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Article

Characterization of the Complete Mitochondrial Genome of the Elongate Loach and Its Phylogenetic Implications in Cobitidae

by
Zhenlin Ke
1,2,
Kangqi Zhou
3,
Mengdan Hou
1,2,
Hui Luo
1,2,
Zhe Li
3,
Xianhui Pan
3,
Jian Zhou
4,
Tingsen Jing
1,2 and
Hua Ye
1,2,*
1
Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, College of Fisheries, Southwest University, Chongqing 402460, China
2
Key Laboratory of Aquatic Science of Chongqing, Chongqing 400175, China
3
Guangxi Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture, Guangxi Academy of Fishery Sciences, Nanning 530021, China
4
Fisheries Institute, Sichuan Academy of Agricultural Sciences, Chengdu 611731, China
*
Author to whom correspondence should be addressed.
Animals 2023, 13(24), 3841; https://doi.org/10.3390/ani13243841
Submission received: 25 October 2023 / Revised: 1 December 2023 / Accepted: 2 December 2023 / Published: 13 December 2023
(This article belongs to the Section Aquatic Animals)
Browse Figures
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Abstract

:

Simple Summary

The complete mitochondrial genome has been widely used in phylogenetics-related studies, as it offers valuable insights into evolutionary relationships. In this study, we reported the complete mitogenome of the elongate loach (Leptobotia elongata) and conducted a detailed analysis of its characteristics which was employed to infer phylogenetic relationships. These findings reveal that both the gene arrangement and composition of mitochondrial genes in the elongate loach are comparable to those found in other bony fishes. Our study further demonstrated that the Cobitidae species under investigation could be grouped into two distinct clades, with elongate loach showing a sister relationship with L. microphthalma. Collectively, our research enhanced the understanding of the mitochondrial genome structure and contributed to the phylogenetic analysis of the elongate loach.

Abstract

The elongate loach is an endemic fish in China. Previous studies have provided some insights into the mitochondrial genome composition and the phylogenetic relationships of the elongate loach inferred using protein-coding genes (PCGs), yet detailed information about it remains limited. Therefore, in this study we sequenced the complete mitochondrial genome of the elongate loach and analyzed its structural characteristics. The PCGs and mitochondrial genome were used for selective stress analysis and genomic comparative analysis. The complete mitochondrial genome of the elongate loach, together with those of 35 Cyprinidae species, was used to infer the phylogenetic relationships of the Cobitidae family through maximum likelihood (ML) reconstruction. The results showed that the genome sequence has a full length of 16,591 bp, which includes 13 PCGs, 22 transfer RNA genes (tRNA), 2 ribosomal RNA genes (rRNA), and 2 non-coding regions (CR D-loop and light chain sub-chain replication origin OL). Overall, the elongate loach shared the same gene arrangement and composition of the mitochondrial genes with other teleost fishes. The Ka/Ks ratios of all mitochondrial PCGs were less than 1, indicating that all of the PCGs were evolving under purifying selection. Genome comparison analyses showed a significant sequence homology of species of Leptobotia. A significant identity between L. elongata and the other five Leptobotia species was observed in the visualization result, except for L. mantschurica, which lacked the tRNA-Arg gene and had a shorter tRNA-Asp gene. The phylogenetic tree revealed that the Cobitidae species examined here can be grouped into two clades, with the elongate loach forming a sister relationship with L. microphthalma. This study could provide additional inferences for a better understanding of the phylogenetic relationships among Cobitidae species.

