Abstract
The catfish genus Pseudecheneis consists of small freshwater catfish that live at the bottom of fast-flowing streams. The genetic information carried by extant freshwater fish can provide new insights into deciphering the evolutionary processes of rivers. However, existing studies often rely on limited molecular markers, which are insufficient to thoroughly resolve genetic information and complex evolutionary relationships. We have deciphered the first high-quality Telomere-to-Telomere (T2T) chromosome-level genome of Pseudecheneis, with a genome size of 574.82 Mb and a contig N50 of 22.76 Mb. A total of 19,787 protein-coding genes were annotated, of which 98.4% were successfully functionally annotated. This study provides important references for clarifying the phylogenetic relationships and evolutionary history of Pseudecheneis, and also offers valuable data support for exploring the interrelationships among structure, function, and adaptation.
Background & Summary
The catfish genus Pseudecheneis belongs to the Family Sisoridae in the Order Siluriformes. All species are small freshwater catfish, dwelling at the bottom of fast flowing streams1. The fish genus Pseudecheneis likely originated in the Hengduan Mountain region of China. In conjunction with the uplift of the Qinghai-Tibet Plateau, the genus underwent multiple differentiation events within this region and subsequently dispersed westward and southward2. Currently, 21 valid species have been recorded within this genus, primarily distributed in Yunnan and Tibet, China, and neighboring countries (https://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp). Their distribution extends westward to India, Bangladesh, and Nepal, southward to Myanmar, Laos, and Vietnam, and northward to the Brahmaputra River, while the Yuanjiang–Red River constitutes the eastern limit of their range3. Interestingly, this ichthyofaunal boundary represented by the Yuanjiang–Red River is also observed in multiple fish genera2,4, bearing remarkable implications for zoogeography and the uplift events of the Qinghai–Tibet Plateau. However, the biogeographical significance of this zoogeographic distribution pattern remains to be fully investigated. Moreover, the issue of river capture in the Honghe River has long been subject to debate and lacks a consensus understanding.
The genetic information carried by extant freshwater fishes provides biological insights into fluvial evolutionary processes5. Drainage reorganization can drive species divergence and faunal differentiation6, while fish dispersal and isolation shape genetic and phylogenetic structures, thereby preserving genetic signals that offer robust evidence for reconstructing hydrological history and interpreting biogeographic patterns7. Previously, only one species of Pseudecheneis, Pseudecheneis paviei (P. paviei) has been recorded in the Yuanjiang–Red River drainage8. It represents a critical system for understanding the diversity, geographic distribution, and evolutionary history of Pseudecheneis. However, previous studies relying on limited molecular markers, while revealing partial insights, remain insufficient in providing decisive genetic evidence or resolving complex evolutionary relationships8. With advances in sequencing technologies, high-quality genomes have become increasingly integral to fundamental biological research9. Yet, the absence of publicly available genomic data for Pseudecheneis has significantly impeded a comprehensive understanding of the group’s biogeography and evolutionary trajectory. Therefore, constructing a high-quality genome for Pseudecheneis will establish a crucial foundation for clarifying phylogenetic relationships and evolutionary history within the genus, while also serving as a valuable resource for exploring structure–function–adaptation interrelationships.
In this study, a high-quality chromosome-level Telomere-to-telomere (T2T) genome was generated by combining Illumina, PacBio, ONT, and Hi-C data. The assembled genome had a size of 574.82 Mb, with a contig N50 of 22.76 Mb, and was anchored to 26 chromosomes. A total of 19,787 protein-coding genes were predicted, of which 98.4% were successfully functionally annotated. The high-quality T2T genome deciphered in this study provides a solid reference for clarifying the phylogenetic relationships and evolutionary history within Pseudecheneis, while also filling a gap in the genomic resources of fish.
Methods
Ethics statement
The Experimental Animal Care and Ethics Committee of Southwest Forestry University approved all experimental protocols.
Sample collection and sequencing
The samples for genome assembly were collected in 2025 from the Lvchun County, Honghe Hani and Yi Autonomous Prefecture, Yunnan province, China (Fig. 1). A single P. paviei individual was dissected to obtain fresh samples from various organs and tissues and these samples were promptly placed in liquid nitrogen until DNA and RNA extraction. Among them, genomic DNA was extracted from muscle tissue according to the TIANamp DNA Kit (Tiangen, Beijing, China) manufacturer’s instructions for whole-genome sequencing. To assist genome annotation and facilitate protein-coding genes prediction, total RNA from pooled samples was extracted using the TRIzol reagent and subjected to transcriptome sequencing. Qubit Fluorometer and 1% agarose gel electrophoresis were used to assess the quality of the extracted DNA and RNA.
