Introduction

Wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) stem rust, caused by Puccinia graminis Pers.:Pers. f. sp. tritici Eriks. & E. Henn. (abbreviated as Pgt), is one of the most devastating diseases of wheat. P. graminis f. sp. tritici race TTKSK (known as Ug99), which was detected in Uganda in 1998 (Pretorius et al. 2000) and has virulence to most stem rust resistance (Sr) genes previously deployed in wheat cultivars, is a major threat to global wheat production (Singh et al. 2008, 2011). Since 2007, an enormous effort, under the coordination of the Borlaug Global Rust Initiative (http://www.globalrust.org), has been undertaken to identify TTKSK-effective Sr genes from wheat and its relatives and deploy them into wheat cultivars in the targeted regions. So far, at least 23 Sr genes cataloged in wheat have been identified as effective against TTKSK (Singh et al. 2011; McIntosh et al. 2013). However, a majority of these genes, such as Sr24, Sr25, Sr26, Sr32, Sr36, Sr37, Sr39, Sr40, Sr43, Sr44, Sr47, Sr50, Sr51, Sr52, and Sr53, were transferred from related species in the secondary and tertiary gene pools of wheat (Dundas et al. 2007; Anugrahwati et al. 2008; Liu et al. 2011a, b, 2013; Niu et al. 2011, 2014; Qi et al. 2011; Klindworth et al. 2012; Mago et al. 2013; McIntosh et al. 2013).

Among these TTKSK-effective alien genes, only four, including Sr24, Sr25, and Sr26 derived from tall wheatgrasses (Thinopyrum ponticum (Podp.) Barkworth & D.R. Dewey) and Sr36 from Triticum timopheevii (Zhuk.) Zhuk. have been deployed in wheat cultivars (McIntosh et al. 1995; Jin and Singh 2006; Tsilo et al. 2008). Two new Ug99 variant races TTKST and TTTSK, which are virulent to Sr24 and Sr36, respectively, were identified in Kenya in 2006 and 2007 (Jin et al. 2008, 2009). Recently, alien chromosome segments carrying five other genes, including Sr32 (Mago et al. 2013), Sr39 (Niu et al. 2011), and Sr47 (Klindworth et al. 2012) from goatgrass species Aegilops speltiodes, Sr43 (Niu et al. 2014) from Th. ponticum, and Sr50 (Anugrahwati et al. 2008) from rye (Secale cereale L.) have been substantially reduced by chromosome engineering. However, the effects of residual chromatin from Ae. speltoides, Th. ponticum and rye on wheat yield and quality have not been evaluated. For deploying Sr genes into new wheat cultivars, it is recognized that pyramiding several Sr genes can lead to increased durability of resistance (Singh et al. 2006, 2011). However, stacking several alien genes into one genotype will increase the total amount of alien chromatin, which may result in potential negative effects on cultivar yield stability and end-use quality.

Contrary to the genes derived from the secondary and tertiary gene pools, the useful genes from the primary gene pool of wheat are rarely associated with the deleterious linkage drags. Therefore, TTKSK-effective Sr genes from the wheat primary gene pool are highly desirable for accelerating development of TTKSK-resistant cultivars through gene pyramiding. So far, only 14 Sr genes cataloged in wheat (McIntosh et al. 2013), including Sr2, Sr13, Sr21, Sr22, Sr28, Sr33, Sr35, Sr42, Sr45, Sr46, Sr55, Sr56, Sr57, and SrWeb derived from primary gene pool, have been identified to be resistant to TTKSK and/or its variant races (Jin et al. 2007; Hiebert et al. 2010, 2011; Rouse et al. 2011a, 2012; Rouse and Jin 2011; Simons et al. 2011; Singh et al. 2011, 2013; Ghazvini et al. 2012; McIntosh et al. 2013; Bansal et al. 2014; Herrera-Foessel et al. 2014). Some of these genes such as Sr2, Sr55, Sr56, and Sr57 have been characterized to confer adult plant resistance (Singh et al. 2013; Bansal et al. 2014; Herrera-Foessel et al. 2014). They are effective only at the adult plant stage or confer inadequate resistance to stem rust when they are present in the cultivars alone (Singh et al. 2011; Rouse et al. 2014). The utility of Sr21, Sr28, Sr42, Sr45, and SrWeb should be limited to gene pyramids because these genes condition resistance to TTKSK, but are ineffective to many other races (Rouse et al. 2011a, 2012; Rouse and Jin 2011; Ghazvini et al. 2012). Therefore, the TTKSK-effective Sr genes from the primary gene pool are very limited. Further efforts are needed to identify more TTKSK-resistant Sr genes from untapped germplasm collections in the primary gene pool.

