{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,4]],"date-time":"2026-10-04T10:44:35Z","timestamp":1791110675429,"version":"4.1.0"},"reference-count":25,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2019,6,25]],"date-time":"2019-06-25T00:00:00Z","timestamp":1561420800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MPs"],"abstract":"<jats:p>Next-generation sequencing (NGS) is a revolutionary advancement allowing large-scale discovery of functional molecular markers that has many applications, including plant breeding. High-quality genomic DNA (gDNA) is a prerequisite for successful NGS library preparation and sequencing; however, few reliable protocols to obtain such plant gDNA exist. A previously reported nuclear pellet (NP) method enables extraction of high-yielding gDNA from fresh leaf tissue of maize (Zea mays L.), but the quality does not meet the stringent requirements of NGS. In this study, we optimized the NP method for whole-genome sequencing of rice (Oryza sativa L.) through the integration of simple purification steps. The optimized NP method relied on initial nucleus enrichment, cell lysis, extraction, and subsequent gDNA purification buffers. The purification steps used proteinase K, RNase A, phenol\/chloroform\/isoamyl alcohol (25:24:1), and chloroform\/isoamyl alcohol (24:1) treatments for protein digestion and RNA, protein, and phenol removal, respectively. Our data suggest that this optimized NP method allowed extraction of consistently high-yielding and high-quality undegraded gDNA without contamination by protein and RNA. Moreover, the extracted gDNA fulfilled the quality metrics of NGS library preparation for the Illumina HiSeq X Ten platform by the TruSeq DNA PCR-Free Library Prep Kit (Illumina). We provide a reliable step-by-step guide to the extraction of high-quality gDNA from fresh leaf tissues of rice for molecular biologists with limited resources.<\/jats:p>","DOI":"10.3390\/mps2020054","type":"journal-article","created":{"date-parts":[[2019,6,25]],"date-time":"2019-06-25T10:52:31Z","timestamp":1561459951000},"page":"54","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Optimized Nuclear Pellet Method for Extracting Next-Generation Sequencing Quality Genomic DNA from Fresh Leaf Tissue"],"prefix":"10.3390","volume":"2","author":[{"given":"Md Masud","family":"Rana","sequence":"first","affiliation":[{"name":"Department of Life and Food Sciences, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan"},{"name":"Agronomy Division, Bangladesh Rice Research Institute, Gazipur-1701, Bangladesh"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2677-404X","authenticated-orcid":false,"given":"Murat","family":"Aycan","sequence":"additional","affiliation":[{"name":"Institute of Science and Technology, Niigata University, Niigata 950-2181, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takeshi","family":"Takamatsu","sequence":"additional","affiliation":[{"name":"Department of Life and Food Sciences, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan"},{"name":"Institute of Science and Technology, Niigata University, Niigata 950-2181, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kentaro","family":"Kaneko","sequence":"additional","affiliation":[{"name":"Department of Life and Food Sciences, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9165-8830","authenticated-orcid":false,"given":"Toshiaki","family":"Mitsui","sequence":"additional","affiliation":[{"name":"Department of Life and Food Sciences, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan"},{"name":"Institute of Science and Technology, Niigata University, Niigata 950-2181, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5341-8086","authenticated-orcid":false,"given":"Kimiko","family":"Itoh","sequence":"additional","affiliation":[{"name":"Department of Life and Food Sciences, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan"},{"name":"Institute of Science and Technology, Niigata University, Niigata 950-2181, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2019,6,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"175","DOI":"10.3732\/ajb.1200020","article-title":"Applications of next-generation sequencing in plant biology","volume":"99","author":"Egan","year":"2012","journal-title":"Am. J. Bot."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1186\/1746-4811-10-21","article-title":"Protocol: A simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species","volume":"10","author":"Healey","year":"2014","journal-title":"Plant Methods"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1186\/s13007-018-0336-1","article-title":"Protocol: A versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing","volume":"14","author":"Anderson","year":"2018","journal-title":"Plant Methods"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Henry, R.J. (2008). DNA extraction from plant tissue. Plant Genotyping II SNP Technol, CAB International.","DOI":"10.1079\/9781845933821.0000"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s13007-016-0152-4","article-title":"Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize","volume":"13","author":"Osman","year":"2017","journal-title":"Plant Methods"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1016\/j.gpb.2015.08.002","article-title":"PacBio Sequencing and Its Applications","volume":"13","author":"Rhoads","year":"2015","journal-title":"Genomics. Proteomics Bioinf."