Fig. 1.
Schematic diagram of sample collection. The left figure represents a picture of P. paviei for assembly and annotation. From top to bottom are the front, side, and back, respectively. The right figure shows the schematic diagram of the sample collection site.
High-throughput sequencing was performed on the P. paviei. Specifically, the short-insert (350 bp) library was constructed using the Illumina Nextera DNA Preparation Kit and sequenced by the Illumina Xplus platform. A PacBio SMRTbell library (20 kb) was constructed using SMRTbell Express Template Prep Kit 2.0 (PacBio, Menlo Park, CA, USA). Briefly, 10 µg of the input DNA was sheared using a Megaruptor 3. The concentration of the sheared sample was measured using a Qubit dsDNA HS Assay Kit. 5ug of the sheared sample was taken to prepare HiFi SMRTbell® Libraries using the SMRTbell Express Template Prep Kit 2.0, including DNA damage repair, and SMRTbell adapter ligation. The constructed library was sequenced by the Pacific Biosciences platform. For ONT ultra-long sequencing, a Nanopore sequencing library was constructed using Ligation Sequencing Kit (SQK-LSK114, Oxford Nanopore Technologies, UK) and sequenced by the PromethION48 platform. Moreover, to assemble the genome at the chromosomal level, an Hi-C library was constructed by 1% formaldehyde crosslinking, nuclear separation, chromatin digestion, DNA fragment ligation, DNA purification and fragmentation, adapter ligation, and PCR amplification. The constructed Hi-C library was sequenced using the Illumina X-TEN platform. Finally, the extracted RNA was used to construct an RNA-seq library using NEBNext Ultra RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions, and then sequenced by Illumina sequencing platform. Fastp (v1.0.1) was used to conduct the quality control with the default parameters. As a result, a total of 46.58 Gb of Illumina reads, 51.22 Gb of PacBio HiFi reads, 60.02 Gb of Hi-C reads, 26.11 Gb of Ultra-long reads and 6.62 Gb of RNA reads were generated (Table 1).
Table 1.
Sequencing data statistics for genome assembly.
| Libraries | Total data (G) | Sequence coverage (X) |
|---|---|---|
| Illumina reads | 46.58 | 81.15 |
| PacBio reads | 51.22 | 89.23 |
| Ultra-long reads (>100 kbp) | 26.11 | 45.49 |
| HiC | 60.02 | 104.56 |
| RNA-seq | 6.62 | \ |
T2T Genome assembly and Hi-C analysis
PacBio HiFi reads and Hi-C reads were first assembled using Hifiasm (v0.25.0)10 with its default parameters. BWA-MEM v0.7.1711 was employed for the alignment of the paired-end Illumina reads to the corrected Hifiasm assembly. Beyond its application in correcting assembly errors, Illumina sequencing data serves as a valuable resource to estimate genome size, characterize genomic features, and evaluate the completeness, consistency, and accuracy of the genome assembly. To correct assembly errors, the draft genome was then polished using Pilon v1.212. Hi-C reads were aligned to contigs by HiC-Pro (v3.1.0)13 with the default parameters, and the LIGATION_SITE was set to ‘GATC’. 3D-DNA was employed for the correction of misassemblies and for the anchoring, ordering, and orienting of DNA fragments. HiCPlotter (v0.5.1)14 was used to visualize the results. To further improve the continuity and completeness of the genome assembly and achieve a T2T gap-free genome, ONT ultralong reads were employed for gap closure by leveraging their long span advantage. Specifically, the tgs-gapcloser software (v1.2.1) was used to fill gaps in the assembled genome with ONT ultralong reads, which played a critical role in optimizing assembly quality and ultimately generating the high-quality T2T genome assembly. Finally, Benchmarking Universal Single-Copy Orthologs (BUSCO)15 was used to validate the completeness and accuracy of the genome assembly with Vertebrata_odb10. Based on the alignment results generated by BWA, single nucleotide polymorphism (SNP) calling was performed using SAMtools (http://samtools.sourceforge.net/), which involved sorting by chromosome coordinates, removing duplicate reads, and conducting statistical analysis of the raw sequencing data. Finally, the quality value (QV) of genomic bases was evaluated using the Merqury-mash module implemented in Merqury (v1.3).
