Introduction

Bread wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is one of the most important cereal crops providing approximately 20% of the food calories and protein for more than 4.5 billion people (Gupta et al. 2008). Reducing yield losses caused by fungal diseases is an effective way to enhance wheat production. Puccinia triticina Eriksson (Pt) and Puccinia striiformis f. sp. tritici (Pst), the causal agents of wheat leaf rust and stripe rust, respectively, are two devastating fungal diseases threatening global wheat production. In recent years, Pt and Pst pathogens have become increasingly problematic due to the emergence of widely virulent races (Boshoff et al. 2018; Han et al. 2015; Hovmøller et al. 2015; Milus et al. 2009; Omara et al. 2021).

New Pst races virulent on wheat genotypes with Yr5 have been reported in various countries, including China, Australia, India, and Turkey (Tekin et al. 2021; Zhang et al. 2022). Race CYR32 was responsible for severe stripe rust epidemics in China in 2001/2002, which led to significant yield losses across approximately 6.6 million hectares of wheat area (Wan et al. 2004). This strain continues to be one of the most predominant races in China even today (Wang et al. 2018). Race CYR34 was first isolated in Sichuan Province, China, in 2008 and was virulent to Yr24/Yr26 and Yr10, resulting in many bread wheat cultivars carrying these Yr genes becoming susceptible (Liu et al. 2010; Wang et al. 2019). The Pt races THTT, THTS, THJT, THJS, PHTT, and PHJT were the most common races in China and showed virulence to many Lr genes, including Lr1, Lr2a, Lr2b, Lr2c, Lr3, Lr3bg, Lr10, Lr11, Lr14a, Lr14b, Lr16, Lr17, Lr26, Lr32, LrB, Lr33, and Lr50 (Zhang et al. 2020). China encountered severe leaf rust epidemics in the years 2012, 2013, and 2015, leading to significant reductions in yield (Zhang et al. 2020). Although fungicides are available for controlling these rust diseases, they are expensive and may pose risks to human health and the environment. Hence, more Pt and Pst resistance genes are needed to diversify the combinations of deployed resistance genes to minimize the risks associated with relying on limited sources of resistance.

So far, approximately 83 leaf rust resistance (Lr) genes and 86 stripe rust resistance (Yr) genes have been cataloged in wheat and its wild relatives (Kolmer et al. 2023; Zhu et al. 2023). Among these, all-stage resistance (ASR) and adult-plant resistance (APR) genes are the two major types of rust resistance genes (Chen 2005). Most of these Lr and Yr genes are ASR genes, which exhibit efficacy in both seedling and adult-plant stages. However, owing to the size and complexity of wheat genomes, only 11 Lr genes (Lr1, Lr9/Lr58, Lr10, Lr13, Lr14a, Lr21, Lr22a, Lr34, Lr42, Lr47, and Lr67) and ten Yr genes (Yr5/YrSP, Yr7, Yr27, Yr15, Yr18, Yr36, Yr46, Yr28, YrU1, and Yr10/YrNAM) have been cloned to date (Li et al. 2023; Ni et al. 2023) either by map-based cloning or by rapid gene-cloning methods, including MutChromSeq, MutRenSeq, MutIsoSeq, and STAM (Ni et al. 2023; Sánchez-Martín et al. 2016; Steuernagel et al. 2016; Wang et al. 2023b). Among the cloned Lr and Yr genes, Lr34/Yr18/Sr57/Pm38, Lr67/Yr46/Sr55/Pm46, and Yr36 are APR genes encoding a putative ATP-binding cassette transporter, a hexose transporter, and a kinase-START protein, respectively (Krattinger et al. 2009; Moore et al. 2015; Fu et al. 2009).

Wild relatives of wheat have previously been utilized for transferring Lr and Yr genes into common wheat varieties, including Yr34, QYrtm.pau-2A, QYrtb.pau-5A, LrPI119435, and Lr63 from T. monococcum (Chen et al. 2021; Chhuneja et al. 2008; Kolmer et al. 2010; Wang et al. 2023a); Yr15, Yr36, and Lr64 from T. dicoccoides (Klymiuk et al. 2018; Ren et al. 2023); Lr21, Lr22a, Lr32, Lr39-Lr43, and Yr28 from Ae. tauschii (Athiyannan et al. 2022b; Ren et al. 2023); Lr25, Lr26, Lr45, Yr9, and Yr83 from Secale cereale (Li et al. 2020; Spetsov and Daskalova 2022); Lr28, Lr35, Lr36, Lr47, Lr51, and Lr66 from Ae. speltoides (Li et al. 2023; Marais et al. 2010b); Lr62 and Yr42 from Ae. neglecta (Marais et al. 2009); and Lr56 and Yr38 from Ae. sharonensis (Marais et al. 2010a). A recent study showed that the leaf and stripe rust resistance gene Lr/Yr548 was originated from Ae. sharonensis and Ae. longissima, which are closely related diploid species of the section Sitopsis (Sharon et al. 2023).

The two linked all-stage resistance genes Lr53 and Yr35 were introduced from T. dicoccoides accession 479 into hexaploid wheat and were mapped on chromosome arm 6BS using monosomic analyses and telocentric mapping (Marais et al. 2003, 2005a). Lr53 confers high resistance to at least 55 individual Pt races and five inoculum mixtures of Pt from North America, South Africa, India, and Australia, and Yr35 exhibits effectiveness against 11 Pst races from the same regions (Dadkhodaie et al. 2011; Dong et al. 2017; Marais et al. 2018, 2005a; Raghunandan et al. 2022). Leaf and stripe rust isolates virulent on Lr53 and Yr35 have not yet been identified, making them potentially useful for breeding rust-resistant wheat cultivars. The objectives of this study were to: (1) test whether Lr53 and Yr35 confer resistance against Pt and Pst races prevalent in China, (2) verify the origin of the introgressed chromosomal segment carrying these two genes, and (3) generate precise genetic maps and identify potential candidate genes associated with Lr53 and Yr35.

