Introduction

Wheat (Triticum L.) is widely grown among the cereals with 215.9 million hectares and a total production of 766 million tons worldwide (FAO 2020) due to its wide adaptability and main energy source in human nutrition. In addition, wheat provides substantial amounts of a number of components, which are essential for health, especially protein, vitamin B, dietary fibers, and minerals. In Turkey, it is grown on an area of 6.9 million hectares with a total production of 20.5 million tons. Three million tons of this production belongs to durum wheat (Triticum turgidum var. durum) (TUIK 2020).

Wheat production can be negatively affected by many biotic and abiotic stress factors. Among the biotic stresses, yellow rust caused by Puccinia striiformis f.sp. tritici (Pst) is a destructive disease of wheat in many regions of the world and can cause significant yield losses during severe epidemics, especially in humid, temperate, and cool environments (Schwessinger 2017). Stripe rust epidemics have occurred every 2 of 5 years in over 25% of the wheat growing areas of some countries including Canada, Russia, Kazakhstan, Iran, Iraq, Turkey, etc. (Chen 2020). In 1991, serious yield loss due to yellow rust occurred up to 62.5% in the Seri-82 variety containing Yr9 resistance gene which was widely cultivated in coastal regions of Turkey (Braun and Saari 1992; Mamluk et al. 1997). In the following years, Düşünceli et al. (1999) reported that yield loss between 26.5 and 50% due to yellow rust epidemic was recorded in Central Anatolia region of Turkey. Furthermore, severe epidemics have continued to occur in Turkey in recent years (Cat et al. 2021).

Pst is highly aggressive and unstable and new Pst races derived from mutation, somatic recombination and sexual production can break the resistance genes (Schwessinger 2017). In order to develop resistance to yellow rust disease in wheat, many studies are carried out to identify Pst races and resistance genes. More than 80 resistance genes to stripe rust have been characterized in wheat (McIntosh et al. 2020). Many of the Yr genes have been identified as race-specific, so these genes provide resistance only against Pst isolates carrying the avirulence gene (Goutam et al. 2015). Many resistance genes have also been introgressed from different wild species into bread wheat (Kuraparthy et al. 2007; Chhuneja et al. 2008). Although the majority of these resistance genes are generally identified in a hexaploid background, the major resistance genes Yr5, Yr10 and Yr15 were characterized on genome B. The Yr5 gene was first identified in 1966 in Triticum spelta var. album and Macer (1966) localized it on chromosome 2B. The Yr10, known as race-specific gene, is derived from a Turkish wheat line (PI 178383) and many studies showed that this gene was located on chromosome 1B (Metzger and Silbaugh 1970; Payne et al. 1986). Lastly, the Yr15 gene was first identified in a wild emmer (Triticum dicoccoides Korn) accession G-25 (Gerechter-Amitai et al. 1989) and McIntosh and Silk (1996) showed that it is located on the short arm of chromosome 1B.

The deployment of genetically resistant varieties is the most effective and economically sound approach to control stripe rust. One of the best strategies to improve resistance to stripe rust is to utilize the genetic resources of the wild relatives including wild emmer wheat (Triticum turgidum var. dicoccoides) (Peng et al. 2011). Wild emmer has a wide range genotypic variation in many important traits like stripe rust resistance (Tene et al. 2022). In addition to Yr15, Yr36 as one of the important resistance genes was first identified in wild emmer (Uauy et al. 2005). At the same time, it is fully compatible with durum wheat and can be easily crossed with bread wheat. Genetic advances in wheat to date have generally relied on breeding activities within a relatively narrow gene pool (Hao et al. 2019). Since this gene pool has also a narrow genetic base for resistance to rust diseases (Olivera et al. 2018), wild emmer genotypes are worth screening for presence of other Yr resistance genes.

