of the mitochondrial genome (60.0%) is much greater than
G+C content (40.0%) (Table 3) which is similar to Cobitis lutheri, R. borapetensis and R.
steineri (Cui et al., 2013; Zhang et al., 2014; Chang et al., 2013). The A+T content of
protein-coding genes (60.6%) and control region (66.5%) differ by a slight 5.9%.
Interestingly, the overall base composition of the entire mitochondrial genome and
overall protein-coding genes did not deviate much from each other: 34.0% for A, 25.2%
for C, 14.8% for G, 26% for T in terms of overall genome; 33.7% for A, 25.9% for C, 13.4%
for G, 26.9% for T in total of 13 protein-coding genes.
Protein-Coding Gene Features
The gene group that made up almost 68.3% of the entire T. pauciperforatum
mitochondrial genome is none other than the protein-coding gene group with a total of
11,412 bp coverage over 13 genes. With the translation capacity of up to 3801 amino
acids, the protein-coding gene group incorporates genes with size ranging between 165
bp (ATP8) and 1830 bp (ND5). All three overlaps found in this group are located on the
H-strand.
The start codon usage of all 12 protein-coding genes are generally ATG, except for
the GTG which is found exclusively in COI gene. These phenomena can be seen
commonly occurring in Brama japonica, R. steineri, R. trilineata, R. argyrotaenia, R.
borapetensis, R. aprotaenia and R. lateristriata (Chen et al, 2016; Chang et al., 2012;
Kusuma et al., 2017; Ho et al., 2014; Zhang et al., 2014; Kusuma & Kumazawa, 2015).
Looking at the termination codon usage, TAA is used by ND1, COI, ATP8, ND4L, ND5
and Cytb; TAG is utilized by ND6; whereas the others (ND2, COII, ATP6, COIII, ND3
and ND4) terminate with incomplete codons. This stop codon pattern is similar as seen
in R. steineri (Chang et al., 2013). However, the termination codon usage is slightly
varied across B. japonica, R. trilineata, R. argyrotaenia, R. borapetensis, R. aprotaenia
and R. lateristriata (Chen et al, 2016; Kusuma et al., 2017; Ho et al., 2014; Zhang et al.,
2014; Kusuma & Kumazawa, 2015) and this dissimilarity is deemed typical among the
vertebrate mitogenomes (Ojala et al., 1981). The base composition of all protein-coding
genes is depicted in Table 3.
Transfer and Ribosomal RNA Gene Features
Out of the 22 tRNA genes identifies in this study, 63.6% (14) of them are encoded by Hstrand while L-strand is responsible for encoding the other 8 tRNA genes. The anticodons of all tRNA genes are highly conserved across other fish metagenome such as
R. borapetensis and B. japonica (Zhang et al., 2014; Chen et al., 2016). The 22 tRNA
genes made up nucleotide length of 1552 bp with A+T content of 57.1%, the tRNAAla
topped the group with A+T content of 69.2% whereas the tRNAThr bottomed the list with
A+T content of 48.6%.
Occupying a sum of 15.7% (2624 bp) of the entire mitochondrial genome of T.
pauciperforatum, both rRNA genes (12S rRNA and 16S rRNA) are 71 bp apart on the
H-strand with tRNAVal gene sandwiched in between them. The A+T content of 16S rRNA
gene (58.1%) is slightly greater than that of 12S rRNA gene (54.2%), both contributing
5
to the overall total rRNA A+T content of 56.6% and base composition as displayed in
Table 3: 35.9% for A, 23.7% for C, 19.6% for G and 20.7% for T.
Non-Coding Region
Excluding the light strand origin and control region, the other non-coding regions are
relatively miniature from 1 to 11 bp. The light strand origin (OL) and the control region
are the two large non-coding regions to be highlighted among the 16 non-coding regions
identified. The light strand origin was located between tRNAAsn and tRNACys in the T.
pauciperforatum mitochondrial genome. This 37 bp region has the stem-loop secondary
structure forming capability with the allocation of 11 complementary nucleotide pairs
contributing to the stem whilst the loop conformation takes up to 15 nucleotides arranged
in closed circle (Figure 2).
