Development of high-resolution melting markers for assessment of Mauremys sinensis and Mauremys reevesii hybridization in South Korea

Development of high-resolution melting markers for assessment of Mauremys sinensis and Mauremys reevesii hybridization in South Korea | 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 Development of high-resolution melting markers for assessment of Mauremys sinensis and Mauremys reevesii hybridization in South Korea Hae-jun Baek, Soo-In Lee, Chang-Deuk Park, Ju-Duk Yoon This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4877974/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Jan, 2025 Read the published version in Conservation Genetics Resources → Version 1 posted 7 You are reading this latest preprint version Abstract South Korea is home to two Mauremys species: M. sinensis and M. reevesii . M. sinensis is an invasive alien species, whereas M. reevesii is a legally protected species endemic to South Korea. The sympatric distributions of these species and their hybrids have recently been identified in South Korea, posing a serious threat to the conservation of M. reevesii . Therefore, we developed a genotyping marker for high-resolution melting (HRM) analysis that targets the intron I region of the nuclear R35 gene to facilitate the rapid identification of hybrids. Three distinct HRM curves, representing M. reevesii , M. sinensis , and their hybrids, were obtained. These results were identical to those observed for R35 , with the hybrid possessing both alleles. This demonstrates that HRM markers can effectively distinguish Mauremys species from their hybrids. The present study provides a valuable tool for rapid and accurate identification of hybrids for future monitoring. Mauremys spp. Hybridization HRM marker R35 Figures Figure 1 Figure 2 Full Text Mauremys is a globally traded species group that was newly identified in 2012 in the South Korean wild (National Institute of Environmental Research 2012) and was designated as an invasive alien species in South Korea in 2020. The National Institute of Ecology (2018) confirmed the sympatric distribution of M. sinensis and M. reevesii , and hybrids as well were identified, indicating a serious threat to the conservation of domestically protected species. Therefore, this study aimed to develop genotypic markers for application in high-resolution melting (HRM) analysis for the rapid and reliable identification of M. sinensis , M. reevesii , and their hybrids. HRM analysis enables genotyping by determining DNA variations, such as single nucleotide polymorphisms (SNPs), based on the melt transition (Tm) shape of real-time PCR products (Reed et al. 2007; Wittwer 2009; Wittwer et al. 2003). It has been applied extensively in hybridization studies and species identification (Buglione et al. 2020; Ganopoulos et al. 2013; Minett et al. 2021; Ouso et al. 2020; Park et al. 2023). Therefore, this study aimed to develop HRM markers that can distinguish M. sinensis , M. reevesii , and their hybrids for future monitoring. Thirteen blood and tissue samples were used to assess HRM marker development (Online Resource 1). The samples belonged to three, six, and four individual M. sinensis , M. reevesii , and hybrids, respectively. DNA was extracted using a Qiagen DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For molecular identification, the R35 region of nuclear DNA intron 1 was amplified using MAU-R35-1f (5′-CAAAAGTCATTCTCTGGCTTC-3′), MAU-R35-2f (5′-GTCAGACTTCTTTGCATATTTGTAA-3′), and MAU-R35-1r (5′-CAACTATGTGCTGGACAG-3′), which were designed in the present study. The sequences of the R35 fragments were edited and aligned using Geneious 5.3.6 (BIOMATTERS, Auckland, New Zealand) and Multiple-sequence alignments were performed using CLUSTAL X (Thompson et al. 1997). We analyzed the DNA sequence data of 10 Mauremys species from the NCBI GenBank database to design a primer combination for PCR amplification of the HRM molecular marker in the intron 1 region of the