Molecular markers for resistance genes Yr5, Yr10 and Yr15 have been widely used in wheat breeding programs worldwide (Zeng et al. 2014; Zheng et al. 2017). In particular, Yr5 and Yr15 are still effective against the Turkish Pst races (Sharma-Poudyal et al. 2013; Cat et al. 2021). The aim of the present study was to detect the Yr5, Yr10 and Yr15 resistance genes in 54 Turkish durum wheat varieties and 11 wild emmer genotypes at molecular level. Additionally, field trials were conducted during 2019-2020 under natural epidemic conditions in Antalya to determine phenotypic reactions of these genotypes against stripe rust.

Materials and methods

Genetic material

A total of 54 durum wheat (Triticum turgidum var. durum) varieties registered in Turkey and 11 wild emmer (Triticum turgidum var. dicoccoides) genotypes supplied from Turkish Gene Bank were used as genetic material in this study (Table S1). Durum wheat varieties were registered from 1963 to 2012. In addition, three positive control lines, AvsNILYr5, AvsNILYr10 and AvsNILYr15, were used to confirm whether the genotypes carry the Yr5, Yr10 and Yr15 genes or not.

Field testing under natural infection conditions

Field tests were conducted in the cropping seasons of 2019 and 2020 under natural infection conditions at the experimental station of the Akdeniz University, Antalya. The seeds of each genotype were sown as two rows with 100 cm long and susceptible bread wheat variety “Morocco” was also sown as spreader in two rows for every 10 rows and around the plots to increase disease pressure. The trials were sprinkler-irrigated to guarantee a moist environment for high pathogen development. The top three leaves were visually scored thrice at late booting (Z45), heading (Z55) and dough stages (Z65), respectively (Zadoks et al. 1974) as Morocco plants reached 70% infection at least. Infection type (IT) and disease severity (DS) were evaluated using a Modified-Cobb scale (Peterson et al. 1948). In addition, IT and DS data were combined into a single value called the coefficient of infection (CI) to rank or easily compare the genotypes. CI was calculated by multiplying IT and DS for each genotype as described by Roelfs et al. (1992). The highest IT and DS values observed at dough stages (Z65) were used to calculate CI: where R= 0.2, MR= 0.4, MS= 0.8, and S=1.0.

DNA extraction, PCR amplification and electrophoresis

At least three seeds of each variety were sown in pots (7 × 7 × 10 cm) with mixture of soil and peat in 1:1 ratio and fresh leaves were collected from seedlings of each variety at two-leaf stage. Genomic DNAs were extracted using the CTAB method (Doyle and Doyle 1990). Quality of genomic DNAs was checked by the agarose gel electrophoresis and stored until use at -20 ℃. To detect resistance genes, different molecular markers developed by Chen et al. (2003), Singh et al. (2009) and Murphy et al. (2009) were used. Information about these markers is given in Table 1. Polymerase chain reactions (PCR) were performed in a thermal cycler (T100, Bio-Rad, USA). The total volume of the reaction mixture was 15 µL containing 1X PCR buffer (50 mmol KCl, 10 mmol Tris-HCI, pH 8.3) (Thermo Fisher Scientific, USA), 2.0-2.5 mM MgCl2 (Thermo Fisher Scientific, USA), 0.2 mM of dNTP (Thermo Fisher Scientific, USA), 0.75 U of Taq DNA polymerase (Thermo Fisher Scientific, USA), 0.4–1.0 µM of each primer and 100 ng of template DNA.

Table 1 The information about molecular markers used in this study
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Amplifications were performed under the following conditions for STS7/8: initial denaturation at 94 °C for 5 min, followed by 45 cycles (each consisting of 30 s at 94 °C, 30 s at 45 °C, 45 s at 72 °C), final extension for 10 min at 72 °C; for Yr10F/R: initial denaturation at 94 °C for 4 min, followed by 35 cycles (each consisting of 30 s at 94 °C, 30 s at 64 °C, 1 min at 72 °C), final extension for 10 min at 72 °C and lastly for Xbarc8: initial denaturation at 94 °C for 4 min, followed by 40 cycles (each consisting of 30 s at 94 °C, 30 s at 50 °C, 1 min at 72 °C), final extension for 10 min at 72 °C.

