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Article

Telomere-to-Telomere Gap-Free Genome Assembly and Comparative Analysis of the Opsariichthys bidens (Cypriniformes: Xenocyprididae)

by
Xinyue Wang
1,2,†,
Qi Liu
3,†,
Denghua Yin
2,
Pan Wang
2,
Min Jiang
2,
Jie Liu
2,
Ning Sun
1,
Yunzhi Yan
1,* and
Kai Liu
1,2,*
1
School of Ecology and Environment, Anhui Normal University, Wuhu 241000, China
2
Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture and Rural Affairs, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China
3
College of Fisheries, Southwest University, Chongqing 400715, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to the research.
Biology 2025, 14(11), 1544; https://doi.org/10.3390/biology14111544
Submission received: 10 October 2025 / Revised: 29 October 2025 / Accepted: 31 October 2025 / Published: 3 November 2025
(This article belongs to the Special Issue Global Fisheries Resources, Fisheries, and Carbon-Sink Fisheries)
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Simple Summary

Opsariichthys bidens is a widely distributed freshwater fish uniquely adapted to the challenging conditions of stream environments, which are typically characterized by high dissolved oxygen and sustained high-flow velocities. To thrive in such habitats, stream fishes often evolve enhanced capabilities in oxygen utilization, energy metabolism, and sustained swimming performance. In this study, we constructed the telomere-to-telomere (T2T) reference genome for O. bidens. Genomic analyses identified several expanded gene families and pathways associated with these adaptive traits. This high-quality genome assembly provides a foundational resource for further research into the genetic mechanisms underlying ecological adaptation in stream-dwelling fishes.

Abstract

Stream-dwelling fishes face diverse hydrological pressures, making the broadly distributed Opsariichthys bidens an ideal model for analyzing adaptive evolution. To elucidate its adaptation to a high-dissolved-oxygen and high-flow-velocity stream environment, a high-quality genome with comprehensive annotation is essential. In this study, we present the first telomere-to-telomere (T2T) reference genome for O. bidens, constructed using PacBio HiFi, Oxford Nanopore Ultra-long, and Hi-C technologies. The assembled genome spans 841.96 Mb, comprising 38 chromosomes, each in a single contig (contig N50 = 22.42 Mb, 2.5-fold higher than the previous version), achieving a gap-free standard with 99.34% BUSCO completeness. Additionally, 38 centromeric sequences, 37 double-telomeric sequences, and 1 single-telomeric sequence were successfully identified, providing essential molecular markers. Phylogenetic analysis revealed a divergence time of 13.5 million years between O. bidens and its closely related species Z. platypus, with collinearity analysis confirming their high genomic conservation. Gene family analysis revealed 350 expanded families enriched in pathways associated with adaptation to high-dissolved-oxygen environments (e.g., antioxidant defense, oxidative phosphorylation, mitochondrial electron transport chain) and high-flow-velocity environments (e.g., exercise endurance, myocardial contraction, actin binding). Positive selection analysis further identified multiple pathways and key genes involved in mitochondrial optimization, oxygen utilization, and metabolic regulation. The T2T assembly greatly improves assembly continuity and enabling precise identification of centromeres and telomeres for O. bidens. These results provide a robust foundation for studying its adaptive evolution to stream environment.

1. Introduction

Opsariichthys bidens belongs to the order Cypriniformes, family Xenocyprididae, and genus Opsariichthys [1,2]. This species exhibits a broad geographic distribution across China, ranging from Hainan Island in the south to Heilongjiang in the north, the Sichuan Basin in the west, and the eastern coastal regions. O. bidens is a typical stream-dwelling fish, preferring fast-flowing, oxygen-rich clear streams. Due to its wide ecological adaptability, high trophic level, and early phylogenetic position, O. bidens represents a key species for investigating the structure and function of stream ecosystems [3].
The stream environment is characterized by typical hydrological dynamics, including high flow velocity, elevated dissolved oxygen levels, and seasonal fluctuations in water volume [4]. This complex environment presents significant challenges for the ecological adaptation of stream-dwelling fish. In oxygen-rich waters, these fish must not only efficiently utilize oxygen to enhance their metabolic capacity but also maintain redox homeostasis to prevent oxidative damage caused by high oxygen concentrations [5]. Additionally, high flow velocity requires the ability to swim continuously to resist the impacts of water flow and to sustain long-term exercise endurance [6]. Biological evidence has highlighted the morphological and physiological adaptations of fish to such specialized environments, including streamlined body shapes, well-developed red muscle tissues, and specialized fin structures [7,8]. However, the molecular mechanisms underlying the environmental adaptation of stream-dwelling fish remains limited. This gap primarily includes a lack of key genes involved in phenotypic adaptation, an incomplete exploration of genomic characteristics under environmental selection pressures, and insufficient information on the molecular pathways that facilitate adaptation to high-flow velocity and high-oxygen environments. These limitations have prevented a comprehensive and in-depth understanding of the environmental adaptation mechanisms in stream fishes.
Current research on O. bidens primarily focuses on its biology and ecology. Studies have confirmed that O. bidens exhibits significant adaptive responses to environmental changes, particularly in terms of body characteristics, life history strategies, and nutritional ecology [9,10,11]. At the molecular systematics level, existing studies primarily rely on mitochondrial sequences and limited nuclear gene markers. These studies have provided initial insights into the phylogenetic relationships [12], genetic diversity characteristics [13], and population historical dynamics [14]. However, due to the use of short fragment sequences, these studies face limitations in fully revealing the genomic structural characteristics and their associated environmental adaptation mechanisms. Although genomic studies have achieved chromosome-level assembly [15,16] and have begun to explore the genetic basis of sexual dimorphism [17], the relationship between karyotype polymorphism and Robertsonian translocation [18], there remains a significant gap in research of the molecular mechanisms underlying environmental adaptability of O. bidens. Notably, the existing genome assemblies still contain scaffold gaps and have not yet reached the complete T2T standard, particularly lacking precise annotations of key genomic structures such as telomeres and centromeres. Therefore, constructing a complete T2T-level reference genome will provide a crucial foundation for advancing adaptive evolution research at the whole-genome level.
In summary, O. bidens, as a representative stream-dwelling species, holds significant reference value for studies on environmental adaptability. However, the genetic basis of how this species adapts to stream environments remains unclear. To address this, the present study integrates PacBio HiFi, Nanopore ultra-long reads, and Hi-C technologies to comprehensively analyze the T2T-level genomic structural features of O. bidens, especially the complete information of telomeres and centromeres. Based on a comprehensive comparative genomics analysis, this study aims to systematically elucidate the genetic adaptations of O. bidens to high-dissolved-oxygen and high-flow-velocity stream environments. The results will provide essential resources and a theoretical basis for research on the adaptive evolution of stream fish and ecological genomics.