The largest non-coding region of the T. pauciperforatum mitochondrial genome, the
control region, has A+T content of 66.5%, depicting higher A+T content than that of the
overall mitogenome (60.0%), which was similarly detected in mitogenome of B. japonica
(Chen et al., 2016). On the side note, the base composition of this control region is as
below: 34.0% for A, 20.9% for C, 12.6% for G and 32.5% for T respectively as shown in
Table 3. Besides, the terminal associated sequence (TAS), central conserved sequence
block (CSB-F, CSB-D and CSB-E) as well as variable sequence block (CSB-1, CSB-2
and CSB-3) were all traced within the control region of this species.
Phylogenetic Relationship Analysis
A maximum likelihood tree was constructed to unravel the phylogenetic relationship of
T. pauciperforatum and its closely related species with the whole mitogenome now
available (Figure 3). The R. aprotaenia, R. lateristriata, R. sumatrana and R. steineri
form a distinctive cluster with bootstrap value of 100%. Besides, the T.
heteromorpha and T. espei pair as well as the R. argyrotaenia and R. borapetensis pair
also scored 100% bootstrap possibilities which also in agreement to the findings by
Kusuma & Kumazawa (2015) as well as Kusuma et al. (2017). T. pauciperforatum
diverged from the basal region of the major clade, where its evolutionary relationships
with B. maculatus, R. cephalotaenia and R. daniconius are poorly resolved as
suggested by the low bootstrap values there. The phylogeny is rooted (indicated by the
dashed line) by the outgroups Acheilognathus typus and Danio rerio.
Comparing to the morphology based phylogenetic tree constructed by Liao et al.
(2010) on 29 species of Rasbora with 41 morphological characters investigated, some
distinctive dissimilarities were observed. For instances, R. lateristriata, R. cephalotaenia
and R. trilineata were found to share the same clade when characterized morphologically
(Liao et al., 2010) but that is not the case in this study. The T. pauciperforatum reside on
the same clade as T. heteromorpha and R. vaterifloris when scored morphologically but
in this study all three of them are located far apart. Some comparisons across the results
of these two trees are not possible yet due to the absence of some species in both
analysis. R. borapetensis was observed to be closely related to R. rubrodorsalis and
both of them formed clade with R. cf. beauforti and R. semilineata (Liao et al., 2010)
whereas in this study, R. borapetensis is closely related to R. argyrotaenia in which R.
argyrotaenia was not included in the analysis by Liao et al. (2010). T. pauciperforatum
was discovered as the closest neighbour to its only genus counterpart, T. gracile beside
6
sharing the clade with other members like B. brigittae, Rasbosoma spilocerca and
Horadandia atukorali which four of them were not included in this study because of the
lack of the whole mitogenome sequences (Liao et al., 2010).
Another comparison of phylogenetic tree was done to that from Kusuma et al.
(2016) and the input sequences used are COI, Cytb, RAG1 and opsin gene sequences.
One of the similarities detected is that R. lateristriata was grouped closely with R.
aprotaenia and R. sumatrana. The grouping of R. borapetensis and R. agryrotaenia
inside the same clade is the other similar scenario observed and the only difference is
that in the tree constructed by Kusuma et al. (2016), R. dusonensis was found to be
related closer to R. agryotaenia than R. borapetensis. The tree from Kusuma et al. (2016)
depicted a strong clade with members like T. pauciperforatum, T. gracile, Kottelatia
brittani, B. merah and R. kalbarensis, with B. merah being the closest to T.
pauciperforatum. However, due to the absence of mitogenome sequences from the
abovementioned species that shares the same clade with T. pauciperforatum, this
analysis cannot be conducted in this study.
