Genome Refinement in Pacific Bluefin Tuna (Thunnus orientalis): Chromosome-level Assembly Using Hybrid Linkage Data

Genome Refinement in Pacific Bluefin Tuna (Thunnus orientalis): Chromosome-level Assembly Using Hybrid Linkage Data | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome Refinement in Pacific Bluefin Tuna (Thunnus orientalis): Chromosome-level Assembly Using Hybrid Linkage Data Xi Fu, Daiki Saka, Kazutoshi Yoshitake, Taiju Saito, Milos Havelka, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7883235/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Apr, 2026 Read the published version in BMC Genomics → Version 1 posted 10 You are reading this latest preprint version Abstract Background: High-quality reference genomes are fundamental for selective breeding and genetic engineering for improving aquaculture productivity. Here, we report an improved chromosome-level genome assembly of Pacific bluefin tuna ( Thunnus orientalis ), a commercially valuable species consumed worldwide. By integrating genetic linkage information from interspecific hybrids of Euthynnus affinis and T. orientalis , we anchored and oriented T. orientalis scaffolds into 24 pseudochromosomes. Results: The updated assembly achieved a scaffold N50 of 34.19 Mb, representing a notable improvement from 13.3 Mb, and contained 22,640 predicted protein-coding genes. Conclusions: This resource provides a robust genomic foundation for future genetic and genomic studies aimed at advancing the selective breeding of T. orientalis in aquaculture. Pacific bluefin tuna aquaculture genome annotation linkage map genome improvement Figures Figure 1 Figure 2 Figure 3 1. Background Tuna ( Thunnus spp.) is among the most valuable fish for human consumption, with bluefin tuna being particularly prized for its high market demand and value [ 1 ]. Full-cycle aquaculture of bluefin tuna has been achieved through advances in larval rearing and juvenile grow-out technologies [ 2 ], yet production and sustainability challenges remain [ 3 ]. Genetic improvement through selective breeding and genetic engineering has emerged as a promising strategy for enhancing production efficiency in fish farming [ 4 ]. Thus, a high-quality species genome assembly is critical to support these genetic improvement efforts. Although recent progress in sequencing technologies has provided valuable genomic insights applicable to basic fish science [ 5 ] and aquaculture practices [ 4 ], genomic information on tuna remains limited. For example, as a model of tuna species, the current Pacific bluefin tuna, Thunnus orientalis (NCBI: txid8238) genome assembly (GenBank accession: GCA_021601225.2, hereafter referred to as the "TUNAREF2023") [ 6 ] remains fragmented (scaffold N50 = 13 Mb) and is not assembled to the chromosome level. High-resolution linkage maps are essential tools in genetics and genomics and support applications such as comparative genome analysis and genome scaffolding [ 7 ]. One of the main challenges in linkage analysis is accurately distinguishing haplotypes, which typically requires high-depth sequencing (over 10× coverage) across numerous individuals to reliably resolve heterozygosity. In contrast, haploid individuals, such as those produced through gynogenesis or androgenesis, require only a single read per polymorphic site to determine the haplotype, thereby substantially reducing the sequencing effort required [ 8 ]. Similarly, in interspecific hybrids, sequence divergence between parental genomes enables the clear assignment of sequencing reads to either maternal or paternal origin. This haploid-equivalent resolution of single-nucleotide polymorphisms (SNPs) facilitates straightforward haplotype determination and allows the independent construction of linkage maps for both diploid parents [ 9 , 10 ]. Here, we leveraged F1 hybrids of two interspecific species, T. orientalis and Euthynnus affinis , to anchor and orient scaffolds of TUNAREF2023 onto 24 pseudochromosomes, producing a chromosome-level assembly with a scaffold N50 of 34.19 Mb with 192 scaffolds, and 22,640 predicted protein-coding genes. This improved resource will support future studies on the genomics, selective breeding, and conservation of T. orientalis . 2. Materials and Methods 2.1 Generation and sequencing of hybrids Hybrids of E. affinis and T. orientalis were generated to establish haploid linkage evidence. Specifically, eggs from a single E. affinis female were artificially inseminated with cryopreserved T. orientalis sperm. After 1–2 hours, eggs undergoing cleavage were selected and transferred to a hatching tank maintained at 24°C. Approximately 36–40 h post fertilization, 200 hatched individuals were collected and preserved in 100% ethanol [ 11 ]. The animal study protocol was reviewed and approved by the Animal Care Committee of Ehime University and the Animal Experimentation Committee of the Graduate School of Agricultural and Life Sciences, University of Tokyo. Genomic DNA was extracted from each F1 hybrid using a NucleoSpin Tissue XS kit (Macherey-Nagel, Düren, Germany). Sequencing libraries were constructed using the Nextera DNA Library Preparation and Nextera Index kits (Illumina, San Diego, CA, USA) following the manufacturer's protocol. Sequencing was conducted on an Illumina HiSeq X Ten sequencing platform to obtain paired-end reads of 150 bp each. Reads were then aligned with BWA mem v.0.7.15 [ 12 ] using default parameters to a reference genome generated by combining the E. affinis genome (GenBank accession: GCA_019973915.1) with the "TUNAREF2023" reference. The aligned reads were sorted using SAMtools v.1.17 [ 13 ], and variant calling was conducted using BCFtools [ 13 ], using the mpileup and call commands with multiallelic-caller and variant-only flags (-mv). 2.2 Genome elongation using linkage analysis Informative SNPs were extracted from specific regions by omitting regions where reads were mapped to male T. orientalis and female E. affinis genomes. Next, the SNPs were confirmed to be heterozygous in the T. orientalis genome. SNPs present in less than 30% of hybrids were removed, with those in which the minor allele was found in less than 30% of the hybrids (i.e., with the major allele appearing in more than 70% of the hybrids). Heterozygous SNPs in more than 10% of the individuals were suspected to be multi-copy genes and were excluded. The extracted SNPs were input into SELDLA (v.2.3.0) for linkage analysis using the following options: DP = 0 --GQ = 0–r 1000 --cs = 2 --mode = crossbreed. 2.3 Repeat annotation Homology-based and de novo strategies have been used to identify repetitive sequences in the SELDLA-extended T. orientalis genome. A de novo repeat library was generated using RepeatModeler v. 2.0.5 [ 14 ] with default parameters. The SELDLA-extended T. orientalis assembly was subsequently screened for repeats using RepeatMasker (version 4.1.7) in two runs using (i) a de novo -generated repeat library and (ii) the Dfam database (release 3.8) under the vertebrate category [ 15 , 16 ]. The results of both runs were integrated into a repetitive sequence library. Using this repeat database, repetitive sequences were identified by homology searches using RepeatMasker and rmBLASTn. 