Analysis Of Stripe-rust Response
To characterize seedling resistance to stripe rust, we recorded the IT response to the predominant Pst races CYR32 and CYR34 at the seedling stage for the wheat landrace panel. The susceptible check ‘MingXian169’ was rated with IT = 4 for the two races tested. The majority of accessions in this panel showed a high frequency of susceptibility to CYR32 (95.8%) and CYR34 (93.7%), respectively. Based on the IT, the resistant accessions had four in approximately 3% of the accessions for both Pst races (Fig. 1a, Additional file 1).
The responses of the 143 wheat landraces to mixed races of Pst were evaluated in five environments in the field (designated CZ16, CZ17, CZ18, MY16 and MY17). Based on BLUP values, a Pearson correlation analysis revealed significant correlations (P < 0.01) for IT, FDS and AUDPC that were observed among the five environments with correlation coefficients ranging from 0.58 to 0.89, 0.57 to 0.89 and 0.60 to 0.92 at the adult-plant stage, respectively (Additional file 2). The H2 values for IT, FDS and AUDPC were high across the five environments and BLUP values; the H2 values were 93.98%, 94.07% and 94.02%, respectively (Table 1). The panel showed a higher frequency of resistance reactions under field conditions than that observed in the seedling tests. With regard to IT, 48.3–75.5% of the accessions displayed resistance to the mixture Pst races in all five environments at the adult-plant stage (Fig. 1b, Additional file 1). Similarly, 63.6–89.5% of the accessions displayed resistance with low values of FDS (FDS < 60%) under the five environments (Fig. 1c, Additional file 1). Under the five environments, the phenotypic performance of the panel varied from 0 to 14 for AUDPC (Fig. 1d, Additional file 1). A total of 17 accessions showed stable high-level resistance to stripe rust across all environments under field tests. These accessions originated from Sichuan (6), Yunnan (6), Gansu (3), Guizhou (1) and Shaanxi (1) (Additional file 1), respectively. Among them, two accessions (Lushanmai (from Sichuan) and Guangtoumai (from Guizhou)) showed ASR resistance to the Pst races CYR32 and CYR34 at the seedling stage and field resistance in all environments. In addition, Bendiyoumangxiaomai (from Yunnan) and Liulengmai (from Guizhou) likely showed ASR resistance to the single Pst race CYR32 or CYR34, respectively (Additional file 1).
Table 1
Summary of the stripe rust response among five environments
Traits | Trials | Minimum | Maximum | Mean | Heritability (%) |
IT a | CZ16 | 0 | 4 | 2.22 | 93.98 |
MY16 | 0 | 4 | 2.28 |
CZ17 | 0 | 4 | 1.80 |
MY17 | 0 | 4 | 1.49 |
CZ18 | 0 | 4 | 2.40 |
BLUP | 0.24 | 3.85 | 2.09 |
FDS b (%) | CZ16 | 0 | 100 | 34.62 | 94.07 |
MY16 | 0 | 100 | 29.86 |
CZ17 | 0 | 100 | 17.87 |
MY17 | 0 | 100 | 16.24 |
CZ18 | 0 | 100 | 31.72 |
BLUP | 3.59 | 87.51 | 26.64 |
AUDPC c | CZ16 | 0 | 14 | 3.11 | 94.02 |
MY16 | 0 | 13.3 | 3.03 |
CZ17 | 0 | 13.02 | 2.11 |
MY17 | 0 | 6.02 | 0.90 |
CZ18 | 0 | 12.46 | 2.31 |
BLUP | 0.28 | 9.47 | 2.27 |
a, infection type b, final disease severity c, the area under disease progress curve |
Genetic Diversity Analysis
After filtering, a total of 6404 polymorphic markers (including 5898 polymorphic DArT-seq markers and 506 polymorphic allele variations from SSR markers) were retained for the 143 accessions. Among these markers, 2120, 3229 and 1055 markers were located in the A, B and D subgenomes, respectively. Chromosome 2B (709) carried the most markers, whereas chromosome 4D (52) carried the fewest markers. Gene diversity, PIC and MAF for the entire genome ranged from 0.2879 to 0.3653, 0.2355 to 0.2916 and 0.2070 to 0.2800 with averages of 0.3288, 0.2664 and 0.2390, respectively. Subgenome B showed the highest gene diversity, PIC and MAF values (0.3307, 0.2674 and 0.2407, respectively). Subgenome D exhibited the lowest gene diversity, PIC and MAF values (0.3232, 0.2630 and 0.2319, respectively). Among individual chromosomes, chromosome 6A carried 376 markers and showed the highest genetic diversity, PIC and MAF values, whereas chromosome 2D carried 270 markers and exhibited the lowest genetic diversity, PIC and MAF values (Table 2).
