Introduction

Wheat stripe (yellow) rust is a typical air-borne disease. This disease is a serious threat to wheat production in Asia, North America, Europe and other wheat-growing areas. China is the largest epidemic region of wheat stripe rust in the world and its occurrence, and damage are especially frequent and severe (Wan et al. 2004). Stripe rust reduces wheat yields by damaging plants and shrinking the grain. Many severe epidemics of wheat stripe rust have occurred since 1950, including the nation-wide extremely severe epidemics in 1950, 1964, 1990 and 2002 which caused multimillion tons of yield losses in these epidemic years. Highly virulent races and widely grown susceptible cultivars were attributed to the epidemics.

Due to the appearance of new races CYR30, CYR31, CYR32 and CYR33 since 1995, many wheat cultivars of the Fan 6 derivatives, Mianyang series and Shuiyuan-derived cultivars have become susceptible. In recent years, a new group of Guinong 22-virulent races that infect wheat cultivars with Yr24 (=Yr26) has been rapidly increasing in frequency and distribution. The predominant races CYR32, CYR33 and new races have accelerated the turnover frequency of wheat cultivars. This has put forward higher requirement of developing wheat cultivars with effective resistance to stripe rust in China.

The use of resistance genes against stripe rust is essential in wheat breeding programmes. Stripe rust resistance genes in wheat have been designed with the symbol ‘Yr’ (Lupton and Macer 1962). Numerous genes for stripe rust resistance have been identified, including 78 Yr permanently named genes (McIntosh et al. 2017) and hundreds of temporarily named genes. Many resistance genes were identified in common wheat, while some came from wheat-related species, such as Yr5 from Triticum spela album (Macer 1966);Yr7, Yr53, Yr64 and Yr65 from T. durum (Macer 1966; Xu et al. 2013; Cheng et al. 2014); Yr9 from Secale cereale (Macer 1975); Yr15, Yr35 and Yr36 from T. dicoccoides (Gerechter-Amitai et al. 1989; Marais et al. 2005a; Uauy et al. 2005);Yr24 (=Yr26) from T. turgidum L. (McIntosh and Lagudah 2000; Ma et al. 2001); Yr8, Yr17, Yr28, Yr37, Yr38, Yr40 and Yr42 from different Aegilops species (Riley et al. 1968; Robert et al. 1999; Singh et al. 2000; Marais et al. 2005b, 2006; Kuraparthy et al. 2007; Marais et al. 2009) and Yr50 from Thinopyrum intermedium (Liu et al. 2013). Most of the genes with resistance to stripe rust were on B group chromosomes. Due to quick development of new races of Puccinia striiformis f. sp. tritici (Pst), wheat cultivars with race-specific resistance usually become susceptible about 3–5 years after release. Currently, most reported seedling resistance genes are ineffective against race CYR32 in China (Yang et al. 2003). Although, a lot of efforts have been made to identify genes for resistance and develop stripe rust-resistant cultivars, it is still a great challenge to have enough wheat cultivars with effective and long-lasting resistance. Therefore, the best strategy for controlling wheat stripe rust is to introduce new resistance genes into wheat-breeding programmes, broaden the genetic basis, and develop cultivars with durable resistance and many other excellent traits.

Zhengmai 7698 with high quality and yield potential was developed by the Molecular Breeding Laboratory, Wheat Research Center, Henan Academy of Agricultural Sciences. It has been grown in more than 13 million acres since its release in 2011. The cultivar has resistance to stripe rust. The objective of this study was to identify the resistance gene(s) through genetic analysis and molecular mapping.

Materials and methods

Plant materials and Pst inoculum

Stripe rust resistant cultivar Zhengmai 7698 was developed from a cross between Zhengmai 9405 and 4B269, and then crossed with Zhoumai 16. Spring wheat Taichung 29 was susceptible to all known Chinese races of Pst. An \(\hbox {F}_{{2}}\) population derived from crossing Zhengmai 7698 with the susceptible cultivar Taichung 29 was used for genetic analysis and molecular mapping. In addition, \(\hbox {F}_{{1}}\) plants were backcrossed with Taichung 29 to develop a \(\hbox {BC}_{{1}}\) population to confirm the \(\hbox {F}_{{2}}\) results. A highly susceptible wheat cultivar, Mingxian 169, was used as a disease spreader in the field tests.

