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Article

The First Genome-Wide Survey Analysis of the Tibetan Plateau Tetraploid Schizothorax curvilabiatus Reveals Its Microsatellite Characteristics and Phylogenetic Relationships

by
Bingjian Liu
1,2,†,
Luxiu Gao
1,2,†,
Yifan Liu
1,2,
Kai He
3,
Hongchi Li
3,
Taobo Feng
1,2,
Mingzhe Han
1,2 and
Chi Zhang
3,*
1
National Engineering Research Center for Marine Aquaculture, Zhejiang Ocean University, Zhoushan 316022, China
2
National Engineering Laboratory of Marine Germplasm Resources Exploration and Utilization, Zhejiang Ocean University, Zhoushan 316022, China
3
Institute of Fisheries Science, Tibet Academy of Agricultural and Animal Husbandry Sciences, Lhasa 850000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Genes 2025, 16(5), 491; https://doi.org/10.3390/genes16050491
Submission received: 2 April 2025 / Revised: 23 April 2025 / Accepted: 23 April 2025 / Published: 25 April 2025
(This article belongs to the Section Animal Genetics and Genomics)
Browse Figures
Versions Notes

Abstract

:
Background/Objectives: Schizothorax curvilabiatus, a typical highland polyploid species within the subfamily Schizothoracinae, holds economic value and ecological research significance. Currently, there are no related genomic studies. To obtain its genetic information and lay the foundation for subsequent whole-genome map construction, this study conducted a genome survey analysis, preliminary genome assembly, microsatellite identification, repeat sequence annotation, mitochondrial genome characterization, and phylogenetic relationship research. Methods: DNA was sequenced on a DNBSEQ-T7 platform to obtain paired-end genomic data. The genome was analyzed using GCE, and the draft genome was assembled with SOAPdenovo. Microsatellites were identified using MISA, and the mitochondrial genome was assembled with NOVOPlasty. Genome features were analyzed, and phylogenetic trees were constructed using PhyloSuite and MEGA. Results: The genome size was estimated at 2.53 Gb, with a heterozygosity of 6.55% and 47.66% repeat sequences. A 1.324 Gb preliminary genome draft was obtained, with repeat sequences comprising 47.17%, the majority being DNA transposons (24.64%). Dinucleotide repeats were most abundant (46.91%), followed by mononucleotide repeats (38.31%), with A/T and AC/GT being the most frequent. A complete mitochondrial genome of 16,589 bp was assembled, and a 939 bp D-loop was annotated. Phylogenetic relationships among genera in the Schizothoracinae subfamily were also clarified. Conclusions: This study provides the latest molecular data for analysis of the S. curvilabiatus genome and its related populations, and for the first time offers genomic resources for research on genomic adaptive evolution and polyploidization in high-altitude environments.

