The Complete Mitochondrial Genome of Critically Endangered Painted Terrapin, Batagur borneoensis (Testudines: Geoemydidae)

Nor Ainsyafikah Madiran; Han Ming Gan; Shahreza Md Sheriff; Aliyu Garba Khaleel; Muhammad Zaid Nasir; Chik Maslinda Omar; Mohamad Zulkarnain Mohd Dali; Muhammad Syafiq Aiman Mohd Nasir; Norshida Ismail; Ahmad Syazni Kamarudin

doi:10.22146/jtbb.13529

Nor Ainsyafikah Madiran School of Animal Science, Aquatic Science and Environment, Faculty of Bioresources and Food Industry, UniSZA Besut Campus, 22200 Besut, Terengganu, Malaysia https://orcid.org/0000-0003-4255-3060
Han Ming Gan Patriot Biotech Sdn Bhd, Bandar Sunway, 47500, Subang Jaya, Selangor, Malaysia https://orcid.org/0000-0001-7987-738X
Shahreza Md Sheriff Faculty of Fisheries and Food Sciences, Universiti Malaysia Terengganu, Kuala Nerus, Terengganu, Malaysia https://orcid.org/0000-0002-3511-2323
Aliyu Garba Khaleel Department of Animal Science, Faculty of Agriculture and Agricultural Technology, Kano University of Science and Technology, Wudil, P.M.B. 3244 Kano State, Nigeria https://orcid.org/0000-0002-8135-2045
Muhammad Zaid Nasir WWF Setiu Field Office, Lot 10575, Tanah Lot, Kg. Pagar Besi, 21020 Kuala Terengganu, Terengganu, Malaysia
Chik Maslinda Omar WWF Setiu Field Office, Lot 10575, Tanah Lot, Kg. Pagar Besi, 21020 Kuala Terengganu, Terengganu, Malaysia
Mohamad Zulkarnain Mohd Dali School of Animal Science, Aquatic Science and Environment, Faculty of Bioresources and Food Industry, UniSZA Besut Campus, 22200 Besut, Terengganu, Malaysia https://orcid.org/0000-0001-7056-7959
Muhammad Syafiq Aiman Mohd Nasir School of Animal Science, Aquatic Science and Environment, Faculty of Bioresources and Food Industry, UniSZA Besut Campus, 22200 Besut, Terengganu, Malaysia https://orcid.org/0000-0001-5418-6793
Norshida Ismail School of Animal Science, Aquatic Science and Environment, Faculty of Bioresources and Food Industry, UniSZA Besut Campus, 22200 Besut, Terengganu, Malaysia https://orcid.org/0000-0001-8178-2726
Ahmad Syazni Kamarudin School of Animal Science, Aquatic Science and Environment, Faculty of Bioresources and Food Industry, UniSZA Besut Campus, 22200 Besut, Terengganu, Malaysia https://orcid.org/0000-0002-5529-2974

DOI: https://doi.org/10.22146/jtbb.13529

Keywords: Batagur borneoensis, Conservation, Genetic diversity, Mitogenome, Next-generation sequencing

Abstract

Characterising mitochondrial genomes is a key to studying evolution in vertebrates including turtles. This study employed Next-Generation Sequencing (NGS) to characterise mitochondrial DNA sequences in Batagur borneoensis (Schlegel & Muller, 1844). We reported the nearly complete mitogenome to clearly characterise the gene sequence of B. borneoensis which has been deposited in GenBank under the accession number PP228865. Phylogenetic analyses using Maximum Likelihood (ML) on the 13 protein-coding genes were conducted with MEGA X Version 11 software. This study presents the second in-depth analysis of the B. borneoensis mitochondrial genome, spanning 16,397 base pairs and containing 13 protein-coding genes, 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs), and a major non-coding region, two non-coding regions: L-strand origin replication (OL) and control region (OH). The sequence length and organisation of this species' mitochondrial genome fall within the typical range and gene arrangement found in vertebrate species. Most genes, except for seven tRNAs and nad6, were encoded on the primary DNA strand. All protein-coding genes (PCGs) began with an ATG initiation codon, except for cox1 and trnF which started with GTG codon, and nad3_0, started with a TTA codon. These findings enhanced our understanding of nucleotide composition and molecular evolution in the genus Batagur. Phylogenetic analyses identified vulnerable and ecologically important species, aiding biodiversity and ecosystem protection. They also expanded the dataset for comparative studies within the Geoemydidae family. Additionally, this research may help develop primers and conservation strategies for future studies.