Table 2
Summary of genetic diversity of 143 wheat accessions on sub-genomes and chromosomes
Chromosome | Number of markers | PIC a | Gene Diversity | Minor Allele Frquency |
1A | 265 | 0.2603 | 0.3188 | 0.2260 |
2A | 485 | 0.2875 | 0.3620 | 0.2800 |
3A | 241 | 0.2605 | 0.3203 | 0.2315 |
4A | 344 | 0.2696 | 0.3332 | 0.2435 |
5A | 134 | 0.2634 | 0.3258 | 0.2403 |
6A | 376 | 0.2916 | 0.3653 | 0.2755 |
7A | 275 | 0.2580 | 0.3164 | 0.2265 |
A genome | 2120 | 0.2687 | 0.3324 | 0.2443 |
1B | 540 | 0.2777 | 0.3456 | 0.2540 |
2B | 709 | 0.2741 | 0.3418 | 0.2570 |
3B | 642 | 0.2649 | 0.3272 | 0.2381 |
4B | 192 | 0.2647 | 0.3269 | 0.2349 |
5B | 521 | 0.2487 | 0.3028 | 0.2123 |
6B | 341 | 0.2638 | 0.3245 | 0.2323 |
7B | 284 | 0.2782 | 0.3463 | 0.2563 |
B genome | 3229 | 0.2674 | 0.3307 | 0.2407 |
1D | 125 | 0.2631 | 0.3219 | 0.2267 |
2D | 270 | 0.2355 | 0.2879 | 0.2070 |
3D | 144 | 0.2589 | 0.3162 | 0.2188 |
4D | 52 | 0.2828 | 0.3492 | 0.2513 |
5D | 112 | 0.2547 | 0.3126 | 0.2277 |
6D | 161 | 0.2807 | 0.3497 | 0.2644 |
7D | 191 | 0.2652 | 0.3251 | 0.2274 |
D genome | 1055 | 0.2630 | 0.3232 | 0.2319 |
Whole genome | 6404 | 0.2664 | 0.3288 | 0.2390 |
a polymorphism information content | | |
Population Structure, Kinship And Ld Analyses
The population structure (Q-matrix) was calculated by means of Bayesian clustering using the 6404 polymorphic markers for the 143 accessions, which were divided into two subgroups, subgroup 1 (Gp1) and subgroup 2 (Gp2) (Additional file 3a). Gp1 contained 67 accessions, which originated from Sichuan (52), Yunnan (7), Shaanxi (5), Gansu (2) and Guizhou (1) provinces. Gp2 consisted of 76 accessions that predominantly originated from Fujian (6), Gansu (5), Guangdong (12), Guangxi (4), Guizhou (14), Hunan (1), Jiangxi (1), Shaanxi (1), Sichuan (18) and Yunnan (14) provinces. On the basis of IT scores, Gp1 contained a higher number of accessions (33) with resistance to stripe rust than Gp2 (12) in all five environments (Additional file 1). All accessions in Gp1 formed a single cluster and the second cluster included all accessions of Gp2 (Additional file 3b). The extent of LD and average rate of LD decay of the 143 genotypes was graphically displayed based on pairwise LD squared correlation coefficients (r2) for all intra-chromosomal markers against the genetic distance (Additional file 4). The LD declined to half (r2 = 0.25) of its initial value at approximately 4 cM for all population sizes. Hence, the significant associated loci on the same chromosome within the confidence interval of ± 4 cM were considered as being located in the same QTL block.
Marker-trait Associations At The Seedling Stage
Using data for the 6404 polymorphic markers, a GWAS analysis was performed for stripe-rust IT to a single Pst race (CYR32 or CYR34) at the seedling stage based on MLM. The GWAS for IT identified a total of 18 DArT-seq markers and one SSR marker within 17 QTLs on 11 chromosomes to be significantly associated (P < 0.001) with seedling resistance; these markers were located on chromosomes 1A, 1B, 2A, 2B, 3B, 4A, 5B, 6A, 6B, 7B and 7D (Fig. 2). The phenotypic variation explained (PVE) by the marker-trait associations (MTAs) ranged from 8.71–17.94% (Table 3). Based on the LD decay distance observed in this study, significant markers within 4 cM were combined as a QTL, hence 17 QTL regions were detected with IT. Of these QTLs, 10 QTLs were significantly associated with ASR to CYR32 and 7 QTLs were significantly associated with ASR to CYR34. Fifteen of these QTLs corresponded with previously reported genes or QTLs and two potentially novel QTLs associated with seedling response were identified on chromosomes 3B and 6A (Fig. 3).