1. Introduction

Schizothorax curvilabiatus (Cypriniformes: Cyprinidae: Schizothoracinae: Schizothorax) is a Tibetan Plateau–endemic fish species primarily distributed in the middle and lower reaches of the Yarlung Tsangpo River (e.g., Motuo and Yigong Lake) and its tributaries, including the Chayu River and Danba Qu in Tibet, China. It is typically found in nearshore rapids or river forks [1]. As a highland species, S. curvilabiatus exhibits distinctive biological traits such as slow growth, relatively long lifespan, late sexual maturity, and low fecundity [1,2,3], and is particularly sensitive to overexploitation of riverine resources and environmental degradation [4]. Recognized for its dual ecological and economic significance, this species not only plays a crucial role in maintaining river ecosystem stability, but also holds potential value for aquaculture development.
The advancement of next-generation sequencing (NGS) technologies has enabled the sequencing of genomes for an increasing number of species, including animals [5], plants [6], and fungi [7]. According to data published in the NCBI (National Center for Biotechnology Information) and IGSR (International Genome Sample Resource) databases, as well as related studies, approximately 100,000 species have been sequenced to date, including over 2000 fish species encompassing model organisms and commercially important varieties. As a polyploid species endemic to high-altitude environments, S. curvilabiatus likely exhibits unique genomic traits such as duplicated gene retention, repeat element expansion, and chromosomal restructuring, which may underpin its adaptation to extreme conditions like hypoxia and temperature fluctuations. However, the impact of polyploidization on its genome organization remains unexplored, limiting our understanding of its evolutionary mechanisms and conservation strategies.
Genome survey analysis based on NGS technologies, employing K-mer analysis to estimate genome size, heterozygosity, and GC content, serves as an effective approach for elucidating the genetic characteristics of organisms. These findings provide critical data for designing subsequent library construction and high-quality genome assembly strategies, while also playing a critical role in the development of genome-wide microsatellite markers (simple sequence repeats, SSRs) [8]. Here, we conduct the first genome survey of S. curvilabiatus to characterize its polyploid genome architecture, including repeat elements, microsatellite distribution, and mitochondrial genome features, and to explore how polyploidy may shape genomic evolution within Schizothoracinae fishes.
S. curvilabiatus has been identified as a typical polyploid fish species endemic to high-altitude environments [9]. Conducting genome survey analysis on this species would provide critical insights into its genomic evolutionary characteristics and phylogenetic relationships, thereby serving as a reference for subsequent genetic studies and aquaculture applications. In recent years, due to habitat destruction, environmental pollution, and climate change, its population has declined significantly, leading to its classification as a priority aquatic species for conservation. Current research on S. curvilabiatus has been limited to preliminary descriptions of growth classification, nutritional composition, and habitat-related dietary habits [10,11,12,13,14]. Furthermore, the sole published mitochondrial genome, which lacks comprehensive analysis, dates back to an earlier period [15], leaving the S. curvilabiatus genome largely unexplored.
Simple sequence repeats (SSRs), also referred to as microsatellites or minisatellites, are hypervariable DNA motifs composed of tandem repeats of short nucleotide patterns, exhibiting mutation rates approximately 10-fold higher than single-nucleotide polymorphisms [16]. SSRs are uniformly distributed across eukaryotic genomes and have been widely applied in studies of population genetic diversity and kinship identification, as well as in genetic map construction [17,18]. In polyploid genomes, SSRs may exhibit distinct distribution patterns due to genome duplication–induced redundancy, potentially affecting functional evolution and adaptability. Investigating SSRs in S. curvilabiatus could therefore reveal polyploidy-specific genomic signatures and adaptation mechanisms. Compared to conventional SSRs development workflows, SSRs mining based on high-throughput sequencing data offers advantages of low cost, high efficiency, and enhanced throughput. Furthermore, SSRs are hypothesized to serve as critical functional elements facilitating evolutionary adaptation and rapid environmental responsiveness [19]. However, the genomic distribution and characteristics of SSRs in S. curvilabiatus remain poorly understood.
This study aimed to address critical gaps in understanding the genome of S. curvilabiatus by conducting the first genome survey to systematically characterize its polyploid genome architecture, including repeat element dynamics, microsatellite (SSR) distribution patterns, and mitochondrial genome features. We further investigate the evolutionary mechanisms through which polyploidy may underpin its adaptation to extreme high-altitude environment. By developing a comprehensive SSR marker suite, this work seeks to support future population genetic studies and conservation management strategies. Additionally, the foundational genomic data generated herein will inform both evolutionary hypotheses and practical frameworks for the development of plateau-adapted aquaculture practices.

2. Materials and Methods

2.1. Sample Collection and DNA Extraction

Muscle tissue samples were taken from S. curvilabiatus collected from the Chayu River (97°02′49.81″ E, 28°27′20.56″ N) in Zhuwagen Town, Chayu County, Tibet, China, in January 2024. Genomic DNA was extracted from the muscle tissue using a standard phenol-chloroform method [20]. DNA concentration and integrity were assessed using a Pultton DNA/Protein Analyzer (Plexech) and 1% agarose gel electrophoresis, respectively. The muscle tissue specimens were deposited in the Laboratory of Marine Germplasm Resource Exploration and Utilization, School of Marine Science and Technology, Zhejiang Ocean University, under the voucher number CY2024001.

2.2. Sequencing and Data Quality Control

Paired-end whole-genome libraries were constructed from quality-checked DNA samples using an MGIEasy FS DNA Prep Kit (BGI, Shenzhen, China) according to the standard workflow of the DNBSEQ-T7 platform (BGI), with an insert size of 350 bp. Sequencing was performed on the DNBSEQ-T7 platform following library quality verification. Raw sequencing data were processed using Fastp v 0.24.0 [21] and FastQC v0.11.5 [22] to remove adapter sequences and low-quality reads. The raw data were deposited in the Sequence Read Archive (SRA) database (http://www.ncbi.nlm.nih.gov/sra/) under the accession number SRR32816543. Sequencing services were provided by Xi’an Haorui Gene Technology Co., Ltd. Xi’an, China.

2.3. Genome Evaluation and De Novo Assembly

Sequencing data from S. curvilabiatus were analyzed using GCE v1.0.2 [23] to estimate genome size, GC content, and heterozygosity. A K-mer distribution analysis was performed by simulating K-mer frequencies across the genome under a Poisson distribution model with the recommended parameter K = 17. The depth and total count of K-mers were utilized for genome characterization. De novo genome assembly was performed using SOAPdenovo2 vr242 [24] based on the effective sequencing data, with Contig and Scaffold sizes calculated (K = 17).