2. Materials and Methods

2.1. Sample Collection

In May 2023, a male specimen of O. bidens (126.42 mm in length, 32.5 g in weight, Figure 1B) was collected from the Jingde segment of the Huishui River in Anhui Province, China (30.274341° N, 118.530638° E, Figure 1A). The species in good condition was collected as part of the Monitoring of Aquatic Resources in Key Waters of Anhui Province Project (ZF2022-18-0399), under a special fishing permit ([2023]002) issued by the Department of Agriculture and Rural Affairs of Anhui Province. The specimen in good condition was randomly selected and anesthetized with MS-222. Under aseptic conditions, tissue samples-including muscle, liver, eyes, brain, spleen, gonads, gills, heart, and kidney-were dissected and immediately snap-frozen in liquid nitrogen. In subsequent procedures, DNA extraction was specifically performed on muscle and liver tissues to facilitate whole-genome assembly, while RNA was extracted from nine tissue samples for genome annotation.

2.2. DNA Extraction, Library Construction and Sequencing

Genomic DNA was first extracted for all samples. For MGI short-read and PacBio sequencing, genomic DNA was extracted using the CTAB method after grinding the tissue in liquid nitrogen. DNA quality was assessed by 0.75% agarose gel electrophoresis, NanoDrop spectrophotometry (Thermo Fisher Scientific, Waltham, MA, USA), and Qubit fluorometry (Thermo Fisher Scientific, Waltham, MA, USA). For Oxford Nanopore sequencing (ONT), high-molecular-weight DNA was extracted using the SDS method. DNA concentration and purity were measured by NanoDrop and Qubit, while DNA integrity was evaluated by pulsed-field gel electrophoresis. For Hi-C library preparation, tissue samples were crosslinked with 2% formaldehyde to preserve native chromatin conformation.
Library construction and sequencing were then performed. MGI short-read libraries were constructed using the MGIEasy Universal DNA Library Prep Kit V1.0 (CAT#1000005250, MGI) (MGI Tech Co. Ltd., Shenzhen, China) and sequenced on the DNBSEQ-T7 platform with 2 × 150 bp reads. Raw reads were filtered using fastp (v0.23.2) [19] with default parameters to remove low-quality reads, short reads, adapter sequences, and duplicates, retaining only paired-end reads longer than 50 bp. PacBio High Fidelity (HiFi) libraries were constructed with the SMRTbell® prep kit 3.0 (PacBio) (Pacific Biosciences of California, Inc., Menlo Park, CA, USA) and sequenced on the Revio platform in Circular Consensus Sequencing (CCS) mode. CCS reads were generated using CCS v6.0.0 with default parameters to yield high-accuracy long reads. ONT ultra-long libraries were prepared using the SQK-LSK110 kit (Oxford Nanopore Technologies) (Oxford Nanopore Technologies plc, Oxford, UK) and sequenced on the Oxford Nanopore PromethION platform. High-throughput chromosome conformation capture (Hi-C) libraries were prepared using a combination of Biotin-14-dCTP (Invitrogen) (Thermo Fisher Scientific, Waltham, MA, USA), T4 DNA polymerase (NEB), and streptavidin magnetic beads (Invitrogen), and sequenced on the DNBSEQ-T7 platform with 2 × 150 bp reads.

