SeedDNA: Optimized Seed-Based Genomic DNA Extraction Protocol for High-throughput Genotyping and Sequencing in Legume Crops

SeedDNA: Optimized Seed-Based Genomic DNA Extraction Protocol for High-throughput Genotyping and Sequencing in Legume Crops | 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 Article SeedDNA: Optimized Seed-Based Genomic DNA Extraction Protocol for High-throughput Genotyping and Sequencing in Legume Crops Sri Swathi Manda, Vinay Sharma, Jalaja Naravula, Naseem Fathima, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8507597/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 Background: Efficient and cost-effective genomic DNA extraction from legume seeds, such as groundnut, chickpea, and pigeonpea, is important for molecular biology studies, large-scale genotyping, and marker-assisted breeding programs. These seeds are rich in inhibitory compounds such as polysaccharides, polyphenols, and secondary metabolites, which make DNA extraction challenging. The DNA isolation from leaves is very common; however, achieving high-quality DNA from seeds remains a challenge in many plant species, although it is very useful in saving resources and time. Results: In this context, we optimized the protocol for high-quality DNA extraction from seeds of these three legume species. This optimized protocol yielded DNA with high purity (A260/A280 ratio of ~1.8–2.0) and yielded up to 60 ng/µL for all three legumes. As compared to the protocol used for leaf samples, key modifications in this protocol include the use of proteinase K, repeating the phase separation and washing process twice, and overnight incubation with RNAse at 37°C to remove the RNA contamination. This protocol improved DNA yield and purity while minimizing contaminants, with a minimal isolation cost of $ 0.35 per sample. PCR amplification with crop-specific primers performed well on the genomic DNA extracted from both seeds and leaves. The results were identical, showing the reliability of seed-derived DNA for genotyping and other downstream applications. Furthermore, the PCR amplification confirmed that the DNA isolated from seeds using this protocol can be used for genetic purity assessment in seed lots and for genotyping applications in marker-assisted selection. Conclusion : A high-throughput DNA extraction protocol was developed and validated for legume seeds, such as groundnut, chickpea, and pigeonpea. The optimized protocol is reliable, cost-effective, and can be applied for large-scale genotyping and marker-assisted breeding programs. Using this protocol, one person can process four 96-well plates per day, with an estimated cost of $ 0.35 per sample, including consumables. Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Genetics Biological sciences/Molecular biology Biological sciences/Plant sciences DNA extraction legume seeds groundnut chickpea pigeonpea genotyping marker-assisted selection Full Text Additional Declarations No competing interests reported. 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-8507597","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":580305868,"identity":"a29b323f-4e57-44f9-8ab2-25706fdad792","order_by":0,"name":"Sri Swathi Manda","email":"","orcid":"","institution":"International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)","correspondingAuthor":false,"prefix":"","firstName":"Sri","middleName":"Swathi","lastName":"Manda","suffix":""},{"id":580305869,"identity":"4d18338f-9564-45a4-a980-4279cf1a1330","order_by":1,"name":"Vinay Sharma","email":"","orcid":"","institution":"International Crops 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selection","lastPublishedDoi":"10.21203/rs.3.rs-8507597/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8507597/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Efficient and cost-effective genomic DNA extraction from legume seeds, such as groundnut, chickpea, and pigeonpea, is important for molecular biology studies, large-scale genotyping, and marker-assisted breeding programs. These seeds are rich in inhibitory compounds such as polysaccharides, polyphenols, and secondary metabolites, which make DNA extraction challenging. The DNA isolation from leaves is very common; however, achieving high-quality DNA from seeds remains a challenge in many plant species, although it is very useful in saving resources and time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this context, we optimized the protocol for high-quality DNA extraction from seeds of these three legume species. This optimized protocol yielded DNA with high purity (A260/A280 ratio of ~1.8–2.0) and yielded up to 60 ng/µL for all three legumes. As compared to the protocol used for leaf samples, key modifications in this protocol include the use of proteinase K, repeating the phase separation and washing process twice, and overnight incubation with RNAse at 37°C to remove the RNA contamination. This protocol improved DNA yield and purity while minimizing contaminants, with a minimal isolation cost of $ 0.35 per sample. PCR amplification with crop-specific primers performed well on the genomic DNA extracted from both seeds and leaves. The results were identical, showing the reliability of seed-derived DNA for genotyping and other downstream applications. Furthermore, the PCR amplification confirmed that the DNA isolated from seeds using this protocol can be used for genetic purity assessment in seed lots and for genotyping applications in marker-assisted selection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: \u0026nbsp;A high-throughput DNA extraction protocol was developed and validated for legume seeds, such as groundnut, chickpea, and pigeonpea. The optimized protocol is reliable, cost-effective, and can be applied for large-scale genotyping and marker-assisted breeding programs. 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