Table 3
The summary of QTLs and significant markers associated with stripe rust seedling response for CYR32 and CYR34 in the panel
QTLName | Races | Trait | Marker | Chromosome | Position(cM) | −log 10 (P) | Marker R2 (%) | References |
QYrsicau-1A | CYR32 | IT | 1279571 | 1A | 39.29 | 3.24 | 11.14 | [38] |
| CYR32 | IT | 1067220 | 1A | 42.17 | 4.01 | 13.96 | |
QYrsicau-2B.1 | CYR32 | IT | 1055456 | 2B | 0.98 | 5.03 | 17.81 | [39, 45] |
QYrsicau-2B.2 | CYR32 | IT | 1687674 | 2B | 74.14 | 4.36 | 15.28 | [46] |
QYrsicau-3B.1 | CYR32 | IT | 4989942 | 3B | 53.54 | 4 | 13.91 | [20] |
QYrsicau-3B.2 | CYR32 | IT | 3953802 | 3B | 116.07 | 3.12 | 10.7 | |
QYrsicau-6A.1 | CYR32 | IT | 1721876 | 6A | 29.3 | 5.07 | 17.94 | [49] |
QYrsicau-6A.2 | CYR32 | IT | 1103920 | 6A | 84.01 | 3.3 | 11.36 | |
QYrsicau-6B.1 | CYR32 | IT | 3533808 | 6B | 24.83 | 3.18 | 10.93 | [30, 31, 50–53] |
QYrsicau-7B | CYR32 | IT | 1121184 | 7B | 129.77 | 3.41 | 11.74 | [55, 56] |
QYrsicau-7D | CYR32 | IT | Xgwm111 | 7D | | 3.22 | 8.71 | [30] |
QYrsicau-1B.1 | CYR34 | IT | 5325193 | 1B | 50.15 | 3.83 | 13.3 | [38, 76] |
| CYR34 | IT | 1261119 | 1B | 51.29 | 3.61 | 12.5 | |
QYrsicau-1B.2 | CYR34 | IT | 1094760 | 1B | 111.34 | 3.08 | 10.56 | [44] |
QYrsicau-2A | CYR34 | IT | 993667 | 2A | 73.88 | 3.67 | 12.7 | [30, 38] |
QYrsicau-3B.3 | CYR34 | IT | 1143801 | 3B | 70.64 | 3.5 | 12.07 | [47] |
QYrsicau-4A | CYR34 | IT | 2288912 | 4A | 29.37 | 3.04 | 10.43 | [31, 39] |
QYrsicau-5B | CYR34 | IT | 4408847 | 5B | 68.21 | 3.59 | 12.43 | [30, 31, 36] |
QYrsicau-6B.2 | CYR34 | IT | 1206552 | 6B | 31.49 | 3.08 | 10.55 | [31, 54] |
Marker-trait Associations At The Adult-plant Stage
Following the same procedure, the GWAS analysis was also performed for IT, FDS and AUDPC of stripe rust against the mixed Pst races within five environments at the adult-plant stage. A total of 32 markers (31 DArT-seq markers and one SSR marker) within 15 QTLs on seven chromosomes were identified to be significantly associated (P < 0.001) with APR in at least two environments; these markers were located on chromosomes 1B, 2A, 2B, 3B, 4A, 5B and 6A (Fig. 2). The PVE by the MTAs ranged from 8.34–23.77% (Table 4). On chromosomes 1B, 2B and 4A, 5 markers were only associated with one of the traits (IT, FDS and AUDPC), respectively. In addition, 27 markers represented loci significantly associated with stripe rust FDS and AUDPC on chromosomes 1B, 2A, 2B, 3B, 5B and 6A. The PVE explained for the FDS and AUDPC loci were in the ranges 8.34–20.92% and 10.04–23.77%, respectively. Based on the LD decay distance observed in this study, significant markers within 4 cM were combined as a QTL, hence a total of 15 QTL regions were detected with IT, FDS and AUDPC. Chromosomes 1B contained four QTLs, chromosomes 3B and 5B carried three QTLs each, chromosome 2B included two QTLs and one QTL was detected on each of chromosomes 2A, 4A and 6A. Among these QTLs, 11 QTLs linked to one marker were associated with IT, FDS, or AUDPC, respectively. QYrsicau-5B.3 linked to 1108002 and 1223817 was associated with both FDS and AUDPC and the PVE was 13.75–20.08% and 14.39–23.3%, respectively. QYrsicau-2B.1 and QYrsicau-5B.2 were linked to three and six markers, respectively. Notably, QYrsicau-3B.3 was linked to ten markers, of which 1129542 was associated with both FDS and AUDPC in three and five environments and the PVE was 19.66% and 19.29%, respectively. Fourteen QTLs corresponded with previously reported genes or QTLs, while QYrsicau-6A may be a potential novel QTL associated with the adult-plant stage response (Fig. 3). Notably, four QTLs (QYrsicau-1B.2, QYrsicau-2B.1, QYrsicau-3B.2 and QYrsicau-5B.3) on chromosomes 1B, 2B, 3B and 5B were detected at the seedling and adult-plant stages for which the PVE ranged from 15.28–23.30%, respectively.