2.3. Genome Assembly and Evaluation

Subsequently, genome assembly was carried out. Initial contigs were assembled from HiFi reads using Hifiasm (v0.19.6) (https://github.com/chhylp123/hifiasm, accessed on 23 July 2024) with the default purge_haplotigs module [20]. A non-redundant contig interaction matrix was generated using the HiCUP pipeline (v0.7.2) [21], followed by chromosome anchoring with the 3D-DNA workflow (180,922) [22]. Manual corrections of misassemblies such as inversions and translocations were performed using Juicebox Assembly Tools (1.11.08) [23]. TGS-GapCloser (v1.2.0) [24] was employed to fill the gaps between contigs by leveraging the coverage relationship between Oxford Nanopore ultra-long reads and pre-assembled contigs, thereby extending the contigs. Pilon (1.23) [25] was utilized to correct the extended and gap-filled genome using short-read sequencing data, resulting in a high-quality T2T-level genome assembly. The depths were calculated as the total data output divided by the genome size. Centromere regions were identified based on the distribution of tandem repeats using the multi-model prediction module in QuarTeT (1.2.1) [26].
Finally, the quality of the O. bidens genome assembly was evaluated in terms of accuracy, consistency, and completeness. To verify the correspondence between the assembly results and the target species, the genome was segmented into 10 kb fragments and aligned against the NCBI Nucleotide Database (NT database). The short-read data, PacBio long-read data, and ONT ultra-long-read data were mapped to the genome using bwa (0.7.12-r1039) [27] and minimap2 (2.24-r1122) [28] software, with sequence consistency evaluated based on alignment rate and coverage. The k-mer based consistency quality value was assessed using mercury (v1.3) [29]. The completeness of the genome assembly was evaluated using BUSCO (v5.7.1) [30] and Compleasm (v0.2.6) [31]. Both of them are based on the highly conserved OrthoDB database, which was constructed by sampling hundreds of genomes and selecting single-copy orthologous genes with a conservation rate exceeding 90% across six major phylogenetic branches.

2.4. Gene Prediction and Annotation

The annotation of repetitive sequences was conducted using a combined approach of homology-based prediction and de novo prediction. The former involved using RepeatMasker (v4.0.9) [32] and RepeatProteinMask (v4.1.0) (http://www.repeatmasker.org, accessed on 10 August 2024) to query the Repbase database [33] with the genomic sequence, followed by integration with the de novo transposable element (TE) library. The latter utilized LTR_FINDER_parallel (v1.0.7) [34] and RepeatModeler (v1.0.11) [35] to construct the repetitive sequence library.
Protein-coding gene annotation was performed using a comprehensive approach integrating ab initio prediction, homology comparison, and RNA-Seq-assisted annotation. Ab initio predictions were performed using Augustus (v3.3.2) [36] and Genscan [37], with species-specific models established based on the organism’s taxonomic classification. Homology-based predictions were executed using protein sequences from closely related species: Danio rerio (GenBank Assembly: GCF_000002035.6), Hypophthalmichthys molitrix (GenBank Assembly: 12618884), Opsariichthys bidens (GenBank Assembly: GWHBJYU00000000), and Zacco platypus (GenBank Assembly: GCA_034642465.1). Following TBlastN filtering (E-value ≤ 1 × 10−5), gene structures were refined using miniprot (v0.11-r234) [38] and liftoff (v1.6) [39]. RNA-Seq-assisted annotation was performed through transcriptome data integration from various tissues, with sequence alignment conducted using HISAT2 (v2.1.0) [40] and assembly performed with StringTie (v1.3.5) [41]. Finally, all gene models were consolidated and redundant entries were eliminated using MAKER2 (v2.31.10) [42] with default parameters and HiFAP (https://www.onemore-tech.com/, accessed on 30 August 2024).
Functional annotation of proteins was carried out based on sequence similarity and domain architecture. Gene sequences were aligned against multiple databases including NCBI non-redundant protein database (NR) [43], Swiss-Prot (http://www.gpmaw.com/html/swiss-prot.html, accessed on 17 September 2024), TrEMBL (http://www.uniprot.org, accessed on 17 September 2024), Eukaryotic Orthologous Groups (KOG) (https://ftp.ncbi.nih.gov/pub/COG/KOG/, accessed on 17 September 2024), and Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/, accessed on 17 September 2024) using DIAMOND (v2.0.14) [44], with an E-value threshold of 1 × 10−5. Protein domain analysis was performed using InterProScan (v5.61-93.0) [45] and hmmscan3 (3.3.1) [46].
The identification of rRNA, miRNA, and snRNA were performed using INFERNAL based on Rfam (v14.8) [47] and miRBase (http://www.mirbase.org/, accessed on 6 November 2024) [48], while tRNA was annotated using tRNAscan-SE (v1.3.1) [49].

2.5. Genome Comparison and Identification of Newly Assembled Genes

The self-assembled genome of O. bidens was subjected to synteny analysis against the published genome (GCA_037194315.1) with a consistent karyotype (2n = 76) using the Syri (v0.1) [50] software. Chromosomal alignment between the two genome versions was performed using Winnomap (2.03) [51], followed by comprehensive comparison. BEDTools (v2.30.0) [51] was employed to identify and summarize previously unresolved regions (PURs) in the published genome. Corresponding genes within the PURs were extracted and subsequently analyzed for functional enrichment.

