Genome-wide discovery, characterization, and validation of novel SSR markers in clove (Syzygium aromaticum) and their application in genetic analysis

Genome-wide discovery, characterization, and validation of novel SSR markers in clove (Syzygium aromaticum) and their application in genetic analysis | 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-wide discovery, characterization, and validation of novel SSR markers in clove (Syzygium aromaticum) and their application in genetic analysis Muhammed Azharudheen TP, Muhammed Nissar VA, Anees K, Lijo Thomas, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8249733/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Genetic improvement of clove ( Syzygium aromaticum ), an economically significant spice and medicinal crop, has been constrained by limited molecular resources. The recent availability of a reference genome enabled the first comprehensive, genome-wide characterization and development of simple sequence repeats (SSRs). An in-silico survey of the 367.77 Mb genome assembly identified 189,127 SSRs at an overall frequency of 514.25 loci/Mb. Mononucleotides (50.30%) and dinucleotides (40.68%) were the most abundant repeat types, exhibiting a strong A/T-rich bias and a notable scarcity of CG/GC motifs. Although SSRs were uniformly distributed at the whole-chromosome level (R² = 0.99 relative to chromosome length), high-resolution analysis revealed significant intra-chromosomal heterogeneity, including distinct hotspots (> 600loci/Mb) and coldspots (< 400 loci/Mb). A total of 11,107 hypervariable Class I loci were identified, which were uniquely dominated by dinucleotide repeats (92.3%), pinpointing them as the primary source of hypervariability. From these findings, a comprehensive resource of 28,147 high-quality primer pairs targeting Class I and II loci was developed and curated into a publicly accessible, searchable Clove SSR Marker Database. Experimental validation of 55 primer pairs across 19 germplasm accessions demonstrated high amplification success (96.4%) and polymorphism rates (90.6%). The validated markers revealed moderate genetic diversity (mean He​= 0.22, PIC = 0.18), indicative of a potential introductory founder effect in the analyzed germplasm. These markers successfully resolved the 19 accessions into distinct phylogenetic clades, identifying a highly divergent accession (Acc. 9833). The practical utility of the markers was further confirmed by verifying the F 1 hybrid status of high-yielding plants, identifying hybrids at the seedling stage within open-pollinated progeny, and confirming co-dominant 1:2:1 Mendelian segregation in an F 2 population. This comprehensive, validated SSR marker toolkit and database provide a foundational resource to accelerate clove breeding programs, germplasm management, and future genetic mapping studies. Clove Genetic Diversity Hybrid Identification Marker Development Microsatellite SSR Syzygium aromaticum Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 05 Jan, 2026 Reviews received at journal 01 Jan, 2026 Reviews received at journal 29 Dec, 2025 Reviews received at journal 24 Dec, 2025 Reviewers agreed at journal 19 Dec, 2025 Reviewers agreed at journal 16 Dec, 2025 Reviewers agreed at journal 16 Dec, 2025 Reviewers invited by journal 16 Dec, 2025 Editor invited by journal 08 Dec, 2025 Editor assigned by journal 05 Dec, 2025 Submission checks completed at journal 05 Dec, 2025 First submitted to journal 01 Dec, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-8249733","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":561462260,"identity":"f9ae8932-ec02-4b2d-9887-e96570554803","order_by":0,"name":"Muhammed Azharudheen 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analysis","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Clove, Genetic Diversity, Hybrid Identification, Marker Development, Microsatellite, SSR, Syzygium aromaticum","lastPublishedDoi":"10.21203/rs.3.rs-8249733/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8249733/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGenetic improvement of clove (\u003cem\u003eSyzygium aromaticum\u003c/em\u003e), an economically significant spice and medicinal crop, has been constrained by limited molecular resources. The recent availability of a reference genome enabled the first comprehensive, genome-wide characterization and development of simple sequence repeats (SSRs). An \u003cem\u003ein-silico\u003c/em\u003e survey of the 367.77 Mb genome assembly identified 189,127 SSRs at an overall frequency of 514.25 loci/Mb. Mononucleotides (50.30%) and dinucleotides (40.68%) were the most abundant repeat types, exhibiting a strong A/T-rich bias and a notable scarcity of CG/GC motifs. Although SSRs were uniformly distributed at the whole-chromosome level (R\u0026sup2; = 0.99 relative to chromosome length), high-resolution analysis revealed significant intra-chromosomal heterogeneity, including distinct hotspots (\u0026gt;\u0026thinsp;600loci/Mb) and coldspots (\u0026lt;\u0026thinsp;400 loci/Mb). A total of 11,107 hypervariable Class I loci were identified, which were uniquely dominated by dinucleotide repeats (92.3%), pinpointing them as the primary source of hypervariability. From these findings, a comprehensive resource of 28,147 high-quality primer pairs targeting Class I and II loci was developed and curated into a publicly accessible, searchable Clove SSR Marker Database. Experimental validation of 55 primer pairs across 19 germplasm accessions demonstrated high amplification success (96.4%) and polymorphism rates (90.6%). The validated markers revealed moderate genetic diversity (mean He​= 0.22, PIC\u0026thinsp;=\u0026thinsp;0.18), indicative of a potential introductory founder effect in the analyzed germplasm. These markers successfully resolved the 19 accessions into distinct phylogenetic clades, identifying a highly divergent accession (Acc. 9833). The practical utility of the markers was further confirmed by verifying the F\u003csub\u003e1\u003c/sub\u003e hybrid status of high-yielding plants, identifying hybrids at the seedling stage within open-pollinated progeny, and confirming co-dominant 1:2:1 Mendelian segregation in an F\u003csub\u003e2\u003c/sub\u003e population. This comprehensive, validated SSR marker toolkit and database provide a foundational resource to accelerate clove breeding programs, germplasm management, and future genetic mapping studies.\u003c/p\u003e","manuscriptTitle":"Genome-wide discovery, characterization, and validation of novel SSR markers in clove (Syzygium aromaticum) and their application in genetic analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-19 03:36:37","doi":"10.21203/rs.3.rs-8249733/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-05T07:01:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-02T02:43:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-29T06:47:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-24T19:46:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"193251212154763566351208955063643024087","date":"2025-12-19T10:51:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"109284986128218017380306639830053353562","date":"2025-12-17T02:00:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331250881095119248581629524168550007697","date":"2025-12-17T01:00:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-16T21:03:35+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-08T19:10:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-05T07:47:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-05T07:46:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-12-01T11:09:12+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":"c1892414-755c-406d-8b58-2c86fcb0f107","owner":[],"postedDate":"December 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-20T18:08:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-19 03:36:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8249733","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8249733","identity":"rs-8249733","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}