Identiﬁcation of Apple Varieties Resistant to Fire Blight ( Erwinia amylovora ) Using Molecular Markers

: Fire blight of fruit crops is one of the most dangerous diseases for apple trees and other plants of the Rosaceae family, and in Kazakhstan, it is subject to quarantine. To study the spread of ﬁre blight, a phytopathological evaluation of 59 apple varieties of domestic and foreign breeds was carried out in various regions of the south and southeast of Kazakhstan while also considering climatic conditions. The susceptibility of an apple tree to ﬁre blight is inﬂuenced by the climatic conditions prevailing in a particular fruit region of Kazakhstan. Samples were collected from various varieties of apple trees with ﬁre blight symptoms for molecular genetic analysis. The phytopathological evaluation and results of the PCR analysis made it possible to identify the causative agent of the disease and its spread to apple varieties in the main fruit regions of Kazakhstan. A molecular study of the resistance to the ﬁre blight pathogen was carried out using the most effective molecular markers. A set of 10 (FBE-1_Y320; FBE-2_Y192; FBE-2_Y495; FBE-2_Y551; FB-MR5-K35; FB-MRS-R240; FB-MR5-R249; FB-MR5-rp16k15_M106; RLP1a; and RLP1b) SNPs was selected, including SNPs reported to be associated with three trait loci, as well as the two markers AE10-375 and GE-8019. Interestingly, the SNP analysis revealed that for all ten markers linked to ﬁre blight resistance, the genotypes of all 59 apple cultivars were identical. No differences in the presence or absence of these markers were observed among the studied varieties. The 26 apple varieties of domestic and foreign breeds most resistant to ﬁre blight were identiﬁed in the molecular analysis using the markers AE-375 and GE-8019. Among the studied 59 apple varieties, 23 varieties were identiﬁed using the AE-375 marker and 7 varieties with the GE-8019 marker. Samuret, Honeycrisp, Pinova, and Red Topaz were found to be resistant using markers AE-375 and GE-8019. The most promising apple varieties for further breeding for resistance to ﬁre blight programs were selected.


