Background

Leaf rust (LR) (Puccinia triticina Eriks, Pt) and stripe rust (YR) (Puccinia striiformis f. sp. tritici, Pst) stand as the two most pervasive foliar diseases significantly impacting global wheat yield. With nearly one-third of the world’s population reliant on wheat as a source of staple carbohydrates and protein [1] and the ever-increasing population on earth (https://www.un.org), it is essential to confront these persistent fungal diseases to sustain food security. The LR outbreaks have been reported to result in yield losses up to 50%, while YR has been documented to incur higher yield losses reaching up to 70%, due to their detrimental effects on the photosynthetic and grain-filling capacity of the crop [2, 3]. The degree of damage, however, is also influenced by the time of infection, environmental conditions, cultivar susceptibility and aggressiveness of the pathogen [1, 4, 5]. India, being one of the leading wheat-producing countries, faces a high incidence of these rusts, particularly in the Northern parts known as the breadbasket of India, due to suitable environmental conditions for pathogens [5,6,7]. The success of the green revolution with the breeding of superior yielding disease-resistant varieties has undeniably resulted in a significant boost in agricultural productivity. However, wheat has succumbed to domestication bottlenecks because of narrow genetic diversity in cultivated germplasm, thereby making it vulnerable to rising biotic and abiotic threats [8, 9]. Therefore, improvement of germplasm through genetic reshuffling is imperative to confront the evolving nature of rusts.

Wild germplasm contains a vast array of genetic diversity that can be harnessed to enhance crop resilience [10]. Incorporation of genetic resistance through wild introgressions offers a more environmentally friendly, cost-effective, and reliable strategy for mitigation of fungal infestation in comparison to chemical control with fungicides [11]. Numerous LR and YR resistance genes have been identified from different wild species such as Lr53/Yr35, Lr64, Yr15, Yr36 from Triticum dicoccoides [12,13,14,15], Lr18, Lr50 from Triticum timopheevii [16, 17], Lr9, Lr76/Yr70 from Aegilops umbellulata [18, 19], Lr54/Yr37 from Aegilops kotschyi [20], Lr56, Yr38 from Aegilops sharonensis [21], Lr57/Yr40 from Aegilops geniculata [22], Lr59, LrAp from Aegilops peregrina [23, 24], Lrtri, Yrtri from Aegilops triuncialis [25], LrAc, YrAc from Aegilops caudata [26] and many more [27].

Aegilops speltoides (2n = 14, SS), a close diploid relative of the B genome in the secondary gene pool of wheat, is an important wild species harbouring a plethora of genes for wheat improvement [28]. It has been previously reported to contribute resistance against several biotic stresses like leaf rust (Lr28, Lr35, Lr36, Lr47, Lr51, Lr65, Lr66), stem rust (Sr32, Sr39, Sr47, Sr54), powdery mildew (Pm1d, Pm12, Pm32, Pm53) and green bug (Gb5) [29,30,31,32,33,34,35].

Among the wild germplasm collection maintained by Punjab Agricultural University (PAU), Ae. speltoides (SS) accession pau 3603 was found resistant against prevalent LR and YR pathotypes in Punjab. The resistance against these two rusts has been transferred into hexaploid wheat background and an introgression line named ILsp3603 with resistance to both rusts has been developed. In the present experiment, the ILsp3603 was crossed with LR and YR susceptible wheat, WL711 and a mapping population was developed to understand the genetics of introgressed resistance and mapping of genomic regions harbouring rust resistance using 35K SNP array.

Materials and methods

Introgression line ILsp3603 and mapping population

The plant material consists of LR and YR resistant introgression line ILsp3603, LR and YR susceptible wheat line WL711 and 152 F2:F6 mapping population developed by their intercrossing. Introgression line ILsp3603 was developed at the School of Agricultural Biotechnology, PAU, Ludhiana, by initially crossing LR and YR resistant diploid S genome wheat progenitor, Ae. speltoides accession pau 3603 (2n = 14) with tetraploid durum wheat, PDW233 (2n = 28). Thereafter, the resulting progeny was crossed and backcrossed with LR and YR susceptible hexaploid wheat, PBW621 (2n = 42), followed by continued selfing for five generations. The introgression line ILsp3603, having resistance to LR and YR, and chromosome number 2n = 42 was selected by screening selfed progenies against LR and YR in each generation. The ILsp3603 × WL711 cross was made to develop F6 and F7 RILs to understand the genetics of LR and YR resistance transferred from Ae. speltoides and their mapping onto specific chromosomes (Fig.S1).