2.4. Microsatellite Identification and Repeat Sequence Annotation

Following genome assembly, SSRs loci were identified from the sequences using the MISA script with default parameters (minimum repeat thresholds for mono- to hexanucleotide motifs: 10, 6, 5, 5, 5, and 5, respectively). SSRs primers were batch-designed using Primer3 v 2.6.0 software [25] and subsequently synthesized by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China. The validity of the primers and SSRs was verified through polymerase chain reaction (PCR) amplification followed by agarose gel electrophoresis analysis.
Two approaches were employed to annotate repetitive sequences in the S. curvilabiatus genome: homology prediction and de novo prediction. For homology prediction, the genome sequences were first partitioned by chromosome using the para_RepeatMasker script provided by GETA v2.5.7 (https://github.com/chenlianfu/geta). Parallelized RepeatMasker v4.1.7-p1 [26] was then executed for homology repeat annotation, with the reference species (--RM_species) specified as Cypriniformes and the Repbase database utilized as the reference. For de novo prediction, a custom repeat database was constructed using the BuildDatabase program in RepeatModeler v2.0.5 [27] and BLAST v2.15.0 [28] to generate database indices. RepeatModeler was subsequently run to identify species-specific repetitive elements. Finally, repeat annotations from both methods were merged using the merge_repeatMasker_out.pl script

2.5. Genomic Features and Phylogenetic Analysis

The complete mitochondrial genome of S. curvilabiatus was assembled using NOVOPlasty v4.3.5 [29] with paired-end sequencing data and the previously published mitochondrial genome (GenBank accession no. NC_035994) as a reference. The assembled mitochondrial genome was annotated using the MITOS2 web server [30] (http://mitos2.bioinf.uni-leipzig.de/index.py), followed by manual curation of the annotation results in BioEdit v7.2.6 [31]. A mitochondrial genome map was generated using the CGView Server [32]. Genomic features, including nucleotide composition, base bias (AT skew = [A − T]/[A + T]; GC skew = [G − C]/[G + C]), amino acid composition, and relative synonymous codon usage (RSCU), were analyzed using PhyloSuite v1.2.0 [33]. tRNA genes were predicted and visualized using tRNAscan-SE v2.0 [34]. The complete mitochondrial genome sequence was deposited in the NCBI GenBank database under accession number PV287704.
Mitochondrial genome sequences of 23 species spanning 14 genera within the subfamily Schizothoracinae were retrieved from the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) for phylogenetic analysis with the study species (Table 1). Thirteen protein-coding genes (PCGs) were aligned using MAFFT v7.520 [35], followed by trimming of ambiguous regions with Gblocks v0.91b [36]. Phylogenetic trees were reconstructed using two approaches: (a) Bayesian inference (BI), implemented in PhyloSuit v1.2.0 [33] under the JC+I+G model, with Markov Chain Monte Carlo (MCMC) parameters set to 600,000 generations; and (b) maximum likelihood (ML), conducted in MEGA11 [37] under the GTR+G+I model, with 1000 bootstrap replicates. The resulting phylogenetic trees were visually refined using the iTOL web server (https://itol.embl.de/) [38].

3. Results

3.1. Genome Sequencing, K-Mer Analysis, and De Novo Assembly

High-throughput sequencing on the DNBSEQ-T7 platform generated 117.71 Gb of raw data, with 109.48 Gb of high-quality data retained after quality control. The GC content, Q20, and Q30 values were 38.38%, 98.88%, and 96.19%, respectively (Table 2), indicating robust sequencing quality. K-mer analysis (k = 17) yielded a total of 270,100,730,872 K-mers. Based on the average coverage depth (107×), the genome size of S. curvilabiatus was estimated as approximately 2.53 Gb, with a heterozygosity of 6.55% and a repetitive sequence content of 47.66% (Figure 1), consistent with its status as a highly heterozygous polyploid species. De novo assembly produced a genome draft of 1,743,555,986 bp in contigs and 1,841,773,848 bp in scaffolds, with contig N50 and scaffold N50 values of 33,205 bp and 33,343 bp, respectively. The overall GC content of the assembled genome was 37.57% (Table 3).

3.2. Genomic SSR Distribution

A total of 1,768,499 SSRs loci were identified in the de novo assembly of the S. curvilabiatus genome. Dinucleotide repeats accounted for the highest proportion (46.91%, 829,582), followed by mononucleotide (38.31%, 677,463), trinucleotide (7.52%, 132,881), tetranucleotide (6.31%, 111,590), pentanucleotide (0.77%, 13,159), and hexanucleotide repeats (0.22%, 3824). The SSRs motifs encompassed four distinct mononucleotide types, 12 dinucleotide types, 60 trinucleotide types, 230 tetranucleotide types, 676 pentanucleotide types, and 628 hexanucleotide types. The most frequent motifs were A/T for mononucleotides, AC/GT for dinucleotides, AAT/ATT for trinucleotides, AGAT/ATCT for tetranucleotides, AATAT/ATATT for pentanucleotides, and AACCCT/AGGGTT for hexanucleotides (Figure 2).

3.3. Genomic Repeat Sequence Annotation

Repeat sequences in the S. curvilabiatus genome assembly were annotated through homology and de novo approaches, identifying 868,837,324 bp of repetitive sequences, accounting for 47.17% of the genome (Table 4). Non-interspersed repeats comprised simple repeats (3.28%), satellite sequences (1.89%), and low-complexity regions (0.45%). Among interspersed repeats, DNA transposons constituted the largest proportion (24.64%), followed by long terminal repeats (LTRs, 3.04%), long interspersed nuclear elements (LINEs, 2.47%), short interspersed nuclear elements (SINEs, 0.56%), and retroposons (0.02%).