2.6. Gene Family and Phylogenomic Analysis

In addition to the self-assembled genome of O. bidens, the genome assembly and annotation data of 11 teleost species were retrieved from NCBI and Ensembl (Table S1). For genes with multiple transcripts, the longest transcript was retained, while those encoding proteins shorter than 30 amino acids, containing internal stop codons, or exhibiting length errors were filtered out. OrthoMCL (v2.0.9) [52] (inflation parameter 1.5) was employed to cluster the results based on protein sequence similarity (E-value < 1 × 10−5). MAFFT (v7.525) [53] was subsequently utilized to align single-copy orthologous genes shared among all fish species. A maximum likelihood phylogenetic tree was then constructed from this alignment using RAxML (v8.2.12) [54] under the GTRGAMMA model with 1000 bootstrap replicates, rooted with Gasterosteus aculeatus (Figure S5). Based on the phylogenetic tree and divergence times, gene family expansion and contraction were analyzed in CAFE4 (v4.2) and CAFE5 (v5.0.0) [55,56] with a significance threshold of 0.05, followed by enrichment analysis. Divergence times were estimated using the MCMCMCTREE (v4.9) [55] program in PAML [57].

2.7. Gene Positive Selection Analysis

Multiple sequence alignment of gene-protein sequences within single-copy gene families was performed using the MAFFT software (v7.525) [53]. Likelihood ratio tests (LRTs) were conducted via the CodeML (v4.9) [55] module in PAML (FDR < 0.05). Subsequently, Bayes empirical Bayes method (BEB) was employed to detect positive selection acting on protein-coding sequences. Generally, sites with a posterior probability greater than 0.95 are considered to be significantly under positive selection.

3. Results and Discussion

3.1. Genome Sequencing and Gap-Free Assembly

K-mer analysis estimated the genome size of O. bidens to be approximately 847 Mb, with a heterozygosity rate of 0.49% (Figure 2 and Figure S1). By integrating PacBio HiFi, Oxford Nanopore ultra-long reads, and Hi-C technologies, we generated an initial assembly of 872.22 Mb, comprising a maximum contig length of 42.22 Mb and a contig N50 of 22.42 Mb (Table S2). The depths of 32.97× for PacBio HiFi, 102.52× for ONT, and 100.72× for Hi-C sequencing. After scaffolding and anchoring, the final assembly was assigned to 38 chromosomes, with an anchoring rate of 98.09% (Table S3). Hi-C interaction mapping (Figure 3B) revealed excellent intra-chromosomal signals along the diagonal and minimal inter-chromosomal noise in off-diagonal regions, supporting the accuracy of chromosome-level scaffold ordering and the absence of major misassemblies. Quality assessments using both short-read (99.94% genome coverage) and long-read data (99.96%) indicated high base-level accuracy (Tables S4 and S5). The homozygous rate of SNP and InDel was 0.001%, reflecting a highly accurate and contiguous genome assembly (Table S7). Collectively, these results demonstrate that the assembled genome possesses strong structural integrity and high assembly quality (Figure 3A).
We generated a chromosome-scale, gap-free genome assembly of O. bidens (841.96 Mb) by using Oxford Nanopore ultra-long reads to bridge inter-contig gaps and extend contig lengths based on coverage relationships with pre-assembled contigs (Figure 4 and Table 1). Both the contig N50 and scaffold N50 reached 25.74 Mb. The identical values suggests that the assembled genome is gap-free, with no additional joins introduced during scaffolding. Taking advantage of the long-read capabilities of PacBio sequencing, we were able to assemble repetitive regions including centromeric and telomeric sequences. Telomeric repeats were detected at both ends of all chromosomes except for chr31, which may lack one telomere due to its single-ended configuration or highly repetitive content (Table 2). Centromere positions were identified using quarTeT-based predictions, Hi-C contact signal patterns, and element density profiles (Figure S2). The enhanced assembly continuity and completeness significantly improved our ability to resolve regions with high mutation rates. For example, in Gasterosteus aculeatus, PacBio sequencing increased genome continuity by more than fivefold, raising the N50 from 91.7 kb to 510.8 kb and filling gaps in highly repetitive regions such as telomeres and centromeres [58]. Furthermore, systematic evaluation of PacBio HiFi and ONT platforms using high-quality human reference specimens (HG002 and HG00733) demonstrated that structural variant (SV) detection based on high-quality assemblies substantially outperformed read-mapping-based methods in both sensitivity and precision, especially for SVs exceeding 10 kb at sequencing depths greater than 12× [59]. These findings highlight that improvements in genome assembly quality can markedly enhance the resolution of single nucleotide polymorphisms (SNPs) and SVs, particularly in structurally complex genomic regions.
Compared with the previously published genome assembly of O. bidens (GCA_037194315.1) [18], which shares the same karyotype (Figure 5), the newly assembled genome exhibits a high degree of synteny. Most chromosomal segments show clear collinearity, indicating strong syntenic conservation and substantial genomic integrity between the two assemblies. Furthermore, the gap-filled regions were significantly enriched in key pathways and functions. These include processes related to transcriptional regulation, protein degradation, mitochondrial metabolism, lipid transport, and immune responses (Table S8). This enrichment indicates that these previously unassembled regions play critical roles in fundamental biological processes. Relative to the earlier version (Table 3), the contig N50 has increased from 9.01 Mb to 22.42 Mb, and the total number of contigs has been reduced from 98 to 38. Single-contig assembly was achieved for each chromosome. Meanwhile, the BUSCO completeness assessment reached 99.34% (Table S6), which represents the current highest level, indicating that almost all functional gene regions were completely covered. Together, these improvements represent a substantial enhancement in genome continuity and completeness. Importantly, this study presents the first complete genome assembly for a representative species of the Opsariichthyinae subfamily, a phylogenetically controversial and ecologically significant lineage. The new assembly resolves previously ambiguous telomeric and centromeric regions. As a member of a basal lineage within Cyprinidae, this high-quality reference genome provides a valuable resource for studying the molecular basis of environmental adaptation and reconstructing the ancestral genome architecture of the family.