Materials and methods

Plant materials and mapping populations

As a source of the rust resistance genes Lr53 and Yr35, we used wheat accessions 98M71 (PI 648417; pedigree: T. dicoccoides-479/4*CS//3*CS-S/3/CS) and Thatcher-Lr53 (PI 682091; pedigree: CS*4/T. dicoccoides-479//3*CS-S/3/CS/4/5*Thatcher). These accessions are near-isogenic lines to wheat varieties Chinese spring (CS) and Thatcher, respectively (Marais et al. 2018, 2005a). The introgression line 98M71 was crossed with the susceptible wheat line Avocet-S and the CS ph1b mutant (CSph1b) to generate two mapping populations. PCR Markers Xwgc2049 and Xwgc2111 were used to confirm the absence of the Ph1 gene (Gyawali et al. 2019). We evaluated a subset of 136 F2 plants from the 98M71 × Avocet-S cross using Pt race PHQS, and another subset of 117 F2 plants from the same population with Pst race CYR34. The second population (98M71 × CSph1b), which included 1,946 plants derived from selected F3 families that were homozygous for the ph1b mutation and segregating for the introgressed alien segment carrying Lr53 and Yr35, was used to construct the genetic linkage maps. Eight Ae. longissima and two Ae. sharonensis accessions obtained from the Chinese Crop Germplasm Resources Information System (https://www.cgris.net/) were evaluated using PCR markers derived from the introgressed segment of 98M71. Finally, we used a collection of 87 accessions of T. turgidum (including T. dicoccon, T. dicoccoides, and T. durum) and 149 accessions of T. aestivum to determine the value of the tightly linked PCR markers identified in the present study for marker-assisted selection.

Leaf rust and stripe rust assays

The leaf rust and stripe rust seedling assays for both the parental lines and the mapping populations were conducted at the Peking University Institute of Advanced Agricultural Sciences, Weifang, China. The avirulence/virulence profiles of the Pt races (PHQS, THDB, PHRT, PHTT, and FHJR) and the Pst races (CYR32 and CYR34) used in this study can be found in Supplementary Table 1. Seedlings at the three-leaf stage were subjected to challenge with fresh urediniospores of Pt or Pst (1:30 talcum powder) using the shaking off method (Chen et al. 2021). The inoculation, incubation, and scoring of disease responses followed established procedures detailed in previous studies (Chen et al. 2021; Stakman et al. 1962). For plants carrying recombination events within the candidate region, we performed progeny tests including approximately 25 plants from each F3:4 family inoculated with Pt or Pst races. The infection types (ITs) of wheat plants were then scored using a 0–4 scale (Chen et al. 2021).

Sequencing and bioinformatics analysis

RNA-seq of 98M71 was carried out at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). The raw sequencing data have been deposited at the National Genomics Data Center (NGDC) under the BioProject accession number PRJCA022411. Exome-capture data for the hexaploid wheat accession Avocet-S were downloaded from the T3/Wheat database (https://triticeaetoolbox.org/wheat/). The published reference genomes of T. dicoccoides (Zavitan) (Avni et al. 2017), T. durum (Svevo) (Maccaferri et al. 2019), T. urartu (G1812) (Ling et al. 2018), T. monococcum (PI 306540) (Wang et al. 2023a), Aegilops tauschii (AL8/78) (Luo et al. 2017), five Sitopsis species of Aegilops (TS01, TE01, TB01, TH02, TL05, AS_1644, and AEG-6782-2) (Avni et al. 2022; Li et al. 2022), and T. aestivum (CS, ArinaLrFor, Attraktion, Fielder, Jagger, Julius, Kariega, Kenong 9204, LongReach Lancer, CDC Landmark, Mace, Norin61, Renan, CDC Stanley, and SY Mattis) were used for comparative analysis (Athiyannan et al. 2022a; Sato et al. 2021; Shi et al. 2022; The International Wheat Genome Sequencing Consortium 2018; Walkowiak et al. 2020). Raw reads of 98M71 were quality-trimmed using Trimmomatic v0.32 (Bolger et al. 2014). The trimmed reads were then aligned to the reference genome of CS using STAR v2.7.10a (Dobin et al. 2013). Freebayes v1.3.6 and BCFtools v1.14 were used for variant calling and filtering (Garrison and Marth 2012). Single-nucleotide polymorphisms (SNPs) were utilized to determine the size of the alien chromosome segment introgressed into bread wheat. Sequences were aligned using Muscle as implemented in software Mega v7.0 (Kumar et al. 2016). A phylogenetic tree was generated using the neighbor-joining method, and the resulting tree was visualized using Interactive Tree Of Life (iTOL) v5.0 (https://itol.embl.de/).

Development of PCR markers

To amplify gene regions harboring putative polymorphisms, genome-specific primer pairs were designed using the Primer3 software (https://bioinfo.ut.ee/primer3-0.4.0/primer3/). The identified polymorphic sites were then utilized for developing two types of markers: cleaved amplified polymorphic sequence (CAPS) and insertion–deletion (InDel) markers (Bhattramakki et al. 2002; Konieczny and Ausubel 1993). PCR reactions were carried out in a Veriti 96-Well Fast Thermal Cycler (Applied Biosystems, USA). The PCR products that exhibited the expected sizes were subjected to Sanger sequencing to verify the presence of the expected polymorphisms. Restriction enzymes for digestion of the PCR products were purchased from New England BioLabs Inc. (Hitchin, UK) and employed according to established protocols.

qRT-PCR analysis

At the three-leaf stage, 98M71 plants were inoculated with Pt (race THDB mixed with talcum powder) or with mock (talcum powder) in two independent growth chambers under identical environmental conditions, including a temperature regime of 24 °C during the day and 22 °C at night with a photoperiod of 16 h light and 8 h dark. Meanwhile, 98M71 plants were inoculated with Pst (race CYR34 mixed with talcum powder) or with mock (talcum powder) in another two independent growth chambers set at 18 °C during the day and 15 °C during the night. The Pt-/Pst-inoculated leaves and the mock-inoculated leaves from different plants were collected at 6 days post inoculation (dpi) and immediately stored in liquid nitrogen. Total RNA extraction was performed using the spectrum plant total RNA kit (MilliporeSigma, MA, USA). RNA-seq was carried out at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). Differentially expressed genes (DEGs) between Pt-/Pst-inoculated and mock-treated samples were identified using the edgeR software, employing significance thresholds of FDR < 0.05, p-value < 0.05, and |log2foldchange|> 1 (Robinson et al. 2010). Furthermore, qRT-PCR validation of candidate DEGs was performed using an ABI QuantStudio 5 real-time PCR system (Applied Biosystems, CA, USA). The transcript levels were determined in four biological replicates and quantified as fold-ACTIN levels (Chen et al. 2018; Zhang et al. 2017).