3.4. Mitochondrial Genome Structural Features

The complete mitochondrial genome of S. curvilabiatus exhibited a typical double-stranded closed circular structure with a total length of 16,589 bp, comprising 22 tRNAs, 2 rRNAs, 13 protein-coding genes (PCGs), and one non-coding control region (D-Loop) (Figure 3). Among these 37 genes, trnQ, trnA, trnN, trnC, trnY, trnS2, nad6, trnE, and trnP were located on the light strand (L-strand), while the remaining genes resided on the heavy strand (H-strand). Gene arrangements included intergenic spacers, overlapping regions, and non-coding sequences. Ten overlapping regions (total length: 28 bp) and 16 intergenic spacers (total length: 117 bp) were identified. The longest overlaps (7 bp) occurred between atp8 and atp6 and between nad4L and nad4. The largest intergenic spacer (33 bp) was situated between trnN and trnC (Table 5). The mitochondrial genome displayed nucleotide compositions of 30.1% A, 25.4% T, 27.1% G, and 17.5% C. The overall A+T content of the 13 PCGs was 54.3%, with atp8 exhibiting the highest A+T content (60.6%). The calculated AT skew (0.085) and GC skew (−0.214) indicated a higher mutation rate in A/C bases compared to G/T bases (Table 6).
In this study, the mitochondrial genome of the S. curvilabiatus encoded a total of 3794 amino acids. Among them, trnLeu1 (500, 13.13%) and trnAla (341, 8.98%) exhibited higher usage frequencies, while trnCys (25, 0.65%) and trnSer1 (51, 1.34%) had lower frequencies. Additionally, the usage of synonymous codons showed a distinct bias for most amino acids. A total of 30 codons exhibited a relative synonymous codon usage (RSCU) value ≥1, with the codons CUA (trnLeu1), CCA (trnPro), and CGA (trnArg) having RSCU values greater than 2 (Figure 4). In terms of secondary structure, all 21 tRNAs of S. curvilabiatus, except for trnSer1, were able to fold into a typical cloverleaf structure. Besides the normal base pairings, base mismatches between G and U were also observed (Figure 5).

3.5. Phylogenetic Relationships of S. curvilabiatus

Phylogenetic trees were constructed for 14 genera within Schizothoracinae and Schizopygopsinae using both BI and ML methods. Both trees exhibited consistent topological structures (Figure 6). Within Schizothoracinae, the genera Schizothorax, Oreinus, Percocypris, Schizopyge, and Aspiorhynchus formed a monophyletic group, while the remaining genera, including Schizopygopsis, Ptychobarbus, Gymnocypris, and Gymnodiptychus, clustered into another branch. Additionally, Schizothorax and Oreinus, as well as Gymnodiptychus and Diptychus, formed smaller subgroups. Within Schizothorax, the relationship between S. curvilabiatus and S. oconnori was the closest. The most closely related genera between the two subfamilies were Heerzensteinia and Chuanchia.

