A multiplex assay with 21 single nucleotide polymorphisms for commercial Eucalyptus clones identification

A multiplex assay with 21 single nucleotide polymorphisms for commercial Eucalyptus clones identification | 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 A multiplex assay with 21 single nucleotide polymorphisms for commercial Eucalyptus clones identification Jing-Yi Zhao, Si-Ming Gan, Qi-Jie Weng, You-Jun Wu, Chang-Pin Zhou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7493160/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 Eucalyptus is the most widely cultivated hardwoodacross tropical, sub-tropical and temperate regions worldwide. However, with successive generations of tissue culture and the rapid expansion of plantations, clone mislabeling and quality decline have become increasingly problematic. In this study, 21 highly polymorphic single nucleotide polymorphism (SNP) markers were identified and characterized across 58 Eucalyptus clones using SNaPshot technology. The polymorphic information content ranged from 0.064 to 0.375, observed heterozygosity from 0.068 to 0.703, and expected heterozygosity from 0.067 to 0.503. We further established a single-tube, multicolor fluorescent SNP-detection assay that enables rapid and accurate clone authentication. These findings can be useful for identifying fake or deranged clones, safeguarding germplasm resources, and supporting future genomic and breeding studies. Eucalyptus SNaPshot technology Single nucleotide polymorphism Identify clones Figures Figure 1 Introduction Eucalyptus is the most widely planted hardwood in tropical, sub-tropical and temperate climatic regions around the world, as its fast growth, broad adaptability and multipurpose wood properties (Grattapaglia et al. 2008). The species supplies renewable feedstock for paper, biomaterials and bioenergy, while its leaves yield abundant essential oils for pharmaceutical and industrial applications (Mbabazi et al. 2011). Commercial plantations rely almost exclusively on tissue-cultured clones; however, repeated sub-culturing and large-scale propagation have led to increasing mislabeling and quality deterioration. Reliable clonal authentication is therefore essential to protect growers’ interests and breeders’ intellectual property. Among DNA markers, single-nucleotide polymorphisms (SNPs) combine high polymorphism, genome-wide abundance and compatibility with high-throughput platforms (Chaves et al. 2018). SNP sets have successfully discriminated cultivars of wheat, barley, chum salmon, and have proven powerful in human genomics (Ganal et al. 2009; Tsukagoshi and Ohari 2025). Eucalyptus exhibits exceptional nucleotide diversity, enabling SNPs to resolve phylogenies, build high-density linkage maps and characterize germplasm (Rafalski 2002). The SNaPshot minisequencing assay permits multiplexed, accurate SNP genotyping independent of site polymorphism or sample number (Ye et al. 2009; Zhou et al. 2018). Although some DNA fingerprinting has been applied to Eucalyptus clones (Li et al. 2011; Zhou et al. 2018), a simple and robust SNP-based identification system is still lacking. Here, we present the SNaPshot multiplex tailored to Eucalyptus , comprising 21 highly polymorphic SNPs that enable one-tube clonal verification. Twenty-one SNPs with high polymorphism information content were selected from resequencing data (Silva-Junior et al., 2015). PCR and SBE (Single base extension) primers were designed in Primer Premier 5.0 (Premier Biosoft) (Table 1). Fifty-eight commercial clones widely deployed in China were sampled (Table 2). Genomic DNA was extracted from fresh leaves using a modified CTAB protocol (Gan et al., 2003) and qualified DNA concentration was diluted to 30 ng/μL. Multiplex PCR reactions (20 µL) contained 10 μL 2 × Type-it Multiplex PCR Master Mix (QIAGEN), 3 μL equimolar primer mix (final concentration of each primer 0.037 µM), 3 µL template DNA and 4 µL ddH 2 O. Cycling conditions: 95 °C 5 min; 32 cycles of 95 °C 30 s, 60 °C 90 s, 72 °C 30 