2.4 Gene prediction and functional assignment Gene predictions were conducted using the BRAKER3 pipeline with RNA-seq and protein data [ 17 ]. In detail, RNA sequencing datasets for T. orientalis (Table S1 ) were obtained from the Sequence Read Archive database [ 18 ]. The RNA-Seq datasets were quality-controlled using Trim_Galore v. 0.6.10 ( https://github.com/FelixKrueger/TrimGalore ). The trimmed reads were aligned to the SELDLA-extended T. orientalis genome using STAR v.2.7.11b (STAR, RRID: SCR_004463) [ 19 ]. For homology-based gene prediction, the full genome protein sequences of the four tuna species (Table S2 , upper part) were retrieved from the Ensembl database [ 20 ] or NCBI RefSeq [ 21 ] as sources of homologous proteins. The BRAKER3 pipeline was used, in which GeneMark-ETP integrates both RNA-seq- and protein-based hints to identify ‘high-confidence’ genes. These were subsequently used by AUGUSTUS v.3.5.0 for gene model prediction. Finally, the gene predictions from AUGUSTUS and GeneMark-ETP were merged and refined using TSEBRA [ 22 ]. BRAKER3 was run using the following additional parameters: -gff3-min_contig = 10000. For functional annotation of the predicted genes, protein sequences generated by BRAKER were queried against the Swiss-Prot and NCBI non-redundant (nr) protein databases (downloaded November 2024) using BLASTP with a minimum sequence identity threshold of 30% and an E-value cutoff of 1e-6. Protein domains were predicted using InterProScan v.5.71 [ 23 ] with default parameters, and the associated Gene Ontology (GO) terms were retrieved from the corresponding InterPro entries. Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology (KO) annotations were assigned using KofamKOALA [ 24 ], applying an E-value threshold of 1e-6. To categorize GO terms into three major functional domains (biological processes, molecular functions, and cellular components), the ontology structure was provided in the go-basic.obo file was parsed using the GOATOOLS library [ 25 ]. 2.5 Quality assessment To assess the completeness of the SELDLA-extended T. orientalis assembly, two approaches were used: (1) evaluation based on complete Core Vertebrate Genes (CVGs) [ 26 ] and (2) BUSCO. First, 233 complete CVGs were confirmed using gVolante [ 27 ]. Second, the actinopterygii_obd12 lineage dataset (creation date: 2025-04-11, number of genomes: 75, number of BUSCOs: 7,207) was used to run BUSCO v.5.8.2 [ 28 ] in genome mode for the SELDLA-extended T. orientalis assembly. To assess the accuracy of scaffold arrangement in the 24 pseudochromosomes, the genomic locations of single-copy orthologs were compared between T. orientalis and Japanese medaka ( Oryzias latipes ) (GenBank accession: GCF_002234675.1). The comparison was visualized using Circos v0.69- 8 (Circos, RRID: SCR_011798) [ 29 ]. Only complete and unduplicated orthologs co-identified using BUSCO across the two species were selected for comparison. 2.6 Comparative Genomic Analysis of Orthologs Orthologous relationships were inferred using OrthoFinder v.3.0.1 b1 [ 30 ] by comparing the predicted genes in T. orientalis with those from the following eight representative fish species listed in Table S2 : zebrafish ( Danio rerio ), climbing perch ( Anabas testudineus ), tiger tail seahorse ( Hippocampus comes ), Japanese medaka ( Oryzias latipes ), Yellowtail amberjack ( Seriola lalandi ), three-spined stickleback ( Gasterosteus aculeatus ), southern bluefin tuna ( Thunnus maccoyii ), and yellowfin tuna ( Thunnus albacares ). The results were visualized using ComplexUpset [ 31 ]. 3. Results 3.1 Sequencing summary Hybrid sequencing generated 390 Gb reads. The average mapping rate for hybrids on the combined E. affinis and "TUNAREF2023" reference genome was 94.2% on average. 3.2 Chromosome-level assembly using linkage map data A total of 4,048,459 SNPs specific to T. orientalis were used for linkage analysis using SELDLA software. The final T. orientalis assembly comprised 24 expected scaffolds larger than 10 Mb (Table S3 ), consistent with the established karyotype of T. orientalis (n = 24) [ 32 ], with 168 unplaced scaffolds. The SELDLA-extended assembly had a total N50 of 34.19 Mb, with the longest scaffold measuring 42.02 Mb. The total assembly size was 795.65 Mb, corresponding to 99.3% of the estimated genome size (~ 800 Mb) [ 33 ]. The chromosome-level scaffolds cover 791.84 Mb (99.55%) of the total sequences in the new assembly. This assembly yielded a 3-fold increase in average scaffold length and a 2.6-fold increase in the N50 scaffold compared to the "TUNAREF2023.” To assess the distribution of scaffolds within the pseudochromosomes, each sub-scaffold inherited from the TUNAREF2023 assembly was categorized as either "Located only" or "Located and Oriented" according to our linkage information. In cases where the scaffolds were subdivided into fragments and assigned to different pseudochromosomes, their contributions were normalized against the length of the parent scaffold. The placement of scaffolds from the TUNAREF2023 assembly across the 24 pseudochromosomes is summarized (Table 1 ; Supplementary File S1). For each chromosome, the number of scaffolds assigned, the total sequence length of scaffolds that were both located and oriented versus those that could only be localized, and the proportion of sequences in each category are reported. This provides a genome-wide overview of the anchoring process, highlighting that the vast majority of the sequences were not only placed but also confidently oriented by linkage information. Table 1 Summary of scaffold placement and orientation using linkage analysis Chromosome #TUNAREF 2023 Scaffolds Mb Oriented Mb Located-only Total Mb % Oriented % Located-only LG01 22 38.32 0.76 39.08 98.1 1.9 LG02 11 20.9 0.52 21.42 97.6 2.4 LG03 25 39.61 1.78 41.39 95.7 4.3 LG04 22 35.1 0.79 35.89 97.8 2.2 LG05 14 38.24 0.31 38.55 99.2 0.8 LG06 15 34.59 1.11 35.71 96.9 3.1 LG07 28 34.19 1.79 35.98 95 5 LG08 9 29.38 1.22 30.6 96 4 LG09 45 33.81 2.9 36.71 92.1 7.9 LG10 14 34.94 0.46 35.4 98.7 1.3 LG11 10 30.16 0.54 30.7 98.2 1.8 LG12 12 31.94 0.5 32.44 98.5 1.5 LG13 17 30.71 5.62 36.33 84.5 15.5 LG14 17 32.05 0.51 32.56 98.4 1.6 LG15 5 31.59 0.17 31.76 99.5 0.5 LG16 27 33.62 1.1 34.71 96.8 3.2 LG17 18 31.45 1.39 32.84 95.8 4.2 LG18 30 25.26 2.07 27.33 92.4 7.6 LG19 18 27.89 0.88 28.77 97 3 LG20 16 27.99 0.19 28.18 99.3 0.7 LG21 8 33.82 0.24 34.06 99.3 0.7 LG22 4 30.37 0.13 30.51 99.6 0.4 LG23 17 26.01 0.67 26.68 97.5 2.5 LG24 17 29.68 0.6 30.27 98 2 Total 421 761.62 26.25 787.87 96.7 3.3 For ease of presentation, the chromosome names are shown as LG01, LG02, …. These names correspond to the official chromosome identifiers in the NCBI-submitted assembly (e.g., C0000000001_linkage_scaffold_1, C0000000002_linkage_scaffold_2) as well as those listed in Supplementary File S1. Genome-wide totals are provided in the last row. Detailed per-scaffold information is available in Supplementary File S1. 3.3 Repeat and gene set annotations Repetitive sequences in the T. orientalis genome were annotated using a combination of ab initio - and homology-based methods. As a result, repetitive elements (REs) comprised 30.41% (~ 242 Mb) of the SELDLA-extended T. orientalis assembly (Table 2 ; Table S4 ), predominantly consisting of long interspersed nuclear elements (LINEs) (25.8 Mb, 3.24%) and DNA transposons (17 Mb, 2.14%). These elements were unevenly distributed along the chromosomes. However, the largest proportion of repetitive elements (21.9%) remained “unclassified” and required further identification. A negative correlation was observed between repeat element abundance and gene density across non-overlapping 100-kb windows in the chromosome-scale scaffolds (Fig. 1 , tracks d and e). Table 2 Genome assembly statistics and annotation of the Thunnus orientalis genome Genome assembly Value Number of sequences 192 Total length (bp) 795,647,880 N50 (bp) 34,195,991 Max scaffold length (bp) 42,023,264 GC content (%) 39.67 Number of gaps 665 BUSCO (%) C = 98.3; S = 97.4; D = 0.9 Repeats annotation Value SINEs (bp) 1,533,091 (0.19%) LINEs (bp) 25,797,628 (3.24%) LTR elements (bp) 3,249,944 (0.41%) DNA transposons (bp) 17,004,590 (2.41%) Simple repeats (bp) 17,103,504 (2.15%) Low complexity (bp) 2,638,066 (0.33%) Small RNA (bp) 1,411,561 (0.18%) Unclassified (bp) 174,264,846 (21.9%) Total (bp) 241,985,022 (30.41%) Gene