4. Discussion

The emergence of sequencing technologies led to completion of the first fish genome sequence (Takifugu rubripes) in 2002, marking the beginning of a new era in fish genomics research [39]. Currently, more than 31,000 species of fish inhabit the Earth, including teleosts, jawless fish, and cartilaginous fish, with several species holding significant ecological and economic value. With the launch of initiatives such as the “Chinese Aquatic 10-100-1000 Genomics Program” and the “Fish 10K Project” [40,41], the number of fish genomes available has increased significantly. Notable species include model organisms like Danio rerio [42] and Oryzias sinensis [43], economically important species such as Cyprinus carpio [44] and Ctenopharyngodon idella [45], and ecologically valuable species like the cave fish Astyanax mexicanus [46] and the high-altitude fish S. younghusbandi [47]. However, as of now, the fish genomes published in the NCBI database (https://www.ncbi.nlm.nih.gov/datasets/genome/, assessed on 24 March 2025) account for less than 10% of the global fish species (2832 species).
In this study, a new genome (S. curvilabiatus) was sequenced. S. curvilabiatus, a flagship polyploid species of the Schizothoracinae subfamily and the Schizothorax genus, is an important economic species in Tibet, China and is regarded as an ideal model for understanding the “evolution-geology synergistic mechanism” (ascent with modification) [48,49]. However, only 3 out of the 45 species in the Schizothorax genus have had their genomes sequenced to date. Based on K-mer analysis, we estimate that the genome size of S. curvilabiatus is approximately 2.53 Gb, with the preliminary assembly resulting in a genome size of about 1.84 Gb (Table 3). Compared to previously published genomes of Schizothorax species, the draft genome of S. curvilabiatus is smaller than that of S. oconnori (~2.07 Gb) [50] but larger than that of S. macropogon (~1.56 Gb) [51] and S. lantsangensis (972.4 Mb). It is similar in size to that of O. stewartii (~1.85 Gb) [52]. We hypothesize that the variation in genome size among Schizothorax species may be due to the accumulation of repetitive elements during species genome evolution and the limitations of short-read data from next-generation sequencing. In future studies, long-read sequencing technologies, such as Single Molecule Real-Time (PacBio SMRT), combined with Hi-C (High-throughput chromosome conformation capture) technology, can be used to construct high-quality genome maps. The observed genome size variation (1.56–2.53 Gb) among Schizothorax species may also reflect polyploidy-driven genomic dynamics. Polyploidization events (e.g., whole-genome duplication) typically induce genome expansion through the retention of duplicated chromosomal regions and repetitive element proliferation. For instance, the larger genome size of S. curvilabiatus compared to S. lantsangensis (972.4 Mb) could have resulted from differential diploidization rates post-polyploidization, where some species rapidly purge redundant sequences, while others retain them for adaptive potential [53]. Similar patterns have been documented in tetraploid carp (C. carpio), where genome duplication facilitates environmental adaptation by preserving stress-responsive gene duplicates [54]. The genome survey analysis conducted in this study provides preliminary insights into the genomic characteristics of S. curvilabiatus and lays the foundation for further whole-genome sequencing and assembly, significantly enriching the genomic resources of the Schizothorax genus.
SSRs are widely distributed in both prokaryotic and eukaryotic genomes and are considered efficient molecular markers for genetic differentiation and evolutionary studies due to their high efficiency and polymorphism [18,19]. In this study, 1,768,499 loci were identified in the S. curvilabiatus genome, with dinucleotides being the most abundant repeat sequence (46.91%). This finding is consistent with results from studies on S. waltoni [55], S. biddulphi [56,57], S. prenanti [58], Gymnocypris eckloni, and G. przewalskii ganzihonensis [59]. Additionally, the distribution patterns of nucleotide base repeats also align with these findings, with mononucleotide repeats predominantly consisting of A/T, dinucleotide repeats primarily composed of AC/GT, and trinucleotide repeats largely consisting of AAT/ATT. The complexity of repeat sequences often reflects the DNA mutation rate and evolutionary level of a species [60,61]. The distribution frequency from mononucleotides to trinucleotides was as high as 92.74%, indicating that S. curvilabiatus has accumulated significant genetic variation during its evolutionary process. The predominance of dinucleotide SSRs (46.91%) in S. curvilabiatus may reflect polyploidy-mediated genomic redundancy. In duplicated genomes, simpler repeat motifs (e.g., AC) are less likely to disrupt essential coding regions compared to complex trinucleotide repeats, allowing their accumulation under relaxed purifying selection. This pattern aligns with observations in other polyploid fish, such as the autotetraploid loach (Misgurnus anguillicaudatus), where dinucleotide SSRs dominate due to their tolerance to mutation in redundant genomic regions [62]. Furthermore, the high SSR polymorphism (1,768,499 loci) could enhance adaptive plasticity, a critical trait for survival in the fluctuating environments of the Tibetan Plateau. Based on these SSRs, this study provides valuable resources for future population genetics research.
Repetitive elements, particularly transposons, are critical components of eukaryotic genomes and serve as a source of genetic variation [63,64]. In the assembled genome draft of S. curvilabiatus, a total of 47.17% (868.84 Mb) of the sequence was identified as repetitive, with DNA transposons being the most prevalent type of repeat sequence (453.96 Mb, 24.64%) (Table 4). DNA transposons have also been identified as the dominant type of transposable elements (TEs) in other plateau species, such as S. oconnori [50], Glyptosternon maculatum [65], Triplophysa dalaica [66], G. przewalskii [67], and Hucho bleekeri [68], while non-LTRs, LINEs, and SINEs were found to be less prominent. The high proportion of DNA transposons (24.64%) in S. curvilabiatus may be a direct consequence of polyploidization. Genome duplication events provide “safe havens” for transposon activity, as redundant genomic regions buffer against deleterious insertions. In high-altitude Schizothorax species, transposon-mediated genomic plasticity could facilitate rapid adaptation to hypoxia and temperature extremes by rewiring stress-responsive gene networks [69]. This suggests that DNA transposons play a significant role in the genomic adaptation and evolutionary processes of plateau species.
Mitochondria, as essential energy factories within cells, are primarily responsible for generating most of the energy required for cellular biochemical reactions [70]. Studies have shown that the genetic material carried by mitochondria (mtDNA) plays multiple crucial roles throughout an organism’s lifecycle, involving vital biological processes such as regulation of lifespan, maintenance of reproductive capacity, response to metabolic stress, adaptation to environmental changes, and thermoregulation [71,72]. Additionally, mitochondrial DNA fragments have been proven to be effective molecular markers for phylogenetic and population genetic analyses [73]. In this study, a complete mitochondrial genome of S. curvilabiatus was assembled using nuclear genome sequences, with a total length of 16,589 bp. This length is consistent with that of other species within the Schizothorax genus, while showing slight variation when compared to other species in the Schizothoracinae subfamily (Table 1). Upon comparison, the complete mitochondrial genome assembled in this study is slightly longer than previously reported (16,589 bp vs. 16,578 bp) [15]. The overall nucleotide composition of A, T, G, C bases and the relatively high AT content are consistent with previous findings (Table 6), aligning with the typical base composition characteristics of most teleosts, reflecting a strong mutation tendency, functional regulation, and evolutionary adaptability [74]. Additionally, other structural features of the mitochondrial genome of S. curvilabiatus, such as synonymous codon usage frequency and tRNA secondary structures, are consistent with those of other species within the Schizothorax genus [75,76,77,78,79]. The D-loop region, known as the “control center” of the mitochondrial genome, directly influences the cell’s energy supply, genetic stability, and environmental response by regulating replication, transcription, mutation accumulation, and metabolic adaptation. Its high variability and functional importance make it a key target for mitochondrial biology, medicine, and evolutionary research [80,81]. In our study, a D-loop region of 939 bp was successfully annotated, which has not been previously reported in prior studies. Based on 13 protein-coding genes (PCGs), we provide a more systematic and comprehensive analysis of the phylogenetic relationships among species in the Schizothoracinae subfamily (Figure 6). Clear clustering differentiation was observed among genera, with S. curvilabiatus showing the closest phylogenetic relationship to S. oconnori, consistent with previous studies [15,82,83,84]. Studies have shown that phylogenetic relationships inferred from mitochondrial data may conflict with nuclear genomic divergence patterns [85]. Such incongruence could arise from hybridization events during polyploid speciation, where mitochondrial genomes (maternally inherited) and nuclear genomes (recombined from parental lineages) capture distinct evolutionary histories [86]. Similar mitochondrial–nuclear discordance is widespread in polyploid Cyprinidae species, such as the Carassius auratus complex, where hybrid origins and genome duplication generate conflicting phylogenetic signals [87]. Currently, the phylogenetic relationships of S. curvilabiatus and other Schizothorax species based on nuclear genomic data remain unclear, and whether such conflicts exist within this genus is unknown. Future studies should integrate nuclear gene trees with cytogenetic analyses (e.g., fluorescence in situ hybridization) to resolve the polyploidization history of the Schizothorax genus.