Cytogenetic assays

For genomic in situ hybridization (GISH), the genomic DNA of Ae. longissima (SlSl) and Ae. sharonensis (SshSsh) was labeled using the Atto550 NT labeling kit and Atto488 NT labeling kit (Jena Bioscience, Jena, Germany), respectively. The genomic DNA of CS was used as blocking DNA. For fluorescence in situ hybridization (FISH), the probes Oligo-pSc119.2 (Tang et al. 2014), Oligo-pTa535 (Tang et al. 2014), and Oligo-pTa-713 (Zhao et al. 2016) were employed for the identification of chromosomes of common wheat and Aegilops species. These synthetic oligonucleotides were labeled at the 5′ end with 6-carboxyfluorescein (6-FAM) or 6-carboxytetramethylrhodamine (Tamra) by Sangon Biotech Co. (Shanghai, China). The in situ hybridization procedure was conducted using the methods described previously (Fan et al. 2023). Chromosome preparations were counterstained with DAPI (4′,6-diamidino2-phenylindole) in Vectashield (Vector Laboratories, Burlingame, USA). Images were captured using a BX-63 microscope (Olympus, Japan) equipped with a Photometric SenSys Olympus DP70 CCD camera.

Transferring of the truncated alien segment carrying Lr53 and Yr35 to hexaploid wheat

The recombinant obtained from the 98M71 × CSph1b mapping population, possessing the truncated alien segment but carrying Lr53 and Yr35, was subjected to crosses and backcrosses with the Chinese bread wheat cultivar Yangmai21 (YM21). YM21 is known to be susceptible to many Pt races, including THDB, PHRT, PHTT, PHQS, FHJL, and HCJR (Li et al. 2023). Flanking and completely linked DNA markers were used to validate the presence of the truncated alien segment in each generation. BC1F1 plants heterozygous for the truncated alien segment were self-pollinated. Subsequently, selected BC1F2 plants homozygous for the 98M71 allele were divided into six groups and grown in six independent growth chambers. Each group was inoculated with specific rust races: Pst race CYR34 or Pt races THDB, PHQS, PHTT, PHRT, and FHJR.

Statistical analyses

The polymorphic PCR markers and the rust resistance phenotypes were used to construct the genetic maps of Lr53 and Yr35 using the software MapChart v2.2 (https://www.wur.nl/en/show/Mapchart.htm) (Voorrips 2002). The significance of the differences in transcript levels was estimated using two-sided unpaired t-test.

Results

Characterization of leaf rust and stripe rust resistance in wheat line 98M71

Seedling tests revealed that the introgression line 98M71 exhibited strong resistance (ITs = 0; to 1) against all five Pt races tested, while its recurrent parent CS showed susceptibility with ITs ranging from 3+ to 4 (Fig. 1a). When evaluated against Chinese Pst races CYR32 and CYR34, 98M71 displayed high resistance (ITs = 0; to ;1-) against Pst race CYR34, but was susceptible (ITs = 3+) to the other race CYR32. By contrast, the recurrent parent CS exhibited susceptible infection types (ITs = 3+) to both Pst races (Fig. 1b).

Fig. 1
figure 1

Infection types of 98M71 and its recurrent parent CS in response to Pt and Pst races. a Susceptibility or resistance responses of 98M71 and CS to Pt races PHQS, THDB, PHRT, PHTT, and FHJR. b Susceptibility or resistance responses of 98M71 and CS to Pst races CYR32 and CYR34. The presence or absence of the resistant allele is indicated by + or −, respectively. R, resistant; S, susceptible

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In a subset of 136 F2 plants from the 98M71 × Avocet-S cross inoculated with Pt race PHQS, 91 plants were resistant (ITs = 0; to ;1-) and 45 were classified as susceptible (ITs = 3+ to 4). This observed ratio deviated slightly from the expected ratio of 3:1 (resistant : susceptible), with an excess of susceptible plants (χ2 = 4.74, p = 0.029). In another subset consisting of 117 F2 individuals from the same population inoculated with Pst race CYR34, 79 plants exhibited resistance (ITs = 0; to 1) and 38 were susceptible (ITs = 3+ to 4). Chi-squared analysis of the phenotyping results did not deviate from the expected segregation ratio of 3:1 for a single dominant gene (χ2 = 3.49, p = 0.062).

The origin of the alien segment carrying Lr53 and Yr35

Previous studies have reported that the linked resistance genes Lr53 and Yr35 were introgressed into common wheat from T. dicoccoides and mapped to the short arm of chromosome 6B (Dadkhodaie et al. 2011; Marais et al. 2005a). To verify the origin of the Lr53 and Yr35 segment, a comparison was made between the SNPs identified in the RNA-seq data of the donor line 98M71 and those in T. dicoccoides (Zavitan), T. durum (Svevo), and T. aestivum (15 hexaploid wheat varieties). To our surprise, we found that the chromosome 6B in 98M71 had a large number of rare polymorphisms (11,953 SNPs from the start of the chromosome to 687.0 Mb; Table S2) that were absent in all tetraploid and hexaploid wheat accessions. This observation suggests that the chromosome segment carrying Lr53 and Yr35 may not originate from T. dicoccoides.

To further explore the potential origin of the Lr53 and Yr35 segment, the RNA-seq data of 98M71 were compared with the available genomic sequences of T. monococcum (PI 306540), T. urartu (G1812), Ae. sharonensis (TH02 and AS_1644), Ae. longissima (TL05 and AEG-6782-2), Ae. speltoides (TS01), Ae. searsii (TE01), Ae. bicornis (TB01), Ae. tauschii (AL8/78), as well as T. dicoccoides, T. durum, and T. aestivum. We focused only on the polymorphisms located within the ~ 687.0 Mb segment on chromosome 6B (based on CS RefSeq v1.1 coordinates) that are polymorphic among the 11 wheat species described above. Based on this approach, a total of 9294 SNPs were identified among the different wheat species (Table S3). A neighbor-joining tree based on these SNPs showed that 98M71 is located in a branch encompassing the accessions of Ae. longissima and Ae. sharonensis (Fig. 2a), suggesting that the chromosome segment carrying Lr53 and Yr35 likely originated from either of these two Aegilops species or their derived polyploid species (e.g., Ae. kotschyi or Ae. peregrina, genome UUSS).

