Russian geneticists have developed a protocol for the isolation of flax DNA, which made it possible to obtain a large yield of pure high-molecular-weight DNA even from a single plant. The sequencing of such DNA on the third generation platform made it possible to assemble the flax genome of high length and completeness, which is the basis for further molecular genetic studies and selection of this crop. The results of the study are published in the International Journal of Molecular Sciences.
The scientific team of the Institute of Molecular Biology. V. A. Engelhardt (IMB) RAS, together with researchers from the Federal Scientific Center for Bast Crops and the Moscow Institute of Physics and Technology (MIPT), developed a protocol for the isolation of flax DNA, including the stage of isolation of cell nuclei, which makes it possible to obtain pure high-molecular DNA. Using the developed protocol, it was possible to isolate enough DNA even from one plant to achieve a high yield of long reads on the Oxford Nanopore Technologies (ONT) platform, which is extremely sensitive to the quality of the material. From the obtained data, the scientists assembled the flax genome of high length and completeness.
Today flax ( Linum usitatissimum L.) is widely used in various fields and is becoming more and more in demand. Flaxseed is rich in lignans, omega-3s, and fiber, making it a useful food supplement as well as a raw material for the pharmaceutical and cosmetic industries. In the production of enamels and paints and varnishes, flax oil is used to increase the durability of the material. Flax fiber is used to create clothing, paper, medical products, insulation, gunpowder and composite materials. Each of these applications requires the cultivation of flax varieties with a specific set of characteristics. To obtain plants with specific traits, selection is used, while knowledge of the genetic bases that determine these traits can significantly increase the efficiency of creating specialized varieties. Affordable high-quality plant genome assembly is an essential tool for studying valuable crop traits. Obtaining a genome from material from a single plant makes it possible to analyze the genotype and phenotype together. Unfortunately, there is no universal approach to constructing a high-quality genome assembly. The final result depends on the very structure of the genome, the chosen platform, the volume and quality of sequencing data.
During the study, flax seeds of line 3896 were germinated in Petri dishes, after which they were planted in pots with soil. When the plant reached the age of three to four weeks, it was partially covered with a dark cloth that protected from light, and grown for another week. This procedure makes it possible to reduce the amount of metabolites in plant cells in order to isolate more pure DNA. After growth in the dark, leaves were collected and immediately frozen in liquid nitrogen. The collected material was stored at –70°C until the moment of isolation of cell nuclei followed by DNA isolation.
“We have developed a protocol for the isolation of flax nuclei followed by DNA extraction, which allows us to isolate about 5 μg of pure high-molecular DNA from 0.5 g of leaves. Such a volume of material can be collected even from a single plant and more than 30 billion nucleotides can be obtained by long readings of the ONT,” said Ekaterina Dvoryaninova, an employee of the Laboratory of Comparative Genomics and Transcriptomics of the IMB RAS, a graduate student at the Moscow Institute of Physics and Technology.
For sequencing, the platform of the third generation ONT was used, which makes it possible to obtain reads up to millions of nucleotides in length, which is extremely important for the assembly of extended genomes. Since genome assembly applications are developed based on different algorithms, the result of their work depends on the quality and quantity of the obtained sequencing data, as well as on the structure of the genome itself.
Elizaveta Sigova, graduate student at Moscow Institute of Physics and Technology, added: “Based on the obtained sequencing data, we conducted a comparative analysis of the results of the work of various genomic assemblers and chose the most complete and extended assembly of the 3896 line flax genome.”
Eight popular assembly applications were tested to select the most appropriate assembly method. The quality of the assemblies was assessed by scientists in terms of completeness and continuity – the optimal assembly of the genome should be complete, that is, contain all the obligatory conservative regions, and extended, that is, consist of as few parts as possible. Next, the so-called “polishing” procedure was applied, which is used to improve the accuracy of the resulting genomic assembly. To select the optimal “polishing” scheme, a comparative analysis of applications in demand today was also carried out. As a result, an algorithm for creating a high-quality assembly of the flax genome has been developed: from growing plants to bioinformatic processing of sequencing data.
“Our study highlights the influence of the chosen genome construction strategy on the obtained assembly parameters and its suitability for future molecular genetic studies,” sums up Alexey Dmitriev, head of the Laboratory for Comparative Genomics and Transcriptomics, IMB RAS.
The genomes of representatives of the same plant species can differ significantly from each other. High-quality genomic assemblies make it possible to identify such differences and move on to the pangenome stage – the totality of nucleotide sequences of all representatives of a species, which takes the study of this species to a new level. The algorithm developed by scientists for obtaining a high-quality flax genome is necessary for creating the pangenome of this crop and will contribute to the selection of varieties with a given set of characteristics and even wider use of this valuable plant.