Among the various wheat-related species in the primary gene pool, Aegilops tauschii Cosson (2n = 2x = 14, DD), the D-genome donor of hexaploid wheat, is an excellent source of unique genes for resistance to many biotic and abiotic stresses. Numerous genes for resistance to the major wheat diseases and insects identified in Ae. tauschii have been transferred into common wheat through production of synthetic hexaploid wheat (SHW) or by direct hybridization (see reviews by Ogbonnaya et al. 2013). For resistance to stem rust, genes Sr33 (Kerber and Dyck 1979), Sr45 (Marais et al. 1998), and Sr46 (Evans Lagudah, unpublished; McIntosh et al. 2008) were identified in Ae. tauschii and transferred into hexaploid wheat. These three genes are all effective against TTKSK (Jin et al. 2007; Olson et al. 2013a; Periyannan et al. 2013). Both Sr33 and Sr45 have been mapped to chromosome arm 1DS at approximately, 10 cM from each other (Sambasivam et al. 2008). Recently, Sr33 was isolated by map-based cloning and the gene was found to encode a coiled-coil, nucleotide-binding, leucine-rich repeat protein (Periyannan et al. 2013).

The result from recent evaluation of 456 unique Ae. tauschii accessions for stem rust resistance showed that 22.2 % of accessions were resistant to TTKSK with diverse TTKSK infection types and diverse infection type patterns to other races (Rouse et al. 2011a), suggesting that there are more unique genes conferring resistance to TTKSK in the Ae. tauschii germplasm collections. The three new Sr genes, designated as SrTA1662, SrTA10187, and SrTA10171 have been identified in three TTKSK-resistant Ae. tauschii accessions, including TA 1662, TA 10187, and TA 10171, respectively, through molecular mapping (Olson et al. 2013a, b). SrTA1662 was mapped to the same position as Sr33, but race specificity suggested that SrTA1662 is different from Sr33 (Olson et al. 2013a). SrTA10187 and SrTA10171 are located on chromosome 6DS and 7DS, respectively (Olson et al. 2013b).

Among the TTKSK-resistant accessions identified by Rouse et al. (2011a), CIae 25 has a moderate level of resistance to TTKSK and several other races. This accession and its derived SHW line have been extensively used in our program to map the Hessian fly-resistant gene H26 (Yu et al. 2009). A large F2 population was developed from a cross between Ae. tauschii accessions CIae 25 and AL8/78. The objectives of this study were to identify any Sr gene(s) conferring the resistance to TTKSK in CIae 25 and to develop molecular markers for marker-assisted selection for the gene(s) in CIae 25.

Materials and methods

Plant materials

Ae. tauschii accession CIae 25 is maintained in USDA-ARS National Plant Germplasm System (http://www.ars-grin.gov/npgs/). The accession was previously identified as resistant to TTKSK and several other races including TRTTF, QTHJC, and TPMKC (Rouse et al. 2011a) while Ae. tauschii accession AL8/78 was susceptible to TTKSK, TRTTF and all other races that have been tested (Friesen et al. 2008; Zhang 2013). A large F2 population was developed from a cross between CIae 25 and AL8/78 to map the Sr gene(s) in CIae 25.

Stem rust resistance evaluation

The parental accessions CIae 25 and AL8/78 were first tested against three locally-maintained US Pgt races, including TPMKC (isolate TNMKsp1), QCCJB (isolate QCC-2, Zhong et al. 2009), and JCMNC (isolate gb-121; Sun and Steffenson 2005). The large F2 population (710 plants) was then tested against TPMKC. The evaluations of parental lines for resistance to the three local races and the F2 population to TPMKC were performed at the USDA-ARS, Northern Crop Science Laboratory (NCSL), Fargo, ND using similar procedures as described by Niu et al. (2011). Two seeds were sown in a super-cell cone (Stuewe and Sons, Inc., Corvallis, OR) filled with Sunshine SB100 mix (Sun Gro Horticulture Distribution Inc., Bellevue, WA) with an application of Osmocote Plus 15-19-12 fertilizer (Scotts Sierra Horticultural Product Company, Marysville, OH). The seedlings were grown in the greenhouse at 20–23 °C with 16/8 h (day/night) photoperiod. Ten days after sowing, seedlings were inoculated with urediniospores of P. graminis f. sp. tritici. Infection types were recorded for each plant at 14 days after the inoculation, using the infection type (IT) scales described by Stakman et al. (1962), where 0 = immune,; = necrotic flecks, 1 = small necrotic pustules, 2 = small to medium-sized chlorotic pustules with green island, 3 = medium-sized chlorotic pustules, and 4 = large pustules without chlorosis. The seedling plants of the F2 population with IT 2 or lower were considered as resistant while the plants scored with IT 3 and higher were considered susceptible. After the F2 plants were scored, they were moved to a vernalization chamber at 2–3 °C for about 6 weeks and then grown to maturity in the greenhouse at 20–23 °C with 16/8 h (day/night) photoperiod.

