2. The development of the initial material of spring and winter wheat with several Sr genes resistant to Puccinia graminis f. sp. tritici

#### 2.1. The modern phytopathological stem rust situation in CFDR and possible threats

The situation in the CFDR reflects the general trend observed in the populations of P. graminis in all areas of pathogen distribution; the fungus actively evolves. Differences concern only the speed and genes of the pathogen virulence, depending on the geographical location. In the case of Ug99 (TTKSK), the process is very fast (in 18 years, 13 biotypes of the fungus appeared); on the other hand, in the territory of CFDR, the change of the dominant races took place for 57 years. The phytopathological situation is complicated by the proximity of the CFDR to European countries, where aggressive pathogens of P. graminis have been identified recently. Six races (TKTTF, TKKTF, TKPTF, TKKTP, PKPTF and MMMTF) were retrieved from 48 isolates, obtained from the P. graminis population in 2013 in Germany [2]. The detection of the TKKTP race causes concern because of its virulence to the Sr24, SrTmp and Sr1RSAmigo genes, although it has been determined that none of these races belongs to the race group TTKSK (Ug99), and the German isolates of the TKTTF race are phenotypically different from the TKTTF race that caused plant disease epidemic in Ethiopia in 2013/2014. It is known that 55% of North American and international cultivars and selection lines resistant to the race TTKSK (Ug99) are susceptible to the TKKTP race [2]. On the Italian island of Sicily, a new race of stem rust, the TTTTF, hit several thousand hectares of durum wheat in 2016, leading to the largest outbreak of stem rust in Europe in recent decades. TTTTF is a newly identified race of stem rust that can soon spread over long distances along the Mediterranean basin and the Adriatic coast [3] (http://www.fao.org/news/story/en/item/469467/icode/).

The analysis of the racial composition of R. graminis f. sp. tritici in CFDR was held annually since 1960. During this time, significant changes occurred in the composition of the dominant races. In the 1960s–1970s, the population of stem rust included physiological races 21, 17 and 34 according to Stakman's nomenclature [4]. Races 11 and 14 were detected regularly, but were not widely distributed. In the 1960s–1970s, only the resistance genes Sr7b and Sr9g were completely ineffective. Virulence to the genes Sr5, Sr21, Sr9e, Sr11, Sr6, Sr8a, Sr36, Sr9b, Sr30 and Sr17 was low or absent [5]. In subsequent years, the fungal pathotypes, virulent to the resistance genes Sr5, Sr21, Sr6, Sr8a and Sr17, appeared. Races of the pathogen MKCT, MKCK, MKBK, MKBS, MKBT, RKCT and RKBS dominated in CFDR in 2004 [6]. During this period, the Sr9e, Sr11, Sr36, Sr9b and Sr30 genes were effective. Over the past decade, the structure of the population on the basis of virulence has changed toward the predominance of several aggressive virulent races, including races that are virulent to genes Sr5, Sr21, Sr9e, Sr7b, Sr6, Sr8a, Sr9g, Sr36, Sr30, Sr9a, Sr9d, Sr10 and SrTmp. Among samples from the European part of the Russian Federation, the races of stem rust MKBT and MRLT in 2002 and TKNT, TKST, TTNT in 2005 dominated [7]. The race composition of P. graminis f. sp. tritici populations in the CFDR in the period 2000–2009 is presented in the work of Skolotneva et al. [8]. They analyzed 387 isolates of the fungus using the North American set of differentiators. Samples were obtained from cereals (wheat and barley), wild herbs and barberry. As a result of the study, 45 races of P. graminis f. sp. tritici were identified. The predominant races TKNT and TKNTF were isolated. The Ug99 race and its derivatives were not found in the Russian Federation.

plants by morphotype with parallel identification of Sr genes. Sixth stage: Testing the progeny of individual spring wheat plants against the infectious background for the North Caucasian and West Siberian populations of stem rust, and plants of winter wheat for the North Caucasian population of stem rust (infectious background) and natural epidemic development of stem rust in the Moscow Oblast. Seventh stage: Evaluation of the economically valuable traits of selected stable lines in the Moscow Oblast conditions in comparison with standard cultivars,

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Schematically, the process of breeding lines with several effective Sr genes can be represented

The identification of the resistance sources to the race Ug99 of stem rust was started by an employee of FSBSI VIZR Anna Anisimova together with the scientists from Minnesota University (USA) in 2010. At the seedling stage, 386 accessions of bread wheat from the collection of VIR and the "Arsenal" collection from Moscow Sc. Res. Inst. of Agr. "Nemchinovka" were evaluated, six accessions of winter wheat and one accession of spring wheat with resistance to this disease were selected (type of reaction during pathogen penetration from 0 to 2) [11]. It is the selection line GT 96/90 (hereinafter referred to as line 96) from Bulgaria with genetic material of the species T. timopheevii, a cultivar of winter wheat Donskaya polukarlikovaya

selection of the best genotypes for competitive testing.

in the following sections.

