Phenotypic evaluation
XN3517 was resistant to PST-Lab.1 (IT 2–3), PST-Lab.2 (IT 2–3), and PST-V26 (IT 4–5) at both the greenhouse seedling test and at the adult plant stage in the field tests, whereas AvS was susceptible (IT 8–9). Both IT and maximum disease severity (MDS) data for the RILs showed normal distributions (Fig. S1), indicating that resistance in XN3517 was quantitatively inherited. Pearson’s correlation coefficients of pairwise comparisons for IT and DS ranged from 0.68-0.89 and 0.72-0.90 (P <0.001) (Table 1), respectively. Broad-sense heritabilities for both IT and DS were 0.94 (Table 2). P values in the ANOVA for IT and DS values showed significant variation (P <0.0001) among RILs, environments, and line × environment interactions. However, the lack of significant variation between replicates suggested that resistance was the main source of phenotypic variation (Table 2). These results indicated that the expression of QTL controlling ASR and APR in XN3517 was consistent across all five environments.
Genetic linkage map
Of the 20,995 SNPs, used for analysis 5,187 (24.71%) were found polymorphic in the RIL population. Using the “BIN” function in QTL IciMapping version 4.2, redundant polymorphic SNPs were removed which had >10% missing data, distorted segregation and wrong combinations. The remaining 1,300 SNPs were used to construct 31 genetic linkage groups spanning 5,134.96 cM. The A, B, and D genomes included 519 (39.92%), 638 (49.08%), and 143 (11.00%) markers covering lengths of 1,604.16, 1,617.91, and 1,550.81 cM with average marker intervals of 1.16, 1.26, and 0.42 cM, respectively. Chromosomes 1A, 1B, 3D, 4B, 4D, 5A, 5B, 5D, 6A, 6D, 7A, 7B, and 7D each had a single linkage group; the other chromosomes had two or three groups (Table S2).
QTL analysis
IT and DS data from the five field environments were used to perform marker trait associations to detect significant QTL. Four stable QTL on chromosome arms 1BL, 2AL, 2BL, and 6BS, designated QYrxn.nwafu-1BL, QYrXN3517-2AL, QYrXN3517-2BL, and QYrXN3517-6BS, respectively, were identified in all field environments, whereas in the seedling test using PST-Lab.1, PST-Lab.2, and PST-V26, races only the stable QTL on 2BL was detected (Table3, Table S3). Among these QTL, QYrXN3517-1BL with the largest effect, closely linked to markers 16k-2430 and 16k-2443, explained 19.2-35.7% and 19.8-35.9% of the variation in IT and DS, respectively. QYrsn.nwafu-2AL located in a 1 cM interval spanned by 16k-4442 and 16k-4471, explained 4.0–10.0% and 5.8–12.9% of the phenotypic variation in IT and DS across environments, respectively. The QTL on chromosome 2BL, flanked by 16k-5754 and 16k-5738, explained 7.5-16.5% and 9.1-16.4% in the tests with IT and DS data, respectively. QYrsn.nwafu-6BS, linked to AX-110602591 and AX-110199811, explained 6.5–18.5% (IT) and 7.1–15.1% (DS) of the phenotypic variation, respectively. Using the △SNP-indices from BSE-seq data we detected the same target regions as for the ICIM method (Fig. 2). All the QTL were derived from XN3517 (Table 3).
QTL combinations
The effects of individual QTL and QTL combinations were investigated by classifying the RILs into 12 genotypic groups based on the field tests. RILs with four QTL QYrXN3517-1BL, QYrXN3517-2AL, QYrXN3517-2BL, and QYrXN3517-6BS were more resistant (lower IT and DS) than all others groups displaying almost similar levels of resistance to XN3517. When present alone or in pyramids QYrXN3517-1BL showed the highest reductions in IT and DS (Fig. 4, Table S4).
A high-density genetic map of QYrXN3517-1BL and marker-assisted selection
QYrXN3517-1BL, flanked by markers 16k-2430 (668,939,708) and 16k-2443 (680,192,269), was initially mapped at 10.8 cM following genotyping by the GenoBaits Wheat 16K Panel (Fig. 3A). We then identified many RILs with recombination events in the region. Based on the data of BSE-Seq and 660K array, nine new significant AQP markers were used for fine mapping (Fig. 3B, 2C). We also added the previously identified markers csLV46G22 (Yr29) and ucw.k31 (QYr.ucw-1BL) to the genetic map. The QYrXN3517-1BL and QYr.ucw-1BL or Yr29 were in different genetic and physical regions (Figs 2C, 2D) (Cobo et al., 2018). The genotypes of 16 recombinant plants with their phenotypes in the genetic interval between markers ucw.k31 and AX-110534659 are shown in Fig. 5, indicated the resistance of 1BL candidate region involved has high recombination frequency. Finally, QYrXN3517-1BL, flanked by the closest markers AX-89744149 and nwafu.a5 located in a genetic interval of 1.7 cM that corresponded to a 336 kb interval in IWGSC RefSeq version 1.0. AQP markers nwafu.a5, were used to genotype the 759 wheat cultivars/breeding line panel. Forty seven wheat cultivars/breeding lines with higher resistance carried the same allele of nwafu.a5 and included Shaanxi cultivars Xinong 1376 (one of the parents for XN3517), Xiaoyan 81, Xinong 889 and Xinong 223, suggesting that the maker nwafu.a5 can be used for developing new cultivars with high-level of durable resistance to stripe rust.
Validation of the causal candidate location and marker-assisted selection
SNP markers tightly linked to QYrXN3517-1BL were converted to the high-throughput, cost-effective SNP genotyping format known as AQP for use by geneticists and breeders. To determine the robustness of identifed marker nwafu.a5 for QYrXN3517-1BL in CW357-9, genotyping of the 853-accession panel suggested it was signifcantly associated with stripe rust response (DS-Mean) of the wheat panel during 2018-2019 cropping season in YL, TS, and JY (Fig. 6a; Table S1). The KASP markers AX-89744149 and nwafu.a5 sequences are given in Table S6.
Annotated genes in the QYrXN3517-1BL candidate region and expression analysis
Based on wheat 16K single nucleotide polymorphism (SNP) array, 660K array and exome capture data, we mapped QYrXN3517-1BL within an interval of 1.7 cM [336 kb in International Wheat Genome Sequencing Consortium (IWGSC) RefSeq version 1.0] on chromosome 1BL. The 336-kb candidate region between the markers AX-89744149 and nwafu.a5 included 12 high confident (HC) annotated genes (Fig. 3D). The proteins produced by these 12 genes included one nucleotide-binding (NB) and leucine-rich repeat (LRR) proteins, one GDSL-like lipase/acylhydrolase superfamily protein, four additional lipid transfer proteins, two additional Polyol transporters, one B-block binding subunit of TFIIIC, one agenet and bromo-adjacent homology (BAH) domain-containing protein, one pfkB-like carbohydrate kinase family protein, and one cardiolipin synthase B.