Phenotypic evaluation
The CW357-9 seedlings were resistant (IT 1-2) to PST-Lab.1 andPST-Lab.2, but susceptible (IT 8–9) to PST-V26. CW357-9 was highly resistant (IT 1–2, DS ≤5%) in field whereasAvS was highly susceptible (IT 8–9) in all experiments.Based on these results, CW357-9 possessed both seedling resistance to two isolates and APR to the mixture of the three isolates used in the field.In the field experiments, both IT and maximum disease severity (MDS) data for RILs showed continuous distributions (Fig. 1), indicating that resistance in CW357-9 was quantitatively inherited. Pearson’s correlation coefficients of pairwise comparisonsofIT and DS ranged from 0.60-0.85 and 0.58-0.88 (P <0.001) (Table 1), respectively. Broad-sense heritabilities forboth IT and DS were 0.92 (Table 2). P values in the ANOVA for IT and DS were highly significant (P<0.0001) forRILs, environments, and line × environment interactions. Lack of significant variation between the replicates suggested that genetically controlledresistance was the main source of phenotypic variation in the RIL population (Table 2). These results indicated that the QTL conferring resistance was effective inthe five environments.
Genetic linkage map
Of the 20,995 SNPs, 5,104 (24.4%) showed polymorphism between the parents. By using the “BIN” function in QTL IciMapping 4.2, redundant polymorphic SNPs were removed showing>10% missing data anddistorted segregation. Finally, 841 SNPs were chosen to construct the genetic linkage map; they were distributed in 22 linkage groups spanning 3,533.11cM. The A, B, and D genomes included 290 (34.48%), 374 (44.47%), and 177 (21.05%) markers covering lengths of 1,268.96, 1,356.53, and 1,012.27 cM with average marker intervals of 4.38, 3.63, and 5.72 cM, respectively. Only chromosome 2D had two linkage groups; the other chromosomes were each represented bya single linkage group (Table S1).
Additive QTL for stripe rust resistance
Two QTLfor seedling resistance to race PST-Lab.1 were detected on chromosomes arms 2BL and 4DL, however did not confer resistance in field indicating that isolate PST-V26 was prevalent in field experiments. Both IT and DS data from the field environments were used to detect QTLat the adult plant stage. Four consistent QTL on chromosome arms 1BL, 2AL, 3DL, and 6BS, designated as QYrCW357-1BL, QYrCW357-2AL, QYrCW357-3DL, and QYrCW357-6BS, respectively, were identified in all five environments using the ICIM method in QTL IciMapping 4.2. All detected QTL were derived from the resistant parent CW357-9 (Table 3; Fig. 2A and B). QYrCW357-1BL with the largest effect wasclosely linked to markers AX-110020417 and 16K-16852 and explained 19.8-28.8% and 23.9-29.1% of variation inIT and DS, respectively (Table 3; Fig. 2A). QYrCW357-2AL located in a 1.3 cM interval spanned by markers 16K-4252and 16K-4207, explained 4.0–10.0% and 5.8–12.9% of the phenotypic variation in IT and DS, respectively,across environments (Table 3; Fig. 2B).QYrCW357-6BS, linked to 16K-15955 and AX-109914318, explained 2.8–8.1% (IT) and 2.6–8.3% (DS) of the phenotypic variances, respectively. QTL on 3DL,flanked by 16K-9333and16K-9526, explained 2.8-8.1% and 9.1-16.4% of the variation in IT and DS, respectively. All QTL had additive effects (Table 3).
Epistatic interaction detected byQTLNetwork version 2.1
Significant epistatic interactions were detected across all field traits using QTL Network version 2.1. Two different intervals on 1BL and 2AL corresponding to the markers AX-110020417-16K-16852 (QYrCW357-1BL or Yr29) and 16K-4252-AX-110974948 (QYrCW357-2AL), showed an estimated additive by additive interaction (A*A) effects of 0.24-0.34 and 3.24-4.50 in IT and DS, respectively (Table 4).Although the QTL on 1BL and 2AL showed significant interaction for reducedstripe rust severity, the presenceof the 3DL and 6BS QTL and some additional environmentally sensitiveQTL were necessary to reach the consistently low response ofCW357-9 (Table 3).
QTL combinations and interaction
In order to investigate the effects of QTL combinations, RILs were classified into five genotypic groups based on allfield tests (Table S2). RILs with all four QTL QYrCW357-1BL, QYrCW357-2AL, QYrCW357-3DL, and QYrCW357-6BS were more resistant (lower IT and DS) than all others, displaying almost similar resistance levels to CW357-9 (Fig. 3A, B; Table S2). Amongthese genes the combination ofQYrCW357-1BL and QYrCW357-2AL showed the most significant effect inreducing stripe rust severity (Fig. 3C, D). RILs with none of the four stable QTLs hadmean IT and DS values of 8.2 and 88.6%, respectively; RILs with only oneQTL(1BL or 2AL) had mean values of 6.9 and 71.0% for the 1BL locus (similar toAvocet-Yr29 in Table S3) and 7.2 and 76.1% for 2AL, respectively (Fig. 3C, D). The group combining QYrCW357-1BL and QYrCW357-2AL, with mean IT and DS of 4.6 and 36.0%, respectively, showedsignificant effectin reducing IT and DS (Fig. 3C, D, E and F).
Marker-assisted selection (MAS) based on AHQ markers
Genotypingof the 238-accession panel for polymorphicAQP markers AX-110020417, AX-110974948,AX-109466386and AX-109995005represented forQYrCW357-1BL,QYrCW357-2AL,QYrCW357-3DL, and QYrCW357-6BS, respectively, suggested these markers can be used in MAS (Table S4). Wheat lines with both QYrCW357-1BL(or Yr29) and QYrCW357-2AL were on the average more resistant than lines without them, but some accessions containing the QTL were very susceptible, indicating that the effects of the two QTL alone were influenced by genetic background and environment. However,wheat linescombining all four loci had the lowest average DS in Yangling and Tianshui(Figure 5). Sequences for the AQP markers AX-110020417, AX-110974948, AX-109466386, and AX-109914318areprovided in Table S4.