Introduction
In Kazakhstan, as in most of the world's countries, the most significant fruit crop is the apple tree (Malus domestica Borkh.), which has been a leader in the increase in fruit production.Orchard areas account for 47.18 thousand hectares in which pome and stone fruit crops are grown, and 75% of this area is used for apple orchards [1].The State Register of breeding achievements approved for use in the Republic of Kazakhstan, for 2023, includes 73 varieties of apple trees of various ripening periods, 29% of which are varieties of local breeds, obtained from the Kazakh Research Institute of Horticulture, and 38% of which are varieties whereby the originator is not registered.
Fire blight, caused by the bacterium Erwinia amylovora, is one of the most dangerous diseases for Malus domestica and other species of the Rosaceae family.This disease is difficult to control because of the limited or completely abandoned application of antibiotics.Therefore, genetic resistance is thought to be the most sustainable approach to managing fire blight.E. amylovora is subject to quarantine in Kazakhstan.Fire blight, since 2010, has caused significant damage to the apple and pear orchards of Kazakhstan, causing large crop losses and the death of trees.According to Vanneste et al. [2], it affects more than 180 species of fruit and tree shrubs of the Rosaceae family around the world.It has been reported that approximately five hundred thousand fruit trees have been destroyed in Italy [2].According to Norelli et al. [3], more than 240 hectares of apple trees in southwestern Michigan State, USA, have been affected, with significant economic losses, because of fire blight.In Turkey, fire blight was first detected in a pear orchard in 1985 [4].Therefore, identifying varieties resistant to the disease as quickly as possible has become a major and worldwide issue [5].
In Kazakhstan, the first symptoms of fire blight were detected in 2008, and by 2010 it had begun to cause significant damage to apple and pear orchards in several areas of the Almaty Region.On some peasant farms, the proportion of affected trees in apple orchards reaches 50-60%, or more, with a high degree of disease symptom development [6,7].According to the results of a study conducted by Gritsenko et al. [8] under such conditions in Kazakhstan, among the 30 varieties collected from three orchards in the Almaty Region, 23 varieties were infected by the pathogen, including 15 varieties without visible symptoms, and 8 varieties were infected by the pathogen [8].
Currently, a rather extensive range of fruit crops is recommended in Kazakhstan.The most important condition for increasing the economic efficiency of horticulture is the constant improvement of the varietal composition.New varieties should have advantages over existing analogs in terms of productivity, resistance to abiotic and biotic stressors, and fruit quality, and they should be distinguished by novelty, competitiveness, and quick return on investment [9].The resistance of fruit crop varieties to the disease is the most important indicator that determines their market value.Despite numerous studies conducted in different countries, many issues related to the resistance of fruit crop varieties to fire blight have not been sufficiently studied.The creation of new varieties of perennial plants consists of several cycles of hybridization and can last, depending on the crop, an average of 20-25 years.Over the last decade, real prerequisites for the transition from traditional breeding techniques based on phenotypic traits to approaches that involve genomic characteristics, such as the identification of "trait-locus" associations, have appeared.
E. amylovora strains differ in virulence, which can lead to varying degrees of fire blight severity and complicate the determination of the genetic basis for the degree of susceptibility to the disease [10][11][12][13].Most of the resistant forms are concentrated among the wild forms of the apple tree, such as Malus robusta, M. sublobata, M. atrosanguinea, M. prunifolia, and M. fusca.High resistance was also noted in other species: M. baccata [14], M. robusta var.persicifolia, and M. sieversii [15].
In the development of organic farming, the greatest priority with the most environmentally friendly direction is the cultivation of disease-resistant varieties and rootstocks of apple trees.An increased level of resistance to diseases is one of the most important requirements for modern varieties of agricultural plants, including fruit crops.Currently, the priority is to create varieties of fruit crops with genetic resistance to the most harmful diseases; in particular, fire blight.Resistance to this pathogen is an important goal of many apple breeding programs [16,17].Therefore, optimal methods for assessing genetically determined resistance to pathogens will always be in demand.Breeding apple tree varieties with genetic resistance offers a solution to fire blight [18].
Based on the results of a study conducted in 2007, two dominant SCAR markers were created [19].By inoculating plants with the pathogen, it was found that genotypes that carried both markers were more resistant than those that lacked them.According to research conducted by Drenova and others, in Russia, it was found that out of 54 analyzed samples, 11 varieties and 1 hybrid form (Avgusta, Zhelannoe, Zaslavskoye, Cinnamon, Orlinka, Skryzhapel, Svezhest, Stroevskoye, Apple Spas, Honey Crisp, Fire Sid, and 376-106) are potentially resistant to the disease and can be used as gene sources of the trait in breeding programs [6].Omasheva et al. [20] identified apple varieties of Kazakh breeds, which were tested for the presence of alleles associated with fire blight resistance using AE10-375 and GE-8019 markers.According to the researchers, only two of the studied varieties (Alatau Column and Maximus) showed a positive result for both markers.The AE10-375 and GE-8019 markers flank the FBF7 QTL; the AE10-375 marker is located at a distance of 4 cm from the QTL peak, and the GE-8019 marker is located at a distance of 6 cm from the QTL peak.The CH-F7-Fb1 marker maps to the same side of the QTL as AE10-375 and is used to improve the reliability of molecular genetic analyses [21].
The aim of this research was to analyze domestic and foreign apple varieties using SNP and SCAR markers to identify genetic sources of fire blight resistance.

Sample Collection
For the timely detection of fire blight, regular examinations of apple plantations in the south and southeast of Kazakhstan were carried out during the growing season according to the methods for detecting and identifying the fire blight agent of fruit trees [22,23].The monitoring of the diseases and sampling for the molecular genetic analysis was carried out in a total of 8 orchards, including the pomological orchard of the Kazakh Research Institute of Horticulture and commercial and farm orchards in the main commercial areas of horticulture: Turkestan (2 orchards), Zhambyl (1 orchard), and Almaty Regions (5 orchards).In this study, 59 apple varieties approved for use in the Republic of Kazakhstan were used, including 24 varieties of Kazakhstani breeds, as well as 35 varieties of foreign breeds.In addition, five varieties not approved for use in the Republic of Kazakhstan were studied.The samples were collected from various varieties of apple trees that displayed symptoms of fire blight, including browned and necrotic young shoots, as well as those with the characteristic shape of a "shepherd's crook", and samples were also taken from trees without visible symptoms.The selected samples of the 59 studied apple varieties were delivered to the laboratory and kept in a freezer (−80 • C) until the DNA was isolated (Table 1).Created by the Purdue, Rutgers, and University of Illinois Agricultural Experimental Station in the 1980s, the Redfree apple was designed to be a disease-resistant summer apple.Obtained as a result of crossing a domestic apple tree and a clone of Malus floribunda 821.

