Chromosome-level genome assembly of the Golden trevally (Gnathanodon speciosus)

Chromosome-level genome assembly of the Golden trevally (Gnathanodon speciosus) | 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 data-descriptor Chromosome-level genome assembly of the Golden trevally (Gnathanodon speciosus) Feng Li, Jinghui Wu, Guangli Li, Zhendong Qin, Chunhua Zhu, Li Lin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8159327/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The golden trevally ( Gnathanodon speciosus ) is an economically vital marine species in tropical and subtropical fisheries and aquaculture, yet its genomic resources remain underdeveloped. This study presents the first haplotype-resolved, chromosome-level genome assembly of G. speciosus , combining PacBio HiFi long-read sequencing, Hi-C scaffolding, and short-read data. The assembled genome spans 600.31 Mb​ (N50 = 21.45 Mb) and is anchored to 24 chromosomes with a 97.3% anchoring rate. Genome annotation revealed 22,413 protein-coding genes, 22.71% repetitive sequences, and 10,233 non-coding RNAs. Comparative genomic analyses uncovered significant gene family expansions/contractions and positively selected genes enriched in environmental adaptation and developmental processes. Divergence time estimation placed G. speciosus within the Carangidae lineage. This genome provides a foundational resource for evolutionary studies, aquaculture breeding, and adaptation mechanisms in carangid fishes. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background & summary​ The golden trevally ( Gnathanodon speciosus ), also known as the golden kingfish, is a commercially valuable marine species widely distributed in tropical and subtropical waters 1 . It plays a significant role in aquaculture and fisheries due to its rapid growth, adaptability to farming conditions, and high market demand 2 . Despite its economic importance, genomic resources for G. speciosus have remained limited, hindering efforts to understand the genetic mechanisms underlying key biological traits, such as sex determination, environmental adaptation, and growth performance 3 . Recent advances in genomics have enabled the assembly of high-quality reference genomes for non-model species, providing insights into evolutionary biology and trait-associated genes. For instance, haplotype-resolved chromosome-level genomes have been generated for several aquatic species, revealing details of sex determination regions (SDRs) and adaptive genetic variations 4 . In G. speciosus , preliminary studies suggest the presence of a ZW sex determination system, with a candidate master sex-determining gene, cyp19a1a, identified through genome and transcriptome analyses 3 , 5 . However, a comprehensive, chromosome-level genome assembly is necessary to validate these findings and explore broader genomic features. In this study, we present the first haplotype-resolved, chromosome-level genome assembly of G. speciosus . Our aims were to: (1) generate a high-quality reference genome using PacBio HiFi and Hi-C technologies; (2) characterize the genomic architecture; and (3) unravel the phylogenetic relationship. This genomic resource serves as a foundation for understanding the evolution in carangid fishes and supports future efforts in genetic improvement and sustainable aquaculture of golden trevally. Methods Sample collection and sequencing A Gnathanodon speciosus individual was collected at the Agro-Tech Extension Center of Guangdong Province. The fish was handled in accordance with the guidelines for the Care, Ethics, and Safety Inspection of Guangdong Ocean University. Ethical approval was obtained from the Institutional Review Board. Genomic DNA was extracted from fresh muscle tissue of a single G. speciosus individual ( Fig 1 ) using a modified CTAB protocol, followed by RNase A treatment to remove residual RNA 6 . DNA integrity was assessed by electrophoresis on a 0.8% agarose gel and purity was assessed with a NanoDrop 2000. Subsequently, three types of sequencing libraries were constructed as described below. Short-insert sizes of 350bp were prepared with the Genomic DNA Sample Preparation kit and sequenced on the BGI platform, generating 143.66 Gb raw sequence data of paired-end reads. For the long-read PacBio HiFi sequencing, approximately 15 µg DNA was sheared with a Covaris g-TUBE, size-selected on a BluePippin system (Sage Science) to enrich fragments larger than 15 kb, and converted to SMRT Bell libraries with the SMRTbell Express Template Prep Kit 2.0 7 . The quantification of size-selected libraries was assessed by using both Femto Pulse and a Qubit fluorometer (Life Technologies). Sequencing was performed on a PacBio Sequel II system using 8M SMRT Cells (Pacific Biosciences), with 30-hour movie acquisition per cell 8 , yielding 40.77 Gb of CCS reads. Hi-C libraries were employed to achieve chromosome-level genome assembly. Finally, 151.55 Gb of 150-bp paired-end reads were obtained. ( Table 1 ) Library type Insert size (bp) Clean data (Gb) Depth ( × ) Short-insert 350 143.66 239 PacBio HiFi 15,000 40.77 67.9 Hi-C 150 151.55 252 Table 1. Summary statistics of sequencing data generated for G. speciosus. Genome assembly and evaluation The genome size was estimated using the k-mer method before genome assembly. The k-mer distribution was calculated from short reads using JCVI based on k-mer (k = 17) 9 . Finally, the estimated genome size of G. speciosus was predicted to be approximately 569Mb ( Fig 2 ). A total of 40.77 Gb PacBio HiFi reads were assembled with hifiasm (v0.19.5) 10 under the parameter of -l3, producing 378 contigs that sum to 600.31 Mb with an N50 of 21.45 Mb. Totally 151.55 Gb of paired-end reads generated from Hi-C libraries were quality-trimmed with Trimmomatic (v0.40) 11 and aligned to the contigs through juicer (v1.6) 12 to calculate the contact frequency. These contigs were subsequently anchored to chromosomes with Hi-C data, achieving an anchoring rate of 97.3% and yielding 584.08 Mb of the final 600.31 Mb assembly positioned on chromosomal scaffolds ( Table 2, Fig 3 ). Hi-C reads were aligned to the contigs using Juicer (v1.6), and chromosomal scaffolds were constructed with 3D-DNA (v180922) 13 followed by manual curation in Juicebox. Assembly Gnathanodon speciosus Assembly length (Mb) 600,308,495 bp Number of contigs 378 Contig N50 (Mb) 21,447,889 bp Sequences anchored to chromosomes (Mb) 584,075,371 bp Anchor ratio (%) 97.3 Short reads mapping rate (%) 99.67 Short reads coverage (%) 99.94 HiFi reads mapping rate (%) 99.88 HiFi reads coverage (%) 99.99 GC content (%) 42.46 BUSCOs (%) 98.0 QV 43.0153 Table 2. Summary statistics of genome assembly and genome completeness evaluation of G. speciosus. To assess assembly completeness and sequencing uniformity, short-insert and PacBio HiFi reads were aligned to the final assembly using BWA-MEM (v0.7.17) 14 and minimap2 (v2.21) 15 , respectively. Mapping rates, genome-wide coverage and depth distributions were calculated to evaluate completeness and coverage evenness across the genome. Consequently, short reads realigned at 99.67% with 99.94% genome-wide coverage, whereas HiFi reads achieved 99.88% mapping and 99.99% coverage, affirming the completeness and accuracy of the assembly ( Table 2 ). Meanwhile, a GC content of 42.46% was obtained, further, we