5. Conclusions

In summary, a genome survey analysis of the tetraploid species S. curvilabiatus from the Tibetan Plateau revealed a complex genome with relatively large genome size, high heterozygosity, and abundant repetitive sequences. A de novo genome draft was assembled, and its size is consistent with that of other species within the Schizothorax genus. Repetitive sequence annotation of the assembled genome revealed that TEs constitute a significant proportion of the genome, which may contribute to the genomic evolution of S. curvilabiatus, enabling it to better adapt to the plateau environment. Additionally, a large number of SSRs were identified, providing foundational information for the development of molecular markers. Furthermore, a complete mitochondrial genome was assembled from the sequencing data. A comprehensive analysis of its gene structure and codon usage was conducted, and the mitochondrial gene sequences obtained in this study were compared with the previously published mitochondrial genome of S. curvilabiatus. The results provide updated reference data for the mitochondrial genome analysis of S. curvilabiatus and its related population genetics studies. The phylogenetic tree based on the mitochondrial genome further clarifies the evolutionary positions of the genera within the Schizothoracinae subfamily, with a clearer understanding of the phylogenetic relationship of S. curvilabiatus. These findings provide genomic resources for the adaptive evolution and genome polyploidization of S. curvilabiatus in high-altitude environments and lay the foundation for constructing a high-quality genome map of S. curvilabiatus in future studies.

Author Contributions

Data curation, L.G., Y.L., and T.F.; formal analysis, K.H. and T.F.; funding acquisition, C.Z.; investigation, K.H. and H.L.; methodology, B.L., L.G., Y.L., K.H., M.H., and C.Z.; resources, K.H. and H.L.; software, T.F.; supervision, B.L. and H.L.; validation, Y.L., H.L., and M.H.; visualization, M.H.; writing—original draft, L.G.; writing—review & editing, L.G. and C.Z. All authors will be updated at each stage of manuscript processing, including submission, revision, and revision reminder, via emails from our system or the assigned Assistant Editor. All authors have read and agreed to the published version of the manuscript.

Funding

The study was supported by the Key Science and Technology Program of Xizang Autonomous Region (No. XZ202301ZY0012N) and the National Key R&D Program of China (grant no. 2024YFD12007000).