3.2. Genome Annotation and Repetitive Element Analysis

Repetitive element annotation revealed that 48.39% of the O. bidens genome consists of non-redundant transposable elements (TEs) (Tables S9 and S10). Among these, DNA transposons, long interspersed nuclear elements (LINEs), short interspersed nuclear elements (SINEs), and long terminal repeats (LTRs) accounted for 26.90%, 4.23%, 0.98%, and 11.02% of the genome, respectively. The proportion of repetitive sequences varies widely across teleost genomes. In evolutionarily conserved species such as Siniperca kneri (5.52%) and S. chuatsi (16.33%) [60], repetitive content tends to be low. In contrast, most teleost species maintain moderate levels of repetitive sequences, such as Gadus morhua (25.4%) [61] and Gasterosteus aculeatus (25.2%) [62]. In ancient polyploid taxa, repetitive elements may constitute a large proportion of the genome, as in Salmo salar (60%) [63]. The proportion of repetitive sequences in O. bidens genome thus represents a relatively high level among diploid teleosts and presumably may be related to genomic structural stability and sequence diversity.
Gene prediction based on both de novo and homology-based approaches identified 29,492 and 36,737 protein-coding genes, respectively. Homology-based predictions across four representative teleost genomes yielded gene counts ranging from 34,153 to 65,986. After integration and removal of redundant entries, a final non-redundant consensus gene set comprising 29,816 protein-coding genes was obtained, with an average gene length of 13,047 bp. Structural features of the annotated genes showed high consistency with those of closely related species. Functional annotations were successfully assigned to 27,169 genes (91.12%) using multiple public databases, providing a comprehensive foundation for downstream analyses of gene function, molecular pathways, and comparative genomic analyses (Figures S3 and S4; Tables S11 and S12).
Annotation of non-coding RNAs identified four major classes in the O. bidens genome, including 1165 microRNAs (miRNAs), 8681 transfer RNAs (tRNAs), 22,396 ribosomal RNAs (rRNAs), and 1698 small nuclear RNAs (snRNAs) (Table S13). These annotations provide an essential foundation for understanding the regulatory landscape of gene expression in this species.
Gene set completeness was evaluated using both BUSCO and Compleasm, which identified 3542 (97.31%) and 3545 (97.39%) complete BUSCOs, respectively (Table S14). These results indicate that the annotated gene set is highly complete and suiTable for downstream functional and evolutionary analyses.