generated a GC-content versus sequencing-depth density plot to assess potential GC bias and possible sample contamination ( Table 2, Fig 4 ). To assess the completeness of the gene catalogue, we ran BUSCO analyze against the actinopterygii_odb10 lineage set to predict the completeness, fragmentation level, and estimated loss rate of the genes 16 . The analysis classified 98.0% of orthologs as complete while 97.1% as single-copy, 0.2% as fragmented, and 1.7% as missing, indicating high gene catalogue completeness ( Table 2 ). To evaluate base-level accuracy, the final assembly was assessed with Merqury using a k-mer-based approach 17 . This yielded a genome-wide consensus quality value (QV) of 43.0153 and confirming the high fidelity of the assembled genome of G. speciosus ( Table 2 ). Genome annotation Repeat annotation We identified repetitive sequences including tandem repeats and transposable elements (TEs), by combining ab initio approach and homology-based approach. Tandem repeats were first detected and annotated with Tandem Repeats Finder (TRF, v4.09.1) 18 using the following parameters 2 7 7 80 10 50 2000 -d -h. For the interspersed repeats, RepeatModeler (v2.0.1) 19 and LTR_FINDER (v1.0.7) 20 were used to build an ab initio repeat library. Together with the Repbase database 21,22 , was then supplied to RepeatMasker (v4.1.2) 23 for DNA-level TE identification. At the protein level, RepeatProteinMask 23 was employed to search against the TE protein database. Repetitive sequences accounted for 136.3 Mb which presented 22.71% of the assembly, and were partitioned into tandem repeats at 6.69% and interspersed repeats at 21.43%. The interspersed repeats included DNA transposons at 11.02%, LINEs at 5.56%, LTR retrotransposons at 5.03% and SINEs at 0.42%, while 2.67% elements were identified as unclassified ( Table 3 ). Classifications Repeat Size (bp) Percentage in genome (%) Tandem Repeat 40,168,677 6.69 Interspersed Repeat DNA 66,188,421 11.02 LINE 33,397,656 5.56 SINE 2,520,953 0.42 LTR 30,227,434 5.03 Other 5,076 0 Unknown 16,023,589 2.67 Total TE 128,636,350 21.43 Table 3. Summary statistics of repeated sequences for G. speciosus. Gene annotation Protein-coding genes were predicted by integrating homology-based, ab initio and transcriptome-assisted annotation. Related-species proteins sequences were aligned to the genome with TBlastN (v2.11.0+) 24 , further filtered and refined with Exonerate (v2.4.0) 25 to establish gene structures. RNA-seq reads mapped with HISAT2 (v2.2.1) 26 were assembled genome-guided with StringTie (v2.1.7) 27 and de novo with Trinity (v2.8.5) 28 before integration via PASA (v2.4.1) 29 to capture splice isoforms and UTRs. We performed Augustus (v3.4.0) 30–32 together with Genscan (v1.0) 33 to provide ab initio predictions. Further, all evidence was consolidated with MAKER (v3.01.03) 34 and updated with PASA (v2.4.1) to yield the final consensus gene set. The annotation comprises 22,413 protein-coding genes with an average length in 14.8 kb and 1.76 kb in CDS, while average 10.5 exons per gene ( Table S1 ). Functional assignment was performed by mapping to the varies database including NR, KEGG 35 , Gene Ontology 36 , TrEMBL 37 and Swiss-Prot 37 , which succeeded for 21,825 genes (97.38%), with 91.56% matching SwissProt, 96.76% TrEMBL, 96.99% KEGG, 87.65% InterPro and 65.09% Gene Ontology. However, 588 genes were unannotated ( Table 4, Fig 5 ). Classifications Number Percent (%) Total 22,413 NA InterPro 19,645 87.65 GO 14,588 65.09 KEGG_ALL 21,738 96.99 KEGG_KO 16,057 71.64 Swissprot 20,522 91.56 TrEMBL 21,686 96.76 NR 21,806 97.29 Annotated 21,825 97.38 Unannotated 588 2.62 Table 4. Summary statistics of the numbers of predicted protein coding genes in the assembled genome of G. speciosus . Annotation of non-coding RNA genes Non-coding RNAs were annotated using tRNAscan-SE (v2.0.9) 38 to identify the genes associated tRNAs. RNAmmer (v1.2) 39 were used to predict rRNAs. MiRNAs and snRNAs were identified by using Infernal (v1.1.4) 40 against the Rfam (v14.6) 41 database. This approach captured a range of non-coding RNA types, providing a comprehensive profile of these elements in the genome. Thereinto, 1,336 tRNAs and 876 rRNAs were annotated with an average length of 75.78 bp and 440.91, respectively. Meanwhile, we discovered 1,023 miRNAs with average 79.02 in length and 520 snRNAs with average length of 151.82bp in several types including CD-box, HACA-box, splicing and scaRNA ( Table 5 ). Classifications Copy Average length(bp) Total length(bp) % of genome miRNA 1,023 79.02 80,837 0.013464 tRNA 1,336 75.78 101,237 0.016862 rRNA rRNA 876 440.91 386,234 0.064332 18S 46 1,820.26 83,732 0.013947 28S 48 4,443.67 213,296 0.035527 5S 782 114.07 89,206 0.014858 snRNA snRNA 520 151.82 78,945 0.013149 CD-box 94 107.01 10,059 0.001675 HACA-box 50 158.82 7,941 0.001323 splicing 371 161.04 59,745 0.009951 scaRNA 5 240.00 1,200 0.000200 Table 5. Summary statistics of noncoding RNAs in the assembled genome of G. speciosus . Annotation results assessment Annotation completeness was evaluated using BUSCO (v5.2.2) 42 , which sampled hundreds of genomes and selected single - copy orthologs with >90% presence to build gene sets for six major phylogenetic branches. The assessment showed that 98.0% of BUSCO genes were complete, with 97.1% founded as single-copy and 0.9% as duplicated. Only 1.7% were missing, indicating high completeness and quality of the annotation ( Table 6 ). Classifications Assembly No. Assembly ratio (%) Annotation No. Annotation ratio (%) Complete BUSCOs 3,569 98.00 3,525 96.90 Complete and single-copy BUSCOs 3,535 97.10 3,489 95.90 Complete and duplicated BUSCOs 34 0.90 36 1.00 Fragmented BUSCOs 8 0.20 25 0.70 Missing BUSCOs 63 1.70 90 2.50 Total BUSCO groups searched 3,640 100.00 3,640 100.00 Table 6. Summary statistics of gene-model completeness and functional annotation in G. speciosus . Comparative genomic analysis To investigate the adaptive evolution of G.speciosus species, we conducted a comparative genomic analysis following the workflow. Firstly, gene family clustering was performed using OrthoFinder2 43 and Diamond 44 based on longest-transcript protein sequences. The results revealed a total of 2,690 single-copy orthologous gene families shared across all 15 analyzed species, providing the basis for downstream evolutionary analyses ( Fig 6 ). Secondly, a phylogenetic tree was constructed using RAxML 45 based on the aligned single-copy orthologous genes and showing clear divergence patterns among species ( Fig 7a ). Divergence time estimation was then carried out using r8s 46 and PAML (mcmctree) 47 with time calibration points derived from the literature 48 . The estimated divergence times indicated G. speciosus splits from its closest relatives within a range of approximately 90–220 million years ago according to the phylogenetic node examined ( Fig 7b ). Subsequently, CAFE 49 analysis conducted on gene families revealed significant expansion or contraction (Q < 0.05) in G. speciosus ( Fig 8a ). Functional enrichment profiling revealed that these gene families converge on fundamental biological processes and pathways, implying a potential genomic basis for ecological adaptation in G. speciosus ( Fig 8b, Fig 8c ). Finally, positive selection analysis using the branch-site model in PAML (codeml) 47 