International Journal of Aquaculture, 2025, Vol.15, No.3, 116-134 http://www.aquapublisher.com/index.php/ija 118 2 Mitochondrial Genome Structure of Marble Goby 2.1 Organization and main characteristics of mitochondrial genome The mitochondrial genome of marble goby determined in this study shows that its full length is about 16 556 bp (Xu et al., 2016) (comparable to the size of mitochondrial genomes of most bony fishes), and it is a typical circular double-stranded DNA molecule. The genome contains 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs) and 2 ribosomal RNA genes (12S rRNAand 16S rRNA), as well as non-coding regions such as the control region (D-loop) and the light chain replication initiation region. The order and direction of each gene in the genome are consistent with the typical fish mitochondrial genome, and no rearrangement occurs. For example, the gene order in the mitochondrial genome of marble goby starts from 12S rRNA, passes through 16S rRNA, tRNA^Val, etc., and reaches the arrangement of coding genes such as COI and COII, which is consistent with the arrangement of mitochondrial genomes of other reported bony fishes (such as pufferfish). The coding region of the mitochondrial genome of the marble goby shows some characteristics consistent with bony fish. Most protein-coding genes use the typical ATG as the start codon, and the stop codon is mostly complete TAA or TAG. It is worth noting that in some fish, the COI gene often starts with the unconventional start codon GTG; according to our analysis of the marble goby mitochondrial sequence, its COI start codon also has this phenomenon (specific sequencing results show that the COI gene starts with GTG), which is consistent with reports from other fish such as the spotted pufferfish (Zhu et al., 2024). In addition, some coding genes (such as cox2 and nad4) use incomplete terminators (single "T"), which is a common phenomenon caused by the mitochondrial post-transcriptional polyadenylation mechanism. Except for tRNA^Ser(AGY) which lacks the DHU arm in the secondary structure, the other 22 tRNAgenes of the marble goby can fold into a typical cloverleaf structure, which is consistent with most vertebrate mitochondrial tRNAs. The mitochondrial control region is located between the tRNA^Pro and tRNA^Phe genes. It is the fastest-changing region in the whole genome. It is about 900 bp long, rich in AT and contains typical repetitive sequences and regulatory elements, which provides a high-variable marker for the future analysis of population genetic structure and maternal genetic diversity. 2.2 Comparative analysis with closely related species To see if there was anything special about the mitochondrial genome of the mullet, we compared it with some closely related fish. The fish selected for comparison included other sharp-pointed tang fish in the same genus, such as the high-fin sharp-pointed tang fish (O. altipinna) living in Papua, Indonesia, and more common fish in the same family, such as the widely distributed common tang fish (Eleotris oxycephala). In addition, several fish from other families belonging to the suborder of goby were selected, such as Hypseleotris cyprinoides, which is a species of the genus Hypseleotris in the family Hypseleotris. The comparison results showed that the mitochondrial genome length of these fish was similar, about 16.5 kb. They all have 37 genes, and the types and order of genes are exactly the same, without any rearrangement (Pan et al., 2023). This shows that in bony fish, the structure of the mitochondrial genome is very stable from a large classification point of view and does not change much. In terms of nucleotide composition, the mitochondrial genomes of marble goby and closely related fishes showed a clear AT bias, with A+T accounting for about 55~57%, which is also a common feature of animal mitochondrial genomes. The average base composition of the mitochondrial coding sequence of marble goby (A≈30%, T≈25%, C≈27%, G≈18%) is basically consistent with that of other Channa fish, showing a biased pattern of C-rich light chains and A-rich heavy chains. Codon usage preference analysis of protein-coding gene sequences showed that marble goby and other comparative species prefer codons ending in A or T, and the two codons with the highest preference are also the same among species (for example, CGA of Arg and UCU of Ser have RSCU values >2 in many fish). This consistency in codon preference illustrates similar selection pressures on the mitochondrial translation system and genome evolution of related fishes. Although the overall structure of the mitochondrial genome of the marble goby and its close relatives is very similar, there are still some differences in the specific sequences. These differences reflect their differentiation
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