Amplified PCR products with the pairs STS7/8 were digested with the restriction enzyme DpnII (New England Biolabs, USA) (Chen et al. 2003). The total volume of reaction mixture for enzymatic digestion was 15 μL containing 8 μL of PCR product, 0.25 μL DpnII, 2 μL of 10× NEBuffer and 4.75 μL distilled water. Enzymatic digestion was performed in a thermo-shaker (Biosan, Latvia) under the following conditions: 37 ℃ for 1h, 65 ℃ for 20 min and holding at 10 ℃ for 5 min and the digested products were separated in 2% agarose gel stained with 0.5 μg/mL ethidium bromide with 85 V for 1 h. The fragments were visualized under UV light using a gel imaging system (UVP UVsolo touch, Analytik Jena, Germany).

Results

Evaluation of tetraploid wheat genotypes for stripe rust resistance at adult plant stage

The 54 durum wheat varieties and 11 wild emmer genotypes were evaluated for stripe rust resistance at adult plant stage. Infection types and severity of stripe rust disease were recorded during 2019 and 2020 growing seasons and data are given in Tables 2 and 3. According to the observations in 2019, 45 varieties showed resistance reaction ranging from trace (TR) to moderately resistant (MR) and disease severity was observed with varying intensities from 0 to 10%. Unlike this, 9 varieties were highly susceptible with disease severities ranging from 5 to 40% (Table 2). However, all wild emmer genotypes showed a high level of susceptible reaction and disease severity ranged from 70 to 100% (Table 3). Based on IT and DS for each genotype, coefficient of infection (CI) was calculated. While the highest CI values were determined in the varieties Kunduru 414/44 and Eyyubi, 35 varieties had the lowest CI with zero (Table 2). Akbaşak 073/144, Sarıçanak 98, Altıntoprak 98, Gediz-75, Pınar-2001 and Zenit had also the low CI values. Unlike these, CI values determined in wild emmer genotypes ranged from 70 to 100 (Table 3).

Table 2 Disease severity (DS), infection type (IT) and coefficient of infection (CI) values on durum wheat varieties in 2019, and identification of resistance gene(s) using molecular markers
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Table 3 Disease severity (DS), infection type (IT), and coefficient of infection (CI) values on 11 wild emmer genotypes, and identification of resistance gene(s) using molecular markers
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In 2020 growing season, unfavorable weather conditions such as high temperature and low humidity limited the pathogen development, and no disease symptoms were observed on durum wheat varieties. However, wild emmer genotypes showed susceptible reaction ranging from 40 to 70% with moderately susceptible (MS) to susceptible (S) and therefore CI values were determined between 24 and 70 (Table 3).

Identification of the resistance genes Yr5, Yr10 and Yr15 in tetraploid wheat genotypes

The 54 registered durum wheat varieties and 11 wild emmer genotypes were analyzed for the stripe rust resistance genes Yr5, Yr10 and Yr15 using linked molecular markers shown in Table 1. After enzymatic digestion, genotypes with Yr5 yielded 308 bp fragment as expected, whereas non-Yr5 genotypes had 181 bp fragment. The dominant marker Yr10F/R produced 543 bp fragment in genotypes with Yr10. In addition, the genotypes with Yr15 had 250 bp fragment and non-Yr15 genotypes yielded 280 bp. Sample electrophoretograms of different markers linked to Yr5, Yr10 and Yr15 are given in Figure S1.

According to these data, Yr5 was not detected in all tested genotypes. While Yr10 was detected in 12 durum wheat varieties (Berkmen 469, Çakmak 79, Kızıltan 91, Altın 40/98, Yılmaz 98, Çeşit-1252, İmren, Kunduru 1149, Altıntaş 95, Dumlupınar, Selçuklu-97 and Sham-1), none of wild emmer genotypes had Yr10 gene. Additionally, Yr15 gene was determined in 17 durum wheat varieties (Berkmen 469, Kızıltan 91, Altın 40/98, Yılmaz 98, Çeşit-1252, Mirzabey 2000, Eminbey, İmren, Kunduru 1149, Fata (Sel), Meram-2002, Tunca 79, Gökgöl 79, Şölen 2002, Pınar 2001, Svevo and Maestrale) (Table 2).

