Mitochondrial genome annotation and comparative characterization of the Andamani goat

Introduction

Goats are among the earliest domesticated livestock species and are believed to be domesticated around 10,000 years ago in the Fertile Crescent (Zeder and Hesse, 2000) [12] . They play an important role in rural livelihood, meat production and smallholder farming systems, especially in developing countries. Indigenous goat populations are valuable animal genetic resources because they are often adapted to local climatic conditions, management systems and ecological pressures. India possesses diverse goat genetic resources distributed across different agro-climatic zones. Among these, island goat populations are of special interest because their genetic background may be influenced by geographical isolation, local management, founder effects and adaptation to coastal environments. The Andaman and Nicobar Islands represent a unique island ecosystem in the Bay of Bengal. The region has a hot and humid climate, high rainfall and coastal environmental conditions. The Andamani goat is an indigenous goat population of this region and is reared mainly under local island production systems. Previous descriptions of goat populations from the Andaman and Nicobar Islands have highlighted their local importance, farming practices and adaptation to the island environment (Sunder et al., 2019) [10] . Mitochondrial DNA is widely used in livestock genetic studies because of its high copy number, compact genome structure and maternal inheritance. It has been used extensively for studies on genetic diversity, domestication and evolutionary history in domestic goats (Luikart et al., 2001) [1] . Annotation of the mitochondrial genome provides basic information on genome organization and serves as a useful reference for future molecular and conservation genetic studies. Mitochondrial genome retrieval and annotation provide a simple and useful approach for developing breed-specific mitogenome resources. GetOrganelle has been widely used for de novo assembly and retrieval of organelle genomes from genome sequencing data (Jin et al., 2020) [5] . MITOS2 provides automated annotation of metazoan mitochondrial genomes, including protein-coding genes, rRNA genes and tRNA genes (Donath et al., 2019) [3] , while GenomeVx enables visual representation of circular organellar genomes (Conant and Wolfe, 2008) [1] . The present study was undertaken to retrieve, annotate and comparatively characterize the mitochondrial genome of Andamani goat. This work provides a preliminary mitochondrial genome annotation resource for this indigenous island goat population.

Materials and Methods

Mitochondrial genome retrieval

The mitochondrial genome sequence of the goat RefSeq ARS1.2 assembly (GCF_001704415.2) was downloaded from NCBI and used as the reference sequence for mitochondrial genome recovery and comparison. Available sequencing reads of Andamani goat were used for mitochondrial genome retrieval. The mitochondrial genome sequence was recovered using GetOrganelle (Jin et al., 2020) [5] , and the recovered sequence was used for downstream annotation, visualization and comparative analysis.

Mitochondrial genome annotation and visualization

The retrieved mitochondrial genome sequence was annotated using MITOS2 (Donath et al., 2019) [3] in Galaxy server (usegalaxy.org). Annotation included identification of protein-coding genes, transfer RNA genes, ribosomal RNA genes and the control region. The annotated mitochondrial genome was then visualized as a circular genome map using GenomeVx (Conant and Wolfe, 2008) [1] .

Comparative mitochondrial genome analysis

For comparative assessment, complete Capra hircus mitochondrial genome sequences were downloaded from the NCBI Nucleotide database in FASTA format. The downloaded sequences were used for comparison of general mitogenome features. The Andamani goat mitogenome was compared with the downloaded complete goat mitochondrial genomes for genome length and base composition. Sequence length was calculated from the FASTA files, and GC content was estimated as the percentage of guanine and cytosine bases in the complete mitochondrial sequence. AT content was calculated as 100-GC%.

Reference-based mitochondrial sequence variation

The assembled Andamani goat mitochondrial genome was aligned against the goat mitochondrial reference sequence to identify sequence-level differences. Variants were called using bcftools (Danecek et al., 2021) [2] .

Results and Discussion

Retrieval of the Andamani goat mitochondrial genome GetOrganelle successfully recovered a circular mitochondrial genome sequence of Andamani goat. The final mitochondrial genome was recovered from the K105 assembly graph and was reported as a circular animal mitochondrial genome. The average mitochondrial k-mer coverage and base coverage were 94.0× and 322.6×, respectively, indicating reliable mitochondrial genome recovery.

Mitochondrial genome annotation

The recovered Andamani goat mitochondrial genome was annotated using MITOS2. The GFF annotation identified the mitogenome as a circular mitochondrial DNA molecule of 16,643 bp. A total of 37 mitochondrial genomic features were annotated, including 13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. The annotated protein-coding genes included seven NADH dehydrogenase genes (nad1, nad2, nad3, nad4, nad4l, nad5 and nad6), three cytochrome c oxidase genes (cox1, cox2 and cox3), two ATP synthase genes (atp6 and atp8) and one cytochrome b gene (cob). The annotation also identified 22 tRNA genes distributed across the mitochondrial genome. The two ribosomal RNA genes, rrnS and rrnL, were located toward the terminal region of the circularized annotation. Most of the annotated features were located on the positive strand, while nad6 and several tRNA genes were annotated on the negative strand, which is consistent with the typical strand distribution of mammalian mitochondrial genomes.