[49] Williams Pride
Was developed by the well-respected "co-op" breeding program of Purdue, Rutgers, and Illinois (PRI) universities in the 1970s, and originally known as Co-op 23.Its ancestry is similar to that of many of the PRI varieties, including Rome Beauty and Malus floribunda, but its early-season character probably comes from Mollie's Delicious and Julyred.The disease distribution index (DDI) was assessed in mid-July using a 5-point scale according to the following classification: 0-asymptomatic; 1-sporadic symptoms of fire blight, with less than 10% of trees affected; 2-10% to 20% of trees affected by pathogen; 3-20% to 50% of trees affected by the pathogen; 4-50% to 100% of trees damaged by infection.The DDI was calculated by dividing the number of fire-blight-affected trees of 1.000 randomly examined trees in the field multiplied by 100 [72]: DDI = (fire blight shoots/total shoots) × 100 The disease severity index (DSI) was calculated using the following formula: The DDI and DSI were evaluated using only three apple varieties, namely Golden Delicious, Idared, and Aport, because these varieties are more often infected by fire blight.Over the last 5 years, these varieties have increasingly become rapidly infected, and according to the results, a detailed analysis was conducted for these main susceptible varieties.

DNA Isolation
Total DNA was isolated with a silica gel membrane and resuspended in TE buffer (10 mM Tris; 0.1 mM EDTA).The DNA concentration was determined using a nanospectrophotometer and normalized to 20 ng/µL [19].
The DNA extracts (20 ng each) were mixed with an equal volume of TaqMan ® OpenArray ® Genotyping Master Mix (Life Technologies Corporation-Pleasanton, 6055 Sunol Boulevard, Pleasanton, CA, USA) according to the instructions and amplified using the QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific).
The results were analyzed using Quantstudio ® Design and Analysis software and Taqman ® Genotyper (Thermo Fisher Scientific).Because the assays were newly designed, each genotyping result was manually verified by viewing the real-time trace and fluores-cence endpoints.Any manual changes were saved using the Taqman ® Genotyper Software and exported as a genotype matrix for each individual sample.