annotation Number of predicted genes 22,640 Average gene length (bp) 12,400 Average CDS length (bp) 1,807 Average exon per transcript 11 Percent of the genome covered by genes 35 BUSCO C = 88.5; S = 69; D = 19.5 Functional annotation total 22,410 (98.98%) Swissport 19,942 (88.07%) TrEMBL 22,144 (97.81%) NCBI nr 21,207 (93.67%) InterPro 20,374 (89.99%) ໿ BUSCO = benchmarking universal single-copy orthologs; C = complete; S = complete and single copy; D = complete and duplicated; F = fragmented; M = missing; LINEs = long interspersed nuclear elements; LTR = long terminal repeat; SINEs = short interspersed nuclear elements; CDS = coding DNA sequence A total of 22,640 putative protein-coding genes were identified, accounting for 35% of the T. orientalis genome (Table 2 ). Nearly all these genes (22,410; 98.98%) were functionally supported by at least one database, including 22,272 genes (98.37%) that matched the investigated protein databases using InterProScan (Table S5). A total of 13,587 predicted genes were annotated using KO terms, representing 8,509 unique KO genes (Supplementary File S2). Similarly, InterProScan assigned at least one GO term to 18,137 genes, corresponding to 6,901 unique GO terms (Supplementary File S3). The number of predicted protein-coding genes in T. orientalis was comparable to that of closely related species, such as yellowfin tuna (24,623 genes), eastern little tuna (23,059 genes), and southern bluefin tuna (24,659 genes) (Table S2 ). Although it is lower than the previously reported annotation for T. orientalis (26,433 genes) [ 34 ], the current assembly represents a substantial quality improvement (N50 increased from 8.2 kb to 34.19 Mb). 3.4 Data validation First, the SELDLA-extended T. orientalis assembly contained 230 of 233 (98.7%) complete CVGs [ 26 ]. Second, the assembly showed 7,085 (98.3%) complete BUSCOs, of which 7,020 (97.4%) were single copies and 65 (0.9%) were duplicates (Table 2 ), indicating completeness. Further, 6,727 of the 6,773 single-copy orthologs were localized on the same chromosomes in both T. orientalis and O. latipes (Fig. 2 ), suggesting high consistency between the two genomes. Based on the comparison results, 24 scaffolds of > 10Mb in the SELDLA-extended T. orientalis assembly were numbered concordantly with the O. latipes chromosomes (Figure S1 ). The completeness of the predicted gene set was also evaluated using BUSCO, which identified 6,378 (88.5%) of the 7,207 actinopterygian single-copy orthologs (Table 2 ). In total, 218,379 genes from the nine fish species were clustered into 21,668 orthogroups, of which 11,437 were conserved across all species (Fig. 3 ). Of the 22,640 predicted genes in T. orientalis , 21,428 (94.6%) were assigned to 17,807 orthogroups. Species of Scombridae, T. albacares , T. maccoyii , and T. orientalis , included 323 unique orthogroups. A total of 761 orthogroups not detected in T. orientalis were identified in all other species, suggesting that although the corresponding genes may not have been annotated, they were likely present in its genome. Conclusions The new chromosome-level T. orientalis genome assembly presented herein has a scaffold N50 of 34.19Mb, 98.3% BUSCO completeness, and 22,640 annotated genes, representing a step advance in the previously published assembly. Of the 22,640 protein-coding genes, 4,677 (20.65%) contained putative, alternative, spliced transcripts. The availability of chromosome-level genome assemblies is expected to support applied and fundamental research, particularly the genetic improvement of this commercially valuable fish species. Abbreviations NCBI = National Center for Biotechnology Information; BUSCO = benchmarking universal single-copy orthologs; LINEs = long interspersed nuclear elements; LTR = long terminal repeat; SINEs = short interspersed nuclear elements; CDS = coding DNA sequence Declarations Authors' contributions SA and TI designed this study. DS conducted the experiments. The Ehime members, TS, ON, TM, RG, MH ( E. affinis ), and TI ( T. orientalis ) provided resources and contributed to the generation of hybrid fish. KY developed SELDLA software. XF and KY analyzed the data. KY and SA performed data analysis. XF, KY, and SA drafted the manuscript. All the authors have read and approved the final version of the manuscript. SA and TI conceived of and designed the study. DS performed the experiments. TS, ON, TM, RG, MH, and TI provided resources and contributed to the generation of the hybrid fish. KY developed SELDLA software. XF and KY conducted data analysis with guidance from KY and SA. XF and SA drafted the manuscript. All authors reviewed and approved the final version of the manuscript. Acknowledgements We thank Director Tokihiko Okada and the staff of the Aquaculture Technology and Production Center of Kindai University for providing tuna testes. Funding This study was supported in part by the Japan Society for the Promotion of Science KAKENHI (Grant nos. 20H00429, 23H00342). Availability of data and materials The sequencing dataset and genome assembly are stored in publicly available databases. The hybrid short-read sequencing data used for the genome assembly were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) repository (submission PRJNA1277492). All data and analysis codes supporting the findings of this study are available in Zenodo with the identifier 10.5281/zenodo.17239627. Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests The authors declare no competing interests. References Benetti D, Partridge G, Buentello A. Advances in Tuna Aquaculture: From Hatchery to Market. Academic Press; 2015. Higuchi K, Takashi T, Okita K, Hayashida T, Nyuji M, Gen K. Effect of a long photoperiod on the timing of spawning in the Pacific bluefin tuna, Thunnus orientalis. Aquac Rep. 2024;36:102062. Ahmad A, Sheikh Abdullah SR, Hasan HA, Othman AR, Ismail N ’izzati. 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Supplementary Files SupplementaryMaterials.docx SupplementaryFileS3.xlsx SupplementaryFileS1.xlsx SupplementaryFileS2.xlsx Cite Share Download PDF Status: Published Journal Publication published 20 Apr, 2026 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 18 Feb, 2026 Reviews received at journal 17 Feb, 2026 Reviews received at journal 09 Feb, 2026 Reviewers agreed at journal 19 Jan, 2026 Reviewers agreed at journal 14 Jan, 2026 Reviewers invited by journal 22 Oct, 2025 Editor invited by journal 22 Oct, 2025 Editor assigned by journal 21 Oct, 2025 Submission checks completed at journal 21 Oct, 2025 First submitted to journal 17 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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16:23:42","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":103871,"visible":true,"origin":"","legend":"","description":"","filename":"1da036aae3054c748bcccae6f26bb0ed1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7883235/v1/12bd7144318c44cbf5eb7300.xml"},{"id":95117071,"identity":"8dd5d75d-465f-43eb-8c46-92d75c4d3672","added_by":"auto","created_at":"2025-11-04 13:13:15","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113130,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7883235/v1/6e22387e0f2b14530c98ae5d.html"},{"id":95117057,"identity":"61bf1c88-67a2-4d26-9b58-bcb7773e0587","added_by":"auto","created_at":"2025-11-04 13:13:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":654604,"visible":true,"origin":"","legend":"\u003cp\u003eCircos plot depicting genome features across the 24 \u003cem\u003eThunnus orientalis\u003c/em\u003e chromosomes. (a) \u003cem\u003eT. orientalis\u003c/em\u003e chromosomes (LG01–LG24 on an Mb scale). (b) Short-read coverage plot. Coverage within 2 SD of the mean is shown as grey circles. Abnormal sequence coverage (± 2 SD from the mean) is indicated with a red square