A large non-coding interval of approximately 1.2 kb was observed between trnP and trnF in the circularized mitochondrial genome annotation. This region likely corresponds to the mitochondrial control-region interval in the present genome orientation. Overall, the MITOS2 annotation confirmed that the Andamani goat mitogenome contains the standard caprine mitochondrial gene complement and showed typical mammalian mitochondrial genome organization.

Visualization of the annotated mitochondrial genome

The MITOS2 annotation was visualized using GenomeVx, which generated a circular map of the Andamani goat mitogenome. The map represented a mitochondrial genome length of 16,643 bp and displayed the annotated proteincoding genes, tRNA genes, rRNA genes and control-region interval in their respective genomic positions.

Comparative mitochondrial genome features

The Andamani goat mitogenome was 16,643 bp in length and showed an AT-rich nucleotide composition. The base composition included 33.56% adenine, 27.32% thymine, 13.09% guanine and 26.04% cytosine. The overall AT and GC contents were 60.87% and 39.13%, respectively. The Andamani goat mitogenome was compared with 10 complete Capra hircus mitochondrial genomes downloaded from NCBI. The published goat mitogenomes ranged from 16,622 bp to 16,643 bp in length, while the Andamani goat mitogenome was 16,643 bp. The GC content of the downloaded goat mitogenomes ranged from 39.11% to 39.36%, and the Andamani goat mitogenome showed a comparable GC content of 39.13%. Thus, the Andamani [13,4,6,8] .

Reference-based mitochondrial sequence differences

Reference-based comparison of the assembled Andamani goat mitochondrial genome with the goat mitochondrial reference sequence identified two single nucleotide differences. These included a T>C transition at position 979 and a G>A transition at position 13,964. The first variant was located within the nad1 gene, whereas the second variant was located in the non-coding interval between trnP and trnF.

Comparison with published goat mitochondrial genomes

The Andamani goat mitogenome showed the typical organization reported for domestic goat mitochondrial genomes, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and a control region. Similar mitochondrial gene content has been reported in Indian Black Bengal goat (Sahu et al., 2024) [8] , Bangladeshi Black Bengal goat (Siddiki et al., 2019) [9] , Yimeng black goat (Liu et al., 2020) [6] , Tibetan goat (Zhang et al., 2016) [13] and Xinong Saanen goat (Jia et al., 2016) [4] . The mitogenome length observed in the present study was 16,643 bp, which was within the range reported for published complete Capra hircus mitochondrial genomes. For example, Tibetan goat, Yimeng black goat and Xinong Saanen goat mitogenomes were reported to be approximately 16.64 kb in length (Zhang et al., 2016; Liu et al., 2020; Jia et al., 2016) [13,6,4] . This indicates that the recovered Andamani goat mitochondrial sequence represents a complete and structurally typical goat mitogenome. The Andamani goat mitogenome showed an AT-rich nucleotide composition, with 60.87% AT and 39.13% GC content. Similar AT-biased composition has been reported in other goat mitochondrial genomes, including Tibetan goat and Xinong Saanen goat (Zhang et al., 2016; Jia et al., 2016) [13,4] . Such AT richness is a common feature of 2001) [1] . Therefore, annotation of the Andamani goat mitogenome provides a useful baseline resource for future molecular characterization of this indigenous island goat population. Since island livestock populations may be influenced by geographic isolation, founder effects and local adaptation, generation of such genomic resources is important for breed documentation and conservation planning.

Utility of mitochondrial genome retrieval and annotation

The workflow used in the present study demonstrates that mitochondrial genome retrieval, annotation and visualization can be efficiently performed using standard bioinformatic tools. GetOrganelle enabled recovery of a circular Andamani goat mitogenome, while MITOS2 provided automated annotation of mitochondrial genes and GenomeVx supported circular genome visualization (Jin et al., 2020; Donath et al., 2019; Conant and Wolfe, 2008) [5,3,1] . This approach can be applied to other indigenous livestock populations for developing baseline mitochondrial genome resources useful for molecular characterization and conservation-oriented studies.

Conclusion

The present study reports mitochondrial genome annotation and comparative characterization of the Andamani goat. The annotated mitogenome was 16,643 bp in length and showed the typical caprine mitochondrial genome structure, consisting of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and a control region. The genome showed AT-rich nucleotide composition and was comparable to published complete Capra hircus mitogenomes in terms of genome length and base composition. This work provides a preliminary mitochondrial genome resource for Andamani goat and may support future molecular characterization and conservation genetic studies of this indigenous island goat population.

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