Considering all molecular results, none of wild emmer genotypes had resistance genes examined (Table 3). However, 7 durum wheat varieties (Berkmen 469, Kızıltan 91, Altın 40/98, Yılmaz 98, Çeşit-1252, İmren and Kunduru 1149) carry both Yr10 and Yr15 resistance genes (Table 2).

Discussion

In this study, we evaluated resistance reactions of Turkish durum wheat varieties and wild emmer genotypes to stripe rust disease under natural infection conditions, and these genotypes were also molecularly screened to investigate presence of major Yr genes. With the genotypic studies, we show that seven durum wheat varieties carry both Yr10 and Yr15 resistance genes (Table 2). In phenotypic studies, for durum wheat varieties, we recorded more meaningful data only in 2019 growing season (Table 2); however, unfavorable weather conditions such as high temperature and low humidity limited the pathogen development, and no disease symptoms were observed on durum wheat varieties in 2020 growing season.

Growing resistant varieties is the most efficient and sustainable approach to control the wheat stripe rust disease. In general, this disease caused by Puccinia striiformis f. sp. tritici can be controlled with Yr5 and Yr15 resistance genes worldwide (Sharma-Poudyal et al. 2013; Ali et al. 2018). Additionally, although the prevalence of virulent races to the Yr10 gene was increasing (Afshari 2013; Gharbarnia et al. 2021; Cat et al. 2021), this gene has still provided resistance to many races in the world (Sharma-Poudyal et al. 2013).

It is known that especially the Yr5 gene is used in the development of new varieties resistant to stripe rust disease in wheat breeding programs (Sun et al. 2002; Murphy et al. 2009; Zhang et al. 2022). Although Yr5 and Yr15 have been still known to be effective to Pst races at global level, several races virulent to Yr5 have been reported in India (Nagarajan 1986), Australia (Wellings and McIntosh 1990) and most recently in China (Zhang et al. 2020), Syria (Kharouf et al. 2021) and Turkey (Tekin et al. 2021). Additionally, it has been known that number of genotypes carrying these major resistance genes in wheat genetic germplasm are quite low. Tabassum et al. (2010) reported that the Yr5 resistance gene was not detected in any of the 100 cultivars registered in Pakistan. Huang et al. (2019) also reported that none of 53 Hungarian wheat cultivars had the major resistance genes Yr5, Yr10 and Yr15. Zeng et al. (2014) indicated that there were only two genotypes (0.4%) carrying Yr5 resistance gene among 494 wheat germplasm in China. In another study, Li et al. (2016) determined that 5 (4.3%) of 115 wheat cultivars in China carry Yr5 resistance gene (Li et al. 2016). The results obtained from the mentioned studies related to the Yr5 resistance gene are in agreement with our findings that Yr5 resistance gene is not found among durum wheat varieties. On the other hand, these frequencies related to presence of Yr5 are not consistent with the frequency reported by Mukhtar et al. (2015), who stated that 14 (35.9%) among a total of 39 wheat cultivars and breeding lines had resistance gene Yr5.

Similar to the Yr5 gene, there is no study investigating the presence of the Yr10 and Yr15 genes in durum wheat varieties in Turkey. However, many studies were conducted to detect these genes in wheat germplasm in many regions of the world. Zheng et al. (2017) reported that 16.67% of breeding lines, 38.82% of landraces and 13.57% of modern varieties in China carry the Yr10 resistance gene. Contrary to this, Zeng et al. (2014) stated that none of the 494 Chinese wheat entries have Yr10 and Yr15. Gebreslasie et al. (2020) also reported that none of the Ethiopian wheat varieties and breeding lines have Yr5, Yr8, Yr10 and Yr15. Similarly, Tabassum et al. (2010) showed that none of the 100 Pakistani wheat cultivars have Yr15 resistance gene. Considering all these studies, it is very promising that the Yr10 gene was found in 12 and the Yr15 gene in 17 durum wheat cultivars in Turkey (Table 2). In addition, it was determined that the varieties Berkmen 469, Kızıltan 91, Altın 40/98, Yılmaz 98, Çeşit-1252, İmren and Kunduru 1149 have both Yr10 and Yr15 genes. However, none of wild emmer genotypes had resistance genes examined (Table 3).