Single urediniospore isolates of eight Pst races (CYR17, CYR26, CYR27, CYR29, CYR31, CYR32, CYR33 and V26) maintained at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, China, were used for evaluating wheat cultivars. The isolate of race CYR32, which is prevalent throughout China, and virulent on all Chinese differential cultivars except for Zhong 4 and all world differential cultivars except for Moro, was used to phenotype the segregating populations for genetic analysis and molecular mapping. Fresh urediniospores of an isolate were produced on susceptible wheat cultivar Mingxian 169.

Resistance evaluation to wheat stripe rust

Seedlings of Zhengmai 7698 were grown in a greenhouse under controlled conditions of \(20^{\circ }\hbox {C}\), humidity 80% and 14 h daily light. Plants at the fully expanded one-leaf stage were inoculated with fresh urediniospores suspended in aqueous solution containing 0.05% v/v Tween 20. The inoculated seedlings were placed in a dew chamber at \(10^{\circ }\hbox {C}\) for 24 h and then transferred into a controlled greenhouse with a diurnal cycle of 14 h of light at \(18^{\circ }\hbox {C}\) and 10 h of dark at \(12^{\circ }\hbox {C}\). \(\hbox {F}_{{1}}\), \(\hbox {BC}_{{1}}\) and \(\hbox {F}_{{2}}\) plants were inoculated with CYR32. After 14–15 days, when the susceptible cultivar Mingxian 169 was fully sporulating, infection type (IT) data were recorded according to the scale of 0–4 (McIntosh et al. 1995). ITs 0–2 were considered resistant whereas ITs 3–4 were susceptible.

Field tests were conducted in the Langfang Experimental Station in the Hebei province. \(\hbox {F}_{{2}}\) plants were grown in rows with 15–20 seeds planted in a 1-m row. Mingxian 169 was planted as a susceptible control around the field and inoculated at the jointing stage with urediniospores of CYR32 suspended in 0.05% v/v Tween 20. The inoculated plants were covered with plastics overnight to facilitate dew formation for Pst infection. The plastics were removed in the following morning. ITs were scored when Mingxian 169 had more than 80% severity. The IT data were classified as resistant (R, IT 0–2) and susceptible (S, IT 3–4).

DNA extraction, PCR amplification, electrophoresis and gel visualization

Genomic DNA was extracted from leaf tissues of each \(\hbox {F}_{{2}}\) plant and the parents using a modified method of cetyltrimethylammonium bromide (CTAB) (Rogers and Bendich 1985) and dissolved in \(100\,\mu \hbox {L}\) TE-buffer (10 mM Tris, pH 8.0, 1 mM EDTA). The DNA was quantified with a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, USA) and assessed in 1% agarose gel. DNA samples were diluted to a concentration of 50 ng/\(\mu \hbox {L}\) before storing at \(-20^{\circ }\hbox {C}\).

To identify the DNA markers that are associated with stripe rust resistance, the bulked segregant analysis (BSA) method was used. DNA samples from 10 R (IT 0 0;) and 10 S (IT 4) \(\hbox {F}_{{2}}\) plants were mixed to construct the R and S bulks, respectively and then evaluated with simple sequence repeat (SSR) markers together with the parents. SSR markers in the WMC, GWM, BARC, CFA, CFD and GDM series (http://wheat.pw.usda.gov/ggpages/SSR) were chosen across the 21 wheat chromosomes according to the consensus map of Somers et al. (2004).

Table 1 ITs of Zhengmai 7698 and Taichung 29 inoculated with P. striiformis f. sp. tritici races.
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Fig. 1
figure 1

Seedling IT of the parents and some \(\hbox {F}_{{2}}\) plants from cross Taichung 29/Zhengmai 7698 inoculated with race CYR32 of P. striiformis f. sp. tritici. ZM, Zhengmai 7698; T29, Taichung 29.

