Stripe rust response of the parents and RILs
Plants of GX were susceptible (IT = 8) to CYR32 and CYR34 at the seedling stage (Fig. 1a), but exhibited strong resistance (IT = 3, FDS < 10%) to mixed Pst races at the adult-plant stage in three crop seasons from 2018 to 2020 (Fig. 1b, Fig. 2, Table S1). These results indicated that GX showed non-race-specific APR to stripe rust. The frequency distributions of RILs for FDS were continuous with a pronounced skewness towards resistance, and the average FDS of RILs for GX × TC 29 was 12.5%–15.7% in the field tests, suggesting the presence of a large-effect QTL in the RIL population (Fig. 1c, Fig. 2, Table S1). Broad-sense heritability (H2) was 96.7% for FDS in all tests (Table 1). Correlation coefficients (R2) for FDS of the RILs among the different environments were significant (P < 0.01) and ranged from 0.82 to 0.95 (Table S2).
Table 1
The summary of final disease severity (FDS) data for the RILs population from the Gaoxianguangtoumai (GX) × Taichung 29 (TC 29) recorded in fields at Chongzhou in 2018-2020
Environments
|
Parents
|
RIL population
|
|
GX
|
TC 29
|
Min-max
|
Mean
|
SD
|
CV
|
H2 (%)
|
CZ2018 (%)
|
5.0
|
80.0
|
0-100
|
14.0
|
25.2
|
1.8
|
|
CZ2019 (%)
|
6.7
|
80.0
|
0-100
|
12.5
|
23.7
|
1.9
|
|
CZ2020 (%)
|
5.0
|
80.0
|
0-100
|
15.7
|
27.0
|
1.7
|
|
BLUP (%)
|
7.1
|
73.2
|
2.1-91.0
|
14.6
|
21.2
|
1.5
|
96.7
|
SD,standard deviation; CV,coefficient of variation; H2,broad-sense heritability
|
Linkage map construction and QTL analysis
A total of 1,871 bin markers were used to construct the linkage map for the GX × TC 29 population (Table S3). The total length of the map covered 2,799.12 cM with an average interval of 1.50 cM between adjacent markers and spanned 911.04, 855.71, and 1,032.37 cM in the A, B, and D genomes, with a density of 1.34, 1.28, and 1.98 cM per marker, respectively (Table S3). The map consisted of 21 linkage groups defined with representatives from each of the 21 chromosomes.
Two major QTL conferring APR to stripe rust were detected from the resistant parent GX in each of the three field tests and BLUP data (Table 2, Fig. 3a, b). The most highly significant QTL, designated QYr.GX-2AS, was mapped to the short arm of chromosome 2AS and explained up to 27.0% of the phenotypic variation with a LOD score of 8.1 (Table 2, Fig. 3a). A second QTL, QYr.GX-7DS, was flanked by the SNP markers AX-109379249 and AX-110431109 on chromosome 7DS and overlapped with Yr18, and explained 9.6%–15.6% of the phenotypic variation in all trials and BLUP data (Table 2, Fig. 3b). We concluded that it was highly likely that QYr.GX-7DS corresponded to Yr18.
To determine the effects of the QTL, the RILs were divided into four groups based on the presence/absence of the most closely linked flanking markers of QYr.GX-2AS and QYr.GX-7DS. Clearly, the RILs that carried one of the QTL showed a lower FDS than those without any QTL (average FDS = 63.4%). In particular, the RILs carrying only QYr.GX-2AS showed only 9.3% of the average FDS, which was similar to the effect of both QTL in combination (average FDS = 7.1%) (Fig. 3c). This result indicated that QYr.GX-2AS had a large effect on stripe rust resistance and provided relatively stronger resistance than QYr.GX-7DS.