identified a set of genes with significant selection signals (p < 0.05, FDR-corrected). Functional enrichment profiling of these genes indicated over-representation of fundamental biological processes and pathways, suggesting that adaptive evolution may have occurred in G. speciosus ( Fig 9 ). Together, these genomic data provide a comparative resource for studying gene family evolution and candidate genes under selection in G. speciosus . Declarations Data availability Raw PacBio, Illumina, Hi-C, and transcriptome sequencing data of G. speciosus have been deposited in the Genome Sequence Archive (GSA) with accession number PRJCA051301 (https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA051301). The assembled genome is available under GSA accession GWHHJEF00000000.1 (https://ngdc.cncb.ac.cn/gwh/Assembly/109454/show). Technical Validation Sequence accuracy and completeness of the G. speciosus assembly were examined at three independent levels. Short-insert reads with 143.66 Gb data realigned with BWA-MEM at 99.67% mapping rate and 99.94 % genome-wide coverage; PacBio HiFi reads with 40.77 Gb data mapped with minimap2 at 99.88% rate and 99.99% coverage. Merqury k-mer analysis yielded a genome-wide QV of 43.0153, corresponding to more than 99.99% base-level accuracy. The final assembly anchors 584.08 Mb of the 600.31 Mb genome to 24 chromosomal scaffolds with an anchoring rate of 97.3%. BUSCO genome assessment against the actinopterygii_odb10 lineage set classified 98.0 % of orthologs as complete (97.1% single-copy, 0.9% duplicated), 0.2% as fragmented and 1.7% as missing, indicating high chromosome-scale completeness and completeness of the gene catalogue. BUSCO protein mode recovered 94.6 % complete orthologs among the 22,413 predicted protein-coding genes. Functional annotation succeeded for 21,825 genes (97.38%) against SwissProt, NR, KEGG, InterPro and GO databases. A total of 10,233 non-coding RNAs were annotated, including 1,336 tRNAs and 876 rRNAs, confirming a comprehensive gene set. Code availability No specific script was used in this work. All commands and pipelines used in data processing were executed according to the manual and protocols of the corresponding bioinformatic software. Software/packages with their versions and settings for genome annotation were listed: tandem Repeats Finder (v4.09.1) using the following parameters 2 7 7 80 10 50 2000 -d -h; RepeatModeler (v2.0.1) and LTR_FINDER (v1.0.7); RepeatMasker (v4.1.2); TBlastN (v2.11.0+); Exonerate (v2.4.0); HISAT2 (v2.2.1); StringTie (v2.1.7) and Trinity (v2.8.5); PASA (v2.4.1); Augustus (v3.4.0); Genscan (v1.0); MAKER (v3.01.03); tRNAscan-SE (v2.0.9); RNAmmer (v1.2); Infernal (v1.1.4); Rfam (v14.6). Acknowledgements This project was supported by Project for Breakthrough in Key Technologies of Suitable Species in Modern Marine Pasture in Guangdong Province (no.2024HYMC-40). Author contributions Chunhua Zhu and Li Lin conceived this project. Feng Li and Jinghui Wu participated in data analysis. Guangli Li and Zhendong Qin collected the samples. Feng Li assembled the genome. Feng Li and Zhendong Qin generated the annotation set. Feng Li wrote the manuscript. Chunhua Zhu and Li Lin revised themanuscript. 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Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable.docx Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8159327","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"data-descriptor","associatedPublications":[],"authors":[{"id":597802466,"identity":"5fb9803d-5549-496b-bff8-20e4acbc85c0","order_by":0,"name":"Feng Li","email":"","orcid":"","institution":"Guangdong Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Li","suffix":""},{"id":597802471,"identity":"18538035-f5e6-4b8d-ac76-3a297b5b9361","order_by":1,"name":"Jinghui Wu","email":"","orcid":"","institution":"Agro-Tech Extension Center of Guangdong Province","correspondingAuthor":false,"prefix":"","firstName":"Jinghui","middleName":"","lastName":"Wu","suffix":""},{"id":597802475,"identity":"e0df3c2e-bd13-47ef-9de4-e3c658c97f7e","order_by":2,"name":"Guangli Li","email":"","orcid":"","institution":"Guangdong Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Guangli","middleName":"","lastName":"Li","suffix":""},{"id":597802482,"identity":"541e6dba-1d9e-4480-8ceb-b41633b3c7d0","order_by":3,"name":"Zhendong Qin","email":"","orcid":"","institution":"Zhongkai University of Agriculture and Engineering","correspondingAuthor":false,"prefix":"","firstName":"Zhendong","middleName":"","lastName":"Qin","suffix":""},{"id":597802483,"identity":"c1486ba8-0b8e-47ee-a10f-ca9c63859864","order_by":4,"name":"Chunhua Zhu","email":"","orcid":"","institution":"Guangdong Ocean University","correspondingAuthor":false,"prefix":"","firstName":"Chunhua","middleName":"","lastName":"Zhu","suffix":""},{"id":597802484,"identity":"0a128d56-dbab-4422-a50f-2944667ac108","order_by":5,"name":"Li Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYBACPmYGxgMJFQwJEiAeDzFa2JgZGA4knCFJCxAfYGwjSQs774EDD+fV5UnOSGB88LaNQd6csMP4Eg4kbmMrlpZIYDac28ZguLOBoBYeA6AWnsR5Egls0rxAFxocIErLHAmQFvbfJGhpMEicDbSFmXgtCccSEmf2PGyWnHNOwnADIS38/GcMH/6oqUuccTz54Ic3ZTbyBG1BAowNQEKCePWjYBSMglEwCnADAK5jOZxYXKR1AAAAAElFTkSuQmCC","orcid":"","institution":"Guangdong Ocean University","correspondingAuthor":true,"prefix":"","firstName":"Li","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2025-11-20 01:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8159327/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8159327/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104400558,"identity":"5922cfd0-8ef6-4bf0-968e-bedca2c3fde2","added_by":"auto","created_at":"2026-03-11 12:10:20","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":45317,"visible":true,"origin":"","legend":"\u003cp\u003eImages of the sequenced Reeve’s G. speciosus.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8159327/v1/7b70e0f382ffd2f2d9f90141.jpeg"},{"id":103778236,"identity":"17022357-c955-4a15-b16b-3b51e02ca17c","added_by":"auto","created_at":"2026-03-02 19:36:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94291,"visible":true,"origin":"","legend":"\u003cp\u003eGenome assessment of \u003cem\u003eG. speciosus. \u003c/em\u003eGenome survey at \u003cem\u003e17-mer \u003c/em\u003eof \u003cem\u003eG. speciosus\u003c/em\u003eestimated by JCVI.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8159327/v1/3ace740c5c35c00ee77798f4.png"},{"id":103778241,"identity":"d23da07b-33f0-48cc-a0b4-82ee101dc94b","added_by":"auto","created_at":"2026-03-02 19:36:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":870218,"visible":true,"origin":"","legend":"\u003cp\u003eHi-C interaction heatmap of \u003cem\u003eG. speciosus\u003c/em\u003e. Colour intensity (light to dark) indicates increasing interaction strength. Both axes represent genomic N-bin positions; each tile corresponds to an individual chromosome.