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PCR was performed in a 10-\(\mu \hbox {L}\) solution containing 2 \(\mu \hbox {L}\) of 50 ng/\(\mu \hbox {L}\) template DNA, 1 \(\mu \hbox {L}\) of \(10\times \) PCR buffer, 0.2 \(\mu \hbox {L}\) of 5 U/\(\mu \hbox {L}\) Taq DNA polymerase, 2.5 mM of each dNTP, 5 \(\mu \hbox {mol/L}\) forward and reverse primer solutions and sterilized double distilled \(\hbox {H}_{{2}}\)O. Amplification was performed in a GeneAmp PCR System 9700 programmed as follows: 5 min at \(94^{\circ }\hbox {C}\) for initial denaturation; 35 cycles each consisting of 1 min at \(94^{\circ }\hbox {C}\) for denaturation, 1 min at \(50^{\circ }\hbox {C}\), \(55^{\circ }\hbox {C}\) or \(60^{\circ }\hbox {C}\) for annealing depending on individual primers, 1 min at \(72^{\circ }\hbox {C}\) for extension and finally a 10 min extension step at \(72^{\circ }\hbox {C}\). Six microlitre formamide loading buffer (98% formamide, 10 mM EDTA (pH 8.0), 0.5% (w/v) xylene cyanol and 0.5% (w/v) bromophenol blue) was added to each PCR product. After 5 min denaturation at \(94^{\circ }\hbox {C}\), 5 \(\mu \hbox {L}\) of the PCR product and loading buffer mixture for each sample was loaded for electrophoresis in a 6% polyacrylamide gel in 1\(\times \) TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3) (Bassam et al. 1991). Electrophoresis was carried out at 1400 V for 1.0–1.5 h. Gel staining and visualization was done as previously described (Chen et al. 1998). Polymorphic markers were used to genotype the \(\hbox {F}_{{2}}\) population. Genotype data were used to construct a genetic map and locate the resistance gene.

Table 2 Seedling and adult plants of ITs of parents and progenies of cross Taichung 29/Zhengmai 7698 inoculated with race CYR32 of P. striiformis f. sp. tritici and chi-squared analysis.
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Fig. 2
figure 2

PAGE patterns of (a) Xwmc786, (b) Xwmc398, (c) Xwmc179 and (d) Xbarc198 on(1) Zhengmai 7698, (2) Taichung 29, (3) resistance bulk and (4) susceptible bulk.

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Mapping and data analysis

A linkage map was established using software JoinMap ver. 3.0 (van Ooijen and Voorrips 2001). A regression mapping algorithm and Kosambi’s function were utilized to convert recombination fractions into map distances and order the markers (Kosambi 1943).

Chi-square \((\chi ^{2})\) tests were applied to evaluate the \(\hbox {F}_{{2}}\) segregation ratios of R/S plants to determine the goodness of a theoretical ratio.

Results

Response of Zhengmai 7698 to Pst races

When tested in the seedling stage in the greenhouse, Zhengmai 7698 was resistant (IT 0;) with necrotic spots without any uredinia to all tested races except for V26, while Taichung 29 was susceptible (IT 4) to all races (table 1).

Reactions of progenies to CYR32

In the progeny test with race CYR32, Taichung 29 was susceptible (4) and Zhengmai 7698 was resistant as described above. All \(\hbox {F}_{{1}}\) plants were resistant and \(\hbox {F}_{{2}}\) plants segregated into resistant and susceptible reactions (figure 1), indicating dominant resistance of Zhengmai 7698. The \(\hbox {F}_{{2}}\) segregation fits a 3 resistant:1 susceptible ratio \((\chi ^{2}\,{=}\,1.73, P\,{=}\,0.19)\), indicating one gene for resistance (table 2). The 32 tested \(\hbox {BC}_{1}\) plants were divided into 15 resistant and 17 susceptible plants. The observed segregation ratio was consistent with the expected ratio of 1:1 \((\chi ^{2}=0.13, P\,{=}\,0.72)\). These results showed that Zhengmai 7698 has a dominant gene conferring resistance to race CYR32.

