Identification of Wheat Septoria tritici Resistance Genes in Wheat Germplasm Using Molecular Markers

Zymoseptoria tritici (Z. tritici) is the main threat to global food security; it is a fungal disease that presents one of the most serious threats to wheat crops, causing severe yield losses worldwide, including in Kazakhstan. The pathogen leads to crop losses reaching from 15 to 50%. The objectives of this study were to (1) evaluate a wheat collection for Z. tritici resistance during the adult plant and seedling growth stages, (2) identify the sources of resistance genes that provide resistance to Z. tritici using molecular markers linked to Stb genes, and (3) identify potentially useful resistant wheat genotypes among cultivars and advanced breeding lines. This study evaluated 60 winter and spring wheat genotypes for Z. tritici resistance. According to the field reactions, 22 entries (35.7%) showed ≤10% disease severity in both years. The resistant reaction to a mix of Z. tritici isolates in the seedling stage was associated with adult plant resistance to disease in four wheat entries. The resistance of Rosinka 3 was due to the presence of Stb8; Omskaya 18 showed an immune reaction in the field and a moderately susceptible reaction in the seedling stage, possibly provided by a combination of the Stb7 and Stb2 genes. The high resistance in both the adult and seedling stages of Omskaya 29 and KR11-03 was due to the Stb4 and Stb2 genes and, possibly, due to the presence of unknown genes. A linked marker analysis revealed the presence of several Stb genes. The proportion of wheat entries with Stb genes was quite high at twenty-seven of the genotypes tested (45.0%), including four from Kazakhstan, nine from Russia, nine from the CIMMYT-ICARDA-IWWIP program, and five from the CIMMYT-SEPTMON nursery. Among the sixty entries, ten (16.7%) carried the resistance genes Stb2 and Stb8, and the gene Stb4 was found in seven cultivars (11.6%). Marker-assisted selection can be efficiently applied to develop wheat cultivars with effective Stb gene combinations that would directly assist in developing durable resistance in Kazakhstan. Resistant genotypes could also be used as improved parents in crossing programs to develop new wheat cultivars.


Introduction
Winter wheat (Triticum aestivum L.) is an important crop in the region of Central and West Asia (CWA), stretching from Kazakhstan and Afghanistan to Iran and Turkey.The crop is grown under both irrigated and rainfed conditions on ~13 Mha, and its average grain yield is ~2.5 t ha −1 , which is far below the potential of 4-5 t ha −1 [1].There are several reasons for poor wheat grain yield in the region, including changes in cultural practices including shifts from conventional tillage and stubble burning to reduced tillage practices, breaking of the technology of fertilizing, violation of or non-compliance with crop rotation, and shallow tillage and wheat monoculture involving the cultivation of susceptible cultivars.However, diseases and pests also play an important role in yield reduction [2].
Kazakhstan plays an important role in regional and global food security since most of the grain produced is sold in these regions.According to experts from the Food and Agriculture Organization of the United Nations (FAO), the world's population will double by 2050.Ensuring food security is the most important priority of the economic strategy of Kazakhstan, as worsening the situation can deform the process of political and economic reforms and become a threat to the internal security of the state.World grain production has been increasing in recent years; at the same time, wheat losses from diseases account for about 10% of the potential crop around the world [3].
The widespread introduction of zero and minimal wheat cultivation technologies contributes to the development and harmfulness of leaf spot diseases (LSDs), the infection of which remains on crop residues.In the period 2000-2015, epiphytotic developments of leaf rust separately or together with Septoria occurred eight times in Kazakhstan [4].Zymoseptoria tritici epidemics in the North of Kazakhstan occur five times every 10 years.According to the monitoring of Z. tritici in the Akmola region, there is a trend of increased development and severity of the disease, and, in recent years, the morbidity rate has reached a critical level.The disease manifested itself even during the years of hard drought (2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010).A strong development of Z. tritici was observed in 2013, 2014, and 2016 [5].In Kazakhstan, five species of Septoria fungi were registered on wheat; the dominant species among them are Parastagonospora nodorum (Berk.)Quaedvl., Verkley et Crous and Zymoseptoria tritici (Desm.)Quaedvl.et Crous [4].Septoria tritici blotch (STB) caused by the ascomycete fungus Zimoseptoria tritici is the major devasting foliar disease that causes significant yield loss in the wheat-growing regions of Kazakhstan.The life cycle of Zymoseptoria tritici in Kazakhstan is the same as in other regions.The life cycle of Z. tritici is broadly divided into two distinct stages, namely the symptomless latent phase and the necrotrophic stage.The latent phase can be further subdivided into three stages: transition, ingress, and colonization [4].Yield losses of grain crops due to these two diseases amount to 9 million tons worldwide [6].The fungus P. nodorum was discovered in 1960 in the Akmola region of Kazakhstan on soft wheat; since then, the disease has been observed in all regions of the country.Septoria tritici blotch and Stagonospora nodorum blotch are a major threat to global food security [7].The average loss from pathogens can reach from 15 to 50% [4].Leaf blotch diseases are a problem in wheat production in the Pacific Northwest and Northern Great Plains of the USA, Europe, and Central and West Asia [6][7][8][9][10][11][12].Apart from leaf blotch diseases, leaf rust (Puccinia triticina Erikss.)[2,13,14], yellow rust (Puccinia striiformis Westend.)[15][16][17], and stem rust (Puccinia graminis f. tritici Erikss.& Henning) [18,19] are also widespread in Kazakhstan.
The disease reduces the assimilation surface of the leaves, causes underdevelopment of the ear, and significantly reduces the yield and quality of the grain [1].To prevent losses, crops are treated with fungicides, which require additional costs.Comprehensive disease control strategies (cultivating resistant varieties, crop rotation) are the most effective, environmentally friendly, and economical means to combat wheat Septoria.Hence, identifying the sources of STB genes with minor-to-moderate effects will be used as an effective way to determine durable and resistant wheat varieties for STB.However, traditional breeding methods are not always effective, as the development of resistant varieties is a laborious and lengthy process.In this regard, the use of the marker-assisted selection (MAS) approach accelerates the cultivation of new cultivars.MAS breeding and the identification of target genes associated with disease resistance make the process of developing and introducing wheat varieties more accurate and reliable.
The objectives of this study were to (1) evaluate a wheat collection for Zymoseptoria tritici resistance during the adult plant and seedling growth stages, (2) identify the sources of resistance genes that provide resistance to Z. tritici using molecular markers linked to Stb genes, and (3) identify potentially useful resistant wheat genotypes among cultivars and advanced breeding lines.

