Mitogenome structure
The complete mitogenome of P. caeruleostictus was determined in this study. This mitogenome contains 37 genes (Table 1) which include 22 transfer RNA genes, 2 ribosomal RNA genes, 13 protein-coding genes in addition to a control region according to a characteristic vertebrate mitochondrial DNA gene [10, 22].
Table 1: 37 genes present in P. caeruleostictus
Name
|
Position
|
Strand
|
Length (bp)
|
Start codon
|
Stop codon
|
Anticodon
|
Intergenic nucleotide
|
trnF(ttc)
|
1-68
|
N
|
68
|
|
|
GAA
|
|
rrnS
|
69-1022
|
N
|
954
|
|
|
|
-1
|
trnV(gta)
|
1022-1093
|
N
|
72
|
|
|
TAC
|
39
|
rrnL
|
1133-2790
|
N
|
1658
|
|
|
|
|
trnL2(tta)
|
2791-2863
|
N
|
73
|
|
|
TAA
|
|
nad1
|
2864-3838
|
N
|
975
|
ATG
|
TAA
|
|
5
|
trnI(atc)
|
3844-3913
|
N
|
70
|
|
|
GAT
|
-1
|
trnQ(caa)
|
3913-3983
|
J
|
71
|
|
|
TTG
|
-1
|
trnM(atg)
|
3983-4052
|
N
|
70
|
|
|
CAT
|
|
nad2
|
4053-5099
|
N
|
1047
|
ATG
|
TAA
|
|
-1
|
trnW(tga)
|
5099-5168
|
N
|
70
|
|
|
TCA
|
1
|
trnA(gca)
|
5170-5238
|
J
|
69
|
|
|
TGC
|
1
|
trnN(aac)
|
5240-5313
|
J
|
74
|
|
|
GTT
|
4
|
trnC(tgc)
|
5349-5414
|
J
|
66
|
|
|
GCA
|
|
trnY(tac)
|
5415-5484
|
J
|
70
|
|
|
GTA
|
1
|
cox1
|
5486-7048
|
N
|
1563
|
GTG
|
AGG
|
|
-9
|
trnS2(tca)
|
7040-7110
|
J
|
71
|
|
|
TGA
|
2
|
trnD(gac)
|
7113-7185
|
N
|
73
|
|
|
GTC
|
8
|
cox2
|
7194-7884
|
N
|
691
|
ATG
|
T(AA)
|
|
|
trnK(aaa)
|
7885-7958
|
N
|
74
|
|
|
TTT
|
1
|
atp8
|
7960-8127
|
N
|
168
|
ATG
|
TAA
|
|
-10
|
atp6
|
8118-8801
|
N
|
684
|
ATG
|
TAA
|
|
-1
|
cox3
|
8801-9586
|
N
|
786
|
ATG
|
TAA
|
|
-1
|
trnG(gga)
|
9586-9657
|
N
|
72
|
|
|
TCC
|
|
nad3
|
9658-10008
|
N
|
351
|
ATG
|
TAG
|
|
-2
|
trnR(cga)
|
10007-10077
|
N
|
71
|
|
|
TCG
|
|
nad4l
|
10078-10374
|
N
|
297
|
ATG
|
TAA
|
|
-7
|
nad4
|
10368-11748
|
N
|
1381
|
ATG
|
T(AA)
|
|
|
trnH(cac)
|
11749-11817
|
N
|
69
|
|
|
GTG
|
|
trnS1(agc)
|
11818-11885
|
N
|
68
|
|
|
GCT
|
6
|
trnL1(cta)
|
11892-11964
|
N
|
73
|
|
|
TAG
|
|
nad5
|
11965-13803
|
N
|
1839
|
ATG
|
TAA
|
|
-4
|
nad6
|
13800-14321
|
J
|
522
|
ATG
|
TAA
|
|
|
trnE(gaa)
|
14322-14390
|
J
|
69
|
|
|
TTC
|
4
|
Cob
|
14395-15535
|
N
|
1141
|
ATG
|
T(AA)
|
|
|
trnT(aca)
|
15536-15609
|
N
|
74
|
|
|
TGT
|
-1
|
trnP(cca)
|
15609-15678
|
J
|
70
|
|
|
TGG
|
|
The circle genome is 16,653 bp in length (Figure 2) which falls within the range of a typical vertebrate with the base composition of 27.92 % A, 28.67 % C, 16.47 % G, and 26.94% T [23]. The complete genome skewed to AT at 0.018 and to GC at -0.27.