nuclear R35 gene belonging to M. sinensis and M. reevesii . These sequences were aligned with the newly produced sequence data using ClustalW in BioEdit 7.2.5 (Özvegy et al. 2015), and a DNA sequence matrix was created. Based on these results, the ProFlex PCR System (Thermo Fisher Scientific, Waltham, MA, USA) was used to design new PCR primer combinations containing SNPs that could distinguish between M. sinensis , M. reevesii, and their hybrids. The parameters included an 18–20-mer length of the oligonucleotide sequence, 40–55% GC content, and a Tm of 50–60 °C. The novel PCR primers for HRM analysis were MAU-R36-0658f (5′-TCAGCTTCTCAGCTTCTTTC-3′) and MAU-R36-0713r (5′-CAGTGCCAGGCAGGATT-3′) (Fig 1). PCR amplification was carried out using the QuantStudio5 Real-Time PCR System (Thermo Fisher Scientific) with MeltDoctor HRM Master Mix (Thermo Fisher Scientific) to perform HRM analysis. The holding stage consisted of an enzyme-activation step at 95 °C for 10 min, whereas the cycling stage consisted of a 40-cycle denaturation step at 95 °C for 15 s and an annealing/elongation step at 60 °C for 1 min. The melt curve/dissociation stage included denaturation at 95 °C for 10 s, annealing at 60 °C for 1 min, HRM at 95 °C for 15 s, and annealing at 60 °C for 15 s. SNPs that could distinguish between M. sinensis , M. reevesii , and their hybrids were examined in intron 1 of R35 . Novel molecular markers, including PCR primer combinations, were subsequently established. The forward and reverse primers were highly conserved, with no genetic variation between M. sinensis and M. reevesii . In addition, the SNPs showed a 1-bp genetic variation between the two species (Fig 1). Thirteen specimens were subjected to HRM analysis and divided into three genotypes: M. sinensis (A), M. reevesii (G), and M. sinensis × M. reevesii (R). HRM analysis using the genotyping markers revealed that three specimens produced a single peak with a Tm of 74.9 °C that corresponded to M. sinensis (A). Moreover, six specimens with a Tm of 75.4–75.7 °C corresponded to M. reevesii (G), whereas four specimens with a Tm of 75.2 °C corresponded to M. sinensis × M. reevesii (G) (Online Resource 1). Further analysis using the melting curves produced visibly different plot shapes (Fig 2). These results were consistent with those observed for R35 (Online Resource 1). In the present study, we successfully developed HRM markers to distinguish the hybrids from M. sinensis and M. reevesii . The HRM markers developed in this study will be useful for the rapid identification of M. sinensis and interspecific hybrids. However, they had limitations in the identification of other turtles in the genus Mauremys . Therefore, further studies should be conducted to develop markers that can compensate for these limitations. Declarations Acknowledgments We would like to thank the members of the Invasive Alien Species Team at the National Institute of Ecology and the students at the Ewha Woman’s University for helping us to carry out the research. We would also like to thank AquaGenTech Co., Ltd. for their great help in the development of the markers. Funding : This work was supported by the National Institute of Ecology, which is funded by the Ministry of Environment of Republic of Korea (grant number NIE-A-2024-09). Competing interests : The authors have no relevant financial or non-financial interests to disclose. Availability of data and material : All data generated or analysed during this study are included in this published article and its supplementary information files. Code availability : Not applicable. Authors' contributions : Conceptualization, data curation, visualization, writing–original draft, H.-J.B.; conceptualization, writing–original draft, S.-I.L. investigation, writing–review and editing, C.-D.P. writing–review and editing, J.