Institutional Review Board Statement

All animal study protocols were approved by the Laboratory of Marine Germplasm Resource Exploration and Utilization, School of Marine Science and Technology, Zhejiang Ocean University (CY2024001), 15 January 2024.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are freely available in SRA and GenBank of NCBI (https://www.ncbi.nlm.nih.gov/). The raw data accession number is SRR32816543. The complete mitochondrial genome sequence accession number is PV287704.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Depth profile of K-mer (17-mer) distribution in the S. curvilabiatus genome. The first peak (frequency=9) is usually in the region of lower frequency caused by sequencing errors at low frequencies K-mer, while the main peak (frequency = 107 in the higher frequency region) usually corresponds to the true genome sequence. It can be expressed as the average depth of coverage of sequencing.
Figure 1. Depth profile of K-mer (17-mer) distribution in the S. curvilabiatus genome. The first peak (frequency=9) is usually in the region of lower frequency caused by sequencing errors at low frequencies K-mer, while the main peak (frequency = 107 in the higher frequency region) usually corresponds to the true genome sequence. It can be expressed as the average depth of coverage of sequencing.
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Figure 2. Frequency distribution of microsatellite loci in S. curvilabiatus. (a) Mononucleotides; (b) dinucleotides; (c) trinucleotides; (d) tetranucleotides; (e) pentanucleotides; (f) hexanucleotides.
Figure 2. Frequency distribution of microsatellite loci in S. curvilabiatus. (a) Mononucleotides; (b) dinucleotides; (c) trinucleotides; (d) tetranucleotides; (e) pentanucleotides; (f) hexanucleotides.
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Figure 3. Mitochondrial genome structure of S. curvilabiatus.
Figure 3. Mitochondrial genome structure of S. curvilabiatus.
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Figure 4. Amino acid composition and RSCU usage in the mitochondrial genome of S. curvilabiatus.
Figure 4. Amino acid composition and RSCU usage in the mitochondrial genome of S. curvilabiatus.
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Figure 5. Secondary structure of tRNAs in the mitochondrial genome of S. curvilabiatus.
Figure 5. Secondary structure of tRNAs in the mitochondrial genome of S. curvilabiatus.
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Figure 6. Phylogenetic tree of S. curvilabiatus based on 13 PCGs. Note: (a) BI; (b) ML.
Figure 6. Phylogenetic tree of S. curvilabiatus based on 13 PCGs. Note: (a) BI; (b) ML.
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Table 1. List of species and corresponding mitochondrial genomes with references.
Table 1. List of species and corresponding mitochondrial genomes with references.
SubfamilyGenusSpeciesAccession No.Length (bp)
SchizothoracinaeSchizothoraxSchizothorax curvilabiatusPV28770416,589
Schizothorax oconnoriNC_020781.116,590
Schizothorax waltoniNC_020606.116,589
OreinusOreinus myzostomusMW786773.116,578
Oreinus dulongensisNC_058201.116,579
PercocyprisPercocypris pingiNC_018601.116,586
SchizopygeSchizopyge nigerNC_022866.116,585
AspiorhynchusAspiorhynchus laticepsNC_022855.116,591
SchizopygopsinaeSchizopygopsisSchizopygopsis anteroventrisNC_029190.116,620
Schizopygopsis malacanthusNC_024880.116,677
Schizopygopsis kessleriNC_045935.116,767
PtychobarbusPtychobarbus dipogonNC_024537.116,787
Ptychobarbus kaznakoviNC_025303.116,842
Ptychobarbus chungtienensisNC_034230.116,970
GymnocyprisGymnocypris namensisNC_021420.116,674
Gymnocypris scleracanthusNC_036349.116,679
Gymnocypris dobulaNC_021419.116,720
GymnodiptychusGymnodiptychus pachycheilusNC_023793.116,586
Gymnodiptychus dybowskiiNC_028544.116,677
DiptychusDiptychus maculatusNC_025650.116,859
OxygymnocyprisOxygymnocypris stewartiiNC_022718.116,646
PlatypharodonPlatypharodon extremusNC_029171.116,668
HeerzensteiniaHerzensteinia microcephalusNC_033403.116,726
ChuanchiaChuanchia labiosaNC_029181.116,705
Table 2. Sequencing data statistics of Schizothorax curvilabiatus.
Table 2. Sequencing data statistics of Schizothorax curvilabiatus.
Data TypeTotal Bases(G)GC (%)Q20 (%)Q30 (%)
Raw data117.7138.3798.9196.28
Clean data109.4838.3898.8896.19
Table 3. De novo assembly of the S. curvilabiatus genome.
Table 3. De novo assembly of the S. curvilabiatus genome.
K = 17NumberTotal Size (bp)GC (%)
Genome//37.57
Scaffold N50/33,343/
Contig N50/33,205/
Scaffolds7,272,5781,841,773,848/
Contigs8,853,8631,743,555,986/
Longest size163,798/
Sequences ≥ 1 kbp213,379384,572,145/