1. Introduction

The elongate loach (Leptobotia elongata), belonging to Cobitidae of Cypriniformes, is indigenous to the middle and upper reaches of the Yangtze River in China [1]. It is characterized by rapid growth and exceptional ornamental value [2,3]. However, the wild population resources of the elongate loach have experienced a significant decline since the 1980s due to overfishing, dam construction, and destruction of feeding and spawning grounds [4]. As a result, it has been classified as vulnerable grade (VU) in the China Red Book of Endangered Animals-Fish [5].
The family Cobitidae was originally proposed by Regan [6]. In this family, extensive research focused on morphological characteristics and mitochondrial genes has been conducted for over a century [7,8,9,10,11]. Currently, many scientists tend to divide Cobitidae into three subfamilies: Nemacheilinae, Botiinae, and Cobitinae [12]. In order to maintain consistency between the phylogenetic relationship and the natural classification of Cobitidae fishes, Tang et al. [13] elevated these three subfamilies to the family level, which aligns with the classification of Liu et al. [9]. As the second-largest group of Cypriniformes, Cobitidae is a key element in resolving the phylogenetic relationships of Cypriniformes. Investigating the phylogenetic relationships of the elongate loach, one of the youngest species in the Cobitidae, is beneficial to resolve the taxonomic ambiguity of Cobitidae fishes. Previous studies only focused on biological characteristics [14], artificial breeding [15], embryo development, and genetic diversity [16,17]. However, its research on the phylogenetic relationships of L. elongate is limited [18]. Studies dealing with the phylogenic status of the elongate loach addressed questions about inter-family relationships in Cobitidae [6,11,12,13], but research into species phylogenetic relationships within the family remain lacking. Therefore, a reevaluation of the phylogenetic relationships of the elongate loach, involving additional genes and a broader range of species, could provide more data for the conservation of the elongate loach’s wild population resources.
Mitochondrial DNA (mtDNA) is present in the cells of all eukaryotes [19]. Compared to nuclear genes, mtDNA evolves at a faster rate, allowing for a more accurate representation of phylogenetic relationships. Therefore, mtDNA is widely utilized as a molecular marker in phylogenetic studies [20,21,22,23]. In fish phylogeny research, genes such as cytochrome b (cytb), cytochrome oxidase (cox), and 16 S rRNA are commonly employed at the species-to-family level [22,24,25]. However, relying solely on a single mitochondrial gene may lead to misleading phylogenetic data due to limited information capacity and homogenization effects [26]. In contrast, utilizing the complete mitochondrial genome could provide a more comprehensive set of phylogenetic information [27].
In this study, we sequenced the mitochondrial genome, analyzed the structural information of the elongate loach, and compared the structures and complete mitochondrial genome with some of the determined Leptobotia species. Additionally, we reconstructed phylogenetic trees using complete mitochondrial genome sequences to analyze the evolutionary relationships of the elongate loach in the Cobitidae family. This study might provide further insight into the structure of L. elongate and improve our understanding of the evolutionary relationships of L. elongate which will be helpful to resolve uncertainties within the Cobitidae family.

2. Materials and Methods

2.1. Sample Collection and DNA Extraction

The elongate loach is adorned with a combination of brown and yellow hues throughout its body. Its head and sides are embellished with irregular spots of various shades of color. There are striking black stripes in the elongate loach. The smaller eyes are in the lateral upper position, and the horseshoe-shaped fissure is in the lower position with two pairs of kissing whiskers and one pair of mouth beards (Figure S1). An elongate loach sample was collected from a breed pond at the Sichuan Fisheries Research Institute of Chengdu (103°54′53.740″ E, 30°45′26.956″ N), Sichuan Province, China, in October of 2020. The pectoral fin of one elongate loach was collected and stored in 95% ethanol at −20 °C. Genomic DNA was isolated from the pectoral fin using the phenol-chloroform method and the quality and integrity of DNA samples were assessed using an Agilent 2100 Bioanalyzer 2.2. Mitochondrial genome sequencing and assembly.
After qualifying the DNA sample, the DNA was mechanically fragmented using ultrasonic interruption. The fragmented DNA underwent fragment purification, end-repair, addition of A at the 3′ end, connection of sequencing adapters, and selection of fragments of different sizes using agarose gel electrophoresis. PCR amplification was then performed to generate a sequencing library [18]. The qualified PCR products were sequenced on the Illumina HiSeq 2500 platform.
Prior to assembly, low-quality data, including the reads of average quality value < 5 or N content > 5, were filtered using Fasta software (version 0.20.0), and the sequences linker and primer sequence were trimmed from the reads. The mitochondrial genome assembly was carried out using the following methodology. First, clean reads were assembled using SPAdes (version 3.10) [28] to obtain SEED sequences, and the seed sequences were iterative extended in GCE (version 1.0.2) to obtain contig sequences. Second, contigs were connected to generate scaffold sequences using SSPACE (version 2.0 (https://www.baseclear.com/services/bioinformatics/basetools/sspace-standard/, accessed on 8 September 2022), and Gaps in the scaffold sequences were filled using Gapfiller (version 2.1.1 (https://sourceforge.net/projects/gapfiller/, accessed on 8 September 2022) until a complete pseudo genome sequence was assembled. Lastly, the sequencing results were mapped onto the assembled pseudo genome sequence to identify and correct any incorrect bases, and the complete mitochondrial circular genome sequence was obtained by coordinate remaking.

