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Mitochondrial DNA B Resour
Mitochondrial DNA B Resour
Mitochondrial DNA. Part B, Resources
2380-2359
Taylor & Francis

10.1080/23802359.2024.2405539
2405539
Version of Record
Data Note
Mitogenome Report
The mitochondrial genome sequence of Manchurian Hare (Lepus mandshuricus)
C. Lin et al.
Lin Chen
Fan Jiale
Bai Suying
College of Wildlife and Protected Area, Northeast Forestry University, Harbin, China
Supplemental data for this article can be accessed online at https://doi.org/10.1080/23802359.2024.2405539.

CONTACT Suying Bai sybai@nefu.edu.cn College of Wildlife and Protected Area, Northeast Forestry University, Harbin 150040, China
23 9 2024
2024
23 9 2024
9 9 12541257
10 5 2024
11 9 2024
KnowledgeWorks Global Ltd.21 9 2024
published online in a building issue23 9 2024
© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

Abstract

The Manchurian hare (Lepus mandshuricus) is widely distributed in eastern Russia and northeastern China, but due to limited research, its taxonomic status remains somewhat ambiguous. The mitochondrial genome of the Manchurian hare was 16,705 bp in length, which was consisted of 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes and one control region. The overall nucleotide composition is 31.7% A, 29.4% T, 13.3% G, and 25.6% C, indicating a high AT content. Phylogenetic analysis reveals a closer relationship of the Manchurian hare with the Korean hare (Lepus coreanus) and the Iberian hare (Lepus granatensis), while its relationship with the Hainan rabbit (Lepus hainanus), European hare (Lepus europaeus) and the snowshoe hare (Lepus americanus) is more distant. The mitochondrial genome of the Manchurian hare is of vital importance for the phylogenetic analysis of lagomorphs and provides valuable data for deeper evolutionary inquiries.

Keywords

Lepus mandshuricus
mitochondrial genome
phylogenetic relationship
National Forestry and Grassland Administration 10.13039/501100007825 2020070209 This work was supported by the Rare and Endangered Species Investigation, Supervision, and Industry Standardization Project of the National Forestry and Grassland Administration: Wildlife Identification Technology Support [2020070209].
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pmcIntroduction

Lepus mandshuricus Radde, 1861, commonly referred to as the Manchurian Hare, is found in the eastern Russia and northeastern China. Its habitat extends eastward from the Ussuri River region in Russia, covering areas such as Heilongjiang and Jilin provinces in northeastern China, and further into the northern Korea. It is considered as one of the significant wild lagomorphs in northeastern China. The Manchurian Hare predominantly resides in forested areas, exhibiting a preference for mixed forests over coniferous ones. It tends to avoid open landscapes and steers clear of human settlements. The elevation across its habitat can reach up to 900 m (2953 ft) above sea level.

Currently, there is no study characterizing the mitochondrial genome of the Manchurian hare (L. mandshuricus). In this study, we first sequenced the mitochondrial genome of the Manchurian hare (L. mandshuricus) and explored its phylogenetic relationships within the family of Leporidae. The mitochondrial genome of the Manchurian hare will provide valuable genetic resources for future studies of ecology and evolution for this species, as well as its relative species in the order of Lagomorpha.

Materials and methods

A single biological sample of Lepus mandshuricus (Figure 1) was collected postmortem from Erdaogou, Qingfeng Forest Farm, Luobei County, Hegang City, Heilongjiang Province, China (48.242472°N, 130.169694°E). Sterile tools were used during collection, and the sample was immediately preserved. Upon arrival at the laboratory, it was stored at −20 °C to ensure DNA integrity. This study received ethical approval from the Institutional Animal Care and Use Committee of Northeast Forestry University. A specimen was deposited at the Forensic Identification Institute of Northeast Forestry University (contact person: Yue Ma, email: mayue@nefu.edu.cn) under the voucher number sj2021-70-1.Genomic DNA was extracted from muscle tissue samples using the TIANamp Micro DNA Kit (DP316) from Tianjin Tiangen Biotech Co., Ltd. Genomic sequencing was carried out using the DNBSEQ-T1 sequencer at the China National GeneBank (Shenzhen, China). The process utilized paired-end 150 bp reads (PE150). Instead of focusing exclusively on mitochondrial DNA, the entire genomic DNA was sequenced. The mitochondrial genome sequence was assembled using NOVOPlasty v4.3.1 (Dierckxsens et al. 2017). Annotation of the mitochondrial genome sequence was conducted using the Chlorobox web service (Tillich et al. 2017). The phylogenetic tree was constructed using IQ-Tree v1.6.6 (Schmidt et al. 2015) with the maximum-likelihood (ML) method. This analysis included 1,000 ultrafast bootstraps (Hoang et al. 2018) and 1,000 SH-aLRT replicates (Guindon et al. 2010). The best-fit partitioning schemes and substitution models were determined using ModelFinder (Kalyaanamoorthy et al. 2017). We constructed a maximum likelihood tree using the mitochondrial genome sequences, including the D-loop region, of L. mandshuricus and 25 other species within the order Lagomorpha (Supplementary Material Table S1). The circular mitochondrial genome map of L. mandshuricus was constructed by OrganellarGenomeDraw (OGDRAW) (Greiner et al. 2019).

Figure 1. The studied specimen of Lepus mandshuricus (voucher no.: sj2021-70-1). Photograph by Suying Bai.