2.2.1. First stage

According to the data obtained in 2013 [9], during assessing the collection of lines with known Sr genes, the effective genes of resistance to stem rust in CFDR were the genes Sr2, Sr9e, Sr13, Sr25, Sr26, Sr31, Sr32, Sr36, Sr44, SrWld and the combination of genes Sr17 + Sr13, Sr31 + Sr38. According to the data of Skolotneva et al. [8], the resistance genes Sr9e and Sr36 in 2009 were ineffective in the Central region of Russia. Differences in the data are probably due to a change in the composition of the pathogen population. Thus, in the same work of Skolotneva, a change in the percentage of fungal isolates virulent to the Sr17 gene was noted from 92.5% in 2000 to 0% in 2008, and the genes of Sr31 and Sr24 remained effective against all local races of stem rust. Observations of the pathogen development in the period 2013–2017, conducted in the All-Russian Research Institute of Phytopathology, showed the annual development of stem rust. The development of the disease on the susceptible genotype of Khakasskaya in 2017 reached 100% [10].

#### 2.2. Development of bread wheat lines with several resistance Sr genes

The process of creating the initial material of bread wheat with several resistance genes passed in several stages. First stage: Isolation of resistance sources in the seedling stage; evaluation of the spring bread wheat line against the background of Ug99 natural infection in Ethiopia. Second stage: Identification of resistance Sr genes using specific molecular markers and isolation of resistance donors. Third stage: Selection of pairs for crossing and hybridization of donors among themselves, obtaining of the segregative population of hybrids F2. Fourth stage: Performing backcrossing by one of the recurrent parents or stepwise hybridization of individual plants by the third parent in the field against the infectious background of leaf rust; subsequent self-pollination or repeated backcrossing with test of progeny against infectious background of leaf rust. At this stage, work with spring and winter plants was carried out in parallel at different plots and with different planting times. Fifth stage: Selection of individual plants by morphotype with parallel identification of Sr genes. Sixth stage: Testing the progeny of individual spring wheat plants against the infectious background for the North Caucasian and West Siberian populations of stem rust, and plants of winter wheat for the North Caucasian population of stem rust (infectious background) and natural epidemic development of stem rust in the Moscow Oblast. Seventh stage: Evaluation of the economically valuable traits of selected stable lines in the Moscow Oblast conditions in comparison with standard cultivars, selection of the best genotypes for competitive testing.

Schematically, the process of breeding lines with several effective Sr genes can be represented in the following sections.

#### 2.2.1. First stage

34 according to Stakman's nomenclature [4]. Races 11 and 14 were detected regularly, but were not widely distributed. In the 1960s–1970s, only the resistance genes Sr7b and Sr9g were completely ineffective. Virulence to the genes Sr5, Sr21, Sr9e, Sr11, Sr6, Sr8a, Sr36, Sr9b, Sr30 and Sr17 was low or absent [5]. In subsequent years, the fungal pathotypes, virulent to the resistance genes Sr5, Sr21, Sr6, Sr8a and Sr17, appeared. Races of the pathogen MKCT, MKCK, MKBK, MKBS, MKBT, RKCT and RKBS dominated in CFDR in 2004 [6]. During this period, the Sr9e, Sr11, Sr36, Sr9b and Sr30 genes were effective. Over the past decade, the structure of the population on the basis of virulence has changed toward the predominance of several aggressive virulent races, including races that are virulent to genes Sr5, Sr21, Sr9e, Sr7b, Sr6, Sr8a, Sr9g, Sr36, Sr30, Sr9a, Sr9d, Sr10 and SrTmp. Among samples from the European part of the Russian Federation, the races of stem rust MKBT and MRLT in 2002 and TKNT, TKST, TTNT in 2005 dominated [7]. The race composition of P. graminis f. sp. tritici populations in the CFDR in the period 2000–2009 is presented in the work of Skolotneva et al. [8]. They analyzed 387 isolates of the fungus using the North American set of differentiators. Samples were obtained from cereals (wheat and barley), wild herbs and barberry. As a result of the study, 45 races of P. graminis f. sp. tritici were identified. The predominant races TKNT and TKNTF were isolated. The Ug99 race and its derivatives were not found in the Russian Federation.

