Unravelling Codon Usage Bias in the VTE4 Gene governing γ- tocopherol methyltransferase in the Oilseed Brassica

Unravelling Codon Usage Bias in the VTE4 Gene governing γ- tocopherol methyltransferase in the Oilseed Brassica | 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 Unravelling Codon Usage Bias in the VTE4 Gene governing γ- tocopherol methyltransferase in the Oilseed Brassica Vijay Kamal Meena, Kapil Choudhary, Subhash Chand, Prashant Yadav, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9409680/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Codon usage bias (CUB) is a phenomenon that shows variation both within the genes of a species and across different species, wherein particular codons are preferred over their synonymous counterparts. Genomic nucleotide configuration provides insights into the molecular basis of genes and their evolutionary relationships in distinct plant species. In this study, the VTE4 gene, responsible for enhancing the nutritional quality and oil stability of oilseed Brassica , sequences from six Brassica species were extracted through the NCBI GenBank database. Subsequently, various CUB-related parameters, including nucleotide composition (AT and GC content), relative synonymous codon usage (RSCU), effective number of codons (ENC), frequency of optimal codons (Fop), relative codon usage bias (RCBS), neutrality plot (GC12 vs. GC3), parity rule-2 [(A3/(A3 + T3) vs. (G3/(G3 + C3)], and correspondence analysis, were assessed to analyse codon biasness within the U’s triangle of Brassica species. The results showed AT bias across the Brassica species and moderate codon usage frequency for specific amino acids based on RSCU values in the VTE4 gene. An evolutionary study confirmed a similarity in codon usage preference among species clustered together. The elevated ENC value, along with low Fop and RSCU values, indicated a low level of gene expression and a moderate bias in codon usage preference within the Brassica genus. Moreover, the results from the neutrality plot, parity rule, and correspondence analysis revealed that natural selection pressure had a more significant influence on shaping codon usage bias (CUB) in the VTE4 gene than mutation pressure. This research contributes to understanding codon optimization, enhancing exogenous gene expression, and advancing transgenic engineering for improved α-tocopherol content profiling in Brassica species to enhance seed oil quality. Brassicas VTE4 α-tocopherol content parity rule-2 neutrality plot codon usage bias Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 28 Apr, 2026 Reviews received at journal 25 Apr, 2026 Reviewers agreed at journal 24 Apr, 2026 Reviewers invited by journal 23 Apr, 2026 Editor assigned by journal 23 Apr, 2026 Editor invited by journal 22 Apr, 2026 Submission checks completed at journal 21 Apr, 2026 First submitted to journal 21 Apr, 2026 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-9409680","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633550046,"identity":"4c599fb6-cb08-4248-8f59-6bbc6fb07f5d","order_by":0,"name":"Vijay Kamal Meena","email":"","orcid":"","institution":"Agriculture University Jodhpur","correspondingAuthor":false,"prefix":"","firstName":"Vijay","middleName":"Kamal","lastName":"Meena","suffix":""},{"id":633550048,"identity":"f449430f-7dcb-41cc-8a2f-008b2c853a45","order_by":1,"name":"Kapil Choudhary","email":"","orcid":"","institution":"Agriculture University Jodhpur","correspondingAuthor":false,"prefix":"","firstName":"Kapil","middleName":"","lastName":"Choudhary","suffix":""},{"id":633550049,"identity":"1d96f065-beb0-409d-9ff0-7ccd4be875ca","order_by":2,"name":"Subhash Chand","email":"data:image/png;base64,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","orcid":"","institution":"Indian Grassland and Fodder Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Subhash","middleName":"","lastName":"Chand","suffix":""},{"id":633550050,"identity":"e4d1ee8f-fe78-438d-9b57-1b01add8b9a3","order_by":3,"name":"Prashant Yadav","email":"","orcid":"","institution":"Directorate of Rapeseed-Mustard Research","correspondingAuthor":false,"prefix":"","firstName":"Prashant","middleName":"","lastName":"Yadav","suffix":""},{"id":633550051,"identity":"97be00e8-d110-4282-a54c-59b621ae2b51","order_by":4,"name":"Bharath Kumar Alam","email":"","orcid":"","institution":"Central Research Institute for Dryland Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Bharath","middleName":"Kumar","lastName":"Alam","suffix":""},{"id":633550052,"identity":"af58fd14-7c5e-4a31-91f2-d5efe4de993d","order_by":5,"name":"Ashish Sheera","email":"","orcid":"","institution":"Sri Karan Narendra Agriculture University, Jobner","correspondingAuthor":false,"prefix":"","firstName":"Ashish","middleName":"","lastName":"Sheera","suffix":""},{"id":633550053,"identity":"706fae62-da90-4822-a47d-db8b5ca41951","order_by":6,"name":"Rakesh Choudhary","email":"","orcid":"","institution":"Rani Lakshmi Bai Central Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Rakesh","middleName":"","lastName":"Choudhary","suffix":""},{"id":633550059,"identity":"2a511801-9ff2-4fd3-b61a-91397f77b8d7","order_by":7,"name":"Harsh Vardhan Singh Shekhawat","email":"","orcid":"","institution":"Agriculture University Jodhpur","correspondingAuthor":false,"prefix":"","firstName":"Harsh","middleName":"Vardhan