or triangle, respectively. (c) GC content (percentage). (d) Distribution of repeat elements: DNA transposons (light orange bar), LINEs (yellow bar), LTRs (green bar), and SINEs (black bar). (e) Gene density. A window size of 1 Mb was used for tracks (d) and (e), 0.1 Mb for track (c), and 0.2 Mb for track (b)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7883235/v1/b9f27f736aa9643f3b28e8cc.png"},{"id":95226106,"identity":"d3bbc0e9-85ea-4855-9dc0-87fb6ff2a0a3","added_by":"auto","created_at":"2025-11-05 16:26:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":738215,"visible":true,"origin":"","legend":"\u003cp\u003eChromosome-level synteny between \u003cem\u003eThunnus orientalis\u003c/em\u003e (right, LG01–LG24) and Japanese medaka, \u003cem\u003eOryzias latipes\u003c/em\u003e (left, O.01–O.24) based on 6,773 complete single-copy orthologs\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7883235/v1/63802b05187826b85f594154.png"},{"id":95117059,"identity":"8932430b-164b-411a-90a4-fc3259cffddf","added_by":"auto","created_at":"2025-11-04 13:13:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":148431,"visible":true,"origin":"","legend":"\u003cp\u003eOrthogroup size and proportion of genes assigned to orthogroups per species. Left: Bar plot showing orthogroup size metrics for each species. Yellow indicates the total number of genes; blue represents the number of genes assigned to orthogroups; green denotes the number of orthogroups containing each species; red shows the number of unassigned genes. Right: UpSet plot showing the intersections of orthogroups among eight fish species. Bars represent the number of orthogroups shared by the species marked by connected dots below the \u003cem\u003ex\u003c/em\u003e-axis. A total of 11,437 orthogroups were conserved across all species. Orthogroups shared exclusively by the three tuna species \u003cem\u003eThunnus albacares\u003c/em\u003e, \u003cem\u003eT. maccoyii\u003c/em\u003e, and \u003cem\u003eT. orientalis\u003c/em\u003e are highlighted in red (n = 323). Notably, \u003cem\u003eT. orientalis\u003c/em\u003e lacks 761 orthogroups that are present in all other species, highlighted in light blue. Intersections comprising fewer than 100 orthogroups are omitted for visual clarity.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7883235/v1/3fd38c413b562078835c455d.png"},{"id":107927756,"identity":"65d3fc47-f7c9-4a47-88bd-4449a44c00b7","added_by":"auto","created_at":"2026-04-27 16:03:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1671568,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7883235/v1/0be930e4-893b-41fe-99fc-4a1556acffc6.pdf"},{"id":95225744,"identity":"f65ad16f-503d-4d61-94da-0b128007d8da","added_by":"auto","created_at":"2025-11-05 16:25:28","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":198366,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7883235/v1/3791ad403deb3b2e482f0d6b.docx"},{"id":95117064,"identity":"3aa8983f-90ca-4268-aa09-319b9c96e245","added_by":"auto","created_at":"2025-11-04 13:13:15","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":145666,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7883235/v1/ff811c1d6c5f8562f5f8f6e1.xlsx"},{"id":95117077,"identity":"06e3d93b-744f-4bc6-b65c-44ba1d68b3b6","added_by":"auto","created_at":"2025-11-04 13:13:15","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10442,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7883235/v1/e04fbda161acb4df89f1b00d.xlsx"},{"id":95117067,"identity":"234f293b-892c-450c-8e9a-ea4aaba8969d","added_by":"auto","created_at":"2025-11-04 13:13:15","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":303643,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7883235/v1/70a6a4e337928a11b6245142.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome Refinement in Pacific Bluefin Tuna (Thunnus orientalis): Chromosome-level Assembly Using Hybrid Linkage Data","fulltext":[{"header":"1. Background","content":"\u003cp\u003eTuna (\u003cem\u003eThunnus\u003c/em\u003e spp.) is among the most valuable fish for human consumption, with bluefin tuna being particularly prized for its high market demand and value [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Full-cycle aquaculture of bluefin tuna has been achieved through advances in larval rearing and juvenile grow-out technologies [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], yet production and sustainability challenges remain [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGenetic improvement through selective breeding and genetic engineering has emerged as a promising strategy for enhancing production efficiency in fish farming [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Thus, a high-quality species genome assembly is critical to support these genetic improvement efforts. Although recent progress in sequencing technologies has provided valuable genomic insights applicable to basic fish science [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and aquaculture practices [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], genomic information on tuna remains limited. For example, as a model of tuna species, the current Pacific bluefin tuna, \u003cem\u003eThunnus orientalis\u003c/em\u003e (NCBI: txid8238) genome assembly (GenBank accession: GCA_021601225.2, hereafter referred to as the \"TUNAREF2023\") [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] remains fragmented (scaffold N50\u0026thinsp;=\u0026thinsp;13 Mb) and is not assembled to the chromosome level.\u003c/p\u003e\u003cp\u003eHigh-resolution linkage maps are essential tools in genetics and genomics and support applications such as comparative genome analysis and genome scaffolding [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOne of the main challenges in linkage analysis is accurately distinguishing haplotypes, which typically requires high-depth sequencing (over 10\u0026times; coverage) across numerous individuals to reliably resolve heterozygosity. In contrast, haploid individuals, such as those produced through gynogenesis or androgenesis, require only a single read per polymorphic site to determine the haplotype, thereby substantially reducing the sequencing effort required [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Similarly, in interspecific hybrids, sequence divergence between parental genomes enables the clear assignment of sequencing reads to either maternal or paternal origin. This haploid-equivalent resolution of single-nucleotide polymorphisms (SNPs) facilitates straightforward haplotype determination and allows the independent construction of linkage maps for both diploid parents [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHere, we leveraged F1 hybrids of two interspecific species, \u003cem\u003eT. orientalis\u003c/em\u003e and \u003cem\u003eEuthynnus affinis\u003c/em\u003e, to anchor and orient scaffolds of TUNAREF2023 onto 24 pseudochromosomes, producing a chromosome-level assembly with a scaffold N50 of 34.19 Mb with 192 scaffolds, and 22,640 predicted protein-coding genes. This improved resource will support future studies on the genomics, selective breeding, and conservation of \u003cem\u003eT. orientalis\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Generation and sequencing of hybrids\u003c/h2\u003e\u003cp\u003eHybrids of \u003cem\u003eE. affinis\u003c/em\u003e and \u003cem\u003eT. orientalis\u003c/em\u003e were generated to establish haploid linkage evidence. Specifically, eggs from a single \u003cem\u003eE. affinis\u003c/em\u003e female were artificially inseminated with cryopreserved \u003cem\u003eT. orientalis\u003c/em\u003e sperm. After 1\u0026ndash;2 hours, eggs undergoing cleavage were selected and transferred to a hatching tank maintained at 24\u0026deg;C. Approximately 36\u0026ndash;40 h post fertilization, 200 hatched individuals were collected and preserved in 100% ethanol [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The animal study protocol was reviewed and approved by the Animal Care Committee of Ehime University and the Animal Experimentation Committee of the Graduate School of Agricultural and Life Sciences, University of Tokyo.