Genome organization and base composition
The complete mitochondrial genome of Bos frontalis is a 16,347 bp closed circular and double-stranded molecule containing 13 PCGs (COX 1–3, ND1-6, ND4L, CYTB, ATP6 and ATP8), 22 tRNA genes (one for each amino acid and two each for leucine and serine), 2 rRNA genes (rrnS or 12S and rrnL or 16S), and a major (CR). The heavy (H) strand encodes 28 of these 37 genes, whereas the light (L) strand encodes the remaining nad6 and 8 tRNAs (Table 1; Fig. 2). As a result, there is no significant difference in the lengths of PCGs, rRNAs and tRNAs within the mitogenomes of different Bos species (Fig. 3). This finding supports the consistency of the genome size and gene architecture of Bangladeshi Bos frontalis in comparison to the mitochondrial genomes of Indian Mithun and Gaur, which have previously been reported The mitochondrial genome's AT and GC content are 60.92% and 39.08% respectively, indicating that the nucleotide composition is overall biased towards adenine and thymine (Fig. 4). Previously described Bos species have shown a similar patterns Furthermore, the mitochondrial genome on average has positive AT (0.085) and CG (0.40) skews (Fig. 4), indicating that adenine and cytosine are more abundant than their corresponding nucleotides, guanine and thymine. Moreover, it contains a total of 90bp of intergenic spacer (IGS) sequences, which are interspersed at 33 regions across the mitochondrial genome with varying ranges from 1 to 33 bp. Among these, four different overlapping regions have been speculated, ranging from 2 to 39 bp; while ATPase subunit 8 (ATP8) and ATPase subunit 6 (ATP6) having the most overlapped area (39 bp). The putative control region (D-loop) of the mitochondrial genome is located between the tRNA-Pro and tRNA-Phe, consisting of 921 bp. Other closely related bovine species, such as Indian Mithun and Bos gaurus, have shown similar result.
Table 2
Amino acid’s codon frequency and relative synonymous codon usage (RSCU) are taken into 4 decimal points. Cat represents color legends used for codons.
Amino Acid
|
Codon
|
Frequency
|
RSCU
|
Cat
|
Amino
Acid
|
Codon
|
Frequency
|
RSCU
|
Cat
|
Ala
|
GCG
|
0.0017
|
0.0458
|
a
|
Leu1
|
CUU
|
0.0181
|
0.8289
|
c
|
GCA
|
0.0121
|
0.0321
|
b
|
CUC
|
0.0241
|
1.1053
|
d
|
GCU
|
0.0088
|
0.9466
|
c
|
Lys
|
AAG
|
0.0089
|
0.4592
|
a
|
GCC
|
0.0149
|
1.5878
|
d
|
AAA
|
0.0298
|
1.5407
|
b
|
Arg
|
CGG
|
0.0032
|
0.6667
|
a
|
Met
|
AUG
|
0.0115
|
0.4969
|
a
|
CGA
|
0.0063
|
1.3333
|
b
|
AUA
|
0.0347
|
1.5031
|
b
|
CGU
|
0.0046
|
0.9697
|
c
|
Phe
|
UUU
|
0.0192
|
0.8428
|
a
|
CGC
|
0.0049
|
1.0303
|
d
|
UUC
|
0.0264
|
1.1572
|
b
|
Asn
|
AAU
|
0.0313
|
1.0531
|
a
|
Pro
|
CCG
|
0.0109
|
0.5188
|
a
|
AAC
|
0.0281
|
0.9469
|
b
|
CCA
|
0.0247
|
1.1097
|
b
|
Asp
|
GAU
|
0.0077
|
0.7941
|
a
|
CCU
|
0.0292
|
1.3925
|
c
|
GAC
|
0.0118
|
1.2059
|
b
|
CCC
|
0.0192
|
0.9147
|
d
|
Cys
|
UGU
|
0.0077
|
0.9643
|
a
|
Ser1
|
AGU
|
0.0089
|
0.4581
|
a
|
UGC
|
0.0083
|
1.0357
|
b
|
AGC
|
0.0192
|
0.9901
|
b
|
Gln
|
CAG
|
0.0158
|
0.6790
|
a
|
Ser2
|
UCG
|
0.0103
|
0.5320
|
a
|
CAA
|
0.0307
|
1.3210
|
b
|
UCA
|
0.0270
|
1.3892
|
b
|
Glu
|
GAG
|
0.0131
|
0.9200
|
c
|
UCU
|
0.0230
|
1.1823
|
c
|
GAA
|
0.0155
|
1.0800
|
d
|
UCC
|
0.0281
|
1.4483
|
d
|
Gly
|
GGG
|
0.0026
|
0.5000
|
a
|
Thr
|
ACG
|
0.0080
|
0.3544
|
a
|
GGA
|
0.0077
|
1.5000
|
b
|
ACA
|
0.0304
|
1.3418
|
b
|
GGU
|
0.0040
|
0.7778
|
c
|
ACU
|
0.0255
|
1.1266
|
c
|
GGC
|
0.0063
|
1.2222
|
d
|
ACC
|
0.0267
|
1.1772
|
d
|
His
|
CAU
|
0.0261
|
1.1166
|
a
|
Trp
|
UGG
|
0.0055
|
0.5352
|
a
|
CAC
|
0.0207
|
0.8834
|
b
|
UGA
|
0.0149
|
1.4648
|
b
|
Ile
|
AUU
|
0.0298
|
1.0400
|
a
|
Tyr
|
UAU
|
0.0319
|
1.1385
|
a
|
AUC
|
0.0275
|
0.9600
|
b
|
UAC
|
0.0241
|
0.8615
|
b
|
Leu2
|
UUG
|
0.0132
|
0.6052
|
a
|
Val
|
GUG
|
0.0017
|
0.3529
|
a
|
UUA
|
0.0270
|
1.2368
|
b
|
GUA
|
0.0100
|
2.0588
|
b
|
Leu1
|
CUG
|
0.0092
|
0.4211
|
a
|
GUU
|
0.0034
|
0.7059
|
c
|
CUA
|
0.0393
|
1.8026
|
b
|
GUC
|
0.0043
|
0.8824
|
d
|
Protein-coding genes (PCGs) and codon usage
The mitochondrial genome encodes 13 PCGs totaling 11379 bp in size, accounting for 69.61% of the mitochondrial genome. The AT and GC concentrations are almost 62% and 38%, respectively (Figure 4), demonstrating that the PCGs have nucleotide compositional inclination towards adenine and thymine. Furthermore, the positive AT (0.085) and CG (0.400) skews of PCGs show that adenine is more abundant than thymine, while cytosine is more abundant than guanine.
