RU2806563C1 - Method for identifying mutation in the fifth exon of the fad3a gene leading to decrease in the content of linolenic acid in flaxseed oil - Google Patents

Abstract

FIELD: biotechnology; agriculture.

SUBSTANCE: method for identifying a mutation in the fifth exon of the FAD3A gene, leading to a decrease in the content of linolenic acid in flaxseed oil. The method is based on amplification of a fragment of the FAD3A gene of the test sample and two standards: without mutation and with mutation, followed by analysis of high-resolution melting curves.

EFFECT: invention allows, starting from the early stages of the breeding process, to highly accurately select flax plants with the desired mutation of the FAD3A gene in a homozygous state or without it, which will have a certain content of linolenic acid in the oil.

1 cl, 1 dwg, 3 ex

Description

Field of technology to which the invention relates

The present invention relates to the field of biotechnology for use in agriculture, in particular breeding and seed production, as well as plant genetics, and can be used to identify flax genotypes with a reduced content of linolenic acid in oil.

State of the art

Common flax (Linum usitatissimum L.) is one of the oldest plants cultivated by humans, the seeds of which are traditionally used to obtain edible oil [Weiss E., Zohary D., Hopf M. 2012. Domestication of Plants in the Old World - The Origin and Spread of Domesticated Plants in South-west Asia, Europe, and the Mediterranean Basin. Oxford University Press, New York]. Flax seeds are one of the richest plant sources of healthy ω-3 fatty acids, and also contain a number of other compounds beneficial to humans (primarily lignans), therefore, consuming flax seed products prevents the occurrence and development of cardiovascular, cancer and other diseases [Muir A.D., Westcott N.D. 2003. Flax: The genus Linum. CRC Press, London; Goyal A., Sharma V., Upadhyay N., et al. 2014. Flax and flaxseed oil: an ancient medicine & modern functional food. J Food Sci Technol. 51, 1633-1653; Imran M., Ahmad N., Anjum F.M., et al. 2015. Potential protective properties of flax lignan secoisolariciresinol diglucoside. Nutr J. 14.71; Abdelhamid A.S., Martin N., Bridges C, et al. 2018. Polyunsaturated fatty acids for the primary and secondary prevention of cardiovascular disease. Cochrane Database Syst Rev. 7, CD012345; Kezimana P., Dmitriev A.A., Kudryavtseva A.V., et al. 2018. Secoisolariciresinol Diglucoside of Flaxseed and Its Metabolites: Biosynthesis and Potential for Nutraceuticals. Front Genet. 9, 641; Locke A., Schneiderhan J., Zick S.M. 2018. Diets for Health: Goals and Guidelines. Am Fam Physician. 97, 721-728; Parikh M., Netticadan T., Pierce G.N. 2018. Flaxseed: its bioactive components and their cardiovascular benefits. Am J Physiol Heart Circ Physiol. 314, Н146-Н159; Campos J.R., Severino P., Ferreira C.S., et al. 2019. Linseed Essential Oil - Source of Lipids as Active Ingredients for Pharmaceuticals and Nutraceuticals. Curr Med Chem. 26, 4537-4558; Cullis C.A. 2019. Genetics and Genomics of Linum. Springer, Cham; Mali A.V., Padhye S.B., Anant S., et al. 2019. Anticancer and antimetastatic potential of enterolactone: Clinical, preclinical and mechanistic perspectives. Eur J Pharmacol. 852, 107-124]. Oil flax seeds are used in the production of environmentally friendly paints, linoleum, composite materials, food products, dietary supplements, pharmaceuticals, as well as functional animal feeds [Kouba M., Mourot J. 2011. A review of nutritional effects on fat composition of animal products with special emphasis on n-3 polyunsaturated fatty acids. Biochimie. 93, 13-17; Singh K.K., Mridula D., Rehal J., et al. 2011. Flaxseed: a potential source of food, feed and fiber. Crit Rev Food Sci Nutr. 51, 210-222; Corino C, Rossi R, Cannata S, et al. 2014. Effect of dietary linseed on the nutritional value and quality of pork and pork products: systematic review and meta-analysis. Meat Sci. 98, 679-688; Goyal A., Sharma V., Upadhyay N., et al. 2014. Flax and flaxseed oil: an ancient medicine & modern functional food. J Food Sci Technol. 51, 1633-1653; Campos J.R., Severino P., Ferreira C.S., et al. 2019. Linseed Essential Oil - Source of Lipids as Active Ingredients for Pharmaceuticals and Nutraceuticals. Curr Med Chem. 26, 4537-4558; Fombuena V., Petrucci R., Dominici F., et al. 2019. Maleinized Linseed Oil as Epoxy Resin Hardener for Composites with High Bio Content Obtained from Linen Byproducts. Polymers (Basel). 11, 301]. The main fatty acids of flaxseed oil are unsaturated linolenic acid (ω-3, on average about 50%), linoleic acid (ω-6, 15%), oleic acid (ω-9, 24%), as well as saturated stearic acid (4%) and palmitic acid. (6%). The fatty acid composition of the oil varies significantly among different varieties; for example, the difference in the content of linolenic acid can reach 10 times or more, and it is the content of a particular fatty acid that determines the direction of use of the oil [Muir A.D., Westcott N.D. 2003. Flax: The genus Linum. CRC Press, London; Cloutier S., Ragupathy R., Niu Z., et al. 2011. SSR-based linkage map of flax (Linum usitatissimum L.) and mapping of QTLs underlying fatty acid composition traits. Molecular Breeding. 28, 437-451; Dmitriev A.A., Kezimana P., Rozhmina T.A., et al. 2020. Genetic diversity of SAD and FAD genes responsible for the fatty acid composition in flax cultivars and lines. BMC Plant Biol. 20, 301]. Oil with a high content of linolenic acid (more than 50%) is used in the pharmaceutical, paint and varnish and polymer industries, but rapid “rancidity” limits its use in the food industry, where the use of seeds with a reduced content of linolenic acid is more promising [Muir A.D., Westcott N.D. 2003. Flax: The genus Linum. CRC Press, London; Goyal A., Sharma V., Upadhyay N., et al. 2014. Flax and flaxseed oil: an ancient medicine & modern functional food. J Food Sci Technol. 51, 1633-1653; Chen Y., Zhou X. R., Zhang Z. J., et al. 2015. Development of high oleic oil crop platform in flax through RNAi-mediated multiple FAD2 gene silencing. Plant Cell Rep. 34, 643-653; Brach N.B., Porokhovinova E.A., Shelenga T.B. 2016. Prospects for the creation of oilseed flax varieties for specialized purposes Agrarian Bulletin of the South-East 1-2, 50-52; Cullis S.A. 2019. Genetics and Genomics of Linum. Springer, Cham; Fombuena V., Petrucci R., Dominici F., et al. 2019. Maleinized Linseed Oil as Epoxy Resin Hardener for Composites with High Bio Content Obtained from Linen Byproducts. Polymers (Basel). 11, 301].

