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Project titleGenome-wide association studies and flax resistance mechanisms to Fusarium wilt

Keywordsflax, GWAS, fusarium wilt, genome assembly, transcriptome, genetic diversity, host-pathogen interactions, gene networks, breeding

Annotation
In this project we will use state-of-the-art genomics, big-data analysis, and phenotyping to characterize the extensive flax collection of the ALL-Russian Flax Institute (VNIIL) and identify genes or genomic regions that control important agronomic traits and plant response to the Fusarium infection, which would be uniquely suitable for improving this strategic both for Russia and world wide culture. We also plan to assemble the Fusarium oxysporum f. lini genome, characterize its genetic variability at the population level, characterize the pathogen specific transcripts associated with the infection, and reconstruct the interaction network of virulence genes and the genes of the host plant associated with the infection. We will study the role of DNA methylation in the response of flax plants to the infection. All this will allow to develop an effective strategy for long-term control of the disease by selecting the optimal combinations of resistance genes. A defining challenge of the 21st century is meeting the nutritional demands of a growing human population with limited land and water resources, under the specter of climate change. Agriculture must intensify, become more sustainable, and gain greater resilience to pests and climate. . However, as a result of Green Revolution, genetic diversity, a traditional mean by which plants, or other organisms, adapt to meet new challenges, was lost and food producers have therefore lost the traditional resource used in breeding. As a rule current crop varieties are created by crossing relatively closely related genotypes, using a variety of selection methods depending on the crop’s biology. These breeding schema very rarely involve genetically more diverse but less productive landraces. A prime example of this is flax. For example, the sturdy forms of flax resulted from the plant selection by Russian peasants for several hundred years, which, along with the unsurpassed quality parameters of the flax fiber, possess coadaptive gene blocks, are not used because of their low productivity. Due to genetic depletion of the cultivated flax varieties, both the adaptive potential and the quality of the flux product reduce. There is a danger of reducing the resistance of the crop to stress, including diseases, the manifestation of which is intensified due to climate change Flax is one of the most important crops, both in Russia and in other countries. Fibre flax is a source of natural fiber raw materials with unique sanitary-hygienic and consumer properties. In modern conditions, fibre flax is considered as a strategic crop in Russia, since it is able to replace raw cotton, which has moved into the category of imported raw materials. Oil flax is used for the production of seeds, which are characterized by valuable nutritional, therapeutic, prophylactic, and other properties. Given this perspective there is a compelling need to re-introduce genetic variation into flax lines. Here we focus on a solution, one that will not impose the political burden imposed by GMO, is based on mobilization of biodiversity and, once again, is at the frontier of agricultural science. То achive this we propose to use unique data collected in the VNIIL collection in combination with modern methods of genomics and bioinformatics. Importantly, the flax collection created at VNIIL is one of the largest in the world and covers almost all the genetic diversity of this crop. The collection was screened for resistance to Fusarium wilt and highly resistant samples were identified. The pathogen strains were also studied for cultural, morphological, and virulent properties. Fusarium wilt of flax is the most destructive and widespread disease, both in the Russian Federation and in other flax-growing countries of the world. The main causative agent of this disease is the fungus F. oxysporum, which is among the ten most dangerous plant pathogens. Most modern domestic flax varieties are characterized by high or moderate resistance to Fusarium wilt. However, there is a risk of developing disease due to the instability of the racial composition of the pathogen population and due to the substantial genetic homogeneity of the varietal material. This situation is intensified by the increasing development of crop diseases associated with climate change. The expected global warming may lead to increased aggressiveness of individual pathogen races and the loss of resistance by varieties determined by one or two genes. In this regard, it is necessary to take proactive steps, namely, to characterize the racial composition of the pathogen population at the genomic level, in order to create varieties of fibre and oil flax with different effective pathogen resistance genes and with optimal combinations of these genes. Priority directions of flax breding are to increase the fiber content and quality, create varieties with different chemical composition of seeds, resistance to biotic and abiotic stresses, create flax varieties adapted to new areas of growth, mainly to northern territories with a short photoperiod. To solve these problems we will apply GWAS, an innovative breeding method. This method, based on genomics and mathematical statistics, makes it possible, to identify genes and gene regions controlling the formation and quality of the fiber, to expand our knowledge of the allelic diversity and regulation of genes involved in the synthesis of fatty acids, to reveal genes and allels controlling flax resistance to Fusarium oxysporum f. sp. lini, as well as to get understanding of fusariam wilt disease at a systems level. We will also investigate very important scientific problems that have not been studied sufficiently to this day. The results of the phytopathological assessment will be compared with the data of genotyping. As a result, new genes will be found that are donors of resistance to fusarium. The genetic control of morphological and agronomic traits will be studied, and our knowledge on the diversity of genes controlling fiber quality and the biosynthesis of fatty acids and other important agronomical traits will be expanded. We will also understand the organizational principles of the Fusarium oxysporum f. lini genome, characterize its genetic variability, understand how the host plant protects itself from infection and how the pathogen infects the plant, reconstruct the network of interaction between virulence genes and the genes of the host plant. The novelty of our project also lies in the fact that we will reveal the genetic features of the co-evolution of virulence and resistance genes associated with the cultivation and regional diversification of flax. In addition, the role of DNA methylation in the response of flax to infection will be investigated, which in the future may help to involve the epigenetic modifications inherited over several generations into the selection process. The use of the data obtained in the project in the flax selection will put it on a qualitatively new level and will be a breakthrough technology.