The parental accessions and 116 F2:3 families with adequate seeds harvested from the TPMKC-susceptible F2 plants and a subset of 14 F2:3 families from the TPMKC-resistant F2 plants were evaluated for their resistance to race TTKSK (isolate 04KEN156/04) at the USDA-ARS Cereal Disease Laboratory (CDL), St. Paul, MN. The evaluation of the parental accessions and their F2:3 families against TTKSK were performed according to the procedure described by Rouse et al. (2011b). For F2:3 family tests, 20 seeds per family were planted in trays filled with vermiculite. Plants were inoculated with urediniospores 8 days after sowing and were grown in a greenhouse at 22 ± 2 and 18 ± 2 °C (day and night, respectively) with supplemental lighting for a photoperiod of 16 h. Plants were scored at 14 days after inoculation using the Stakman et al. (1962) IT scales as described above. Chi-square (χ 2) goodness-of-fit tests were performed on segregating families.

SSR marker analysis

Leaf tissue was sampled from the parental accessions and each susceptible F2 plant. DNA was extracted from freeze-dried leaf samples using the procedure of Dellaporta et al. (1983). Bulked segregant analysis (BSA) was used to identify SSR markers linked to the stem rust resistance gene. Two bulks of DNA were made by pooling equal amounts of DNA from eight homozygous susceptible plants and the eight most resistant plants with the lowest IT scores. After the SSR markers linked to the Sr gene were identified, they were further analyzed on a subpopulation composed of 179 susceptible F2 plants. The polymerase chain reaction (PCR) amplification was carried out as described by Yu et al. (2009, 2010). The PCR products were separated on 6 % poly-acrylamide gels using the procedure of Yu et al. (2009). The gels were stained with Gel-Red, and then scanned with a Typhoon 9410 imager (Molecular Dynamics, Ithaca, NY, USA). The band sizes in the gel images were analyzed using Kodak 1D Image Analysis Software version 3.6.5 K2 (Eastman Kodak, Rochester, NY).

Sequence tagged site (STS) marker development

After the Sr gene was mapped to the distal region of chromosome 2D short arm (2DS5-0.47-1.00) based on the genetic linkage analysis with SSR markers, a total of 194 expressed sequence tags (ESTs) (http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi) were used to develop sequence tagged site (STS) markers. The genomic sequences of the 2DS collinear region in the Brachypodium (http://www.phytozome.org/cgi-bin/gbrowse/brachy/) and rice genomes (http://www.phytozome.org/cgi-bin/gbrowse/rice/) were also employed for STS marker development. The program Primers3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3) (Koressaar and Remm 2007; Untergrasser et al. 2012) was used to design STS primers. The STS marker analysis was conducted in the same way as for SSR markers.

Genetic linkage analysis and identity confirmation of the stem rust resistance gene

A total of 21 polymorphic SSR and STS markers around the stem rust resistance gene were analyzed on the 179 F2 plants susceptible to TPMKC. In this homozygous susceptible subpopulation, the F2 plants carrying the marker alleles from the resistant parent CIae 25 were identified as recombinants. The recombination frequencies were calculated using the program Map Manager QTX (Manly et al. 2001) with the algorithm for an F2 population. The genetic linkage map was constructed using the program Map Manager QTX (Manly et al. 2001). A probability of 0.001 (equivalent to LOD = 3) was used for genetic linkage map construction. The Kosambi mapping function (Kosambi 1944) was used to calculate the genetic distance. The map was drawn using the program Mapchart 2.2 (Voorrips 2002).

The genetic linkage analysis showed that the Sr gene in CIae 25 was located in the same chromosomal region as Sr46, which was previously identified from Ae. tauschii accession AUS 18913 by Evans Lagudah (see McIntosh et al. 2008). AUS 18913 is an accession maintained in the Australian Winter Cereals Collection, Tamworth, New South Wales, Australia (http://www2.dpi.qld.gov.au/extra/asp/AusPGRIS/Centres.asp). To confirm that the Sr gene in CIae 25 was Sr46, we surveyed the origin of CIae 25 and AUS 18913 by examining accession details in the USDA-ARS National Plant Germplasm System (http://www.ars-grin.gov/cgi-bin/npgs/acc/display.pl?1000898) and AusPGRIS—Australian Plant Genetic Resource Information Service (http://www2.dpi.qld.gov.au/extra/asp/auspgris/), respectively. We also tested CIae 25, AUS 18913, and TA 1703 (synonymous with AUS 18913) for their seedling reactions to six Pgt races and genotyped them with the SSR markers previously mapped to seven D-genome chromosomes. The original seeds of AUS 18913 were obtained from the Commonwealth Scientific and Industrial Research Organization (Canberra, Australia) and the seed sample used in this study was currently maintained in the CDL. The seeds of TA 1703 were provided by Jon Raupp, Wheat Genetics and Genomics Resource Center (Manhattan, Kansas, USA).