It is known that the key genes involved in the synthesis of flax fatty acids encode desaturases that introduce double bonds into the hydrocarbon chain. FAD3A and FAD3B (fatty acid desaturases) catalyze the conversion of linoleic acid to linolenic acid [Vrinten R., Hu Z., Munchinsky M.A., et al. 2005. Two FADS desaturase genes control the level of linolenic acid in flax seeds. Plant Physiol. 139, 79-87; Banik M., Duguid S., Cloutier S. 2011. Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax {Linum usitatissimum L.) and their role in linolenic acid accumulation. Genome. 54, 471-483]. The length of the FAD3A and FAD3B paralogs is about 3 thousand nucleotides, they have 6 exons [Thambugala D., Duguid S., Loewen E., et al. 2013. Genetic variation of six desaturase genes in flax and their impact on fatty acid composition. Theor Appl Genet. 126, 2627-2641; Raj wade A.V., Kadoo N.Y., Borikar S.P., et al. 2014. Differential transcriptional activity of SAD, FAD2 and FADS desaturase genes in developing seeds of linseed contributes to varietal variation in alpha-linolenic acid content. Phytochemistry. 98, 41-53]. It has been shown that polymorphisms of the FAD3A and FAD3B genes are associated with the content of linoleic and linolenic acids in flaxseed oil, for example, a mutation in the fifth exon of the FAD3A gene (replacement of C with T in the site CP027631.1:16090340 in the flax genome assembly GCA_000224295.2/ASM22429v2 from the database NCBI, https://www.ncbi.nlm.nih.gov/), which leads to the replacement of arginine with a stop codon (the result of replacing the triplet CGA with TGA), determines the reduced content of linolenic acid in the oil [Vrinten R., Hu Z ., Munchinsky M.A., et al. 2005. Two FAD3 desaturase genes control the level of linolenic acid in flax seeds. Plant Physiol. 139, 79-87; Banik M., Duguid S., Cloutier S. 2011. Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax (Linum usitatissimum L.) and their role in linolenic acid accumulation. Genome. 54, 471-483; Thambugala D., Duguid S., Loewen E., et al. 2013. Genetic variation of six desaturase genes in flax and their impact on fatty acid composition. Theor Appl Genet. 126, 2627-2641; Rajwade A.V., Kadoo N.Y., Borikar S.P., et al. 2014. Differential transcriptional activity of SAD, FAD2 and FADS desaturase genes in developing seeds of linseed contributes to varietal variation in alpha-linolenic acid content. Phytochemistry. 98, 41-53; You F.M., Xiao J., Li P., et al. 2018. Genome-Wide Association Study and Selection Signatures Detect Genomic Regions Associated with Seed Yield and Oil Quality in Flax. Int J Mol Sci. 19; Porokhovinova E.A., Shelenga T.V., Matveeva T.V., et al. 2019. Polymorphism of genes controlling low level of linolenic acid in lines from VIR flax genetic collection. Ecological Genetics. 17, 5-19; Dmitriev A.A., Kezimana P., Rozhmina T.A., et al. 2020. Genetic diversity of SAD and FAD genes responsible for the fatty acid composition in flax cultivars and lines. BMC Plant Biol. 20, 301].