Rust screening

Seedling stage

Seedlings of parental lines and mapping population were tested against prevalent Puccinia triticina (Pt) pathotype 77−9 (121R60-1) having avirulence/virulence formula: Lr2a, Lr2b, Lr2c, Lr9, Lr19, Lr24, Lr25, Lr28, Lr32, Lr39, Lr42, Lr45, Lr47/ Lr1, Lr3, Lr10, Lr11, Lr12, Lr13, Lr14a, Lr14b, Lr14ab, Lr15, Lr16, Lr17a, Lr17b, Lr18, Lr20, Lr21, Lr22a, Lr22b, Lr23, Lr26, Lr27 + 31, Lr30, Lr33, Lr34, Lr35, Lr36, Lr37, Lr38, Lr44, Lr46, Lr48, Lr49 [36]. Seeds were sown in rows in plastic boxes (25 cm × 10 cm), with 8 rows/box and 10–12 seeds per row, with the first row as susceptible check WL711 in each box. For achieving uniform spread and the desired concentration of urediniospores, one week old seedlings were inoculated with a mixture of pathotype 77−9 and talcum powder, incubated overnight under 100% humidity in the dark and then shifted to a glasshouse maintained at 18–20 °C. Leaf rust infection types (ITs) were scored 14 days after inoculation following modified Stakman 0–4 scale [37].

Adult plant stage

At the adult plant stage, parental lines and mapping population were planted in two separate sets and evaluated for LR and YR, respectively for two cropping seasons, 2021-22 (F6) and 2022-23 (F7). A mixture of Puccinia striiformis (Pst) pathotypes, 46S119 (46E159), 110S119 (110E159) and 238S119 (238E159) was used for testing against YR while a mixture of Pt pathotypes, 77−5 (121R63-1) and 77−9 (121R60-1) was used to screen against LR (Table S1). Scoring against both rusts was executed by assessing the percentage of leaf area covered, with ITs assigned following modified Cobb’s scale [38].

DNA extraction and 35K SNP array genotyping

Genomic DNA was extracted from young leaves using a modified CTAB protocol [39] and the concentration and purity of DNA were evaluated on NanoDrop Spectrophotometer. For bulked segregant analysis (BSA), two sets of contrasting bulks were prepared against both LR and YR. Twenty homozygous resistant (HR) and 20 homozygous susceptible (HS) F6 progenies were selected each for LR and YR. Single plant DNA from 10 selected F6 progenies was pooled, and four bulks were formed, each for LR (LrRB1, LrRB2, LrSB1, LrSB2) and YR (YrRB1, YrRB2, YrSB1, YrSB2). Genotyping of these bulks and parental lines was performed using 35K Axiom wheat breeder’s SNP array (AFFYMETRIX) (Fig. S2). Raw data was processed by filtering SNPs with missing data points or unassigned chromosomes. In the finally selected data, polymorphic SNPs were identified between contrasting parental lines and bulks.

Primer designing and linkage mapping

Axiom IDs associated with potentially linked SNPs were used for designing KASP assays using the pipeline Polymarker [40]. Two allele-specific forward primers (each labelled with HEX 5-GAAGGTCGGAGTCAACGGATT-3 and FAM 5-GAAGGTGACCAAGTTCATGCT-3 sequences), and a common reverse primer were synthesized. PCR assays were carried out in 4 µl reaction volume, containing 2 µl genomic DNA (30 ng/µl), 1.96 µl 2X KASP reaction mix and 0.054 µl KASP assay mix (containing 3 primers) in 384-well plate on thermocycler (Applied Biosystems Veriti) at 95 °C for 15 min; 10 touchdown cycles of 95 °C for 10 s, 61 –57 °C for 1 min (0.6 °C drop per min); 25 cycles at 95 °C for 20 s and annealing at 57 °C for 1 min. Fluorescence intensity was recorded by Tecan infinite F200 PRO plate reader, and results were imported to Kluster Caller software (LGC Biosearch Technologies v3.4.1.36).

