Marker-assisted selection and use of molecular markers in sunflower breeding for resistance to diseases and parasites

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Introduction
There are two main approaches in genetic studies -"direct" and "reverse" genetics.Since Gregor Mendel's discoveries of (1865), classical or "direct" genetics has been studying the inheritance of traits (phenotypes) in living organisms for several generations.Taking the phenotype as a starting point, "direct" genetics identifies genetic factors that affect the expression of any trait.Thus, "direct" genetics is directed from phenotype to genotype.Production of bulky mutant populations for further search for phenotypic changes in them is a key stage in the experimental application of this approach (Sulima, Zhukov, 2015).
Since the 1980s, the knowledge gained over the past period (discovery of DNA as a carrier of genetic information, decoding of the genetic code, development of sequencing and genome-modifying methods) (Inge-Vechtomov, 2010) opened a new approach that involves analyses of DNA sequences and the effects that are exerted by changes in these sequences (mutations) rather than analyses of phenotypes and their genetic control.This concept is called "reverse" genetics (Struhl, 1983;Reski, 1998).
Modern genetics has entered the "post-genomic era", when information about the genome structure of a wide assortment of organisms has become available.Nowadays, it is especially important to identify biological functions of genes, the sequences of which are already known (Eisenberg et al., 2000;Griffiths, Stotz, 2006;Hsiao, Kuo, 2009).
The object of "reverse" genetics is usually a gene with an unknown function (which was detected by Expressed Sequence Tag (EST) sequencing), the entire genome, or a separate part of it, etc.The research strategy is in altering the gene structure or activity and subsequent analyzing associated changes in the phenotype.With the development of large-scale genomic sequencing technologies, "reverse" genetics has received significant support, taking a leading position both in fundamental science and in applied research (Alonso, Ecker, 2006;Barrangou et al., 2007;Small, 2007;Boutros, Ahringer, 2008;Hirochika, 2010;Bolle et al., 2011;Upadhyaya et al., 2011;Liu, Fan, 2014).
Traditional breeding focuses mainly on phenotypic selection and is unable to distinguish effects of the environment or other factors related to growing conditions from genetic characteristics.This problem can be solved by using molecular markers in direct selection (MAS).The trait of interest is marked by a molecular marker that is closely linked to the gene determining this trait or to the gene that affects the trait expression.
MAS allows selecting in accordance with the genotype, regardless of environmental effects.In addition, MAS is used as a tool that complements and significantly facilitates traditional breeding.
The European Technology Platform 'Plants for the Future' in its proposed strategic research agenda for plant genomics and biotechnology until 2025 regards a rise in the selection efficiency as a priority.The molecular approach, namely Marker-Assisted Breeding (MAB) or Marker-Assisted Selection (MAS) is a promising way to solve this problem (Kozhukhova, 2011).
Literature Review, Summarization of Basic Provisions.The term "Marker-Assisted Selection" was first used in the literature in 1986 to describe the possible use (Beckmann, Soller, 1983).The first ground-breaking article on MAS in plant breeding using DNA markers was devoted to resistance of soybean to nematodes (Heterodera glycines Ichinohe) (Concibido et al., 1996).According to the Glossary of Biotechnology for Food and Agriculture of the Food and Agriculture Organization of the United Nations (FAO), MAS is the use of DNA markers to increase the selection efficiency, basing on the detection of markers of breeding traits (Zaid et al., 2007).
The MAS principle is as follows: if the localization of a gene that affects the expression of an agronomically important trait is known, inheritance of the gene that controls it, or the presence of the required allele in breeding material (not the expression of this trait) is monitored (Kozhukhova, 2011).
Available molecular markers are a necessary prerequisite for any MAS project.A molecular marker can be any DNA fragment used to detect a polymorphism and having a close genetic association with the gene responsible for the trait under investigation (Kalendar, Glazko, 2002).
The rapid development of new methods of molecular biology, including automation and computerization of different processes, development of appropriate methods and software for statistical processing and creation of available databases needed to study DNA polymorphism contribute to the arsenal of molecular markers and their increasing use in various fields of fundamental and applied biology (Kozhukhova, 2011).