The two Ae. tauschii accessions AUS 18913 and TA 1703 carrying Sr46 previously showed different seedling infection types to races QTHJC and RKQQC tested in different locations (Rouse et al. 2011a). To determine if the variation can be attributed to different environments, we tested the reaction of lines with Sr46 to Pgt races in three environments. The three Ae. tauschii accessions and checks were tested independently for reactions to six Pgt races RKQQC (isolate 99KS76A-1), QTHJC (isolate 75ND717C), QFCSC (isolate 06ND76C), MCCFC (isolate 59KS19), TPMKC (isolate 74MN1409), and TTTTF (isolate 01MN84A-1-2) at the CDL and five races RKQQC (isolate CRL-3, Zhong et al. 2009), QTHJC [isolate 64E(1)sp1], QFCSC (isolate 370C), MCCFC (isolate A-5), and TPMKC (isolate TNMKsp1) at the NCSL. At each location, the three Ae. tauschii accessions and checks were evaluated in the greenhouse and at two temperature (low and high) conditions in growth chambers. The greenhouses were maintained at 21 °C at the NSCL and 22 °C during the day and 19 °C during the night at the CDL with supplemental lighting for a photoperiod of 16 h. The growth chambers at both locations were set to 15 and 18 °C in the dark (8 h) and light (16 h), respectively, for the experiments at low temperature and to 20 and 24 °C in the dark (8 h) and light (16 h), respectively, for high temperature. The common wheat cultivar ‘Chinese Spring’, Ae. tauschii AL8/78, and the durum line Rusty (PI 639869) (Klindworth et al. 2006) were used as susceptible checks. The durum cultivar ‘Langdon’ (CItr 13165), which is known to have Sr13 (Simons et al. 2011), was used as a resistant check. Two SHW lines SW8 (PI 639730) (Xu et al. 2006) and SW73 derived from the crosses of Langdon and Rusty with CIae 25 were also included. In addition, common wheat line LMPG-6 was included as a susceptible check in the experiments conducted at the CDL. The stem rust inoculation and scoring in the experiments were performed using the same procedures described above except that the plants at high temperature conditions were scored at 11 and 13 days after the inoculation at the CDL and the NCSL, respectively.

For marker genotyping analysis, the three Ae. tauschii accessions CIae 25, AUS 18913, and TA 1703, along with checks (AL8/78, SW8, SW73, Langdon, and Rusty), were genotyped using 18 SSR markers that were previously mapped to chromosomes 1D through 7D (Somers et al. 2004) and one STS marker closely linked to the Sr gene in CIae 25 developed in this study. The 18 SSR markers (Xwmc222, Xwmc336, and Xwmc432 on chromosome 1D, Xgwm210 and Xwmc111 on 2D, Xgdm99 and Xgwm191 on 3D, Xgwm213 and Xwmc182 on 4D, Xcfa2141, Xgwm16, Xwmc289, and Xwmc405 on 5D, Xcfd49, Xcfd75, and Xgdm132 on 6D, Xcfa2040, Xcfd21, and Xwmc273 on 7D, and Xgwm133 on 4D and 6D) were selected based on evaluation of 29 markers for their polymorphism between CIae 25 and AL8/78. The procedure for marker genotyping was the same as described above.

Molecular marker validation for marker-assisted selection

A set of 31 common wheat cultivars and a breeding line were used to validate the markers closely-linked to the stem rust resistance gene for marker-assisted selection (MAS). Among them, seven are eastern Chinese winter wheat cultivars, 23 are the hard red spring wheat cultivars developed in the states of Minnesota, North Dakota, and South Dakota in the U.S., and two are the hard red winter wheat cultivar (‘Newton’) and breeding line (IL06-14262) from Kansas and Illinois, respectively. Ae. tauschii accessions CIae 25, AUS 18913, and TA 1703 and two SHW lines SW8 and SW73 derived from CIae 25 were included as positive checks and Ae. tauschii AL8/78 and durum wheat Langdon and Rusty were used as negative checks, The DNA extraction and marker analysis were performed using the procedures described above.

Results

Resistance of Ae. tauschii accession CIae 25 to TTKSK and other P. graminis f. sp. tritici races

Evaluation of the two parental Ae. tauschii accessions CIae 25 and AL8/78 showed that CIae 25 was resistant to races TTKSK (IT 2), TPMKC (IT 1−), QCCJB (IT 1+), and JCMNC (IT 1+) while AL8/78 showed susceptible reactions to these races (IT 3−, 3+, 3, and 3+, respectively). Because CIae 25 and AL8/78 exhibited a stronger differential reaction to race TPMKC than the other races (Fig. 1), we used this race to evaluate 710 F2 plants from the cross between CIae 25 and AL8/78. From the 710 F2 plants, we initially identified 180 susceptible plants. To verify if the resistance gene detected in the F2 population using race TPMKC was the same gene for resistance to race TTKSK, 116 F2:3 families with adequate seeds derived from the F2 plants susceptible to TPMKC and a sample set of 14 F2:3 families from the F2 plants resistant to TPMKC were evaluated for their reactions to TTKSK. Among the 14 F2:3 families derived from TPMKC-resistant F2 plants, 10 families (16–20 plants per family) were homozygous for resistance to TTKSK (IT 2) and four families segregated for resistant (IT 2) and susceptible (ITs 3 to 3+) plants (Table 1). The segregation of resistant and susceptible plants in the four families fit a 3:1 ratio (Table 1), indicating that the stem rust resistance in CIae 25 is controlled by a single dominant gene. Among the 116 F2:3 families derived from the race TPKMC-susceptible F2 plants, 104 families were homozygous for susceptibility to race TTKSK (IT 3–3+), seven families showed all the plants with intermediate ITs (ITs 2+3 or 32+), four families showed a mixture of intermediate (IT 2+3) and susceptible reactions, and one family segregated for resistant and susceptible reactions. The family segregating for resistant and susceptible plants should be derived from a heterozygous F2 plant which was misclassified as susceptible to TPMKC. The F2:3 family tests substantiated that the Sr gene in CIae 25 confers the resistance to both TPMKC and TTKSK.