Monitoring
Observation of the disease distribution index (DDI) and disease severity index (DSI) of fire blight in apple trees in the main fruit regions of Kazakhstan.
The routine inspection of apple orchards, to determine the DDI and DSI of the disease, was carried out over two years (2021-2022) on several varieties on various farms in the south and southeast of Kazakhstan, including Almaty Region (Almalyk village, Baibulak village, Kyzylsharik village, Bayseit village, Koram village), Turkestan Region (Akzhar village, Shakpak baba village), and Zhambyl Region (Merke village) located in different climatic areas.
During the visual inspection of apple trees, the presence of symptoms characteristic of fire blight was noted: sudden wilting and drying of blooming flowers; drying of the tops of young shoots, the tips of which are bent in a hook-like manner; leaf necrosis; brown spots on unripe fruits, which gradually mummy; characteristic "marbling" on the cut of the bark; wedge-shaped ulcers on the bark; the release of exudate on affected organs (Figures 1 and 2).
The study area's apple commercial and farm orchards (Turkestan, Zhambyl, and Almaty Regions) are favorable for horticulture under conditions of irrigation.The main unfavorable factor for fruit crops, in particular apple trees, is the return of spring frosts in April and early May, which have become more frequent in recent decades.
Meteorological stations collecting climate data are located in the city of Talgar and the foothills, covering the orchards of the Almaty Region; in the city of Merke, covering the orchards of the Zhambyl Region; and in the city of Shakpak Baba, covering the orchards of the Turkestan Region.Merke, in the Zhambyl Region, is described as more arid with sharp temperature fluctuations between day and night [76].The climatic conditions of the Turkestan Region are characterized by pronounced continentality, aridity, hot and dry summers, and mild winters.Data of the meteorological observations for the regions in the main fruit-growing area of Kazakhstan, for 2021 and 2022, are presented in Figure 3. Figure 4 presents the correlation between the disease severity index (DSI) and climatic conditions (temperature, precipitation, and humidity) by using linear regression analysis.According to the analysis, the DSI decreases with increasing temperature, whereas the DSI increases with increasing precipitation and humidity.The study area's apple commercial and farm orchards (Turkestan, Zhambyl, and Almaty Regions) are favorable for horticulture under conditions of irrigation.The main unfavorable factor for fruit crops, in particular apple trees, is the return of spring frosts in April and early May, which have become more frequent in recent decades.
Meteorological stations collecting climate data are located in the city of Talgar and the foothills, covering the orchards of the Almaty Region; in the city of Merke, covering the orchards of the Zhambyl Region; and in the city of Shakpak Baba, covering the orchards of the Turkestan Region.Merke, in the Zhambyl Region, is described as more arid  The study area's apple commercial and farm orchards (Turkestan, Zhambyl, and Almaty Regions) are favorable for horticulture under conditions of irrigation.The main unfavorable factor for fruit crops, in particular apple trees, is the return of spring frosts in April and early May, which have become more frequent in recent decades.
Meteorological stations collecting climate data are located in the city of Talgar and the foothills, covering the orchards of the Almaty Region; in the city of Merke, covering the orchards of the Zhambyl Region; and in the city of Shakpak Baba, covering the orchards of the Turkestan Region.Merke, in the Zhambyl Region, is described as more arid 3. Figure 4 presents the correlation between the disease severity index (DSI) and climatic conditions (temperature, precipitation, and humidity) by using linear regression analysis.According to the analysis, the DSI decreases with increasing temperature, whereas the DSI increases with increasing precipitation and humidity.3. Figure 4 presents the correlation between the disease severity index (DSI) and climatic conditions (temperature, precipitation, and humidity) by using linear regression analysis.According to the analysis, the DSI decreases with increasing temperature, whereas the DSI increases with increasing precipitation and humidity.The weather conditions in 2021 had a number of differences from the long-term averages in 2022.In the first half of spring 2021, the weather was cool.At the end of April, frosts were observed to be reduced by −3.90 • C, and in the second half of spring, the weather was warm.In addition, for all regions, in April 2022, a slight change was found, on average, in the form of an increase in temperature by +4.2 • C compared to 2021.The humidity across all regions increased by at least 10% in late spring and early summer 2022, while the precipitation doubled.The summer was dry and hot, and the average daily air temperature in the summer months of 2021 was 2-4 • C higher than the long-term values, which adversely affected the vegetation and productivity of trees and also helped to stop the development of the disease.
The inspection of apple plantations in the main commercial area of horticulture (Almaty, Turkestan, and Zhambyl Regions) showed that fire blight was quite widespread.A focal distribution of the disease in the apple orchards of eight inspected farms was established.During a visual observation of the commercial orchards and farms among 59 varieties, symptoms of fire blight were found on most varieties: Idared, Aport, Golden Delicious, Gala, Granny Smith, Maksat, Voskhod, Renet Burkhardt, Starkrimson, Rubin, and Fuji.Disease symptoms were noted among the following foreign apple varieties: Pinova, Pink Lady, Rashida, Konfetnoye, Sinap Almaty, Red Topaz, Deljohns, Santana, and Wilton Star.Asymptomatic samples were collected in the laboratory for identification of the pathogen.
The study of plant reaction to the pathogen was focused on three of the most susceptible varieties Golden Delicious, Idared, and Aport, due to the susceptibility of these genotypes to fire blight in 2021 and 2022, whereas other varieties only experienced sporadic infections.Using these genotypes as examples, it was able to demonstrate the varying levels of resistance/susceptibility to fire blight in different regions of Kazakhstan.As a result of assessing the intensity of the development of fire blight on the apple varieties Idared, Golden Delicious, and Aport in different regions, certain zonal foci in the manifestation of the disease were noted.As can be seen from the data in Figure 5, the Idared variety was highly affected in all regions.The DDI of fire blight reached 36.0%with a DSI of 9.6%.The most severe damage to all varieties of apple trees with fire blight was noted in the plantations of the Almaty Region.
The DDI ranged from 22.0 to 36.0% and DSI from 5.2 to 9.6%.The disease's development was weaker in the Zhambyl Region, which has a more arid climate, and an even lower DDI was noted in the Turkestan Region, where the prevalence of the disease was between 12.0 and 20.0% and the DSI from 3.0 to 5.2%.This is probably because of the different natural and climatic conditions of these regions.The climatic conditions of the Turkestan Region are characterized by pronounced continentality and aridity, which adversely affect the development of infection.At the same time, in the Almaty Region with higher air humidity, favorable conditions are created for the development and spread of the disease.The two-year monitoring period made it possible to identify apple varieties susceptible and resistant to fire blight under field conditions, taking into account the climatic conditions of the main fruit regions of Kazakhstan.
Idared, Golden Delicious, and Aport in different regions, certain zonal foci in the ma festation of the disease were noted.As can be seen from the data in Figure 5, the Idar variety was highly affected in all regions.The DDI of fire blight reached 36.0%with a D of 9.6%.The most severe damage to all varieties of apple trees with fire blight was not in the plantations of the Almaty Region.Prima ( 1)