Methods of molecular genetics have been widely used in various biological branches: botany, entomology, phytopathology, genetics, etc.The idea of markers is not new, as it was formulated by AS Serebrovskiy as a method of signals in the 1930s.Genetic and breeding achievements in agricultural plants are often attributed to different marker systems.After all, to optimize and accelerate the breeding process, one has first to identify genes of the desired traits in staring and breeding materials.Traditional methods of their detection (hybridological analysis) are labor-and time-consuming.MAS, a novel comprehensive combining traditional breeding and achievements of such new disciplines as genomics and bioinformatics, offers exciting possibilities for the creation of harmful organism-resistant plant varieties and hybrids (Sivolap et al., 2011).
Marker as a general concept in breeding is a trait that is easily recognizable and is closely associated with a gene of breeders' interest.That is, the presence of the marker is a signal that the important for breeding trait is present too.Markers make it possible to conduct the desired trait-oriented selection (for example, for resistance) and to add fundamentally new genes to the plant genome.Data on markers of resistance genes allow researchers to quickly find the desired resistance genes and their combinations in wild and domestic plants as well as to monitor the presence of this trait while creating resistant or tolerant varieties and hybrids.The method of biochemical markers was developed and investigated in the late 1970s.It was found that alleles of genes that determine the synthesis of storage proteins or isoenzymes are linked to genes of wheat resistance to fungal pathogens (Lisova, 1999).Due to these findings, individual proteins can be used as markers in breeding for resistance.
Markers for plant breeding began to gain popularity in the early 1980s, when isozyme markers were used to accelerate the introgression of monogenic traits from exotic germplasm into cultivated background (Tanksley, Rick, 1980;Tanksley, 1983).In the mid-1980s, the first use of restriction fragment length polymorphism (RFLP) markers in agricultural crop improvement was described, including theoretical issues related to marker-assisted backcrossing (MABC) to improve quality triats (Beckmann, Soller, 1983).
Marker systems can be categorized into three groups: morphological, biochemical (storage proteins and isoenzymes) and genetic (DNA markers).The first two groups of markers, morphological and biochemical ones (markers based on the polymorphism of storage proteins and some enzymes), were widely used in genetic and breeding studies of agricultural crops until the 1990s.Lande and Thompson (1990) first conducted theoretical studies of MAS for quantitative traits, thus motivating other scientists to a number of modeling studies in the 1090s (Zhang, Smith, 1992, 1993;Gimelfarb, Lande, 1994a, 1994b, 1995;Hospital, Charcosset, 1997;Whittaker et al., 1997).In the early 2000s, additional theoretical discussions were held on the application of MAS and strategies for pyramiding the necessary alleles by recurrent crosses (Hospital et al., 2000;Frisch, Melchinger, 2001, 2005;Hospital, 2002;Servin et al., 2004;Bernardo et al., 2006).These studies have answered many key genetic questions about MAS systems, such as sample size, number and type of markers, population type, and genome size (Avise, 2004;Guimaraes et al., 2007).
In MAS, when one marker is used, the selection reliability increases from 95% to 99.5%, and when two flanking markers are used, it is 100% provided that this marker is located within the gene (Collard, Mackill, 2008).The markers to be used must be close to the target gene (<5 recombination units).This is mandatory to ensure that only a small percentage of specimens will be recombinant.Typically, two markers are used rather than one, to reduce the probability of an error caused by homologous combination.That is, the first step in MAS is to map the gene(s) or quantitative trait locus(loci) (QTL(s)) of interest by different techniques.The recombination frequency between the target locus and the first marker is approximately 5%.Thus, the recombination between the target locus and the marker can occur in approximately 5% of the offspring.The recombination probability between marker 1 and marker 2 (i.e.double crossingover) is much lower than for one marker (about 0.4%).Thus, the selection reliability is much higher when one use flanking markers (Kozhukhova, 2011).
MAS in breeding is used for almost all major crops in four broad directions, namely: -Traits which are difficult to manage through traditional phenotypic selection because of significant resource costs or complex heredity; -Traits, the expression of which depends on the specific environmental conditions or on stages of development, which affects the target phenotype; -To accelerate backcross breeding and maintain recessive alleles in this breeding; -To pyramide several QTLs for one target trait with complex heredity (drought resistance or other adaptive traits) or several monogenic traits (qualitative traits and resistance to diseases and pests) (Xu, Crouch, 2008).