To saturate the genetic region localized by SNP markers, we also used some already reported SSR markers [41], PCR-based landmark unique genes (PLUG) [42] and also designed new SSRs from the targeted region using the MIcroSAtellite identification tool, MISA v2.1 [43]. These markers were amplified in 10 µl PCR reaction mixture having 2 µl (60ng) DNA, 4 µl 2X EmeraldAmp PCR Mastermix, and 1 µl (0.01mM) forward and reverse primer each. PCR amplification was carried out in a 96-well thermocycler for 35 cycles at 94 °C for 1 min, 53–61 °C for 1 min, 72 °C for 2 min. PCR products were separated on 3.0-3.5% agarose gel. Polymorphic SNPs, SSRs and PLUG markers were amplified onto the entire mapping population.

Homoeologue confirmation for leaf rust and stripe rust resistance

To determine the presence of linked markers on chromosome 6B, these markers were amplified on nullisomic-tetrasomic stocks, CS-N6A-T6D, CS-N6B-T6A, CS-N6D-T6A and CS-N6D-T6B available in cultivar Chinese Spring background (acquired from Wheat Genetics Resource Center, Kansas State University, Manhattan).

Statistical analysis

Chi-square analysis was carried out to determine the goodness of fit of observed genotypic and phenotypic data with the expected genetic ratios using the following formula:

$${{\rm{\chi }}^{\rm{2}}}\,{\rm{ = }}\,\sum {{{\left( {{\rm{O - E}}} \right)}^{{\rm{2}}}}{\rm{/}}{\rm{E}}}$$

where χ2 = chi-squared, test statistic, O = observed phenotype in each category, E = expected phenotype in each category. Linkage analysis was conducted using QTL ICi mapping software v4.2. Kosambi mapping function was used to calculate recombination frequencies and estimate map distances in centimorgans (cM). A LOD score value of 3 was set, and the ‘GROUPING’, ‘ORDERING’ and ‘RIPPLING’ commands were carried out sequentially to yield the final marker order. Following, linkage map was constructed using the Microsoft excel software, MapDrawJZ-v2 [44].

Physical map construction and target annotation

Primer sequences corresponding to mapped markers were used for extracting physical positions through blast search against Chinese Spring reference genome assembly (IWGSC RefSeq v2.1). The DNA sequence within the target region, bordered by flanking markers, was further used for retrieving gene IDs.

Results

Rust response against LR and YR pathotypes

Leaf rust

At the seedling stage, ILsp3603 was resistant (IT=;) to Pt pathotype 77 − 9 while WL711 was susceptible (IT = 3) (Fig. 1). Similarly, at the adult plant stage, ILsp3603 had only traces (TR) of LR severity while WL711 was susceptible with a severity of 60–80 S. In F6, there were 77 Homozygous Resistant (HR), 14 Segregating (Seg) and 61 Homozygous Susceptible (HS) progenies with χ² (1.875:0.25:1.875) = 3.51, p-value = 0.173 while in F7, there were 82 HR: 7Seg: 63HS progenies (χ² (1.9375:0.125:1.9375) = 3.55, p-value = 0.169) at both the seedling and adult plant stages. The LR data at both the growth stages indicated the inheritance of a single LR resistance gene, effective throughout the life of the plant. This LR resistance gene is an all-stage resistance gene and is temporarily designated as Lrsp3603 (Table 1).

Fig. 1
figure 1

Infection type against leaf rust pathotype 77 − 9 (121R60-1) at seedling stage (a) ILsp3603 (b) WL711 (c, d) resistant F6 progenies (e, f) susceptible F6 progenies

Full size image
Table 1 Segregation of F6 and F7 progenies from cross ILsp3603× WL711 as HR (Homozygous Resistant), Seg (Segregating) and HS (Homozygous Susceptible)
Full size table

Stripe rust

ILsp3603 was resistant to YR at the seedling stage with IT of “ ; ”, and at the adult plant stage, it showed YR severity of traces (TR). WL711 was susceptible with IT = 3 at the seedling stage and YR severity of 80S at the adult plant stage (Fig. 2). Out of 152 F6 progenies, 77 were HR, 13 were found to be Seg, while the remaining 62 were HS with χ² (1.875:0.25:1.875) = 2.55, p-value = 0.279. In F7 generation, there were 85 HR: 5Seg: 62HS progenies (χ² (1.9375:0.125:1.9375) = 3.61, p-value = 0.164). Segregation of progenies at seedling and adult plant stages at F6 and F7 generation revealed that the YR segregation pattern also adhered to the monogenic inheritance of an all-stage resistance gene (Table 1). This YR resistance gene has been temporarily designated as Yrsp3603.