Reaction of the Wheat Collection to Zymoseptoria tritici
Analysis of variance (ANOVA) revealed highly significant effects for wheat genotypes on resistance to Zymoseptoria tritici in both field and laboratory tests (Table 1).A total of 60 cultivars and breeding lines were analyzed over 2 years for field trials and 3 replicates for laboratory screening of seedling resistance.The resistance score (AUDPC, field/Z.tritici, seedling) to Z. tritici in ANOVA was taken as a variable; the year of field testing (n = 2, for APR) or replication (n = 5, for ASR) and genotype (n = 60) were taken as factors.There was significant variation among genotypes and disease severity (p < 0.001) in both growing seasons of field trials (p < 0.001).The ANOVA results suggest that genotype is the predominant source of variation in resistance to Z. tritici in the collection, explaining 94.44% of the total variance in field trials and 74.24% of the variance in seedling resistance.All genotypes exhibited a high broad-sense heritability (0.94 and 0.74) for all the traits, indicating that resistance to Z. tritici Septoria can be improved by breeding (Table 1).
The resistance score with the AUDPC values for 2020 and 2021 for each genotype is presented in Figure 1.This clarifies whether the quantification is consistent across years.The Pearson correlation analysis revealed a significant positive correlation between the two years of field trials (r = 0.94; p < 0.001).No correlation was found between the resistance of adult plants and seedlings.
The resistance score with the AUDPC values for 2020 and 2021 for each genotype is presented in Figure 1.This clarifies whether the quantification is consistent across years.The Pearson correlation analysis revealed a significant positive correlation between the two years of field trials (r = 0.94; p < 0.001).No correlation was found between the resistance of adult plants and seedlings.The genetic parameters of 60 winter wheat genotypes are presented in Table 2. Wide variability exists for Z. tritici resistance in the wheat collection for field and seedling resistance as evidenced by the presence of highly significant genotypic variance (Table 2).The CV ranged from 30.3% (Z.tritici, seedling) to 80% (AUDPC in 2020).The distribution of accessions concerning Z. tritici infection types in the adult plant stage (a) and seedling stage (b) with an indication of the standard error is presented in Figure 2. Septoria development varied greatly among the wheat entries.Most of the entries showed higher levels of resistance in the adult stage under field conditions than as seedlings (Table 3, Figure 2).According to the field reactions in 2020, the majority of the studied genotypes (86.6%) had highly resistant (RR) and resistant (R) reactions to Z. tritici; in 2021, 49 wheat entries (81.6%) had RR and R reactions, indicating that these wheat entries are promising sources for adult plant resistance (Figure 2).Among the 60 genotypes, 8 (13%) and 11 (18.3%)wheat entries were susceptible in 2020 and 2021, respectively.The most susceptible cultivar over the two years was wheat cultivar Egemen, with AUDPC values of 507.5 and 595 in 2020 and 2021, respectively (Table 3).Twenty-two genotypes (Severyanka, Rosinka The genetic parameters of 60 winter wheat genotypes are presented in Table 2. Wide variability exists for Z. tritici resistance in the wheat collection for field and seedling resistance as evidenced by the presence of highly significant genotypic variance (Table 2).The CV ranged from 30.3% (Z.tritici, seedling) to 80% (AUDPC in 2020).The distribution of accessions concerning Z. tritici infection types in the adult plant stage (a) and seedling stage (b) with an indication of the standard error is presented in Figure 2. Septoria development varied greatly among the wheat entries.Most of the entries showed higher levels of resistance in the adult stage under field conditions than as seedlings (Table 3, Figure 2).According to the field reactions in 2020, the majority of the studied genotypes (86.6%) had highly resistant (RR) and resistant (R) reactions to Z. tritici; in 2021, 49 wheat entries (81.6%) had RR and R reactions, indicating that these wheat entries are promising sources for adult plant resistance (Figure 2).Among the 60 genotypes, 8 (13%) and 11 (18.3%)wheat entries were susceptible in 2020 and 2021, respectively.The most susceptible cultivar over the two years was wheat cultivar Egemen, with AUDPC values of 507.5 and 595 in 2020 and 2021, respectively (Table 3).Twentytwo genotypes (Severyanka, Rosinka 3, Omskaya 18, Omskaya 28, Omskaya 29, Omskaya 35, Omskaya 36, Mironovskaya 808, Pamyati Azieva, KR11-40, KR11-03, NANJTNG82149/KAUZ, CROC 1AE.SQUARROSA, GAN/AE.437.SQUARROSA, TRAP#1/BOW, EFED/F5.83 7792(BAJAS), Batyr, Bayandy, Samgay, and Sapaly) showed ≤10% disease severity in both years and were considered as resistant in the adult plant stage under field conditions.CROC 1AE.SQUARROSA, GAN/AE.437.SQUARROSA, TRAP#1/BOW, EFED/F5.83 7792(BAJAS), Batyr, Bayandy, Samgay, and Sapaly) showed ≤10% disease severity in both years and were considered as resistant in the adult plant stage under field conditions.The seedling reactions of 60 wheat genotypes to the mix of Z. tritici isolates differed greatly.The wheat genotypes showed arrays of patterns in their responses to Z. tritici (Table 3).Most wheat cultivars and breeding lines were generally moderately susceptible (53.3%) and susceptible (16.6%) to Z. tritici.Of the 60 wheat entries, 18 had average disease reaction scores of less than two as seedlings, and 5 of these had reaction scores equal to one, indicating that these wheat entries were resistant.Eighteen wheat entries (Saratovskaya 42, Saratovskaya 55, Omskaya 29, Omskaya 35, KR11-9025, KR12-07, KR11-03, KR11-9014, P83-5112/V82274, NANJTNG82149/KAUZ, CROC_1/AE.SQUARROSA (205)//BORL95/3/2/*Milan, JAC161/TEMU51.80,BR14/CEP847-2, ECHA/LI115, Celinnaya 50, Manshyk, Sapaly, and Steklovidnaya 24) were resistant to Z. tritici.Reactions to Z. tritici isolates were associated with adult plant resistance to Septoria in four wheat entries, including Rosinka 3, Omskaya 18, Omskaya 29, and KR11-03.These genotypes were highly resistant both under greenhouse and field conditions.
SSR marker WMS44 was used to screen the 60 wheat entries for Stb5 gene detection.The expected size of the fragment amplification for locus WMS44 coupled to the resistant allele of the Stb4 gene was 182 bp.Out of 60 genotypes tested for Stb5, the expected PCR product was not amplified in any wheat entry.
