Chromosome-scale assembly of the Xenocypris davidi using PacBio HiFi reads and Hi-C technologies

Abstract

Xenocypris davidi is a benthic fish species widely distributed in the water systems south of the Yellow River in China, playing a significant role in aquatic ecosystems. Despite its ecological and economic importance, genomic resources for X. davidi are limited, hindering a comprehensive understanding of its evolutionary adaptations and genetic improvements. This study presents the first chromosome-level genome assembly of X. davidi, utilizing PacBio long-reads, Illumina short reads, and Hi-C sequencing data. The genome assembly spans 1.05 Gb with a scaffold N50 length of 33.99 Mb, and 95.12% of the genome sequence was successfully anchored onto 24 pseudochromosomes. We identified 27,360 protein-coding genes, of which 26,672 were functionally annotated. This genome sequence provides a valuable resource for exploring the molecular basis of agronomic traits in X. davidi and will facilitate its genetic enhancement.

Background & Summary

Xenocypris davidi Bleeker, 1871, a species of fish extensively distributed throughout the water systems south of the Yellow River in China, is commonly found in rivers, lakes, and reservoirs, and is classified as a benthic species. It primarily feeds on bottom-dwelling algae and detritus. These fish typically inhabit the mid-to-upper reaches of rivers and migrate upstream to shallow rapids for spawning during the breeding season¹. Owing to its identity as a benthic scraper, it actively contributes to the enhancement of aquatic ecological environments and the amelioration of water quality, rendering it an ideal candidate for artificial propagation and release, as well as for pond polyculture practices. Research on the dietary habits of X. davidi in a certain reservoir has revealed that adult fish primarily consume a substantial amount of detritus, supplemented by a small number of sessile diatoms and oscillatorian cyanobacteria. This diet does not compete with that of other economically important fish species such as silver carp and bighead carp in the reservoir, indicating that X. davidi is a preferred species for integrated aquaculture². Studies by Tang et al. have demonstrated that X. davidi has significant potential in managing the overgrowth of filamentous algae³. Appreciated for its delicate texture and savory taste, X. davidi has become one of the specialty fish species favored by consumers. Recent research has also explored the dietary nutritional requirements of X. davidi broodstock and the impact of adding bee pollen and glutamine to their feed on the growth of this species^4–6. In the field of toxicology, it has been discovered that X. davidi exhibits a low tolerance to heavy metal copper ions. A hepatic transcriptome database for X. davidi has been constructed to analyze gene expression profiles, providing molecular insights into the species’ response to environmental pollutants⁷.

X. davidi, classified within the order Cypriniformes, family Cyprinidae, subfamily Xenocyprininae, and genus Xenocypris, has been the subject of evolutionary studies primarily based on the analysis of the complete mitochondrial genome^8,9. Assessments in the field of population genetics, utilizing microsatellite markers, have evaluated the impact of stocking activities on wild populations of X. davidi in the Qiantang River, with results indicating that these activities have not posed genetic risks to the wild populations¹⁰. Additionally, research has identified genetic diversity variations among different aquaculture populations of X. davidi¹¹. When analyzing wild populations from various water systems, significant genetic divergence was observed between populations in Qiandao Lake and the Yangtze River¹². Nonetheless, the scant genomic data resources limit the understanding of X. davidi’s evolutionary adaptation and molecular mechanisms of its traits, restricting our full appreciation and exploitation.

In this research, we have achieved the construction of a chromosome-level genome assembly for X. davidi, representing the initial case of jointly employing PacBio long-reads, Illumina short reads, and Hi-C sequencing data. The obtained genome assembly has a total length of 1.05 Gb. The scaffold N50 length reaches 33.99 Mb, and a remarkable 95.12% of the genome sequence is effectively anchored to 24 pseudochromosomes. By using a combined method including de novo gene predictions, RNA-seq data, and homologous protein evidence, a sum of 27,360 protein-coding genes have been identified, among which 26,672 have been functionally annotated. The genome sequence is a valuable asset for understanding X. davidi’s agronomic trait molecular basis and facilitating its genetic improvement.

