Ionic liquid-assisted seed genomic DNA extraction for advanced sequencing applications.

IF 4.4 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Plant Methods Pub Date : 2025-07-16 DOI:10.1186/s13007-025-01417-1

Shashini De Silva, Philip C Bentz, Cecilia Cagliero, Morgan R Gostel, Gabriel Johnson, Jared L Anderson

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引用次数: 0

Abstract

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.

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离子液体辅助种子基因组DNA提取用于高级测序应用。

背景：现代植物育种策略依赖于大量使用先进的基因组工具来加速改良作物品种的开发。与新鲜叶片组织相比，从作物种子中提取基因组DNA消除了种植植物的需要；然而，由于存储化合物的存在和基质的复杂性，它仍然可能是一个瓶颈。环境友好的胆碱离子液体（ILs）与DNA的相互作用为提高从种子中提取的DNA质量提供了一种创新的方法。虽然之前基于il的植物DNA提取工作流程主要支持聚合酶链反应（PCR）和基于定量PCR的应用，但它们对高通量测序（HTS）的适用性仍然很大程度上未被探索。本研究探讨了il辅助方法提取大豆（Glycine max）种子基因组DNA的有效性，解决了il在HTS中应用有限的问题。结果：优化后的DNA提取方法，采用25% （w/v）甲酸胆碱提取，可回收高纯度DNA，片段大小为bb0 ~ 20kb，适用于PCR、全基因组扩增（WGA）、SSR扩增和Illumina高通量测序等下游应用。利用商业植物DNA提取试剂盒，以十六烷基三甲基溴化铵（CTAB）和十二烷基硫酸钠（SDS）作为裂解剂，对il法和二氧化硅结合法进行了DNA产率、纯度、丰富的DNA片段大小分布和完整性的比对。此外，从该方法分离的DNA成功地扩增了来自核和质体基因组的标记，并在Illumina （PE150）测序reads中获得了bb0 99%的全基因组覆盖率。结论：这是已知的第一个从ILs提取的DNA中产生全基因组序列的实例。这些发现标志着将ILs作为传统种子DNA提取方法的一个重要里程碑，在第三代（长读）测序实验中具有潜在的实用性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Plant Methods 生物-植物科学

CiteScore

9.20

自引率

3.90%

发文量

121

审稿时长

2 months

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.