-D.Y.; All authors have read and approved the final manuscript. Ethics approval : The Animal Care and Use protocol was reviewed and approved by the Institutional Animal Care and Use Committee at National Institute of Ecology Center, South Korea (approval numbers: NIEIACUC-2021-027, NIEIACUC-2022-03). Consent to participate : Not applicable. Consent for publication : Not applicable. References Buglione M, Petrelli S, Notomista T, de Filippo G, Gregorio R, Fulgione D (2020) Who is who? High resolution melting analysis to discern between hare species using non-invasive sampling. Conserv Genet Resour 12:727–732. https://doi.org/10.1007/s12686-020-01153-9 Ganopoulos I, Aravanopoulos F, Madesis P, Pasentsis K, Bosmali I, Ouzounis C, Tsaftaris A (2013) Taxonomic identification of Mediterranean pines and their hybrids based on the high resolution melting (HRM) and trnL approaches: from cytoplasmic inheritance to timber tracing. PLOS ONE 8:e60945. https://doi.org/10.1371/journal.pone.0060945 Lee DH, Kim YC, Chang MH, Kim S, Kim D, Kil J (2016) Current status and management of alien turtles in Korea. J Environ Impact Assess 25:319–332. https://doi.org/10.14249/eia.2016.25.5.319 Lee Y, Lin JW, Tseng SP, Chen TS, Lin SM (2019) Human disturbance as a possible cause of genetic introgression from exotic into native Mauremys turtles. Anim Conserv 22:556–567. https://doi.org/10.1111/acv.12494 Minett JF, Garcia de Leaniz C, Brickle P, Consuegra S (2021) A new high-resolution melt curve eDNA assay to monitor the simultaneous presence of invasive brown trout ( Salmo trutta ) and endangered galaxiids. Environ DNA 3:561–572. https://doi.org/10.1002/edn3.151 National Institute of Ecology (2018) Investigating ecological risk of alien species , vol V. Seocheon, Republic of Korea, p 21–33 National Institute of Environmental Research (2012) Detailed studies on invasive alien species and their management , vol VII. Incheon, Republic of Korea, p 23–26 Ouso DO, Otiende MY, Jeneby MM, Oundo JW, Bargul JL, Miller SE et al. (2020) Three-gene PCR and high-resolution melting analysis for differentiating vertebrate species mitochondrial DNA for biodiversity research and complementing forensic surveillance. Sci Rep 10:4741. https://doi.org/10.1038/s41598-020-61600-3 Özvegy J, Marinković D, Vučićević M, Gajić B, Stevanović J, Krnjaić D, Aleksić-Kovačević S (2015) Cytological and molecular identification of Haemogregarina stepanowi in blood samples of the European pond turtle ( Emys orbicularis ) from quarantine at Belgrade Zoo. Acta Vet 65:443–453. https://doi.org/10.1515/acve-2015-0037 Park CD, Kim KS, Kim KY, Heo JS, Oh HS, Park SM (2023) Development of the genotyping marker for Reeves’ turtle ( Mauremys reevesii ) using high-resolution melting (HRM) analysis. Conserv Genet Resour 15:149–152. https://doi.org/10.1007/s12686-023-01314-6 Reed GH, Kent JO, Wittwer CT (2007) High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics 8:597–608. https://doi.org/10.2217/14622416.8.6.597 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882. https://doi.org/10.1093/nar/25.24.4876 Wittwer CT (2009) High‐resolution DNA melting analysis: advancements and limitations. Hum Mutat 30:857–859. https://doi.org/10.1002/humu.20951 Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49:853–860. https://doi.org/10.1373/49.6.853 Additional Declarations No competing interests reported. Supplementary Files ESM1.pdf Cite Share Download PDF Status: Published Journal Publication published 29 Jan, 2025 Read the published version in Conservation Genetics Resources → Version 1 posted Editorial decision: Revision requested 28 Oct, 2024 Reviews received at journal 12 Sep, 2024 Reviewers agreed at journal 23 Aug, 2024 Reviewers invited by journal 21 Aug, 2024 Editor assigned by journal 08 Aug, 2024 Submission checks completed at journal 08 Aug, 2024 First submitted to journal 08 Aug, 2024 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. 