Fig. 1
figure 1

Stem rust symptoms on Ae. tauschii accessions CIae 25 and AL8/78 and their F2 plants resistant and susceptible to race TPMKC of P. graminis f. sp. tritici

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Table 1 Infection type (IT) of Ae. tauschii accessions CIae 25 and AL8/78 and their 14 F2:3 families derived from the TPMKC-resistant F2 plants to race TTKSK
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Molecular mapping of the stem rust resistance gene in Ae. tauschii CIae 25

Molecular mapping was performed using a population of 179 F2 plants that were homozygous susceptible to race TPMKC (the F2 plant which was misclassified as susceptible to TPMKC was excluded). Since stem rust resistance genes are frequently identified on chromosome 2B of wheat and group 2 chromosomes of wild relative species such as Ae. speltoides, we started BSA with the SSR markers previously mapped to chromosome 2D. The result showed that 11 SSR markers in the distal region of the short arm of chromosome 2D were polymorphic between the two parents as well as between the resistant and susceptible bulks. The 11 SSR markers were then analyzed on the 179 F2 susceptible plants used as a mapping population. The initial linkage analysis showed that the Sr gene was proximal to Xbarc124 and Xgwm210, and distal to Xcfd36, Xgwm455, and Xgwm296, which are all mapped to the distal deletion bin 2DS5-0.47-1.00 (Sourdille et al. 2004), indicating that the gene is located within this deletion bin.

Based on the preliminary linkage analysis, we used the sequences of the ESTs mapped to the distal deletion bin (2DS5-0.47-1.00) and co-linearity with Brachypodium or rice to develop new markers linked to the Sr gene in CIae 25. Five co-dominant STS markers were developed and designated as Xrwgs33 to Xrwgs37 (Table 2). Xrwgs36 amplified two DNA fragments in estimated sizes of 1,200 and 1,240 bp, respectively, while the other four STS markers, including Xrwgs33, Xrwgs34, Xrwgs35, and Xrwgs37, all amplified polymorphic bands of less than 500 bp (Fig. 2). Four STS markers, including Xrwgs33, Xrwgs35, Xrwgs36, and Xrwgs37, amplified highly intense bands. Two STS markers Xrwgs33 and Xrwgs34 amplified the bands with a large size difference between the parents (Fig. 2). To determine the relationship between Sr46 and Sr6 on chromosome arm 2DS (Tsilo et al. 2010), the SSR markers around Sr6 were also screened for polymorphism between CIae 25 and AL8/78 and additional five polymorphic SSR markers in the region were mapped with the mapping population.

Table 2 Primers for newly developed STS markers linked to the stem rust resistance gene in Ae. tauschii CIae 25
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Fig. 2
figure 2

Gel images of five new codominant sequence-tagged site (STS) markers linked to the stem rust resistance gene in Ae. tauschii accession CIae 25. Amplicons of five codominant STS markers were separated on polyacrylamide gels and generated polymorphic bands distinguishing the CIae 25 (lane 1) and AL8/78 (lane 2) parents of the CIae 25 × AL8/78 F2 population. Lanes 3 through 8 contain samples from the population with the parental marker genotypes. The numbers on the left and right indicate size of polymorphic bands and DNA ladder (lane L) in base pair (bp), respectively

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For determining the identities of CIae 25 and AUS 18913 in which Sr46 was originally identified, we found that CIae 25 was originally collected from Gilan, Iran in 1955 with original accession number 2147 designated by Kyoto University, Kyoto, Japan (USDA-ARS, National Genetic Resources Program. Available: http://www.ars-grin.gov/cgi-bin/npgs/acc/display.pl?1000898. Accessed on November 23 2014). In the AusPGRIS, AUS 18913 has additional names listed as CIae 25 and 2147 (Supplementary material 1), clearly indicating that AUS 18913 and CIae 25 are the same accession that was assigned different names in the two germplasm systems in Australia and the United States, respectively. The genotyping analysis revealed that CIae 25, AUS 18913, and TA 1703 produced identical amplicons from the STS marker Xrwgs33 linked to the Sr gene in CIae 25 and 18 SSR markers that were polymorphic between CIae 25 and AL8/78. As shown in Fig. 3, four illustrative SSR markers Xwmc432, Xgwm210, Xwmc405, and Xcfa2040 produced bands with sizes of 217 and 238, 178, 217, and 284 and 316 bp, respectively, from the three Ae. tauschii accessions TA 1703, AUS 18913, and CIae 25 and the two SHW lines SW8 and SW73. The results from the stem rust evaluation showed that CIae 25 had the same or similar reactions to the six Pgt races as AUS 18913 and TA 1703 when they were tested at the various environmental conditions (Table 3; Fig. 4; Supplementary material 2–6). Therefore, marker genotyping and stem rust responses confirmed that the CIae 25 has the same genotype as AUS 18913 and TA 1703 and that the Sr gene in CIae 25 is the gene Sr46 identified in AUS 18913.