Evaluation of the fatty acid composition of flaxseed oil is usually carried out using the chromatographic method [Ermakov A.I., Arasimovich V.V., Yarosh N.P. 1987. Methods for biochemical research of plants. L.: Agropromizdat, Leningrad; Bjelkova M., Nozkova J., Fatrcova-Sramkova K., et al. 2012. Comparison of linseed (Linum usitatissimum L.) genotypes with respect to the content of polyunsaturated fatty acids. Chemical Papers. 66, 972-976] and has a number of disadvantages: it does not allow the identification of samples with mutations that determine the content of fatty acids in the heterozygous state; requires a significant number of seeds for analysis; analysis of a pool of seeds heterogeneous in linolenic acid content leads to an incorrect understanding of the sample. Due to the above features, the assessment of fatty acid composition is often carried out only at late stages of the breeding process, which makes it difficult to effectively create varieties with the required linolenic acid content. The use of DNA markers will solve these problems - it will allow analysis of individual plants using only a few leaves to isolate DNA, making it possible to perform other studies of this genotype and obtain seeds from it, as well as distinguish between the desired mutations in heterozygous and homozygous states, which significantly increases accuracy analysis and allows for the selection of valuable genotypes, starting from the early stages of the breeding process (for example, from the generation of F ₂ hybrids).

Thus, the development of a reliable and easy-to-use DNA test system for identifying a mutation in the fifth exon of the FAD3A gene, leading to a decrease in linolenic acid content, will open up new opportunities for marker-based selection of flax in order to create varieties for food, pharmaceutical or industrial purposes, as well as control of varietal purity during seed production.