The assembled genome of P. paviei was 574.82 Mb in size with a contig N50 of 22.76 Mb, and was successfully anchored to 26 chromosomes with an anchoring rate of 99.79% (Fig. 2a,b, Table 2), demonstrating a high assembly continuity. The GC content of the assembled genome was 41.5%. Furthermore, BUSCO assessment results demonstrated the high completeness of the genome assembly. Specifically, 96.5% of conserved genes were identified as complete BUSCOs, including 95.7% complete and single-copy BUSCOs and 0.8% complete and duplicated BUSCOs (Table 3). To further assess the integrity of the assembly, reads from the small-fragment library were aligned to the assembled genome using BWA (0.7.17-r1188), followed by the calculation of alignment rate, genome coverage, and read depth distribution. The read alignment rate was approximately 99.03%, and the genome coverage rate was approximately 99.75% (Table 4). The heterozygous SNP ratio of the P. paviei genome was 0.253846%, and the homozygous SNP ratio was 0.000224% (Table 5), suggesting high base-level accuracy of the assembled genome. Additionally, the QV in the present study was 44.6852. These metrics collectively demonstrate the chromosomal integrity and high continuity of the P. paviei genome assembly.
Fig. 2.
Overview of the P. paviei T2T genome. The left picture shows circos plot of the P. paviei genome assembly. (a) chromosome length. (b) Protein-coding genes present in the scaffolds, red represents genes on forward strand and green for genes on reverse strand. (c) GC content windows of 500 kb. (d) Distribution of gene density with sliding windows of 500 kb. (e) Distribution of repeat element density with sliding windows of 500 kb. (f) Distribution of trf element density with sliding windows of 500 kb. The right picture shows Hi-C intra-chromosomal contact map of the P. paviei genome assembly.
Table 2.
The statistical results of assembled chromosome length.
| Chromosome | Sequences Length (bp) |
|---|---|
| Chr1 | 30,931,218 |
| Chr2 | 29,539,444 |
| Chr3 | 28,950,378 |
| Chr4 | 28,635,456 |
| Chr5 | 27,634,588 |
| Chr6 | 26,094,986 |
| Chr7 | 24,978,321 |
| Chr8 | 24,776,138 |
| Chr9 | 23,570,041 |
| Chr10 | 22,979,024 |
| Chr11 | 22,579,461 |
| Chr12 | 22,485,279 |
| Chr13 | 21,901,608 |
| Chr14 | 21,507,004 |
| Chr15 | 21,492,618 |
| Chr16 | 20,787,895 |
| Chr17 | 20,224,154 |
| Chr18 | 20,182,419 |
| Chr19 | 18,849,351 |
| Chr20 | 18,693,381 |
| Chr21 | 17,454,085 |
| Chr22 | 15,743,280 |
| Chr23 | 15,719,345 |
| Chr24 | 15,268,969 |
| Chr25 | 14,793,742 |
| Chr26 | 13,189,109 |
Table 3.
BUSCO assessment of the T2T genome of P. paviei.
| Genomic characteristic | Percentage |
|---|---|
| BUSCO | n = 3354 |
| Complete BUSCOs | 96.5% |
| Complete and single-copy BUSCOs | 95.7% |
| Complete Duplicated BUSCOs | 0.8% |
| Fragmented BUSCOs | 0.7% |
| Missing BUSCOs | 2.8% |
Table 4.
Statistics on the coverage of genome reads of P. paviei.
| Reads | Mapping rate (%) | 99.03 |
|---|---|---|
| Genome | Coverage (%) | 99.75 |
| Coverage at least 4X (%) | 99.51 | |
| Coverage at least 10X (%) | 99.12 | |
| Coverage at least 20X (%) | 97.88 |
Table 5.