Fig. 3
figure 3

Gel images of four Ae. tauschii accessions and controls analyzed with four illustrative simple sequence repeat (SSR) markers. The numbers on the top of each image indicate the samples for Ae. tauschii accessions AL8/78 (lane 1), TA 1703 (lane 2), AUS 18913 (lane 3), and CIae 25 (lane 4), synthetic hexaploid wheat lines SW8 and SW73 having pedigrees of Langdon/CIae 25 (lane 5) and Rusty/CIae 25 (lane 6), respectively, and durum wheat line Rusty (lane 7) and cultivar Langdon (lane 8). The numbers on the left of each image indicate size of polymorphic bands between AL8/78 and the other three Ae. tauschii accessions (TA 1703, AUS 18913, and CIae 25) in base pair (bp). The numbers on the right of each image indicate size of DNA ladder (lane L). The four SSR markers Xwmc432, Xgwm210, Xwmc405, and Xcfa2040 were previously mapped onto chromosome 1D, 2D, 5D, and 7D, respectively (Somers et al. 2004). They produced identical amplicons from the three Ae. tauschii accessions TA 1703, AUS 18913, and CIae 25

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Table 3 Infection types of Ae. tauschii accessions CIae 25, AUS 18913, TA 1703, and checks to six races of P. graminis f. sp. tritici (Pgt) in the greenhouse experiments conducted at USDA-ARS Cereal Disease Laboratory (CDL), St. Paul, MN and USDA-ARS, Northern Crop Science Laboratory (NCSL), Fargo, ND, respectively
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Fig. 4
figure 4

Seedling reactions of four Ae. tauschii accessions AL8/78, CIae 25, TA 1703, and AUS 18913, common wheat cultivar ‘Chinese Spring’ (susceptible check), and durum wheat cultivar ‘Langdon’ (resistant check) to races RKQQC and QTHJC of P. graminis f. sp. tritici in greenhouse experiment. The three Ae. tauschii accessions CIae 25, TA 1703, and AUS 18913 showed the similar reactions to the two races

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Stem rust tests at different environmental conditions showed that Sr46 is a temperature-sensitive gene. In the greenhouse experiments, the three Ae. tauschii accessions exhibited similar moderate or high levels of resistance to races RKQQC, QTHJC, QFCSC, TPMKC, and TTTTF and intermediate reactions to race MCCFC (Table 3; Fig. 4). At the low temperature environment (15/18 °C), the accessions displayed susceptible or intermediate reactions to races RKQQC, QTHJC, and MCCFC and they also had susceptible reactions or decreased levels of resistance to races QFCSC and TTTTF (Supplementary material 2–6). At the high temperature environment (20/24 °C), the three Ae. tauschii accessions had susceptible reactions only to RKQQC and MCCFC in the CDL test. They had intermediate or susceptible reactions to RKQQC at the NCSL and TTTTF at the CDL. The data suggest Sr46 was less effective at the low temperature environment compared to the high temperature environment and that Sr46 was most effective in the greenhouse tests at both the CDL and the NCSL. Infection type data also showed that the SHW line SW8 was resistant to all six races but SW73 was susceptible to five of the six races except for TPMKC in all the experiments (Table 3; Supplementary material 2–6), suggesting that the resistance of Sr46 is suppressed in the SHW having Rusty background. Because SW8 had a similar level of resistance compared to its durum parent Langdon in all the experiments, the resistance in SW8 may be due to the presence of Sr13 or other genes from Langdon.

The linkage map of Sr46 consists of 16 SSR and five STS markers and its total length was 64.0 cM (Fig. 5). The average genetic distance between two markers was 3.2 cM. Among the 16 SSR markers, six, including Xbarc124, Xgwm210, Xcfd36, Xgwm455, Xgwm296, and Xcfd43, were previously mapped to deletion bin 2DS5-0.47-1.00; marker Xgwm102 was mapped to deletion bin 2DS1-0.33-0.47; two SSR markers, Xgwm261 and Xgwm484, were mapped to deletion bin C-2DS1-0.33; and the other nine SSR markers are unassigned (Sourdille et al. 2004). Based on the genetic linkage map, several markers were identified to be closely linked to Sr46 in the distal region of chromosome arm 2DS (Fig. 5). On the distal side of the gene, there are two SSR markers, Xgwm210 and Xbarc124, linked to the gene at a distance of 3.9 and 7.7 cM, respectively (Fig. 5). On the proximal side of the gene, there are three SSR markers and one STS marker, including Xcfd36, Xwmc111, and Xgwm455, and Xrwgs33, linked to the gene at distances of 5.6, 5.9, 7.6, and 7.9, respectively.