Disclosure of the invention

The purpose of the invention was to develop a DNA test system that makes it possible to identify a mutation in the fifth exon of the FAD3A gene (replacement from C to T in the site CP027631.1:16090340 in the flax genome assembly GCA_000224295.2/ASM22429v2 from the NCBI database), leading to a reduced content linolenic acid in the oil, and makes it possible to select flax plants with the presence of the desired mutation in a homozygous state or its absence and, accordingly, the necessary fatty acid composition of the oil, even at the early stages of the selection process. To identify a mutation in the FAD3A gene leading to a decrease in the content of linolenic acid, the authors performed an analysis of the polymorphism of the FAD3A gene based on deep sequencing data for more than 400 varieties, lines and hybrids of flax, differing in the content of linolenic acid in oil, and found that the mutation in the fifth exon of this gene, leading to the replacement of arginine with a stop codon, determines the reduced content of linolenic acid, and other DNA polymorphisms in the FAD3A gene were identified, information about which is necessary for the successful selection of primers for creating a DNA test system. Based on the data obtained, specific primers were developed to amplify a region of the FAD3A gene containing the site of the desired mutation and not containing sites of other DNA polymorphisms, for subsequent evaluation of the resulting amplicons by analyzing high-resolution melting curves (HRM, High Resolution Melting), as well as two standard, representing a section of the FAD3A gene, one of which carries a mutation, and the other is absent.

The present invention relates to a new method for identifying flax genotypes with a reduced content of linolenic acid in oil, which is a high-resolution melting curve analysis of the fifth exon region of the FAD3A gene. The shift in the melting curve when analyzing a section of the FAD3A gene carrying a mutation in the fifth exon, compared to the same section of the gene without a mutation, serves as a marker of a reduced content of linolenic acid in flaxseed oil.

Another aspect of the present invention is the possibility of using the melting curve shape of the fifth exon region of the FAD3A gene as an indicator for determining the linolenic acid content in the oil of various flax samples.

The method for determining a mutation includes the following steps:

a) obtaining flax plant material (for example, several leaves);

b) DNA extraction;

c) carrying out an amplification reaction of a region of the FAD3A gene using a pair of gene-specific primers and a template (DNA obtained in step b), or standards representing a region of the FAD3A gene, one of which carries a mutation, and the other does not);

d) analysis of high-resolution melting curves of the amplified fragment of the FAD3A gene for the test sample and two standards;

e) comparison of the obtained melting curve for the flax genotype under study with the melting curves for two standards; the similarity of the melting curve for the analyzed sample and the standard carrying the mutation indicates the presence of the mutation in the studied genotype in a homozygous state; the similarity of the melting curve for the analyzed sample and a standard that does not have a mutation indicates the absence of a mutation in the genotype being studied; the shape and position of the melting curve for the analyzed sample, different from those for the two standards, indicate the presence of a mutation in the studied genotype in a heterozygous state.

In a particular preferred embodiment of the present invention, a set of primers is used to perform a polymerase chain reaction (PCR) to subsequently determine the presence of a mutation in the fifth exon of the FAD3A gene having the sequences SEQ ID NO: 1 and 2 (Sequence Listing).

In yet another embodiment of the claimed method, DNA sequences SEQ ID NO: 3 and 4 (Sequence Listing) are used as standards in step c).

Brief description of figures and tables

Figure 1. High-resolution amplicon melting curves for two standards - reference FAD3A_E5_ref (without mutation, right) and alternative FAD3A_E5_alt (with mutation, left), as well as a sample obtained by mixing these two standards in a 1:1 ratio (mutation in heterozygous condition, center) in three technical replicates.

Carrying out the invention

This invention provides a method for identifying flax genotypes with a certain content of linolenic acid in oil, based on the analysis of high-resolution melting curves of an amplicon from a region of the FAD3A gene carrying a mutation that leads to the replacement of arginine with a stop codon.

Tissue samples for analysis

Various tissues of the flax plant can be used as material for analysis, but the preferred option is to use several leaves, which makes it possible to perform other studies of this genotype and obtain seeds from the plant.

DNA extraction

Methods for isolating DNA from plant tissues are well known and, as a rule, include the following stages: grinding plant material, cell lysis, DNA isolation and purification, checking DNA quality by electrophoresis in a 1% agarose gel in the presence of ethidium bromide dye or its analogues, as well as spectrophotometric determination of DNA quantity. Homogenization of the material can be done manually using pestles or using homogenizers such as MagNA Lyser (Roche, Switzerland) or TissueLyser II (Qiagen, Germany) in tubes with ceramic, glass or steel balls. Various protocols or reagent kits can be used to isolate DNA. Classical methods for extracting DNA from plants use a buffer containing guanidine chloride or CTAB (cetyltrimethylammonium bromide) as a lysis agent, followed by extraction with chloroform. Commercial kits are also available for DNA extraction from plants, for example, DNA-Extran-3 (Syntol, Russia) and DNeasy Plant Mini Kit (Qiagen, Germany).