\u003c/p\u003e\u003cp\u003eGenomic DNA was extracted from each F1 hybrid using a NucleoSpin Tissue XS kit (Macherey-Nagel, D\u0026uuml;ren, Germany). Sequencing libraries were constructed using the Nextera DNA Library Preparation and Nextera Index kits (Illumina, San Diego, CA, USA) following the manufacturer's protocol. Sequencing was conducted on an Illumina HiSeq X Ten sequencing platform to obtain paired-end reads of 150 bp each. Reads were then aligned with BWA mem v.0.7.15 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] using default parameters to a reference genome generated by combining the \u003cem\u003eE. affinis\u003c/em\u003e genome (GenBank accession: GCA_019973915.1) with the \"TUNAREF2023\" reference. The aligned reads were sorted using SAMtools v.1.17 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and variant calling was conducted using BCFtools [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], using the \u003cem\u003empileup\u003c/em\u003e and \u003cem\u003ecall\u003c/em\u003e commands with multiallelic-caller and variant-only flags (-mv).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Genome elongation using linkage analysis\u003c/h2\u003e\u003cp\u003eInformative SNPs were extracted from specific regions by omitting regions where reads were mapped to male \u003cem\u003eT. orientalis\u003c/em\u003e and female \u003cem\u003eE. affinis\u003c/em\u003e genomes. Next, the SNPs were confirmed to be heterozygous in the \u003cem\u003eT. orientalis\u003c/em\u003e genome. SNPs present in less than 30% of hybrids were removed, with those in which the minor allele was found in less than 30% of the hybrids (i.e., with the major allele appearing in more than 70% of the hybrids). Heterozygous SNPs in more than 10% of the individuals were suspected to be multi-copy genes and were excluded. The extracted SNPs were input into SELDLA (v.2.3.0) for linkage analysis using the following options: DP\u0026thinsp;=\u0026thinsp;0 --GQ\u0026thinsp;=\u0026thinsp;0\u0026ndash;r 1000 --cs\u0026thinsp;=\u0026thinsp;2 --mode\u0026thinsp;=\u0026thinsp;crossbreed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Repeat annotation\u003c/h2\u003e\u003cp\u003eHomology-based and \u003cem\u003ede novo\u003c/em\u003e strategies have been used to identify repetitive sequences in the SELDLA-extended \u003cem\u003eT. orientalis\u003c/em\u003e genome. A \u003cem\u003ede novo\u003c/em\u003e repeat library was generated using RepeatModeler v. 2.0.5 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] with default parameters. The SELDLA-extended \u003cem\u003eT. orientalis\u003c/em\u003e assembly was subsequently screened for repeats using RepeatMasker (version 4.1.7) in two runs using (i) a \u003cem\u003ede novo\u003c/em\u003e-generated repeat library and (ii) the Dfam database (release 3.8) under the vertebrate category [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The results of both runs were integrated into a repetitive sequence library. Using this repeat database, repetitive sequences were identified by homology searches using RepeatMasker and rmBLASTn.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Gene prediction and functional assignment\u003c/h2\u003e\u003cp\u003eGene predictions were conducted using the BRAKER3 pipeline with RNA-seq and protein data [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In detail, RNA sequencing datasets for \u003cem\u003eT. orientalis\u003c/em\u003e (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were obtained from the Sequence Read Archive database [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The RNA-Seq datasets were quality-controlled using Trim_Galore v. 0.6.10 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/FelixKrueger/TrimGalore\u003c/span\u003e\u003cspan address=\"https://github.com/FelixKrueger/TrimGalore\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The trimmed reads were aligned to the SELDLA-extended \u003cem\u003eT. orientalis\u003c/em\u003e genome using STAR v.2.7.11b (STAR, RRID: SCR_004463) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For homology-based gene prediction, the full genome protein sequences of the four tuna species (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, upper part) were retrieved from the Ensembl database [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] or NCBI RefSeq [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] as sources of homologous proteins. The BRAKER3 pipeline was used, in which GeneMark-ETP integrates both RNA-seq- and protein-based hints to identify \u0026lsquo;high-confidence\u0026rsquo; genes. These were subsequently used by AUGUSTUS v.3.5.0 for gene model prediction. Finally, the gene predictions from AUGUSTUS and GeneMark-ETP were merged and refined using TSEBRA [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. BRAKER3 was run using the following additional parameters: -gff3-min_contig\u0026thinsp;=\u0026thinsp;10000.\u003c/p\u003e\u003cp\u003eFor functional annotation of the predicted genes, protein sequences generated by BRAKER were queried against the Swiss-Prot and NCBI non-redundant (nr) protein databases (downloaded November 2024) using BLASTP with a minimum sequence identity threshold of 30% and an E-value cutoff of 1e-6. Protein domains were predicted using InterProScan v.5.71 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] with default parameters, and the associated Gene Ontology (GO) terms were retrieved from the corresponding InterPro entries. Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology (KO) annotations were assigned using KofamKOALA [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], applying an E-value threshold of 1e-6. To categorize GO terms into three major functional domains (biological processes, molecular functions, and cellular components), the ontology structure was provided in the go-basic.obo file was parsed using the GOATOOLS library [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Quality assessment\u003c/h2\u003e\u003cp\u003eTo assess the completeness of the SELDLA-extended \u003cem\u003eT. orientalis\u003c/em\u003e assembly, two approaches were used: (1) evaluation based on complete Core Vertebrate Genes (CVGs) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and (2) BUSCO. First, 233 complete CVGs were confirmed using gVolante [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Second, the actinopterygii_obd12 lineage dataset (creation date: 2025-04-11, number of genomes: 75, number of BUSCOs: 7,207) was used to run BUSCO v.5.8.2 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] in genome mode for the SELDLA-extended \u003cem\u003eT. orientalis\u003c/em\u003e assembly.\u003c/p\u003e\u003cp\u003eTo assess the accuracy of scaffold arrangement in the 24 pseudochromosomes, the genomic locations of single-copy orthologs were compared between \u003cem\u003eT. orientalis\u003c/em\u003e and Japanese medaka (\u003cem\u003eOryzias latipes\u003c/em\u003e) (GenBank accession: GCF_002234675.1). The comparison was visualized using Circos v0.69- 8 (Circos, RRID: SCR_011798) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Only complete and unduplicated orthologs co-identified using BUSCO across the two species were selected for comparison.