Evaluation of Wheat Germplasm for Resistance to Leaf Rust (Puccinia triticina) and Identification of the Sources of Lr Resistance Genes Using Molecular Markers

Leaf rust, caused by Puccinia triticina (Ptr), is a significant disease of spring wheat spread in Kazakhstan. The development of resistant cultivars importantly requires the effective use of leaf rust resistance genes. This study aims to: (i) determine variation in Ptr population using races from the East Kazakhstan, Akmola, and Almaty regions of Kazakhstan; (ii) examine resistance during seedling and adult plant stages; and (iii) identify the sources of Lr resistance genes among the spring wheat collection using molecular markers. Analysis of a mixed population of Ptr identified 25 distinct pathotypes. Analysis of these pathotypes using 16 Thatcher lines that are near-isogenic for leaf rust resistance genes (Lr) showed different virulence patterns, ranging from least virulent “CJF/B” and “JCL/G” to highly virulent “TKT/Q”. Most of the pathotypes were avirulent to Lr9, Lr19, Lr24, and Lr25 and virulent to Lr1, Lr2a, Lr3ka, Lr11, and Lr30. The Ptr population in Kazakhstan is diverse, as indicated by the range of virulence observed in five different races analyzed in this study. The number of genotypes showed high levels of seedling resistance to each of the five Ptr races, thus confirming genotypic diversity. Two genotypes, Stepnaya 62 and Omskaya 37, were highly resistant to almost all five tested Ptr pathotypes. Stepnaya 62, Omskaya 37, Avangard, Kazakhstanskaya rannespelaya, and Kazakhstanskaya 25 were identified as the most stable genotypes for seedling resistance. However, most of the varieties from Kazakhstan were susceptible in the seedling stage. Molecular screening of these genotypes showed contrasting differences in the genes frequencies. Among the 30 entries, 22 carried leaf rust resistance gene Lr1, and two had Lr9 and Lr68. Lr10 and Lr28 were found in three and four cultivars, respectively. Lr19 was detected in Omskaya 37. Two single cultivars separately carried Lr26 and Lr34, while Lr37 was not detected in any genotypes within this study. Field evaluation demonstrated that the most frequent Lr1 gene is ineffective. Kazakhstanskaya 19 and Omskaya 37 had the highest number of resistance genes: three and four Lr genes, respectively. Two gene combinations (Lr1, Lr68) were detected in Erythrospermum 35 and Astana. The result obtained may assist breeders in incorporating effective Lr genes into new cultivars and developing cultivars resistant to leaf rust.


Introduction
Central Asia, including Kazakhstan, is a significant player in regional and global food security, producing most of the grain traded in the region, with total area sown to wheat in Kazakhstan representing over 85% of total cereal production [1]. One of the main reasons for the reduction in the yield of wheat in Kazakhstan is the disease with airborne infection. lines against Ptr races in order to monitor resistance breakdown and plan the replacement of susceptible cultivars with resistant cultivars.
Identification and selection of resistant genes through gene postulation and other plant protection and breeding strategies are time-consuming and cannot be employed if no different fungal isolates are available [31,32]. The molecular marker technology-the most accurate and efficient tool to screen wheat material against various genes, conferring resistance to rust pathogens, and for developing disease-resistant cultivars-is needed to overcome these problems [31][32][33][34].
Understanding the nature of resistance and prevention of genetic erosion leading to a rapid efficiency loss of used genes requires screening of new material. The screening should include the identification of resistance genes using molecular markers, as well as the study of the response of seedlings and adult plants of commercial varieties and advanced breeding lines to the Ptr pathotypes.
There is an understanding of adult plant resistance to leaf rust in winter wheat [4,27], but there is limited information available on Lr genes present in commercial cultivars and advanced breeding lines of spring wheat from Kazakhstan.
Several recent reports listed a number of released cultivars and advanced breeding lines of wheat in Kazakhstan and Russia that were resistant to leaf rust [27,30,35], but their reactions to the diverse Ptr races of Kazakhstan is not known. This study aims to: (i) determine variation in Ptr population using races from East Kazakhstan, Akmola, and Almaty regions of Kazakhstan; (ii) examine seedling and adult plant stage resistance; and (iii) identify the sources of resistance among the spring wheat collection using molecular markers.

