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

Copyright © 2023 The Authors. Applications in Plant Sciences published by Wiley Periodicals LLC on behalf of Botanical Society of America.

This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Associated Data

Appendix S1. Sample and voucher information for the taxa used for high‐molecular‐weight DNA extraction.

Appendix S2. Integrity and size distribution of the high‐molecular‐weight DNA extractions from the selected samples.

All supporting data are provided in the article and the Supporting Information.

Abstract

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 (A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ > 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.

Ferns were one of the last major plant lineages for which a whole genome was sequenced. The de novo assembly of typical fern genomes is particularly challenging due to their large size (Clark et al., 2016; Kuo and Li, 2019; Fujiwara et al., 2023) and surprising complexity (Zhong et al., 2022). The genomes of ferns in the order Salviniales were the first to be sequenced because of their unusually small size (Li et al., 2018), and long‐read sequencing has made the assembly of additional fern genomes feasible (Fang et al., 2022; Huang et al., 2022; Marchant et al., 2022; Rahmatpour et al., 2023). The assembly of complex fern genomes requires a considerable amount of intact and pure DNA, namely high‐molecular‐weight (HMW) DNA. A PacBio library, for example, usually requires over 10 μg of HMW DNA averaging 30–50 kbp in size (Li and Harkess, 2018); therefore, establishing protocols to extract sufficient quantities of high‐quality HMW DNA is essential for fern genome research.

For decades, researchers have observed that DNA extraction from ferns is particularly difficult. Dempster et al. (1999) noticed that DNA extraction methods used to tackle challenging flowering plants performed poorly on the maidenhair fern (Adiantum capillus‐veneris L.). Previous DNA barcoding studies of ferns (e.g., De Groot et al., 2011) have relied on commercial DNA isolation kits to ensure the quality of DNA extracts from diverse fern lineages; however, while the incorporation of spin columns in most kits improves DNA purity, it also causes DNA fragmentation. Instead of using commercial kits, modified cetyltrimethylammonium bromide (CTAB)‐ or sodium dodecyl sulfate (SDS)‐based methods have been adopted for HMW DNA extraction in various plant groups (Aboul‐Maaty and Oraby, 2019; Li et al., 2020; Jones et al., 2021); however, none of these established protocols have been tested on ferns, whose tissues are usually rich in secondary metabolites (Vetter, 2018). Moreover, due to the small body size of many fern species, there is often a limited amount of tissue per individual. For genome sequencing, acquiring a considerable amount of DNA from a single individual or clone is necessary to avoid mixing DNA from multiple individuals with non‐identical genotypes, which usually complicates the downstream genome assembly.

In this study, we demonstrate two modified CTAB‐based protocols for HMW DNA extraction in ferns, referred to as “standard” and “nuclei isolation,” and apply them to samples from 33 diverse fern species across 19 families, as well as two lycophyte species. In contrast with a typical CTAB procedure (Doyle and Doyle, 1987; reviewed by Schenk et al., 2023), our protocols include modifications to minimize mechanical disruption (e.g., pulverization, pipetting, or vortexing) during the suspension or lysis steps (e.g., mixing with a CTAB buffer). In particular, we keep the ground tissue frozen and gently transfer it into CTAB or nuclei isolation buffer instead of grinding the tissue in the presence of these lysis buffers. Tissue debris is then removed through a filter. These modifications significantly reduce DNA shearing. The DNA yield of the standard protocol exceeded 8 μg of DNA and used less than 0.5 g of fresh leaf tissue. The nuclei isolation protocol adopts an initial step to isolate nuclei, which accommodates a greater input amount and ensures a high yield (>10 μg) of DNA. With both protocols, we successfully obtained pure HMW DNA products (as measured by the ratio of absorbance at 260 and 230 nm [A₂₆₀/A₂₃₀] and at 260 and 280 nm [A₂₆₀/A₂₈₀]; A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ > 1.8) with mean sizes longer than 50 kbp.