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2003","DOI":"10.2144\/000114460","article-title":"Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules","volume":"61","author":"Mayjonade","year":"2016","journal-title":"Biotechniques"},{"key":"ref_8","first-page":"11","article-title":"A rapid DNA isolation procedure for small quantities of fresh leaf tissue","volume":"19","author":"Doyle","year":"1987","journal-title":"Phytochem. Bull."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1007\/BF02772759","article-title":"A protocol for high-throughput extraction of DNA from rice leaves","volume":"23","author":"Xu","year":"2005","journal-title":"Plant Mol. Biol. Report."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"736","DOI":"10.1111\/j.1755-0998.2007.02074.x","article-title":"A high-throughput protocol for extracting high-purity genomic DNA from plants and animals","volume":"8","author":"Whitlock","year":"2008","journal-title":"Mol. Ecol. Resour."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"8086","DOI":"10.3390\/ijms12118086","article-title":"An efficient method for genomic DNA extraction from different molluscs species","volume":"12","author":"Pereira","year":"2011","journal-title":"Int. J. Mol. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1186\/1179-5395-43-3","article-title":"Extraction of high purity genomic DNA from pine for use in a high-throughput Genotyping Platform","volume":"43","author":"Telfer","year":"2013","journal-title":"New Zeal. J. For. Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"610","DOI":"10.13158\/heia.29.2.2016.610","article-title":"A Fast and Inexpensive High-Throughput Protocol for Isolating High Molecular Weight Genomic DNA from Lichens","volume":"29","author":"Werth","year":"2016","journal-title":"Herzogia"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1111\/j.1574-6968.2011.02424.x","article-title":"Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures","volume":"325","author":"Minas","year":"2011","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Simbolo, M., Gottardi, M., Corbo, V., Fassan, M., Mafficini, A., Malpeli, G., Lawlor, R.T., and Scarpa, A. (2013). DNA Qualification Workflow for Next Generation Sequencing of Histopathological Samples. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0062692"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1007\/BF00338389","article-title":"Regulation of Mu element copy number in maize lines with an active or inactive Mutator transposable element system","volume":"211","author":"Walbot","year":"1988","journal-title":"Mol. Gen. Genet."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"274","DOI":"10.1038\/338274a0","article-title":"Fertile transgenic rice plants regenerated from transformed protoplasts","volume":"338","author":"Shimamoto","year":"1989","journal-title":"Nature"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"266","DOI":"10.3389\/fpls.2018.00266","article-title":"Optimized Method of Extracting Rice Chloroplast DNA for High-Quality Plastome Resequencing and de Novo Assembly","volume":"9","author":"Takamatsu","year":"2018","journal-title":"Front. Plant Sci."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Rana, M.M., Takamatsu, T., Baslam, M., Kaneko, K., Itoh, K., Harada, N., Sugiyama, T., Ohnishi, T., Kinoshita, T., and Takagi, H. (2019). Salt Tolerance Improvement in Rice through Efficient SNP Marker-Assisted Selection Coupled with Speed-Breeding. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20102585"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2114","DOI":"10.1093\/bioinformatics\/btu170","article-title":"Trimmomatic: A flexible trimmer for Illumina sequence data","volume":"30","author":"Bolger","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"338","DOI":"10.1038\/nbt.4060","article-title":"Nanopore sequencing and assembly of a human genome with ultra-long reads","volume":"36","author":"Jain","year":"2018","journal-title":"Nat. Biotechnol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1038\/nature20098","article-title":"De novo assembly and phasing of a Korean human genome","volume":"538","author":"Seo","year":"2016","journal-title":"Nature"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1101\/gr.214007.116","article-title":"Discovery and genotyping of structural variation from long-read haploid genome sequence data","volume":"27","author":"Huddleston","year":"2017","journal-title":"Genome Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"14789","DOI":"10.1038\/s41598-017-13712-6","article-title":"Nanopore-based single molecule sequencing of the D4Z4 array responsible for facioscapulohumeral muscular dystrophy","volume":"7","author":"Mitsuhashi","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Colle, M., Leisner, C.P., Wai, C.M., Ou, S., Bird, K.A., Wang, J., Wisecaver, J.H., Yocca, A.E., Alger, E.I., and Tang, H. (2019). Haplotype-phased genome and evolution of phytonutrient pathways of tetraploid blueberry. Gigascience, 8.","DOI":"10.1093\/gigascience\/giz012"}],"container-title":["Methods and Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2409-9279\/2\/2\/54\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:01:01Z","timestamp":1760187661000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2409-9279\/2\/2\/54"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,6,25]]},"references-count":25,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2019,6]]}},"alternative-id":["mps2020054"],"URL":"https:\/\/doi.org\/10.3390\/mps2020054","relation":{},"ISSN":["2409-9279"],"issn-type":[{"value":"2409-9279","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,6,25]]}}}