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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-4877974","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":345080454,"identity":"00cfb9d3-3dfa-4cb2-a73f-b34ec02e46e8","order_by":0,"name":"Hae-jun 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hybrids\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4877974/v1/17eb6aef930966cfd9ff933e.png"},{"id":63906819,"identity":"568ff39e-6b7e-4c62-974f-4f427223eda6","added_by":"auto","created_at":"2024-09-03 15:28:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":332906,"visible":true,"origin":"","legend":"\u003cp\u003eDerivative melt curves (a) and difference plot curves (b) produced through HRM analysis of \u003cem\u003eM. sinensis, M. reevesii\u003c/em\u003e, and \u003cem\u003eM. sinensis × M. reevesii \u003c/em\u003ehybrids\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4877974/v1/87a4b6ba789186f871e1b02c.png"},{"id":75351952,"identity":"11e13d90-3b4b-4f5d-8341-d9d93eb8fcd2","added_by":"auto","created_at":"2025-02-03 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Text","content":"\u003cp\u003e\u003cem\u003eMauremys\u003c/em\u003e is a globally traded species group that was newly identified in 2012 in the South Korean wild (National Institute of Environmental Research 2012)\u0026nbsp;and\u0026nbsp;was designated as an invasive alien species in South Korea in 2020. The National Institute of Ecology (2018) confirmed the sympatric distribution of \u003cem\u003eM. sinensis\u003c/em\u003e and \u003cem\u003eM. reevesii\u003c/em\u003e,\u0026nbsp;and\u0026nbsp;hybrids\u0026nbsp;as\u0026nbsp;well\u0026nbsp;were identified, indicating a serious threat to the conservation of domestically protected species. Therefore, this study aimed to develop genotypic markers for application in high-resolution melting (HRM) analysis for the rapid and reliable identification of \u003cem\u003eM. sinensis\u003c/em\u003e, \u003cem\u003eM. reevesii\u003c/em\u003e, and their hybrids.\u003c/p\u003e\n\u003cp\u003eHRM analysis enables genotyping by determining DNA variations, such as single nucleotide polymorphisms (SNPs), based on the melt transition (Tm) shape of real-time PCR products (Reed et al. 2007; Wittwer 2009; Wittwer et al. 2003).\u0026nbsp;It has been applied extensively in hybridization studies and species identification (Buglione et al. 2020; Ganopoulos et al. 2013; Minett et al. 2021; Ouso et al. 2020; Park et al. 2023). Therefore, this study aimed to develop HRM markers that can distinguish \u003cem\u003eM. sinensis\u003c/em\u003e, \u003cem\u003eM. reevesii\u003c/em\u003e, and their hybrids for future monitoring.\u003c/p\u003e\n\u003cp\u003eThirteen blood and tissue samples were used to assess HRM marker development (Online Resource\u0026nbsp;1). The samples belonged to three, six, and four individual \u003cem\u003eM. sinensis\u003c/em\u003e, \u003cem\u003eM. reevesii\u003c/em\u003e, and hybrids, respectively. DNA was extracted using a Qiagen DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions. For molecular identification, the \u003cem\u003eR35\u003c/em\u003e region of nuclear DNA intron 1 was amplified using MAU-R35-1f (5\u0026prime;-CAAAAGTCATTCTCTGGCTTC-3\u0026prime;), MAU-R35-2f (5\u0026prime;-GTCAGACTTCTTTGCATATTTGTAA-3\u0026prime;), and MAU-R35-1r (5\u0026prime;-CAACTATGTGCTGGACAG-3\u0026prime;), which were designed in the present study. The sequences of the \u003cem\u003eR35\u003c/em\u003e fragments were edited and aligned using Geneious 5.3.6 (BIOMATTERS, Auckland, New Zealand)\u0026nbsp;and\u0026nbsp;Multiple-sequence alignments were performed using CLUSTAL X (Thompson et al. 1997).