If one combines simultaneous selections for large number of target traits in traditional breeding, it will lead to a general weakening of the resulting material and increase the number of selection cycles to obtain final accessions.On the other hand, MAS ensures fewer selections and fewer losses when breeders build up several target traits in the same genotype.It should be noted that the improvement of complex traits, such as resistance to diseases and pests, is complicated by large numbers of additional genes with unpredictable epistatic effects, impacts of different environmental factors and weak heredity (Kozhukhova, 2011).Modern methods based on the achievements of DNA technologies enable searching for resistance genes in starting material more accurately and quickly.At present, in the breeding practice, it is DNA markers that are successfully used: short fragments of DNA that are closely linked with a gene that is responsible for a particular trait or directly characterizes the target gene.MAS, which is based on such markers, is suitable in the selection for different agronomic traits, including resistance to pathogens and pests.The main advantage of DNA markers is that it is possible to study almost any part of the DNA molecule, while there is no need for repeated reseeding of breeding material on infection backgrounds, which significantly shortens the time of creation of resistant varieties and hybrids.
Of molecular methods used in marker-assisted plant breeding, polymerase chain reaction (PCR), which is widely applied to identify most DNA markers, is the most effective tool of DNA analysis.PCR was invented by American biochemist Kary Mullis, and he won the 1993 Nobel Prize in chemistry for this invention (Korovaeva, Popova, 2015).
The PCR method is based on the identification of specific DNA (RNA) fragments in the test material, their selective synthesis to a concentration at which they are easy detected and subsequent determination of amplicons (amplification reaction products) (Fedorenko et al., 2007).Except for RNA viruses, DNA is a unique carrier of genetic information in all organisms on Earth (Glazko, 2003).
DNA has a unique property -the ability to double after unraveling the helix and the separation of DNA strands (replication).DNA replication is catalyzed by enzymes called DNA polymerases using the complementarity principle.To start replication, this enzyme needs an initial double-stranded DNA fragment.Such a fragment is formed when a short single-stranded DNA fragment (primer) interacts with a complementary region of the corresponding parental strand of DNA.Two strands of DNA are replicated, but they grow in opposite directions.As a result, two double-stranded molecules are synthesized from one double-stranded DNA molecule, each of which contains one strand from the parental molecule of DNA and the other daughter, newly synthesized strand (Oleshchuk et al., 2014).
Each DNA replication cycle includes three main stages: -The DNA helix is unraveled and the double-stranded DNA template is separated into single strands (denaturation); -Primers anneal, or bind, to the DNA template; -A daughter strand of DNA is synthesized.
In the PCR machine, these processes are cycled in vitro.The transition from one stage of the reaction to another is achieved by changing the temperature of the incubated mixture (Lopukhov, Eldeinshtein, 2000;Fedorenko et al., 2007;Kutyrev et al., 2010).To perform PCR analysis, it is necessary to prepare a sample of biomaterial (DNA or RNA extraction), complete PCR (amplification) and to detect the PCR products (amplified nucleic acid) (Romanenko et al., 1998).The requirements for PCR laboratories are formulated and summarized in corresponding regulations and guidelines (Edwards et al., 2004;Kutyrev et al., 2010;Stehnii et al., 2010;Kalachniuk et al., 2012).
Polymerase chain reaction allows one to selectively synthesize certain (target) DNA sites of several hundred to thousands of nucleotide pairs (usually not longer than 2 kilobases (kb)), using any DNA sample as a template, including a sample of degraded DNA.Today, the PCR principles remain unchanged despite numerous modifications.They consist in DNA amplification via synthesis of complementary strands catalyzed by DNA polymerase.To start replication, this enzyme needs two artificially synthesized single-stranded oligonucleotide primers.Primers are normally between 18 and 30 nucleotides in length.The primers are oriented in opposite directions with their 3' ends pointing towards each other.so that the elongation reaction proceeds from 5′ to 3′ across the region between the two primers, i.e. the distance between the primers determines the length of DNA fragments amplified during PCR.As a result of amplification, new DNA fragments of a certain size are synthesized (they appear after cycle 2).
Polymerase chain reaction is a cyclic process and usually consists of 30-40 cycles.Starting from cycle 2, the newly synthesized DNA molecules serve as templates for further synthesis of the target DNA region.Therefore, PCR will lead to an exponential increase in the number of copies of the DNA region of interest, which was flanked on both sides by primers.The number of amplicons can be approximately estimated by formula 2", where n is the number of cycles.Correspondingly, the target DNA region can be 2 20 -fold amplified in 20 cycles (Patrushev, 2004).