In the field evaluation, Taichung 29 was susceptible (IT 4), Zhengmai 7698 was resistant (IT 0;) and \(\hbox {F}_{{1}}\) plants were also resistant (IT 0;). The \(212 \hbox { F}_{{2}}\) plants segregated into 158 resistant and 54 susceptible fitting the ratio of 3:1 \((\chi ^{2}=0.03, P\,{=}\,0.86)\), indicating that resistance under field conditions was also controlled by a single-dominant gene (table 2). The segregation of resistant and susceptible \(\hbox {BC}_{1}\) plants fit the 1:1 ratio \((\chi ^{2}=0.04, P\,{=}\,0.84)\). The field results confirmed a single-dominant gene for resistance to CYR32 and the gene was tentatively designated as YrZM.

Screening of polymorphic markers

From 240 tested SSR markers, which were selected to cover the 21 common wheat chromosome 5 markers, Xwmc786, Xwmc398, Xwmc179, Xbarc198 and Xwmc597, were polymorphic between the parents, and between the resistant and susceptible bulks (figure 2), indicating that these markers were linked to YrZM. The five markers were all from chromosome 6B, suggesting that YrZM was likely located on chromosome 6B.

Linkage analysis and mapping resistance gene

The associations of the five polymorphic SSR markers with the resistance gene were tested with 212 \(\hbox {F}_{{2}}\) plants. Because the dominant marker Xwmc597 was present in Taichung 29 and susceptible bulk, it was not used to construct the linkage map. Other four markers were present in Zhengmai 7698 and the resistant bulk. Figure 3 shows the amplification results of marker Xbarc198 for the two parents and some \(\hbox {F}_{{2}}\) plants. Three of the markers were codominant and one was dominant (table 3). Codominant markers Xwmc398, Xwmc179 and Xbarc198 segregated at the expected 1:2:1 ratio for homozygous resistant, heterozygous and homozygous susceptible in the \(\hbox {F}_{{2}}\) population \((\chi ^{2}=0.03\,{-}\,0.46, P\,{=}\,0.50\,{-}\,0.86)\). The segregation of marker Xwmc786 fit the expected ratio 3:1 \((\chi ^{2}=0.91, P\,{=}\,0.34)\) for presence and absence in the \(\hbox {F}_{{2}}\) population (table 3).

Fig. 3
figure 3

Amplification of \(\hbox {F}_{{2}}\) plants of cross Taichung 29/Zhengmai 7698 using the primers of SSR marker Xbarc198. \(\hbox {P}_{{1}}\), Zhengmai 7698; \(\hbox {P}_{{2}}\), Taichung 29. A, homozygous resistant; B, homozygous susceptible; H, heterozygote. The band associated with Yr is indicated by the arrow.

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Table 3 Segregations of SSR markers in the \(\hbox {F}_{{2}}\) population of cross Taichung 29/Zhengmai 7698.
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A linkage map was constructed with the four segregating markers (figure 4). YrZM was flanked by markers Xwmc179 and Xbarc198 at genetic distances of 2.7 and 2.4 cM, respectively.

Fig. 4
figure 4

Genetic linkage map of stripe rust resistance gene YrZM on chromosome 6BL. The left side shows the map distances in centi-Morgans and the right side indicates the corresponding locations on the genetic map between the two loci and the YrZM locus.