According to the data obtained in 2013 [9], during assessing the collection of lines with known Sr genes, the effective genes of resistance to stem rust in CFDR were the genes Sr2, Sr9e, Sr13, Sr25, Sr26, Sr31, Sr32, Sr36, Sr44, SrWld and the combination of genes Sr17 + Sr13, Sr31 + Sr38. According to the data of Skolotneva et al. [8], the resistance genes Sr9e and Sr36 in 2009 were ineffective in the Central region of Russia. Differences in the data are probably due to a change in the composition of the pathogen population. Thus, in the same work of Skolotneva, a change in the percentage of fungal isolates virulent to the Sr17 gene was noted from 92.5% in 2000 to 0% in 2008, and the genes of Sr31 and Sr24 remained effective against all local races of stem rust. Observations of the pathogen development in the period 2013–2017, conducted in the All-Russian Research Institute of Phytopathology, showed the annual development of stem rust. The development of the disease on the susceptible genotype of Khakasskaya in 2017

The process of creating the initial material of bread wheat with several resistance genes passed in several stages. First stage: Isolation of resistance sources in the seedling stage; evaluation of the spring bread wheat line against the background of Ug99 natural infection in Ethiopia. Second stage: Identification of resistance Sr genes using specific molecular markers and isolation of resistance donors. Third stage: Selection of pairs for crossing and hybridization of donors among themselves, obtaining of the segregative population of hybrids F2. Fourth stage: Performing backcrossing by one of the recurrent parents or stepwise hybridization of individual plants by the third parent in the field against the infectious background of leaf rust; subsequent self-pollination or repeated backcrossing with test of progeny against infectious background of leaf rust. At this stage, work with spring and winter plants was carried out in parallel at different plots and with different planting times. Fifth stage: Selection of individual

2.2. Development of bread wheat lines with several resistance Sr genes

reached 100% [10].

186 Global Wheat Production

The identification of the resistance sources to the race Ug99 of stem rust was started by an employee of FSBSI VIZR Anna Anisimova together with the scientists from Minnesota University (USA) in 2010. At the seedling stage, 386 accessions of bread wheat from the collection of VIR and the "Arsenal" collection from Moscow Sc. Res. Inst. of Agr. "Nemchinovka" were evaluated, six accessions of winter wheat and one accession of spring wheat with resistance to this disease were selected (type of reaction during pathogen penetration from 0 to 2) [11]. It is the selection line GT 96/90 (hereinafter referred to as line 96) from Bulgaria with genetic material of the species T. timopheevii, a cultivar of winter wheat Donskaya polukarlikovaya (hereinafter referred to as D) in the pedigree of which Aegilops tauschii was present (accessions from the collection of VIR). From the collection, "Arsenal" lines with translocations from Ae. speltoides were selected: 9/00w (2n = 42), disomic addition lines of Ae. speltoides: 19/95w and 141/97w (2n = 44); wheat-Ae. speltoides-rye line 119/4-06rw (2n = 42), hereinafter referred to as line 119. The only stable accession of spring wheat 113/00i-4 (2n = 42) (in the text accession 113), obtained from crossing the spring cultivar Rodina with irradiated pollen of the species Ae. triuncialis [12] and then crossed with the line with the genetic material of T. kiharae, showed immunity to the natural infection of stem rust race Ug99 in Ethiopia at the stage of an adult plant [13].

3 h in 0.5 TBE buffer. As markers of molecular weights, 50 bp, 100 bp and 1 kb GeneRulerTM DNA Ladder from "Fementas" were used. The results of gene identification in new sources are

Genetic Improvement of Bread Wheat for Stem Rust Resistance in the Central Federal Region of Russia: Results…

We explain a wide range of identified genes in donors from the "Arsenal" collection by multiple alien translocations of the genetic material of species Aegilops speltoides, Ae. triuncialis, Triticum kiharae, Secale cereale, arising during the irradiation of pollen, and in the selection line GT 96/90 by the presence of translocations from the species T. timopheevii. The use of such donors, even in paired crosses, can lead to the creation of plant forms with an unusual combination of resistance Sr genes due to the recombination of genes in meiosis. However, since we were faced with the task of obtaining the initial material for the selection process, we had to take into account not only the level of donors ploidy but also the presence of economically valuable traits. It should be noted that despite the positive identification of the Sr22 gene in the wheat-Aegilops lines (9/00w, 141/97w and 119/4-06rw) using the Xbarc121 and Xcfa2123 markers, the absence in the pedigree of these lines of the genetic material T. monococcum leaves