Table 2
Quantitative trait loci (QTL) for stripe rust resistance detected in the RILs population from the Gaoxianguangtoumai (GX) × Taichung 29 (TC 29) using final disease severity (FDS) data across three environments and BLUP values
QTL
|
Environment
|
Trait
|
Chromosome
|
LeftMarker
|
RightMarker
|
Distance (cM)
|
LOD
|
PVE (%)
|
Resistance source
|
QYr.GX-2AS
|
CZ2018
|
FDS
|
2AS
|
AX-109957471
|
AX-110026721
|
0.2
|
8.1
|
27.0
|
GX
|
|
CZ2019
|
|
|
AX-109957471
|
AX-110026721
|
0.2
|
7.1
|
17.1
|
|
|
CZ2020
|
|
|
AX-109957471
|
AX-110026721
|
0.2
|
5.2
|
15.5
|
|
|
BLUP
|
|
|
AX-109957471
|
AX-110026721
|
0.2
|
7.7
|
21.8
|
|
QYr.GX-7DS
|
CZ2018
|
FDS
|
7DS
|
AX-109379249
|
AX-89737284
|
9.6
|
3.3
|
9.6
|
GX
|
|
CZ2019
|
|
|
AX-110502471
|
AX-109303704
|
9.6
|
6.6
|
15.6
|
|
|
CZ2020
|
|
|
AX-109303704
|
AX-110431109
|
9.6
|
4.9
|
13.6
|
|
|
BLUP
|
|
|
AX-89737284
|
AX-89378255
|
9.6
|
4.4
|
10.9
|
|
Haplotype analysis of QYr.GX-2AS
To assess the distribution of QYr.GX-2AS among 325 Chinese wheat landraces, the favorable haplotype was identified by haplotype analysis and seven SNP markers tightly linked to QYr.GX-2AS were screened from the Wheat 55K or 660K SNP arrays (Fig. 4a, b, c). Eight major haplotypes (n > 10) were detected in the panel (Fig. 4a, b). GX and 15 other accessions clustered with Hap1 (Table S4), which showed a frequency of about 5.3% in the total population (Fig. 4a). Almost all accessions carrying Hap1, except one from Henan, were collected from Sichuan. The accessions carrying Hap1 showed 18.4% of the average FDS and thus were more strongly resistant to stripe rust than those accessions carrying other haplotypes (Hap2 = 37.2%, Hap3 = 24.1%, Hap4 = 47.7%, Hap5 = 21.5%, Hap6 = 39.0%, Hap7 = 27.0%, Hap8 = 47.6%) (Fig. 4c). These results revealed that the favorable haplotype of QYr.GX-2AS was Hap1, which was relatively rare among the Chinese wheat landraces.
Validation and mapping of QYr.GX-2AS
To further validate and map the location of QYr.GX-2AS, the SNPs in the target region for QYr.GX-2AS identified by exome capture sequencing and the Wheat 55K array were selected for conversion to KASP markers. Eleven markers were confirmed to be polymorphic between GX and TC 29 (Table S5). Combined with the KASP markers for QYr.GX-2AS, a NIL-derived population of 130 individuals (F2) for QYr.GX-2AS was developed from a residual heterozygous plant in the F8 generation of RILs. No significant phenotypic differences were observed in the NIL-derived population, except for APR to stripe rust (Fig. 5a). With regard to stripe rust response in the field test, the F2 plants of the NIL-derived population were clearly classifiable into 97 resistant (IT ≤ 6) and 33 susceptible (IT ≥ 7) individuals, which fits the expected ratio (3:1) for a single Mendelian factor (chi-square goodness-of-fit test, χ2 = 0.01, P = 0.92) (Table S6). Using the newly developed eleven KASP markers (Table S6) to construct the genetic map, QYr.GX-2AS was localized to a 1.37 Mb interval between KP2A_36.85 and KP2A_38.22, and co-segregated with the KASP marker KP2A_37.09 (Fig. 5b).
Validation of KASP markers for marker-assisted selection
To check the specificity and polymorphism of the linked marker of QYr.GX-2AS for marker-assisted selection, a set of 109 Chinese wheat cultivars was tested with the markers KP2A_36.85, KP2A_37.09, and KP2A_38.22 (Fig. S1, Table S7). Most of the lines amplified the susceptible-specific alleles in the three markers, which showed 85.3%, 99.1%, and 95.4% polymorphism, respectively, in the cultivars (Table S7). Thus, these KASP markers can be used as the specificity markers for marker-assisted selection of QYr.GX-2AS in the vast majority of Chinese wheat cultivars.