On the other hand, in field testing, it was found that most of the tested varieties showed resistance reactions to stripe rust, while only 9 varieties were susceptible (Table 2). As we mentioned above, due to unfavorable weather conditions, no disease symptoms were observed on durum wheat varieties in 2020 growing season. However, all wild emmer genotypes had susceptible reactions in both years (Table 3). As mentioned above, frequencies of major Yr genes and gene combination (Yr10+Yr15) examined in this study were quite low among durum wheat varieties. However, most varieties were observed to be phenotypically resistant against Pst race(s) under natural infection conditions as given in Table 2. These varieties can have other Yr genes not examined in this study, effective to Pst race(s), except for the Yr5, Yr10, and Yr15. Moreover, it is considered that the combination of genes conferring partial resistance may provide resistance to Pst race(s). Zheng et al. (2017) reported that the number of pyramided Yr genes positively correlated with Pst resistance (R2 > 0.8, p < 0.01) based on field resistance evaluation, and significant additive effects were observed in some gene combinations such as Yr9+Yr18 and Yr30+Yr46. On the other hand, some varieties with Yr10 (Selçuklu 97, Sham-1) or the combination Yr10+Yr15 (Berkmen 469) gave surprisingly susceptible reactions (Table 2). In such studies carried out under natural infection conditions, the virulence formula of the races infecting the genotypes is not known since race analysis was not performed. Cat et al. (2021) first detected Yr10-virulent races with moderate frequency (25%) in the coastal regions of Turkey where this study was also conducted. Therefore, susceptible genotypes with Yr gene/gene combinations may have been infected by more than one Pst races or Yr10-virulent races. It is considered that the phenotypical differences between resistant varieties without Yr5, Yr10, Yr15 genes and susceptible varieties carrying Yr10 and/or Yr15, may have resulted from these factors.

It is known that the Yr10 was derived from a Turkish wheat line (PI 178383) (Metzger and Silbaugh 1970) and the Yr15 gene was first identified in a wild emmer accession G-25 (Gerechter-Amitai et al. 1989). Ozkan et al. (2002) stated that wild emmer domesticated from western Fertile Crescent including southeastern Turkey. Therefore, it is considered that there is a wide variation in wild emmer accessions for many important traits like stripe rust resistance. However, it was determined that none of 11 wild emmer genotypes had resistance genes in this study. This finding is supported by He et al. (2020), who reported that only 13.6% of 361 wild emmer accessions was positive for the Yr15 gene-specific markers, and 21 accessions from Lebanon, Syria, and Turkey populations were Yr15 negative. In durum wheat production, southeastern region of Turkey ranks first among the regions with approximately 1.6 million tons (TUIK 2020). In this region, many wild relatives of cereals such as Aegilops spp. and Hordeum spp. distribute naturally (Özkan et al. 2020), and these are known as super spreaders of wheat stripe rust pathogen (Tekin et al. 2020). Therefore, susceptible wild emmer accessions like these wild relatives have also the potential to be super-spreading and threaten durum wheat production in western Fertile Crescent including Turkey, Lebanon, Jordan, Syria, and Israel.

To sum up, this is the first report to identify major Yr-genes in Turkish durum wheat varieties and wild emmer genotypes. None of durum wheat varieties had Yr5 gene but seven durum wheat varieties have both Yr10 and Yr15 genes. The pedigrees of these varieties, containing resistance gene(s), (Table S1) show that old durum wheat landraces such as Üveyik, Berkmen 469 and Kunduru 414/44 are one of the common parents of them. It is considered that these parents and varieties containing both Yr10 and Yr15 genes can be used in breeding studies to be conducted for resistance to stripe rust. Additionally, much attention should be paid to the role of susceptible wheat genotypes in stabilization of the Pst races and the spread of new Pst races from susceptible wild relatives.