Expected results
In this project, we will use information about genome, population structure and phenotypic traits of flax in order to create promising starting material for breeding of new varieties based on genome analysis and predictive analytics technologies. We will apply GWAS to link genetic polymorphism with variability of both agronomically important traits and environmental factors (climate and biotic stresses). This comprehensive and rational mining of the allelic richness of the VNIIL collection 1) will deliver efficient and rapid marker-assisted and genomic selection breeding strategies using previously untapped genetic material and 2) will make it possible for domestic flax-growing to become a leader in the world, since VNIIL collection is the largest in the world and contains over 1200 races of flax pathogens and no other country and no other company even have similar resource. We will also assemble the genome of Fusarium oxysporum f. sp. lini and characterize its genetic variability at the population level, analyze the host plant and fungus transcripts, reconstruct the network of interaction between flax and Fusarium oxysporum f. sp. lini genes. lini, and investigate the role of DNA methylation in the expression of flax genes during infection. This will allow not only to develop an effective long-term disease control strategy by selecting optimal combinations of resistance genes, but also to answer many fundamental questions about the mechanisms of interaction of flax and pathogen: Which fungus genomic regions and which genes are involved in the infection? How does Fusarium oxysporum f. sp. lini infect plants? What is the variability of infection related genomic regions and how do they co-evolve with the plant resistance genes? How does the host plant protect itself against infection and what are its resistance mechanisms? Which host plant genomic regions are involved in the resistance mechanism? How do the fungus virulence genes and plant resistance genes co-evolve? Does DNA methylation play an important role in the response of flax plants to infection? The answers to these questions, in turn, will make it possible to create sustainable flax lines due to targeted actions, for example, on pathogen-recognizing receptors, or MAP/His kinases, which are central regulators of the fungus virulence genes, or by targeting editing the fungus genome using CRISPR/Cas9 technology, or by selecting epigenetic changes in a number of generations.