We rejected the use of disomic addition lines with chromosomes of Aegilops speltoides in the selection of pairs for crossing, since the supplemented alien chromosome with which we bind resistance was rarely conjugated to wheat chromosomes and was lost in the process of division in meiosis. The remaining donors had an euploid number of chromosomes, but were different according to the morphophysiological and agronomic characteristics (terms of ear formation, height, susceptibility to powdery mildew). The D cultivar and the GT 96/90 line had a very short stem (60–70 cm), early ear formation (late May to early June) and were affected by powdery mildew to a high degree (severity 30–50%), remaining resistant to leaf rust. For donors from the "Arsenal" collection, on the contrary, later ear formation, long stem, but the high resistance to powdery mildew and leaf rust were characteristic. Parent pairs for crossing were selected with alternative development of traits (short stem, early ear formation,

mildew

Sr32, Sr44, Sr9a, Sr17, Sr19 50 5/2 60–70 1.3 40.0

Table 2. Results of the identification of Sr genes in resistance donors to stem rust [32] and their economically useful traits.

cm

Leaf rust

weight, g Powdery

Grain weight per ear, g

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1000 grains

Line, cultivar Identified Sr genes Severity, % Height,

9/00w Sr22, Sr32, Sr44, Sr15 0–5 0 70–80 1.5 40.0 141/97w Sr22, Sr44 0–10 0 90–110 1.4 36.0 113/00i-4 Sr2, Sr36, Sr39, Sr40, Sr44, Sr47, Sr15 0–1 0 90–110 1.4 41.0 119/4-06rw Sr22, Sr32, Sr44, Sr9a, Sr17, Sr19 0–1 10/1 80–100 1.5 42.0 GT 96/90 Sr24,Sr36, Sr40, Sr47, Sr15, Sr17, Sr31 30 0 60–70 1.5 42.0

given in Table 2 [32].

doubt in the presence of this gene.

2.2.3. Third stage

Donskaya polukarlikovaya

### 2.2.2. Second stage

Identification of resistance Sr genes was carried out using molecular markers both to effective genes against the Ug99 race (Sr2, Sr22, Sr24, Sr25, Sr26, Sr32, Sr35, Sr36, Sr39, Sr40, Sr44, Sr47) and to ineffective ones: Sr9a, Sr15, Sr17, Sr19 and Sr31, but providing resistance to local populations of the pathogen. The list of molecular markers is given in Table 1. For each primer, the most optimal PCR conditions were selected; the conditions given in the original studies are taken as basis. Separation of amplification products was performed by electrophoresis in 2% agarose and 8% polyacrylamide gels stained with ethidium bromide at a voltage of 100 V for


Table 1. Specific primers used to identify Sr genes.

3 h in 0.5 TBE buffer. As markers of molecular weights, 50 bp, 100 bp and 1 kb GeneRulerTM DNA Ladder from "Fementas" were used. The results of gene identification in new sources are given in Table 2 [32].

We explain a wide range of identified genes in donors from the "Arsenal" collection by multiple alien translocations of the genetic material of species Aegilops speltoides, Ae. triuncialis, Triticum kiharae, Secale cereale, arising during the irradiation of pollen, and in the selection line GT 96/90 by the presence of translocations from the species T. timopheevii. The use of such donors, even in paired crosses, can lead to the creation of plant forms with an unusual combination of resistance Sr genes due to the recombination of genes in meiosis. However, since we were faced with the task of obtaining the initial material for the selection process, we had to take into account not only the level of donors ploidy but also the presence of economically valuable traits. It should be noted that despite the positive identification of the Sr22 gene in the wheat-Aegilops lines (9/00w, 141/97w and 119/4-06rw) using the Xbarc121 and Xcfa2123 markers, the absence in the pedigree of these lines of the genetic material T. monococcum leaves doubt in the presence of this gene.