s; final extension 60 °C 30 min. PCR products were purified with 2 μL SAP-IT (Applied Biosystems, USA) at 37 °C 1 h, followed by 75 °C 15 min to inactivate the enzyme. SNaPshot single-base extension with 5’ tail-length SBE primer was performed according to the manufacturer’s protocol (Applied Biosystems, USA). Total volume of 10.55 μL including 1 μL treated products, 9.25 μL Hi-Di formamide and 0.3 μL GeneScan Size Standards-120LIZ (Applied Biosystems, USA) was denatured at 95 °C for 5 min, 4 °C for 4 min. The fluorescence and size of the extended products were determined by capillary electrophoresis on ABI 3130xl Genetic Analyzer (Applied Biosystems, USA). Alleles were scored using GeneMapper v4.1. For validated loci, statistics including polymorphism information content ( PIC ), observed heterozygosity ( Ho ) and expected heterozygosity ( He ) were computed in MSA software (Dieringer and Schlotterer 2003). A total of 21 SNP markers were successfully validated across 58 Eucalyptus clones, yielding clear and reproducible peaks in a single-tube, 21-plex SNaPshot assay (Fig. 1, Table 3). The panel distinctly resolved the DH32-29 and A13 reference clones (Fig. 1), confirming its utility for clone authentication. The summary statistics of genetic diversity were shown in Table 1. The Ho from 0.068 to 0.703, He from 0.067 to 0.503, and PIC ranged from 0.064 to 0.375, 18 of the 21 SNPs exhibited moderate to high polymorphism ( PIC > 0.25; Table 1). The genotype characteristics were summarized in Table 3. Overall, the newly developed 21-plex multicolor fluorescent system provides a rapid, cost-effective tool for authenticating planting material, detecting off-types, and protecting proprietary Eucalyptus germplasm. This marker set will serve as a robust reference for quality control in commercial forestry and as a foundation for future genomic studies. Declarations Acknowledgements We thank the staff of the Guangxi Forestry Research Institute for maintaining the experimental materials and for laboratory support—specifically, Changrong Li and Shengkan Chen. Author Contributions CPZ and SMG conceived and designed the study, contributed to the collection of the samples and the drafted the manuscript. JYZ contributed to the analysis and writing. QJW and YJW participated in the data collection, purification, and management of the samples for sequencing and genotyping. All authors have reviewed and approved the manuscript. Funding This work was supported by the Science and Technology Program from Forestry Administration of Guangdong Province (Grant No. 2023KJCX013), the Fundamental Research Funds of Chinese Academy of Forestry (Grant No. CAFYBB2021QD001-3), and the Guangzhou Science and Technology Plan Project (Grant No. 202102080550). Data availability The data that supports the findings of this study are available in this article. Competing Interests The authors declare no competing interests. References Chaves CL, Blanc-Jolivet C, Sebbenn AM, Mader M, Meyer-Sand BRV, Paredes-Villanueva K et al . (2019) Nuclear and chloroplastic SNP markers for genetic studies of timber origin for Hymenaea trees. Conserv Genet Resour 11: 329–331. https://doi.org/10.1007/s12686-018-1077-1 Dieringer D, Schlotterer C (2003) MICROSATELLITE ANALYSER (MSA): a platform independent analysis tool for large microsatellite data sets. Mol Ecol Notes 3:167–169. https://doi.org/10.1046/j.1471-8286.2003.00351.x Ganal MW, Altmann T, Röder MS (2009) SNP identification in crop plants. Curr Opin Plant Biol 12: 211-217. https://doi.org/10.1016/j.pbi.2008.12.009 Gan S, Shi J, Li M, Wu K, Wu J, Bai J (2003). Moderate-density molecular maps of Eucalyptus urophylla s. t. Blake and E. tereticornis smith genomes based on RAPD markers. Genetica 118: 59–67. https://doi.org/10.1023/A:1022966018079 Grattapaglia D, Kirst M (2008) Eucalyptus applied genomics: from gene sequences to breeding tools. New phytol 179: 911–929. https://doi.org/10.1111/j.1469-8137.2008.02503.x Li F, Gan S, Zhang Z, Weng Q, Xiang D, Li M (2011) Microsatellite-based