2.2. Mitochondrial Genome Annotation and Analysis

The newly assembled sequences were annotated in the Mitos web server (http://mitos2.bioinf.uni-leipzig.de, accessed on 8 September 2022) [29] with the following parameters: E-value Exponent = 5, Maximum Overlap = 100, ncRNA overlap = 100. The annotation results were then compared with those of closely related species. Finally, after manual correction, the final annotation results were obtained.
The secondary structure of tRNAs was obtained from the annotation results. The circular map of the mitochondrial genome was generated using OGDRAW (version 1.3.1) [30]. The relative synonymous codon usage (RSCU) values were analyzed with MEGA (version 6.0). The mitochondrial genome skew values were calculated using the following formula: ATskew = (A − T)/(A + T); GCskew = (G − C)/(G + C) [31]. Mafft (version 7. 310) [32] software was used for gene sequences comparison between the elongate loach and six Leptobotia fishes (L. mantschurica, L. taeniops, L. microphthalma, L. rubrilabris, L. punctata, and L. pellegrini), and the evolutionary rate (Ka/Ks, ω) was calculated using KaKs_Calculator (version 2.0) [33]. If the evolutionary rate is equal to 1, >1, or, <1, the PGCs are expected to be under no selection, positive selective constraint (purifying selection), or diversifying selection [34]. The mitochondrial genome structure was compared between the elongate loach and six Leptobotia fish species in a CGVIEW server [35] with default parameters (http://stothard.afns.ualberta.ca/cgview_server/, accessed on 12 September 2021) and the alignment results were visualized using mauve software (version 2.4.0).

2.3. Phylogenetic Analyses

The phylogenetic tree was reconstructed using the complete mitochondrial genome sequences of 36 Cypriniformes species, with Myxocyprinus asiaticus and Danio rerio used as outgroups (Table 1). All the genome sequences were set to the same start points in the circular sequence. Multiple sequence alignment was performed in MAFFT software (version 7.42) with auto model, and the alignment sequences were trimmed using trimAl (version 1.4. rev15). Subsequently, the RaxML (version 8.2.0) software was used to conduct the rapid bootstrap analysis (bootstrap = 1000) to construct the maximum likelihood evolution tree.

3. Results and Discussion

3.1. Mitochondrial Structural Characteristics

The complete mitochondrial genome of the elongate loach was obtained through high-throughput sequencing technology (OR818399), with a total length of 16,591 bp (Figure 1). It consists of 37 typical animal mitochondrial genes, including 22 tRNA genes, 13 PCGs, 2 rRNA genes, and 2 non-coding regions (D-Loop and OL). Among the mitochondrial genes, nine genes (trnQ, trnP, trnE, nad6, trnS2, trnY, trnC, trnN, trnA) were encoded by the light (L) strand, while the remaining genes were encoded by the heavy (H) strand. The arrangement and content of the mitochondrial genome in the elongate loach were similar to those reported in teleost fishes [20,36,37]. The entire base composition of the elongate loach mitochondrial genes is as follows (Table 2): 30.79% A, 24.77% T, 16.17% G, and 28.27% C, and the AT and GC percentages are 55.56% and 44.44%, respectively, which results in a positive skew value for AT and a subtractive skew value for CG. It was suggested that the occurrence of A and C bases was more frequent in the genome. Previous studies have shown that the bias in base composition plays a crucial role in the replication and transcription of mitochondrial genomes [38].