An analysis of the general distribution of dependent variables of various wheat samples characterized by the presence and absence of Z. trititci resistance genes Stb2, Stb4, Stb7, and Stb8 was conducted (Supplementary Figures S1-S4).For this purpose, the effectiveness of carriers and non-carriers of Stb2, Stb4, Stb7, and Stb8 genes for the Z. trititci score was compared using a non-parametric analog of the analysis of variance and the Kruskal-Wallis test.The wheat samples were grouped into two categories: (1) those with one Stb gene and (2) those without the Stb gene.In the Stb2 groups, no significant difference (p > 0.05) was observed in either the seedling or adult plant stages (Supplementary Figure S1).The analysis revealed that there were no statistically significant differences between the carrier and non-carrier groups of other genes (Stb4, Stb7, and Stb8).This is The presence of the Stb4 gene in wheat genotypes was studied using the SSR marker WMS111.Of the 60 cultivars/lines identified to carry this resistance gene in our study, 7 genotypes (11.6%) amplified 210/220 bp fragments, indicating the presence of the Stb4 resistance gene.These wheat entries include Albidum 31, Omskaya 29, KR11-13, KR12-18, KR11-26, TRAP#1/BOW, and EFED/F5.83-7792(BAJAS)(Table 3).There is no information confirming variations in the Stb4 gene.Adhikari et al., 2004, showed that the Stb4 locus from the wheat cv.Tadinia showed resistance to Z. tritici in both the seedling and adult plant stages [30].Similar results were obtained in our studies.Based on the molecular screening results, all of the seven genotypes containing Stb4 genes showed fairly good to moderate resistance responses during field testing.The Stb4 gene had a large effect on Z. tritici, which provided a high level of field resistance in both years, with a final disease score of 5-10%.These genotypes, also exhibited seedling resistance values from moderately susceptible to susceptible.
SSR marker WMS44 was used to screen the 60 wheat entries for Stb5 gene detection.The expected size of the fragment amplification for locus WMS44 coupled to the resistant allele of the Stb4 gene was 182 bp.Out of 60 genotypes tested for Stb5, the expected PCR product was not amplified in any wheat entry.
An analysis of the general distribution of dependent variables of various wheat samples characterized by the presence and absence of Z. trititci resistance genes Stb2, Stb4, Stb7, and Stb8 was conducted (Supplementary Figures S1-S4).For this purpose, the effectiveness of carriers and non-carriers of Stb2, Stb4, Stb7, and Stb8 genes for the Z. trititci score was compared using a non-parametric analog of the analysis of variance and the Kruskal-Wallis test.The wheat samples were grouped into two categories: (1) those with one Stb gene and (2) those without the Stb gene.In the Stb2 groups, no significant difference (p > 0.05) was observed in either the seedling or adult plant stages (Supplementary Figure S1).The analysis revealed that there were no statistically significant differences between the carrier and non-carrier groups of other genes (Stb4, Stb7, and Stb8).This is indicated by high p values (p > 0.05) for both seedling and adult plants (Supplementary Figures S2-S4).Among the studied genes, the Stb8 gene demonstrated the greatest influence on resistance to Z. trititci, as statistical analysis showed that the lowest p value (0.19) was observed between carriers and non-carriers of the Stb8 gene (Supplementary Figure S4).Hence, the presence of this gene in wheat genotypes significantly enhanced resistance to Z. trititci.
However, the significant impact of the year factor on resistance was also observed (p < 0.05).Supplementary Figures S5-S10 illustrate the level of Z. trititci development in various wheat groups under field conditions across two years (2020 and 2021).The average disease development level in 2020 ranged from 11.4% to 11.8%, whereas in 2021, this figure increased from 15.7% to 16.6% (Supplementary Figures S5-S10).
To clarify the effects of Stb genes on resistance to Z. tritici, we compared the field resistance of positive wheat accessions that contained Stb genes with the resistance of negative wheat cultivars that lacked Stb genes.Statistical parameters were assessed, comparing datasets of a single variable, which consists of two multiple independent levels: (1) Stbpositive (27 wheat genotypes carrying Stb genes, 162 measurements) and (2) Stb-negative (9 genotypes lacking Stb genes, 55 measurements).The general variable showed that the population distribution between the groups falls within the following range: Min.-0.0; 1st Qu and Median-10; Mean-18.7;3rd Qu.-25.0;Max.-65.0 (Supplementary Figure S11).The average severity of Z. tritici in 2020 and 2021 was 32.21% and 37.21%, respectively (Figure 3).The average Z. tritici severity among wheat genotypes in the positive group (Stb gene carriers) was 16.25%, while in the negative group (lack of Stb genes), it was 20.25%.A statistically significant difference was observed between the evaluated levels of the positive and negative genotype groups (p value < 0.01).In the positive genotype group, 19% of plants exhibited immunity to the disease (0% severity).The majority of plants in this group (75%) demonstrated resistance to Z. tritici.The remaining 25% of plants were susceptible to Z. tritici.In the negative genotype group, no wheat varieties exhibited immunity; 29% of plants showed moderate susceptibility, while 71% of plants were susceptible to Z. tritici (Supplementary Figure S11).Thus, a significant influence of genetic background (positive and negative samples concerning the presence of Stb genes) on Z. tritici severity in the adult plant stage (field studies) was detected (p value < 0.01) (Supplementary Figure S11).
Similar calculations were conducted to obtain data confirming that Stb genes influence resistance in the seedling stage.However, no influence of the genetic background factor (positive and negative samples concerning the presence of Stb genes) on Z. tritici severity in the seedling stage was found (p value < 0.9).This suggests that the presence of Stb genes does not necessarily imply a causal relationship.Our statistical analyses demonstrated a significant influence of the presence of Stb genes on enhancing resistance only in the adult plant stage (under field conditions).
Thus, Stb genes have been identified in 27 varieties.Our analyses suggest that the correlation between the presence of Stb genes in these 27 varieties and resistance is supported by the provided field resistance data, but not supported by data on seedling resistance.