genotyping of the commercial Eucalyptus clones cultivated in China. Silvae Genet 60: 216–223. https://doi.org/10.1515/sg-2011-0029 Mbabazi J (2011) Ecosystem goods and services from plantation forests. Int J Environ Stud 68: 249–250. https://doi.org/10.1080/00207233.2011.552230 Rafalski, JA (2002) Novel genetic mapping tools in plants: SNPs and LD-based approaches. Plant Sci 162: 329–333. https://doi.org/10.1016/S0168-9452(01)00587-8 Silva-Junior, Orzenil B, Faria, Danielle A. Grattapaglia, Dario (2015) A flexible multi-species genome-wide 60K SNP chip developed from pooled resequencing of 240 Eucalyptus tree genomes across 12 species. New Phytol 206: 1527-1540. https://doi.org/10.1111/nph.13322 Tsukagoshi H, Ohari,Y (2025) Characterization of 23 SNP markers for the chum salmon ( Oncorhynchus keta ) based on multiplex ISSR genotyping by sequencing (MIG-seq). Conservation Genet Resour 17: 79–82 . https://doi.org/10.1007/s12686-025-01380-y Ye RG, Huang ZG, Wang X, Wang N, Wang SY, Chen X. M (2009) Detection of single nucleotide polymorphism of IL-1B-1473 in atrophic gastritis with SNaPshot technology. World Chinese J. Digestol 12: 1202–1206. https://doi.org/10.11569/wcjd.v17.i12.1202 Zhou C, Weng Q, Gan S, Ji H, Chen S, Wang L, Li F (2018) Application of SNaPshot to detect SNP markers in Eucalyptus . J Nanjing For Univ (Nat Sci Ed) 42: 83–88. https://doi.org/10.3969/j.issn.1000-2006.2017.05055 Tables Tables 1 to 3 are available in the Supplementary Files section Additional Declarations No competing interests reported. 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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-7493160","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":507718590,"identity":"20014a91-bfec-406d-b443-8fb775697074","order_by":0,"name":"Jing-Yi Zhao","email":"","orcid":"","institution":"Research Institute of Tropical Forestry, Chinese Academy of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Jing-Yi","middleName":"","lastName":"Zhao","suffix":""},{"id":507718591,"identity":"bb65f443-21e3-4335-bd8f-5b2d0593de2c","order_by":1,"name":"Si-Ming Gan","email":"","orcid":"","institution":"Research Institute of 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Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYDACCSBmbGCAkB8YGPhJ08I4g4FBsoEELQwMzDzEaJGf3XxM4ucOuzz56Oa2xzY1dhIM7IcfMPzcgVsL45xjaZK9Z5KLDe8cbDfOOZYswcCTZsDYewa3FmaJHDNpxjbmxI0zEtukcxuY6xgYchiYGdtwa2GDaKmHaLFsqJdg4H+DXwsPRMvhxPkSQC2MDYclGCQI2CIhkZZs2dt2PHGDzME2yZ5jxyXYJJ4ZHOzFo0V+RvLBGz/bqhPnz25/JvGjplqCnz/54YOfeLTAgcENmO+A+AARGkDWEaVsFIyCUTAKRiIAAOEjSp8SpwGaAAAAAElFTkSuQmCC","orcid":"","institution":"Research Institute of Tropical Forestry, Chinese Academy of Forestry","correspondingAuthor":true,"prefix":"","firstName":"Chang-Pin","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-08-30 06:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7493160/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7493160/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90466221,"identity":"7489cda8-de83-4841-b2d9-c248799da69c","added_by":"auto","created_at":"2025-09-03 05:33:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":167978,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of DH32-29 and A13 \u003cem\u003eEucalyptus\u003c/em\u003e clones with the single-tube, 21-plex SNaPshot assay. “G” allele is represented by blue, “C” by black, “T” by red, “A” by green colour.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7493160/v1/0cd68a99ac545a929111b8a7.png"},{"id":91040572,"identity":"913a4fab-5490-400c-9ce1-837677ec0885","added_by":"auto","created_at":"2025-09-11 03:46:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":432722,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7493160/v1/83576874-2220-45d8-a546-146f73a4cbb8.pdf"},{"id":90466335,"identity":"15b337b0-9917-4d2a-aec9-689714eaa76e","added_by":"auto","created_at":"2025-09-03 05:33:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":54937,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7493160/v1/4054f127f4cf3ec9f696848f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A multiplex assay with 21 single nucleotide polymorphisms for commercial Eucalyptus clones identification","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eEucalyptus\u003c/em\u003e is the most widely planted hardwood in tropical, sub-tropical and temperate climatic regions around the world, as its fast growth, broad adaptability and\u0026nbsp;multipurpose\u0026nbsp;wood properties (Grattapaglia et al. 2008).