Races of Puccinia triticina and Their Virulence Pattern
Use of 16 TcLr-lines helped to identify 25 virulence phenotypes of P. triticina from leaf rust-infected leaf samples that were collected in East Kazakhstan (East), Akmola (North), and Almaty (Southeast) regions of Kazakhstan during the 2019 and 2020 seasons ( Table 1). The northern population of leaf rust is more numerous (12 pathotypes) and more virulent in comparison to the eastern (8 pathotypes) and the southeastern (4 pathotypes) population. The virulence patterns of the pathotypes ranged from least virulent "CJF/B" and "JCL/G" (virulent on 5 of 16 differentials) to highly virulent "TKT/Q" (virulent on 13 of the 16 differentials). The isogenic lines with Lr24 and Lr25 were immune to 23 and 24 pathotypes, respectively. TcLr-lines with Lr9 and Lr19 were immune to 19 of 25 pathotypes.
The average value of the virulence of pathotypes from the Akmola region was 61.0%, which is 2.7% more than the virulence of the eastern population (58.3%) and 25% more than the southeastern population of P. triticina (36%).
There were some pathotypes with high virulence to Lr-lines in the eastern population of P. triticina. Thus, the virulence of the TKT/Q and TRT/G pathotypes were 81.3% and 75.0%, respectively. The southeastern leaf rust population contains four pathotypes with virulence from 31.3% to 43.7%.
Some isogenic lines (Lr10, Lr23, Lr26, Lr29) in most cases exhibit intermediate resistant reactions (2, 2+), which quickly turn into susceptibility during plant development and/or at the slightest change in plant growth conditions. Consequently, the sources of these genes are inappropriate to use in breeding for immunity due to the variability of their resistance response.

Seedling Test
Spring wheat germplasm was evaluated for response to P. triticina pathotypes. A complete list of this plant material, its origin, and leaf rust reaction are given in Table 2. The seedling reactions of 30 wheat genotypes to the 5 pathotypes of Ptr differed greatly. The wheat genotypes showed arrays of patterns in their responses to the five pathotypes. The most commercial spring wheat varieties in Kazakhstan were generally susceptible to leaf rust pathotypes. The varieties Stepnaya 62 and Omskaya 37 were resistant to almost all tested pathotypes of leaf rust. Omskaya 36 was resistant to two of the five pathotypes. Avangard, Kazakhstanskaya rannespelaya, and Kazakhstanskaya 25 were resistant to one pathotype of leaf rust.
The five pathotypes showed arrays of virulence patterns across 30 wheat genotypes ( Table 2). Based on infection type across 30 genotypes, SBR/H was the most virulent, followed by SBP/C and KHT/B. The pathotypes THT/B and QBQ/G were the least virulent. The analysis based on reactions of all five pathotypes showed that Stepnaya 62 and Omskaya 37 were the most stable resistant genotypes.

Field Evaluation
Arrays of variation for resistance to leaf rust under field conditions ( Table 2) were present. Most wheat varieties (73.33%) were susceptible to leaf rust in the field. Among 30 genotypes, 7 (Akmola 2, Astana 2, Kazakhstanskaya 25, Stepnaya 62, Omskaya 37, Tertsiya, and Chelyaba jubilejnaja) were considered as resistant (≤20% disease severity) in the adult plant stage under field conditions in Kazakhstan ( Table 2). Four genotypes showed ≤5% disease severity in both years. Among the genotypes with the most stable resistance in the seedling stage, Stepnaya 62 and Omskaya 37 were resistant in the adult plant stage under field conditions in both years.

Identification of Leaf Rust Resistance Genes Using Molecular Markers
The occurrence of known Lr genes in the 30 wheat cultivars is shown in Table 2. Molecular screening of these genotypes showed contrasting differences in the frequencies of these genes. The most frequent Lr gene, identified in the material studied individually or in combination, was Lr1. The expected marker fragment associated with Lr1 was found in 22 of the 30