Singh","lastName":"Shekhawat","suffix":""},{"id":633550061,"identity":"a5f883a4-e4d9-4128-be03-81b557f85957","order_by":8,"name":"Jitendar Kumar Sharma","email":"","orcid":"","institution":"Agriculture University Jodhpur","correspondingAuthor":false,"prefix":"","firstName":"Jitendar","middleName":"Kumar","lastName":"Sharma","suffix":""},{"id":633550064,"identity":"09bbbb54-d09a-4a26-bb37-81f3d2039e9c","order_by":9,"name":"Lekha Meel","email":"","orcid":"","institution":"Agriculture University Jodhpur","correspondingAuthor":false,"prefix":"","firstName":"Lekha","middleName":"","lastName":"Meel","suffix":""},{"id":633550067,"identity":"63ca6086-b034-434f-b414-32508c413065","order_by":10,"name":"Devendra Kumar Yadava","email":"","orcid":"","institution":"Agriculture University Jodhpur","correspondingAuthor":false,"prefix":"","firstName":"Devendra","middleName":"Kumar","lastName":"Yadava","suffix":""}],"badges":[],"createdAt":"2026-04-14 02:53:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9409680/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9409680/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108803785,"identity":"3650edb4-81e2-469d-ad01-d0a3e7556fc8","added_by":"auto","created_at":"2026-05-08 15:06:57","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":598914,"visible":true,"origin":"","legend":"","description":"","filename":"CUBBMCrevised2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9409680/v1_covered_253c7609-ad23-4747-b088-e16563b0a6ed.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unravelling Codon Usage Bias in the VTE4 Gene governing γ- tocopherol methyltransferase in the Oilseed Brassica","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Brassicas, VTE4, α-tocopherol content, parity rule-2, neutrality plot, codon usage bias","lastPublishedDoi":"10.21203/rs.3.rs-9409680/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9409680/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCodon usage bias (CUB) is a phenomenon that shows variation both within the genes of a species and across different species, wherein particular codons are preferred over their synonymous counterparts. Genomic nucleotide configuration provides insights into the molecular basis of genes and their evolutionary relationships in distinct plant species. In this study, the \u003cem\u003eVTE4\u003c/em\u003e gene, responsible for enhancing the nutritional quality and oil stability of oilseed \u003cem\u003eBrassica\u003c/em\u003e, sequences from six \u003cem\u003eBrassica\u003c/em\u003e species were extracted through the NCBI GenBank database. Subsequently, various CUB-related parameters, including nucleotide composition (AT and GC content), relative synonymous codon usage (RSCU), effective number of codons (ENC), frequency of optimal codons (Fop), relative codon usage bias (RCBS), neutrality plot (GC12 vs. GC3), parity rule-2 [(A3/(A3\u0026thinsp;+\u0026thinsp;T3) vs. (G3/(G3\u0026thinsp;+\u0026thinsp;C3)], and correspondence analysis, were assessed to analyse codon biasness within the U\u0026rsquo;s triangle of Brassica species. The results showed AT bias across the \u003cem\u003eBrassica\u003c/em\u003e species and moderate codon usage frequency for specific amino acids based on RSCU values in the \u003cem\u003eVTE4\u003c/em\u003e gene. An evolutionary study confirmed a similarity in codon usage preference among species clustered together. The elevated ENC value, along with low Fop and RSCU values, indicated a low level of gene expression and a moderate bias in codon usage preference within the \u003cem\u003eBrassica\u003c/em\u003e genus. Moreover, the results from the neutrality plot, parity rule, and correspondence analysis revealed that natural selection pressure had a more significant influence on shaping codon usage bias (CUB) in the \u003cem\u003eVTE4\u003c/em\u003e gene than mutation pressure. This research contributes to understanding codon optimization, enhancing exogenous gene expression, and advancing transgenic engineering for improved α-tocopherol content profiling in Brassica species to enhance seed oil quality.\u003c/p\u003e","manuscriptTitle":"Unravelling Codon Usage Bias in the VTE4 Gene governing γ- tocopherol methyltransferase in the Oilseed Brassica","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 17:50:04","doi":"10.21203/rs.3.rs-9409680/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"35576443486476316687904998933320450413","date":"2026-04-28T07:36:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-25T04:25:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125362733724695895967908218581225035563","date":"2026-04-24T15:18:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-23T07:58:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-23T07:55:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-22T04:09:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-21T17:21:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-04-21T16:29:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9c781ef0-c46c-4184-9799-0e74811ab472","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T17:50:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 17:50:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9409680","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9409680","identity":"rs-9409680","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}