Sequences ≥ 2 kbp49,706166,415,365/
Sequences ≥ 3 kbp20,55696,556,207/
Table 4. Landscape of repetitive elements in the de novo assembly of the S. curvilabiatus genome.
Table 4. Landscape of repetitive elements in the de novo assembly of the S. curvilabiatus genome.
Repeat NumberRepeat Size (bp)Percentage (%)
Total1,194,665868,837,32447.17%
Non-interspersed
Repeats		Simple_repeat230,35370,680,1923.83%
Satellite20,12225,517,5461.38%
Low_complexity30,47310,538,4990.57%
Interspersed RepeatsLTRs24,20356,086,4123.04%
LINEs42,39445,536,8232.47%
SINEs17,39010,472,0530.56%
Retroposon814485,0540.02%
DNA transposons437,981453,962,39624.64%
Unknown360,488139,173,0427.56%
Rolling-circles62846,379,7370.35%
rRNA569441,0390.02%
tRNA16,0543,400,6640.18%
snRNA4153899,6680.05%
scRNA3088634,7960.03%
Table 5. Features of the mitochondrial genome of S. curvilabiatus. H and L represent the heavy chain and light chains, respectively; T-- represents incomplete stop codons; – represents base overlap.
Table 5. Features of the mitochondrial genome of S. curvilabiatus. H and L represent the heavy chain and light chains, respectively; T-- represents incomplete stop codons; – represents base overlap.
GenePositionLength (bp)CodonIntergenic RegionStrand
FromToStartStop
trnF16969 0H
rrnS701023954 0H
trnV1026109772 2H
rrnL112027491630 22H
trnL22775285076 25H
nad128523826975ATGTAA1H
trnI3831390272 4H
trnQ3901397171 –2L
trnM3974404269 2H
nad2404350891047ATGTAG0H
trnW5088515871 –2H
trnA5161522969 2L
trnN5231530373 1L
trnC5337540367 33L
trnY5403547371 –1L
cox1547570251551GTGTAA1H
trnS27026709671 0L
trnD7099717072 2H
cox271847874691ATGT--13H
trnK7875795076 0H
atp879528116165ATGTAG1H
atp681108793684ATGTAA–7H
cox387939578786ATGTAA–1H
trnG9578964972 –1H
nad3965010,000351ATGTAG0H
trnR999910,06870 –2H
nad4L10,06910,365297ATGTAA0H
nad410,35911,7391381ATGT--–7H
trnH11,74011,80869 0H
trnS111,80911,87668 0H
trnL111,87811,95073 1H
nad511,95413,7771824ATGTAA3H
nad613,77414,295522ATGTAA–4L
trnE14,29614,36469 0L
cytb14,36915,5091141ATGT--4H
trnT15,51015,58172 0H
trnP15,58115,65070 –1L
D-loop15,65116,589939 0H
Table 6. Nucleotide composition and skewness of S. curvilabiatus.
Table 6. Nucleotide composition and skewness of S. curvilabiatus.
RegionSize (bp)T(U) (%)C (%)A (%)G (%)AT (%)GC (%)GT (%)ATskew (%)GCskew (%)
PCGs11,41232.822.221.623.654.345.856.3−0.2100.026
atp668427.827.330.314.658.141.942.40.043−0.303
atp816527.326.733.312.760.639.440.00.1.00−0.354
cox1155129.425.826.118.755.544.548.1−0.059−0.159
cox269128.224.930.416.558.641.444.70.037−0.203
cox378626.728.127.617.654.345.744.30.016−0.231
cytb114128.727.926.317.255.045.145.9−0.043−0.237
nad197525.130.825.518.650.649.443.70.008−0.247
nad2104722.731.428.916.951.648.339.60.120−0.300
nad335127.629.129.613.757.242.841.30.035−0.360
nad4138126.128.728.816.454.945.142.50.050−0.274
nad4L29726.931.025.616.552.547.543.4−0.026−0.305
nad5182425.129.530.614.855.744.339.90.100−0.332
nad652238.915.914.830.553.746.469.4−0.4500.314
rrnL163019.723.436.520.456.243.840.10.299−0.067
rrnS95420.825.930.822.551.648.443.30.195−0.069
rRNAs258420.124.334.421.254.545.541.30.263−0.068
tRNAs156227.620.627.524.455.144.951.9−0.0030.084
Full genome16,58925.427.130.117.555.544.642.90.085−0.214
D-Loop93928.823.430.617.259.440.646.00.030−0.152
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Liu, B.; Gao, L.; Liu, Y.; He, K.; Li, H.; Feng, T.; Han, M.; Zhang, C. The First Genome-Wide Survey Analysis of the Tibetan Plateau Tetraploid Schizothorax curvilabiatus Reveals Its Microsatellite Characteristics and Phylogenetic Relationships. Genes 2025, 16, 491. https://doi.org/10.3390/genes16050491

AMA Style

Liu B, Gao L, Liu Y, He K, Li H, Feng T, Han M, Zhang C. The First Genome-Wide Survey Analysis of the Tibetan Plateau Tetraploid Schizothorax curvilabiatus Reveals Its Microsatellite Characteristics and Phylogenetic Relationships. Genes. 2025; 16(5):491. https://doi.org/10.3390/genes16050491

Chicago/Turabian Style

Liu, Bingjian, Luxiu Gao, Yifan Liu, Kai He, Hongchi Li, Taobo Feng, Mingzhe Han, and Chi Zhang. 2025. "The First Genome-Wide Survey Analysis of the Tibetan Plateau Tetraploid Schizothorax curvilabiatus Reveals Its Microsatellite Characteristics and Phylogenetic Relationships" Genes 16, no. 5: 491. https://doi.org/10.3390/genes16050491

APA Style

Liu, B., Gao, L., Liu, Y., He, K., Li, H., Feng, T., Han, M., & Zhang, C. (2025). The First Genome-Wide Survey Analysis of the Tibetan Plateau Tetraploid Schizothorax curvilabiatus Reveals Its Microsatellite Characteristics and Phylogenetic Relationships. Genes, 16(5), 491. https://doi.org/10.3390/genes16050491

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