Identification of carriers of the wheat genotypes stem rust ( Puccinia graminis f.sp. tritici ) resistance genes

. Stem rust in recent years has acquired an epiphytotic character, causing significant economic damage for wheat production in some parts of Kazakhstan. On the basis of a race composition study of the stem rust populations collected in 2016-2017 in South-Eastern and northern regions of Kazakhstan were identified. The race differentiation of stem rust using a tester set of 6 Sr genes differentiator lines was carried out. In different conditions of Kazakhstan, the Sr22 and Sr25/Lr19 gene sources are effective. As a result of PCR analysis, the Sr22 gene was found in 5 lines of 6 samples taken for the study: Line C-19SB, Lutescens 7-04-4, Lutescens 220-03-45, GA961662-1-7/TAM107, Line D 25 77. Sr25/Lr19 is a carrier of sources: Lutescens 220-03-45, Advance, Line D 25 77. The Sr22 and Sr25/Lr19 gene was Lutescens 220-03-45 and Line D 25 77. These sources are recommended for inclusion in the breeding process for developing stem rust resistant varieties in the region.


Introduction
The wheat belt of Western Siberia and Northern Kazakhstan covers more than 15 million ha.While moisture stress is the main factor limiting production, rusts also represent a major challenge, especially in years with higher rainfall.Stem rust was not considered economically important until 2015 when a local epidemic occurred in the Omsk region of Russia and neighboring areas of Kazakhstan and affected more than 1 million ha.It occurred again in 2016 though the spread, severity and losses were less.
The spring wheat production region of Northern Kazakhstan and Western Siberia covers around 15 million ha, in a belt 600-1000 km wide between the desert of central Kazakhstan and boreal forests of Siberia (latitude 45-47 north).The crop is grown under continental Severe epidemics of wheat stem rust caused by Puccinia graminis f. sp.tritici (Pgt) have been observed in recent years in major spring wheat producing regions in Kazakhstan.However, information on the virulence structure and race composition of Pgt is currently not available.Stem rust samples were collected in 2015-2018 in three regions of Kazakhstan to determine the virulence diversity and race distribution in the Pgt populations.A total of 203 single-pustule isolates were derived and evaluated on the stem rust differential and supplemental lines and 38 races were identified.Among them, the races QHHSF and THMTF were found in all the regions and in all the years.The races RFRTF, RHMRF, TKRPF and MHCTC were the most common races in the Akmola and Kostanay regions, and the races LHCSF, QKCSF and LKCSF were only widely distributed in East Kazakhstan.The virulence complexity (i.e., number of Sr genes on which the races were virulent) ranged from 5 to 16, with about 40% of the races having 14 or more virulence.The stem rust resistance genes Sr11, Sr13, Sr22, Sr26, Sr31, Sr33 and Sr35 were found to confer resistance to all the races identified during the study period.Hence, these genes can be used as sources of resistance in wheat breeding programmes in Kazakhstan.(Rsaliyev et al. 2020).

Materials and methods
This research was carried out at the fields of the Kazakh Research Institute of Agriculture and Crop Production.Sixteen genotype varieties taken from the International CIMMYT Research Center were selected as the object of the study, and Sonalika entries were selected as the control variety.The local population of Pgt was used as an epidemic material for the Almaty region.In the study of plant resistance to rust during vegetation, the Stackman scale was used (Table 1).CIMMYT Research Center were selected as the object of the study, and Sonalika samples were selected as the control variety.The local population of Puccinia graminis f. sp tritici was used as an epidemic material of the Almaty region.
McIntosh et al in assessing the rate of disease development in cultivated areas.According to this method, 5 types of reaction are considered: 0-immune, without various symptoms of the lesion.Absence of damage based on the physiological incompatibility of the plant and the pathogen.R-resistance, the presence of the ability to resist pathogenic microorganisms (small pustules surrounded by necrosis).MR-normal (small and mediumsized pustules with chlorotic and necrotic zones).MS is moderately intolerant (mediumsized pustules, there is no necrosis, but there may be areas of chlorosis).S-intolerant (large pustules have chlorosis in both regions).(McIntosh et al. 1995).A polymerase chain reaction was isolated from five-day-old seedlings of wheat plants by a well-known method using cetyltrimethylmoniumbromide (CTAB) (Murray et al. 1980).To determine the tolerance genes, the molecular brands of 2 Sr genes (Sr22, Sr25) were used in the work.The conditions of PCR are given in the work.The most optimal conditions were selected for each marker.The amplification products were divided into 2% agarose gel colored with ethidium bromide.GeneRulerTM 100bp polymerase chain reaction (Fermentas) was used as a molecular weight marker.Isogenic lines and varieties with known Sr genes served as a positive control.For PCR amplification, the reaction mixture was 10 µl, it contained 0.2 mm dNTP, 1 × QIAGEN PCR buffer, 0.