Polymerase chain reaction has been widely used in medicine, veterinary science, biology, criminology, history, archeology and other branches of human activities.This method is supersensitive and specific in diagnostics of infections.Thanks to PCR, a lot of modern scientific challenges are successfully solved; organisms are genotyped; genetic diseases are diagnosed and liability to them is evaluated.PCR can accurately test family relations, identify individuals, analyze ancient remains and expose GMOs (Rybicki, 2005;Mahony, 2008).
To date, a lot of modifications of classical PCR analysis have been developed, among which the following types of PCR can be distinguished: Real-time PCR.This approach is able to determine the number of DNA copies or mRNA in the sample under study.Today, real-time PCR is the most common method used in different sectors.This method is based on the quantitative determination of the PCR product content in the reaction mixture in each cycle of the reaction.Fluorescent-labeled oligonucleotides are used to quantify the PCR products.When performing real-time PCR, one should compare the obtained graphs of fluorescence accumulation between several samples, for example, between a standard sample and a test sample.
Real-time PCR is now widely used in medicine and plant production to determine viral load in living organisms, to elucidate transcript levels, to assess mononucleotide polymorphism and to quantitatively determine the content of a foreign DNA molecule in organisms and food (the presence of genetically modified sources).
Multiplex PCR.Several primers specific for different genetic loci are added to the reaction mixture simultaneously.
Polymerase chain reaction with reverse transcription (RT-PCR).cDNA is synthesized on the RNA template by reverse transcriptase, and the resulting DNA sequence is used for classical PCR.RT-PCR is used for the following purposes: -To study the differential expression of genes at the transcription level; -In DNA diagnostics of infectios.PCR in situ.This technique is designed to amplify DNA or RNA sequences directly on fixed slides of tissues, cells or chromosomes.
Allele-specific PCR.It is used to detect mutations in genomic DNA with allele-specific primers, which are complementary to the mutant sequences, while the wild type (norm) is not amplified (Patrushev, 2004).
Different molecular methods are used to detect DNA markers of the trait under investigation.Gupta et al. (2008) grouped DNA markers according to the detection technique as follows: 1) DNA markers based on restriction fragment length polymorphism (RFLP).RFLP markers are detected after treatment of genomic DNA with restriction endonucleases.
2) DNA markers, which are detected by various types of PCR analysis.These include the following types of markers: -RAPD (random amplified polymorphic DNA) -randomly amplified polymorphic DNA; RAPD markers include DNA sequences obtained through amplification with arbitrary primers (9-11 bp); -ISSR (inter simple sequence repeats) -intermicrosatellite sequences; during amplification, a DNA fragment located between intermicrosatellite loci is replicated; -AFLP (amplified fragment length polymorphism) -DNA regions are identified by treating the DNA with two restriction enzymes, ligating adapters to the ends of the target nucleic acid and further amplifying with primers that are complementary to the adapter nucleotides; -SSR (simple sequence repeat) -simple repeating sequences or microsatellites; these are tandem repeats of 2 -6 bp in length, for example, (A) n, (AT) n, (GA) n, where n varies between 10 and 80 bp; -EST (expressed sequences tags) -expressed DNA sequences, anonymous sequences, or sequences of unknown function, obtained from sequencing cDNA libraries; -SCAR (sequence characterized amplified region) -a sequence that characterizes the amplification region; first, the RAPD fragment is excised from the gel, cloned and sequenced, and then specific primers are designed for this site with a length of 14-20 bp; -CAPS (cleaved amplified polymorphic sequence) -amplification polymorphic sequences that are cleaved; amplification products are treated with endonucleases; -IRAP (inter-retransposon amplified polymorphism) -amplification polymorphism of interretrotransposon sequences; amplification occurs between primers that are complementary to the sequence of two adjacent LTR regions of the retrotransposon; -STS (sequence tagged site) -a sequence that characterizes the locus.A fragment of genomic DNA obtained by amplification with primers that are specific to the primary structure of a known locus.
3) DNA markers, which are detected by sequencing and using DNA chips.Single nucleotide polymorphism (SNP).SNPs are sites in the genome at which more than one nucleotide is found in a population, and the frequency of the rare allele should be at least 1% (Malyshev, Kartel, 1997;Lesk, 2009).