Fig. 2
figure 2

The origin of the alien segment carrying Lr53 and Yr35. a Phylogenetic analysis based on 9,294 SNPs (Table S3) identified within the ~ 687.0 Mb segment on chromosome 6B across 11 wheat species. The neighbor-joining method was employed for inferring the evolutionary history, and the resulting tree was visualized using the interactive tree of life (iTOL) v5. The wheat line 98M71 is highlighted in red. b Distribution of 8,322 putative Ae. sharonens/Ae. longissima-specific SNPs on chromosome 6B. The introgressed segment in 98M71 spans approximately 687.0 Mb, extending from the start of the chromosome to 687.0 Mb. 1, AEG-6782-2; 2, TL05; 3, AS_1644; 4, TH02; 5, 98M71; 6, Zavitan; 7, Svevo; and 8-22, sequenced T. aestivum accessions CS, ArinaLrFor, Attraktion, Fielder, Jagger, Julius, Kariega, Kenong9204, LongReach Lancer, CDC Landmark, Mace, Norin61, Renan, CDC Stanley, and SY Mattis. This figure was generated using the Integrative Genomics Viewer (IGV) software v2.8.9. Vertical lines in blue represent Ae. sharonens/Ae. longissima-specific SNPs whereas lines in light gray are normal wheat SNPs. Coordinates are based on CS RefSeq v1.1 (color figure online)

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To gain a deeper understanding of the Lr53 and Yr35 chromosomal segment, our analysis focused on the specific SNPs that were present in two accessions of Ae. longissima (TL05 and AEG-6782-2) and two accessions of Ae. sharonensis (TH02 and AS_1644), but absent in other polyploid wheat accessions (T. dicoccoides, T. durum, and T. aestivum). These SNPs are hereafter referred to as Ae. longissima/Ae. sharonensis-specific SNPs. Through this comparison, we identified 8,322 putative Ae. longissima/Ae. sharonensis-specific SNPs that were shared with 98M71 on chromosome 6B (Table S4). To visualize the distribution of these SNPs, we plotted these Ae. longissima/Ae. sharonensis-specific SNPs as blue vertical lines, while other normal wheat SNPs shown in gray (Fig. 2b). This figure revealed that the alien chromosome segment in 98M71 spans a length of approximately 687.02 Mb (from 0 to 687.02 Mb in CS RefSeq v1.1; Fig. 2b). The translocation breakpoint was located between SNPs at positions 687,016,683 bp and 688,295,787 bp (Table S4).

Using the reference genomes of TL05 (Ae. longissima) and TH02 (Ae. sharonensis), we developed 17 genome-specific primer pairs targeting the 6S/6B genomes across the 687.02-Mb introgressed chromosomal segment (Tables S5 and S6). These primers were used to amplify PCR products from 98M71, which were subsequently sequenced using the Sanger method. The obtained sequences from 98M71 were then subjected to BLASTN searches against the reference genomes of the 11 wheat species mentioned above. All the sequences from 98M71 exhibited greater similarity to Ae. longissima/Ae. sharonensis than to the other nine wheat species, including T. dicoccoides (Table S6). Furthermore, we evaluated ten Ae. longissima and Ae. sharonensis accessions with five markers derived from the introgressed S segment of 98M71. Most of these accessions exhibited haplotypes identical to those of 98M71 (Table S7). These findings strongly confirm that the chromosome segment carrying Lr53 and Yr35 indeed originated from either Ae. longissima or Ae. sharonensis, or their derived species.

Six PCR markers pku6B97F (6.03 Mb; CS RefSeq v1.1), pku6B1059 (85.79 Mb), pku6B1851 (209.24 Mb), pku6B2389 (415.87 Mb), pku6B2836 (512.19 Mb), and pku6B613M (613.11 Mb; Fig. S1) were used to genotype the selected 136 and 117 F2 plants from the 98M71 × Avocet-S cross described above. Among the 253 plants evaluated with the six markers, recombination events were observed only between PCR markers pku6B1851 (209.24 Mb) and pku6B2389 (415.87 Mb) at a frequency of 1.0% (Tables S8 and S9). These recombination events are probably due to centromeric (Robertsonian) translocations. However, no recombination was detected among the markers pku6B97F (6.03 Mb), pku6B1059 (85.79 Mb), and pku6B1851 (209.24 Mb) on chromosome arm 6BS in the presence of the Ph1 gene (Tables S8 and S9). These three markers were completely linked to the phenotypes, confirmed that Lr53 and Yr35 were located on chromosome arm 6BS. The translocation breakpoint in 98M71 was defined using PCR markers and pinpointed between markers pku6B4135 (687.02 Mb) and pku6B4191 (690.96 Mb; Fig. S1).

We then explored the presence of the alien translocation in PI 682091 (Thatcher-Lr53), a Thatcher near-isogenic line carrying Lr53 and Yr35 (Marais et al. 2018). Sanger sequencing was performed on seven 6S/6B-genome-specific PCR markers (pku6B165, pku6B97F, pku6B756, pku6B1059, pku6B2836, pku6B613M, and pku6B4135; Table S5) across the introgressed segment. The results confirmed that PI 682091 carries the same allele and length of the alien segment as observed in 98M71.

To validate the findings described above, GISH experiments were conducted. The GISH analyses confirmed the presence of the Ae. longissimi or Ae. sharonensis translocation in 98M71 on chromosome 6B (Fig. 3a–c and S2). Approximately 95% of the physical length of chromosome 6B was replaced by 6S chromatin, while the small wheat 6BL telomeric end accounted for the remaining 5% observed in the images (Fig. 3a–c). This original recombinant chromosome was hereafter referred to as 6SS.6SL-6BL. Further karyotype analysis using FISH revealed strong Oligo-pSc119.2 signals in the telomeric region of the alien chromosome arm 6SS (marked with yellow arrows) in 98M71 (Fig. 3d). In contrast, this pSc119.2 signal was absent in the telomeric region of chromosome arm 6BS in the recurrent parent CS (Fig. 3e), further confirming the presence of the alien translocation in 98M71. Another Oligo-pSc119.2 signal was detected in the telomeric region of chromosome arm 6BL in both 98M71 and CS, indicated by white arrows (Fig. 3d, e).