Methods

Ethics statement

The Experimental Animal Care and Ethics Committee of Xinyang Agriculture and Forestry University approved all fish sampling experiment procedures.

Samling and genome survey

A healthy female specimen of X. davidi, weighing 659.36 grams, was collected from the Pohe Reservoir in Guangshan County, Xinyang City, Henan Province, China. Following euthanasia with eugenol at an anesthetic concentration of 16 mg L^-3¹³, the fish was rapidly rinsed three times with sterile physiological saline and dabbed dry with sterile cotton. Tissues including those from the muscle, heart, spleen, liver, and kidney were promptly dissected and promptly immersed in liquid nitrogen for conservation, and then stored at -80 °C until the DNA extraction process. High-quality genomic DNA from the muscle was extracted with the PureLink™ Genomic DNA Mini Kit (K182001, Thermo Fisher Scientific, USA). Meanwhile, RNA from diverse tissues was isolated by TRIzol™ Reagent (15596026CN, Thermo Fisher Scientific, USA). The quality and concentration of DNA were evaluated via 1% agarose gel electrophoresis and a Qubit 2.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, USA). In contrast, for RNA, its purity and integrity were further appraised using a NanoDropTM One spectrophotometer (Thermo Fisher Scientific, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., USA).

In the context of the genomic survey, a 10 μg DNA sample was utilized to construct a 350 bp library, employing a paired-end 150 bp (PE150) sequencing strategy on the Illumina Novaseq 6000 platform. A total of 50.51 Gb of raw data and 168,353,928 read pairs were generated. The Jellyfish¹⁴ (version 2.2.7) was used to construct the 17-mer frequency depth distribution and estimate the genome size. Genome assembly was performed using SOAPdenovo2¹⁵. Ultimately, survey analysis conducted with a Kmer size of 17 estimated the genome size to be 1,020.95 Mbp, which was refined to 1,004.32 Mbp. The heterozygosity rate was determined to be 0.52%, with the proportion of repetitive sequences constituting 53.91% (Table 1). Assembly with a Kmer size of 41 yielded a contig N50 of 2,166 bp, with a total length of 876,892,788 bp, and a scaffold N50 of 3,222 bp, with a total length of 899,312,128 bp. (Table 2).

Table 1.

Kmer = 17 Analysis of genomic characteristic statistics.

Kmer	Depth	N Kmer	Genome size (M)	Revised genome size (M)	Heterozygous rate (%)	Repeat_rate (%)
17	37	37,775,268,357	1,020.95	1,004.32	0.52	53.91

Table 2.

Statistical summary of the genome survey assembly results for X. davidi.

Title	Total_length	Total_number	Max_length	N50_length	N90_length
Contig	876,892,788	1,361,752	79,659	2,166	183
Scaffold	899,312,128	1,111,026	104,922	3,222	258

PacBio and Hi-C based whole-genome sequencing

Regarding PacBio sequencing, high-quality DNA samples (with the main band being greater than 30 kb) were fragmented into segments ranging from 15 to 18 kb by utilizing a Covaris ultrasonic disruptor. Afterwards, the large DNA fragments were enriched and purified through magnetic beads. Subsequently, the fragmented DNA underwent damage repair as well as end repair. Hairpin sequencing adapters were then ligated to both ends of the DNA fragments, and exonucleases were used to eliminate any fragments that had unsuccessful ligation. The properly prepared library was then sequenced on the PacBio Sequel IIe platform in the CCS mode. After filtering polymerase reads, raw subreads were acquired, and these were further processed using SMARTLink (version 11.0, parameters: filter_min_qv = 20) to generate HiFi reads. Ultimately, a total of 27.86 Gb HiFi reads were obtained, along with a read number of 1,908,298 and a mean read length of 14,601 bp (as shown in Table 3 and Table 4).

Table 3.

Statics of different types of sequencing reads.