#### 2.2.3. Third stage

(hereinafter referred to as D) in the pedigree of which Aegilops tauschii was present (accessions from the collection of VIR). From the collection, "Arsenal" lines with translocations from Ae. speltoides were selected: 9/00w (2n = 42), disomic addition lines of Ae. speltoides: 19/95w and 141/97w (2n = 44); wheat-Ae. speltoides-rye line 119/4-06rw (2n = 42), hereinafter referred to as line 119. The only stable accession of spring wheat 113/00i-4 (2n = 42) (in the text accession 113), obtained from crossing the spring cultivar Rodina with irradiated pollen of the species Ae. triuncialis [12] and then crossed with the line with the genetic material of T. kiharae, showed immunity to the natural infection of stem rust race Ug99 in Ethiopia at the stage of an adult

Identification of resistance Sr genes was carried out using molecular markers both to effective genes against the Ug99 race (Sr2, Sr22, Sr24, Sr25, Sr26, Sr32, Sr35, Sr36, Sr39, Sr40, Sr44, Sr47) and to ineffective ones: Sr9a, Sr15, Sr17, Sr19 and Sr31, but providing resistance to local populations of the pathogen. The list of molecular markers is given in Table 1. For each primer, the most optimal PCR conditions were selected; the conditions given in the original studies are taken as basis. Separation of amplification products was performed by electrophoresis in 2% agarose and 8% polyacrylamide gels stained with ethidium bromide at a voltage of 100 V for

Sr gene Chromosome Marker References Sr2 3B Xgwm533 [14] Sr9a 2BL Xgwm47 [15, 16] Sr15 7AL STS638 [17] Sr17 7BL Wpt5343 [18] Sr19 2BS Wpt9402 [18, 19] Sr22 7AL Xbarc121, cfa2123 [20, 21] Sr24/Lr24 3DL/1BS Sr24#12, Sr24#50 [22] Sr25/Lr19 7DL Gb [23] Sr26 6AL Sr26#43 [22] Sr31 1R/1B Scm9 [24] Sr32 2AS,2B Xbarc55, Xstm773 [19, 25, 26] Sr35 3AL Xcfa2170, BE485004 [27] Sr36 2BS Xwmc477, Xstm773-2 [28] Sr39 2BS Sr39#22 [29] Sr40 2BS Xgwm344 [30] Sr44 7DS Wpt2565 [18] Sr47 T2BL-2SL2SS Xgwm501 [31]

plant [13].

188 Global Wheat Production

2.2.2. Second stage

Table 1. Specific primers used to identify Sr genes.

We rejected the use of disomic addition lines with chromosomes of Aegilops speltoides in the selection of pairs for crossing, since the supplemented alien chromosome with which we bind resistance was rarely conjugated to wheat chromosomes and was lost in the process of division in meiosis. The remaining donors had an euploid number of chromosomes, but were different according to the morphophysiological and agronomic characteristics (terms of ear formation, height, susceptibility to powdery mildew). The D cultivar and the GT 96/90 line had a very short stem (60–70 cm), early ear formation (late May to early June) and were affected by powdery mildew to a high degree (severity 30–50%), remaining resistant to leaf rust. For donors from the "Arsenal" collection, on the contrary, later ear formation, long stem, but the high resistance to powdery mildew and leaf rust were characteristic. Parent pairs for crossing were selected with alternative development of traits (short stem, early ear formation,


Table 2. Results of the identification of Sr genes in resistance donors to stem rust [32] and their economically useful traits.

susceptibility to powdery mildew) x (long stem, later ear formation, resistance to powdery mildew). The first crossings were conducted in 2010 in the greenhouse. The following pairs of direct crossing and backcrossing were successful: (GT 96/90 113/00i-4), (119/96rw GT 96/ 90), (113/00i-4 119/96rw). In the conditions of the greenhouse, the D cultivar was found to be the earliest ripening, and it was not possible to hybridize with it because of the mismatch of the flowering periods. Later, this cultivar was used in stepwise hybridization. F1 plants were also grown in the greenhouse. The fact of the segregation of future F2 populations into winter and spring genotypes from the crossing of winter lines with the spring line 113/00i-4 was taken into account when planning crossings. Crossing with the productive wheat-Ae. speltoides line 145/05i (grain weight from ear is 1.9 g, weight of 1000 grains is 49.0 g), which had group resistance to powdery mildew and leaf rust, but was susceptible to Ug99, was additionally planned in order to shift the formative process toward the isolation of productive spring forms of plants.