References

1. Bernt, M., Donath, A., Jühling, F., Externbrink, F., Florentz, C., Fritzsch, G., Pütz, J., Middendorf, M., & Stadler, P. F. (2013). MITOS: Improved de novo metazoan mitochondrial genome annotation. Molecular Phylogenetics and Evolution, 69(2), 313-319. https://doi.org/10.1016/j.ympev.2012.08.023
2. Boore J. L. (1999). Animal mitochondrial genomes. Nucleic Acids Res. 27: 1767-1780
3. Chen, Y., Chen, Y., Shi, C., Huang, Z., Zhang, Y., Li, S., Li, Y., Jia, Y., Yu, C. H., Li, Z., Zhang, X., Wang, J., Yang, H., Fang, L., & Chen, Q. (2017). SOAPnuke: a MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data. GigaScience, 7(1). https://doi.org/10.1093/gigascience/gix120
4. Darwin C. On the origin of species. London: John Murray. 1859; 502 p.
5. Fernandez-Silva P, Enriquez JA, Montoya J, (2003). Replication and transcription of mammalian mitochondrial DNA. Experimental Physiology, 88: 41-56.
6. Hasegawa M, Kishino H, Yano T (1985). Dating the human-ape split by a molecular clock of mitochondrial DNA. J Mol Evol 22: 160– 174.
7. Hernawan, E., Basuni, S., Masy’ud, B., & Kusrini, M. D. (2018). Literature Review: Distribution, Ecology, History and Conservation of Painted Terrapin (Batagur borneoensis Schlegel and Muller 1845) in Indonesia. International Journal of Sciences: Basic and Applied Research, 42(5), 199-209. https://gssrr.org/index.php?journal=JournalOfBasicAndApplied&page=article&op=view&path%5B%5D=9296
8. Iwasaki W., Fukunaga T, Isagozawa R, Yamada K, Maeda Y, Satoh TP, Sado T, et al. (2013). MitoFish and MitoAnnotator: A mitochondrial genome database of fish with an accurate and automatic annotation pipeline. Mol Biol Evol 30:2531–40.
9. Kolandaiveloo, V., Kalaiselvam, R., Fong, M. W. C., Mustapha, M. S., Souce, R. M., Sugnaseelan, S., Watanabe, M., Lau, M. M., Sharma, D. S. K., Sharma, R. S. K. (2020). Endoscopy gender determination and reproductive hormone profiles of Painted Terrapins (Batagur borneoensis) subjected to ex situ incubation. The Journal of Veterinary Medical Science. 82(4). 497-502.
10. Kumar S, Stecher G, Tamura K (2016). MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33: 1870– 1874.
11. Kundu S, Kumar V, Tyagi K, Chandra K (2020) The complete mitochondrial genome of the endangered Assam Roofed Turtle, Pangshura sylhetensis (Testudines: Geoemydidae): Genomic features and phylogeny. PLoS ONE 15(4): e0225233. https://doi.org/10.1371/journal. pone.0225233
12. Lee, P. S., Sing, K. W., Wilson, J. J. (2015). Reading mammal diversity from flies: the persistence period of amplifiable mammal mtDNA in blowfly guts (Chrysomya megacephala) and a new DNA mini-barcode target. PLoS One. 10: e0123871.
13. Li, D., Liu, C., Luo, R., Sadakane, K., & Lam, T. W. (2015). MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics, 31(10), 1674-1676. https://doi.org/10.1093/bioinformatics/btv033
14. Lourenco J. M., Claude J, Galtier N., Chiari Y. Dating cryptodiran nodes: origin and diversification of the turtle superfamily Testudinoidea. Mol Phylogenet Evol. 2012; 62: 496–507. https://doi.org/10.1016/j. ympev.2011.10.022 PMID: 22100825
15. Luo, X., & Kang, X. (2022b). Benchmarking datasets used in the manuscript “Enhancing long read based strain-aware metagenome assembly.” Zenodo (CERN
European Organization for Nuclear Research. https://doi.org/10.5281/zenodo.5830706
Oberacker, P., Stepper, P., Bond, D. M., Höhn, S., Focken, J., Meyer, V., Schelle, L., Sugrue, V. J., Jeunen, G., Moser, T., Hore, S. R., Von Meyenn, F., Hipp, K., Hore, T. A., & 16. Jurkowski, T. P. (2019). Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation. PLOS Biology, 17(1), e3000107. https://doi.org/10.1371/journal.pbio.3000107
17. Rand DM (1993). Endotherms, ectotherms, and mitochondrial genome-size variation. J. Mol. Evol. 37: 281-295.
18. Sahoo, Prabhati & Goel, Chirag & Kumar, Rohit & Dhama, Nisha & Ali, Shahnawaz & Sarma, Dandadhar & Nanda, Prasanta & Barat, Ashoktaru. (2015). The Complete mitochondrial genome of threatened chocolate mahseer (Neolissochilus hexagonolepis) and its phylogeny.. Gene. 570. 10.1016/j.gene.2015.07.024.
19. Sturk-Andreaggi, K., Ring, J., Ameur, A., Gyllensten, U., Bodner, M., Parson, W., Marshall, C., & Allen, M. (2022). The Value of Whole-Genome Sequencing for Mitochondrial DNA Population Studies: Strategies and Criteria for Extracting High-Quality Mitogenome Haplotypes. International Journal of Molecular Sciences, 23(4), 2244. https://doi.org/10.3390/ijms23042244
20. Toomey, D. (2016). Pulling the stunningly unique painted terrapin back from the brink. Mongabay News & Inspiration from Nature’s Frontline.
21. Zhang, L., Sun, K., Csorba, G. et al. Complete mitochondrial genomes reveal robust phylogenetic signals and evidence of positive selection in horseshoe bats. BMC Ecol Evo 21, 199 (2021). https://doi.org/10.1186/s12862-021-01926-2