Molecular Screening Using SCAR Markers
The identification of variants of QTL FBF7 linked to fire blight resistance is commonly carried out using the SCAR markers AE10-375 and GE-8019.The resistant genotypes are indicated by the dominant alleles of markers AE10-375 and GE-8019, which have lengths of 375 bp and 397 bp, respectively [19,75].The presence of resistance alleles for both markers has previously been revealed in the genotypes with high resistance to the pathogen compared with the genotypes bearing only the resistance allele for one of the two markers [19].In the present research, we identified 23 varieties (Starkrimson, Aport, Fuji, Golden Delicious, Granny Smith, Red Delicious, Aigul, Jeromini, Kandil Sinap, Kandil Kirghiz, Starks Earliest, Scarlet Spur, Summer red, Nicole Grain, Gold spur, Honeycrisp, Camspur, Gala Anna, Jonagold, Synap Almatinsky, Wilton Star, Pinova, Red Topaz) and 7 varieties (Quinti, Summer red, Williams Pride, Honeycrisp, Elstar, Pinova, Red Topaz) carrying resistant alleles of AE10-375 and GE-8019, respectively, as shown in Table 4.However, according to DDI and DSI analyses, Golden Delicious, Idared, and Aport were infected by the fire blight pathogen in all evaluated orchards.Additionally, Honeycrisp, Red Topaz, Pinova, and Samuret varieties bear resistant alleles of both markers.

Discussion
The DDI and DSI of fire blight are highly dependent on environmental conditions in Almaty Region (Almalyk village, Baibulak village, Kyzylsharik village, Bayseit village, Koram village), Turkestan Region (Akzhar village, Shakpak baba village), and Zhambyl Region (Merke village) (e.g., temperature, humidity, and rainfall), host factors (e.g., tree vigor), E. amylovora strain virulence, various host-strain interactions, and host quantitative stability, which adds to the challenge of phenotypic resistance/susceptibility to fire blight [10,[77][78].Different phenotyping methods can produce variable and often uncorrelated results [79,80].In addition, genetic markers can reduce the cost of breeding new varieties through early selection.The number of plants and duration of the evaluation can be reduced, and the varieties, even those with combined resistance, can become commercially available in a short time [81].
However, seedling phenotypic breeding is a cost-effective and relatively efficient approach used in apple tree breeding programs (e.g., WABP) to create breeding populations with low susceptibility to fire blight.The use of DNA information in breeding decisions (i.e., DNA-based breeding), which has become common for several traits (e.g.,

RLP1a
RLP1b AE-375 GE-8019 Remarkably, all 59 apple varieties exhibited identical genotypes for all ten markers associated with fire blight resistance.There was no variation observed among the studied varieties in terms of the presence or absence of these markers.This outcome indicates a lack of genetic diversity for fire blight resistance within the analyzed panel of apple varieties.