Selection of specific primers

The selection of specific primers was carried out using data on FAD3A gene polymorphisms obtained by the authors and other researchers, as well as NCBI Primer-BLAST programs [Ye J., Coulouris G., Zaretskaya I., et al. 2012. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 13, 134] and MethyMer [Krasnov G.S., Melnikova N.V., Lakunina V.A., et al. 2018. MethyMer: Design of combinations of specific primers for bisulfite sequencing of complete CpG islands. J Bioinform Comput Biol. 16, 1840004]. The selection of primers for amplification of the FAD3A gene region in which the mutation leading to the replacement of arginine with a stop codon is localized for the purpose of further analysis of high-resolution melting curves was carried out in accordance with standard recommendations and method requirements. In addition, the primers were selected in such a way that the amplicon did not contain sites of DNA polymorphisms other than the site of the desired mutation. In a preferred embodiment, the primers used are SEQ ID NOs: 1 and 2 (Sequence Listing).

Determination of a mutation in the fifth exon of the FAD3A gene, leading to the replacement of arginine with a stop codon

The method of analysis of high-resolution melting curves (HRM, High Resolution Melting) is widely used for identifying DNA polymorphisms and genotyping samples [Montgomery J.L., Sanford L.N., Wittwer ST. 2010. High-resolution DNA melting analysis in clinical research and diagnostics. Expert Rev Mol Diagn. 10, 219-240; Druml V., Cichna-Markl M. 2014. High resolution melting (HRM) analysis of DNA—its role and potential in food analysis. Food Chem. 158, 245-254; Simko I. 2016. High-Resolution DNA Melting Analysis in Plant Research. Trends Plant Sci. 21, 528-537; Tamburro M., Ripabelli G. 2017. High Resolution Melting as a rapid, reliable, accurate and cost-effective emerging tool for genotyping pathogenic bacteria and enhancing molecular epidemiological surveillance: a comprehensive review of the literature. Ann Ig. 29, 293-316]. The present invention uses high-resolution melting curve analysis of a 95 bp amplicon generated using primers FAD3A_E5_F (SEQ ID NO: 1) and FAD3A_E5_R (SEQ ID NO: 2) (Sequence Listing) by polymerase chain reaction for flax plant samples (PCR).

Selection of comparison samples

To compare the results of the analysis of high-resolution melting curves obtained for flax samples in order to identify a mutation in the fifth exon of the FAD3A gene, leading to the replacement of arginine with a stop codon, two standards are used, the first of which is a variant of the DNA sequence that does not contain the desired mutation, and the second is a variant of the DNA sequence in which the desired mutation is present. In a preferred embodiment, the standards FAD3A_E5_ref (SEQ ID NO: 3) and FAD3A_E5_alt (SEQ ID NO: 4) (Sequence Listing) are used.

The present invention is further illustrated in detail using specific examples representing preferred embodiments of the invention. At the same time, the invention is not limited to the described embodiments, but includes any alternative options and modifications, the use of which is possible taking into account the essence of the invention.

Example 1: Sample Preparation

Seeds of flax varieties and lines are treated with a 3% sodium hypochlorite solution, washed with distilled water and germinated in Petri dishes on filter paper moistened with water. Leaves of 7-day-old seedlings are used for DNA extraction.

Example 2: DNA Isolation

The leaves are crushed using a TissueLyser II homogenizer (Qiagen, Germany), after which DNA extraction is carried out using the DNA-Extran-3 kit (Synthol, Russia) according to the manufacturer’s protocol. The quality of DNA is assessed by electrophoresis in a 1% agarose gel. DNA concentration is determined using a Qubit fluorimeter (Thermo Fisher Scientific, USA) or a NanoDrop spectrophotometer (Thermo Fisher Scientific).