#### 2.2.4. Fourth stage

Beginning with F2, the work with spring and winter forms of plants was carried out against the infectious background of leaf rust at different seeding times. Half of the seeds were sown in February in the heated plot after snow melting. After the emergence of shoots, the heating of the soil was switched off, and the plants passed vernalization at natural low temperatures and natural snow cover. In this case, spring plants perished, and winter plants formed the ear. The second half of the seeds were sown in the field in spring. Under these conditions, spring plants formed the ear, and winter crops remained in the tillering phase. Backcrossing of plants resistant to leaf rust, beginning with F2, was conducted by recurrent spring parent or line 145/05i (when working with spring genotypes) and winter recurrent parent or D cultivar (when creating winter wheat lines). The infectious background of leaf rust was created using all races characteristic for the Moscow Oblast. For further hybridization, only plants resistant to leaf rust were selected. The second backcrossing or self-pollination was carried out under the conditions of a greenhouse, the progeny was sown on the appropriate soil background, and the process of backcrossing on the infectious background of leaf rust was repeated again. Then self-pollination of plants was carried out. The scheme of the selection process for obtaining spring and winter lines with several resistance Sr genes is shown in Figure 2.

#### 2.2.5. Fifth stage

In the progeny of self-pollinated plants, which were sown as lines of different generations BC1F3, BC2F2, BC3F2, F4, F5, individual plants were selected by morphotype with parallel identification of Sr genes by PCR analysis. During the selection, attention was drawn to the habitus of the plant (bush form, the number of productive shoots), the location of the leaves, the shape of the ear, the presence of marker morphological features (the presence of awns and anthocyanin on different parts of the plant such as stem, ear, anther), the date of ear formation and the degree of severity of affection by powdery mildew and leaf rust were taken into account. Preference was given to plants with group resistance to diseases, with optimal plant height (80–110 cm), early ripening and an ear with 19–21 developed spikelets, that is, the selection of individual plants was not accidental, but aimed to combining economically valuable traits. In such plants, a piece of leaf was taken to isolate DNA and to identify Sr genes. In total 200 spring plants and more than 200 winter plants were selected for PCR analysis. The

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In spring plants, the genes Sr2, Sr44, Sr36 and Sr40 were found most often in the homozygous state (71, 89, 78 and 26%, respectively, of the number of plants tested). The Sr22 gene, which was originally identified in the winter donor 119/4-06rw using two markers Xbark 121 and cfa 2123, was detected in the progeny of selected spring plants at a frequency of 20%, when the donor was used for backcrossing and the resulting progeny was self-pollinated. The Sr39 and

spectrums of identified effective Sr genes in spring and winter plants differed.

Figure 2. Scheme of development of spring and winter wheat lines with several resistance Sr genes.

Genetic Improvement of Bread Wheat for Stem Rust Resistance in the Central Federal Region of Russia: Results… http://dx.doi.org/10.5772/intechopen.75379 191

susceptibility to powdery mildew) x (long stem, later ear formation, resistance to powdery mildew). The first crossings were conducted in 2010 in the greenhouse. The following pairs of direct crossing and backcrossing were successful: (GT 96/90 113/00i-4), (119/96rw GT 96/ 90), (113/00i-4 119/96rw). In the conditions of the greenhouse, the D cultivar was found to be the earliest ripening, and it was not possible to hybridize with it because of the mismatch of the flowering periods. Later, this cultivar was used in stepwise hybridization. F1 plants were also grown in the greenhouse. The fact of the segregation of future F2 populations into winter and spring genotypes from the crossing of winter lines with the spring line 113/00i-4 was taken into account when planning crossings. Crossing with the productive wheat-Ae. speltoides line 145/05i (grain weight from ear is 1.9 g, weight of 1000 grains is 49.0 g), which had group resistance to powdery mildew and leaf rust, but was susceptible to Ug99, was additionally planned in order to shift the formative process toward the isolation of productive spring forms

Beginning with F2, the work with spring and winter forms of plants was carried out against the infectious background of leaf rust at different seeding times. Half of the seeds were sown in February in the heated plot after snow melting. After the emergence of shoots, the heating of the soil was switched off, and the plants passed vernalization at natural low temperatures and natural snow cover. In this case, spring plants perished, and winter plants formed the ear. The second half of the seeds were sown in the field in spring. Under these conditions, spring plants formed the ear, and winter crops remained in the tillering phase. Backcrossing of plants resistant to leaf rust, beginning with F2, was conducted by recurrent spring parent or line 145/05i (when working with spring genotypes) and winter recurrent parent or D cultivar (when creating winter wheat lines). The infectious background of leaf rust was created using all races characteristic for the Moscow Oblast. For further hybridization, only plants resistant to leaf rust were selected. The second backcrossing or self-pollination was carried out under the conditions of a greenhouse, the progeny was sown on the appropriate soil background, and the process of backcrossing on the infectious background of leaf rust was repeated again. Then self-pollination of plants was carried out. The scheme of the selection process for obtaining spring and winter lines with several resistance Sr genes is