Fig. 5
figure 5

Alignment of genetic and physical location of Sr46 and its linked markers on chromosome arm 2DS. a Genetic map of 2DS on CIae 25 × AL8/78 F2 population. b Physical map of 2DS of Chinese Spring, which was reproduced from Sourdille et al. (2004). Deletion bins are indicated by rectangle boxes

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Validation of markers for marker-assisted selection

Three markers, including SSR markers Xgwm210 and Xwmc111 and STS marker Xrwgs33, which are closely linked to Sr46, were analyzed on a panel of 31 common wheat cultivars and a breeding line (Table 4; Supplementary material 7). Because flanking marker Xcfd36 produced multiple bands, it was not used for the validation. Marker Xgwm210 amplified a 178-bp band from CIae 25 and a 174-bp band from AL8/78 and all the wheat cultivars and line IL06-14262. This marker also amplified a 176-bp band from IL06-14262 and a 172-bp band from all the cultivars except for Jinan 177 from which a 173-bp band was produced. Marker Xwmc111 amplified two bands with sizes of 385 and 404 bp from CIae 25 and two bands with sizes of 394 and 410 bp from AL8/78. Among the 32 wheat cultivars and line, nine cultivars had the marker allele of AL8/78 (394 + 410 bp) and all other cultivars and the line had different amplicons from both CIae 25 and AL8/78. This result suggests that SSR markers Xgwm210 and Xwmc111 can be effectively used in deployment of Sr46 into diverse backgrounds of common wheat germplasm.

Table 4 Amplified fragment size (bp) from Ae. tauschii accessions CIae 25 and AL8/78 and 31 wheat cultivars and a breeding line at the three marker loci closely linked to stem rust resistance gene Sr46 in Ae. tauschii CIae 25
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Marker Xrwgs33 generated a 226-bp band from CIae 25 and a 200-bp band from AL8/78. It produced 200-bp bands from six of the seven Chinese cultivars, a 224-bp band from three cultivars, and a 151-bp band from the remaining 22 cultivars and line IL06-14262 (Table 4; Supplementary material 7). Thus, Xrwgs33 produced amplicons with a large size difference (75 bp) between the marker allele in CIae 25 and the alleles of a majority of the wheat cultivars and line. However, its usefulness in marker-assisted selection for Sr46 may need to be further tested because it amplified two bands (approximately, 226 and 228 bp) from Rusty and Langdon and a band (225 bp) from the line IL06-14262 and 28 cultivars with the same or similar size as the 226-bp band from CIae 25.

Discussion

Stem rust evaluation results from Rouse et al. (2011a) and this study suggest that Ae. tauschii accession CIae 25 conferred moderate levels of resistance to races TTKSK, TRTTF, QTHJC, and RKQQC and high levels of resistance to TPMKC, QCCJB, and JCMNC. Phenotyping of the CIae 25 × AL8/78 F2 population indicated that a single gene in CIae 25 confers resistance to races TTKSK and TPMKC. Using a population of 179 susceptible F2 plants derived from the cross between CIae 25 and AL8/78, we mapped the Sr gene to the distal region of the deletion bin 2DS5-0.47-1.00 on the short arm of chromosome 2D with 16 SSR and five newly developed STS markers. The population of 179 susceptible F2 plants is genetically equivalent to 358 gametes or 358 doubled haploid lines. The genetic linkage map developed from such a population should be more accurate than the ones based on smaller populations. The order of most SSR markers on the map is consistent with that of the wheat SSR consensus map (Somers et al. 2004). However, several SSR markers are not in the same order as those on the consensus SSR map. The inconsistency between the Sr gene map and the SSR consensus map might be caused by difference in 2D chromosomes between bread wheat and Ae. tauschii because the SSR consensus map was developed primarily based on hexaploid wheat (Somers et al. 2004).

The map location of the Sr gene in CIae 25 identified from our study prompted us to examine its relationship with gene Sr46 previously assigned to chromosome arm 2DS from Ae. tauschii accession AUS 18913. Based on the survey on CIae 25 and AUS 18913 in the USDA-ARS National Plant Germplasm System and AusPGRIS, respectively, we found that AUS 18913 and CIae 25 are actually the same accession originally collected from Iran. In addition, evidences from cross-referenced marker and stem rust test information also support our conclusion that CIae 25 carries Sr46. The information in the Catalog of Gene Symbols for Wheat indicates that Sr46 co-segregated with RFLP marker Xpsr649 at both the diploid and hexaploid levels and a PCR-based marker, csSC46, was developed from a BAC clone containing Xpsr649 (Evans Lagudah, unpublished; McIntosh et al. 2013). Xpsr649 is 0.3 cM from another RFLP marker Xpsr908 on the RFLP map of chromosome 2D (Devos et al. 1993; http://wheat.pw.usda.gov/cgi-bin/cmap/viewer?mapMenu=1&featureMenu=1&corrMenu=1&displayMenu=1&advancedMenu=1&ref_map_accs=Ta-Gale-2D&sub=Draw+Selected+Maps&ref_map_set_acc=Ta-Gale-2D&data_source=GrainGenes), whereas Xpsr908 is closely linked to the SSR marker Xgwm210 on the wheat composite map of chromosome 2D (http://wheat.pw.usda.gov/cgi-bin/cmap/viewer?ref_map_set_acc=Wheat_Composite_2004&ref_map_accs=Wheat-Composite2004-2D&ref_species_acc=1&data_source=GrainGenes), suggesting that Xgwm210 is closely linked to Sr46 identified in AUS 18913. Xgwm210 is physically located in the deletion bin 2DS5-0.47-1.00 (Somers et al. 2004) and it is closely linked to the Sr gene in CIae 25, indicating that the Sr gene in CIae 25 and Sr46 are located in the same chromosome region on the short arm of chromosome 2D.

