Isolating Linum usitatissimum L. Nuclear DNA Enabled Assembling High-Quality Genome

Area of Science:

• Plant genomics
• Crop science
• Molecular biology

Background:

• High-quality genome sequences are crucial for understanding biological processes and species evolution.
• Previous high-quality genome assemblies for flax (Linum usitatissimum L.) using Oxford Nanopore Technologies (ONT) data were limited due to challenges in isolating pure, high-molecular-weight DNA.

Purpose of the Study:

• To develop a reliable protocol for obtaining high-molecular-weight DNA from flax for genome sequencing.
• To achieve a contiguous genome assembly for L. usitatissimum using Nanopore sequencing data.

Main Methods:

• A novel protocol for flax nuclei isolation and subsequent DNA extraction was developed, yielding approximately 5 μg of pure high-molecular-weight DNA from 0.5 g of leaves.
• Comparative analysis of various genome assemblers and polishers was performed on the generated ONT data.
• The Canu-Racon (two iterations)-Medaka combination was utilized for genome assembly.

Main Results:

• The optimized protocol successfully produced sufficient high-molecular-weight DNA for extensive sequencing, generating over 30 Gb of ONT data from two MinION runs.
• A final genome assembly of 447.1 Mb for the L. usitatissimum line 3896 was achieved, consisting of 1695 contigs with an N50 of 6.2 Mb.
• The assembly demonstrated high completeness, with 93.8% of BUSCOs from eudicots_odb10 identified.

Conclusions:

• The developed DNA extraction protocol overcomes previous limitations in flax genome sequencing.
• The resulting high-quality genome assembly provides a valuable resource for future genomic studies in flax.
• The study underscores the importance of genome construction strategy in achieving robust assembly parameters.