3.2. Protein Coding Genes

The PCGs account for 68.89% of the total length of the elongate loach mitochondrial genome. As expected (Table 3), most PCGs started with the regular codon ATG, except for the cox1 which started with GTG. Among the PCGs, there were seven genes that shared the complete stop codon TAA, while six genes shared incomplete stop codons (TA- or T--) which exists in many teleosteans as shown in numerous studies: L. microphthalma with seven incomplete stop codons [39], Cobitis macrostigma with seven incomplete stop codons [40], Pelteobagrus fulvidraco with five incomplete stop codons [20], Parabotia kiangsiensis with three incomplete stop codons [41], etc. The presence of tRNA sequences at the 3’ end of these genes is responsible for the incomplete stop codons [42], and these incomplete stop codons can be converted to TAA through post-transcriptional polyadenylation [43].
Three overlapping regions between certain PCGs (ATPase8-ATPase6, ND4-ND4L, and ND5-ND6) were also identified in this study. These overlapping regions were 4–10 bp in length, with the largest overlapping occurring between ATP8 and ATP6, which was common among Cobitidae species [44]. These overlapping regions contribute to the variation in mitochondrial genome length among closely related species [45]. The relative synonymous codon usage (RSCU) values of PCGs are revealed in Table 4 and Figure 2. In the protein-coding region, a total of 2012 codons were used. According to the degeneracy of codons, serine and leucine were encoded by six codons, while the remaining amino acids were encoded by either four or two codons. In the coded passwords, CUA (leucine), AUU (isoleucine), GCC (Aminopropanoic), and GCA (Aminopropanoic) are the most common, while AAA (Lysine) and CUA (leucine) have the highest RSCU values. Therefore, PCGs preferred the codons using adenine at the third codon. The codon usage varied between different species, which was more prominent between species with a more distant evolutionary relationship [46]. It is relevant to gene length, mutation bias, GC composition, amino acid composition, tRNA abundance, and translational selection [47,48,49,50,51,52].

3.3. Genome Comparative Analysis

The nonsynonymous substitution ratio (Ka) and synonymous substitution ratio (Ks) were calculated to evaluate selective pressures during the evolutionary process of PCGs among Leptobotia species. It was shown that the average Ka was similar among the six fishes (0.0089–0.0114), with nd5 exhibiting the highest average Ka (Figure 3A; Table S1), indicating that it might be under positive selection across species. The Ks of L. microphthalma was significantly lower than the other species (Figure 3B; Table S2). There were more synonymous substitutions per synonymous sites in nd4 and atp6, exhibiting the high polymorphic nature of the genes in these fishes. nd4 has also been confirmed to be polymorphic among sharks [53] and blue-spotted maskray [45]. The Ka/Ks ratio (ω) is a means to examine molecular adaption [54,55], which could be used to estimate the evolutionary rate among Cobitidae species. In this study, the Ka/Ks ratios of all PGCs were less than 1, indicating that purifying selection possesses the leading role in the evolution of these PGCs (Figure 3C; Table S3). Therein, cox3 (0.0076) and nd4l (0.0087) were evolving under a strong purifying selection, whereas nd4 (0.0549), nd5 (0.0782), and nd2 (0.0784) were evolving under comparatively relaxed mutational constraints. Currently, selective pressure in mitochondrial PCGs has been poorly studied in other Cobitidae species [13,18,39,40,56,57,58], while the same pattern of widespread purifying selection has been discovered in several other decapod crustaceans [59].
The comparison of the mitochondrial genome sequences between the elongate loach and six Leptobotia species showed a significant sequence homology within the Leptobotia genus (Figure 4 and Figure 5). The elongate loach showed a higher identity with the other five species, except for L. microphthalma, which lacked the tRNA-Arg and a shorter tRNA-Asp, indicating that the arrangement of genes of the Leptobotia species is comparatively conserved.

