Xie Pei-Jun, Ke Ya-Ting, Kuo Li-Yaung
Institute of Molecular and Cellular Biology National Tsing Hua University Hsinchu City Taiwan.
Appl Plant Sci. 2023 Jun 14;11(3):e11526. doi: 10.1002/aps3.11526. eCollection 2023 May-Jun.
Efficient protocols for extracting high-molecular-weight (HMW) DNA from ferns facilitate the long-read sequencing of their large and complex genomes. Here, we perform two cetyltrimethylammonium bromide (CTAB)-based protocols to extract HMW DNA and evaluate their applicability in diverse fern taxa for the first time.
We describe two modified CTAB protocols, with key adjustments to minimize mechanical disruption during lysis to prevent DNA shearing. One of these protocols uses a small amount of fresh tissue but yields a considerable quantity of HMW DNA with high efficiency. The other accommodates a large amount of input tissue, adopts an initial step of nuclei isolation, and thus ensures a high yield in a short period of time. Both methods were proven to be robust and effective in obtaining HMW DNA from diverse fern lineages, including 33 species in 19 families. The DNA extractions mostly had high DNA integrity, with mean sizes larger than 50 kbp, as well as high purity (A/A and A/A > 1.8).
This study provides HMW DNA extraction protocols for ferns in the hope of facilitating further attempts to sequence their genomes, which will bridge our genomic understanding of land plant diversity.
从蕨类植物中提取高分子量（HMW）DNA的高效方案有助于对其庞大而复杂的基因组进行长读长测序。在此，我们首次采用两种基于十六烷基三甲基溴化铵（CTAB）的方案来提取HMW DNA，并评估它们在不同蕨类植物类群中的适用性。
我们描述了两种改良的CTAB方案，通过关键调整以尽量减少裂解过程中的机械破坏，防止DNA剪切。其中一种方案使用少量新鲜组织，但能高效产生大量HMW DNA。另一种方案可处理大量输入组织，采用细胞核分离的初始步骤，从而确保在短时间内获得高产率。这两种方法都被证明在从包括19个科的33个物种在内的不同蕨类植物谱系中获取HMW DNA方面是稳健且有效的。所提取的DNA大多具有高完整性，平均大小大于50 kbp，且纯度高（A/A和A/A > 1.8）。
本研究为蕨类植物提供了HMW DNA提取方案，希望有助于进一步尝试对其基因组进行测序，这将增进我们对陆地植物多样性的基因组理解。