Results
Research work in some farms of the fields of spring wheat of the north of Kazakhstan (2020-2022 year), the stem rust (Puccinia graminis f.sp tritici) it can be seen that the pathogen has become more wide spread and developed at a high level over the years.Phytosanitary observations conducted in some farms of Kostanay region in 2020 showed that the prevalence of stem rust was 74.1 %, the development was 26.7%.According to the monitoring carried out in some farms of Akmola region in 2021, the average development of classes was 7.8%, and the prevalence reached an average of 35.9%.In 2022, the incidence of stem rust spread to an average of 44.7% in the first milkweed maturation period, and development increased to an average of 17.7% (Figure 2).3).Thus, as a result of determining the indicators of the biomass index of wheat varieties, we found that the indicators of the biomass index of spring wheat varieties gave an average value.The Lutescens 1062 variety, which had a minimum value during the spike period, showed a scale of 20 S, because the damage to the stem with rust disease was average.The biomass index of the Aktobe 10 Variety, infected with rust on the scale of 90 S, reached 0.34 at the stage of milkweed maturation, the reason for being higher than that of the Lutescens 1062 Variety, the maturation period was late.Carberry, Advance, Uralo-Siberian, Muchmore, Line D 25 genotypes had a resistivity to stem rust disease, the reason for which the NDVI index was maximum (Table 3).
Under the conditions of Kazakhstan, most wheat varieties are intolerant for the local stem rust population, and most of them accept two or more pathotypes if they are resistant to one stem rust pathotype.At the same time, the effectiveness of SR-tolerance genes is gradually being lost.In connection with these urgent problems, Puccinia graminis f.sp.tritici in all periods of vegetation of the plant, it is important to find tolerance genes and gene owners (sorgenotypes) that allow pathotypes to be effectively protected.For this purpose, 7 wheat samples were selected from the international CIMMYT Research Center.The disease was studied in laboratory conditions and in an introduction nursery (Table 4).
As a result of the study of the resistance of wheat samples in a greenhouse to stem rust during germination of Puccinia graminis f. sp tritici 1 wheat samples to the pathogen were identified as immunodeficient: GA961662-1-7/tam107, samples Line c-19sb, Lutescens 7-04-4, Lutescens 220-03-45, Advance and Line D 25 77, which showed 1 point of reaction to the disease, were found to be resistant.
Puccinia graminis f in an artificial epidemic environment in a field.GA961662-1-7/The TAM107 models were distinguished by immunity.Puccinia graminis f. sp tritici it is possible to note the samples Lutescens 7-04-4 and Advance which showed resistance to the pathogen with the reaction index "R".The classes showed the average endurance of the samples Line c-19sb, Lutescens 220-03-45, Line D 25 77 and Sonalika the indicator of infection with the disease of which was 5 MR.One of the ways to strengthen the result of breeding programs is the use of molecular markers along with classical methods.The use of molecular markers significantly expands the ability to assess plant diseases and tolerance genes.The method of using molecular markers associated with genes that ensure plant resistance to the pathogen increases the speed of the selection process.With this method, research work can be carried out at any stage of plant development and independently of the state of the environment.During the research work, work was carried out on identification of wheat E3S Web of Conferences 463, 01003 (2023) EESTE2023 https://doi.org/10.1051/e3sconf/202346301003varieties resistant to the disease of stem rust.To achieve this goal, the rust stalk was conducted screening of polymerase chain reaction genotypes of wheat varieties using markers associated with rust resistance genes.
As a result of the correlation analysis of spring wheat genotypes between the length of the main ears and the number of ears, the correlation coefficient R=0.63 in these two indicators was positively correlated.

Discussion
In conclusion, phytopathological and molecular genetic studies of 6 wheat samples taken from the CIMMYT International Research Center were carried out.In the Almaty region, the population of Puccinia graminis F. sp tritici during the wheat germination period, 5 wheat samples were isolated as immune, these are GA961662-1-7/TAM107.
Rust on the stem in an artificial epidemic environment in field conditions GA961662-1-7/The TAM107 models were distinguished by immunity.PCR analysis was performed to identify rust-resistant sources of the Sr22 and Sr25/Lr19 genes.As a of the study, it was found that 6 samples are carriers of the Sr22 gene they, Line c-19SB, Lutescens 7-04-4, Lutescens 220-03-45, GA961662-1-7/tam107 and Line D 25 77.The tasmanian of the Sr25 gene also revealed 3 samples: Lutescens 220-03-45, Advance and Line D 25 77.

Conclusion
Stem rust is common in the regions of Kazakhstan where wheat is grown.Wheat Puccinia graminis f.sp.protection against the pathogen tritici, despite all the studies of this disease, remains an urgent problem.Chemical control of the disease is ineffective and does not give accurate results, the only effective way to combat the disease is to produce samples and varieties that are resistant to the disease.Currently, about 50 genes that show resistance to this disease are known.In different conditions of Kazakhstan, the Sr22 and Sr25/Lr19 gene E3S Web of Conferences 463, 01003 (2023) EESTE2023 https://doi.org/10.1051/e3sconf/202346301003sources are effective.Molecular screening of SSR and STS type markers (Sequence Tagged Sites) was performed using Xgwm533 and PSY-E1.As a result of PCR analysis, the Sr22 gene was found in 5 lines of 6 samples taken for the study: Line C-19SB, Lutescens 7-04-4, Lutescens 220-03-45, GA961662-1-7/TAM107, Line D 25 77.Sr25/Lr19 is a carrier of H sources: Lutescens 220-03-45, Advance, Line D 25 77.These lines can be used in Marker Assisted Selection programs to increase rust resistance in the stem.

Fig. 2 .
Fig. 2. Diagram of distribution of spring wheat varieties by susceptibility to stem rust during (2020-2022).Normalized Difference Vegetation Index (NDVI) is a commonly used metric to quantify the density and health of vegetation.Its values range from -1 to 1, with negative values indicating water or bare soil, values near zero indicating sparse vegetation, and higher values indicating denser and healthier vegetation (Table3).

Fig. 3 .
Fig. 3. Correlation indicator of spring wheat genotypes between the height of the main plant and the number of spikelets.The correlation between the main length of the ears and the number of ears of spring wheat varieties, between the height of the plant and the number of ears (R=0.63 and R=0.42) was positive and moderately positive.

Table 1 .
Signs of infestation of wheat varieties with stem rust disease.

Table 2 .
Description of the PCR program and molecular markers used in gene identification.

Table 4 .
Resistance of wheat genotypes to rust stem using the Sr molecular marker identification of resistant genes.