Annotation of the results obtained in 2021
3 genomes of weakly virulent strains of Fusarium oxysporum f.sp. lini were sequenced and assembled. To ensure consistency in the analysis, genomes of the previously assembled 5 strains sequenced using only short reads were reassembled using the updated RagTag software. New algorithms for predicting coding sequences implemented in the BUSCO program (metaEUK algorithm as an alternative to Augustus used in previous versions) have significantly improved the quality of annotation of genomes and coding regions. We present the genetic characterization of a flax collection of 306 samples provided by the Russian Federal Research Center for Bast Fiber Crops. The collection includes flax varieties from Eurasia with a large proportion of inherited local Russian forms, which distinguishes it from the collections used in previous genetic studies. 3,416,829 biallelic SNPs have been identified, and the genetic variations identified are far more abundant as compared to those reported previously. We observed a significant population differentiation between fiber and linseed accessions. Fiber flax accessions have a strong relationship with each other, which is especially noticeable for kryazhs. Higher nucleotide diversity was observed in linseed accessions as compared to fiber flax accessions. The LD decay in flax is significantly lower than in soy and rice, which means that the screening process for random genes that regulate important agronomic traits will be simplified, which is important for breeding programs. An analysis was carried out to identify regions of the genome with recent selection signals, and the identified loci in fiber flax and linseed accessions differ noticeably. In this study, for the first time, we attempted to extensively characterize kryazhs, Russian heritage landraces, to shed light on their breeding history and their relationship with modern fiber flax varieties. Our results provide significant evidence of introgression of genetic material from Asian, Russian, and, importantly, European landraces into kryazhs (Fig. 5 File with additional information), supporting the hypothesis of mixed origin of kryazhs from both Indo-Afghan diversity center and Fertile Crescent. The ancestors of fiber flax cultivars include landraces of various origins, but the contribution of Russian landraces and kryazhs is the most significant. As a first step to uncover the genetic factors associated with resistance to Fusarium wilt disease, we performed a genome-wide association study (GWAS) using 297 accessions from the collection of the Federal Research Centre of the Bast Fiber Crops, Torzhok, Russia. These genotypes were infected with a highly pathogenic Fusarium oxysporum f.sp. lini MI39 strain; the wilt symptoms were documented in the course of three successive years. Six different single-locus models implemented in GAPIT3 R package were applied to a selected subset of 72,526 SNPs. A total of 15 QTNs (Quantitative Trait Nucleotides) were detected during at least two years of observation, while eight QTNs were found during all three years of the experiment. Of these, ten QTNs occupied a region of 640 Kb at the start of chromosome 1, while the remaining QTNs mapped to chromosomes 8, 11 and 13. All stable QTNs demonstrate a statistically significant allelic effect across 3 years of the experiment. Importantly, several QTNs spanned regions that harbored genes involved in the pathogen recognition and plant immunity response, including the KIP1-like protein (Lus10025717) and NBS-LRR protein (Lus10025852). Our results provide novel insights into the genetic architecture of flax resistance to Fusarium wilt and pinpoint potential candidate genes for further in-depth studies. The association analysis was performed for all 11 fiber-related traits. Single trait GWAS results identified 206 significantly associated QTNs, of which 21 were associated with several traits. Most QTNs were associated with technical length (69 QTNs), elementary fibre length (49 QTNs) and fiber content (33 QTNs). Analysis of genes around QTNs revealed 9 potential candidate genes associated with fiber quality. GWAS results using multitrait model implemented in GEMMA package identified 354 QTNs. Analysis of genes around QTNs, taking into account the LD value for each chromosome, revealed 30 potential candidate genes associated with fiber quality. 405 samples of the species Linum usitatissimum L. were tested in field conditions, sowing was carried out with a feeding area of 1 plant for fiber flax 2.5 x 2.5 cm and seed flax - 2.5 x 5.0 cm. As a result of the assessment of a representative samples from the world gene pool of culture, which included domestic and foreign varieties, selection lines of fiber flax and seed flax, local forms and krjazhi of various ecological and geographical origin, the following range of variability of the main morphological characters that determine the productivity of seeds and fiber was established: plant height – 45.9…104.8 cm, weight of the technical part of 1 plant - 112 ... 995 mg, fiber mass – 19.4 ... 173.2 mg, fiber content in the stem – 14.4 ... 31.8 %, the number of boll - 1.4 ... 22.6 pcs. and seeds on a plant - 8.0 ... 156.1 pcs. For the first time, the screening of the world gene pool of culture was carried out according to "non-traditional traits" that determine the quality of the fiber, and their variability was revealed depending on the genotype: "sbeg" - 0.20 ... 0.81 mm and "myklost" of the stem - 160.1 ... 637.1 units, the length of the elementary fiber is 8.0 ... 16.5 mm, which allows us to consider this culture as an additional source of fibrous raw materials. For the first time, samples from the gene pool of seed flax were identified that are not inferior to the standard - fiber flax variety Cesar in terms of "plant height", "technical stem length" and "fiber weight", which allows us to consider this crop as an additional source of fibrous raw materials. At the same time, the samples Modares (Romania) and k-2784 (Georgia), which have improved spinning properties due to the increased length of the elementary fibers and the "myklost" of the stem, are of particular value. As a result of evaluating flax samples for resistance to fusarium wilt, the following were identified: resistant (damage from 0 to 20%) - 190 (46.9%), slightly susceptible - 20 (4.9%), moderately susceptible - 63 (15.6%), highly susceptible - 132 (32.6%) samples. Of the 233 samples of fiber flax analyzed, the share of highly resistant genotypes is 44.7%, and of 172 samples of seed flax - 50.0%. These are mainly varieties and breeding lines of fiber flax from Russia, R. Belarus, Poland, France, Japan and China, as well as seed flax from Canada and the USA, which indicates the high efficiency of the selection of spinning and seed flax for resistance to this most harmful disease in these flax-growing countries. Induction of genotypic variability was revealed in breeding lines and varieties of fiber flax grown at different temperature ranges on selective backgrounds differing in mineral nutrition: option 1 - control (without fertilization), option 2 - introduction of a 1.5% solution of triple superphosphate into the soil and option 3 - 1.5% ammonium sulfate solution, where the main control was the option with the sowing of the studied varieties reproduced under favorable agro-climatic conditions. When growing the offspring of the studied genotypes on an optimal agrochemical background, in option 2, compared with the main control, there was a significant decrease in the height of plants and the weight of the technical part in the breeding forms Liral Prince (Great Britain), D-1444-66 (Ukraine), Nike, (Poland) and G-1071/7 (Russia), which, depending on the genotype, was 13.2 ... 26.4 and 30.7 ... 49.6%, respectively. In genotypes Liral Prince and D-1444-66 in variant 3, an increase was observed in comparison with the main control of the growing season - by 3 and 6 days, plant height - by 4.9 and 6.8%, as well as the weight of the technical part of the stem - by 19.7 and 25.6%, respectively. At the same time, the fiber flax variety Atlant showed genetic stability, deviations from control in variants 2 and 3 were not significant. Two linseed flax cultivars, resistant cultivar Atalante and susceptible cultivar LM 98 were infected with the highly virulent MI39 strain of Fusarium oxysporum f.sp. lini and transcriptomes of both plant and fungus were analyzed on the 3rd and 5th day of infection. It was found that the response to infection in the resistant cultivar begins earlier than in the susceptible one. Likewise, the genes of the fungus begin to be differentially expressed in a resistant plant earlier in comparison with suseptible one. A significant part of plant genes with differential expression during infection with a pathogen encodes various enzymes, presumably involved in neutralizing the infection (for example, chitinases), as well as general factors of the stress response. Evaluation of the expression of 13 candidate genes identified in GWAS and potentially involved in various processes associated with the response to the pathogen showed that all of these genes, with the exception of one, are differentially expressed when comparing plants infected with the MI39 strain and control plants. This result confirms the functional significance of the candidates and makes it possible to prioritize the most interesting genes for subsequent detailed functional analysis. In 100 well-sequenced flax genomes, two types of structural variation were analyzed: gene presence/absence variation (PAV) and copy number variation (CNV). In comparison with the reference genome we observed (1) deletions of DNA sequences, (2) insertions of new sequences, (3) copy number amplifivation in individual flax varieties. The functional annotation of the corresponding regions in the reference genome and new sequences showed that they encode proteins involved in the plant response to biotic and abiotic stresses, in energy metabolism, viral and transposon activity, and in the formation of cell membranes. The revealed functions may indicate adaptation of varieties to regional growing conditions.

