Bioscience Methods 2025, Vol.16, No.3, 137-153 http://bioscipublisher.com/index.php/bm 139 copy number of tandem repeat sequences. Studies have found that there are some repeat motifs and microsatellite sequences in the control region of abalone. The unstable expansion and contraction of these repeat units lead to polymorphism in the length of the control region. At the same time, the control region sequence evolves rapidly and often accumulates interspecies diagnostic mutation sites. For example, by comparing the control region sequences of multiple geographical populations of Haliotis discus, it was found that the base substitution frequency in the hypervariable region was significantly higher than that in the coding gene region, which can be used to identify subtle population genetic differentiation. In addition to the control region, there are also some extremely short non-coding spacers (usually only a few bp to tens of bp) in the abalone mitochondrial genome. Despite the short length of these spacers, base insertion/deletion (InDel) variations have also been observed between some species. For example, a comparison of mitochondrial genes of H. diversicolor from two geographical populations showed that there was a 5 bp spacer between the tRNA-His and nad5 genes, and a 1 bp insertion mutation was detected only in Vietnamese individuals (Xin et al., 2011). In general, the variation of abalone mitochondrial non-coding regions (especially control regions) is higher than that of coding regions, and the high polymorphism of their sequence length and composition provides useful markers for population genetic diversity analysis and discrimination of closely related species. However, since control region sequences are usually difficult to align and analyze, concatenated sequences of 13 protein-coding genes are more often used in phylogenetic studies to construct reliable phylogenetic trees. 2.3 Structural differences in tRNAs, rRNAs, and protein-coding genes The abalone mitochondrial genome contains 22 tRNA genes, most of which are between 60-71 bp in length and have a typical cloverleaf secondary structure. The mitochondrial tRNA gene sequences of different abalone species are highly conserved, and most of the differences are point mutations in the mid-loop or anti-codon loop. However, the typical DHU arm phenomenon of individual tRNA genes losing was also observed in a few species, which is a common secondary structure simplification pattern of mitochondrial tRNA in mollusks. The lengths of the two mitochondrial rRNA genes (12S and 16S rRNA) do not vary much among abalone species, generally about 0.96 kb for 12S and about 1.3 kb for 16S. Their sequence conservation is relatively high, but rRNA genes of different species can still accumulate certain substitutions, especially in some highly exposed loop regions. Studies have shown that the partial sequence of the mitochondrial 16S rRNA gene can be used to distinguish different abalone species along the coast of China, and the sequence differences are significant (Zhang et al., 2022). In contrast, due to the functional constraints of amino acids, the evolution of most sites of the 13 protein-coding genes remains conservative, and there are fewer non-synonymous substitutions between species. For example, the cytochrome oxidase subunit I gene (COI) is often used as a DNA barcode, and its sequence differs by more than 10% among closely related abalone species, but the intraspecific difference is usually less than 2%, which can effectively distinguish species. In the genus Haliotis, the lengths of each protein-coding gene are completely consistent or only slightly different between species, suggesting that these genes have hardly undergone frameshift mutations or large fragment deletions since their common ancestor. However, there are differences in the evolutionary rates of different coding genes: genes such as ATP synthase subunit 8 (ATP8) and NADH dehydrogenase 6 (NAD6) tend to evolve faster and accumulate relatively more non-synonymous substitutions, while genes such as COI and cytochrome b (CYT b) are more conservative. For example, by calculating the non-synonymous/synonymous substitution rate ratio (Ka/Ks) of the four species of the genus Haliotis, it was found that the Ka/Ks of the NAD2, NAD6, and ATP8 genes were significantly higher than those of other genes, suggesting that these genes may have experienced relatively loose selection constraints or directional selection pressures. This result shows that different mitochondrial genes bear different functional constraints in the evolution of abalone, and the structural differences and variation patterns of some genes may be related to environmental adaptation (Tshilate et al., 2023). The components of the abalone mitochondrial genome differ to varying degrees among species: tRNA and rRNA genes are highly conserved, protein-coding genes are generally conserved but have mutation hotspots, and non-coding regions, especially control regions, are rich in variation, providing important clues for interspecies and population identification.
RkJQdWJsaXNoZXIy MjQ4ODYzNA==