Library types	Sample	Platform	Bases (Gb)	Reads Count	Mean Length (bp)	N50 (bp)
SMRT Bell	muscle	PacBio Sequel IIe (HiFi)	27.86	1,908,298	14,601	15,148
Hi-C	muscle	Illumina Novaseq 6000	49.22	344,604,444	150	150
Short-read	muscle	Illumina Novaseq 6000	50.51	168,353,928	150	150
RNA-seq	muscle	Illumina Novaseq 6000	7.01	23,352,108	150	150
RNA-seq	heart	Illumina Novaseq 6000	6.33	21,090,726	150	150
RNA-seq	spleen	Illumina Novaseq 6000	6.48	21,598,661	150	150
RNA-seq	liver	Illumina Novaseq 6000	6.99	23,304,023	150	150
RNA-seq	kidney	Illumina Novaseq 6000	7.22	24,060,364	150	150

Table 4.

A statistical analysis of the sequencing data obtained from Hi-Fi.

Type	HiFi reads
Read_base (bp)	27,863,295,623
Read_Number	1,908,298
Read_length(max) (bp)	56,349
Read_length(mean) (bp)	14,601
Read_length(N50) (bp)	15,148

In the case of Hi-C sequencing, muscle tissue was processed with paraformaldehyde to stabilize the intracellular DNA conformation. After cell lysis, the crosslinked DNA was digested by the restriction enzyme MboI to create sticky ends. Subsequently, the DNA termini were biotinylated, and DNA ligase was used to connect the DNA fragments. Thereafter, proteases were applied to reverse the crosslinking of DNA. The purified DNA was then randomly fragmented into pieces ranging from 300 to 500 bp and utilized to construct a Hi-C library^16,17. Hi-C sequencing was carried out on the Illumina Novaseq 6000 platform following a paired-end 150 bp (PE-150) sequencing strategy. The raw sequence data obtained were processed through the HiCUP¹⁸ (v 0.8.3), which includes hicup_truncater for identifying and mapping chimeric sequences, hicup_filter for filtering mapped reads, and hicup_deduplicator for removing duplicate contacts. After conducting the aforementioned quality assessments on the Hi-C data, a total of 5,564,221 Total Reads Pairs were obtained, with 2,754,693 Total Paired (mapped) reads (accounting for 49.51%), 2,334,774 Valid Pairs, and 2,237,596 Unique di-Tags (Table 5).

Table 5.

Statistical analysis of sequencing data from Hi-C.

Type	Counts
Total Reads Pairs	5,564,221
Total Paired (mapped)	2,754,693
Total Paired Ratio (%)	49.51
Valid Pairs	2,334,774
Unique di-Tags	2,237,596
Effect Rate (%)	40.21

Genome assembly

The genomic assembly of X. davidi was facilitated by employing the default settings within the Hifiasm software¹⁹ (v 0.16.1-r375). This assembly approach commences from the uncollapsed genomic data, thereby maximizing the retention of haplotype information. Hifiasm was provisioned with HiFi long reads to produce a monoploid assembly and a pair of contig graphs that resolve haplotypes. Consequently, the assembly process resulted in the construction of 134 contigs with a combined length of 1.05 Gb. This genomic assembly size is marginally larger than what was initially anticipated based on the survey results. The average length, maximum contig size, and N50 were 7.8, 57.52, and 33.99 Mb (Table 6), respectively.

Table 6.

Statistics for assembly at the contig level.

Type	Contig
Total length (bp)	1,048,230,699
Total number	134
Average length (bp)	7,822,617
Max length (bp)	57,517,301
Min length (bp)	17,878
N50 length (bp)	33,985,674
N50 number	12
N90 length (bp)	12,284,600
N90 number	32

Hi-C assembly and Chromosome anchoring

The Hi-C technology was employed to differentiate contigs or scaffolds into distinct chromosomes based on the higher probability of intra-chromosomal interactions compared to inter-chromosomal interactions. Additionally, it facilitated the ordering and orientation of contigs or scaffolds on the same chromosome, as the interaction probability decreases with increasing interaction distance along the chromosome. After the Hi-C corrected contigs were integrated into the ALLHiC pipeline²⁰ for pruning, partition, rescue, optimization, and building, a substantial 95.12% of the assembled sequences were anchored to 24 pseudochromosomes²¹ (Fig. 1), with chromosome lengths varying from 31.29 Mb to 60.28 Mb (Table 7). This outcome aligns with the karyotype results derived from cytological observations²², which are consistent with the chromosome numbers of 2n = 48 observed in several Xenocyprididae fish species, such as Plagiognathops microlepis²³, Chanodichthys erythropterus²⁴, M. amblycephala²⁵, and Ctenopharyngodon idella²⁶. Moreover, we manually refined the Hi-C scaffolding based on the chromatin contact matrix within Juicebox²⁷ (Fig. 2). The 24 pseudochromosomes can be clearly identified on the heatmap, and there is a strong signal intensity around the diagonal, which suggests the high quality of the genome assembly. After Hi-C correction, the final assembled genome had a total span of 1.05 Gb, along with a scaffold N50 of 40.13 Mb (Table 8).