Discussion
Leaf rust is a perennial problem for spring and winter wheat in Central Asia, including Kazakhstan [14,15,35], and Ptr populations are diverse and highly virulent [27,30].
Previous studies have reported variations in Ptr populations in Kazakhstan [4,27,30]. It is therefore necessary to periodically evaluate resistant cultivars and advanced breeding lines against Ptr races in order to monitor resistance breakdown and plan the replacement of susceptible cultivars with resistant ones.
This study provides additional information by presenting 25 diverse Ptr races with wide virulence patterns on 16 international differentials of leaf rust and 30 genetically diverse genotypes in both seedling and adult plant stages. Each of the 25 races showed diverse reaction patterns on the wheat genotypes, with virulence varying from resistant to highly susceptible. Lr25 and Lr24 were the most effective genes in Kazakhstan. In this study, the isogenic lines with Lr24 and Lr25 were immune to 23 and 24 pathotypes, respectively. TcLr-lines with Lr9 and Lr19 were immune to 19 of 25 pathotypes from Kazakhstan. Most of the pathotypes were avirulent to Lr9, Lr19, Lr24, and Lr25 and virulent to Lr1, Lr2a, Lr3ka, Lr11, and Lr30. The study of the virulence of the Russian South Ural population of P. triticina showed that all isolates were avirulent to Tc-lines with gene Lr16, Lr19, Lr24, Lr28, and Lr29 and virulent to Lr1, Lr3a, Lr3bg, Lr3ka, Lr14a, Lr14b, Lr17, and Lr18 [30]. This suggests that at least two genes (Lr19 and Lr24) have been found to be effective against pathogen populations from both Kazakhstan and Russia.
The number of genotypes showed high levels of seedling resistance to each of the 5 Ptr races, thus confirming genotypic diversity. Two genotypes (Stepnaya 62 and Omskaya 37) were highly resistant to almost all five tested Ptr pathotypes, while Omskaya 36 was resistant to more than two races. Therefore, Stepnaya 62, Omskaya 37, Avangard, Kazakhstanskaya rannespelaya, and Kazakhstanskaya 25 were identified as the most stable genotypes for seedling resistance. Omskaya 37 also showed a stable type of reaction at the seedling stage to Russian isolates [28]. However, most of the varieties from Kazakhstan were susceptible in the seedling stage.
In previous studies, the sources of Lr resistance genes (Lr19, Lr26, Lr37, Lr34, Lr1, Lr26, Lr34, Lr10, Lr37, Lr19, and Lr68 genes) were identified in winter wheat breeding material [4,[36][37][38]. In this research, molecular screening of spring wheat cultivars showed contrasting differences in the frequencies of nine important Lr genes. Among the 30 entries, 22 carried leaf rust resistance gene Lr1, 6 had Lr19, 2 had Lr9, and Lr68, Lr10, and Lr28 were found in 3 and 4 cultivars, respectively. Two single cultivars separately carried Lr26 and Lr34, while Lr37 was not detected in any genotypes in this study. Field evaluation demonstrated that the most frequent Lr1 gene to be ineffective. Kazakhstanskaya 19 and Omskaya 37 had the highest number of resistance genes, three and four Lr genes, respectively. Two gene combinations (Lr1 and Lr68) were detected in Erythrospermum 35 and Astana.
The wheat genotypes from Kazakhstan and Russia differed greatly in leaf rust severity recorded at the adult plant stage in the field in Kazakhstan. This supports previous reports on varietal resistance and variation among Ptr populations in Kazakhstan [27] and in Russia [30]. However, a high genetic similarity was shown in virulence and phenotypic composition between the Omsk (Russia) and North Kazakhstan [28]. This indicates the possibility of joint breeding programs to improve leaf rust resistance in these countries. Several cultivars (Stepnaya 62, Omskaya 37, Avangard, Kazakhstanskaya rannespelaya, and Kazakhstanskaya 25) showed low leaf rust severity, suggesting their potential value as sources of resistance. The resistance of Omskaya 37 was due to the rye translocation 1AL.1RS in combination with ineffective Lr1 and Lr10 and, possibly, by the presence of unknown genes. Kazakhstanskaya 19 showed field resistance provided by partial resistance gene Lr34 in combination with Lr1, Lr28. These findings support previous reports that leaf rust resistance improved wheat germplasm are becoming increasingly available in Kazakhstan [4,14].
A number of varieties (Akmola 2, Chelyaba jubileynaya, Astana 2, and Kazakhstanskaya 19) were susceptible in the seedling stage but moderately resistant in the adult plant stage. Such varieties are valuable in terms of potential sources of adult plant resistance [39]. If the presence of both seedling stage resistance and adult plant resistance (APR) and only APR represent major and minor gene control of resistance, respectively, the set of 30 genotypes of this study are indicative of both types of resistance.
The Ptr population in Kazakhstan is diverse, as indicated by the range of virulence shown by the five different races analyzed in this study. Wheat cultivars possess a range of variability for response to Ptr races, and a number of genotypes differed in their level of disease severity in Kazakhstan, suggesting that Ptr populations differ in various regions of Kazakhstan. This study identified some wheat genotypes highly resistant to leaf rust that may contribute to the improvement of leaf rust resistance. The cultivation of new leaf rust-resistant varieties could help reduce disease epidemics in Kazakhstan. Resistant genotypes could also be used as improved parents in crossing programs to develop new varieties.