Discussion
The main agricultural crop, wheat, is affected by pathogens of leaf spot diseases (LSDs), including Septoria.Z. tritici is the main threat to global food security.Losses due to this disease can reach up to 50% in epidemic years and often vary between 5 and 20% depending on the environment and the cultivar of wheat [4].The species composition of pathogen populations of wheat Septoria spot in Northern Kazakhstan in 2018-2019 comprised three species of Septoria fungi: Zimoseptoria tritici, Septoria nodorum, and Septoria avenae f. sp.triticea.Soft spring wheat was mainly affected by Z. tritici.During the two-year study of species diversity of Septoria spot pathogens, Z. tritici was predominant, followed by S. nodorum and S. avenae f. sp.triticea [5].This study has significance for future wheat breeding in Kazakhstan.There is not a single variety in our country that is resistant to Z. tritici among the varieties approved for use [5].As a result of our study, identifying the sources of STB genes with minor-to-moderate effects will evolve as an effective way to have durable and resistant wheat varieties for STB.
The cultivation of resistant varieties is the most reliable, environmentally friendly, and cost-effective method of combating diseases.However, traditional breeding methods are not always effective, as the development of resistant varieties is a laborious and lengthy process.In this regard, the use of the marker-assisted selection (MAS) approach accelerates the development of new cultivars.Molecular markers are useful for identifying lines with multiple genes and for pyramiding multiple resistance genes, which is difficult and sometimes impossible to achieve using only phenotypic data [31].Diversity in Stb genes in commercial cultivars could play an important role in managing frequent leaf blotch disease epidemics in the region.In the present study, linked marker analysis revealed the presence of several genes.Five SSR markers, WMS389, WMS111, WMS44, WMC313, and WMS577, linked with Septoria resistance genes Stb2, Stb4, Stb5, Stb7, and Stb8 were used to confirm these markers in wheat genotypes.
In different previous studies, the sources of Stb resistance genes were identified in wheat breeding material [32][33][34][35][36][37][38][39][40].As a consequence of molecular genetic tagging of resistance genes in 36 varieties of wheat, it was determined that the majority of the analyzed samples carried resistance genes ineffective against Z. tritici in their genotype.The result of a study by Babkenova et al. (2017) showed moderately effective resistance of the Stb2 gene [32]; eight wheat entries with this gene were identified among the studied cultivars.Research conducted by Pakholkova et al., 2016 [33], showed that six of the eight known resistance genes (Stb1-Stb5, Stb7) had only partial functionality in natural populations of Z. tritici in Russia; the Stb6 gene was highly effective against five populations of Z. tritici, and the Stb8 gene was efficient in an absolute sense against all tested isolates.They also, as in the present study, report the usefulness of screening for the presence of Stb genes for the breeding of Septoria-resistant cultivars.
In this study, 60 wheat cultivars and breeding lines were tested with linked markers for some Stb genes.The proportion of wheat entries with Stb genes was quite high at twentyseven of the genotypes tested (45.0%), including four from Kazakhstan, nine from Russia, nine from the CIMMYT-ICARDA-IWWIP program, and five from the CIMMYT-SEPTMON nursery.Molecular screening of these genotypes showed contrasting differences in the gene's frequencies.Among the 60 entries, 10 (16.7%) carried resistance genes Stb2 and Stb8.Gene Stb4 was found in seven cultivars (6.7%), while Stb5 was not detected in any genotypes in this study.Omskaya 18 (Stb2 and Stb7), EFED/22150 and Egemen (Stb2 and Stb8), and EFED/F5.837792(BAJAS) (Stb4 and Stb7) had the highest number of resistance genes, with two-gene combinations.Genotypes Omskaya 18, EFED/22150, EFED/F5.83 7792(BAJAS), and Egemen were identified with a maximum of two Stb genes, followed by one Stb gene in twenty-four genotypes.The used SSR markers did not identify Stb genes in 32 genotypes.None of the five Stb genes were detected in a few resistant and moderately resistant genotypes (JAC161/TEMU51.80,NANJTNG82149/KAUZ, KR11-9025, KR12-07, P83-5112/V82274, Sapaly, and BR14/CEP847-2), suggesting that additional Stb genes confer resistance to Septoria in these genotypes.This demonstrates the diversity of Stb genes in the gene pool comprising cultivars and advanced breeding lines of wheat in the studied collection.
The wheat genotypes from Kazakhstan, Russia, and CIMMYT differed greatly in Septoria severity recorded in the adult plant stage in the field in Kazakhstan.This supports reports on varietal resistance and variation among Z. tritici populations in Kazakhstan and Russia [4,33,[41][42][43][44][45][46].Zeleneva and Konkova (2023a) evaluated the resistance of winter and spring wheat cultivars to detect resistance to Septoria blotch and studied the populations of Parastagonospora nodorum and P. pseudonodorum in the territory of the Saratov region of Russia to detect the presence of effector genes; eleven wheat genotypes were selected as resistant to different species of Septoria blotch (Zymoseptoria tritici, P. nodorum, P. pseudonodorum) [41].
It was found that the year significantly influences the development of Z. trititci in field conditions between the carrier and non-carrier groups of Stb2, Stb4, Stb7, and Stb8 genes.This indicates that the influence of genetic factors may vary depending on the conditions of the year.The average level of Z. trititci in 2021 was higher than in 2020 for both carriers and non-carriers of Stb resistance genes.This may suggest an increase in the infection rate in the latter year, regardless of the presence or absence of specific genetic variants.
The result of a recent study on screening the resistance of spring bread wheat cultivars and lines from Russia to Septoria leaf blotch allowed us to identify 23 accessions with high levels of resistance to Z. tritici [47].In our study, twenty-two genotypes (Severyanka, Rosinka 3, Omskaya 18, Omskaya 28, Omskaya 29, Omskaya 35, Omskaya 36, Mironovskaya 808, Pamyati Azieva, KR11-40, KR11-03, NANJTNG82149/KAUZ, CROC 1AE.SQUARROSA, GAN/AE.437.SQUARROSA, TRAP#1/BOW, EFED/F5.83 7792(BA-JAS), Batyr, Bayandy, Samgay, and Sapaly) showed ≤10% disease severity in both years.Resistant reactions to Z. tritici isolates in the seedling stage were associated with adult plant resistance to disease in four wheat entries, including Rosinka 3, Omskaya 18, Omskaya 29, and KR11-03.These genotypes were highly resistant under both greenhouse and field conditions.The resistance of Rosinka 3 was due to the presence of Stb8.Omskaya 18 showed an immune reaction in the field and an MS reaction to Z. tritici in the seedling stage, possibly provided by the combination of Stb7 and Stb2 genes.The high resistance in both the adult and seedling stages of Omskaya 29 and KR11-03 was due to Stb4 and Stb2 genes and, possibly, by the presence of unknown genes.The deployment of specific gene combinations provides durable and improved resistance versus using single genes because a single specific gene is subject to becoming susceptible due to genetic shifts in the pathogen [48].Given that the assessed entries comprised germplasm from Russia and CIMMYT, developed within breeding programs aimed at enhancing resistance to Septoria, it is probable that they possess diverse constitutions of resistance genes.Among these sources, twenty-two entries showed disease resistance, suggesting their potential value as sources of resistance, and can be used directly in breeding programs to improve the Septoria resistance of wheat.Marker-assisted selection can be efficiently applied to develop wheat cultivars with effective Stb gene combinations that would directly assist in developing durable resistance in Kazakhstan.Resistant genotypes could also be used as improved parents in cross-breeding programs to develop new varieties.