CONCLUSION
The complete mitogenome of T. pauciperforatum has been unravelled with the
completion of the sequencing and characterization process. Besides, this study had also
revealed the close molecular phylogenetic relationship between this species and 13
other closely related members of the Danioninae subfamily (from Rasbora genus and
other species previously classified under Rasbora genus). This study also serves as an
enrichment towards the complete mitochondrial genome count within the Trigonopoma
genus in terms of evolution and conservation genetics.
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Zhang, S., Cui, J., Li, C. Y., Mahboob, S., Al-Ghanim, K., Xu, P., & Sun, J. (2014). The
complete mitochondrial genome of Rasbora borapetensis (Cypriniformes:
Cyprinidae: Rasbora). Mitochondrial DNA, 27(2), 1-2.
9
Figure 1. Circular genome map of T. pauciperforatum. Genes encoded on heavy and
light strand are depicted in outer and inner circle respectively. The inner ring displays
the GC percent per every 5 bp where the darker lines represent higher GC percent. The
size of the complete mitogenome of T. pauciperforatum is 16,707 bp with the contribution
from 22 tRNA genes, 13 protein-coding genes, two rRNA genes and a control region.
10
Figure 2. The predicted secondary structure of light strand origin which is situated
between tRNAAsn and tRNACys genes of R. T. pauciperforatum. The part of the tRNACys
gene sequence is in the box.
Figure 3. Phylogenetic tree of T. pauciperforatum with other Rasbora genus members
and outgroups, based on 12 protein-coding genes (except ND6 gene) via the GTR+G
(General Time Reversible model with Gamma distributed rates among sites) Maximum
Likelihood (ML) analysis with bootstrap of 1000 replicates. The tree was rooted
(represented by dashed line) by the outgroups Acheilognathus typus and Danio rerio.
11
Table 1. Primers used for the amplification of the T. pauciperforatum mitogenome.
Primer
name
SF1
SR1
LF1
LR1
Primer sequence
Tm (°C)
GTGCTTCCTCTACACCAC
TGATGTTGAGAAGGCTAC
CCTATCTTACCGAGAAAG
GAGGCCTTCCCATCTAGA
55.3
Amplification length
(bp)
8923
48.6
9990
Table 2. Features of the whole T. pauciperforatum mitogenome.
Gene
tRNAPhe
12S rRNA
tRNAVal
16S rRNA
tRNALeu (UUA)
ND1
tRNAIle
tRNAGln
tRNAMet
ND2
tRNATrp
tRNAAla
tRNAAsn
tRNACys
tRNATyr
COI
tRNASer (UCA)
tRNAAsp
COII
tRNALys
ATP8
ATP6
COIII
tRNAGly
ND3
tRNAArg
ND4L
ND4
tRNAHis
tRNASer (AGC)
tRNALeu (CUA)
ND5
ND6
tRNAGlu
Cytb
tRNAThr
tRNAPro
D-loop
Position (5’-3’)
Start
End
1
70
1021
1092
2765
2841
3821
3961
3963
4032
5077
5218
5292
5391
5463
5465
7086
7088
7164
7855
7932
8090
8773
9558
9629
9978
10048
10338
11720
11789
11858
11931
14278
14347
14354
15495
15645
15646
69
1020
1091
2764
2839
3815
3892
3891
4031
5076
5148
5151
5220
5327
5393
7015
7016
7157
7854
7929
8096
8772
9557
9628
9977
10047
10344
11719
11788
11856
11930
13760
13757
14279
15490
15564
15576
16707
Codon
Start
Stopa
Amino
acid
Anticodon
Intergenic
nucleotideb
(bp)
Strandc
GAA
0
0
0
0
1
5
-2
1
0
0
2
1
34
1
1
0
1
6
0
2
-7
0
0
0
0
0
-7
0
0
1
0
-4
0
6
4
11
0
H
H
H
H
H
H
H
L
H
H
H
L
L
L
H
H
L
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
L
L
H
H
L
-
TAC
TAA
ATG
TAA
325
GAT
TTG
CAT
ATG
T--
348
TCA
TGC
GTT
GCA
GTA
GTG
TAA
517
TGA
GTC
ATG
T--
230
ATG
ATG
ATG
TAA
TATA-
55
227
261
ATG
T--
116
ATG
ATG
TAA
TA-
99
460
TTT
TCC
TCG
GTG
GCT
TAG
ATG
ATG
TAA
TAG
610
174
ATG
TAA
379
TTC
TGT
TGG
a
TA- and T—indicate incomplete stop codons; b Numbers indicate interspaced nucleotides and negative numbers
indicate overlapping nucleotides; c H and L indicate heavy or light strand respectively.
