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

Area of Science:

• Plant genomics
• Molecular biology
• Biotechnology

Background:

• Ferns possess large, complex genomes that are challenging to sequence.
• High-molecular-weight (HMW) DNA is essential for long-read sequencing technologies.
• Existing DNA extraction methods are often inefficient for ferns.

Purpose of the Study:

• To develop and evaluate efficient protocols for extracting HMW DNA from diverse fern species.
• To optimize DNA extraction for long-read sequencing applications in ferns.
• To facilitate genomic studies of fern diversity.

Main Methods:

• Two modified cetyltrimethylammonium bromide (CTAB)-based DNA extraction protocols were employed.
• Key adjustments were made to minimize mechanical disruption and DNA shearing during lysis.
• One protocol optimized for small tissue amounts, the other for large amounts with nuclei isolation.

Main Results:

• Both CTAB protocols successfully extracted HMW DNA from 33 fern species across 19 families.
• Extracted DNA exhibited high integrity (mean size >50 kbp) and purity (A260/A230 and A260/A280 > 1.8).
• One protocol yielded considerable HMW DNA quantity with high efficiency from small tissue samples.

Conclusions:

• The developed CTAB protocols are robust and effective for obtaining HMW DNA from diverse ferns.
• These protocols provide a foundation for advancing fern genome sequencing efforts.
• This work will contribute to a broader genomic understanding of land plant diversity.