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Comparative Genomic Analysis of Orthologs\u003c/h2\u003e\u003cp\u003eOrthologous relationships were inferred using OrthoFinder v.3.0.1 b1 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] by comparing the predicted genes in \u003cem\u003eT. orientalis\u003c/em\u003e with those from the following eight representative fish species listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e: zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e), climbing perch (\u003cem\u003eAnabas testudineus\u003c/em\u003e), tiger tail seahorse (\u003cem\u003eHippocampus comes\u003c/em\u003e), Japanese medaka (\u003cem\u003eOryzias latipes\u003c/em\u003e), Yellowtail amberjack (\u003cem\u003eSeriola lalandi\u003c/em\u003e), three-spined stickleback (\u003cem\u003eGasterosteus aculeatus\u003c/em\u003e), southern bluefin tuna (\u003cem\u003eThunnus maccoyii\u003c/em\u003e), and yellowfin tuna (\u003cem\u003eThunnus albacares\u003c/em\u003e). The results were visualized using ComplexUpset [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Sequencing summary\u003c/h2\u003e\u003cp\u003eHybrid sequencing generated 390 Gb reads. The average mapping rate for hybrids on the combined \u003cem\u003eE. affinis\u003c/em\u003e and \"TUNAREF2023\" reference genome was 94.2% on average.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Chromosome-level assembly using linkage map data\u003c/h2\u003e\u003cp\u003eA total of 4,048,459 SNPs specific to \u003cem\u003eT. orientalis\u003c/em\u003e were used for linkage analysis using SELDLA software. The final \u003cem\u003eT. orientalis\u003c/em\u003e assembly comprised 24 expected scaffolds larger than 10 Mb (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), consistent with the established karyotype of \u003cem\u003eT. orientalis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;24) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], with 168 unplaced scaffolds. The SELDLA-extended assembly had a total N50 of 34.19 Mb, with the longest scaffold measuring 42.02 Mb. The total assembly size was 795.65 Mb, corresponding to 99.3% of the estimated genome size (~\u0026thinsp;800 Mb) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The chromosome-level scaffolds cover 791.84 Mb (99.55%) of the total sequences in the new assembly. This assembly yielded a 3-fold increase in average scaffold length and a 2.6-fold increase in the N50 scaffold compared to the \"TUNAREF2023.\u0026rdquo;\u003c/p\u003e\u003cp\u003eTo assess the distribution of scaffolds within the pseudochromosomes, each sub-scaffold inherited from the TUNAREF2023 assembly was categorized as either \"Located only\" or \"Located and Oriented\" according to our linkage information. In cases where the scaffolds were subdivided into fragments and assigned to different pseudochromosomes, their contributions were normalized against the length of the parent scaffold. The placement of scaffolds from the TUNAREF2023 assembly across the 24 pseudochromosomes is summarized (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Supplementary File S1). For each chromosome, the number of scaffolds assigned, the total sequence length of scaffolds that were both located and oriented versus those that could only be localized, and the proportion of sequences in each category are reported. This provides a genome-wide overview of the anchoring process, highlighting that the vast majority of the sequences were not only placed but also confidently oriented by linkage information.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSummary of scaffold placement and orientation using linkage analysis\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChromosome\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e#TUNAREF 2023 Scaffolds\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMb Oriented\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMb Located-only\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTotal Mb\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e% Oriented\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e% Located-only\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e38.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e39.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e98.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e21.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e97.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e39.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e41.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e95.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e35.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e35.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e97.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e38.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e38.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e99.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e34.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e35.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e96.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e34.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e35.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e29.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e30.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e33.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e36.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e92.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e34.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e35.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e98.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e30.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e98.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e31.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e32.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e98.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e36.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e84.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e32.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e32.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e98.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e31.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e31.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e99.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e33.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e34.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e96.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e31.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e32.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e95.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e25.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e27.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e92.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e28.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e28.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e99.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e33.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e34.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e99.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e30.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e99.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e26.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e26.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e97.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLG24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e29.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e30.