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe analyzed the DNA sequence data of\u0026nbsp;10\u0026nbsp;\u003cem\u003eMauremys\u003c/em\u003e species from the NCBI GenBank database to design a primer combination for PCR amplification of the HRM molecular marker in the intron 1 region of the nuclear \u003cem\u003eR35\u003c/em\u003e gene belonging to \u003cem\u003eM. sinensis\u003c/em\u003e and \u003cem\u003eM. reevesii\u003c/em\u003e. These sequences were aligned with the newly produced sequence data using ClustalW in BioEdit 7.2.5 (\u0026Ouml;zvegy et al. 2015), and a DNA sequence matrix was created. Based on these results, the ProFlex PCR System (Thermo Fisher Scientific, Waltham, MA, USA) was used to design new PCR primer combinations containing SNPs that could distinguish between \u003cem\u003eM. sinensis\u003c/em\u003e, \u003cem\u003eM. reevesii,\u003c/em\u003e and their hybrids. The parameters included an 18\u0026ndash;20-mer length of the oligonucleotide sequence, 40\u0026ndash;55% GC content, and a Tm of 50\u0026ndash;60 \u0026deg;C. The novel PCR primers for HRM analysis were MAU-R36-0658f (5\u0026prime;-TCAGCTTCTCAGCTTCTTTC-3\u0026prime;) and MAU-R36-0713r (5\u0026prime;-CAGTGCCAGGCAGGATT-3\u0026prime;) (Fig 1). PCR amplification was carried out using the QuantStudio5 Real-Time PCR System (Thermo Fisher Scientific) with MeltDoctor HRM Master Mix (Thermo Fisher Scientific) to perform HRM analysis. The holding stage consisted of an enzyme-activation step at 95 \u0026deg;C for 10 min, whereas the cycling stage consisted of a 40-cycle denaturation step at 95 \u0026deg;C for 15 s and an annealing/elongation step at 60 \u0026deg;C for 1 min. The melt curve/dissociation stage included denaturation at 95 \u0026deg;C for 10 s, annealing at 60 \u0026deg;C for 1 min, HRM at 95 \u0026deg;C for 15 s, and annealing at 60 \u0026deg;C for 15 s.\u003c/p\u003e\n\u003cp\u003eSNPs that could distinguish between \u003cem\u003eM. sinensis\u003c/em\u003e, \u003cem\u003eM. reevesii\u003c/em\u003e, and their hybrids were examined in intron 1 of \u003cem\u003eR35\u003c/em\u003e. Novel molecular markers, including PCR primer combinations, were subsequently established. The forward and reverse primers were highly conserved, with no genetic variation between \u003cem\u003eM. sinensis\u003c/em\u003e and \u003cem\u003eM. reevesii\u003c/em\u003e. In addition, the SNPs showed a 1-bp genetic variation between the two species (Fig 1). Thirteen specimens were subjected to HRM analysis and divided into three genotypes: \u003cem\u003eM. sinensis\u003c/em\u003e (A), \u003cem\u003eM. reevesii\u003c/em\u003e (G), and \u003cem\u003eM. sinensis\u0026nbsp;\u003c/em\u003e\u0026times; \u003cem\u003eM. reevesii\u003c/em\u003e (R).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHRM analysis using the genotyping markers revealed that three specimens produced a single peak with a Tm of 74.9 \u0026deg;C that corresponded to \u003cem\u003eM. sinensis\u003c/em\u003e (A). Moreover, six specimens with a Tm of 75.4\u0026ndash;75.7 \u0026deg;C corresponded to \u003cem\u003eM. reevesii\u003c/em\u003e (G), whereas four specimens with a Tm of 75.2 \u0026deg;C corresponded to \u003cem\u003eM. sinensis \u0026times; M. reevesii\u0026nbsp;\u003c/em\u003e(G) (Online Resource 1). Further analysis using the melting curves produced visibly different plot shapes (Fig 2). These results were consistent with those observed for \u003cem\u003eR35\u0026nbsp;\u003c/em\u003e(Online Resource 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the present study, we successfully developed HRM markers to distinguish the hybrids from \u003cem\u003eM. sinensis\u003c/em\u003e and \u003cem\u003eM. reevesii\u003c/em\u003e. The HRM markers developed in this study will be useful for the rapid identification of \u003cem\u003eM. sinensis\u003c/em\u003e and interspecific hybrids. However, they had limitations in the identification of other turtles in the genus \u003cem\u003eMauremys\u003c/em\u003e. Therefore, further studies should be conducted to develop markers that can compensate for these limitations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the members of the Invasive Alien Species Team at the National Institute of Ecology and the students at the Ewha Woman\u0026rsquo;s University for helping us to carry out the research. We would also like to thank AquaGenTech Co., Ltd. for their great help in the development of the markers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e:\u0026nbsp;This work was supported by the National Institute of Ecology, which is funded by the Ministry of Environment of Republic of Korea (grant number NIE-A-2024-09).