Plant Material
This study used 60 winter wheat genotypes comprising both cultivars and breeding lines.The collection included 15 cultivars and breeding lines from Kazakhstan, 15 cultivars from Russia, 15 lines released by the CIMMYT-ICARDA-IWWIP program, and 15 lines released by the CIMMYT-SEPTMON nursery (Table 3).The collection comprised several important wheat genotypes that have been widely used as parents in breeding programs across Kazakhstan and Central Asian countries.The highly susceptible control cultivar Morocco as well as the isogenic lines with known resistance genes (Veranopolis (Stb2), Tadinia (Stb4), CS (Synthetic 7D) (Stb5), Estanzuela Federal (Stb7), and Synthetic W7984 (Stb8)) were included as checks.

Field Evaluation of Wheat Entries against Zymoseptoria tritici
Evaluation of field resistance to Septoria tritici was carried out at the Kazakh Research Institute of Agriculture and Crop Production (KazNIIZiR), (Almalybak, 43 • 13 ′ 09 ′′ N, 76 • 36 ′ 17 ′′ E, Almaty region) in Southeast Kazakhstan, Almaty region, during the 2020 and 2021 cropping seasons.
Each entry was planted in a 1 m 2 plot in mid-September and was harvested in mid-August the following year for two years.Experiments were conducted with a completely randomized design with two replicates in 1 m 2 .The Septoria-susceptible cultivar Morocco was planted in every 10th row and as a spreader border around the nursery to ensure uniform infection.Fertilizer treatments of 60 and 30 kg/ha of N and P 2 O 5 , respectively, and other management practices corresponded to those normally recommended for the region [49].Annual rainfall ranged from 332 to 644 mm during the two years.The growing seasons were favorable for pathogen infection and disease development.The mean daily temperature and relative humidity showed similar trends in both years and conditions were highly conducive for Septoria infection and development.
The experiments on an artificial infectious background were made with naturally infected straw stubbles.Ten Flag-1 leaves were evaluated for each disease assessment of genotypes.The disease was assessed three times.In October, before sowing, the infected straw (1 kg/m 2 ) was incorporated into the soil.For the evaluation of field response, disease severities were assessed on first leaves and flag leaves when all wheat genotypes were near or at Zadoks growth stages Z69 (flowering) and Z75 (milk) [50].The percentage of Septoria-infected leaf area was determined on each leaf and the average value for all evaluated leaves was calculated for each wheat entry to determine the STB score.A rating system based on the % leaf area infected, developed for appraising the foliar intensity of diseases, was used to categorize host reactions to Septoria according to a double-digit scale (00-99) modified from Saari and Prescott [51].According to the degree of damage, the varieties were divided into the following groups: 0-10%-highly resistant (RR; free from infection or with a few isolated lesions on the lowest leaves only); 11-20%-resistant (R; scattered lesions on the second set of leaves with first leaves infected at light intensity); 21-40%-moderately susceptible (MS; moderate-to-severe infection of lower leaves with scattered-to-light infection extending to the leaf immediately below the mid-point of the plant); 41-70%-susceptible (S; severe lesions on lower and middle leaves; moderate to severe infection of upper third of plant; flag leaf infected in amounts more than a trace), 71-100%-highly susceptible (SS; severe infection on all leaves and the spike infected to some degree).
To test the virulence of these 5 isolates, wheat cultivars (durum wheat: Valentina cv; bread wheat: Al'mera and Astet cvs) were inoculated with Z. tritici isolates in a greenhouse environment.After 4 days, leaf segments from seedlings were cut and put on agar in Petri dishes at 20 • C for subsequent culture and monoconidial isolation, identification, and storage.Three weeks later, a high presence of pycnidia was observed.The Z. tritici isolates were pathogenic to both wheat species.The culture of Z. tritici was revitalized by transferring it to fresh PDA medium (potato dextrose agar, 39 g/L HiMedia, Mumbai, India) and incubated at 18 • C with a photoperiod of 12 h over 10 days.After incubation, sterile distilled water was added to each plate and spores were scraped gently with special glass rods.The spores were then transferred to a yeast-sucrose liquid medium (yeast extract 10 g/L; sucrose 10 g/L) and left shaking for 7 days at 18 • C, with permanent light.The spores were collected by centrifugation at 5000 rpm for 5 min at 15 • C, washed twice with sterile distilled water, and resuspended in a MgSO 4 solution (0.01 M) containing Tween 20 surfactant (0.1% v/v).The concentration was adjusted to 10 7 spores/mL [52].The dishes inoculated with Z. tritici pycnidia were placed in a thermostat at a temperature of 21 • C; no lighting was required.All Petri dishes were sealed with a double layer of paraffin.The cultures were monitored daily.On days 8-10, young colonies were transferred to a fresh nutrient medium.The fungi were cultivated for 30 days in a thermostat at a temperature of 21 • C. For inoculation, 30-day-old colonies were used or they were stored in a refrigerator at a temperature of +4 • C [53].