Fig. 2
figure 2

Stripe rust response recorded as disease severity against mixture of yellow rust pathotypes at adult plant stage: (a) ILsp3603 (b) WL711 (c, d) resistant F6 progenies (e, f) susceptible F6 progenies

Full size image

Association between Lr sp3603 and Yr sp3603

LR and YR scores were compared in F7 progenies to determine the association between Lrsp3603 and Yrsp3603. Of the total 152 F7 progenies, 130 (∼ 86%) showed similar reaction for LR and YR, meaning progenies were either HR or Seg or HS for both LR and YR, and the remaining 22 progenies (∼ 14%) showed contrasting phenotype, either resistant for LR and susceptible for YR or vice versa, suggesting recombination between Lrsp3603 and Yrsp3603 genes. This implies that these two genes exist as separate entities though closely located on the same chromosome, with a genetic distance of approximately 14 cM (Table 2).

Table 2 Two-way table showing comparison of number of F7 homozygous resistant (HR), segregating (Seg) and homozygous susceptible (HS) progenies for leaf rust and stripe rust
Full size table

Mapping through BSA-35K SNP data analysis

Leaf rust

The parents, ILsp3603 and WL711 along with LR bulks (LrRB1, LrRB2, LrSB1, LrSB2) were genotyped using 35K Wheat Breeders’ SNP array. After filtering, 24,693 SNPs spanning throughout the genome were retained of which 833 SNPs were polymorphic between contrasting parents and bulks. The highest proportion of these polymorphic SNPs (∼ 60%) was on chromosome 6B, followed by 16.2% on chromosome 6D and 8.4% on chromosome 2D (Fig. 3).

Stripe rust

Similarly, independent processing of 35K SNP array data was carried out for parental lines and bulks prepared for YR (YrRB1, YrRB2, YrSB1, YrSB2), resulting in 26,985 filtered SNPs of which 474 SNPs were polymorphic between contrasting parental lines and bulks. These SNPs were distributed on chromosomes 1A, 1D, 2B, 6A, 6B and 6D, with the maximum number of the polymorphic SNPs lying on chromosome 6B (∼ 69%), followed by 6D (18.6%) and 6A (4.7%) (Fig. 3).

Fig. 3
figure 3

Distribution of polymorphic SNPs from 35K SNP array data on 21 wheat chromosomes. Blue color represents SNPs polymorphic between leaf rust resistant and susceptible bulks while green color represents SNPs polymorphic between stripe rust resistant and susceptible bulks, both the bulks generated using F6 progenies of cross, ILsp3603 and WL711

Full size image

Notably, the maximum number of SNPs showing polymorphism between resistant and susceptible bulks, both for LR (237 SNPs) and YR (148 SNPs), were on chromosome 6B. Of these, 90 SNPs were common among LR and YR, suggesting the presence of both Lrsp3603 and Yrsp3603 genes in the vicinity of these SNPs on chromosome 6B (Fig. S3). A major number of closely positioned SNPs were lying on the short arm of chromosome 6B, spanning a physical interval of ∼ 35 Mbp. From these, 19 SNPs were selected and converted into PCR-based KASP assay for validation. Besides, this region was searched for already known markers from which 8 PLUG and 7 SSRs were selected (Table S2). Also, 15 new SSR markers were designed from the genic regions and were named “Tag-SSRs” (Table S3). All these SNP, SSR and PLUG markers were amplified on contrasting parents and bulks of which 10 markers including 4 SNP based KASP, 1 PLUG and 5 genic-SSRs (Tag-SSRs), were polymorphic.