In the progeny of self-pollinated plants, which were sown as lines of different generations BC1F3, BC2F2, BC3F2, F4, F5, individual plants were selected by morphotype with parallel identification of Sr genes by PCR analysis. During the selection, attention was drawn to the habitus of the plant (bush form, the number of productive shoots), the location of the leaves, the shape of the ear, the presence of marker morphological features (the presence of awns and anthocyanin on different parts of the plant such as stem, ear, anther), the date of ear formation and the degree of severity of affection by powdery mildew and leaf rust were taken into account. Preference was given to plants with group resistance to diseases, with optimal plant height (80–110 cm), early ripening and an ear with 19–21 developed spikelets, that is, the

of plants.

2.2.4. Fourth stage

190 Global Wheat Production

shown in Figure 2.

2.2.5. Fifth stage

Figure 2. Scheme of development of spring and winter wheat lines with several resistance Sr genes.

selection of individual plants was not accidental, but aimed to combining economically valuable traits. In such plants, a piece of leaf was taken to isolate DNA and to identify Sr genes. In total 200 spring plants and more than 200 winter plants were selected for PCR analysis. The spectrums of identified effective Sr genes in spring and winter plants differed.

In spring plants, the genes Sr2, Sr44, Sr36 and Sr40 were found most often in the homozygous state (71, 89, 78 and 26%, respectively, of the number of plants tested). The Sr22 gene, which was originally identified in the winter donor 119/4-06rw using two markers Xbark 121 and cfa 2123, was detected in the progeny of selected spring plants at a frequency of 20%, when the donor was used for backcrossing and the resulting progeny was self-pollinated. The Sr39 and Sr47 genes were rare, with a frequency of 4.4 and 1.4%, respectively. After PCR analysis, 137 individual plants with several Sr genes in the homozygous state were selected from 200 spring plants, namely: with two resistance genes—54 plants, with three—64 plants, with four—15 plants and with five genes—4 plants.

wheat, only 198 spring plants were selected for further testing: 129 plants with identified Sr

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Progeny testing of individual spring wheat plants was carried out against the infectious background for the North Caucasian and West Siberian populations of stem rust and leaf rust, and against the natural background of the disease course in the Moscow Oblast. It should be clarified that in the south of Russia (Krasnodar Krai), most of the known resistance genes are ineffective against the causative agent of stem rust. Genes Sr1, Sr5, Sr6, Sr9a, Sr9e, Sr13, Sr24, Sr27, Sr31, Sr32, Sr35, Sr36 remain effective [33]. One hundred and fifty-eight lines of spring wheat (or 81% of the number of studied lines) showed high resistance to infection (0R) by the North Caucasian population of stem rust, and 160 lines were resistant to

Testing of the same set of spring lines in Western Siberia (Omsk), which were sown with a special late spring sowing (late crops are more affected than those sown in the optimal time) led to the death of some lines, but from the 167 surviving lines, 111 lines (66.5%) with resistance to stem rust were selected. In the year of testing (2015), strong epidemic of stem rust was observed in the region. Under these conditions, only a small group of genes, according to the observations of the researchers, was effective (Sr2, Sr9e, Sr11, Sr12, Sr13, Sr19, Sr24, Sr25, Sr26, Sr27, Sr30, Sr31, Sr35 Sr37), but none of these genes provided full protection against the disease. The severity of lines with known resistance genes varied from 5 to 30% in comparison with 50–60% severity of cultivars without effective genes [34]. Selected in such harsh conditions, stable lines with group resistance to stem and leaf rust are valuable initial material for the selection of spring wheat in this region. Structural analysis performed in comparison with the standard cultivar Omskaya 37 allowed to select 20 lines with the least decrease in productivity in the unfavorable dry conditions of Western Siberia. In 2016, these lines were involved in crosses with the best adapted varieties culti-