Publications

1. Alexander Kanapin, Mikhail Bankin, Tatyana Rozhmina, Anastasia Samsonova and Maria Samsonova Genomic Regions Associated with Fusarium Wilt Resistance in Flax International journal of molecular sciences, Int. J. Mol. Sci. 2021, 22, 12383 (year - 2021) https://doi.org/10.3390/ijms222212383

2. Anastasia Samsonova, Alexander Kanapin, Michael Bankin, Anton Logachev, Maria Gretsova, Tatyana Rozhmina and Maria Samsonova A Genomic Blueprint of Flax Fungal Parasite Fusarium oxysporum f. sp. lini International journal of molecular sciences, Int. J. Mol. Sci. 2021, 22, 2665 (year - 2021) https://doi.org/10.3390/ijms22052665

3. M.A. Duk, А.A. Кanapin, А.А. Samsonova, T.A.Rozhmina, М.G. Samsonova Анализ структурной вариабельности геномов льна Linum usitatissimum L. Биофизика, - (year - 2022)

4. Maria Duk, Alexander Kanapin, Svetlana Surkova, Mikhail Bankin, Tatyana Rozhmina, Anastasia Samsonova and Maria Samsonova The Genetic Landscape of Fiber Flax Frontiers in Plant Science, Front. Plant Sci., 07 December 2021 (year - 2021) https://doi.org/10.3389/fpls.2021.764612

5. Tatyana Rozhmina, Mikhail Bankin, Anastasia Samsonova, Alexander Kanapin, MariaSamsonova A comprehensive dataset of flax (Linum uitatissimum L.) phenotypes Data in brief, Volume 37, August 2021, 107224 (year - 2021) https://doi.org/10.1016/j.dib.2021.107224

6. А.A. Кanapin, А.А. Samsonova, М. P. Bankin, А.A. Logachev, Т.А. Rozhmina, М.G. Samsonova Сборка геномов трех слабовирулентных штаммов Fusarium oxysporum f.sp. lini Биофизика, - (year - 2022)