METHODS AND RESULTS

Standard protocol for HMW DNA extraction

Young and fresh plant tissues, usually newly expanded foliar parts or greenish gametophytes, were used for the HMW DNA extractions. These tissues and organs were preferred because of their nuclei‐rich meristems and relatively fewer secondary metabolites (Moreira and Oliveira, 2011). Additionally, the gametophyte tissues sampled here were clones all derived from a single haploid spore, meaning they were genetically identical and completely homozygous for the ease of whole genome sequencing (WGS) and assembly. We were therefore particularly interested in testing HMW DNA extraction methods on these gametophyte samples. Before the DNA extraction, the leaves or gametophyte tissues were harvested and subjected to darkness for 48 h to reduce the contents of carbohydrates, polysaccharides, and polyphenolic compounds (Li et al., 2020). The equipment, including spatulas, mortars, and pestles, was autoclaved and then baked at 180°C for 8 h before use. Fresh tissues of each sample, typically 0.2 to 0.3 g, were flash‐frozen in liquid nitrogen in a mortar before being ground into powder using a pestle. The frozen tissue powder was transferred into a 5‐mL tube containing 2 mL of CTAB using a spatula (Figure 1 ), rather than mixing CTAB with the tissue powder in the mortar. To remove the tissue debris, the CTAB–tissue mixture was filtered through a 30‐µm filter (Sysmex Partec, Goerlitz, Germany) followed by incubation at 55°C for 10 min (Figure 1 ). These procedures are critical to reduce the physical shearing of DNA caused by repetitive pipetting. The DNA extractions were then washed twice with a 1/2 volume of chloroform (details in Appendix 1). In between the chloroform washes, the aqueous extracts were treated with RNase A (100 mg/mL; Qiagen, Hilden, Germany) and incubated at room temperature for 10 min. After mixing a 1/2 volume of cool isopropanol (pre‐chilled at −20°C) with the extractions, the mixtures were centrifuged under 13,000 × g at 4°C for 10 min to pellet the DNA. The DNA pellets were then washed twice with 70% ethanol and air‐dried at room temperature for 3 min, before being dissolved in nuclease‐free water. If the DNA extracts will be used immediately for sequencing, they should be stored at 4°C to avoid any further freeze‐thawing; otherwise, −80°C is a better condition for their long‐term storage.

Flow diagrams of the two CTAB protocols for high‐molecular‐weight (HMW) DNA extractions. Steps identified as (1) represent the standard protocol with low tissue input and without nuclei isolation. Steps identified as (2) represent the nuclei isolation protocol for large tissue input including nuclei isolation.

Nuclei isolation protocol for HMW DNA extraction

We modified this protocol by increasing the amount of input tissue and adding an initial nuclei isolation step. This is likely to improve DNA purity by first separating the contaminants, such as polysaccharides, polyphenols, and other secondary metabolites, from the tissue (Li et al., 2020). About 6–8 g of fresh tissue per sample was ground as described for the standard protocol above, and the powdered tissue was mixed with 30 mL of nuclei isolation buffer in a 50‐mL tube. We selected the ideal nuclei isolation buffer based on the taxon: either LB01 (Doležel et al., 1989, 2007), Beckman (Ebihara et al., 2005), or general‐purpose buffer (GPB; Loureiro et al., 2007). The buffer selection depended on their flow cytometric performance in the same species or a close relative (e.g., Clark et al., 2016; Fujiwara et al., 2023). The tissue–buffer mixture was filtered through a 250‐mesh (~58 μm) polyester silk screen printing mesh (Figure 1 ), which could accommodate a large flowthrough volume. After removing the tissue debris, the filtered mixture was centrifuged at 100 × g for 15 min at 4°C to pellet the nuclei. After carefully discarding the supernatant, the retained nuclei pellet was dissolved in 4 mL of CTAB solution (details in Appendix 2). The remaining procedures, including the lysis, chloroform wash, RNase treatment, DNA precipitation, and storage, were the same as described in the standard protocol.

Quality and quantity of HMW DNA

The DNA integrity was first evaluated by electrophoresis using a Tris acetate EDTA (TAE) 1% agarose gel. The purity of the DNA extractions was inferred using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) to measure their A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ values. The precise DNA concentration was determined using QuantiFluor (Promega, Madison, Wisconsin, USA). Eight recently extracted samples that had not been subjected to repeated freezing and thawing (Table 1 ) were selected for the examination of their DNA fragment sizes using a Fragment Analyzer 5200 (Agilent Technologies, Santa Clara, California, USA). The DNA recovery rate, defined as the total DNA yield (in micrograms) recovered per gram of fresh input tissue, was also calculated. Using our standard protocol, we extracted HMW DNA from 31 fern species and two lycophyte species (Appendix S1). Notably, due to the limited amounts of fresh tissue per individual, we were unable to test both the standard and nuclei isolation protocols on all samples, except for the two Diplopterygium (Diels) Nakai species.

Table 1

Quality and quantity of the eight selected high‐molecular‐weight (HMW) DNA samples.

Note: NA = not applicable; TAIF = Herbarium of Taiwan Forestry Research Institute.