3.4. Ribosomal RNA and Transfer RNA Genes

The total length of rRNAs was 2638 bp, with an AT skew value of 0.272 and a GC skew value of −0.095. The lengths of 12 S rRNA and 16 S rRNA were 955 bp and 1683 bp, respectively (Table 3). These rRNAs were located between tRNA-Phe and tRNA-Leu and were separated by tRNA-Val, which is consistent with the most reported teleost [60].
There were 22 tRNAs in the mitochondrial genome of the elongate loach, with a total length of 1558 bp. The AT content was 53.89% and the AT skew value was 0.044. Each tRNA has a length of 66–76 bp, with 14 encoded in the H chain and 8 encoded in the L chain. Most of the secondary structure of tRNA genes (Figure 6) in the elongate loach were standard clover-shaped, except for trnS1, which lacked the DHU stem. It was very common to defect DHU stem in metazoan [43]. Additionally, there were 18 false GU pairings in the tRNA sequences of the elongate loach. GU mismatch was frequently observed in teleost fishes and allowed for an expanded chemical and conformational diversity of double-stranded RNA. This diversity provided unique sites that were recognized by amino acids, contributing to a higher genetic diversity for the elongate loach [61]. The base mismatch was essential for the secondary structure of tRNA and played a crucial role in the accurate translation of the genetic code. It also helped minimize errors during mRNA transcription [62].

3.5. Non-Coding Regions

Two common non-coding regions (OL and CR) were identified in the elongate loach mitogenome, the OL region was 39 bp in length and was located between tRNA-Asn and tRNA-Cys. The CR region was located between tRNA-Pro and tRNA-Phe, which is the longest no-coding region in the entire mitochondrial genome with a span distance of 926 bp. It plays a key role in replication and transcription [63]. Similar to other vertebrates [21,64], the CR of the elongate loach exhibited the highest AT content (67.39%) among all regions of the mitochondrial genome. The palindromic sequence motifs ‘tacat’ and ‘atgta’ were related to the termination of H strand replication found in the CR of the elongate loach (Figure 7), which might complete the termination by forming a stable hairpin structure [65].

3.6. Phylogenetic Relationships

Based on the complete mitochondrial genome sequences of the elongate loach and 36 Cyprinidaes species, the phylogenetic tree was constructed. It was shown that the entire phylogenetic tree was grouped into two major clades (Figure 8). The genus Cobitis, Pangio, Triplophysa, and Acanthocobitis formed one clade and matched the subfamily Cobitinae. The Cobitis and the Pangio were sister-lineage, the Triplophysa and the Acanthocobitis were sister-lineage, and the two sister-lineages were sister-lineages to each other. The other clade consisted of Yasuhikotakia, Sinibotia, Chromobotia, Botia, Parabotia, and Leptobotia, corresponding to the subfamily Botiinae. In the subfamily Botiinae, the elongate loach was more closely related to L. microphthalma than to other species.
As a diverse taxa, there was a controversy in the taxonomic relationship of the subfamily Cobitinae. This study exhibited a monophyly of the subfamily Cobitinae which consists of four clades. However, according to Liu et al. [11], there were sisterhoods in many branches. Therefore, the species in Cobitinae cannot form a monophyletic group, the classification of Cobitinae in our study is incomplete, and more taxa should be used in future studies.
It is generally considered that the subfamily Botiinae is a group with a relatively clear taxonomic relationship. In this study, according to their respective genera separately, all individuals except for those of the subfamily Botiinae were clustered into a common branch, which could be confirmed as the monophyly of the subfamily Botiinae. In a previous study, the genera Botia was separated into a separate genus [7] and the genera Botia was divided into three subgenera: Sinibotia, Botia, and Hymenophysa [66]. Others did not further categorize these subgenera, but instead grouped them under the genus Botia [12,67]. In this study, subgenera Botia and subgenera Sinibotia species were clustered separately and formed parallel branches with the species of other genera. Thus, the results supported that subgenera Botia and subgenera Sinibotia should be raised to genus status. Additionally, the phylogenetic tree showed that the elongate loach and L. microphthalma formed a sister group which together formed a sister group of other Leptobotia species. According to Li et al. [18], the elongate loach and L. mantschurica were classified as sister lineages using protein genome sequencing to construct the phylogenetic tree; however, this study was analyzed based on limited taxa sampling, thus lacking sufficient phylogenetic information of the elongate loach.
Tang et al. [13] suggested that the Leptobotia and Parabotia genera were an unnatural group and not reciprocally monophyletic groups as previously hypothesized [13,67,68,69]. They used the species “L. mantschurica” in the phylogenetic analysis which was nested with Parabotia and it shared the same sequences with Parabotia mantschuricus. However, we have not found any detailed explanation taxonomically concerning “Leptobotia mantschurica” and “Parabotia mantschuricus”. Thus, the species “Leptobotia mantschurica” is improper for use in phylogenetic analysis before clear classification. Our phylogenetic tree clearly showed that the Leptobotia and Parabotia genera were a perfect monophyly. Additionally, in the Parabotia species, part of the support value in the branch was low, suggesting that the phylogenetic relationships of these species have not been solved well. Further investigations should be performed to solve this problem.