The Cox1 and
Cox3 genes have a slightly negative AT skew, whereas the other PCGs have a positive AT skew. Being repeatedly observed in other bovine and mammalian species (11,15–17), the PCGs are organized into seven
NADH dehydrogenase subunits (
ND1,
ND2,
ND3,
ND4,
ND4L,
ND5 and
ND6), three
cytochrome c oxidase subunits (
COX1,
COX2 and
COX3), two
ATPase subunits (
ATP8 and
ATP6) and one
cytochrome b (
CYTB). The PCGs vary greatly in size, with
ATP8 (201bp) being the smallest while
ND5 (1821bp) being the largest. Furthermore, four neighboring pairs of PCGs (
ATP8-ATP6,
ATP6-COX3, ND4L-ND4 and
ND5-ND6), as well as vertebrates (11,16,17), share a common gene location. Following the exclusion of stop codons, the relative synonymous codon usage (RSCU) is determined and summarized in Figure 5. The RSCU analysis reveals that GCU (A), GUA (V), and AAA (K) codons are the most often used. Furthermore, proline, threonine, leucine 1, asparagine and serine are the most frequently used amino acids in mitochondrial proteins, in that order.
Transfer RNA (tRNAs) and Ribosomal RNA (rRNAs)
There are a total of 2443bp long 22 tRNA genes in the Bos frontalis mitogenome, ranging in size from 60 bp (tRNA-Ser1) to 75 bp (tRNA-Leu2).Most of the tRNA genes (14) are encoded by the H-strand (tRNA-Thr, tRNA-Phe, tRNA-Val, tRNA-Leu2, tRNA-Ile, tRNA-Met, tRNA-Trp, tRNA-Asn, tRNA-Lys, tRNA-Gly, tRNA-Arg, tRNA-His, tRNA-Ser1, tRNA-Leu1), while the remaining 8 tRNAs (tRNA-Glu, tRNA-Pro, tRNA-Gln, tRNA-Ala, tRNA-Asp, tRNA-Cys, tRNA-Tyr, tRNA-Ser2) are encoded by the L-strand (Table 1). MITOS (5) and tRNA scan-SE (7) are used to determine all tRNA sequences and structures. Except for tRNA-Ser1 and tRNA-Lys which lack a stable dihydrouridine arm loop, rest of the tRNA genes have the characteristic cloverleaf secondary structure (Figure 6). Such aberrant tRNA structures have been observed in mammals in general, including the previously reported Indian Gaur and Mithun (11,12,18). In addition to the conventional Watson-Crick base pairs, these tRNAs contain a total of 9 mismatched base pairs (Figure. 6). G-A (two) and C-A (three) pairs are among them, and they are known as non-canonical pairs generating weak connections in tRNA secondary structures. One C-U and three U-U combinations are among the remaining four mismatches.
There are two rRNA genes, one 1569bp 16S ribosomal RNA gene and one 956bp 12S ribosomal RNA gene, for a total size of 2525 bp. The H (+) strand contains both rRNA genes. The 16S ribosomal RNA is found between tRNA-Val and tRNA-Leu2, and the 12S ribosomal RNA gene is found between tRNA-Phe and tRNA-Val. (Table. 1; Figure 2).
Phylogenetic relationship
The evolutionary analysis uses 26 bovine species from 8 congeneric species, including Bos frontalis, Bos gaurus, Bos javanicus, Bos indicus, Bos primigenius, Bos taurus, Bos grunniens, and Bos mutus. In this study, our sequenced species, Bos frontalis (MW763078.1), clustered with Mithun, Gaur, mutus, javanicus, and grunniens.The presence of Bangladeshi Bos frontalis with Indian mithun (MK279401.1) and Cambodian gaur (NC024818, JN632604) species indicates a tight genetic link between mithun and gaur (Figure 7). This finding clearly supports the unequivocal concept of Mithun being a direct descendant of Gaur, which is supported by the findings of prior research using cytochrome b(CYTB) gene (19,20), 16S ribosomal RNA gene (21), SNP genotyping (22), entire mtDNA (3), and Y chromosomal DNA markers (23). One of the Mithuns, on the other hand, has been paired with Bos javanicus (AB915322.1), a Southeast Asian cattle species known as Banteng or Tembadau. The dispersed grouping of Banteng with Bos frontalis, Bos gaurus and Bos taurus respectively indicates its hybrid nature, likewise reported earlier (24).
In cluster 2, there is a strong phylogenetic relationship between Bos frontalis, Bos Gaurus, Bos Jananicus, Bos Mutus, and Bos Grunniens, and in cluster 1, there is a relationship between Bos Taurus, Bos Javanicus (706), primigenius, and Indicus.
Similar findings have also been obtained in previous studies using different markers (3,25). These findings indicate that hybridization between domestic mithun and cattle occurs (21,25,26) and that it is common in China.