3.3. Comparative Genomics Analysis

Based on gene family clustering (Figure 6A,B and Table S15), 69 unique gene families, 6433 common gene families, and 2933 single-copy orthologs were identified. Using these single-copy genes, the representative support rate of each clade reached 100%, highlighting the effectiveness of single-copy genes in accurately resolving phylogenetic relationships. Using known species divergence times G. aculeatus–C. molitorella (252.2-180.8 MYA), A. fasciatus–X. davidi (124.7-81.0), P. elongata–H. molitrix (58.6-19.3 MYA), and C. auratus subA–C. auratus subB (21.5-17.5 MYA), were used as a correction time to construct phylogenetic trees and determine species developmental relationships (Figure S5). The results indicated that O. bidens and its closely related species Z. platypus shared the closest relationship, with an estimated divergence time of 13.5 MYA based on fossil records. Collinearity analysis further confirmed high homology between the two species (Figure 3C).
For such widely distributed species with strong ecological plasticity, adaptive evolution is often accompanied by complex changes in gene families and selection pressures [64]. Based on the above 12 species, gene family evolution was analyzed using CAFE (v5.0.0), revealing that 350 gene families underwent expansion and 584 gene families underwent contraction (Figure 6C, Figures S6 and S7). Positive selection analysis using CodeML (v4.9) [55] identified 193 positively selected genes (FDR < 0.05, Figure S8).
The expansion of gene families has provided a fundamental genetic reservoir for adaptation to high dissolved oxygen and high flow velocity in O. bidens. Based on annotation results, we observed significant gene enrichment in KEGG pathways associated with high dissolved oxygen adaptation, including the Phagosome pathway (ko04145, p = 1.3 × 10−5) and Oxidative phosphorylation (ko00190, p = 0.0747). Similarly, significantly expanded GO terms were identified, encompassing the Myosin complex (GO:0016459, p = 4.3 × 10−4), Mitochondrial respiratory chain complex IV (GO:0005751, p = 0.152), and Mitochondrial electron transport, cytochrome c to oxygen (GO:0006123, p = 0.469). Notably, the adaptation to high dissolved oxygen also emphasizes the repair of hyperoxic damage, requiring the clearance of damaged cellular components and regulation of redox balance to mitigate ROS accumulation. This phenomenon has been similarly reported in certain fish species inhabiting high-oxygen environments [65], suggesting a conserved adaptive strategy. These enrichment results reveal that O. bidens adapts to hyperoxic stress through antioxidant homeostasis, optimization of energy metabolism, and enhancement of mitochondrial function in high dissolved oxygen environments.
Furthermore, significant gene expansion pathways associated with flow velocity adaptation were identified, highlighting the need for efficient oxygen utilization to enhance metabolic capacity and the maintenance of redox homeostasis to mitigate hyperoxia-induced damage. The KEGG pathways revealed notable expansions in Cardiac muscle contraction (ko04260, p = 0.7278), Vascular smooth muscle contraction (ko04270, p = 0.9972), and Regulation of actin cytoskeleton (ko04810, p = 0.9972). Similarly, significantly enriched GO terms included Myosin complex (GO:0016459, p = 4.3 × 10−4) and Homophilic cell adhesion (GO:0007156, p = 6.3 × 10−7). These enrichment results demonstrate that O. bidens enhances its swimming performance and endurance in rapid-flow environments through strengthened muscle contraction, sustained swimming capacity, regulated cardiovascular energy supply, and dynamic cytoskeletal remodeling. While these mechanisms show certain similarities with those reported in migratory and other stream-dwelling fish species [65,66], the present study provides more comprehensive genomic-level evidence.
Concurrently, positive selection analysis conducted using the CodeML (v4.9) [55] software identified 193 positively selected genes (FDR < 0.05), revealing adaptive signals of O. bidens in high dissolved oxygen and high metabolic demand environments, primarily involving mitochondrial function optimization, oxygen utilization, and metabolic regulation [67,68,69]. These include pathways such as Propanoate metabolism (ko00640, p = 0.8146), Folate biosynthesis (ko00790, p = 0.8146), HIF-1 signaling pathway (ko04066, p = 0.8146), Prolactin signaling pathway (ko04917, p = 0.8146). In addition, genes such as myo18a, slc25a51, slc25a25, mtx2, eral1, coq8b, th, mrpl21 further support its adaptive evolution in high-dissolved-oxygen and high-flow-velocity environments. Further functional validation is essential to confirm the adaptive roles of these genes and pathways.
Based on the T2T-level high-quality genome, we systematically analyzed the genetic adaptation characteristics of O. bidens in response to high-dissolved-oxygen and high-flow-velocity environments. The results showed that O. bidens has developed multidimensional adaptations during evolution, characterized by improved oxygen utilization efficiency and optimized continuous locomotion capacity, consistent with the features of stream environments. It should be noted that although this study identified partial gene family expansions and positive selection signals related to metabolic regulation and the motor system in O. bidens, whether these genomic changes are directly associated with adaptation to high dissolved oxygen and high-flow conditions requires further confirmation through functional validation, comparative population data, and environmental adaptation experiments. These findings provide preliminary clues for understanding the genetic adaptation characteristics of stream-dwelling fish and contribute to further exploration of the ecological adaptation mechanisms of fish in complex hydrological environments.

4. Conclusions

In this study, we constructed and updated the complete genome of O. bidens, and further discussed the genomic characteristics of O. bidens as a typical representative of stream environmental adaptation. Through the dual evolutionary mechanism of gene family expansion and positive selection, O. bidens has gained significant adaptive advantages in oxygen metabolism efficiency and locomotor capacity, enabling successful adaptation to high-dissolved-oxygen and high-flow-velocity stream environments. Our results provide a theoretical basis for the assessment of O. bidens germplasm resources and for studying the environmental adaptability of stream fish under complex ecological pressures, offering genomic resource for understanding biodiversity maintenance and ecosystem stability.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biology14111544/s1, Figure S1. K-mer distribution of different copy numbers in the assembled sequence of O. bidens genome. (A). Short-read-length data. (B). Long-read-length data. Figure S2. Chromosome element density distribution map (using chr1-4 as examples). Figure S3. Comparison of gene length. Figure S4. Venn diagram of the InterPro/GO/KEGG_KO/Swissprot/NR annotation. Figure S5. Phylogenetic tree of species. Figure S6. Enrichment of expanded gene families of O. bidens. Figure S7. Enrichment of contracted gene families of O. bidens. Figure S8. Enrichment of positive selection analysis of O. bidens. Table S1. Species information used in comparative genomic analyses Table S2. The preliminary genome assembly results of O. bidens genome. Table S3. The Hi-C assisted assembly of O. bidens genome. Table S4. Statistics of reads alignment for O. bidens genome reads. Table S5. The consensus quality (QV) assessment of O. bidens genome. Table S6. The evaluation of genome assembly in O. bidens. Table S7. Homozygous and heterozygous rates of assembly for O. bidens genome. Table S8. Pathways were significantly enriched for PURs. Table S9. Statistics of repeated sequence classification for O. bidens. Table S10. The repeat sequence statistics of O. bidens genome. Table S11. Gene prediction of assembly for O. bidens genome. Table S12. The Functional annotation of O. bidens genome. Table S13. Statistics of non-coding RNA annotation for O. bidens genome. Table S14. The evaluation of genome annotation in O. bidens. Table S15. Statistics of the clustered gene families identified from the 12 fish species.