Mapping of Lr sp3603 and Yr sp3603

For linkage analysis and genetic map construction, 152 F7 progenies were amplified with 10 polymorphic markers of which 5 markers including one KASP (AX-94542331), one PLUG (TNAC1674) and three Tag-SSRs (Tag-SSR10, Tag-SSR12 and Tag-SSR14) displayed segregation within the population. Marker AX-94542331 showed distinct cluster formation and amplified the ‘T’ allele in ILsp3603 and resistant progenies, and the ‘C’ allele in WL711 & susceptible progenies (Fig. S4), with 5 recombinants observed with Lrsp3603 and 10 recombinants with Yrsp3603. The only polymorphic PLUG marker TNAC1674 showed 14 and 29 recombinants with Lrsp3603 and Yrsp3603, respectively. It amplified the allele of 628 bp in ILsp3603 and resistant progenies, while an amplicon size of 600 bp was observed in WL711 and susceptible progenies (Fig. S5a). Tag-SSR12 and Tag-SSR14 had 4 and 19 recombinants with Lrsp3603 and Yrsp3603 genes, respectively. The former produced an amplicon size of 140 bp and 178 bp, while the latter amplified alleles of 146 bp and 165 bp in resistant and susceptible parents and progenies (Fig. S5b, c). The Tag-SSR10 amplified a resistant allele of 175 bp and a susceptible allele of 160 bp (Fig. S5d) with 5 and 21 recombinants with Lrsp3603 and Yrsp3603 genes, respectively.

Finally, the linkage map was constructed using these five markers, SNP AX-94542331, PLUG marker TNAC1674 and SSRs Tag-SSR10, Tag-SSR12 and Tag-SSR14. KASP marker AX-94,542,331 was 3.28 cM away from Lrsp3603 and 6.62 cM farther from Yrsp3603. PLUG marker TNAC1674 was mapped distally at 9.37 cM and 19.27 cM from Lrsp3603 and Yrsp3603, respectively. Tag-SSR12 and Tag-SSR14 were co-segregating and mapped closest to Lrsp3603 at 2.42 cM distance but 12.32 cM away from Yrsp3603. Tag-SSR10 was positioned at 0.32 cM from the other two co-segregating SSRs but was mapped 2.81 cM away from the Lrsp3603 (Fig. 4a).

Fig. 4
figure 4

Comparison of (a) genetic map with (b) physical locations (IWGSC v2.1) of chromosome 6B and (c) corresponding annotations associated with disease resistance genes within the target region. Gene IDs marked in RED encode for NBS-LRR genes while GREEN represents other disease resistance-associated domains

Full size image

Physical positions of markers Tag-SSR14 and AX-94,542,331, closely linked with Lrsp3603, delimited the gene within ∼ 1 Megabase pairs (Mbp) (Fig. 4b). Annotation of this region revealed a total of 28 gene IDs among which 10 IDs are known to be associated with disease resistance or plant immunity mechanisms (Fig. 4c, Table S4). Three of these, TraesCS6B03G0098500, TraesCS6B03G0098600 and TraesCS6B03G0101000, encode for NBS-LRR domains representing a major class of R genes, which play a central role in providing host resistance against various pathogens. On the other hand, the candidate region for Yrsp3603 was not precisely defined due to its location at the distalmost end of the linkage map, with markers positioned only on one side. Functional annotation of ∼ 10 Mbp candidate region around Yrsp3603 was carried out which also identified 25 gene IDs involved in plant defence mechanisms including gene ID TraesCS6B03G0114500 which encodes typical NBS-LRR resistance like protein (Table S5). Overall, annotation of ∼ 15 Mbp target chromosomal region revealed 10 NBS/LRR encoding gene IDs which can serve as candidates for LR and/or YR resistance (Table S6).

Confirmation of homoeologous chromosome position of Lr sp3603 and Yr sp3603

To validate the location of Lrsp3603 and Yrsp3603 on chromosome 6B, all markers forming distinct segregation within mapping population were amplified on nullisomic-tetrasomic (NT) stocks of chromosome 6 (CS-N6A-T6D, CS-N6B-T6A, CS-N6D-T6A, CS-N6D-T6B) in Chinese Spring background (Fig. S6). AX-94542331, Tag-SSR10, Tag-SSR12 and Tag-SSR14 amplified a single susceptible allele on all stocks except CS-N6B-T6A, lacking a 6B homoeologue. This indicated the markers were from chromosome 6B only and absent on chromosome 6 A or 6D. TNAC1674 amplified two amplicons of size around 600 bp and 700 bp in CS-N6A-T6D, CS-N6D-T6A and CS-N6D-T6B, but only one allele of ∼ 700 bp in CS-N6B-T6A indicating the absent allele has 6B homoeologue-specific binding (Fig. S5a, S6e).