Fig. 3
figure 3

Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) images of wheat line 98M71. a–c GISH images of mitotic chromosomes in 98M71. Genomic DNA of Ae. longissima (SlSl) and Ae. sharonensis (SshSsh) were labeled with the Atto550 NT labeling kit (red) and Atto488 NT labeling kit (green), respectively. Yellow arrows indicate the introgressed 6S chromosomes. d–e FISH images of 98M71. Probes pSc119.2 (green), pTa535 (red), and pTa713 (yellow) were used for the hybridization experiments. Genomic DNA of CS was used as blocking DNA. Yellow arrows indicate the Oligo-pSc119.2 signals in the telomeric region of the alien chromosome arm 6SS, and white arrows represent the Oligo-pSc119.2 signals in the telomeric region of wheat (CS) chromosome arm 6BL. 6SS.6SL-6BL, the recombinant chromosome in 98M71. All experiments were repeated three times independently with consistent results (color figure online)

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Mapping of Lr53 and Yr35 using ph1b-induced homoeologous recombination

To induce recombination between wheat chromosome 6B and the introgressed 6S chromosome segment, a cross was made between the introgression line 98M71 and the CSph1b mutant. The PCR markers pku6B97F (6.03 Mb), pku6B1851 (209.24 Mb), and pku6B4135 (687.02 Mb) were used to select F2 plants that were heterozygous for the introgressed 6S segment. Subsequently, six F2 plants were obtained which were homozygous for ph1b and heterozygous for the introgressed 6S segment. These plants were self-pollinated to generate F3 seeds for genetic mapping. Among the 188 F3 plants from the 98M71 × CSph1b cross inoculated with Pt race THDB, 132 plants exhibited resistance (ITs = 0; to ;1-) and 56 were susceptible (ITs = 3–4). This distribution corresponded to the segregation ratio of 3:1 expected for a single dominant genetic locus (χ2 = 2.30, p = 0.13). All plants were genotyped using ten 6S/6B-genome-specific PCR markers (Table S5) across the introgressed chromosomal segment. By integrating these polymorphic DNA markers with the leaf rust resistance phenotypes, a genetic linkage map for Lr53 was constructed (Fig. 4a). Based on the linkage results, Lr53 was mapped to the distal region of chromosome arm 6BS, located 0.27 cM distal to pku6B97F, and was completely linked to the marker pku6B165 (Fig. 4a).

Fig. 4
figure 4

Genetic maps for Lr53 and Yr35. a Genetic map for Lr53, constructed using 188 F2 plants and 10 PCR markers. The values to the left of the PCR markers represent the genetic distances in centimorgans (cM). b Genetic map for Yr35 based on 187 F2 plants and 10 PCR markers. The values to the left of the PCR markers indicate the genetic distances in centimorgans (cM). c Colinear genomic region on chromosome 6B of Chinese Spring (RefSeq v1.1). The values to the left of the genes are physical locations in megabases (Mb; CS RefSeq v1.1)

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Similarly, seedlings of another 187 F3 plants inoculated with Pst race CYR34, 130 plants displayed resistance (ITs = 0; to 1), and 57 were susceptible (ITs = 3–4). The observed ratio remained consistent with the expected 3:1 segregation ratio (χ2 = 3.00, p = 0.08). These 187 plants were genotyped using the same PCR markers, resulting in the construction of a genetic map for Yr35 (Fig. 4b). Yr35 was mapped 0.54 cM distal to pku6B97F and was completely linked to pku6B165, within the same interval as Lr53 (Fig. 4a, b).

To determine the localization of Lr53 and Yr35 more precisely, we screened an additional 1,571 F3 plants with the markers pku6B97F (6.03 Mb) and pku6B165 (0.16 Mb; CS RefSeq v1.1). Within this screening, 15 plants carrying informative recombination events were identified. Based on these recombinants and three additional plants found in the previous screening of 375 plants (188 and 187 individuals), the genetic distance between pku6B97F and pku6B165 was reestimated to be 0.46 cM. The progeny of these 18 plants with informative recombination events were subsequently inoculated with Pt race THDB and Pst race CYR34, respectively. Using these recombinants and two newly developed markers within the candidate region [pku6B3127 (3.13 Mb) and pku6B5555 (5.56 Mb); Table S5], both Lr53 and Yr35 were confined to the same 6.03-Mb interval and completely linked to markers pku6B165, pku6B3127, and pku6B5555 (Table S5).

Candidate genes for Lr53 and Yr35 in the colinear regions of sequenced wheat genomes

Comparisons among the published reference genomes of Ae. sharonensis (TH02 and AS_1644), Ae. longissima (TL05 and AEG-6782-2), T. dicoccoides (Zavitan), T. durum (Svevo), and T. aestivum (five hexaploid wheat varieties) revealed potential structural rearrangements and chromosomal inversions within the Lr53 and Yr35 candidate region (Fig. S3). These observed rearrangements and inversions in the candidate region across different wheat genomes provide an explanation for the lack of recombinations detected among markers pku6B165, pku6B3127, and pku6B5555, although a large mapping population consisting of 3,892 gametes was used.

The 6.03-Mb candidate region in CS contains 97 annotated genes with high-confidence (TraesCS6B02G000100–TraesCS6B02G009700). These genes include four annotated NLR genes and 23 receptor-like protein kinases (Table S10). The corresponding regions in the TH02 (Ae. sharonensis) and TL05 (Ae. longissima) reference genomes, spanning 3.28 Mb and 3.37 Mb, respectively, also contain multiple NLR genes and receptor-like protein kinases (Table S11 and S12). These genes hold significant relevance for this project as NLRs and protein kinases are the most prevalent gene classes associated with disease resistance in plants.

