Shashini De Silva, Philip C Bentz, Cecilia Cagliero, Morgan R Gostel, Gabriel Johnson, Jared L Anderson
{"title":"离子液体辅助种子基因组DNA提取用于高级测序应用。","authors":"Shashini De Silva, Philip C Bentz, Cecilia Cagliero, Morgan R Gostel, Gabriel Johnson, Jared L Anderson","doi":"10.1186/s13007-025-01417-1","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Modern plant breeding strategies rely on the intensive use of advanced genomic tools to expedite the development of improved crop varieties. Genomic DNA extraction from crop seeds eliminates the need to grow plants in contrast to fresh leaf tissue; however, it can still be a bottleneck due to the presence of stored compounds and the complexity of the matrix. The interaction of environmentally benign choline-based ionic liquids (ILs) with DNA offers an innovative approach to enhance the quality of extracted DNA from seeds. While prior IL-based plant DNA extraction workflows have primarily supported polymerase chain reaction (PCR) and quantitative PCR-based applications, their suitability for high-throughput sequencing (HTS) remained largely unexplored. This study explores the efficacy of IL-assisted method for genomic DNA extraction from soybean (Glycine max) seeds, addressing the limited application of ILs in HTS.</p><p><strong>Results: </strong>The optimized DNA extraction method, utilizing 25% (w/v) choline formate, enabled the recovery of high-purity DNA with abundant fragment sizes > 20 kb, suitable for downstream applications including PCR, whole genome amplification (WGA), simple sequence repeat (SSR) amplification, and high-throughput Illumina sequencing. The IL-method was benchmarked against a silica-binding method using cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) as lysis agents using a commercial plant DNA extraction kit in terms of DNA yield, purity, abundant DNA fragment size distribution, and integrity. In addition, DNA isolated from this method demonstrated successful PCR amplification of markers from both the nuclear and plastid genomes and yielded > 99% whole genome coverage with Illumina (PE150) sequencing reads.</p><p><strong>Conclusions: </strong>This is the first known instance of a whole genome sequence generated from DNA extracted with ILs. These findings mark a significant milestone in establishing ILs as promising alternatives to conventional methods for seed DNA extraction, with potential utility in third generation (long-read) sequencing experiments.</p>","PeriodicalId":20100,"journal":{"name":"Plant Methods","volume":"21 1","pages":"97"},"PeriodicalIF":4.4000,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12265370/pdf/","citationCount":"0","resultStr":"{\"title\":\"Ionic liquid-assisted seed genomic DNA extraction for advanced sequencing applications.\",\"authors\":\"Shashini De Silva, Philip C Bentz, Cecilia Cagliero, Morgan R Gostel, Gabriel Johnson, Jared L Anderson\",\"doi\":\"10.1186/s13007-025-01417-1\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Modern plant breeding strategies rely on the intensive use of advanced genomic tools to expedite the development of improved crop varieties. Genomic DNA extraction from crop seeds eliminates the need to grow plants in contrast to fresh leaf tissue; however, it can still be a bottleneck due to the presence of stored compounds and the complexity of the matrix. The interaction of environmentally benign choline-based ionic liquids (ILs) with DNA offers an innovative approach to enhance the quality of extracted DNA from seeds. While prior IL-based plant DNA extraction workflows have primarily supported polymerase chain reaction (PCR) and quantitative PCR-based applications, their suitability for high-throughput sequencing (HTS) remained largely unexplored. This study explores the efficacy of IL-assisted method for genomic DNA extraction from soybean (Glycine max) seeds, addressing the limited application of ILs in HTS.</p><p><strong>Results: </strong>The optimized DNA extraction method, utilizing 25% (w/v) choline formate, enabled the recovery of high-purity DNA with abundant fragment sizes > 20 kb, suitable for downstream applications including PCR, whole genome amplification (WGA), simple sequence repeat (SSR) amplification, and high-throughput Illumina sequencing. The IL-method was benchmarked against a silica-binding method using cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) as lysis agents using a commercial plant DNA extraction kit in terms of DNA yield, purity, abundant DNA fragment size distribution, and integrity. In addition, DNA isolated from this method demonstrated successful PCR amplification of markers from both the nuclear and plastid genomes and yielded > 99% whole genome coverage with Illumina (PE150) sequencing reads.</p><p><strong>Conclusions: </strong>This is the first known instance of a whole genome sequence generated from DNA extracted with ILs. These findings mark a significant milestone in establishing ILs as promising alternatives to conventional methods for seed DNA extraction, with potential utility in third generation (long-read) sequencing experiments.</p>\",\"PeriodicalId\":20100,\"journal\":{\"name\":\"Plant Methods\",\"volume\":\"21 1\",\"pages\":\"97\"},\"PeriodicalIF\":4.4000,\"publicationDate\":\"2025-07-16\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12265370/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Plant Methods\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1186/s13007-025-01417-1\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Plant Methods","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1186/s13007-025-01417-1","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Ionic liquid-assisted seed genomic DNA extraction for advanced sequencing applications.
Background: Modern plant breeding strategies rely on the intensive use of advanced genomic tools to expedite the development of improved crop varieties. Genomic DNA extraction from crop seeds eliminates the need to grow plants in contrast to fresh leaf tissue; however, it can still be a bottleneck due to the presence of stored compounds and the complexity of the matrix. The interaction of environmentally benign choline-based ionic liquids (ILs) with DNA offers an innovative approach to enhance the quality of extracted DNA from seeds. While prior IL-based plant DNA extraction workflows have primarily supported polymerase chain reaction (PCR) and quantitative PCR-based applications, their suitability for high-throughput sequencing (HTS) remained largely unexplored. This study explores the efficacy of IL-assisted method for genomic DNA extraction from soybean (Glycine max) seeds, addressing the limited application of ILs in HTS.
Results: The optimized DNA extraction method, utilizing 25% (w/v) choline formate, enabled the recovery of high-purity DNA with abundant fragment sizes > 20 kb, suitable for downstream applications including PCR, whole genome amplification (WGA), simple sequence repeat (SSR) amplification, and high-throughput Illumina sequencing. The IL-method was benchmarked against a silica-binding method using cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) as lysis agents using a commercial plant DNA extraction kit in terms of DNA yield, purity, abundant DNA fragment size distribution, and integrity. In addition, DNA isolated from this method demonstrated successful PCR amplification of markers from both the nuclear and plastid genomes and yielded > 99% whole genome coverage with Illumina (PE150) sequencing reads.
Conclusions: This is the first known instance of a whole genome sequence generated from DNA extracted with ILs. These findings mark a significant milestone in establishing ILs as promising alternatives to conventional methods for seed DNA extraction, with potential utility in third generation (long-read) sequencing experiments.
期刊介绍：
Plant Methods is an open access, peer-reviewed, online journal for the plant research community that encompasses all aspects of technological innovation in the plant sciences.
There is no doubt that we have entered an exciting new era in plant biology. The completion of the Arabidopsis genome sequence, and the rapid progress being made in other plant genomics projects are providing unparalleled opportunities for progress in all areas of plant science. Nevertheless, enormous challenges lie ahead if we are to understand the function of every gene in the genome, and how the individual parts work together to make the whole organism. Achieving these goals will require an unprecedented collaborative effort, combining high-throughput, system-wide technologies with more focused approaches that integrate traditional disciplines such as cell biology, biochemistry and molecular genetics.
Technological innovation is probably the most important catalyst for progress in any scientific discipline. Plant Methods’ goal is to stimulate the development and adoption of new and improved techniques and research tools and, where appropriate, to promote consistency of methodologies for better integration of data from different laboratories.
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