Figure 2: The mitogenome map of P. caeruleostictus
The mitochondrial gene arrangement of P. caeruleostictus indicated that all genes were encoded on heavy strand (OH) except for NADH dehydrogenase subunit 6 (ND6) and eight tRNA genes (tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Glu and tRNA-Pro) which occurred on the light strand (OL). This is in confirmation with other previous studies on vertebrate circular mitogenome sequence [24–27]. A total of thirteen (13) complete stop codons (TAA, TAA, AGG, TAA, TAA, TAA, TAG, TAA, TAA and TAA) and three (3) incomplete stop codons; T(AA), T(AA), and T(AA) were used in the protein-coding genes. Moreover, it consisted of 22 anticodons; GAA, TAC, TAA, GAT, TTG, CAT, TCA, TGC, GTT, GCA, GTA, TGA, GTC, TTT, TCC, TCG, GTG, GCT, TAG, TTC, TGT, and TGG.
Features of 22 tRNA genes
During translation, making a protein from mRNA template, tRNA functions as a transporter of nucleotide from mRNA to amino acids. It functions as the bridge between mRNA and amino acid sequence to the ribosome where proteins are constructed [28]. The mitochondrial genome contained 22 tRNA genes (Table 2). The tRNA genes ranged in size from 65 to 75 nucleotides, which allowed the encoded tRNA to fold into the characteristic clover-leaf secondary structure [29].
Table 2: Present tRNA in the complete mitochondrion DNA of P. caeruleostictus
Product
|
Location
|
Length
|
Atypical pairings
|
tRNA-Phe
|
1-68
|
68
|
|
tRNA-Val
|
1022-1093
|
71
|
|
tRNA-Leu
|
2791-2863
|
72
|
UAA
|
tRNA-Ile
|
3844-3913
|
69
|
|
tRNA-Gln (complement)
|
3913-3983
|
70
|
|
tRNA-Met
|
3983-4052
|
69
|
|
tRNA-Trp
|
5099-5168
|
70
|
|
tRNA-Ala(complement)
|
5170-5238
|
68
|
|
tRNA-Asn (complement)
|
5240-5313
|
73
|
|
tRNA-Cys (complement)
|
5349-5414
|
65
|
|
tRNA-Tyr (complement)
|
5415-5484
|
69
|
|
tRNA-Ser
|
7040-7110
|
70
|
UGA
|
tRNA-Asp
|
7113-7185
|
72
|
|
tRNA-Lys
|
7885-7958
|
73
|
|
tRNA-Gly
|
9586-9657
|
71
|
|
tRNA-Arg
|
10007-10077
|
70
|
|
tRNA-His
|
11749-11817
|
68
|
|
tRNA-Ser
|
11818-11885
|
67
|
GCU
|
tRNA-Leu
|
11892-11964
|
72
|
UAG
|
tRNA-Glu (complement)
|
14322-14390
|
68
|
|
tRNA-Thr
|
15536-15609
|
73
|
|
tRNA-Pro (complement)
|
15609-15678
|
69
|
|
Features of rRNA
Ribosomal RNA (rRNA) combines with enzyme and proteins to form complex structure within the cytoplasm to form ribosomes which serve as protein synthesis site. These complex structures travel along the mRNA molecule during translation and facilitate the assembly of amino acids to form a polypeptide chain [30]. The ribosomal RNA (rRNA) identified within the genome were 12S rRNA located at 69 bp to 1021 bp consisting 952 bp and 16S rRNA which also stationed at 1094 bp to 2790 bp and made up of 1696 bp.
Features of protein-coding genes
As is other vertebrate, a total of 13 protein-coding genes were present in the mitogenome of P. caeruleostictus. Within the obtained 13 protein-coding genes, 4 of them were paired, located adjacently to each other; atp8-atp6, atp6-cox3, nad4l-nad4, and nad5-nad6. Apart from atp6-cox3, all pairs had some overlap genes between them. The length of overlap between these 3 cases, atp8-atp6, nad4l-nad4, and nad5-nad6 were 9 bp, 6 bp and 3 bp respectively (Table 3). All protein genes had an ATG initiation codon except for the cytochrome oxidase subunit I (cox1) gene which had GTG as its initiation codon.