Molecular Screening Using SCAR Markers
The identification of variants of QTL FBF7 linked to fire blight resistance is commonly carried out using the SCAR markers AE10-375 and GE-8019.The resistant genotypes are indicated by the dominant alleles of markers AE10-375 and GE-8019, which have lengths of 375 bp and 397 bp, respectively [19,75].The presence of resistance alleles for both markers has previously been revealed in the genotypes with high resistance to the pathogen compared with the genotypes bearing only the resistance allele for one of the two markers [19].In the present research, we identified 23 varieties (Starkrimson, Aport, Fuji, Golden Delicious, Granny Smith, Red Delicious, Aigul, Jeromini, Kandil Sinap, Kandil Kirghiz, Starks Earliest, Scarlet Spur, Summer red, Nicole Grain, Gold spur, Honeycrisp, Camspur, Gala Anna, Jonagold, Synap Almatinsky, Wilton Star, Pinova, Red Topaz) and 7 varieties (Quinti, Summer red, Williams Pride, Honeycrisp, Elstar, Pinova, Red Topaz) carrying resistant alleles of AE10-375 and GE-8019, respectively, as shown in Table 4.However, according to DDI and DSI analyses, Golden Delicious, Idared, and Aport were infected by the fire blight pathogen in all evaluated orchards.Additionally, Honeycrisp, Red Topaz, Pinova, and Samuret varieties bear resistant alleles of both markers.