Discussion

Wheat, an essential nutrition staple for billions of people, takes the worldwide lead in production volume and cultivated area [45]. However, it is facing numerous biotic and abiotic challenges due to faded resilience in cultivated wheat germplasm. Among all stresses, leaf rust and stripe rusts have remained the most important diseases of wheat since ancient times. In India, breeding for rust resistance was initiated in the 1930s by Dr B.P. Pal, and since then, the rapidly evolving fungal pathogens have never let a pause in resistance breeding. Both the rusts have been identified with multiple variants such as in LR, the groups named 12, 77 and 104 have repeatedly evolved, resulting in the breakdown of many important LR resistance genes [36, 46]. Similarly, virulence against important YR resistance genes such as Yr9, Yr17, Yr18, Yr19, Yr21, Yr22, Yr23, Yr25, Yr27 have been detected in newly evolved pathotypes 238S119, 110S119 and 46S119 of yellow rust [47, 48]. The eroding diversity in cultivated wheat and the evolution of pathogens make it essential to explore the unutilized germplasm to enhance genetic diversity and thus tackle the mainstream problems in wheat [49]. Hence, in this study, we aimed to introduce the LR and YR resistance in wheat from the wild progenitor species Aegilops speltoides.

Ae. speltoides (SS), the diploid wild relative from the secondary gene pool of wheat, is among the five members of the Sitopsis section. It shares close similarities with the B genome of wheat, which originated from a diploid extinct species that diverged from Ae. speltoides around 4.49 million years ago [49]. It has proven to be a valuable genetic resource for enhancing crop resilience against many abiotic [50, 51] and most importantly biotic challenges like leaf rust, stem rust, powdery mildew and green bug. Numerous resistance genes like Lr28, Lr35, Lr36, Lr47, Lr51, Lr65, and Lr66 against leaf rust; Sr32, Sr39, Sr47, and Sr54 against stem rust; Pm1d, Pm12, Pm32, and Pm53 against powdery mildew and Gb5 against green bug have been identified from this species [52, 53].

For this study, we selected one of the LR and YR resistant Ae. speltoides accession pau 3603, which was found to be resistant to LR and YR when screened against these rusts in PAU for multiple years. To make this resistance usable, it was introduced into the hexaploid wheat background and a hexaploid introgression line named ILsp3603 was developed by crossing the wild species first with tetraploid durum wheat as bridging species (to bridge the ploidy gap between diploid Ae. speltoides and hexaploid wheat) and then backcrossed F1 twice with hexaploid wheat to retain chromosome number 2n = 42 and recover wheat background. Then to characterize and map the transferred rust resistance, we created an F6 and F7 RIL mapping population by crossing ILsp3603 with LR and YR susceptible cultivar WL711. The dominant nature of Lrsp3603 and Yrsp3603 resistance genes against both the rusts was evident from the F1 being resistant against both rusts. Inheritance studies were conducted over two cropping years in F6 and F7 generation against prevalent leaf rust (LR) pathotypes, 77−5 (121R63-1), 77−9 (121R60-1) and stripe rust (YR) pathotypes, 46S119 (46E159), 110S119 (110E159), 238S119 (238E159). The single major gene designated as Lrspelt3603 and Yrspelt3603 demonstrated the competence of Ae. speltoides-derived resistance genes in providing host protection against predominant pathotypes under Indian conditions.

A revolution in the mapping of valuable genes came with the rise of high-throughput next-generation sequencing platforms [24, 54, 55]. Furthermore, these advancements have enabled the development of high-density SNP arrays, like the cost-effective 35 K SNP array with 35,143 data points. Unlike more expensive platforms, such as the 820K array, this condensed version is ideal for large-scale mapping studies and marker-assisted selection in wheat breeding [56, 57]. In the present study, we integrated BSA with high throughput 35K SNP genotyping for rapid identification of genomic region introgressed from LR and YR resistance donor, Ae. speltoides. Independent analysis was carried out for LR and YR resistance, both of which pinpointed chromosome 6BS as the target region. Moreover, the amplification of a diverse set of markers like SSRs and PLUG validated the efficacy of this region for providing rust resistance, thereby proving the productiveness of this approach in mapping useful genes. Genotyping success of SNP arrays has also been demonstrated earlier through mapping of LrTs276-2 on chromosome 1DS using Affymetrix 35K SNP array [58], LrP and YrP derived from Aegilops peregrina on chr5DS [53] and Lr81 on chromosome 6A using iSelect 90K SNP array [59].