Fig. 1.

A circus plot of 24 chromosomes of X. davidi. The tracks from outside to inside are: 24 chromosomes, the distributions of transposable element and bar plot for gene density profile.

Table 7.

Statistics of the 24 anchored chromosomes of X. davidi genome.

Chromosome	Cluster Number	Sequeues Length (bp)
Chr1	2	60,275,181
Chr2	2	57,625,182
Chr3	1	55,586,653
Chr4	1	52,022,816
Chr5	6	51,677,919
Chr6	4	44,187,633
Chr7	4	43,435,225
Chr8	2	43,302,729
Chr9	2	42,370,478
Chr10	3	40,516,972
Chr11	2	40,131,624
Chr12	4	38,782,997
Chr13	2	38,256,806
Chr14	2	38,096,289
Chr15	2	38,035,212
Chr16	3	37,432,084
Chr17	2	37,282,341
Chr18	3	35,654,014
Chr19	2	35,628,516
Chr20	3	35,330,893
Chr21	3	34,045,325
Chr22	1	33,397,843
Chr23	3	32,692,433
Chr24	2	31,287,097

Fig. 2.

Hi-C chromatin interaction heatmap of the X. davidi assembly.

Table 8.

Assembly statistics for Hi-C.

Sample ID	Contig length	Scaffold length	Contig number	Scaffold number
Total	1,048,230,699	1,048,234,399	142	105
Max	57,517,301	60,275,181	—	—
Number > = 2000	—	—	142	105
N50	31,018,029	40,131,624	13	11
N60	27,366,945	38,096,289	17	14
N70	24,045,337	37,282,341	21	17
N80	13,754,553	35,330,893	26	20
N90	10,369,524	32,692,433	34	23

Genome annotation

In the annotation of repetitive sequences within the genome of X. davidi, this study employed two complementary approaches: homology-based alignment²⁸ and de novo prediction²⁹. The homology-based alignment method was predicated on the RepBase database³⁰ and leveraged two software tools, RepeatMasker³¹ (version 4.1.0, parameters: -a -nolow -no_is -norna -parallel 4) and RepeatProteinMask (version 4.1.0, parameters: -noLowSimple -pvalue 0.0001 -engine ncbi), to identify sequences with similarity to known repetitive elements. Conversely, the de novo prediction approach commenced with the establishment of a de novo repetitive sequence library utilizing LTR_FINDER³² (version 1.06, parameters: -C -w 2), RepeatScout (version 1.0.5), and RepeatModeler³³ (version 2.0.1, parameters: -engine ncbi -pa 15), followed by the application of RepeatMasker for prediction. Additionally, within the de novo prediction methodology, the software TRF³⁴ (version 4.09, parameters: 2 7 7 80 10 50 2000 -d -h -ngs) was employed to detect tandem repeats within the X. davidi genome. Ultimately, all predicted results were consolidated and duplicates were eliminated, yielding the identification of 533.34 Mb of repetitive sequences, constituting 50.88% of the assembled genome. The predominant element among these repetitive sequences was long terminal repeats (LTR), which accounted for 45.59% (477.87 Mb) of the assembled genome (Table 9), a significant departure from the genome of the fine-scaled gudgeon, where DNA transposons were the most abundant, comprising 31.55%²³. Long interspersed nuclear elements (LINE) constituted 1.81% of the genome, short interspersed nuclear elements (SINE) constituted 0.02% of the genome, and DNA elements constituted 4.56% of the genome, respectively (Table 9).

Table 9.