Annotation of the results obtained in 2019
406 accessions of the species Linum usitatissimum L. were tested in the field, sowing was carried out as a hole nursery with a feeding area of 1 plant for fibre flax 2.5 x 2.5 cm and oil flax 2.5 x 5.0 cm. The assessment of representative accessions from the global gene pool of culture, which includes domestic and foreign varieties, selection lines of spinning and oil flax, local and draft forms of various ecological and geographical origin allows us to estimate ranges of variability of the main morphological characters determining the productivity of seeds and fiber. It has been shown for the first time that the most important source of “runaway” of the stem and “length of the elementary fiber” are the lace forms of flax from Russia. It has been established that the vast majority of oil flax samples have a low “shoot” of the stem (less than 0.5 mm), therefore, its increased value in modern varieties of long flax is the result of artificial selection. The greatest values for this characteristic have predominantly flax varieties of Chinese selection. From the oil flax gene pool, tall specimens with a high fiber content in the stem and an increased length of elementary fibers were identified, which allows us to consider this culture as an additional source of fibrous raw materials. The cultural and virulent properties of the strains of the fungus Fusarium oxysporum f. lini No. 282, 283, 287, 324, 329 and mono-isolate No. 39.2.1 from the “Collection of microorganisms - causative agents of flax diseases”. It was established that these biosamples differ in cultural-morphological characteristics and are highly virulent. As a result of evaluation of accessions of the world flax gene pool from 41 countries for resistance to Fusarium wilting against an infectious background with monoisolate No. 39.2.1 F. oxysporum f. lini revealed we found that 148 accessions are resistant (36.5%), 25 accessions are weakly sensitive (6.1%), 88 accessions - medium-sensitive (21.7%) and 145 accessions highly sensitive (35.7%). Of the 229 fibre flax accessions analyzed ,the share of highly resistant genotypes is 38.9%, and of the 177 samples of oil flax - 33.3%. These are mainly varieties and breeding lines of fibre flax from Russia, R. Belarus, Poland, the Czech Republic, Japan and China, as well as oil flax from China, Canada and the USA, which indicates the high efficiency of selection of spinning and oil flax for resistance to the most harmful disease in these countries. On the territory of the Institute of Flax (Torzhok), from June 25 to 26, 2019, 406 samples of flax (Linum usitatissimum L.) were collected. Genomic DNA has been extracted from leaves of all collected samples. The integrity, purity, concentration and the total volume of DNA will allow for genotyping by sequencing of all samples and genome-wide sequencing of the selected samples. Pure genomic DNA was isolated from highly virulent strains of Fusarium oxysporum f. lini No. 282, 287, 324, 329 and mono-isolate 39. Monoisolate 39 DNA was sequenced at the University of Oregon, Center for Genome Research and Biocomputing using PacBio technology, and DNA from all 5 strains was sequenced using Illumina technology by Novogene (Novogene Corporation Inc.). We analysed the sequencing data of the pathogenic strain F391 Fusarium oxysporum f. sp. lini, sequenced using PacBio and Illumina protocols, and the reference genome was assembled using the hybrid method. Analysis of the assembled fragments showed their high quality (level of coverage of the related genome, low level of duplications and fragmentation) and applicability for further research on the project. The genomes of 4 other virulent strains of Fusarium oxysporum f. sp. lini F329, F32, F282, F287 sequenced using Illumina technology were assembled. High-quality sequences were obtained that will be further used to analyze the structural variation of the fungal genome.