Transcript levels of the candidate genes were analyzed in Pt-inoculated and mock-inoculated 98M71 plants at 6 dpi (Table S13). Out of the 119 annotated genes within the candidate region of the TL05 (Ae. longissima) genome, 42 genes were expressed in wheat leaves infected with Pt race THDB. These expressed genes included one annotated NLR gene and 12 receptor-like protein kinases (Table S13). Among them, transcript levels of Ae.longissima.TL05.6S01G0003700.1 were significantly higher in Pt-inoculated plants (FDR < 0.05, p-value < 0.05, and |log2fold change|> 1) compared to mock-inoculated controls (Table S13). Conversely, the Ae.longissima.TL05.6S01G0003400.1 gene exhibited significantly lower transcript levels in Pt-inoculated plants relative to mock-inoculated plants (Table S13).

DEGs between Pst-inoculated and mock-inoculated 98M71 plants at 6 dpi were also identified using RNA-seq data (Table S14). Among the candidate genes in the target interval in TL05, we found that 49 genes were expressed in wheat leaves infected with Pst race CYR34, which included 16 annotated protein kinases and one NLR gene. A total of eight DEGs were significantly upregulated in Pst-inoculated plants relative to mock-inoculated plants (Table S14).

To validate these findings from RNA-seq data, the expression levels of the genes Ae.longissima.TL05.6S01G0003400.1, Ae.longissima.TL05.6S01G0003700.1, Ae.longissima.TL05.6S01G0003200.1, and Ae.longissima.TL05.6S01G0009000.1 were further determined using qRT-PCR. The results confirmed significant upregulation or downregulation (p < 0.05) of these genes after Pt or Pst inoculation compared to mock inoculation (Fig. S4 and S5), supporting the findings obtained from the RNA-seq analysis. In addition, we detected amino acid changes between 98M71 and TL05 for these four DEGs (Table S15), but it was unknown whether Lr53 and Yr35 were present in TL05.

Validation of the Lr53- and Yr35-linked markers in uncharacterized tetraploid and hexaploid wheat accessions

To determine the value of the tightly linked markers identified in this study for marker-assisted selection, we evaluated a collection of 87 accessions of T. turgidum (T. dicoccon, T. dicoccoides, and T. durum) and 149 of T. aestivum with the flanking marker pku6B97F and three completely linked markers pku6B165, pku6B3127, and pku6B5555. None of these wheat accessions showed haplotypes identical to those of 98M71 and PI 682091 (Table S16). PCR amplification using the marker pku6B3127 at an annealing temperature of 56˚C resulted in the generation of an 1118-bp fragment in 73 (83.9%) of T. turgidum accessions and 53 (35.6%) of the T. aestivum accessions (Table S16). Treatment of the amplified PCR products with the restriction enzyme AvaI generated two bands of 418-bp and 700-bp for the introgression lines 98M71 and PI 682091 carrying Lr53 and Yr35, while a single band of 1118-bp was observed for the other tetraploid and hexaploid wheat accessions (Fig. S6). No PCR product was amplified from 14 T. turgidum and 96 T. aestivum accessions (Table S16), indicating the absence of the introgressed segment in these accessions. These results suggest that the marker pku6B3127 holds significant value in predicting the presence of Lr53 and Yr35 in uncharacterized wheat genotypes.

Transfer of a small alien chromosome segment carrying Lr53 and Yr35 into hexaploid wheat cultivar YM21

Using ph1b-induced homoeologous recombination, we successfully obtained a resistant recombinant line named C580 from the 98M71 × CSph1b mapping population. The procedures for generating C580 and transferring the truncated 6S segment into hexaploid wheat are illustrated in Fig. 5a. In C580, PCR markers pku6B165 (0.16 Mb), pku6B3127 (3.13 Mb), and pku6B5555 (5.56 Mb) showed the 6S (98M71) alleles, whereas markers pku6B97F (6.03 Mb), pku6B126 (12.66 Mb), and pku6B273 (27.33 Mb) exhibited the T. aestivum (CS) alleles (Fig. 5b), indicating the crossover breakpoint occurred between markers pku6B5555 and pku6B97F. Therefore, the size of the truncated 6S chromosome segment in C580 is between 5.56 and 6.03 Mb based on Chinese Spring RefSeq v1.1 coordinates (Fig. 5b). FISH-based karyotype analysis revealed strong Oligo-pSc119.2 signals in the telomeric regions of both chromosome arms 6SS and 6BL in C580 (Fig. S7). The progeny of C580 homozygous for the presence of the truncated alien segment showed high resistance to Pt and Pst races THDB, PHQS, PHTT, PHRT, FHJR, and CYR34 (similar to 98M71; Fig. S8), whereas plants homozygous for the absence of the alien segment displayed susceptible reactions.

Fig. 5
figure 5

Introgression of the truncated alien segment (< 6.03 Mb) carrying Lr53 and Yr35 into the common wheat background. a Procedures for generating C580 and transferring the truncated 6S segment into hexaploid wheat. YM21, bread wheat cultivar Yangmai21. b Markers were used to estimate the length of the truncated 6S chromosomal segment in C580. The crossover breakpoint in C580 was between markers pku5555 (5.56 Mb) and pku6B97F (6.03 Mb; CS RefSeq v1.1). Blue regions indicate the introgressed alien chromatin, while white rectangles represent T. aestivum 6B chromatin. c Infection types observed in the new introgression line with the truncated 6S chromosome segment (< 6.03 Mb), as well as the control YM21 and BC1F2 plants lacking the truncated alien segment. Plants were challenged with five Pt races (THDB, PHQS, PHTT, PHRT, and FHJR) and one Pst race (CYR34). + indicates the presence of the resistant allele, while − denotes the absence of the resistant allele. R, resistant; S, susceptible

Full size image

Recombinant C580 with the truncated 6S chromosome segment was crossed and backcrossed once with the susceptible Chinese bread wheat cultivar YM21 (Fig. 5a). Four DNA markers, pku6B165, pku6B3127, pku6B5555, and pku6B97F, were used to verify the presence of the truncated 6S chromosome segment in the selected BC1F2 plants (Fig. S9). The BC1F2 plants homozygous for the truncated 6S segment exhibited strong levels of resistance (ITs = 0; to 1) against all Pt and Pst races tested (races THDB, PHQS, PHTT, PHRT, FHJR, and CYR34; Fig. 5c). In contrast, the control plants YM21 and BC1F2, which lacked the truncated alien segment, displayed susceptible infection types (ITs = 3–4) when challenged with the same races (Fig. 5c). These results suggest that the truncated alien segment possesses a similar resistance profile to the original introgressed segment in 98M71.