\u0026nbsp;The species supplies renewable feedstock for paper, biomaterials and bioenergy, while its leaves yield abundant essential oils for pharmaceutical and industrial applications (Mbabazi et al. 2011). Commercial plantations rely almost exclusively on tissue-cultured clones; however, repeated sub-culturing and large-scale propagation have led to increasing mislabeling and quality deterioration. Reliable clonal authentication is therefore essential to protect growers\u0026rsquo; interests and breeders\u0026rsquo; intellectual property.\u003c/p\u003e\n\u003cp\u003eAmong DNA markers, single-nucleotide polymorphisms (SNPs) combine high polymorphism, genome-wide abundance and compatibility with high-throughput platforms (Chaves et al. 2018). SNP sets have successfully discriminated cultivars of wheat, barley, chum salmon, and have proven powerful in human genomics (Ganal et al. 2009; Tsukagoshi and Ohari 2025).\u003cem\u003eEucalyptus\u003c/em\u003e exhibits exceptional nucleotide diversity, enabling SNPs to resolve phylogenies, build high-density linkage maps and characterize germplasm (Rafalski 2002). The SNaPshot minisequencing assay permits multiplexed, accurate SNP genotyping independent of site polymorphism or sample number (Ye et al. 2009; Zhou et al. 2018). Although some DNA fingerprinting has been applied to \u003cem\u003eEucalyptus\u003c/em\u003e clones (Li et al. 2011; Zhou et al. 2018), a simple and robust SNP-based identification system is still lacking. Here, we present the SNaPshot multiplex tailored to \u003cem\u003eEucalyptus\u003c/em\u003e, comprising 21 highly polymorphic SNPs that enable one-tube clonal verification.\u003c/p\u003e\n\u003cp\u003eTwenty-one SNPs with high polymorphism information content were selected from resequencing data (Silva-Junior\u0026nbsp;et al., 2015). PCR and SBE (Single base extension) primers were\u0026nbsp;designed in Primer Premier 5.0 (Premier Biosoft) (Table 1). Fifty-eight commercial clones widely deployed in China were sampled (Table 2).\u0026nbsp;Genomic DNA was extracted from fresh leaves using a modified CTAB protocol (Gan et al., 2003) and qualified DNA concentration was diluted to 30 ng/\u0026mu;L.\u0026nbsp;Multiplex PCR\u0026nbsp;reactions (20 \u0026micro;L) contained\u0026nbsp;10 \u0026mu;L 2 \u0026times; Type-it Multiplex PCR Master Mix (QIAGEN), 3 \u0026mu;L\u0026nbsp;equimolar primer mix (final concentration of each primer 0.037 \u0026micro;M), 3 \u0026micro;L template DNA and 4 \u0026micro;L ddH\u003csub\u003e2\u003c/sub\u003eO. Cycling conditions: 95 \u0026deg;C 5 min; 32 cycles of 95 \u0026deg;C 30 s, 60 \u0026deg;C 90 s, 72 \u0026deg;C 30 s; final extension 60 \u0026deg;C 30 min.\u003c/p\u003e\n\u003cp\u003ePCR products were purified with 2\u0026nbsp;\u0026mu;L SAP-IT (Applied Biosystems, USA) at 37 \u0026deg;C 1 h,\u0026nbsp;followed by 75 \u0026deg;C 15 min to inactivate the enzyme. SNaPshot single-base extension with 5\u0026rsquo; tail-length SBE primer was performed according to the manufacturer\u0026rsquo;s protocol (Applied Biosystems, USA).\u0026nbsp;Total volume of 10.55\u0026nbsp;\u0026mu;L including 1 \u0026mu;L treated products, 9.25 \u0026mu;L Hi-Di formamide and 0.3 \u0026mu;L GeneScan Size Standards-120LIZ (Applied Biosystems, USA) was denatured at 95\u0026nbsp;\u0026deg;C for 5 min, 4 \u0026deg;C for 4 min. The fluorescence and size of the extended products were determined by capillary electrophoresis on ABI 3130xl Genetic Analyzer (Applied Biosystems, USA).\u0026nbsp;Alleles were scored using GeneMapper v4.1.