4. Conclusions

In this study, we reported the complete mitogenome of the elongate loach, the structural characteristics of the mitogenome of the elongate loach were analyzed in detail, and the phylogenetic analyses of the elongate loach were inferred using the complete mitogenome. The full length of the genome sequence was 16,591 bp, and the arrangement of the elongate loach mitochondrial genome is similar to most teleost fishes. Almost all 13 PCGs showed the regular start codon ATG, except for gene cox1, which started with GTG. Six PCGs had incomplete stop codons T--. Thirteen PCGs were evolving under purifying selection, and the mitogenome shared a high identity with Leptobotia species. All of the tRNA genes were standard clover-shaped except for the lack of a DHU stem in trnS1. The phylogenetic analysis showed that the elongate loach was more closely related to L. microphthalma than to other species. The Leptobotia and Parabotia genera were monophyly. In this study, we first studied the selection pressure of complete PCGs in the elongate loach. Overall, we have a deeper understanding of the mitochondrial genome structure and phylogenetic analysis of the elongate loach. However, exact information about many Cobitidae fishes is still unknown. Extra taxa should be used for the phylogenetic research of Cobitidae in the future.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ani13243841/s1, Figure S1: Elongate loach; Table S1: Non-synonymous substitution rate of mitochondrial genes in Leptobotia species; Table S2: Synonymous substitution rate of mitochondrial genes in Leptobotia species; Table S3: The ratio of the number of nonsynonymous substitutions per nonsynonymous site (Ka) to the number of synonymous substitutions per synonymous site (Ks) of mitochondrial genes in Leptobotia species.

Author Contributions

Conceptualization, Z.K. and H.Y.; methodology, H.Y. and K.Z.; writing—original draft preparation, Z.K.; writing—review and editing, Z.K., M.H., H.Y., Z.L., J.Z., H.L., T.J., and X.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported with funding by the National Training Program of Innovation and Entrepreneurship for Undergraduates of China (202110635024), and the Chongqing Alliance for Aquatic Science and Technology Innovation (CQFTIU2022-09).

Institutional Review Board Statement

This study was conducted in accordance with the Declaration of Helsinki and approved by the Animal Care and Use Committee of Southwest University, Chongqing, China. (Approval ID was 20190922 and the approval date was on 22 September 2019).

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets presented in this study were submitted to The National Center for Biotechnology Information (NCBI) database.