Summary of the transposable elements in X. davidi genome.

Type	Denovo + Repbase		TE Proteins		Combined TEs
	Length(bp)	% in Genome	Length(bp)	% in Genome	Length(bp)	% in Genome
DNA	39,993,470	3.82	15,440,641	1.47	47,841,916	4.56
LINE	7,002,432	0.67	14,991,786	1.43	19,012,975	1.81
SINE	172,417	0.02	0	0	172,417	0.02
LTR	475,525,141	45.36	23,810,950	2.27	477,886,955	45.59
Unknown	13,215,147	1.26	0	0	13,215,147	1.26
Total	527,762,304	50.35	54,235,314	5.17	533,336,825	50.88

For the prediction of gene structures within the genome of X. davidi, this manuscript employed a triad of methodologies: de novo prediction, homology-based prediction, and annotation using transcriptome data³⁵. The de novo prediction outcomes were derived from the utilization of Augustus³⁶ (version 3.2.3, parameters: --species = pasa1--uniqueGeneId = TRUE --noInFrameStop = TRUE --GFF3 = on --genemodel = complete --strand = both), GlimmerHMM³⁷ (version 3.0.4, parameters: -d pasa1 -f -g), SNAP (version 2013.11.29, parameters: -gff pasa1.hmm), Geneid (version 1.4, parameters: -P homo_sapiens.param -v -G -p geneid), and Genscan (version 1.0, parameters: HumanIso.smat) software. In the homology-based prediction approach, protein sequences from Carassius auratus³⁸ (GenBank: GCA_003368295.1), Cyprinus carpio³⁹ (GenBank: GCA_000951615.2), Ctenopharyngodon idellus²⁶ (GenBank: GCA_019924925.1), Danio rerio⁴⁰ (GenBank: GCA_000002035.4), Onychostoma macrolepis⁴¹ (GenBank: GCA_012432095.1), M. amblycephala²⁵ (GenBank: GCA_018812025.1), and Opsariichthys bidens⁴² (GenBank: GCA_037194245.1) were downloaded from the NCBI database and used to predict gene structures within the genome through alignment software such as Blastall⁴³ (version 2.2.26, parameters: -e 1e-05 -F T -m 8), Solar (version 0.9.6, parameters: -a prot2genome2 -z -f m8), and Genewise⁴⁴ (version 2.4.1, parameters: -tfor -genesf -gff -sum) (Fig. 3). For the transcriptome data annotation method, high-quality RNA from muscle, heart, spleen, liver, and kidney were used to construct RNAseq libraries. Subsequently, these libraries were sequenced on the Illumina Novaseq 6000 platform, and 150 bp paired-end reads were obtained as a result. Post-sequencing, 37.73 Gb of raw data was generated, which was filtered to yield 34.03 Gb of clean data (Table 3). Subsequent de novo assembly was performed using Trinity (version 2.1.1, parameters:–normalize_reads–full_cleanup–min_glue 2–min_kmer_cov 2–KMER_SIZE 25), alignment analysis with Hisat2 (version 2.0.4), and assembly annotation with Stringtie (version 1.3.3). The gene sets predicted by the above-mentioned three methods were combined into a non-redundant gene set with the help of EvidenceModeler (EVW)⁴⁵ (version 1.1.1, parameters:–segmentSize 200000 –overlapSize 20000–min_intron_length 20). Finally, PASA (http://pasa.sourceforge.net/) was utilized, in conjunction with transcriptome assembly results, to refine the EVW annotation, incorporating UTR and alternative splicing information, to arrive at the final gene set. The genomic prediction for X. davidi resulted in 27,360 genes, with an average transcript length of 10,053.32 bp, an average coding region length of 1,121.54 bp, an average of 6.28 introns per gene, an average intron length of 178.57 bp, and an average exon length of 1,691.46 bp (Table 10, Fig. 4A).

Fig. 3.

Comparisons of the genomic elements of closely related species.

Table 10.

Statistical analyses of the gene structure annotation of the X. davidi.