\u0026nbsp;For validated loci, statistics including polymorphism information content (\u003cem\u003ePIC\u003c/em\u003e), observed heterozygosity (\u003cem\u003eHo\u003c/em\u003e) and expected heterozygosity (\u003cem\u003eHe\u003c/em\u003e) were computed in MSA software (Dieringer and Schlotterer 2003).\u003c/p\u003e\n\u003cp\u003eA total of 21 SNP markers were successfully validated across 58 \u003cem\u003eEucalyptus\u003c/em\u003e clones, yielding clear and reproducible peaks in a single-tube, 21-plex SNaPshot assay (Fig. 1, Table 3). The panel distinctly resolved the DH32-29 and A13 reference clones (Fig. 1), confirming its utility for clone authentication. \u0026nbsp;The summary statistics of genetic diversity were shown in Table 1. The\u003cem\u003e\u0026nbsp;Ho\u003c/em\u003e from 0.068 to 0.703, \u003cem\u003eHe\u003c/em\u003e from 0.067 to 0.503, and \u003cem\u003ePIC\u003c/em\u003e ranged from 0.064 to 0.375, 18 of the 21 SNPs exhibited moderate to high polymorphism (\u003cem\u003ePIC\u003c/em\u003e \u0026gt; 0.25; Table 1). The genotype characteristics were summarized in Table 3.\u003c/p\u003e\n\u003cp\u003eOverall, the newly developed 21-plex multicolor fluorescent system provides a rapid, cost-effective tool for authenticating planting material, detecting off-types, and protecting proprietary \u003cem\u003eEucalyptus\u0026nbsp;\u003c/em\u003egermplasm. This marker set will serve as a robust reference for quality control in commercial forestry and as a foundation for future genomic studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWe thank the staff of the Guangxi Forestry Research Institute for maintaining the experimental materials and for laboratory support—specifically, Changrong Li and Shengkan Chen.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCPZ and SMG\u003c/em\u003e\u003cem\u003e\u0026nbsp;conceived and designed the study, contributed to the collection of the samples and the drafted the manuscript.\u0026nbsp;\u003c/em\u003e\u003cem\u003eJYZ\u003c/em\u003e\u003cem\u003e\u0026nbsp;contributed to the analysis and writing.\u0026nbsp;\u003c/em\u003e\u003cem\u003eQJW and YJW\u003c/em\u003e\u003cem\u003e\u0026nbsp;participated in the data collection, purification, and management of the samples for sequencing and genotyping. All authors have reviewed and approved the manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis work was supported by\u0026nbsp;\u003c/em\u003ethe Science and Technology Program from Forestry Administration of Guangdong Province (Grant No. 2023KJCX013), the Fundamental Research Funds of Chinese Academy of Forestry (Grant No. CAFYBB2021QD001-3), and the Guangzhou Science and Technology Plan Project (Grant No. 202102080550).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe data that supports the findings of this study are available in this article.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eChaves CL, Blanc-Jolivet C, Sebbenn AM, Mader M, Meyer-Sand BRV, Paredes-Villanueva K \u003cem\u003eet al\u003c/em\u003e. (2019) Nuclear and chloroplastic SNP markers for genetic studies of timber origin for \u003cem\u003eHymenaea\u003c/em\u003e trees. Conserv Genet Resour 11: 329\u0026ndash;331. https://doi.org/10.1007/s12686-018-1077-1\u003c/li\u003e\n \u003cli\u003eDieringer D, Schlotterer C (2003) MICROSATELLITE ANALYSER (MSA): a platform independent analysis tool for large microsatellite data sets. Mol Ecol Notes 3:167\u0026ndash;169. https://doi.org/10.1046/j.1471-8286.2003.00351.x\u003c/li\u003e\n \u003cli\u003eGanal MW, Altmann T, R\u0026ouml;der MS (2009) SNP identification in crop plants. Curr Opin Plant Biol 12: 211-217. https://doi.org/10.1016/j.pbi.2008.12.009\u003c/li\u003e\n \u003cli\u003eGan S, Shi J, Li M, Wu K, Wu J, Bai J (2003). Moderate-density molecular maps of \u003cem\u003eEucalyptus urophylla\u0026nbsp;\u003c/em\u003es. t. Blake and \u003cem\u003eE. tereticornis\u003c/em\u003e smith genomes based on RAPD markers. Genetica 118: 59\u0026ndash;67. https://doi.org/10.1023/A:1022966018079\u003c/li\u003e\n \u003cli\u003eGrattapaglia