Rouse et al. (2011a) included two Ae. tauschii accessions (TA 1703 and AUS 18913) for Sr46 in the Ae. tauschii germplasm screening for reactions to multiple races of stem rust. The two accessions exhibited consistent reactions to TTKSK, TRTTF, TTTTF, and TPMKC, but they showed variations in reactions to races QTHJC and RKQQC. Rouse et al. (2011a) indicated that variable reactions of TA 1703 and AUS 18913 to QTHJC and RKQQC might be attributed to unstable intermediate reactions to the two races and/or the screening of two accessions in different experiments. The stem rust evaluations conducted in this study showed that the three Ae. tauschii accessions (CIae 25, AUS 18913, and TA 1703) had similar reactions to each of the six Pgt races including RKQQC and QTHJC when they were tested in the same experiments or in the different experiments with the same or similar environmental conditions. The results from genotyping analysis revealed that the three Ae. tauschii accessions had the same alleles at 19 marker loci distributed in seven D-genome chromosomes. Thus, molecular mapping, genotyping analysis, and the stem rust tests performed in this study confirmed that the resistance to stem rust in Clae 25 is conditioned by Sr46. The stem rust tests also showed that Sr46 is a temperature-sensitive gene that can become less resistant or susceptible to several races such as RKOOC, QTHJC, MCCFC, and TTTTF in both low- and high-temperature conditions. The decreased effectiveness of Sr46 in both the high and low temperature environments compared to the greenhouse environment may be due to the difference in light intensity in the growth chambers compared to the greenhouse. Unfortunately, our experimental design did not permit the evaluation of the effects of light intensity per se. Overall, Sr46 was most ineffective at the lower temperature.

Among the 57 known Sr genes (McIntosh et al. 2013), only Sr6 (Tsilo et al. 2009, 2010) and Sr46 (Evans Lagudah, unpublished, McIntosh et al. 2008) were previously mapped to chromosome arm 2DS. We therefore, examined the relationship between Sr6 and Sr46. The gene Sr6, which originated from common wheat ‘Red Egyptian’ (CItr 12345) (Loegering and Harmon 1969), exists in many Australian and North American wheat cultivars (McIntosh et al. 1995) and has been one of the most important Sr genes in North America due to its resistance against a wide range of races, including TPMKC (Tsilo et al. 2009). However, Sr6 is ineffective against TTKSK (Jin et al. 2007). Sr6 was mapped to the interval between SSR markers Xcfd43 and Xgwm102 on chromosome arm 2DS (Tsilo et al. 2010). On the current genetic linkage map, the Sr46 in CIae 25 is 48.6 cM away from marker Xcfd43 (Fig. 5), which is 1.2 and 1.5 cM away from Sr6 in two different populations (Tsilo et al. 2009, 2010). Thus, Sr46 is at least 48.6 cM away from Sr6. Such a genetic distance may allow pyramiding the two genes together into a cultivar.

The D genome of Ae. tauschii has been involved in the origin and evolution of hexaploid wheat. A previous study based on meiotic pairing showed that the D genome of Ae. tauschii remains largely unaltered and homologous to the D genome of common wheat (Kimber and Zhao 1983). Useful genes in Ae. tauschii could easily be incorporated into common wheat through homologous recombination. Therefore, Sr46 can be transferred through direct crossing between CIae 25 and a common wheat line similar to transfer of SrTA1662, SrTA10187, and SrTA10171, which were introduced into hard winter wheat lines from Ae. tauschii using direct crossing (Olson et al. 2013a, b). The two SSR markers closely linked to Sr46, including Xgwm210 and Xwmc111, which differentiate CIae 25 from all the 31 wheat cultivars and a breeding line genotyped in this study, can be used for marker-assisted selection for Sr46 in wheat breeding programs.

Author contribution statement

SSX, GY, and JBR initiated the project, designed the experiment, developed and evaluated the populations, marker development and analysis and prepared the manuscript; QZ performed stem rust test and genotyping for the three Ae. tauschii accessions and controls; SZ and TLF-assisted stem rust test using local races; MNR and YJ conducted stem rust test using TTKSK; ESL provided information on Sr46 originally identified in Ae. tauschii AUS 18913; all authors provided comments and revisions of the manuscript.