	Gene set	Number	Average transcript length(bp)	Average CDS length(bp)	Average exons per gene	Average exon length(bp)	Average intron length(bp)
De novo	Augustus	41,765	10,053.32	1,121.54	6.28	178.57	1,691.46
	GlimmerHMM	106,335	8,754.46	573.66	3.89	147.46	2,830.35
	SNAP	59,634	12,134.09	673.63	4.87	138.4	2,963.41
	Geneid	32,058	19,612.36	1,344.29	6.3	213.33	3,445.94
	Genscan	31,763	22,650.39	1,559.14	8.21	189.9	2,925.14
Homolog	Ctenopharyngodon idellus	29,826	9,959.04	1,251.03	6.87	182.16	1,484.06
	Opsariichthys bidens	26,232	13,465.66	1,449.43	7.96	182.16	1,727.23
	Onychostoma macrolepis	24,228	14,824.04	1,520.60	8.58	177.18	1,754.54
	Megalobrama amblycephala	24,907	14,497.76	1,576.73	8.68	181.66	1,682.52
	Carassius auratus	29,056	10,456.97	1,301.02	7.02	185.33	1,520.97
	Cyprinus carpio	26,439	10,919.42	1,317.09	7.12	184.91	1,568.31
	Danio rerio	23,752	14,024.94	1,547.84	8.39	184.57	1,689.21
RNAseq	PASA	26,100	15,024.86	1,453.96	8.96	162.26	1,704.73
	Transcripts	33,355	26,488.96	2,770.83	9.87	280.77	2,674.37
EVM		38,936	12,542.04	1,232.01	6.93	177.83	1,907.85
Pasa-update*		38,720	12,882.32	1,250.40	7.03	177.78	1,927.93
Final set*		27,360	16,345.93	1,566.90	8.87	176.69	1,878.35

Fig. 4.

Gene prediction and functional annotation of the X. davidi genome. (A) Venn diagram of the gene set prediction. (B) Venn diagram of functional annotation based on different databases.

To conduct the functional annotation of protein-coding genes, this study employed a dual strategy utilizing Blastp⁴⁶ (version 2.2.26, parameters: -max_target_seqs. 1 -evalue 1e-4) and Diamond⁴⁷ (version 0.8.22, parameters:–more-sensitive -k 10 -e 1e-5 -f 6 qseqid qlen qstart qend sseqid slen sstart send pident ppos qcovhsp bitscore evalue–salltitles–threads 10) for aligning the protein-coding genes against the SwissProt⁴⁸, NCBI Non-redundant protein (NR) (https://www.ncbi.nlm.nih.gov/refseq/about/nonredundantproteins/), KEGG⁴⁹, InterPro⁵⁰, Gene Ontology (GO)⁵¹, and Pfam⁵² protein databases. The identification of protein domains and motifs was facilitated through the application of InterProScan⁵³ (version 5.35–74.0, parameters: -cpu 20 -format tsv -appl ProDom, SMART, ProSiteProfiles, PRINTS, Pfam, Panther -iprlookup -dp -goterms). Ultimately, 26,672 (97.50%) of the 27,360 predicted genes received annotations from at least one of the databases (Table 11). Among the functionally annotated proteins, 20,599 (75.29%) were corroborated by annotations from all four databases (Fig. 4B).

Table 11.

Non-coding RNA statistical results of X. davidi.

	Type	Copy number	Average length(bp)	Total length(bp)	% of genome
miRNA		2,009	105.11	211,175	0.020146
tRNA		11,325	76.03	861,093	0.082147
rRNA	rRNA	13,213	147.93	1,954,630	0.19
	18S	322	732.25	235,783	0.022493
	28S	790	387.85	306,403	0.02923
	5.8S	116	156.56	18,161	0.001733
	5S	11,985	116.34	1,394,283	0.13
snRNA	snRNA	2,412	160.5	387,133	0.036932
	CD-box	249	152.23	37,906	0.003616
	HACA-box	92	148.27	13,641	0.001301
	splicing	2,031	162.25	329,527	0.031436
	scaRNA	26	194.35	5,053	0.000482
	Unknown	14	71.86	1,006	0.000096