Example 3. Amplification of the FAD3A gene region in which the mutation leading to the replacement of arginine with a stop codon is localized, and HRM analysis

1. Prepare a reaction mixture for amplification by mixing all reaction components, except DNA, at the rate of 1 reaction with a volume of 20 μl (of which 5 μl is reserved for DNA) for each sample and standard plus 10%. The composition of the reaction mixture for amplification of the FAD3A gene region in which the mutation is localized, leading to the replacement of arginine with a stop codon, is as follows: 1x Taq Turbo buffer (Evrogen, Russia), 250 nM of each deoxynucleoside triphosphate (Evrogen), 350 nM of primer FAD3A_E5_F (SEQ ID NO: 1), 350 nM primer FAD3A_E5_R (SEQ ID NO: 2), 1x EvaGreen dye (Biotium, USA), 1x LowROX dye (Evrogen), 2 units. HS Taq DNA polymerase (Evrogen).

2. Add 15 µl of the prepared reaction mixture without DNA into the wells of the PCR plates.

3. Add 5 μl of DNA into the wells of PCR plates (concentration 5 ng/μl for samples and 5 fg/μl for standards), seal the plate tightly with an optically transparent film.

4. Place the plate in the instrument compartment of the amplifier, set the names of the cells and the program: +95°C - 10 min - primary DNA denaturation; 50 amplification cycles: +95°C - 15 sec - denaturation, +60°C - 1 min - primer annealing and DNA chain elongation (detection on Green channel); HRM analysis (+65°С -+90°С, step 0.1°С, detection on Green channel). Then the program is run on a QuantStudio 5 thermal cycler (Thermo Fisher Scientific).

5. Process the data using High Resolution Melt Analysis software (Thermo Fisher Scientific) and present the results in graphical and/or tabular form. As two comparison samples (with a reference and alternative allele), amplicons obtained from standards represented by the sequences FAD3A_E5_ref (SEQ ID NO: 3) and FAD3A_E5_alt (SEQ ID NO: 4), respectively, are used (Figure 1).

The above detailed description of the invention and its specific embodiments presented in the examples is intended solely to better understand the essence of the claimed invention, but not to limit it. Changes may be made which, however, will remain within the spirit of the present invention as defined by the appended claims.

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List of sequences

<210> 1

<211> 17

<212> FAD3A_E5_F

<213> Artificial sequence

<220>

<223> direct primer

<400> 1

gagggctgac gaccgtc (SEQ ID NO: 1)

<210> 2

<211> 25

<212> FAD3A_E5_R

<213> Artificial sequence

<220>

<223> reverse primer

<400> 2

catttgagg aagagatggt gaata (SEQ ID NO: 2 )

<210> 3

<211> 95

<212>

<213> Artificial sequence

<220>

<223> DNA sequence of the standard for the reference allele FAD3A_E5_ref

<400> 3

gagggctgac gaccgtcgat cgagattacg gggtcatcaa caccatccac catgacattg

gcacccatgt tattcaccat ctcttccctc aaatg (SEQ ID NO: 3 )

<210> 4

<211> 95

<212>

<213> Artificial sequence

<220>

<223> DNA sequence of the standard for the alternative allele FAD3A_E5_alt

<400> 4

gagggctgac gaccgtcgat tgagattacg gggtcatcaa caccatccac catgacattg

gcacccatgt tattcaccat ctcttccctc aaatg (SEQ ID NO: 4 )

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Claims (1)

A method for identifying a mutation in the fifth exon of the FAD3A gene, leading to a decrease in the content of linolenic acid in flaxseed oil, by amplifying a section of the fifth exon of the FAD3A gene carrying the site CP027631.1:16090340 in the flax genome assembly GCA_000224295.2/ASM22429v2, replacing C with T in which, as a result of a change in the triplet CGA to TGA, results in the replacement of arginine with a stop codon, using primers represented by the sequences SEQ ID NO: 1 and SEQ ID NO: 2, followed by analysis of high-resolution melting curves for the flax samples studied in comparison with amplicons , obtained from two standards, which used sequences with the reference allele SEQ ID NO: 3 and the alternative allele SEQ ID NO: 4.

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