Mapping of LR and YR resistance genes on chromosome 6BS involved 1 SNP, 1 PLUG and 3 SSR markers. Chromosomal positions of these markers revealed a physical coverage of ∼ 15 Megabase pairs (Mbp) spanning from 15.5 Mbp to 30.1 Mbp. Lrsp3603 was specifically confined within the ∼ 1 Mbp segment (29.2–30.1 Mbp) of this region flanked by markers Tag-SSR14 and AX-94542331. The identified NLR-coding gene IDs, TraesCS6B03G0098500, TraesCS6B03G0098600 and TraesCS6B03G0101400, serve as promising candidates for Lrspelt3603.

S genome of Ae. speltoides shares maximum sequence identity with the B genome of T. aestivum [60]. Yet gene introgressions from Ae. speltoides into the A and D genomes have been observed, such as Lr28 on 4AL [61], Lr47 on 7AL [62], Lr65 on 2AS [63], Lr66 on 3AS [64], Sr32 on 2DS [32], Gb5 on 7AL [30] which is likely due to the repetitive and homoeologous nature of the wheat genome. Therefore, to validate the homoeologue identity of genes mapped in the current study, we employed nullisomic-tetrasomic (NT) aneuploid stocks developed originally by E.R. Sears in Chinese Spring background [65,66,67]. Amplification of all the markers near Lrsp3603 and Yrsp3603 genes (AX-94542331, Tag-SSR10, Tag-SSR12, TNAC1674 and Tag-SSR14) on the Nullitetrasomic stocks CS-N6A-T6D, CS-N6D-T6A and CS-N6D-T6B and absence of their amplification in CS-N6B-T6A confirmed these markers on 6B homoeologue.

Till date, four designated LR resistance genes have been reported on chromosome 6B viz. Lr36 from Aegilops speltoides, Lr53 from Triticum dicoccoides, Lr59 from Aegilops peregrina, and Lr61 from durum wheat. Lr36 is the only gene that has been mapped on chromosome 6BS as well as derived from Ae. speltoides. The presence of Lr36 in the present study was tested by amplifying linked SSR Xgwm88 [68] which was monomorphic between ILsp3603 and WL711 indicating that the leaf rust resistance gene mapped in the present study is not Lr36. Moreover, pathotypes 77−5 and 77−9 have gained virulence against this gene, while Lrsp3603 resistance mapped in the current study, is effective. Lr53 is effective against all the prevalent Indian leaf rust pathotypes [69] but has been derived from a different progenitor species, T. dicoccoides (AB). Its closely linked marker, Xcfd1 [12] was monomorphic in the ILsp3603 and WL711 hence, an unlikely candidate for Lrsp3603. Lr59, though on chromosome 6B has been introduced from an entirely different origin i.e. Ae. peregrina (UUSS) with S genome originating from Ae. longissima [23, 70]. Moreover, SSR Xgwm518 [71] linked with Lr59 was monomorphic in ILsp3603 and WL711, ruling out this gene as a candidate for Lrsp3603. Lr61 has also been mapped on Chromosome 6BS but has been identified from a non-wild source i.e. Chilean durum wheat cultivar by Herrera-Foessel et al. in 2008 [72].

Besides LR resistance genes, three Yr genes, Yr35 (linked to Lr53), Yr36 and Yr78 have been reported on chromosome 6B. While Yr35 and Yr36 have been reportedly derived from T. dicoccoides, Yr78 has been mapped through validation of QTL identified through GWAS studies [73]. Yr36 is a high-temperature adult plant stage resistance gene [15] but Yrsp3603 is an all-stage resistance gene. The Yr78 gene is again an adult plant resistance gene that has been delimited within markers, CDM88 and CDM103 with corresponding physical locations, 107.4 Mbp to 118.6 Mbp according to RefSeq v2.1 [74]. However, in this study, Yrsp3603 provides all-stage resistance and is mapped closer to AX-94542331 which is physically positioned at 30.1 Mbp, far away from Yr78. Therefore, Yr78 and Yrsp3603 are different genes.

Conclusion

This study utilised an introgression line ILsp3603 which was developed through crossing and backcrossing Aegilops speltoides with susceptible wheat varieties. ILsp3603 carrying LR and YR resistance in the background of hexaploid wheat, was subsequently crossed with susceptible wheat WL711 to generate a mapping population suitable for characterising and mapping rust resistance genes. We identified closely linked markers spanning a genetic distance of 12.65 cM, which could be beneficial for marker-assisted transfer of novel LR and YR resistance genes in wheat breeding programmes.