As it has been mentioned above, MAS is applied to almost all major crops.Below, we describe some molecular markers of resistance genes to biotic factors exemplified by one of the most common oil crops in the world -sunflower.
Downy mildew (Plasmopara helianthi Novot.).Breeding for resistance to downy mildew is a difficult challenge due to the large number of pathogen races and their significant variability.To date, 20 genes (Pl1 -Pl20) are known to determine race-specific resistance to downy mildew.These genes were found in different accessions, and Pl alleles are dominant.Pl1 and Pl2 are the most common genes, which are present in almost all breeding specimens of sunflower.There are published data on marking some of them, which helps to significantly accelerate the selection of valuable genotypes.In search for markers of these genes, scientists extensively use 13 lines -differentiators, which are included in the international standard for the identification of the downy mildew pathogen (Table 1).,300,304,330,334,700,710,714,730,733,Pl13 100,300,304,330,334,700,710,714,730,734,Pl13 100,300,304,330,334,700,710,714,730,734,Pl13 100,300,304,330,334,700,710,714,730,734, 100,300,304,330,334,700,710,714,730,734,Pl2,Pl6 100,300,330,700,710,730,733, PlARG Universally resistant To date, the following genes of resistance to downy mildew are marked: Pl1, Pl2, Pl5-8, Pl13-14, Pl16-20, and PlARG.Carriers of the PlARG gene, which determines the universal resistance against all known races of Plasmopara helianthi, are the most valuable sources of resistance in further sunflower breeding for resistance to downy mildew (Jocić et al., 2010).The PlARG gene was mapped with SSR markers in linkage group (LG) 1 of the sunflower genetic map (Duble et al., 2004), and it was shown to be closely linked with microsatellites ORS716, ORS509, ORS1128, and ORS543 (Wieckhorst et al., 2010).
Note: R = resistance; S = susceptibility.Search for carriers of the Pl6 resistance gene, which determines resistance to 11 races, including races 710, 730 and 330, is no less important in sunflower breeding for resistance to downy mildew (Ramazanova, Antonova, 2018).Table 2 summarizes the characteristics of 13 STS-markers within this locus and three primers that were developed by Bouzidi et al. (2002) and are used by researchers from different countries to identify the Pl6 locus in sunflower genotypes.The nucleotide sequences of these primers are presented in Table 3.
The introgression of the Pl8 locus into breeding accessions is also promising, since this locus is in the same LG with the Pl5 locus, so one can use them in the crop breeding to create universally resistant sunflower lines and hybrids, i.e. with resistance to the most common races.To flank the Pl5 and Pl8 loci, researchers used six primers (Bouzidi et al., 2002), the characteristics of which are summarized in Table 4, and their nucleotide sequences are presented in Table 5.
To successfully breed sunflower for resistance to downy mildew, one should analyze not only plants, but also the pathogen.Hence nowadays, PCR methods are used to determine the molecular genetic variability of P. helianthi.This approach allows elucidating the structure of the pathogen population and the rate of its variability, which significantly accelerates the breeding work to create resistant starting material (Radwan et al., 2008).
The fact that sunflower rust resistance genes quickly lose their effectiveness due to the emergence of new virulent races in a short period of time after the introduction of resistant varieties and hybrids into production poses a serious problem.Therefore, it is important to search for new genes of rust resistance and for molecular markers that can identify these genes as well as to pyramide several resistance genes in one genotype.
Today, R5 is the only sunflower rust resistance gene in LG 2. Qi et al. (2012bQi et al. ( , 2015a) ) identified two SSR-and two SNP-markers that flank the gene (SFW03654, ORS653a, NSA_000267, ORS1197-2).LG 11 contains two rust resistance genes: R12 and R14.The two genes were aligned with markers ORS1227 and ZVG53.Talukder et al. (2014) used five SNP-markers (NSA_000064, NSA_004155, NSA_003426, NSA_008884, NSA_003320) to identify the R12 gene, but only two of these markers (NSA_003426 and NSA004155) were effective in identifying the R12 gene.Qi et al. (2012b) also used previously developed SSR-and SNP-markers to identify homozygous multi-race-resistant genotypes in a population of F2 hybrids derived from crossing BC3F2 accessioncarrier of the R5 gene and HA-R6 accession -carrier of the R13a gene.The offspring obtained from plants selected from this hybrid population were more resistant to races 336 and 777 compared to lines with only one resistance gene.The researchers also pyramidized the R5 and R13a genes in confectionery sunflower using SSR-and SNP-markers (Qi et al. 2015a).They revealed that pyramidation of the R genes could ensure long-term resistance to the causative agent of sunflower rust.Thus, the creation of sunflower genotypes combining several rust resistance genes is a very important objective in and the breeding process can be significantly facilitated and accelerated by using the molecular markers described above.