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8159327/v1/163f89c38d221603600eed21.png"},{"id":103778238,"identity":"37d78c76-b9e4-4026-a2a5-8be7d343aded","added_by":"auto","created_at":"2026-03-02 19:36:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":212082,"visible":true,"origin":"","legend":"\u003cp\u003eGC-content and sequencing-depth distribution density plot: the x-axis indicates GC content, the y-axis indicates coverage depth; the right panel shows the contig-depth distribution, the upper panel shows the GC-content distribution, and the central large panel is a scatter plot drawn from the GC distribution and depth information of the contigs, in which colour intensity reflects the density of the points in the scatter plot. \u003cstrong\u003ea)\u003c/strong\u003e The distribution density plot of short reads; \u003cstrong\u003eb)\u003c/strong\u003e The distribution density plot of HiFi reads.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8159327/v1/dcad107904b113d1d6da1d9e.png"},{"id":103778245,"identity":"22f59cbf-698e-4f26-95d9-6934779def37","added_by":"auto","created_at":"2026-03-02 19:36:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":322661,"visible":true,"origin":"","legend":"\u003cp\u003eComparative gene architecture between the targeted species and closely related taxa, with the target species highlighted in red. \u003cstrong\u003ea)\u003c/strong\u003e Distributions of exon number, intron number, gene-level GC content and CDS-level GC content. \u003cstrong\u003eb)\u003c/strong\u003e Distributions of gene length, CDS length, exon length and intron length.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8159327/v1/996452d567b6b9062d94cf8c.png"},{"id":104400804,"identity":"9ec181fc-6221-48b5-8dd7-38f865a3db32","added_by":"auto","created_at":"2026-03-11 12:11:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":131801,"visible":true,"origin":"","legend":"\u003cp\u003eGene-family clustering among the targeted species and 14 closely related taxa.\u003cstrong\u003e a)\u003c/strong\u003eBar chart summarizing the numbers of clustered gene families shared across all 15 analyzed species; \u003cstrong\u003eb)\u003c/strong\u003e Venn diagram detailing the clustering results for the first four species, showing the number of gene families shared by all four and the numbers unique to each species relative to the other three.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8159327/v1/27b3ca69398e4cc946d7ec8c.png"},{"id":104400059,"identity":"da5e0410-33d6-4d3f-bf23-dff2903509ae","added_by":"auto","created_at":"2026-03-11 12:08:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":200567,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationships and divergence-time estimation. \u003cstrong\u003ea) \u003c/strong\u003eMaximum-likelihood phylogeny constructed with RAxML based on shared single-copy orthologous genes; \u003cstrong\u003eb)\u003c/strong\u003e Divergence-time estimates obtained using r8s and the mcmctree module of PAML, calibrated with time points from TimeTree and published literature.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8159327/v1/c1877ccc63aef82dd444814a.png"},{"id":103778246,"identity":"7d08292f-8aac-4d8b-918a-d17e1f51e8a1","added_by":"auto","created_at":"2026-03-02 19:36:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":291273,"visible":true,"origin":"","legend":"\u003cp\u003eGene-family expansion and contraction analysis with functional enrichment.\u003cstrong\u003e a) \u003c/strong\u003ePhylogenetic tree showing significant gene-family expansion (green) and contraction (red) inferred by CAFE (q \u0026lt; 0.05); \u003cstrong\u003eb)\u003c/strong\u003e GO enrichment bubbles for significantly expanded gene families. Bubble size indicates gene number; \u003cstrong\u003ec)\u003c/strong\u003eGO functional classification of genes within significantly contracted gene families. Bars indicate the number of genes assigned to each GO term.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8159327/v1/e4d6e40f1378ae6ef1634738.png"},{"id":103778243,"identity":"b13ab1aa-2cfb-41fd-a5b1-fe4cd1f9734d","added_by":"auto","created_at":"2026-03-02 19:36:31","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":233938,"visible":true,"origin":"","legend":"\u003cp\u003ePositive selection and functional enrichment. \u003cstrong\u003ea)\u003c/strong\u003e GO functional classification of positively selected genes (PSGs). Bars show gene counts per category; \u003cstrong\u003eb)\u003c/strong\u003e Bubble plot of GO enrichment for PSGs. Bubble size indicates gene number.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8159327/v1/52283cadf1cbaf8f3dd03245.png"},{"id":104408229,"identity":"09ce6b89-35e8-4a4c-8de1-49696a2bfe93","added_by":"auto","created_at":"2026-03-11 12:42:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3060770,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8159327/v1/805fe529-f12c-4737-b7f1-f875fd348ad0.pdf"},{"id":104400127,"identity":"6424b602-2fc5-405f-8f79-2c529fcf16a4","added_by":"auto","created_at":"2026-03-11 12:08:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18532,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-8159327/v1/ab755338826cc5f1a6081b13.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chromosome-level genome assembly of the Golden trevally (Gnathanodon speciosus)","fulltext":[{"header":"Background \u0026 summary​","content":"\u003cp\u003eThe golden trevally (\u003cem\u003eGnathanodon speciosus\u003c/em\u003e), also known as the golden kingfish, is a commercially valuable marine species widely distributed in tropical and subtropical waters\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. It plays a significant role in aquaculture and fisheries due to its rapid growth, adaptability to farming conditions, and high market demand\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Despite its economic importance, genomic resources for \u003cem\u003eG. speciosus\u003c/em\u003e have remained limited, hindering efforts to understand the genetic mechanisms underlying key biological traits, such as sex determination, environmental adaptation, and growth performance\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003eRecent advances in genomics have enabled the assembly of high-quality reference genomes for non-model species, providing insights into evolutionary biology and trait-associated genes. For instance, haplotype-resolved chromosome-level genomes have been generated for several aquatic species, revealing details of sex determination regions (SDRs) and adaptive genetic variations\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eG. speciosus\u003c/em\u003e, preliminary studies suggest the presence of a ZW sex determination system, with a candidate master sex-determining gene, cyp19a1a, identified through genome and transcriptome analyses\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, a comprehensive, chromosome-level genome assembly is necessary to validate these findings and explore broader genomic features.\u003c/p\u003e \u003cp\u003eIn this study, we present the first haplotype-resolved, chromosome-level genome assembly of \u003cem\u003eG. speciosus\u003c/em\u003e. Our aims were to: (1) generate a high-quality reference genome using PacBio HiFi and Hi-C technologies; (2) characterize the genomic architecture; and (3) unravel the phylogenetic relationship. This genomic resource serves as a foundation for understanding the evolution in carangid fishes and supports future efforts in genetic improvement and sustainable aquaculture of golden trevally.