Discussion
The DDI and DSI of fire blight are highly dependent on environmental conditions in Almaty Region (Almalyk village, Baibulak village, Kyzylsharik village, Bayseit village, Koram village), Turkestan Region (Akzhar village, Shakpak baba village), and Zhambyl Region (Merke village) (e.g., temperature, humidity, and rainfall), host factors (e.g., tree vigor), E. amylovora strain virulence, various host-strain interactions, and host quantitative stability, which adds to the challenge of phenotypic resistance/susceptibility to fire blight [10,77,78].Different phenotyping methods can produce variable and often uncorrelated results [79,80].In addition, genetic markers can reduce the cost of breeding new varieties through early selection.The number of plants and duration of the evaluation can be reduced, and the varieties, even those with combined resistance, can become commercially available in a short time [81].
However, seedling phenotypic breeding is a cost-effective and relatively efficient approach used in apple tree breeding programs (e.g., WABP) to create breeding populations with low susceptibility to fire blight.The use of DNA information in breeding decisions (i.e., DNA-based breeding), which has become common for several traits (e.g., apple scab resistance and malic acid content) in apple trees [82], will enable more efficient and accurate breeding for fire blight resistance.The development of apple varieties with long-term fire blight resistance and superior fruit quality can be effectively achieved through DNA breeding; however, progress is hampered by the few DNA tests available that predict symptoms.In the short term, published information on the phenotypic resistance/susceptibility [80] and on the reduced and increased susceptibility alleles for several important parent plants (IBPs) and varieties [83] can be immediately applied to inform parental selection in apple tree breeding programs.The breeding-relevant QTLs that have been previously characterized can be targeted to develop DNA tests for breeders to pyramid favorable alleles and/or combine superior fruit quality with fire blight resistance.The introgression and pyramiding of favorable alleles can be accelerated by fast-cycle breeding techniques [18].The objective of this research was to analyze domestic and foreign apple varieties using SNP and SCAR markers to identify genetic sources of fire blight resistance.Khan et al. (2007) confirmed that genotypes carrying the markers AE10-375 and GE-8019 have, on average, higher resistance to fire blight compared to genotypes that do not contain the markers [19].Previous studies have shown similar results [19,84].
Another study found that fire blight resistance in genotypes carrying AE10-375 and GE-8019 confirms their utility for MAS.The identified genotypes on the basis of this study especially varieties that contain AE10-375 and GE-8019 Honeycrisp, Red Topaz, Pinova, and Samuret will be useful for MAS and breeding programs.The advantage of determining resistance genes is their presence in large-fruited varieties of commercial fruit quality, which can be easily used to breed new varieties.Fire blight resistance scores under controlled conditions have been shown to correlate well with field resistance [85].Many factors, such as host and environmental conditions, influence the DDI and DSI of the disease [86][87][88].
The identification of sources of resistance with high fruit quality (i.e., elites) for use as breeding parents is an important precursor to the development of breeding populations with low susceptibility to fire blight, suggesting moderate to high heritability of traits.As a result of the research, an assessment was made of the stability of currently grown varieties of domestic and foreign breeding in the main commercial farm area of horticulture (south and southeast Kazakhstan).Molecular diagnostic techniques based on DNA markers have revolutionized plant breeding processes by enabling more accurate and efficient selection of desirable traits such as yield forecast, fruit quality, and disease resistance.These techniques provide plant breeders with tools to identify and select plants with the desired traits at an early stage, saving time and resources compared to traditional breeding methods.
Most modern commercial apple varieties are susceptible to fire blight [89][90][91][92], and an updated comparison of resistance/susceptibility levels of 94 IBPs and varieties was provided.As in previous studies, for example [80,[90][91][92], most apple varieties (e.g., Jonathan, Ginger Gold, Sansa, and Sweet Sixteen) have shown high or moderate susceptibility to fire blight [81].Several varieties with medium to high resistance have been confirmed, with eight varieties (e.g., Dolgo, Enterprise, Frostbite, Kidd's Orange Red, Tsugaru, Vista Bella, Wildung, and Williams' Pride) being classified as highly resistant in one year and moderately resistant in another [80].Artificial inoculation of seedlings from crosses with low susceptibility can be used to cull highly susceptible seedlings.Artificial seedling inoculation under greenhouse conditions often overestimates susceptibility and, thus, may not predict field performance [93].
Lyzhin and Saveleva [21] showed that most of the analyzed apple varieties (85.7%) had at least one marker out of three (AE10-375, GE-8019, and CH-F7-Fb1).Two out of three markers were present in five varieties (35.7%): variety Lobo has markers GE-8019 and AE10-375, and varieties Ligol, Skala, Fregat, and Fuji have markers AE10-375 and CH-F7-Fb1.The target fragments of the studied markers are absent in the varieties Antonovka ordinary and Galarina.Three markers were identified in the genome of Bylina, Rozhdestvenskoye, Uspenskoye, and Charodeyka varieties, indicating the presence of resistance to fire blight QTL FBF7.QTL flanking markers GE-8019 and AE10-375 (marker CH-F7-Fb1 was absent) were found in the variety Lobo.Presumably, this also indicates the presence of QTL FBF7 with a lower probability than the presence of three markers.After analyzing the varieties of apple trees for resistance to fire blight using molecular markers, the authors concluded that the markers GE-8019, AE10-375, and CHF7-Fb1, linked to QTL FBF7, were found in the varieties Bylina, Rozhdestvenskoye, Uspenskoye, and Charodeyka.They recommend these varieties for use in breeding for resistance to E. amylovora [21].
A previous study conducted in Kazakhstan by Nurtaza et al. [94] shows that resistant varieties to fire blight using both markers AE10-375 and GE-8019 have not been identified.However, they found that three genotypes from the wild Cherkesai population and five genotypes from Nur-Sultan contain fire blight-resistant alleles according to AE10-375 or GE-8019 [94].Two SCAR marker alleles, AE10-375 and GE-8019, appear to be useful in marker-assisted breeding, but their ability to predict fire blight resistance is not ideal.The very large number of varieties that have only one of the two marker alleles suggests that the simultaneous occurrence of two marker alleles in the same variety, in some cases, is not associated with the presence of QTL.Similarly, the absence of two marker alleles does not necessarily indicate a lack of fire blight resistance.In our study, AE10-375 and GE-8019 alleles were not found in some genotypes.However, these genotypes (Pestrushka, Tulpan, Ainur, Egemen, Zarya Alatau, Esen, Sarkyt, Talgarskoe, Zharkyn, Modi, Braeburn, Mutsu, Redfree, Diana, July Chernenko, Maria Red, Red JonaPrince, and SQ159) were resistant to fire blight in the field.The level of relatedness to "Cox's Orange Pippin" does not appear to have any effect on the strength of the association between DNA marker alleles and fire blight resistance.Another study used marker CH-F7-Fb1 to identify the resistance of rootstocks to fire blight [95].The results of the molecular analysis and susceptibility of plants to metabolites of the causative agent of fire blight of fruit crops were compared.There was no clear relationship between the number of markers present and the degree of plant tissue necrosis in the tested forms.However, studies have shown that the presence of SCAR marker AE10-375 and microsatellite CH-F7-FB1 in the forms of 62-396 and 14-1 provides a phenotypic manifestation of resistance to E. amylovora metabolites [95].However, more varieties need to be investigated before arriving at a general conclusion, which is confirmed by Nybom et al. [96].
Genotyping of genebank material is critical to ensure that genetic variation is maintained and also to make genebank material a useful resource for breeding.In the past, SSR markers have been widely used in apple germplasm research at national, regional, and European levels.More recently, SNP arrays have been used to determine apple diversity [97].In a preliminary study, the gene sequence FBE, MR5, and RLP1 [75] was used which was found to be associated with fire blight resistance.In our study, all 10 pairs of primers and probes were designed based on this gene sequence and screened for 59 apple varieties of Kazakh and foreign breeding programs.Interestingly, all 59 apple varieties exhibited identical genotypes for all ten markers associated with fire blight resistance.There was no variation observed among the studied varieties in terms of the presence or absence of these markers.This outcome indicates a lack of genetic diversity for fire blight resistance within the analyzed panel of apple varieties.This unexpected lack of genetic diversity for fire blight resistance within the analyzed panel of apple varieties may have implications for future fire blight management in apple orchards.Noticeably, a few cases were identified where the SNP data were in conflict with data obtained from the presumed graft-wood source using SSR markers.Understanding the genetic basis of this uniformity is critical for developing effective strategies to enhance fire blight resistance in apple cultivars and maintaining the sustainability of apple production in the face of evolving pathogens.