Discussion

Introgression of rust resistance genes Lr53 and Yr35 from Aegilops species to bread wheat

Marais et al. (2005a) reported that the rust resistance genes Lr53 and Yr35 were introgressed from T. dicoccoides into common wheat. In this study, we found that the introgressed segment carrying Lr53 and Yr35 was introduced from Ae. longissima or Ae. sharonensis or their derived species. Four lines of evidence support this conclusion. First, the analysis of SNPs and sequence comparisons (Tables S2-S4, S6) demonstrated that the introgressed chromosomal segment clustered together with Ae. longissima and Ae. sharonensis. Second, GISH and FISH analyses confirmed the presence of the Ae. longissima or Ae. sharonensis chromatin in 98M71 on the recombinant chromosome 6B/6S (Fig. 3a–e). Third, the 98M71 × Avocet-S mapping population showed suppression of recombination within the 687.02-Mb introgressed region (except for a few putative centromeric translocations), consistent with the characteristic behavior observed in alien introgressions. Finally, 98M71 and Thatcher-Lr53 were found to carry the same length of the alien chromosome segment throughout their breeding history, which agrees with the discovery of Ae. longissima or Ae. sharonensis introgression. The pedigrees of 98M71 and Thatcher-Lr53 suggest that the introgressed segment passed through at least 6 and 5 backcrosses, respectively, along with several generations of self-pollination to reach their current states. This extensive breeding history would imply multiple opportunities for recombination during the meiosis. However, no recombination events were detected within the introgressed segment present in both 98M71 and Thatcher-Lr53 during crossing and backcrossing. We cannot rule out the possibility that the original alien segment carrying Lr53 and Yr35 was spontaneously introgressed into T. dicoccoides from Ae. longissima or Ae. sharonensis or their derived species, and then this alien segment in T. dicoccoides accession 479 was transferred to hexaploid wheat by Marais et al. (2005a).

Previous study by Dadkhodaie et al. (2011) reported that the Lr53 and Yr35 chromosome region showed a segregation distortion favoring the susceptible allele. Similar results were observed in the current study in tests for both stripe rust and leaf rust, as evidenced by an excess of susceptible plants (Tables S8, S9). The presence of the alien introgression likely explains the observed segregation distortion in the chromosomal region carrying Lr53 and Yr35. Although segregation distortion can occur in both introgressed alien segments and segments from the same species, it is more commonly observed in the former (Chen et al. 2021). Various examples of segregation distortion have been reported in relation to alien introgressions carrying disease resistance genes. Notable instances include Lr19 in Ag. elongatum (Prins and Marais 1999), Lr54 and Yr37 in Ae. kotschyi (Marais et al. 2005b), and Yr34 and QYrtb.pau-5A in T. monococcum (Chhuneja et al. 2008; Lan et al. 2017).

Previous studies had indicated that the introgressed alien segment carrying Lr53 and Yr35 exhibited normal recombination with chromosome 6BS of the wheat line Avocet-S (Marais et al. 2005a), with an estimated recombination rate of 3% (11 recombinants out of 186 individuals) between these two genes (Dadkhodaie et al. 2011). The mapping results positioned Lr53 and Yr35 around 3.0 cM apart, approximately 18.9 cM proximal to the SSR marker gwm191 and 1.1 cM distal to cfd1 (Dadkhodaie et al. 2011). Using the sequences of the flanking markers, we successfully determined the location of the marker cfd1, which is located at ~ 40.70 Mb on chromosome arm 6BS in CS RefSeq v1.1, but we were unable to determine the location of the marker gwm191 on chromosome arm 6BS (gwm191 was found on chromosomes 3D and 7A). This mapping region (distal to cfd1: 40.70 Mb) appears to encompass our purposed candidate region for Lr53 and Yr35 (0–6.03 Mb; CS RefSeq v1.1). However, in the presence of the Ph1 gene, we did not observe recombination between the wheat chromosome arm 6BS and the introgressed 6S segment, as indicated by the lack of recombination events among markers pku6B97F-6.03 Mb, pku6B1059-85.79 Mb, and pku6B1851-209.24 Mb (Tables S8, S9). This absence of recombination is consistent with the typical behavior observed in alien introgressions, although it can also be caused by inverted chromosome segments. In addition, our investigation demonstrated complete linkage between Lr53 and Yr35 in a large mapping population (3,892 gametes) derived from the cross between 98M71 and CSph1b (using ph1b-induced homeologous recombination). These contradictory results might be attributed to differences in the susceptible parental lines used. Marais et al. (2005b) reported that the introgressed segment did not pair with chromosome arm 6BS of CS during meiosis. It is possible that the wheat line “Avocet-S” used by Dadkhodaie et al. (2011) carries any gene(s) that promote homoeologous pairing in the presence of Ph1, which could explain the observed recombination events in their study. Another possibility is the existence of additional Lr/Yr genes within the introgression segment that contribute to resistance against different races of Pt and Pst from various regions. An example of this scenario is the case of the original Sr32 introgression, where another Sr gene (SrAes1t) was later discovered to contribute to resistance (Mago et al. 2013). However, this probability is low since the small alien segment (< 6.03 Mb) generated in this study showed the same resistance profile and similar seedling resistance responses as the original introgression (687.02 Mb) in 98M71.

Genetic mapping of Lr53 and Yr35 and identification of their candidate genes

Using our genetic maps and the available reference genomes of hexaploid wheat (CS), Ae. sharonensis (TH02), and Ae. longissima (TL05), we delimited the region of the Lr53 and Yr35 candidate genes to a 6.03-Mb region in CS, a 3.28-Mb region in TH02, and a 3.37-Mb region in TL05, respectively (Tables S10–S12). Comparisons among the reference genomes revealed the presence of chromosomal rearrangements and inversions within the Lr53 and Yr35 candidate region, as illustrated in Fig. S3. These structural variations likely account for the observed suppression of recombination among markers pku6B165, pku6B3127, and pku6B5555 in the presence of the ph1b mutation. However, it is important to consider the possibility that these rearrangements and inversions may also arise from potential assembly errors in the reference genomes for this specific region.