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eSample collection and sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA \u003cem\u003eGnathanodon speciosus\u003c/em\u003e individual was collected at the Agro-Tech Extension Center of Guangdong Province. The fish was handled in accordance with the guidelines for the Care, Ethics, and Safety Inspection of Guangdong Ocean University. Ethical approval was obtained from the Institutional Review Board. Genomic DNA was extracted from fresh muscle tissue of a single \u003cem\u003eG. speciosus\u003c/em\u003e individual (\u003cstrong\u003eFig 1\u003c/strong\u003e) using a modified CTAB protocol, followed by RNase A treatment to remove residual RNA\u003csup\u003e6\u003c/sup\u003e. DNA integrity was assessed by electrophoresis on a 0.8% agarose gel and purity was assessed with a NanoDrop 2000. Subsequently, three types of sequencing libraries were constructed as described below. Short-insert sizes of 350bp were prepared with the Genomic DNA Sample Preparation kit and sequenced on the BGI platform, generating 143.66 Gb raw sequence data of paired-end reads. For the long-read PacBio HiFi sequencing, approximately 15 \u0026micro;g DNA was sheared with a Covaris g-TUBE, size-selected on a BluePippin system (Sage Science) to enrich fragments larger than 15 kb, and converted to SMRT Bell libraries with the SMRTbell Express Template Prep Kit 2.0\u003csup\u003e7\u003c/sup\u003e. The quantification of size-selected libraries was assessed by using both Femto Pulse and a Qubit fluorometer (Life Technologies). Sequencing was performed on a PacBio Sequel II system using 8M SMRT Cells (Pacific Biosciences), with 30-hour movie acquisition per cell\u003csup\u003e8\u003c/sup\u003e, yielding 40.77 Gb of CCS reads. Hi-C libraries were employed to achieve chromosome-level genome assembly. Finally, 151.55 Gb of 150-bp paired-end reads were obtained. (\u003cstrong\u003eTable 1\u003c/strong\u003e)\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"513\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 145px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLibrary type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInsert size (bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClean data (Gb)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDepth (\u003c/strong\u003e\u003cstrong\u003e\u0026times;\u003c/strong\u003e\u003cstrong\u003e)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 145px;\"\u003e\n \u003cp\u003eShort-insert\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e350\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e143.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e239\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 145px;\"\u003e\n \u003cp\u003ePacBio HiFi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e15,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e40.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e67.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 145px;\"\u003e\n \u003cp\u003eHi-C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 125px;\"\u003e\n \u003cp\u003e151.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 124px;\"\u003e\n \u003cp\u003e252\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eSummary statistics of sequencing data generated for \u003cem\u003eG. speciosus.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome assembly and evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome size was estimated using the k-mer method before genome assembly. The k-mer distribution was calculated from short reads using JCVI based on k-mer (k\u0026thinsp;=\u0026thinsp;17)\u003csup\u003e9\u003c/sup\u003e. Finally, the estimated genome size of \u003cem\u003eG. speciosus\u003c/em\u003e was predicted to be approximately 569Mb (\u003cstrong\u003eFig 2\u003c/strong\u003e). A total of 40.77 Gb PacBio HiFi reads were assembled with hifiasm (v0.19.5)\u003csup\u003e10\u003c/sup\u003e under the parameter of -l3, producing 378 contigs that sum to 600.31 Mb with an N50 of 21.45 Mb. Totally 151.55 Gb of paired-end reads generated from Hi-C libraries were quality-trimmed with Trimmomatic (v0.40)\u003csup\u003e11\u003c/sup\u003e and aligned to the contigs through juicer (v1.6)\u003csup\u003e12\u003c/sup\u003e to calculate the contact frequency. These contigs were subsequently anchored to chromosomes with Hi-C data, achieving an anchoring rate of 97.3% and yielding 584.08 Mb of the final 600.31 Mb assembly positioned on chromosomal scaffolds (\u003cstrong\u003eTable 2, Fig 3\u003c/strong\u003e). Hi-C reads were aligned to the contigs using Juicer (v1.6), and chromosomal scaffolds were constructed with 3D-DNA (v180922)\u003csup\u003e13\u003c/sup\u003e followed by manual curation in Juicebox.\u0026nbsp;\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAssembly\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGnathanodon speciosus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAssembly length (Mb)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e600,308,495 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNumber of contigs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e378\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eContig N50 (Mb)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e21,447,889 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSequences anchored to chromosomes (Mb)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e584,075,371 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAnchor ratio (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eShort reads mapping rate (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eShort reads coverage (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHiFi reads mapping rate (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHiFi reads coverage (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGC content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e42.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBUSCOs (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eQV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43.0153\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Summary statistics of genome assembly and genome completeness evaluation of \u003cem\u003eG. speciosus.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo assess assembly completeness and sequencing uniformity, short-insert and PacBio HiFi reads were aligned to the final assembly using BWA-MEM (v0.7.17)\u003csup\u003e14\u003c/sup\u003e and minimap2 (v2.21)\u003csup\u003e15\u003c/sup\u003e, respectively. Mapping rates, genome-wide coverage and depth distributions were calculated to evaluate completeness and coverage evenness across the genome. Consequently, short reads realigned at 99.67% with 99.94% genome-wide coverage, whereas HiFi reads achieved 99.88% mapping and 99.99% coverage, affirming the completeness and accuracy of the assembly (\u003cstrong\u003eTable 2\u003c/strong\u003e). Meanwhile, a GC content of 42.46% was obtained, further, we generated a GC-content versus sequencing-depth density plot to assess potential GC bias and possible sample contamination (\u003cstrong\u003eTable 2, Fig 4\u003c/strong\u003e). To assess the completeness of the gene catalogue, we ran BUSCO analyze against the actinopterygii_odb10 lineage set to predict the completeness, fragmentation level, and estimated loss rate of the genes\u003csup\u003e16\u003c/sup\u003e. The analysis classified 98.0% of orthologs as complete while 97.1% as single-copy, 0.2% as fragmented, and 1.7% as