Soil preparation and inoculation were carried out according to generally accepted methods.The universal substrate was used ("Terra vita", manufactured by OOO "Nord Pflp", a limited liability company under the laws of the Russian Federation).Ten seeds of all 60 wheat accessions were raised in plastic containers with a capacity of 20 cm 3 .Ten seeds of each wheat variety were sown in a pot.All entries were arranged in a randomized complete design with three replications.So, 10 seedlings in a pot and 30 plants of the same variety were evaluated.Each container was considered as an experimental unit, and every single plant of a wheat variety in a container with three lots of ten seedlings served as an entry.The fungal isolates were mixed before inoculation.Thus, ten seedlings of each genotype were inoculated individually at the two-leaf stage against the mix of Z. tritici isolates.In each experiment, replications were treated as random effects, and the wheat accessions as fixed effects.All experiments were conducted in a greenhouse facility at the All-Russian Institute of Plant Protection (ARIPP, St Petersburg, Russia).The cultivar Morocco, susceptible to Z. tritici, was included as a check to monitor the development of infection.Spores produced by fungal colonies in pure culture were used as the inoculum.Inoculation was carried out with an aqueous suspension of spores.One day before inoculation, the viability of the existing inoculum was determined; then, a working suspension of spores was prepared [54].Cells were scraped from the colony surface using a sterile pipet tip and resuspended in a sterile 2 mL microcentrifuge tube containing 1 mL of sterile water.Dilutions (1:10) of the blastospore suspension were made in additional sterile 2 mL microcentrifuge tubes.Tubes were briefly vortexed between dilutions.
The hemocytometer and coverslip were carefully cleaned with 70% ethanol before use and the coverslip was positioned correctly (indicated by the appearance of Newton's rings).An appropriate dilution was selected; the blastospore suspension was briefly vortexed, and 10 µL was pipetted into each compartment of the hemocytometer.A compound microscope was used to observe cells.Cell density was calculated considering the average number of counted cells, the specifications of the hemocytometer in use, and the dilution of the cell suspension that was counted [54].The inoculum was adjusted to the required cell density (e.g., 10 6 or 10 7 cells/mL) in 0.1% Tween ® 20 in sterile water.The final inoculum was kept in a sterile 50 mL Falcon tube and used within a few hours.The suspension was evenly applied to the plants using a spray bottle, after which the cuvettes with flowerpots were placed in a climate chamber (model MLR-352H-PE, "PHCbi", Tokyo, Japan).
To inoculate the plants for each treatment, a cleaned spray gun was used with a pressure of 2.0 bar to inoculate the marked leaf sections of each plant with the Z. tritici inoculum by evenly spraying until runoff.The inoculum was left to dry on the leaf surface (~15 min) and the pots were placed (sorted by treatment) in large plastic bags containing water (~1 L).The bags were sealed using tape or plastic locking clips to generate an environment with maximal relative humidity.The plants were incubated in the bags at ~20 • C (day)/~12 • C (night) and a 16 h day/8 h night cycle under controlled greenhouse conditions.After 48 h, the pots were removed from the bags, and the plants were placed back onto the trays.The randomized placement was ensured.The plants were watered regularly and a relative humidity of 70% to 90% was maintained.The wheat was grown for 21 days post-inoculation (dpi) in the same temperature and light conditions [54,55].The degree of plant damage was determined 20-22 days after inoculation.Seedlings were rated using the rating scale of 0-4 [43], where 0-1 is resistant, 2 is moderately susceptible, 3 is susceptible, and 4 is highly susceptible.According to the methodological recommendations [55], we evaluated 10 seedlings separately in each pot.Since we had 3 replicates, we received 30 ratings per variety.Then, the results were statistically processed.