Plant Material
The object of the study was represented by a collection of 30 spring wheat Triticum aestivum entries, including 22 registered cultivars from Kazakhstan and 8 cultivars from Russia, which were evaluated for Puccinia triticina resistance in greenhouse studies and field experiments. This germplasm is produced or used in breeding programs of Kazakhstan. The highly susceptible control cultivar Morocco as well as the near-isogenic lines (NILs) of cv. Thatcher was also used in both seedling and field tests.

Experimental Site
Evaluation of field resistance to leaf rust was carried out under conditions of 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 2019 and 2020 cropping seasons. Experiments were conducted with a completely randomized design with two replicates in 1 m 2 . The leaf rust susceptible cultivar Morocco was planted in every 10th row and as a spreader border around the nursery to ensure uniform infection. Fertilizer treatments, 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 [40]. Annual rainfall ranged from 332 to 644 mm during the two years. Experimental plants were sown in 1 m 2 plots in mid-April every two experimental years. Weather conditions in Almaty in 2019 and in 2020 were favorable for the development of leaf rust, and the infection on susceptible checks reached 20S and 40S, respectively; however, there was a severe late development of leaf rust reaching 80% on susceptible check Morocco. The growing seasons were favorable for pathogen infection and disease development. Mean daily temperature and relative humidity showed similar trends in both years. The average maximum air temperature for mid-May in 2019 and 2020 reached 31.3 and 32.5 • C, respectively. From April to June 2019, the mean daily temperature was 11.4, 16.6, and 21.6 • C, respectively, and in 2020, 11.4, 16.6, and 21.8 • C. From April to June 2019, the monthly rainfalls and average relative humidity (RH) were 168, 39, and 72 mm, and 59.5%, respectively, and in 2020, 140, 74, and 30 mm, and 57.3% (www.pogodaiklimat.ru/monitor.php accessed 15 June 2021), conditions highly conducive for leaf rust infection and development.

Race Identification
Race identification was performed under controlled greenhouse conditions at the Research Institute for Biological Safety Problems (RIBSP), Gvardeysky, Zhambyl region, Kazakhstan.
Leaf samples infected with leaf rust Puccinia triticina were randomly collected during the main spring wheat-growing season in East Kazakhstan, Akmola, and Almaty regions of Kazakhstan. Eighty to 100 rust-infected leaves with sporulating pustules were collected from the research stations and fields of farmers from susceptible cultivars (Morocco, Pmayat Azieva, etc.). The diseased leaf samples were pressed in the folds of newspaper, placed in an envelope, and stored at 4 • C until further analysis.

Multiplication and Preservation of Inoculum
The inoculum of Puccinia triticina was multiplied and maintained on the susceptible cultivar Morocco. Seeds of Morocco were sown in 11 × 11 × 15 cm plastic pots and placed at room temperature until germination. Upon germination, seedlings were dislocated to the glasshouse under temperatures of 25-30 • C and 19-21 • C during day and night, respectively. The two-leaf stage seedlings of Morocco were disinfected with Maleic hydrazide (5 mg in 50 mL of water per pot) [42] and inoculated with spores from the infected leaf samples. Four pots of Morocco (five plants per pot) were inoculated with urediniospores from individual rust samples. Inoculated seedlings were incubated in a dew chamber for 24 h at 18-20 • C and 90% humidity before being dislocated to the glasshouse at temperatures of 18-24 • C (day) and 19-21 • C (night). Pustules of leaf rust appeared on the leaves 8-10 days after inoculation, from which inoculum was collected on the 14th day using a mechanical cyclone collector in a zero-size capsule. The inoculum was then preserved in a vacuum glass vial and later transferred to a refrigerator (+4 • C) until further use. A separate collector was used for each isolate and multiplication of culture. Spore collection, storage, and reproduction were then conducted in accordance with the methods of Roelfs et al. [43]. Spores of P. triticina were used to determine the pathotypes of leaf rust isolated from wheat leaves in the different regions of Kazakhstan.