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e:\u0026nbsp;The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e: All data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e: Conceptualization, data curation, visualization, writing\u0026ndash;original draft, H.-J.B.; conceptualization, writing\u0026ndash;original draft, S.-I.L. investigation, writing\u0026ndash;review and editing, C.-D.P. writing\u0026ndash;review and editing, J.-D.Y.; All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e:\u0026nbsp;The Animal Care and Use protocol was reviewed and approved by the Institutional Animal Care and Use Committee at National Institute of Ecology Center, South Korea (approval numbers: NIEIACUC-2021-027, NIEIACUC-2022-03).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e:\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: \u0026nbsp;Not applicable.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBuglione M, Petrelli S, Notomista T, de Filippo G, Gregorio R, Fulgione D (2020) Who is who? 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J Environ Impact Assess 25:319\u0026ndash;332. https://doi.org/10.14249/eia.2016.25.5.319\u003c/li\u003e\n\u003cli\u003eLee Y, Lin JW, Tseng SP, Chen TS, Lin SM (2019) Human disturbance as a possible cause of genetic introgression from exotic into native \u003cem\u003eMauremys\u003c/em\u003e turtles. Anim Conserv 22:556\u0026ndash;567. https://doi.org/10.1111/acv.12494\u003c/li\u003e\n\u003cli\u003eMinett JF, Garcia de Leaniz C, Brickle P, Consuegra S (2021) A new high-resolution melt curve eDNA assay to monitor the simultaneous presence of invasive brown trout (\u003cem\u003eSalmo trutta\u003c/em\u003e) and endangered galaxiids. Environ DNA 3:561\u0026ndash;572. https://doi.org/10.1002/edn3.151\u003c/li\u003e\n\u003cli\u003eNational Institute of Ecology (2018) Investigating ecological risk of alien species\u003cem\u003e, \u003c/em\u003evol V. Seocheon, Republic of Korea, p 21\u0026ndash;33\u003c/li\u003e\n\u003cli\u003eNational Institute of Environmental Research (2012) Detailed studies on invasive alien species and their management\u003cem\u003e, \u003c/em\u003evol VII. Incheon, Republic of Korea, p 23\u0026ndash;26\u003c/li\u003e\n\u003cli\u003eOuso DO, Otiende MY, Jeneby MM, Oundo JW, Bargul JL, Miller SE et al. (2020) Three-gene PCR and high-resolution melting analysis for differentiating vertebrate species mitochondrial DNA for biodiversity research and complementing forensic surveillance. Sci Rep 10:4741. https://doi.org/10.1038/s41598-020-61600-3\u003c/li\u003e\n\u003cli\u003e\u0026Ouml;zvegy J, Marinković D, Vučićević M, Gajić B, Stevanović J, Krnjaić D, Aleksić-Kovačević S (2015) Cytological and molecular identification of \u003cem\u003eHaemogregarina stepanowi\u003c/em\u003e in blood samples of the European pond turtle (\u003cem\u003eEmys orbicularis\u003c/em\u003e) from quarantine at Belgrade Zoo. Acta Vet 65:443\u0026ndash;453. https://doi.org/10.1515/acve-2015-0037\u003c/li\u003e\n\u003cli\u003ePark CD, Kim KS, Kim KY, Heo JS, Oh HS, Park SM (2023) Development of the genotyping marker for Reeves\u0026rsquo; turtle (\u003cem\u003eMauremys reevesii\u003c/em\u003e) using high-resolution melting (HRM) analysis. Conserv Genet Resour 15:149\u0026ndash;152. https://doi.org/10.1007/s12686-023-01314-6\u003c/li\u003e\n\u003cli\u003eReed GH, Kent JO, Wittwer CT (2007) High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics 8:597\u0026ndash;608. https://doi.org/10.2217/14622416.8.6.597\u003c/li\u003e\n\u003cli\u003eThompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876\u0026ndash;4882. https://doi.org/10.1093/nar/25.24.4876\u003c/li\u003e\n\u003cli\u003eWittwer CT (2009) High‐resolution DNA melting analysis: advancements and limitations. Hum Mutat 