Acknowledgments

We would like to express our sincere thanks to the Sichuan Fisheries Research Institute for their help in sample collecting.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Mitochondrial genome map of the elongate loach.
Figure 1. Mitochondrial genome map of the elongate loach.
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Figure 2. The relative synonymous codon usage (RSCU) in the mitogenome of the elongate loach.
Figure 2. The relative synonymous codon usage (RSCU) in the mitogenome of the elongate loach.
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Figure 3. Non-synonymous (A) and synonymous (B) substitutional rates and the ratios of KaKs (C) of the protein coding genes of the elongate loach.
Figure 3. Non-synonymous (A) and synonymous (B) substitutional rates and the ratios of KaKs (C) of the protein coding genes of the elongate loach.
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Figure 4. The comparative circle diagram of the genomes structure of Leptobotia species.
Figure 4. The comparative circle diagram of the genomes structure of Leptobotia species.
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Figure 5. The visualized results of the genome comparison of L. elongata.
Figure 5. The visualized results of the genome comparison of L. elongata.
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Figure 6. Putative secondary structure of elongate loach tRNA.
Figure 6. Putative secondary structure of elongate loach tRNA.
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Figure 7. Compositional features of the control region of the elongate loach mitochondrial genome. Palindromic motif sequence ‘TACAT’ and ‘ATGTA’ are marked in yellow and purple, respectively.
Figure 7. Compositional features of the control region of the elongate loach mitochondrial genome. Palindromic motif sequence ‘TACAT’ and ‘ATGTA’ are marked in yellow and purple, respectively.
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Figure 8. ML tree with boostrap values on the nodes constructed by using the nucleotide sequences of the 13 PCGs in the mitogenome of the elongate loach.
Figure 8. ML tree with boostrap values on the nodes constructed by using the nucleotide sequences of the 13 PCGs in the mitogenome of the elongate loach.
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Table 1. Taxonomic information and Genebank accession numbers of all species used in the phylogenetic analysis.
Table 1. Taxonomic information and Genebank accession numbers of all species used in the phylogenetic analysis.
FamilyGenusSpeciesAssession Number
MyxocyprinaeMyxocyprinusMyxocyprinus asiaticusNC_006401.1
CyprinidaeDanioDanio rerioNC_002333.2
CobitidaeLeptobotiaLeptobotia microphthalmaKY307846.1
LeptobotiaLeptobotia elongataOR818399
LeptobotiaLeptobotia pellegriniNC_031602.1
LeptobotiaLeptobotia taeniopsAP013304.1
LeptobotiaLeptobotia rubrilabrisKY307847.1
LeptobotiaLeptobotia punctataMH644033.1
LeptobotiaLeptobotia mantschuricaAB242170.1
ParabotiaParabotia fasciataAP011437.1
ParabotiaParabotia banarescuiNC_026127.1
ParabotiaParabotia kiangsiensisMT850132.1
ParabotiaParabotia lijiangensisMT323118.1
BotiaBotia udomritthirujiAP011349.1
BotiaBotia lohachataKP729183.1
ChromobotiaChromobotia macracanthusAB242163.1
YasuhikotakiaYasuhikotakia morletiNC_031600.1
Yasuhikotakia modestaKY131962.1
SinibotiaSinibotia robustaNC_027853.1
Sinibotia pulchraKT362179.1
Sinibotia reevesaeNC_030322.1
Sinibotia superciliarisJX683724.1
Sinibotia superciliarisKY307848.1
AcanthocobitisAcanthocobitis botiaAP012139.1
Acanthocobitis botiaAP012138.1
TriplophysaTriplophysa pappenheimiNC_033972.1
Triplophysa siluroidesNC_024611.1
PangioPangio kuhliiNC_031599.1
Pangio cuneovirgataNC_031594.1
CobitisCobitis lutheriNC_022717.1
Cobitis melanoleuca granoeiNC_023473.1