12
Table 3. The nucleotide base composition of all genes in the T. pauciperforatum
mitogenome.
Region
A
Protein-coding gene
ND1
ND2
COI
COII
ATP8
ATP6
COIII
ND3
ND4L
ND4
ND5
ND6
Cytb
Overall of protein-coding
gene
tRNA gene
tRNAPhe
tRNAVal
tRNALeu (UUA)
tRNAIle
tRNAGln
tRNAMet
tRNATrp
tRNAAla
tRNAAsn
tRNACys
tRNATyr
tRNASer (UCA)
tRNAAsp
tRNALys
tRNAGly
tRNAArg
tRNAHis
tRNASer (AGC)
tRNALeu (CUA)
tRNAGlu
tRNAThr
tRNAPro
Overall of tRNA gene
rRNA gene
12S rRNA
16S rRNA
Overall of rRNA gene
Control region
Overall of the genome
Base composition (%)
C
G
T
A + T content
(%)
34.5
38.7
28.4
33.7
35.8
33.7
30.4
30.1
29.0
33.6
35.8
44.6
31.7
33.7
26.7
28.1
24.4
22.6
24.2
25.5
25.7
27.2
27.6
26.3
25.2
29.9
26.0
25.9
12.9
10.3
16.8
15.8
8.5
11.1
16.2
14.3
13.8
12.8
12.4
10.7
14.1
13.4
25.9
22.9
30.4
27.9
31.5
29.7
27.6
28.4
29.6
27.3
26.5
14.8
28.2
26.9
60.4
61.6
58.8
61.6
67.3
63.4
58.0
58.5
58.6
60.9
62.3
59.4
59.9
60.6
37.7
28.2
28.0
25.0
35.2
31.9
36.1
36.8
32.9
29.2
31.0
26.8
37.1
34.7
36.6
27.1
34.8
35.3
36.5
34.8
28.6
37.1
32.8
20.3
25.4
24.0
22.2
25.4
30.4
22.2
22.1
27.4
27.7
31.0
28.2
20.0
25.3
22.5
25.7
23.2
19.1
17.66
23.2
28.6
28.6
24.5
20.3
23.9
22.7
26.4
14.1
15.9
22.2
8.8
19.2
23.1
19.7
19.7
14.3
18.7
12.7
21.4
13.0
19.1
17.6
17.4
22.9
11.4
18.4
21.7
22.5
25.3
26.4
25.4
21.7
19.4
32.4
20.5
20.0
18.3
25.4
28.6
21.3
28.2
25.7
29.0
26.5
28.4
24.6
20.0
22.9
24.3
59.4
50.7
53.3
51.4
60.6
53.6
55.5
69.2
53.4
49.2
49.3
52.2
65.7
56.0
64.8
52.8
63.8
61.8
64.9
59.4
48.6
60.0
57.1
33.9
37.1
35.9
34.0
34.0
25.0
23.0
23.7
20.9
25.2
20.8
18.9
19.6
12.6
14.8
20.3
21.0
20.7
32.5
26.0
54.2
58.1
56.6
66.5
60.0
13