missing, indicating high gene catalogue completeness (\u003cstrong\u003eTable 2\u003c/strong\u003e). To evaluate base-level accuracy, the final assembly was assessed with Merqury using a k-mer-based approach\u003csup\u003e17\u003c/sup\u003e. This yielded a genome-wide consensus quality value (QV) of 43.0153 and confirming the high fidelity of the assembled genome of \u003cem\u003eG. speciosus\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eTable 2\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome annotation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRepeat annotation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe identified repetitive sequences including tandem repeats and transposable elements (TEs), by combining \u003cem\u003eab initio\u003c/em\u003e approach and homology-based approach. Tandem repeats were first detected and annotated with Tandem Repeats Finder (TRF, v4.09.1)\u003csup\u003e18\u003c/sup\u003e using the following parameters 2 7 7 80 10 50 2000 -d -h. For the interspersed repeats, RepeatModeler (v2.0.1)\u003csup\u003e19\u003c/sup\u003e and LTR_FINDER (v1.0.7)\u003csup\u003e20\u003c/sup\u003e were used to build an \u003cem\u003eab initio\u003c/em\u003e repeat library. Together with the Repbase database\u003csup\u003e21,22\u003c/sup\u003e, was then supplied to RepeatMasker (v4.1.2)\u003csup\u003e23\u003c/sup\u003e for DNA-level TE identification. At the protein level, RepeatProteinMask\u003csup\u003e23\u003c/sup\u003e was employed to search against the TE protein database.\u003c/p\u003e\n\u003cp\u003eRepetitive sequences accounted for 136.3 Mb which presented 22.71% of the assembly, and were partitioned into tandem repeats at 6.69% and interspersed repeats at 21.43%. The interspersed repeats included DNA transposons at 11.02%, LINEs at 5.56%, LTR retrotransposons at 5.03% and SINEs at 0.42%, while 2.67% elements were identified as unclassified (\u003cstrong\u003eTable 3\u003c/strong\u003e).\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eClassifications\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eRepeat Size (bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage in genome (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTandem Repeat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e40,168,677\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"7\"\u003e\n \u003cp\u003eInterspersed Repeat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e66,188,421\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e11.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLINE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e33,397,656\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSINE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2,520,953\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLTR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e30,227,434\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eOther\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5,076\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eUnknown\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16,023,589\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTotal TE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e128,636,350\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e21.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eSummary statistics of repeated sequences for \u003cem\u003eG. speciosus.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene annotation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein-coding genes were predicted by integrating homology-based, ab initio and transcriptome-assisted annotation. Related-species proteins sequences were aligned to the genome with TBlastN (v2.11.0+)\u003csup\u003e24\u003c/sup\u003e, further filtered and refined with Exonerate (v2.4.0)\u003csup\u003e25\u003c/sup\u003e to establish gene structures. RNA-seq reads mapped with HISAT2 (v2.2.1)\u003csup\u003e26\u003c/sup\u003e were assembled genome-guided with StringTie (v2.1.7)\u003csup\u003e27\u003c/sup\u003e and de novo with Trinity (v2.8.5)\u003csup\u003e28\u003c/sup\u003e before integration via PASA (v2.4.1)\u003csup\u003e29\u003c/sup\u003e to capture splice isoforms and UTRs. We performed Augustus (v3.4.0)\u003csup\u003e30\u0026ndash;32\u003c/sup\u003e together with Genscan (v1.0)\u003csup\u003e33\u003c/sup\u003e to provide ab initio predictions. Further, all evidence was consolidated with MAKER (v3.01.03)\u003csup\u003e34\u003c/sup\u003e and updated with PASA (v2.4.1) to yield the final consensus gene set.\u003c/p\u003e\n\u003cp\u003eThe annotation comprises 22,413 protein-coding genes with an average length in 14.8 kb and 1.76 kb in CDS, while average 10.5 exons per gene (\u003cstrong\u003eTable S1\u003c/strong\u003e). Functional assignment was performed by mapping to the varies database including NR, KEGG\u003csup\u003e35\u003c/sup\u003e, Gene Ontology\u003csup\u003e36\u003c/sup\u003e, TrEMBL\u003csup\u003e37\u003c/sup\u003e and Swiss-Prot\u003csup\u003e37\u003c/sup\u003e, which succeeded for 21,825 genes (97.38%), with 91.56% matching SwissProt, 96.76% TrEMBL, 96.99% KEGG, 87.65% InterPro and 65.09% Gene Ontology. However, 588 genes were unannotated (\u003cstrong\u003eTable 4, Fig 5\u003c/strong\u003e).\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eClassifications\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNumber\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePercent (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e22,413\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eInterPro\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e19,645\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e87.65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14,588\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e65.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eKEGG_ALL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e21,738\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e96.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eKEGG_KO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16,057\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e71.64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSwissprot\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e20,522\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e91.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTrEMBL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e21,686\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e96.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e21,806\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAnnotated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e21,825\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eUnannotated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e588\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSummary statistics of the numbers of predicted protein coding genes in the assembled genome of \u003cem\u003eG. speciosus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnnotation of non-coding RNA genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNon-coding RNAs were annotated using tRNAscan-SE (v2.0.9)\u003csup\u003e38\u003c/sup\u003e to identify the genes associated tRNAs. RNAmmer (v1.2)\u003csup\u003e39\u003c/sup\u003e were used to predict rRNAs. MiRNAs and snRNAs were identified by using Infernal (v1.1.4)\u003csup\u003e40\u003c/sup\u003e against the Rfam (v14.6)\u003csup\u003e41\u003c/sup\u003e database. This approach captured a range of non-coding RNA types, providing a comprehensive profile of these elements in the genome.