Broomrape (Orobanche cumana Wallr.).Broomrape (a plant -parasite)-caused damage significantly reduces sunflower yields.The most reliable way to control this parasite is to create resistant varieties and hybrids with prior studies of the inheritance of resistance to broomrape.The genetics of this trait is studied by Ukrainian and foreign scientists.Sunflower has been bred for resistance to broomrape for almost a century (Shindrova, 2006).Currently, 8 broomrape races with different virulence are known.They are denoted by the Latin letters as A, L, B, C, D, E, F, G, H (Melero-Vara et al., 1989;Alonso et al., 1996;Akhtouch et al., 2002;Shindrova, 2006;Pacureanu et al., 2009;Antonova et al., 2011).Until recently, the first five physiological races of broomrape were spread in all regions of sunflower cultivation; resistance to them is determined by individual Or genes (Sukno, 1999).Studies have confirmed that resistance to races A to E is determined by the genes Or1 to Or5, which are allelic or strongly linked, and resistance to race E is controlled by a single dominant gene, Or5 (Lu et al., 2000;Fernández-Martínez et al., 2000;Fernández-Martínez et al., 2008).
Most molecular analyses were performed to study and create different types of molecular markers to identify the Or5 gene, which determines resistance to races E and below (Tang et al., 2003;Fernández-Martínez et al., 2004;Guchetl et al., 2012).Foreign scientists (Tang et al., 2003) identified some DNA markers in the same linkage group with Or5.The nearest SCAR-marker is mapped at a distance of 5.6 cM from the distal end of Or5 (Table 7).LG 13 Оrab-vl-8 AB-VL-8 ORS683 ORS657 1.5 4.7 Imerovski et all., 2016Tang et al. (2003) identified SSR-markers closely linked to Or5 and mapped this locus in the upper part of LG 3 in the genetic map of SSR loci.The nearest SSR-markers are at distances of 6.2 cM (CRT392) and 7.5 cM (ORS1036) from the Or5 locus.
Search for donors of resistance to highly virulent broomrape race F is urgent today.This race originated at the end of the last century in Spain.Imerovski I. et al. (Imerovski et al., 2016) used line AB-VL-8, resistant to this broomrape race, to map markers of a resistance gene, which was named Оrab-vl-8 by the researchers.Оrab-vl-8 was established to be in LG 13.In their molecular studies, the researchers found the nearest SSR-markers, which are at distances of 1.5 cM (ORS683) and 4.7 cM (ORS652) from the locus.
For traits with weak heredity, typical breeding programs involve the cultivation of millions of individual plants in thousands of populations to achieve greater homozygosity in lines, which occurs in approximately F5-F6 generations.This process requires significant resource costs and a significant time of 5-12 years.Due to the rapid development of agriculture and life in general, breeding programs with their scale, complexity of selection, numbers and sizes of populations, etc., require the latest approaches, which certainly include MAS.

Conclusions
Thus, MAS has been theoretically justified in numerous scientific publications and is implemented in most breeding institutions around the world.This trend in breeding opens new opportunities for studying genetic diversity and relations at the species and genus levels.Molecular marker-based selection is necessary in modern plant breeding, including in sunflower breeding for resistance to biotic factors.
The achievements of scientists in marking genes of resistance to the pathogen of downy mildew and sunflower rust as well as to the plant -parasite, broomrape, opens opportunities of identifying reliable sources of resistance and involving them in breeding programs in order to create valuable starting material.The emergence of new virulent races of these diseases and broomrape forces researchers to carry out deeper studies in finding new resistance genes and identifying molecular markers for these genes.
The effectiveness of molecular markers in sunflower breeding for resistance to diseases and parasites is confirmed by the results of scientific studies.Today, the search for donors of resistance to highly virulent races, which will significantly accelerate the selection of valuable genotypes in breeding for resistance, is urgent.