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e421\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e761.62\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e26.25\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e787.87\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e96.7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e3.3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eFor ease of presentation, the chromosome names are shown as LG01, LG02, \u0026hellip;. These names correspond to the official chromosome identifiers in the NCBI-submitted assembly (e.g., C0000000001_linkage_scaffold_1, C0000000002_linkage_scaffold_2) as well as those listed in Supplementary File S1. Genome-wide totals are provided in the last row. Detailed per-scaffold information is available in Supplementary File S1.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Repeat and gene set annotations\u003c/h2\u003e\u003cp\u003eRepetitive sequences in the \u003cem\u003eT. orientalis\u003c/em\u003e genome were annotated using a combination of \u003cem\u003eab initio\u003c/em\u003e- and homology-based methods. As a result, repetitive elements (REs) comprised 30.41% (~\u0026thinsp;242 Mb) of the SELDLA-extended \u003cem\u003eT. orientalis\u003c/em\u003e assembly (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), predominantly consisting of long interspersed nuclear elements (LINEs) (25.8 Mb, 3.24%) and DNA transposons (17 Mb, 2.14%). These elements were unevenly distributed along the chromosomes. However, the largest proportion of repetitive elements (21.9%) remained \u0026ldquo;unclassified\u0026rdquo; and required further identification. A negative correlation was observed between repeat element abundance and gene density across non-overlapping 100-kb windows in the chromosome-scale scaffolds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e, tracks d and e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGenome assembly statistics and annotation of the \u003cem\u003eThunnus orientalis\u003c/em\u003e genome\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGenome assembly\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eValue\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of sequences\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e192\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal length (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e795,647,880\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN50 (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e34,195,991\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMax scaffold length (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e42,023,264\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGC content (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e39.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of gaps\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e665\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBUSCO (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u0026thinsp;=\u0026thinsp;98.3; S\u0026thinsp;=\u0026thinsp;97.4; D\u0026thinsp;=\u0026thinsp;0.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRepeats annotation\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eValue\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSINEs (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1,533,091 (0.19%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLINEs (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25,797,628 (3.24%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLTR elements (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3,249,944 (0.41%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDNA transposons (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17,004,590 (2.41%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSimple repeats (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17,103,504 (2.15%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow complexity (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2,638,066 (0.33%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSmall RNA (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1,411,561 (0.18%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUnclassified (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e174,264,846 (21.9%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e241,985,022 (30.41%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGene annotation\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of predicted genes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e22,640\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAverage gene length (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12,400\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAverage CDS length (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1,807\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAverage exon per transcript\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePercent of the genome covered by genes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBUSCO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u0026thinsp;=\u0026thinsp;88.5; S\u0026thinsp;=\u0026thinsp;69; D\u0026thinsp;=\u0026thinsp;19.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFunctional annotation total\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e22,410 (98.98%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSwissport\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19,942 (88.07%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTrEMBL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e22,144 (97.81%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNCBI nr\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e21,207 (93.67%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInterPro\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20,374 (89.99%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"2\"\u003e\u003cb\u003e໿\u003c/b\u003e\u003cem\u003eBUSCO\u0026thinsp;=\u0026thinsp;benchmarking universal single-copy orthologs; C\u0026thinsp;=\u0026thinsp;complete; S\u0026thinsp;=\u0026thinsp;complete and single copy; D\u0026thinsp;=\u0026thinsp;complete and duplicated; F\u0026thinsp;=\u0026thinsp;fragmented; M\u0026thinsp;=\u0026thinsp;missing; LINEs\u0026thinsp;=\u0026thinsp;long interspersed nuclear elements; LTR\u0026thinsp;=\u0026thinsp;long terminal repeat; SINEs\u0026thinsp;=\u0026thinsp;short interspersed nuclear elements; CDS\u0026thinsp;=\u0026thinsp;coding DNA sequence\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eA total of 22,640 putative protein-coding genes were identified, accounting for 35% of the \u003cem\u003eT. orientalis\u003c/em\u003e genome (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Nearly all these genes (22,410; 98.98%) were functionally supported by at least one database, including 22,272 genes (98.37%) that matched the investigated protein databases using InterProScan (Table S5). A total of 13,587 predicted genes were annotated using KO terms, representing 8,509 unique KO genes (Supplementary File S2). Similarly, InterProScan assigned at least one GO term to 18,137 genes, corresponding to 6,901 unique GO terms (Supplementary File S3).\u003c/p\u003e\u003cp\u003eThe number of predicted protein-coding genes in \u003cem\u003eT. orientalis\u003c/em\u003e was comparable to that of closely related species, such as yellowfin tuna (24,623 genes), eastern little tuna (23,059 genes), and southern bluefin tuna (24,659 genes) (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Although it is lower than the previously reported annotation for \u003cem\u003eT. orientalis\u003c/em\u003e (26,433 genes) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], the current assembly represents a substantial quality improvement (N50 increased from 8.2 kb to 34.19 Mb).