Results

We yielded a total of 41.58 Gb of raw whole genome sequencing data. The length of the mitochondrial genome of Lepus mandshuricus we assembled in this study was 16,705 bp, which was deposited in the GenBank with the accession number of PP597113. The coverage sequencing depth per base along the mitochondrial genome reached 7,885.98-fold (Supplementary Figure S1). Annotation of the mitochondrial genome revealed 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, 13 protein-coding genes (PCGs) and one control region, with the heavy DNA strand (H-strand) carring 12 PCGs, 2 rRNAs, and 15 tRNAs, while the remaining eight genes were located on the light DNA strand (Figure 2). The nucleotide composition of the mitochondrial genome was 31.7% A, 29.4% T, 13.3% G, and 25.6% C, displaying a high AT bias (61.1%). Our phylogenetic analysis (Figure 3) revealed a close affinity between Lepus mandshuricus and Lepus coreanus or Lepus granatensis, whereas it showed a relatively distant relationship with Lepus hainanus, Lepus europaeus and Lepus americanus.

Figure 2. Mitochondrial genome map of Lepus mandshuricus (PP597113). The inner circle represents GC content, while the outer circle displays gene features. Genes outside the circle represent those located on the H-strand, whereas those inside represent the genes located on the L-strand.

Figure 3. The maximum likelihood phylogenetic tree shows the relationship between the mitochondrial genome of Lepus mandshuricus and other species in the order Lagomorpha. This tree includes branches representing the genus Lepus (blue), genus Oryctolagus (red), genus Pronolagus (green), and family ochotonidae genus Ochotona (orange). Lepus mandshuricus is highlighted with a red box. The following sequences were used: Lepus americanus NC_024043(Melo-Ferreira et al. 2014), Lepus arcticus NC_044769 (Unpublished), Lepus capensis GU937113 (Wang and Yang 2014), Lepus coreanus NC_024259 (Unpublished), Lepus europaeus OR876275 (Riikka Tapanainen et al. 2024), Lepus granatensis NC_024042 (Melo-Ferreira et al. 2014), Lepus hainanus NC_025902 (Unpublished), Lepus oiostolus NC_050983 (Unpublished), Lepus sinensis NC_025316 (Ding et al. 2014), Lepus tibetanus MZ297478 (Unpublished), Lepus timidus OR939643 (Riikka Tapanainen et al. 2024), Lepus tolai NC_025748 (Ding et al. 2014), Lepus townsendii NC_024041 (Melo-Ferreira et al. 2014), Lepus yarkandensis NC_050569 (Unpublished), Oryctolagus cuniculus NC_001913 (Gissi et al. 1998), Romerolagus diazi MK614507 (Unpublished), Ochotona erythrotis NC_037186 (Unpublished), Ochotona koslowi NC_039987 (Unpublished), Ochotona curzoniae NC_011029 (Unpublished), Ochotona dauurica NC_044120 (Unpublished), Ochotona thibetana NC_070362 (Unpublished), Ochotona collaris NC_003033 (Unpublished), Ochotona coreana NC_052873 (Unpublished), Ochotona hyperborea NC_057103 (Unpublished) and Ochotona princeps NC_005358 (Unpublished). The GenBank accession numbers for the sequences are indicated next to the species names. Numbers near nodes indicate maximum-likelihood bootstrap percentages.

Discussion and conclusion

In this study, we sequenced and assembled the mitochondrial genome of the Manchurian hare (Lepus mandshuricus). The arrangement and nucleotide composition of this genome closely resemble those of other species in the Leporidae family (Huang et al. 2019; Kim et al. 2019; Shan et al. 2020; Zhang et al. 2020). Phylogenetic analysis indicated a close relationship between the Manchurian hare and certain geographically related species, while revealing a distant relationship with others. Notably, Lepus hainanus is genetically distant from Lepus mandshuricus. This study is the first to characterize the mitochondrial genome of L. mandshuricus, providing essential data for future research.

We utilized the DNBSEQ-T1 short-read sequencing platform to perform whole-genome sequencing with paired-end 150 bp (PE150) reads. While this method offers high throughput and accuracy, it faces challenges with long tandem repeat regions. The short reads produced by this method can complicate the assembly of these regions, potentially leading to information loss or misassembly. A prominent characteristic of the non-coding regions in Lagomorpha mitochondrial DNA is the presence of long tandem repeats (Casane et al. 1997). In our study, this limitation likely caused the assembled mitochondrial genome of Lepus mandshuricus to miss the long tandem repeat region, which can span up to 1000 base pairs depending on the number of repeats (Riikka Tapanainen et al. 2024).

Future research could incorporate long-read sequencing technologies to produce longer reads that can span entire repeat regions, thus improving the completeness and accuracy of genome assembly. Despite these challenges, our study successfully obtained a high-quality mitochondrial genome sequence for Lepus mandshuricus, providing a valuable foundation for further genomic research.

Supplementary Material

Supplementary Table S1.xlsx

Supplementary Figure S1.pdf

Ethical statement

All animal experiments in this study were approved by the Experimental Animal Management and Ethics Committee of Northeast Forestry University.

Authors’ contributions

The study was conducted collaboratively by Chen Lin, Jiale Fan and Suying Bai. Chen Lin was responsible for sample collection, DNA extraction, and drafting the initial manuscript. Chen Lin and Jiale Fan jointly conducted data analysis and provided a critical review of the manuscript. In addition to contributing to data analysis, Jiale Fan was involved in the critical review, revisions, and final approval of the draft. Suying Bai contributed to the conception and design of the study, and provided critical review, editing, and finalization of the manuscript. Suying Bai also approved the final version for publication. All authors have agreed to take responsibility for all aspects of this research.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data supporting the findings of this study are available at https://www.ncbi.nlm.nih.gov/. GenBank accession No. is PP597113. The associated Bio-Sample, SRA, and BioProject numbers are SAMN41256110, SRR28956761 and PRJNA1108806, respectively, and all accession numbers are activated.
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