\u003c/p\u003e\n\u003cp\u003eThereinto, 1,336 tRNAs and 876 rRNAs were annotated with an average length of 75.78 bp and 440.91, respectively. Meanwhile, we discovered 1,023 miRNAs with average 79.02 in length and 520 snRNAs with average length of 151.82bp in several types including CD-box, HACA-box, splicing and scaRNA (\u003cstrong\u003eTable 5\u003c/strong\u003e).\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eClassifications\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCopy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAverage length(bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTotal length(bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e% of genome\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003emiRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1,023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e79.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e80,837\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.013464\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003etRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1,336\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e75.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e101,237\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.016862\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003erRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003erRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e876\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e440.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e386,234\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.064332\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1,820.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e83,732\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.013947\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e28S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4,443.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e213,296\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.035527\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e782\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e114.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e89,206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.014858\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003esnRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003esnRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e520\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e151.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e78,945\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.013149\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCD-box\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e107.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10,059\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.001675\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHACA-box\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e158.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7,941\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.001323\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003esplicing\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e371\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e161.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e59,745\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.009951\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003escaRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e240.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1,200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.000200\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSummary statistics of noncoding RNAs in the assembled genome of \u003cem\u003eG. speciosus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnnotation results assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnnotation completeness was evaluated using BUSCO (v5.2.2)\u003csup\u003e42\u003c/sup\u003e, which sampled hundreds of genomes and selected single - copy orthologs with \u0026gt;90% presence to build gene sets for six major phylogenetic branches. The assessment showed that 98.0% of BUSCO genes were complete, with 97.1% founded as single-copy and 0.9% as duplicated. Only 1.7% were missing, indicating high completeness and quality of the annotation (\u003cstrong\u003eTable 6\u003c/strong\u003e).\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eClassifications\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAssembly No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAssembly ratio (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAnnotation No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAnnotation ratio (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eComplete BUSCOs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3,569\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3,525\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e96.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eComplete and single-copy BUSCOs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3,535\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3,489\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e95.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eComplete and duplicated BUSCOs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eFragmented BUSCOs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMissing BUSCOs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTotal BUSCO groups searched\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3,640\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3,640\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSummary statistics of gene-model completeness and functional annotation in \u003cem\u003eG. speciosus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative genomic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the adaptive evolution of \u003cem\u003eG.speciosus\u003c/em\u003e species, we conducted a comparative genomic analysis following the workflow. Firstly, gene family clustering was performed using OrthoFinder2\u003csup\u003e43\u003c/sup\u003e and Diamond\u003csup\u003e44\u003c/sup\u003e based on longest-transcript protein sequences. The results revealed a total of 2,690 single-copy orthologous gene families shared across all 15 analyzed species, providing the basis for downstream evolutionary analyses (\u003cstrong\u003eFig 6\u003c/strong\u003e). Secondly, a phylogenetic tree was constructed using RAxML\u003csup\u003e45\u003c/sup\u003e based on the aligned single-copy orthologous genes and showing clear divergence patterns among species (\u003cstrong\u003eFig 7a\u003c/strong\u003e). Divergence time estimation was then carried out using r8s\u003csup\u003e46\u003c/sup\u003e and PAML (mcmctree)\u003csup\u003e47\u003c/sup\u003e with time calibration points derived from the literature\u003csup\u003e48\u003c/sup\u003e. The estimated divergence times indicated\u003cem\u003e\u0026nbsp;G. speciosus\u003c/em\u003e