The annotation of non-coding RNAs encompasses tRNA, rRNA, miRNA, and snRNA. To identify tRNA sequences within the genome, the structural characteristics of tRNA were leveraged using the tRNAscan-SE software⁵⁴ (version 1.4). Given the high conservation of rRNA, the rRNA sequences of closely related species were chosen as reference sequences. Moreover, the identification of rRNA within the genome was made easier through BLAST alignment (v 2.2.26, parameters: -e 1e-10 -v 10000 -b 10000). Furthermore, employing the covariance models from the Rfam family, the INFERNAL software, which is integrated within the Rfam⁵⁵ (v 14.1) suite, can be utilized to predict microRNA (miRNA) and small nuclear RNA (snRNA) sequence information on the X. davidi genome. Ultimately, four types of non-coding RNAs were identified from the X. davidi genome, including 2,009 miRNAs, 11,325 tRNAs, 13,213 rRNAs, and 2,412 snRNAs (Table 12).

Table 12.

Statistical analysis of the functional gene annotations of the X. davidi genome.

	Number	Percent (%)
Total	27,360	—
Swissprot	22,152	81
Nr	26,253	96
KEGG	22,742	83.1
InterPro	25,497	93.2
GO	16,878	61.7
Pfam	20,899	76.4
Annotated	26,672	97.5
Unannotated	688	2.5

Data Records

The raw sequence data of RNAseq data, HiC data, PacBio data and Illumina short reads data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA020814⁵⁶, CRA020817⁵⁷, CRA020818⁵⁸, CRA020819⁵⁹, CRA020820⁶⁰, CRA020821⁶¹, CRA020822⁶², CRA020823⁶³). The whole genome sequence data reported in this paper have been deposited in the GenBank (JBLRZY000000000)⁶⁴ and figshare database⁶⁵. The genome annotation files will already be uploaded and shared publicly in the figshare database⁶⁶.

Technical Validation

Evaluation of the genome assembly and annotation

The quality assessment of the genome assembly and annotation was conducted with meticulous attention to detail. The completeness of the assembled genome was assessed by utilizing BUSCO⁶⁷ (version 5.4.3) with the actinopterygii_odb10 database, yielding a 98.3% complete BUSCO score within the assembled genomes (Table 11), which is a testament to the high degree of completeness of our genomic assemblies. The genomic consistency was further appraised by aligning Illumina short-reads to the assembled genomes with BWA⁶⁸ (version 0.7.17), resulting in exceptionally high mapping rates (99.36%) and coverage (99.96%) against the assembled genomes (Table 13). Employing Merqury⁶⁹ (version 1.4.1), the consensus quality value (QV), indicative of per-base consensus accuracy, was calculated to be 50.458 for the assembled genomes. Additionally, a comparative analysis of the length distributions of genes, coding sequences (CDSs), introns, and exons across the genomes of C. idellus, O. bidens, O. macrolepis, M. amblycephala, C. auratus, C. carpio, and D. rerio was performed, revealing similarities (Fig. 3), which substantiates the reliability of our genome annotation. Collectively, the outcomes from these four methodologies demonstrate the high accuracy and completeness of the final genome assembly.

Table 13.

Completeness and accuracy evaluation of the genome.

Type	Number	Percentage (%)
Complete BUSCOs (C)	3,578	98.3
Single-copy BUSCOs (S)	3,502	96.2
Duplicated BUSCOs (D)	76	2.1
Fragmented BUSCOs (F)	25	0.7
Missing BUSCOs (M)	36	1.0
Total BUSCOs	3,640	—
Short-reads mapping rate	—	99.36
Genome covered by reads	—	99.96
Quality value (QV)	50.458	—

Author contributions

X. G. and X. P. conceived the research project. C. S. and S. Y. collected the samples. L. Z. designed the experiment. T. Y., G. Y. and L. Z. performed data analysis. T. Y and C. S. drafted the manuscript. L. Z. and G. Y. revised this manuscript. All authors have read and approved the manuscript.

Code availability

In this study, no custom-written codes were used. All data processing operations were conducted in accordance with the manuals and protocols of the relevant software. The specific parameters for different software and tools were described in the Methods section. For cases where detailed parameters were not specified, default parameters were adopted.

Competing interests

The authors declare no competing interests.