30:857\u0026ndash;859. https://doi.org/10.1002/humu.20951\u003c/li\u003e\n\u003cli\u003eWittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49:853\u0026ndash;860. https://doi.org/10.1373/49.6.853\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"conservation-genetics-resources","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cogr","sideBox":"Learn more about [Conservation Genetics Resources](https://www.springer.com/journal/12686)","snPcode":"12686","submissionUrl":"https://submission.nature.com/new-submission/12686/3","title":"Conservation Genetics Resources","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Mauremys spp., Hybridization, HRM marker, R35","lastPublishedDoi":"10.21203/rs.3.rs-4877974/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4877974/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSouth Korea is home to two \u003cem\u003eMauremys\u003c/em\u003especies: \u003cem\u003eM. sinensis\u003c/em\u003e and \u003cem\u003eM. reevesii\u003c/em\u003e. \u003cem\u003eM. sinensis\u003c/em\u003e is an invasive alien species, whereas \u003cem\u003eM. reevesii\u003c/em\u003e is a legally protected species endemic to South Korea. The sympatric distributions of these species and their hybrids have recently been identified in South Korea, posing a serious threat to the conservation of \u003cem\u003eM. reevesii\u003c/em\u003e. Therefore, we developed a genotyping marker for high-resolution melting (HRM) analysis that targets the intron I region of the nuclear \u003cem\u003eR35\u003c/em\u003e gene to facilitate the rapid identification of hybrids. Three distinct HRM curves, representing \u003cem\u003eM. reevesii\u003c/em\u003e, \u003cem\u003eM. sinensis\u003c/em\u003e, and their hybrids, were obtained. These results were identical to those observed for \u003cem\u003eR35\u003c/em\u003e, with the hybrid possessing both alleles. This demonstrates that HRM markers can effectively distinguish \u003cem\u003eMauremys\u003c/em\u003especies from their hybrids. The present study provides a valuable tool for rapid and accurate identification of hybrids for future monitoring.\u003c/p\u003e","manuscriptTitle":"Development of high-resolution melting markers for assessment of Mauremys sinensis and Mauremys reevesii hybridization in South Korea","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-03 15:28:15","doi":"10.21203/rs.3.rs-4877974/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-28T09:52:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-12T08:44:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53168889731737646232507329716459012872","date":"2024-08-23T10:18:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-21T07:53:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-08T14:24:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-08T14:23:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Conservation Genetics Resources","date":"2024-08-08T04:12:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"conservation-genetics-resources","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cogr","sideBox":"Learn more about [Conservation Genetics Resources](https://www.springer.com/journal/12686)","snPcode":"12686","submissionUrl":"https://submission.nature.com/new-submission/12686/3","title":"Conservation Genetics Resources","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"20fa8fbc-8505-4682-bada-83f80c3dd6b5","owner":[],"postedDate":"September 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-03T16:10:12+00:00","versionOfRecord":{"articleIdentity":"rs-4877974","link":"https://doi.org/10.1007/s12686-025-01378-6","journal":{"identity":"conservation-genetics-resources","isVorOnly":false,"title":"Conservation Genetics Resources"},"publishedOn":"2025-01-29 15:57:02","publishedOnDateReadable":"January 29th, 2025"},"versionCreatedAt":"2024-09-03 15:28:15","video":"","vorDoi":"10.1007/s12686-025-01378-6","vorDoiUrl":"https://doi.org/10.1007/s12686-025-01378-6","workflowStages":[]},"version":"v1","identity":"rs-4877974","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4877974","identity":"rs-4877974","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}