DNA Extraction and Molecular Screening of Stb Resistance Genes
Each genotype's genomic DNA was extracted using the CTAB method from fresh leaves of individual plants at the two-leaf seedling stage [56].The concentration and purity of the resulting preparation were measured using a NanoDrop One spectrophotometer.The DNA concentration for PCR was adjusted to 20 ng/µL.Primers linked to Stb genes were employed according to certain approved protocols.The polymerase chain reaction (PCR) was conducted using the primers and annealing temperature settings that were specified for each Stb gene in the references (Table 4).A Bio-Rad T100TM Thermal Cycler (Bio-RAD, Hercules, CA, USA) was used to conduct the PCR experiments.The PCR mixture (25 µL) contained 2.5 µL of genomic DNA (30 ng), 1 µL of each primer (1 pM/µL) (Sigma-Aldrich, St. Louis, MO, USA), 2.5 µL of dNTP mixture (2.5 mM, dCTP, dGTP, dTTP and dATP aqueous solution) (ZAO Sileks, Moscow, Russia), 2.5 µL of MgCl2 (25 mM), 0.2 µL of Taq polymerase (5 units µL) (ZAO Sileks, Russia), 2.5 µL of 10X PCR buffer, and 12.8 µL of ddH20.TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8) was used to separate the amplification products, and ethidium bromide was added [57].A 100 bp DNA ladder (Fermentas, Vilnius, Lithuania) was employed to gauge the size of the amplification fragment.The Gel Documentation System (Gel Doc XR+, BIO-RAD, Hercules, CA, USA) was used to visualize the results.Each sample underwent three separate tests.Leaf samples from all 60 entries were genotyped with the SSR marker designed to detect alleles of the Stb genes.Identification of Stb genes resistant to the Z. tritici pathogen was carried out using molecular genetic tagging in selected winter wheat cultivars.To analyze Stb genes of resistance, microsatellite markers linked to the resistance genes were used (Table 3) [20,30,[58][59][60].The isogenic lines with known resistance genes were used as control entries.To investigate the presence of the Stb2 resistance gene, the SSR marker WMS389 was used with the Veranopolis wheat cultivar as a positive control.The presence of Stb4 was studied using the SSR marker WMS111 and Tadinia as positive control.The presence of Stb5 was confirmed using the WMS44 marker when the Stb5-carrying cultivar CS (Synthetic 7D) was used as a positive control.The Stb7 gene was identified using SSR marker WMC313 and cultivar Estanzuela Federal as a positive control, and the presence of the Stb8 gene was confirmed using the WMS577 marker when the Stb8-carrying Synthetic W7984 was used as the positive control.