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Data validation\u003c/h2\u003e\u003cp\u003eFirst, the SELDLA-extended \u003cem\u003eT. orientalis\u003c/em\u003e assembly contained 230 of 233 (98.7%) complete CVGs [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Second, the assembly showed 7,085 (98.3%) complete BUSCOs, of which 7,020 (97.4%) were single copies and 65 (0.9%) were duplicates (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating completeness. Further, 6,727 of the 6,773 single-copy orthologs were localized on the same chromosomes in both \u003cem\u003eT. orientalis\u003c/em\u003e and \u003cem\u003eO. latipes\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e), suggesting high consistency between the two genomes. Based on the comparison results, 24 scaffolds of \u0026gt;\u0026thinsp;10Mb in the SELDLA-extended \u003cem\u003eT. orientalis\u003c/em\u003e assembly were numbered concordantly with the \u003cem\u003eO. latipes\u003c/em\u003e chromosomes (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The completeness of the predicted gene set was also evaluated using BUSCO, which identified 6,378 (88.5%) of the 7,207 actinopterygian single-copy orthologs (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn total, 218,379 genes from the nine fish species were clustered into 21,668 orthogroups, of which 11,437 were conserved across all species (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Of the 22,640 predicted genes in \u003cem\u003eT. orientalis\u003c/em\u003e, 21,428 (94.6%) were assigned to 17,807 orthogroups. Species of Scombridae, \u003cem\u003eT. albacares\u003c/em\u003e, \u003cem\u003eT. maccoyii\u003c/em\u003e, and \u003cem\u003eT. orientalis\u003c/em\u003e, included 323 unique orthogroups. A total of 761 orthogroups not detected in \u003cem\u003eT. orientalis\u003c/em\u003e were identified in all other species, suggesting that although the corresponding genes may not have been annotated, they were likely present in its genome.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe new chromosome-level \u003cem\u003eT. orientalis\u003c/em\u003e genome assembly presented herein has a scaffold N50 of 34.19Mb, 98.3% BUSCO completeness, and 22,640 annotated genes, representing a step advance in the previously published assembly. Of the 22,640 protein-coding genes, 4,677 (20.65%) contained putative, alternative, spliced transcripts. The availability of chromosome-level genome assemblies is expected to support applied and fundamental research, particularly the genetic improvement of this commercially valuable fish species.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNCBI = National Center for Biotechnology Information; BUSCO = benchmarking universal single-copy orthologs; LINEs = long interspersed nuclear elements; LTR = long terminal repeat; SINEs = short interspersed nuclear elements; CDS = coding DNA sequence\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSA and TI designed this study. DS conducted the experiments. The Ehime members, TS, ON, TM, RG, MH (\u003cem\u003eE. affinis\u003c/em\u003e), and TI (\u003cem\u003eT. orientalis\u003c/em\u003e) provided resources and contributed to the generation of hybrid fish. KY developed SELDLA software. XF and KY analyzed the data. KY and SA performed data analysis. XF, KY, and SA drafted the manuscript. All the authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003eSA and TI conceived of and designed the study. DS performed the experiments. TS, ON, TM, RG, MH, and TI provided resources and contributed to the generation of the hybrid fish. KY developed SELDLA software. XF and KY conducted data analysis with guidance from KY and SA. XF and SA drafted the manuscript. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Director Tokihiko Okada and the staff of the Aquaculture Technology and Production Center of Kindai University for providing tuna testes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported in part by the Japan Society for the Promotion of Science KAKENHI (Grant nos. 20H00429, 23H00342).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequencing dataset and genome assembly are stored in publicly available databases. The hybrid short-read sequencing data used for the genome assembly were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) repository (submission PRJNA1277492). All data and analysis codes supporting the findings of this study are available in Zenodo with the identifier 10.5281/zenodo.17239627.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBenetti D, Partridge G, Buentello A. Advances in Tuna Aquaculture: From Hatchery to Market. Academic Press; 2015.\u003c/li\u003e\n\u003cli\u003eHiguchi K, Takashi T, Okita K, Hayashida T, Nyuji M, Gen K. Effect of a long photoperiod on the timing of spawning in the Pacific bluefin tuna, Thunnus orientalis. 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Proc Natl Acad Sci U S A. 2013;110:11061\u0026ndash;6.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pacific bluefin tuna, aquaculture, genome annotation, linkage map, genome improvement","lastPublishedDoi":"10.21203/rs.3.rs-7883235/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7883235/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e High-quality reference genomes are fundamental for selective breeding and genetic engineering for improving aquaculture productivity. Here, we report an improved chromosome-level genome assembly of Pacific bluefin tuna (\u003cem\u003eThunnus orientalis\u003c/em\u003e), a commercially valuable species consumed worldwide. By integrating genetic linkage information from interspecific hybrids of \u003cem\u003eEuthynnus affinis\u003c/em\u003e and \u003cem\u003eT. orientalis\u003c/em\u003e, we anchored and oriented \u003cem\u003eT. orientalis \u003c/em\u003escaffolds into 24 pseudochromosomes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The updated assembly achieved a scaffold N50 of 34.19 Mb, representing a notable improvement from 13.3 Mb, and contained 22,640 predicted protein-coding genes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003eThis resource provides a robust genomic foundation for future genetic and genomic studies aimed at advancing the selective breeding of \u003cem\u003eT. orientalis\u003c/em\u003e in aquaculture.\u003c/p\u003e","manuscriptTitle":"Genome Refinement in Pacific Bluefin Tuna (Thunnus orientalis): Chromosome-level Assembly Using Hybrid Linkage Data","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-04 13:13:10","doi":"10.21203/rs.3.rs-7883235/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-18T10:58:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-17T18:51:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-09T17:54:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27543577318583046215272989816997988824","date":"2026-01-19T17:43:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315992376480181863152532585315267025565","date":"2026-01-14T16:57:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-23T00:38:34+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-22T19:39:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-21T06:01:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-21T06:00:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-10-17T06:11:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6f45a5ce-7387-4e65-a658-82ecde804736","owner":[],"postedDate":"November 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:01:39+00:00","versionOfRecord":{"articleIdentity":"rs-7883235","link":"https://doi.org/10.1186/s12864-026-12856-y","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2026-04-20 15:58:22","publishedOnDateReadable":"April 20th, 2026"},"versionCreatedAt":"2025-11-04 13:13:10","video":"","vorDoi":"10.1186/s12864-026-12856-y","vorDoiUrl":"https://doi.org/10.1186/s12864-026-12856-y","workflowStages":[]},"version":"v1","identity":"rs-7883235","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7883235","identity":"rs-7883235","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}