Author Contributions

K.L., Y.Y. and N.S. Project administration, Q.L., N.S., K.L. and Y.Y. Methodology, M.J. and J.L. Resources, X.W., K.L., Y.Y., M.J. and J.L. investigation, X.W. Writing—original draft, Q.L. and X.W. Formal analysis, D.Y. and P.W. Writing—review & editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Central Public-interest Scientific Institution Basal Research Fund, CAFS (NO. 2023TD11), the Natural Science Foundation of Anhui Province (No. 2208085MC45), the Monitoring of Aquatic Resources in Key Waters of Anhui Province Project (ZF2022-18-0399) and the Youth Project of Anhui Provincial Natural Science Foundation (No. 2408085QC107). The specimen was collected as part of the Monitoring of Aquatic Resources in Key Waters of Anhui Province Project (ZF2022-18-0399), under a special fishing permit ([2023]002) issued by the Department of Agriculture and Rural Affairs of Anhui Province.

Institutional Review Board Statement

The animal study was conducted in accordance with the guidelines of the Chinese National Standard Guidelines for the Ethical Review of Laboratory Animal Welfare (GB/T 35892-2018, https://www.chinesestandard.net/PDF.aspx/GBT35892-2018 (accessed on 28 October 2025)). All animal samples were collected under the official Monitoring of Aquatic Resources in Key Waters of Anhui Province Project (ZF2022-18-0399), which is fully authorized by a Special Fishing License ([2023]002) issued by the Department of Agriculture and Rural Affairs of Anhui Province. This regulatory framework ensured that all field sampling activities complied with Chinese regulations for aquatic resource conservation and management.

Data Availability Statement

The sequencing dataset and genome assembly of O. bidens have been deposited in the NCBI SRA database under project number PRJNA1306202. The data are as follows: Hi-C data (SRR34997172); DNBSEQ-T7 genome sequencing data (SRR34997171); PacBio Revio genome sequencing data (SRR34997170); OXFORD_NANOPORE genome sequencing data (SRR34997168). The assembled genome was deposited in the NCBI Genome with the accession number GCA_046055825. Genome annotations, along with predicted coding sequences and protein sequences, can be accessed through the Figshare (https://doi.org/10.6084/m9.figshare.29848886 (accessed on 28 October 2025)).

Acknowledgments

We extend our gratitude to Wuhan Onemore-tech Co., Ltd. for their technical assistance in genome sequencing and bioinformatics analysis.

Conflicts of Interest

The authors declare that they have no competing interests.