Figure 1 .
Figure 1.The resistance score for Z. tritici with the AUDPC values for 2020 and 2021 for wheat genotypes in the adult plant stage.

3 ,Figure 1 .
Figure 1.The resistance score for Z. tritici with the AUDPC values for 2020 and 2021 for wheat genotypes in the adult plant stage.

Figure 2 .
Figure 2. Summary of infection types among 60 wheat cultivars and breeding lines infected with Z. tritici (a) in the adult plant stage and (b) in the seedling stage.Note: RR-highly resistant; R-resistant; MS-moderately susceptible; S-susceptible.

Figure 2 .
Figure 2. Summary of infection types among 60 wheat cultivars and breeding lines infected with Z. tritici (a) in the adult plant stage and (b) in the seedling stage.Note: RR-highly resistant; R-resistant; MS-moderately susceptible; S-susceptible.

Table 1 .
Analysis of variance of the effect of the plant genotype and pathogen on the resistance of wheat seedlings to Zymoseptoria tritici.

Table 2 .
Genetic parameters of wheat genotypes.

Table 2 .
Genetic parameters of wheat genotypes.

Table 3 .
Disease severity for Z. tritici and detected Stb genes based on linked markers in the collection of wheat genotypes.

Table 4 .
Molecular markers used to identify Stb genes.