splits from its closest relatives within a range of approximately 90\u0026ndash;220 million years ago according to the phylogenetic node examined (\u003cstrong\u003eFig 7b\u003c/strong\u003e). Subsequently, CAFE\u003csup\u003e49\u003c/sup\u003e analysis conducted on gene families revealed significant expansion or contraction (Q \u0026lt; 0.05) in \u003cem\u003eG. speciosus\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFig 8a\u003c/strong\u003e). Functional enrichment profiling revealed that these gene families converge on fundamental biological processes and pathways, implying a potential genomic basis for ecological adaptation in \u003cem\u003eG. speciosus\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFig 8b, Fig 8c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFinally, positive selection analysis using the branch-site model in PAML (codeml)\u003csup\u003e47\u003c/sup\u003e identified a set of genes with significant selection signals (p \u0026lt; 0.05, FDR-corrected). Functional enrichment profiling of these genes indicated over-representation of fundamental biological processes and pathways, suggesting that adaptive evolution may have occurred in \u003cem\u003eG. speciosus\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFig 9\u003c/strong\u003e). Together, these genomic data provide a comparative resource for studying gene family evolution and candidate genes under selection in \u003cem\u003eG. speciosus\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eRaw PacBio, Illumina, Hi-C, and transcriptome sequencing data of \u003cem\u003eG. speciosus\u0026nbsp;\u003c/em\u003ehave been deposited in the Genome Sequence Archive (GSA) with accession number PRJCA051301 (https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA051301). The assembled genome is available under GSA accession GWHHJEF00000000.1 (https://ngdc.cncb.ac.cn/gwh/Assembly/109454/show).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eTechnical Validation\u003c/h2\u003e\n\u003cp\u003eSequence accuracy and completeness of the \u003cem\u003eG. speciosus\u003c/em\u003e assembly were examined at three independent levels. Short-insert reads with 143.66 Gb data realigned with BWA-MEM at 99.67% mapping rate and 99.94 % genome-wide coverage; PacBio HiFi reads with 40.77 Gb data mapped with minimap2 at 99.88% rate and 99.99% coverage. Merqury k-mer analysis yielded a genome-wide QV of 43.0153, corresponding to more than 99.99% base-level accuracy. The final assembly anchors 584.08 Mb of the 600.31 Mb genome to 24 chromosomal scaffolds with an anchoring rate of 97.3%. BUSCO genome assessment against the actinopterygii_odb10 lineage set classified 98.0 % of orthologs as complete (97.1% single-copy, 0.9% duplicated), 0.2% as fragmented and 1.7% as missing, indicating high chromosome-scale completeness and completeness of the gene catalogue. BUSCO protein mode recovered 94.6 % complete orthologs among the 22,413 predicted protein-coding genes. Functional annotation succeeded for 21,825 genes (97.38%) against SwissProt, NR, KEGG, InterPro and GO databases. A total of 10,233 non-coding RNAs were annotated, including 1,336 tRNAs and 876 rRNAs, confirming a comprehensive gene set.\u003c/p\u003e\n\u003ch2\u003eCode availability\u003c/h2\u003e\n\u003cp\u003eNo specific script was used in this work. All commands and pipelines used in data processing were executed according to the manual and protocols of the corresponding bioinformatic software. Software/packages with their versions and settings for genome annotation were listed: tandem Repeats Finder (v4.09.1) using the following parameters 2 7 7 80 10 50 2000 -d -h; RepeatModeler (v2.0.1) and LTR_FINDER (v1.0.7); RepeatMasker (v4.1.2); TBlastN (v2.11.0+); Exonerate (v2.4.0); HISAT2 (v2.2.1); StringTie (v2.1.7) and Trinity (v2.8.5); PASA (v2.4.1); Augustus (v3.4.0); Genscan (v1.0); MAKER (v3.01.03); tRNAscan-SE (v2.0.9); RNAmmer (v1.2); Infernal (v1.1.4); Rfam (v14.6).\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis project was supported by Project for Breakthrough in Key Technologies of Suitable Species in Modern Marine Pasture in Guangdong Province (no.2024HYMC-40).\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eChunhua Zhu and Li Lin conceived this project. Feng Li and Jinghui Wu participated in data analysis. Guangli Li and Zhendong Qin collected the samples. Feng Li assembled the genome. Feng Li and Zhendong Qin generated the annotation set. Feng Li wrote the manuscript. Chunhua Zhu and Li Lin revised themanuscript. All authors have read and approved the final manuscript for publication.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGrandcourt, E. M., Al Abdessalaam, T. Z., Francis, F. \u0026amp; Al Shamsi, A. Population biology and assessment of representatives of the family Carangidae: Carangoides bajad and Gnathanodon speciosus (Forssk\u0026aring;l, 1775), in the Southern Arabian Gulf. \u003cem\u003eFish Res\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e, 331\u0026ndash;341 (2004).\u003c/li\u003e\n\u003cli\u003eTran, T. L. 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CAFE: A computational tool for the study of gene family evolution. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, (2006).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8159327/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8159327/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe golden trevally (\u003cem\u003eGnathanodon speciosus\u003c/em\u003e) is an economically vital marine species in tropical and subtropical fisheries and aquaculture, yet its genomic resources remain underdeveloped. This study presents the first haplotype-resolved, chromosome-level genome assembly of \u003cem\u003eG. speciosus\u003c/em\u003e, combining PacBio HiFi long-read sequencing, Hi-C scaffolding, and short-read data. The assembled genome spans 600.31 Mb​ (N50\u0026thinsp;=\u0026thinsp;21.45 Mb) and is anchored to 24 chromosomes with a 97.3% anchoring rate. Genome annotation revealed 22,413 protein-coding genes, 22.71% repetitive sequences, and 10,233 non-coding RNAs. Comparative genomic analyses uncovered significant gene family expansions/contractions and positively selected genes enriched in environmental adaptation and developmental processes. Divergence time estimation placed \u003cem\u003eG. speciosus\u003c/em\u003e within the Carangidae lineage. This genome provides a foundational resource for evolutionary studies, aquaculture breeding, and adaptation mechanisms in carangid fishes.\u003c/p\u003e","manuscriptTitle":"Chromosome-level genome assembly of the Golden trevally (Gnathanodon speciosus)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-02 19:36:26","doi":"10.21203/rs.3.rs-8159327/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"140c9a34-8480-42f3-a8eb-e5bae6532165","owner":[],"postedDate":"March 2nd, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-04T13:11:13+00:00","index":66,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T10:57:21+00:00","index":65,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T19:36:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-02 19:36:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8159327","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8159327","identity":"rs-8159327","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}