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Biology 14 01544 g001
Figure 1. Specimen Data. (A). Map of sampling sites. (B). Live photo of O. bidens.
Figure 1. Specimen Data. (A). Map of sampling sites. (B). Live photo of O. bidens.
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Figure 2. Assessment of genome assembly quality. (A) K-mer distribution frequency. (B) BUSCO assessment.
Figure 2. Assessment of genome assembly quality. (A) K-mer distribution frequency. (B) BUSCO assessment.
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Figure 3. Genome assembly and gene annotation of O. bidens. (A) Features the O. bidens genome arranged from the outermost. (B) A heatmap of chromosomal interactions in O. bidens. (C) Synteny between genomes of O. bidens and Zacco platypus.
Figure 3. Genome assembly and gene annotation of O. bidens. (A) Features the O. bidens genome arranged from the outermost. (B) A heatmap of chromosomal interactions in O. bidens. (C) Synteny between genomes of O. bidens and Zacco platypus.
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Figure 4. Visualization of T2T genome. Purple in the figure represents telomeres, black squares represent centromeres, and pink represents subtelomeric centromeres.
Figure 4. Visualization of T2T genome. Purple in the figure represents telomeres, black squares represent centromeres, and pink represents subtelomeric centromeres.
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Figure 5. Collinearity analysis of different genome versions.
Figure 5. Collinearity analysis of different genome versions.
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Figure 6. Comparative genomic analysis for O. bidens. (A) Orthologous gene families. (B) Unique and shared gene families. (C) Phylogenetic tree. The green numbers indicate expanded gene families, while the red numbers indicate contracted gene families.
Figure 6. Comparative genomic analysis for O. bidens. (A) Orthologous gene families. (B) Unique and shared gene families. (C) Phylogenetic tree. The green numbers indicate expanded gene families, while the red numbers indicate contracted gene families.
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Table 1. Summary statistics of O. bidens genome assembly.
Table 1. Summary statistics of O. bidens genome assembly.
Contig Length (bp)Contig NumberScaffold Length (bp)Scaffold Number
N9012,343,8323112,343,83231
N8019,053,5432519,053,54325
N7021,089,6842121,089,68421
N6022,405,6811722,405,68117
N5025,739,5001325,739,50013
Total length841,960,764NA841,960,764NA
Number (≥100bp)NA38NA38
Number (≥2kb)NA38NA38
Max length42,194,854NA42,194,854NA
NA: Not Applicable (indicating that a field or parameter is irrelevant in the given context).
Table 2. The details of chromosome and number of telomere characteristic sequences for O. bidens genome.
Table 2. The details of chromosome and number of telomere characteristic sequences for O. bidens genome.
ChromosomeLength (bp)Number of ContigsNumber of GapsNumber of Start Repeat UnitNumber of End Repeat Unit
chr112,090,1871018782521
chr211,846,864102173478
chr342,194,8541023081440
chr433,560,1561025982236
chr528,811,627107251437
chr625,739,50010625123
chr711,611,5101047121556
chr812,343,8321018902057
chr926,899,6041017711723
chr1022,405,6811018981442
chr1127,380,5741013752676
chr1213,984,8261013181118
chr1333,569,5051019531989
chr1413,802,9481016892244
chr1523,521,3441049621668
chr1619,700,78510307432
chr1715,140,455101221674
chr1819,053,5431019401682
chr1936,299,1731036301463
chr2023,626,31210253041
chr2121,089,68410331642
chr2235,847,6891018722997
chr2329,779,2891026083403
chr249,738,4101014171527
chr257,725,8301021611856
chr2610,171,860109631397
chr2735,461,4291022341577
chr2821,077,6291017231847
chr2920,275,87610452229
chr3021,102,6981031387
chr3121,769,7791001951
chr3234,646,3601011952618
chr3321,231,4451032291936
chr3418,000,3211093372
chr3513,313,1691016883629
chr3610,202,2371017361113
chr3725,135,10010781344
chr3831,808,6791014582
Table 3. Comparison of genomes of different versions of O. bidens.
Table 3. Comparison of genomes of different versions of O. bidens.
Statistical IndicatorsGCA_037194365.1GCA_037194315.1GCA_037194245.1GWHBEIO00000000-1GWHBEIO00000000-2This Study
Sexmalefemalefemalefemalemalefemale
Total size of assembled genome (Mb)852.41843.11840.94818.78 Mb992.9841.96
Contig N509.012.95.274.665.225.74
Contig N90NANANANANA12.34
Number of ContigsNANANA403137338
Scaffold N50 (Mb)21.0123.6224.7525.2919.4425.74
Scaffold N90 (Mb)NANANANANA12.34
Scaffold number22845098393838
chromosome number373839393838
Number of gap-free chromosomeNANANANANA38
Number of gapsNANANANANA0
Number of telomeres(pairs/single)NANANANANA37/1
TE size360.05356.29356.64347.06357.31407.46
GC content38.338.338.1NA37.938.4
Chromosome anchoring ratio (%)90.3995.6799.0195.66%89.3198.09
Total chromosome length (Mb)770.52806.65832.70814.71886.81841.96
Gene number26,55625,03626,28323,99236,73829,816
Functional proteins25,383 (95.58%)23,139 (92.42%)24,493
(93.19%)		22,869
(95.4%)		30,922
(84.17%)		27,169 (91.12%)
BUSCO completeness genome (%)97.296.696.896.697.599.34
BUSCO completeness (protein)91.791.894.593.5NA97.31
DNA matching rate (%)98.7899.0598.9898.7699.599.94
NA: Not Available (indicating the absence of data or non-reporting).
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Wang, X.; Liu, Q.; Yin, D.; Wang, P.; Jiang, M.; Liu, J.; Sun, N.; Yan, Y.; Liu, K. Telomere-to-Telomere Gap-Free Genome Assembly and Comparative Analysis of the Opsariichthys bidens (Cypriniformes: Xenocyprididae). Biology 2025, 14, 1544. https://doi.org/10.3390/biology14111544

AMA Style

Wang X, Liu Q, Yin D, Wang P, Jiang M, Liu J, Sun N, Yan Y, Liu K. Telomere-to-Telomere Gap-Free Genome Assembly and Comparative Analysis of the Opsariichthys bidens (Cypriniformes: Xenocyprididae). Biology. 2025; 14(11):1544. https://doi.org/10.3390/biology14111544

Chicago/Turabian Style

Wang, Xinyue, Qi Liu, Denghua Yin, Pan Wang, Min Jiang, Jie Liu, Ning Sun, Yunzhi Yan, and Kai Liu. 2025. "Telomere-to-Telomere Gap-Free Genome Assembly and Comparative Analysis of the Opsariichthys bidens (Cypriniformes: Xenocyprididae)" Biology 14, no. 11: 1544. https://doi.org/10.3390/biology14111544

APA Style

Wang, X., Liu, Q., Yin, D., Wang, P., Jiang, M., Liu, J., Sun, N., Yan, Y., & Liu, K. (2025). Telomere-to-Telomere Gap-Free Genome Assembly and Comparative Analysis of the Opsariichthys bidens (Cypriniformes: Xenocyprididae). Biology, 14(11), 1544. https://doi.org/10.3390/biology14111544

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