In the Moscow Oblast, in 2015, no development of stem and leaf rust was observed even on the highly susceptible line Khakasskaya because of unfavorable weather conditions for the development of these pathogens (low air humidity, lack of dew, strong wind). However, in the Moscow suburbs, the spring lines were evaluated for resistance to powdery mildew. After that, the results of lines estimates at three geographic locations were combined, and genotypes that showed resistance simultaneously to leaf and stem rust in Krasnodar and Omsk and resistance to powdery mildew in the Moscow Oblast (71 genotypes) were selected. In 2016, under the conditions of epidemic development of stem rust in the Moscow Oblast, after negative selection for resistance to diseases, the timing of the ear formation, height and the presence of segregation by morphological features, 40 genotypes were left for further tests. After the statistical evaluation of the productivity elements (yield of grain from 0.3 m2

ductivity of the ear, weight of 1000 grains), 25 best genotypes with a set of economically

, pro-

genes and 69 plants with a set of valuable traits.

vated in this region (Shamanin, personal communication).

valuable traits were selected (see Stage 7).

2.2.6. Sixth stage

leaf rust.

2.2.6.1. Spring wheat

In individual winter plants, selected from the hybrid population represented by the families F3, BC1F2, BC1F3, BC2F3, BC3F2 of different origin, eight genes were identified, which form a row: Sr2 > Sr44 > Sr32 > Sr36 > Sr22 > Sr31 > Sr47 > Sr39 and Sr40 by the frequency of occurrence in progeny. The combination spectrum of the identified genes in winter wheat plants differed from the spectrum of genes identified in spring wheat lines. This is connected with the orientation of backcrossings conducted in winter and spring wheat. The combination of Sr genes compound in the genotypes of winter wheat is more diverse. The plants with the combination of the Sr22, Sr32 and Sr44 genes in the homozygous state were most often encountered. Plants with a unique combination of genes characteristic only of winter plants have been found: Sr2 + Sr22, Sr2 + Sr32, Sr2 + Sr36, Sr36 + Sr44, Sr36 + Sr47, Sr32 + Sr44, Sr22 + Sr44, Sr31 + Sr36, Sr31 + Sr47, Sr31 + Sr44, Sr22 + Sr44 + Sr47, Sr22 + Sr31 + Sr32, Sr22 + Sr31 + Sr44, Sr22 + Sr36 + Sr44, Sr32 + Sr44 + Sr47, Sr31 + Sr36 + Sr47, Sr36 + Sr39 + Sr47, Sr2 + Sr22 + Sr36 + Sr44,Sr2 + Sr31 + Sr36 + Sr44, Sr22 + Sr32 + Sr40 + Sr44, Sr22 + Sr31 + Sr36 + Sr44, Sr2 + Sr22 + Sr32 + Sr44, Sr2 + Sr22 + Sr32 + Sr40 + Sr44. Specific features in transmission of some resistance genes are noted. In particular, no plants with the Sr24 gene were detected. The second feature is associated with the Sr2 gene (the gene was originally identified only in spring wheat 113/ 00i-4). The Sr2 gene was in a heterozygous state in more than 70% of winter plants in which it was identified (Figure 3).

The presence of Sr32, Sr39, Sr40, Sr44 genes, which are poorly studied in relation to other Pgt races and rarely used in selection programs, with the resistance Sr2 gene of an adult plant showing "slow rusting" effect, gives particular value to the selected winter plants. However, the presence of the recessive Sr2 gene of resistance in the heterozygous state in most winter wheat plants will require additional efforts to transfer it to a homozygous state. In particular, we have planned experiments on the production of digaploid lines using androgenesis method. Individual plants with the identified genotype of resistance to stem rust differed greatly in height (75–145 cm), ear productivity (1.0–2.7 g), weight of 1000 grains (36–60 g) and morphological features. For further testing in infectious nurseries of stem and leaf rust, 373 individual winter wheat plants were selected: 199 plants with the identified Sr genes and 174 plants selected for a set of other economically valuable traits. From the populations of spring

Figure 3. Identification of the Sr2 gene using the molecular marker Xgwm533 in winter plants 1–36: M—molecular weight marker of 50 bp "Fermentas", Sr2—positive control Pavon76, K—negative control Saratovskaya 29 cultivar. The arrow indicates a diagnostic fragment with a molecular weight of 120 bp. The amplification products were separated in 2% agarose gel. "+"—presence of the diagnostic fragment; ""—absence of a diagnostic fragment; h—heterozygote.

wheat, only 198 spring plants were selected for further testing: 129 plants with identified Sr genes and 69 plants with a set of valuable traits.