D, Kirst M (2008) \u003cem\u003eEucalyptus\u003c/em\u003e applied genomics: from gene sequences to breeding tools. New phytol 179: 911\u0026ndash;929. https://doi.org/10.1111/j.1469-8137.2008.02503.x\u003c/li\u003e\n \u003cli\u003eLi F, Gan S, Zhang Z, Weng Q, Xiang D, Li M (2011) Microsatellite-based genotyping of the commercial \u003cem\u003eEucalyptus\u003c/em\u003e clones cultivated in China. Silvae Genet 60: 216\u0026ndash;223. https://doi.org/10.1515/sg-2011-0029\u003c/li\u003e\n \u003cli\u003eMbabazi J (2011) Ecosystem goods and services from plantation forests. Int J Environ Stud 68: 249\u0026ndash;250. https://doi.org/10.1080/00207233.2011.552230\u003c/li\u003e\n \u003cli\u003eRafalski, JA (2002) Novel genetic mapping tools in plants: SNPs and LD-based approaches. Plant Sci 162: 329\u0026ndash;333. https://doi.org/10.1016/S0168-9452(01)00587-8\u003c/li\u003e\n \u003cli\u003eSilva-Junior, Orzenil B, Faria, Danielle A. Grattapaglia, Dario (2015) A flexible multi-species genome-wide 60K SNP chip developed from pooled resequencing of 240 \u003cem\u003eEucalyptus\u003c/em\u003e tree genomes across 12 species. New Phytol 206: 1527-1540. https://doi.org/10.1111/nph.13322\u003c/li\u003e\n \u003cli\u003eTsukagoshi H, Ohari,Y (2025) Characterization of 23 SNP markers for the chum salmon (\u003cem\u003eOncorhynchus keta\u003c/em\u003e) based on multiplex ISSR genotyping by sequencing (MIG-seq). Conservation Genet Resour 17: 79\u0026ndash;82 . https://doi.org/10.1007/s12686-025-01380-y\u003c/li\u003e\n \u003cli\u003eYe RG, Huang ZG, Wang X, Wang N, Wang SY, Chen X. M (2009) Detection of single nucleotide polymorphism of IL-1B-1473 in atrophic gastritis with SNaPshot technology. World Chinese J. Digestol 12: 1202\u0026ndash;1206. https://doi.org/10.11569/wcjd.v17.i12.1202\u003c/li\u003e\n \u003cli\u003eZhou C, Weng Q, Gan S, Ji H, Chen S, Wang L, Li F (2018) Application of SNaPshot to detect SNP markers in \u003cem\u003eEucalyptus\u003c/em\u003e. J Nanjing For Univ (Nat Sci Ed) 42: 83\u0026ndash;88. https://doi.org/10.3969/j.issn.1000-2006.2017.05055\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Eucalyptus, SNaPshot technology, Single nucleotide polymorphism, Identify clones","lastPublishedDoi":"10.21203/rs.3.rs-7493160/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7493160/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eEucalyptus\u003c/em\u003e is the most widely cultivated hardwoodacross tropical, sub-tropical and temperate regions worldwide. However, with successive generations of tissue culture and the rapid expansion of plantations, clone mislabeling and quality decline have become increasingly problematic. In this study, 21 highly polymorphic single nucleotide polymorphism (SNP) markers were identified and characterized across 58 \u003cem\u003eEucalyptus\u003c/em\u003eclones using SNaPshot technology. The\u003cem\u003e \u003c/em\u003epolymorphic information content ranged from 0.064 to 0.375, observed heterozygosity from 0.068 to 0.703, and expected heterozygosity from 0.067 to 0.503. We further established a single-tube, multicolor fluorescent SNP-detection assay that enables rapid and accurate clone authentication. These findings can be useful for identifying fake or deranged clones, safeguarding germplasm resources, and supporting future genomic and breeding studies.\u003c/p\u003e","manuscriptTitle":"A multiplex assay with 21 single nucleotide polymorphisms for commercial Eucalyptus clones identification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-03 05:31:27","doi":"10.21203/rs.3.rs-7493160/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":"dd754a7c-a6a7-432b-836a-acd4ce8f4bd6","owner":[],"postedDate":"September 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-11T03:38:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-03 05:31:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7493160","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7493160","identity":"rs-7493160","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}