Single Spore Culture
Morocco seedlings (8-9 days old) were inoculated by spraying the urediniospores previously increased and suspended in light paraffin mineral oil (70 ether: 30 oil). Plants were dried for 1 h before they were placed in a dew chamber overnight at 18-20 • C and then transferred to the greenhouse, where temperatures were maintained at 18-24 • C and 19-21 • C during the day and night, respectively. Seven days after inoculation, leaves were trimmed with scissors so that just a single uredium remained on the trimmed upper edge of the leaves and preserved with purity [43].

Virulence Analysis
To study the resistance of wheat germplasm, the pathotypes representing different regions (1 pathotype from the east, 2 from the north, 2 from the southeast) and possessing approximately average virulence to isogenic lines were used. Seedlings were grown in a greenhouse at 18-20 • C, with 16 h of supplementary lighting. The seedlings were inoculated with individual P. triticina isolates with virulence phenotypes QBQ/G, SBR/H, KHT/B, SBP/C, and THT/B 7 d after planting when the primary leaves were fully emerged. Urediniospores of each of 25 isolates (5 × 10 3 spores) were spray inoculated onto a differential host series consisting of 16 wheat single-gene near-isogenic lines known to possess resistance genes (Lr) in a Thatcher genetic background [41]. The inoculated seedlings were air-dried for at least 30 min and were then placed overnight in a mist chamber at 18 • C and 100% RH. After a period of 12 h of high humidity, inoculated differential lines were placed in a temperature-controlled climatic chamber (20 ± 2 • C, 16 h light/8 h dark). IT was scored on the 0 to 4 scale 10-12 d after inoculation [44]. IT 0 had no hypersensitive flecks, necrosis, or uredinia, IT ";" had distinct hypersensitive flecks; IT 1 had small uredinia surrounded by necrosis; IT 2 had small uredinia surrounded by distinct chlorosis; IT 3 had moderate size uredinia without distinct chlorosis, and IT 4 had large uredinia without distinct chlorosis, with larger and smaller uredinia for each IT were indicated by appending + or −, respectively. Isolates with infection types 0-2 and 3-4 were assumed to be avirulent and virulent, respectively. Leaf rust resistance gene postulations were determined based on the similarity of the Thatcher line IT to the entry, as described in Oelke and Kolmer (2004) [45].
The wheat germplasm was evaluated for Lr genes using similar methods as described above. Plant reaction to leaf rust at the seedling stage was evaluated to the same five P. triticina isolates QBQ/G, SBR/H, KHT/B, SBP/C, and THT/B.

Field Evaluation of Adult Plant Resistance
Field plots were inoculated with mixed races of Ptr obtained from 80 to 100 random infected leaf samples collected from the main spring wheat-growing areas of Kazakhstan. Sampling of spores, their storage, and reproduction was carried out according to the methods of Roelfs et al. [43]. The inoculum was multiplied in the greenhouse on cultivar Morocco, and the collected urediniospores were inoculated by a spore: talc mixture (1:100, 20 mg/m 2 ) applied in the tillering stage in spring.
Infection type and severity data were recorded on flag leaves in late May and early June when plots were at boot and milk stages, respectively. The time of second evaluation was also determined when rust severity on the susceptible control Morocco reached 60-80%. Scoring of leaf rust symptoms was performed according to the method developed at the CIMMYT [43]. The five infection types (IT) were: 0-immune; R-resistant; MR-moderately resistant; MS-moderately susceptible; and S-susceptible. Partial resistance in the field was evaluated at boot and milk stages, respectively, using the modified Cobb scale [46]. Leaf rust severities were recorded using three replications, and the means of the replicated data were calculated.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/plants10071484/s1, Table S1: Seedlings reaction of Thatcher Lr genes isogenic lines to eight Prt pathotypes, Kazakhstan, Table S2: Markers and primers used to identify the presence of Lr genes in wheat germplasm.
Author Contributions: A.K. and S.R. conceived the manuscript and designed the research. A.K. and S.R. analyzed the data and wrote the manuscript. M.A., M.K., A.M., Z.K., D.Z. and A.B. generated the phenotypic and genotyping data. All authors reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.