Publications

Annotation of the results obtained in 2020
We have shown that the genome of F.oxysporum f. sp. lini is subdivided into more than two compartments with different structural characteristics and evolutionary history In contrast to housekeeeping genes, members of SIX gene family demonstrate host-related clustering as revealed by phylogenetic analyses, thus being indicative of strong adaptation to host. In this context SIX7 gene poses a particular interest, as it is located on different chromosomes and is absent from other 4 FOLINI strains sequenced, strongly suggesting a relatively recent gene duplication event. Functional analyses of F. oxysporum f.spp protein repertoire reveals the dominating role of the variable compartment in diversification of the formae specialis. Remarkably, the main contributors to proteome divergence are PFAM domains associated with DNA-binding and mobilization of transposable elements, possibly suggestive of active transcription and ongoing genome reshuffling in variable compartment. Using PacBio and Illumina technologies, the genomes of three weakly virulent strains of F.xysporum f.sp. lini were sequenced. We have successfully sequenced with a different level of coverage 406 flax accessions from the collection. Genetic variants have been obtained, allowing to characterize the genetic diversity of varieties, and a processing pipeline have been developed to search for genetic variants. 405 accessions of the species Linum usitatissimum L. were tested in field conditions, sowing was carried out with a feeding area of 1 plant for fiber flax 2.5 x 2.5 cm and seed flax - 2.5 x 5.0 cm. As a result of the assessment of a representative samples from the world gene pool of culture, which included domestic and foreign varieties, selection lines of fiber flax and seed flax, local forms and krjazhi of various ecological and geographical origin, the following range of variability of the main morphological characters that determine the productivity of seeds and fiber was established: plant height - 46.2 ... 116 cm, weight of the technical part of 1 plant - 128.7 ... 976.7 mg, fiber mass - 32.6 ... 225.2 mg, fiber content in the stem - 17.46 ... 41, 90%, the number of boll - 1.0 ... 16.0 pcs. and seeds on a plant - 8.0 ... 125.8 pcs., the mass of 1000 seeds is 3.60 ... 8.00 g, the oil content in seeds is 34.1 ... 45.60%. For the first time, the screening of the world gene pool of culture was carried out according to "non-traditional traits" that determine the quality of the fiber, and their variability was revealed depending on the genotype: "sbeg" - 0.20 ... 0.81 mm and the "myklost" of the stem - 181.3 ... 861.8 units, the length of the elementary fiber is 9.52 ... 17.26 mm, which is important for creating specialized flax varieties for various purposes. For the first time, tall samples were found from the seed flax gene pool - 81.3 ... 93.5 cm with an increased fiber content in the stem - 25.54 ... 30.11 % and an increased length of elementary fibers 14.20 ... 15.59 mm, which allows us to consider this culture as an additional source of fibrous raw materials. As a result of the assessment of flax samples, the following were identified: resistant (damage by fusarium wilt from 0 to 20%) - 161 (39.8%), weakly susceptible - 22 (5.4%), moderately susceptible - 55 (13.6%), highly susceptible - 167 (41.2%). Of the analyzed 232 samples of fiber flax, the share of highly resistant genotypes is 45.3%, and of 173 samples of seed flax - 45.1%. These are mainly varieties and breeding lines of fiber flax from Russia, Belarus, Poland, the Czech Republic, Japan and China, as well as seed flax from China, Canada and the United States, which indicates the high efficiency of the selection of fiber flax and seed flax for resistance to this most harmful disease in these flax-growing countries. A statistical analysis of the phenotypic traits of the samples of the world collection of flax (Linum usitatissimum L.) was carried out. It has been established that all samples of the world collection of flax, according to the degree of phenotypic similarity, form three groups: the first includes curly flax, mezheumki, large-seeded forms and creeping flax; the second is represented mainly by modern varieties and cultural lines of fiber flax; the third - by ridge and local forms of fiber. In order to induce genotypic variability at different temperature ranges, various varieties of fiber flax were cultivated (experiment 1 - Liral Prince, Northern Ireland; D-1444-66, Ukraine; Nike, Poland; Atlant, G-1071/7, Russia; experiment 2 - Liral Prince, D-1444-66 and Nike) on selective backgrounds differing in mineral nutrition: option 1 - control (without fertilization), option 2 - adding a 1.5% solution of triple superphosphate to the soil and option 3 - 1, 5% ammonium sulfate solution. Seed material has been obtained for further research to identify flax genotrophs. The method of obtaining roots infected with the highly virulent strain of Fusarium oxysporum f. sp. lini of cultivated flax plants (Linum usitatissimum L.) in vitro has been successfully worked out. Infected roots have been successfully used to analyze the expression levels of genes of interest in the plant-pathogen system. PCR was used to demonsterate specifc expression of the F.oxysporum f. sp. lini genes in planta, which allows us to speak about the effectiveness of the selected primers and the selected method of plant infection.

Publications

1. Alexander Kanapin, Anastasia Samsonova, Tatyana Rozhmina, Michael Bankin, Anton Logachev, Maria Samsonova The Genome Sequence of Five Highly Pathogenic Isolates of Fusarium oxysporum f. sp. lini Molecular Plant-Microbe Interactions, Mol Plant Microbe Interact. 2020 Sep;33(9):1112-1115 (year - 2020) https://doi.org/10.1094/MPMI-05-20-0130-SC

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