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Discussion

The first requirement for breeding programmes is to have resistance sources which can be used for developing cultivars with resistance to major diseases and pests. Many genetic studies of wheat stripe rust have identified one, two or more genes with additive effects on disease resistance. Wheat cultivar Zhengmai 7698 was derived from a complex cross involving three parental varieties (Zhengmai 9405/ Zhengmai 4B269/Zhoumai 16). The cultivar is resistant to many Pst races, including predominant races CYR17, CYR26 and CYR32. CYR32, virulent to Yr1, Yr2, Yr3, Yr4, Yr6, Yr7, Yr9, Yr27, YrA, YrAlba, YrCle, YrCV, YrGaby, YrRes, YrSD, YrSP and YrSu circumvents resistance in many wheat cultivars and has caused a severe epidemic in recent decades in China (Kang et al. 2010). In addition to CYR32, CYR33 has become predominant, which has made more cultivars susceptible (Kang et al. 2010). Only a few genes for race-specific all stage resistance, such asYr5 and Yr15 are effective against Chinese Pst races. In this study, Zhengmai 7698 was found to be resistant to Pst races CYR32 and CYR33, indicating that its resistance is effective against the major predominant races. Genetic analysis indicated that resistance of Zhengmai 7698 against CYR32 was conferred by a single dominant gene, tentatively designated as YrZM. The closely linked SSR primers mapped YrZM to the long arm of chromosome wheat 6B. The two flanking markers, Xbarc198 and Xwmc179, should be useful for identifying YrZM in breeding programmes.

Fig. 5
figure 5

Comparison of the YrZM linkage map with the consensus map of Somers et al. (2004) and QTL on 6BL chromosome (William et al. 2006).

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Currently, four formally named Yr gene loci (Yr4, Yr35, Yr36 and Yr78) have been identified on chromosome 6B (Wang and Chen 2017). The typical carriers of these genes are Cappelle-Desprez (Yr4a), Hybrid 46 (Yr4b), 98M71 (Yr35), Glupro (Yr36) and PI 519805 (Yr78). Cappelle-Desprez was first released in France in 1946 and widely cultivated in Western Europe up to 1970s (Lupton and Macer 1962; Worland and Law 1986; Bonjean et al. 2001). It is known to possess the seedling resistance genes Yr4a (de Vallavieille-Pope et al. 1990) on chromosome 6B and Chen et al. (1996) also reported Yr4b in Hybrid 46 on chromosome 6B. However, their specific chromosomal location is unknown. Based on the seedling tests with different Chinese Pst races, Cappelle-Desprez and Hybrid 46 were susceptible to CYR32, indicating that Yr4a and Yr4b were different from YrZM. Yr35 on the short arm of chromosome 6B was transferred from T. turgidum spp. dicoccoides to hexaploid wheat by Marais et al. (2005a). Yr36, also located on chromosome 6BS and from T.turgidum ssp. dicoccoides, confers high-temperature and adult-plant resistance (Uauy et al. 2005). Through pedigree analysis, it was concluded that YrZM was different from Yr35 and Yr36. Yr78 for adult-plant resistance to stripe rust was identified in PI 519805 and several other common wheat cultivars and mapped to chromosome 6BS (Dong et al. 2017). As YrZM is located on chromosome 6BL and resistant to CYR32 and CYR33, it is different from all permanently named Yr genes on chromosome 6B.

There were three regions of importance in chromosome 6B for stripe rust resistance (Rosewarne et al. 2013): QRYr6B.1 referred to the short arm; QRYr6B.2 mainly contained Yr36 and the final region was identified in Pastor (Rosewarne et al. 2012) and Pavon (William et al. 2006). Due to the differences in marker platforms and associated linkage maps, it was difficult to accurately compare the chromosomal location of YrZM with previously identified quantitative trait loci (QTL). By comparing chromosome 6B with other published SSR maps based on shared markers, the YrZM locus is different from previously reported QTL for stripe rust resistance (figure 5). As marker platforms will continue to be developed and improved, and more linkage maps will be published with combinations of SSR and SNP markers, we will be able to compare the YrZM linkage map with maps containing similar chromosomal regions.

To improve the durability of resistance in wheat cultivars and to achieve sustainability of stripe rust management, it is important to diversify the resistance gene in breeding programmes and deploy diverse effective resistance genes in wheat cultivars. As a widely grown cultivar for high yield and stripe rust resistance, Zhengmai 7698 is also a valuable cultivar to be used in breeding programmes. As its stripe rust resistance gene YrZM is race-specific and ineffective against the recently emerged race groups, e.g. V26, caution needs to be taken to use the gene in combinations with other effective genes and especially for nonrace specific adult-plant resistance genes. The flanking markers Xbarc198 and Xwmc179 can be used for marker-assisted selection in pyramiding YrZM with other resistance genes.