SNP statistics of the P. paviei genome.
| Number | Percentage | |
|---|---|---|
| All SNP | 1,152,376 | 0.254071% |
| Heterozygosis SNP | 1,151,358 | 0.253846% |
| Homology SNP | 1,018 | 0.000224% |
Telomere identification
Identification of telomeres was performed using the tidk toolkit (v0.2.65)16 to facilitate the assembly of a high-quality P. paviei T2T genome. Firstly, low-quality fragments and contaminants were removed from the high-quality genome assembly. Secondly, the tidk find module was utilized to identify potential telomeric regions in the genome by providing the species-specific telomeric repeat sequence as input (AACCCT). The tidk search module was then employed to precisely determine the telomeric sequences on both ends of each chromosome. Finally, the T2T genome diagram was generated to illustrate the continuous structure of all chromosomes from one telomere to the other. The results indicate that telomeric regions were identified at both ends of each chromosome in the P. paviei genome, confirming that the assembly has reached the T2T level (Fig. 3).
Fig. 3.
Genome-wide telomere regions of the P. paviei genome. The telomeric regions are marked by inverted triangles, and a light-to-dark color gradient signifies increasing repetitive sequence abundance.
Annotation of repeat element
An integrated approach of homology-based search and ab initio prediction was used to identify repetitive sequences in the P. paviei genome. RepeatModeler (v2.0.4)17 was employed for the identification of repeat families within the P. paviei genome. De novo identification and masking of repeat sequences were conducted with the assistance of RepeatMasker (v4.1.2)18. tRNAscan-SE (v2.0.11) and INFERNAL (http://infernal.janelia.org/) in the Rfam package were used to identify the non-coding RNAs (ncRNAs), including tRNA, microRNA (miRNA), small nuclear RNA (snRNA), and vertebrate ribosomal RNA (rRNA).
The identified repetitive sequences accounted for 33.89% of the P. paviei genome. Among them, SINEs (short interspersed nuclear elements) and LINEs (long interspersed nuclear elements) accounted for 1.03% and 6.10% of the genome, respectively. LTR (long terminal repeats) and DNA elements accounted for 7.96% and 10.71% of the genome, respectively.
Annotation of protein-coding gene
An integrated strategy was employed to annotate protein-coding genes in the P. paviei genome, including de novo prediction, homology-based search, and transcriptome-assisted methods. The ab initio coding gene prediction was performed using Augustus (v3.2.3)19 and SNAP (v2.0.11). Moreover, to facilitate homology-based prediction, we downloaded the protein-coding genes of closely related species from NCBI, including Bagarius yarrelli, Ictalurus punctatus, Tachysurus fulvidraco, Tachysurus vachellii, Pangasianodon hypophthalmus, Silurus meridionalis, and Danio rerio. BLASTP20 was then used to align the genome sequences of closely related species against the P. paviei genome with the parameters “-evalue 1e-5”. Exonerate v2.2.02821 was employed for gene structure prediction. The RNA-seq data was aligned to the assembled genome according to the method described in the Hisat2 (v2.2.1)22 software documentation. The “-multi-read-correct” parameter was employed in Stringtie (v1.3.3)23 for conducting gene model analysis on the transcripts. To generate a final non-redundant gene set, we integrated the data derived from the three aforementioned approaches by EvidenceModeler software (v2.1.0)24. Functional annotation of the genes was performed against the NCBI non-redundant (nr), SwissProt, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases using BLASTP (v2.15.0)20. The E-value was set to 1e-5. InterProScan (v5.68–96.0)25 and HMMER (v3.4)26 were applied to map the protein domains against the InterPro and Pfam27 databases.
The EVM-integrated annotation initially yielded 27,262 protein-coding genes, which were refined to a final set of 19,787 protein-coding genes following PASA-based correction. The average CDS length was 1749.22 bp, with an average of 10.17 exons per gene. The mean lengths of exons and introns were 171.99 bp and 1252.20 bp, respectively. The results showed that a total of 19,469 genes were successfully annotated, accounting for 98.4% of the genome (Table 6).
Table 6.
Summary of functional annotations of P. paviei genome.
| Number | Percent (%) | |
|---|---|---|
| Total | 19,787 | — |
| Swissprot | 18,433 | 93.20 |
| Nr | 19,103 | 96.50 |
| KEGG | 17,109 | 86.50 |
| InterPro | 19,273 | 97.40 |
| GO | 14,377 | 72.70 |
| Pfam | 17,178 | 86.80 |
| Annotated | 19,469 | 98.40 |
Data Records
All data generated for Pseudecheneis paviei in this study have been made publicly available. The Illumina sequencing data, PacBio reads, and ONT sequencing data have been stored in the NCBI SRA with the accession numbers SRR3521067528, SRR3521067429, and SRR3521067330, respectively. The Hi-C reads data and RNA-seq raw data generated by the sequencing platform have been deposited into the National Center for Biotechnology Information (NCBI) SRA database with accession number SRR3521067231 and SRR3756964532, respectively. The genome assembly has been deposited in GenBank under accession number JBWAXU00000000033. The genome annotation files are available in the figshare database34.