Conclusions
In the course of this study, an observation of disease in 59 apple varieties in the general fruit regions of Kazakhstan was carried out, taking into account the climatic conditions.Based on the results obtained in the study, it was found that fire blight has become quite widespread.Conspicuously, the SNP analysis showed that all 59 apple varieties exhibited identical genotypes for all ten markers associated with fire blight resistance.No variation was observed among the studied varieties regarding the presence or absence of these markers.The 26 most resistant apple varieties of domestic and foreign breeds to fire blight were identified using the molecular screening markers AE-375 and GE-8019.Among the studied 59 apple varieties, 23 varieties were identified using the AE-375 marker and 7 varieties with the GE-8019 marker.The varieties Samuret, Honeycrisp, Pinova, and Red Topaz were found to be resistant using markers AE-375 and GE-8019.As a result of the analysis of the genotypes, out of 59 analyzed samples, 26 varieties have distinctive alleles that can be associated with resistance to the disease and can be used as sources of resistance in breeding programs.

22 Figure 1 .
Figure 1.Symptoms of fire blight on the Idared apple variety (Merke, Zhambyl Region): (A) drying of the tops of young shoots and a shepherd's crook; (B) release of bacterial exudate.

Figure 2 .
Figure 2. Symptoms of fire blight on the Golden Delicious apple variety (Baidibek bi village Enbekshikazakh District): (A) withering and drying of blossoming flowers and young leaves; (B affected leaves on young shoots.

Figure 1 . 22 Figure 1 .
Figure 1.Symptoms of fire blight on the Idared apple variety (Merke, Zhambyl Region): (A) drying of the tops of young shoots and a shepherd's crook; (B) release of bacterial exudate.

Figure 2 .
Figure 2. Symptoms of fire blight on the Golden Delicious apple variety (Baidibek bi village Enbekshikazakh District): (A) withering and drying of blossoming flowers and young leaves; (B) affected leaves on young shoots.

Figure 2 .
Figure 2. Symptoms of fire blight on the Golden Delicious apple variety (Baidibek bi village, Enbekshikazakh District): (A) withering and drying of blossoming flowers and young leaves; (B) affected leaves on young shoots.

Figure 3 .
Figure 3. Agroclimatic indicators for April-September according to the weather stations of the Almaty, Zhambyl, and Turkestan Regions, 2021-2022.

Figure 4 .
Figure 4.The correlation between the disease severity index (DSI) and climatic conditions by using linear regression analysis.

Figure 3 .
Figure 3. Agroclimatic indicators for April-September according to the weather stations of the Almaty, Zhambyl, and Turkestan Regions, 2021-2022.

Figure 3 .
Figure 3. Agroclimatic indicators for April-September according to the weather stations of the Almaty, Zhambyl, and Turkestan Regions, 2021-2022.

Figure 4 .
Figure 4.The correlation between the disease severity index (DSI) and climatic conditions by using linear regression analysis.

Figure 4 .
Figure 4.The correlation between the disease severity index (DSI) and climatic conditions by using linear regression analysis.

Figure 5 .
Figure 5. Infection of apple trees with the pathogen under the conditions of the south and southeast of Kazakhstan in 2021 and 2022.Note: DDI-The disease distribution index; DSI-The disease severity index.

Table 1 .
Characteristics of the apple varieties.

Table 2 .
Description of 10 single nucleotide polymorphisms (SNPs) selected to study fire blight resistance.

Table 4 .
Results of the SNP and SCAR analysis of apple varieties.