Table 3: Characteristics of the protein-coding genes
Region
|
A%
|
C%
|
G%
|
T%
|
A+T%
|
G+C%
|
AT skew
|
GC skew
|
Rrns
|
30.19
|
25.79
|
22.54
|
21.49
|
51.68
|
48.32
|
0.168
|
-0.067
|
Rrnl
|
33.78
|
24.43
|
20.99
|
20.81
|
54.58
|
45.42
|
0.238
|
-0.076
|
nad1
|
25.33
|
32.1
|
14.87
|
27.69
|
53.03
|
46.97
|
-0.044
|
-0.367
|
nad2
|
25.69
|
36.1
|
12.51
|
25.69
|
51.38
|
48.62
|
0
|
-0.485
|
cox1
|
24.12
|
26.36
|
18.43
|
31.09
|
55.21
|
44.79
|
-0.126
|
-0.177
|
cox2
|
27.79
|
27.5
|
16.79
|
27.93
|
55.72
|
44.28
|
-0.003
|
-0.242
|
atp8
|
28.57
|
35.12
|
12.5
|
23.81
|
52.38
|
47.62
|
0.091
|
-0.475
|
atp6
|
23.1
|
30.85
|
13.45
|
32.6
|
55.7
|
44.3
|
-0.171
|
-0.393
|
cox3
|
25.95
|
29.9
|
16.79
|
27.35
|
53.31
|
46.69
|
-0.026
|
-0.281
|
nad3
|
16.66
|
34.19
|
15.95
|
30.2
|
49.86
|
50.14
|
-0.211
|
-0.364
|
nad4l
|
21.21
|
34.01
|
15.49
|
29.29
|
50.51
|
49.49
|
-0.16
|
-0.374
|
nad4
|
26.29
|
31.21
|
13.9
|
28.6
|
54.89
|
45.11
|
-0.042
|
-0.384
|
nad5
|
28.98
|
30.29
|
13.32
|
27.41
|
56.39
|
43.61
|
0.028
|
-0.389
|
nad6
|
18.39
|
14.75
|
27.97
|
38.89
|
57.28
|
42.72
|
-0.358
|
-0.309
|
Cob
|
25.07
|
30.24
|
14.99
|
29.71
|
54.78
|
45.22
|
-0.085
|
-0.337
|
Features of D- loop
The D-loop of P. caeruleostictus was positioned at the end of tRNA-Pro (15679 to 16653) with a length of 974 bp. This correlates with other literature on teleost complete mitochondrial sequence [10, 31–33]. However, according to available literature, the D-loop in some vertebrates are located between tRNA-Pro and tRNA-Phe [34–36]. Conserved Sequence Blocks (CSBs) and High Variational Blocks (HVBs) were the findings of the multiple sequence alignment of the D-loops using D. rerio (GenBank Accession number: NC_002333.2) as the reference.
Table 4: Sequence of conserved regions of P. caeruleostictus and other teloest with D. rerio as reference.
Element
|
Species
|
Sequence
|
CSB-1
|
Danio rerio
|
C
|
A
|
A
|
A
|
C
|
C
|
C
|
C
|
C
|
T
|
Pagrus caeruleotictus
|
T
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
C
|
Oryzias curvinotus
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
C
|
Gobiopterus lacustris
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
C
|
Brycon henni
|
T
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
C
|
Trichiurus lepturus
|
T
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
C
|
Lutjanus russellii
|
A
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
C
|
CSB-2
|
Danio rerio
|
G
|
G
|
G
|
A
|
C
|
A
|
T
|
C
|
A
|
A
|
Pagrus caeruleotictus
|
.
|
.
|
.
|
.
|
.
|
.
|
A
|
G
|
.
|
.
|
Oryzias curvinotus
|
.
|
.
|
.
|
.
|
.
|
.
|
A
|
A
|
.
|
.
|
Gobiopterus lacustris
|
.
|
.
|
.
|
.
|
.
|
.
|
A
|
T
|
.
|
.
|
Brycon henni
|
.
|
.
|
.
|
.
|
.
|
.
|
A
|
A
|
.
|
.
|
Trichiurus lepturus
|
A
|
.
|
.
|
.
|
.
|
.
|
G
|
T
|
G
|
.
|
Lutjanus russellii
|
.
|
.
|
.
|
.
|
.
|
.
|
A
|
T
|
.
|
.
|
CSB-3
|
Danio rerio
|
G
|
C
|
A
|
T
|
T
|
T
|
C
|
A
|
T
|
A
|
Pagrus caeruleotictus
|
A
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
C
|
.
|
Oryzias curvinotus
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
C
|
.
|
Gobiopterus lacustris
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
C
|
.
|
Brycon henni
|
A
|
.
|
.
|
.
|
.
|
.
|
G
|
G
|
.
|
C
|
Trichiurus lepturus
|
A
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
G
|
.
|
Lutjanus russellii
|
A
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
G
|
.
|
Note: Dot represent the same nucleotide as the reference species, D. rerio
Phylogenetic analysis
In order to ascertain the evolutionary features of P. caeruleostictus mitogenomes, complete mitochondrial and Cox1 sequences of other species from Order Perciformes were downloaded in FASTA format from the database of National Center for Biotechnology Information (NCBI-Blast) (https://blast.ncbi.nlm.nih.gov) and Barcode of Life Database (BOLD) (http://www.barcodinglife.org) systems respectively, these database contains comprehensive data on species gene sequence for molecular phylogenetic analysis [37]. Using MEGA version 6.0v, nucleic acid sequences were aligned using ClustalW with the Alignment Explorer tool and constructed a neighbor-joining trees at the order-level [38]. As shown in Figure 3 and 4 below, P. caeruleostictus clustered closest with P. auriga as supported by a high bootstrap value (99%) in both cases.
Figure 3: Neighbor-joining phylogenetic tree based on Cox1 sequences.
Figure 4: Neighbor-joining phylogenetic tree based on complete mitochondrial sequences with L. fulviflamma as the out group.