Technical Validation
We assembled a high-quality P. paviei telomere-to-telomere (T2T) chromosome-level genome, which was 574.82 Mb in size with a contig N50 of 22.76 Mb. The Hi-C chromosome interaction intensity corroborates the high assembly continuity. BUSCO was used to assess the genome quality with the Vertebrata_odb10. The results of BUSCO showed a completeness of 96.5%. To further assess the integrity of the genome assembly, Illumina reads were aligned to the assembled genome, followed by the calculation of alignment rate, genome coverage, and read depth distribution. The read alignment rate was approximately 99.03%, and the genome coverage rate was approximately 99.75%. The heterozygous SNP ratio of the P. paviei genome was 0.253846%, and the homozygous SNP ratio was 0.000224%. Collectively, multiple independent validation methods confirmed that the assembled P. paviei genome is consistent, complete, and accurate, which is sufficient to meet the requirements of various subsequent genomic research studies.
Acknowledgements
We acknowledge Yandong Bai (Huanglianshan National Nature Reserve, Honghe, China), Jianping Xiao and Yang Wei (School of Biodiversity Conservation and Utilisation, Southwest Forestry University, Kunming, China) who contributed to specimen collection.
Author contributions
R.H. and X.L. conceived this study. R.L., C.X. and W.D. collected the samples and performed the experiment. R.H. performed the research and analyzed the data. R.H., X.L. and W.D. draft the manuscript. Z.P. revised the manuscript. All authors have read and approved the final manuscript.
Funding
This research was supported by the grant from the First Class Forestry Academic Subject in Yunnan Province (LXXK-2025D04), First Class Forestry Academic Subject in Yunnan Province (51600625), and Key Discipline of Wildlife Conservation and Utilization in Yunnan Province (XKZ200904).
Data availability
The Illumina sequencing data, PacBio sequencing data, ONT sequencing data, Hi-C sequencing data, and transcriptome Illumina sequencing data are deposited in the National Center for Biotechnology Information (NCBI) SRA database under the accession numbers SRR3521067528, SRR3521067429, SRR3521067330, SRR3521067231, and SRR3756964532, respectively. This Whole Genome Shotgun project has been deposited at NCBI under the accession JBWAXU00000000033. This Whole Genome and annotations are available from the Figshare repository (10.6084/m9.figshare.30976780.v1)34.
Code availability
No specialized code was employed in this research. The data analyses complied with the manuals and protocols offered by the developers of the relevant bioinformatics tools, whose parameter settings were specified in the methods section.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
These authors contributed equally: Zhen Peng, Rui Li.
Contributor Information
Chaoyong Xiong, Email: xiongchaoyongae@163.com.
Ruobing Han, Email: hanruobing0413@163.com.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Citations
- NCBI Sequence Read Archivehttps://identifiers.org/ncbi/insdc.sra:SRR35210675 (2026).
- NCBI Sequence Read Archivehttps://identifiers.org/ncbi/insdc.sra:SRR35210674 (2026).
- NCBI Sequence Read Archivehttps://identifiers.org/ncbi/insdc.sra:SRR35210673 (2026).
- NCBI Sequence Read Archivehttps://identifiers.org/ncbi/insdc.sra:SRR35210672 (2026).
- NCBI Sequence Read Archivehttps://identifiers.org/ncbi/insdc.sra:SRR37569645 (2026).
Data Availability Statement
The Illumina sequencing data, PacBio sequencing data, ONT sequencing data, Hi-C sequencing data, and transcriptome Illumina sequencing data are deposited in the National Center for Biotechnology Information (NCBI) SRA database under the accession numbers SRR3521067528, SRR3521067429, SRR3521067330, SRR3521067231, and SRR3756964532, respectively. This Whole Genome Shotgun project has been deposited at NCBI under the accession JBWAXU00000000033. This Whole Genome and annotations are available from the Figshare repository (10.6084/m9.figshare.30976780.v1)34.
No specialized code was employed in this research. The data analyses complied with the manuals and protocols offered by the developers of the relevant bioinformatics tools, whose parameter settings were specified in the methods section.