Modified CTAB protocols for high-molecular-weight DNA extractions from ferns

Premise: 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. Methods and Results: 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 ₂₆₀ /A ₂₃₀ and A ₂₆₀ /A ₂₈₀ > 1.8). Conclusions: 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.