The NLR candidate genes identified within the colinear regions of the CS, TH02, and TL05 reference genomes exhibit both copy number and structural variations (Tables S10–S12). Similar to the Lr53 and Yr35 candidate region, deletions, duplications, and rearrangements of NLR genes were reported for previously cloned rust resistance genes in wheat, such as Lr47 (Li et al. 2023), Sr13 (Zhang et al. 2017), Sr21 (Chen et al. 2018), and SrKN (Li et al. 2021). As many cloned disease resistance genes in wheat and other plant species encode intracellular NLR proteins (Ellis et al. 2000; Marone et al. 2013; Zhang et al. 2017), the NLR genes within the target region are considered strong candidates for Lr53 and Yr35.

In addition to the NLR candidates, numerous receptor-like protein kinases (Tables S10–S12) were detected in the Lr53 and Yr35 candidate region. Protein kinases represent promising candidate genes because they are involved in rust resistance in wheat and its wild relatives, such as Yr15 (Klymiuk et al. 2018), Yr36 (Fu et al. 2009), Sr60 (Chen et al. 2020), and Lr9 (Wang et al. 2023b). We have prioritized these NLR genes and protein kinases for further functional characterization.

Relationship between Lr53 and Yr35 and other Lr/Yr genes on chromosome 6S of Aegilops species

Several leaf rust and stripe rust resistance genes were identified on chromosome 6S of Aegilops species and transferred to the group 6 chromosomes of common wheat, including Lr9 (Wang et al. 2023b), Lr/Yr548 (Sharon et al. 2023), Lr59 (Marais et al. 2008), linked genes Lr62 and Yr42 (Marais et al. 2009), and linked genes Yr38 and Lr56 (Marais et al. 2010a). Lr9, introgressed into bread wheat from Ae. umbellulata (genome UU), is located on chromosome arm 6BL (Wang et al. 2023b), indicating its distinction from Lr53 and Yr35. The rust resistance gene Lr/Yr548, which was identified in Ae. sharonensis and Ae. longissima, confers resistance against both Pt and Pst pathogens (Sharon et al. 2023). Lr/Yr548 was located at position 51.76 Mb on chromosome 6S (Ae. longissima AEG-6782-2 reference), which differs from the location of Lr53 and Yr35 (0–7.31 Mb; AEG-6782-2 v1.0 coordinates). The absence of Lr/Yr548 in the introgression line 98M71 was also confirmed by using a published diagnostic PCR marker for Lr/Yr548 (Sharon et al. 2023) and BLASTN searches in the 98M71 transcriptome database.

Lr59, a leaf rust resistance gene introduced from Ae. peregrina (genome UUSS), was initially mapped to chromosome arm 1AL (Marais et al. 2008). Subsequent studies by Pirseyedi et al. (2015) revealed that the distal end of the original introgression differed structurally from chromosome 1AL and was homoeologous to the telomeric region of chromosome 6BS (Pirseyedi et al. 2015). Linked resistance genes Lr62 and Yr42 were introgressed from tetraploid Ae. neglecta (genome UUMM), with Lr62 being mapped to the distal ends of chromosome arms 6AS and 6BS (Marais et al. 2018). Linked genes Yr38 and Lr56 were transferred from Ae. sharonensis into chromosome 6A of bread wheat (Marais et al. 2006). During the process of reducing the length of the introgressed segment, Lr56 was separated from Yr38 and mapped to the telomeric region of chromosome 6AS (Marais et al. 2018). The leaf rust resistance genes Lr53 (the current study), Lr56, Lr59, and Lr62 have similar locations in the telomeric region of either 6AS or 6BS, as well as the similar resistance profiles and seedling resistance responses (Marais et al. 2018). These results suggest a potential homeo-allelic relationship among these loci. However, the absence of shared markers and high-resolution genetic maps for Lr56, Lr59, and Lr62 has limited our ability to establish the mapping relationship among these genes. Further analysis is required to determine the relationship between Yr35 and the other two Yr genes (Yr38 and Yr42) on chromosome 6S.

Utilization of Lr53 and Yr35 in agriculture

Using the PCR marker pku6B3127, it was shown herein that the introgressed segment carrying Lr53 and Yr35 is absent in all tested tetraploid and hexaploid wheat genotypes, except for the introgression lines 98M71 and Thatcher-Lr53. This finding indicates that the incorporation of the Lr53 and Yr35 segment has the potential to benefit a wide range of commercially cultivated wheat varieties.

Lr53 confers robust resistance against more than 60 individual or mixed Pt races from China (the current study), South Africa (Marais et al. 2005a), North America (Marais et al. 2018), India (Raghunandan et al. 2022), and Australia (Dadkhodaie et al. 2011), indicating broad-spectrum resistance to different Pt races. The strong and broad-spectrum resistance makes Lr53 a valuable genetic resource in wheat breeding. On the other hand, while Yr35 showed effectiveness against 11 Pst races from different regions (Dong et al. 2017; Marais et al. 2018), our study identified one stripe rust race virulent on Yr35. This indicates limited potential for utilizing Yr35 in regions where the CYR32 race is prevalent. Therefore, a combination of Yr35 with other CYR32-effective resistance genes, such as Yr36 (Fu et al. 2009) and Yr15 (Klymiuk et al. 2018), is a preferred strategy for breeding wheat cultivars with good resistance.

The newly developed introgression line, which contains a very small alien chromosome segment (< 6.03 Mb), provides similar levels of resistance as the initial introgression line 98M71 (Fig. 5c) and minimizes potential linkage drag. This truncated introgression segment with Lr53 and Yr35 presents significant interest for breeding programs, as it confers resistance to two distinct wheat pathogens. However, further studies will be needed to test if Lr53 and Yr35 are effective in different bread wheat backgrounds and to test potential pleiotropic effects. If necessary, PCR markers within the candidate region can be used to develop new introgression lines with even smaller alien segments carrying the Lr53 and Yr35 genes.

In summary, our study successfully demonstrated that the chromosomal segment carrying Lr53 and Yr35 were introgressed into bread wheat from Ae. longissima or Ae. sharonensis or their S-genome containing species. The development of the newly introgressed line and the closely linked PCR markers will facilitate map-based cloning of these two rust resistance genes and accelerate their deployment in wheat breeding programs.