Cobitis nalbantiMH349461.1
Cobitis elongatoidesNC_023947.1
Cobitis hankugensisMN841275.1
Cobitis macrostigmaMK156771.1
Cobitis sinensisNC_007229.1
Cobitis matsubaraiNC_029441.1
Table 2. Nucleotide composition and skewness values of the elongate loach mitogenome of H and L strands.
Table 2. Nucleotide composition and skewness values of the elongate loach mitogenome of H and L strands.
L. elongataSize (bp)A%T%G%C%A + T%G + C%AT-SkewGC-Skew
Mitogenome16,59130.7924.7716.1728.2755.5644.440.108−0.272
PCGs11,43028.5626.7715.5529.1355.3344.670.032−0.304
tRNAs155828.1825.823.4922.5353.9846.020.0440.021
rRNAs263834.0419.4821.0425.4453.5346.470.272−0.095
Dloop92635.6431.7513.7118.967.3932.610.058−0.159
Table 3. Summary of the elongate loach mitogenome.
Table 3. Summary of the elongate loach mitogenome.
Position Codon
GeneStandFromToSizeIntergenic LengthStartStop
tRNA-pheH169690
12 S rRNAH7010249550
tRNA-valH10251096720
16 S rRNAH1097277916830
tRNA-leuH27802854750
nd1H285538299750ATGTAA
tRNA-ileH38383909728
tRNA-glnL3908397871−2
tRNA-metH39804048691
nd2H4049509410460ATGTA-
tRNA-trpH50955163690
tRNA-alaL51665234692
tRNA-asnL52365308731
OLL53105340391
tRNA-cysL5339540466−2
tRNA-tyrL54065476711
cox1H5478702815511GTGTAA
tRNA-serL70307100711
tRNA-aspH71037174722
cox2H7188787869113ATGT--
tRNA-lysH78797954760
ATPase8H795681231681ATGTAA
ATPase6H81148797684−10ATGTAA
cox3H879795817851ATGTA-
tRNA-glyH95829653720
nd3H965410,0023490ATGT--
tRNA-argH10,00310,072700
nd4lH10,07310,3692970ATGTAA
nd4H10,36311,7441382−7ATGTA-
tRNA-hisH11,74511,814700
tRNA-serH11,81511,881670
tRNA-leuH11,88311,955731
nd5H11,95613,79418390ATGTAA
nd6L13,79114,312522−4ATGTAA
tRNA-gluL14,31314,381690
cytbH14,38615,52611414ATGT--
tRNA-thrH15,52715,598720
tRNA-proL15,59715,66670−2
CRH15,66616,5919260
Table 4. Relative synonymous codon usage and codon numbers of L. elongata mitochondrial PCGs.
Table 4. Relative synonymous codon usage and codon numbers of L. elongata mitochondrial PCGs.
CodonNo.RSCUCodonNo.RSCUCodonNo.RSCU
UAA()71AAA(K)381.8536CGG(R)50.5
GCA(A)681.4468AAG(K)30.1464CGU(R)50.5
GCC(A)942CUA(L)1232.271AGC(S)241.161
GCG(A)30.064CUC(L)571.0524AGU(S)40.1938
GCU(A)230.4892CUG(L)220.4062UCA(S)502.4192
UGC(C)71.1666CUU(L)530.9786UCC(S)221.0644
UGU(C)50.8334UUA(L)510.9414UCG(S)10.0486
GAC(D)251.3158UUG(L)190.351UCU(S)231.113
GAU(D)130.6842AUA(M)602.0226ACA(T)721.87
GAA(E)361.44AUG(M)280.9438ACC(T)591.5324
GAG(E)140.56GUG(M)10.0336ACG(T)10.026
UUC(F)630.9618AAC(N)541.4594ACU(T)220.5716
UUU(F)681.0382AAU(N)200.5406GUA(V)521.5524
GGA(G)481.4116CCA(P)471.6348GUC(V)230.6864
GGC(G)310.9116CCC(P)471.6348GUG(V)240.7164
GGG(G)391.1472CCG(P)60.2088GUU(V)351.0448
GGU(G)180.5296CCU(P)150.5216UGA(W)451.6364
CAC(H)421.7142CAA(Q)451.9148UGG(W)100.3636
CAU(H)70.2858CAG(Q)20.0852UAC(Y)260.963
AUC(I)590.792CGA(R)232.3UAU(Y)281.037
AUU(I)901.208CGC(R)70.7
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Ke, Z.; Zhou, K.; Hou, M.; Luo, H.; Li, Z.; Pan, X.; Zhou, J.; Jing, T.; Ye, H. Characterization of the Complete Mitochondrial Genome of the Elongate Loach and Its Phylogenetic Implications in Cobitidae. Animals 2023, 13, 3841. https://doi.org/10.3390/ani13243841

AMA Style

Ke Z, Zhou K, Hou M, Luo H, Li Z, Pan X, Zhou J, Jing T, Ye H. Characterization of the Complete Mitochondrial Genome of the Elongate Loach and Its Phylogenetic Implications in Cobitidae. Animals. 2023; 13(24):3841. https://doi.org/10.3390/ani13243841

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Ke, Zhenlin, Kangqi Zhou, Mengdan Hou, Hui Luo, Zhe Li, Xianhui Pan, Jian Zhou, Tingsen Jing, and Hua Ye. 2023. "Characterization of the Complete Mitochondrial Genome of the Elongate Loach and Its Phylogenetic Implications in Cobitidae" Animals 13, no. 24: 3841. https://doi.org/10.3390/ani13243841

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