Table 4
The summary of QTLs for stripe rust resistance identified at the adult plant stage across five experiments in the panel
QTL Name | Marker | Chromosome | Position (cM) | Trait | Environment | −log 10 (P) | Marker R2 (%) | References |
QYrsicau-1B.1 | 1255154 | 1B | 32.28 | AUDPC | CZ16,MY16,MY17 | 3.11–3.92 | 10.31–13.21 | [20, 30, 76, 57] |
QYrsicau-1B.2 | 4537457 | 1B | 51.29 | FDS | CZ17 | 4.44 | 15.04 | [20, 31, 39, 58] |
| | | | AUDPC | CZ16,MY16,CZ17,BLUP | 3.36–3.62 | 11.09–12.27 | |
QYrsicau-1B.3 | Xgwm268 | 1B | | AUDPC | CZ16,MY16 | 3.55–4.48 | 9.4-12.58 | [59, 60] |
QYrsicau-1B.4 | 1161065 | 1B | 286.65 | FDS | CZ17,MY17 | 3.53–4.91 | 9.39–14.14 | [61] |
| | | | AUDPC | CZ17,MY17,BLUP | 3.12–5.01 | 8.23–14.45 | |
QYrsicau-2A | 4004515 | 2A | 60.91 | FDS | MY16,MY17 | 3.41–5.65 | 11.33–19.68 | [30, 39, 62–64] |
| | | | AUDPC | MY16,MY17,BLUP | 3.69–5.05 | 11.91–17.35 | |
QYrsicau-2B.1 | 1263973 | 2B | 71.82 | FDS | CZ16,CZ18 | 3.11–3.38 | 10.62–11.26 | [45, 65–67] |
| 1138058 | 2B | 73.02 | FDS | MY17 | 3.22 | 10.77 | |
| | | | AUDPC | CZ16,BLUP | 3.02–3.32 | 9.99–10.64 | |
| 4663985 | 2B | 74.08 | FDS | CZ17,MY17 | 3.27–3.29 | 10.92–10.93 | |
| | | | AUDPC | MY16,CZ17,BLUP | 3.2–3.96 | 10.76–12.85 | |
QYrsicau-2B.2 | 1254647 | 2B | 107.03 | AUDPC | CZ16,CZ17,MY16,BLUP | 3.05–3.98 | 9.99–13.39 | [20, 55, 68] |
QYrsicau-3B.1 | 3943894 | 3B | 20.8 | FDS | MY16 | 3.21 | 8.34 | [67, 69, 70] |
| | | | AUDPC | CZ17,MY16,MY17 | 3.16–4.08 | 8.25–11.14 | |
QYrsicau-3B.2 | 1133063 | 3B | 68.59 | FDS | CZ17,MY17 | 3.11–3.33 | 10.28–11.15 | [45, 47] |
| | | | AUDPC | CZ16,CZ17,BLUP | 3.18–4.61 | 10.57–15.88 | |
Table 4
QTL Name | Marker | Chromosome | Position (cM) | Trait | Environment | −log 10 (P) | Marker R2 (%) | References |
QYrsicau-3B.3 | 1086466 | 3B | 90.44 | FDS | MY17,BLUP | 3.66–5.65 | 11.94–19.67 | [71, 72] |
| | | | AUDPC | MY17,BLUP | 4.39–5.57 | 14.34–19.31 | |
| 1244635 | 3B | 90.68 | FDS | MY17,BLUP | 4.01–5.66 | 13.16–19.72 | |
| | | | AUDPC | CZ17,MY17,BLUP | 3.11–5.6 | 10.46–19.41 | |
| 1129542 | 3B | 90.68 | FDS | CZ17,MY17,BLUP | 3.12–6.45 | 8.09–19.66 | |
| | | | AUDPC | CZ16,CZ17,MY16,MY17,BLUP | 3.16–6.37 | 8.16–19.29 | |
| 2275715 | 3B | 90.68 | FDS | CZ17,MY17,BLUP | 3.43–5.66 | 11.42–19.71 | |
| | | | AUDPC | CZ17,MY16,MY17,BLUP | 3.065.9 | 10.04–20.58 | |
| 1102869 | 3B | 91.03 | FDS | MY17,BLUP | 3.81–5.65 | 12.44–19.68 | |
| | | | AUDPC | MY16,MY17,BLUP | 3.56–5.61 | 11.77–19.47 | |
| 2279272 | 3B | 91.04 | FDS | MY17,BLUP | 4.32–5.9 | 14.23–20.65 | |
| | | | AUDPC | CZ17,MY16,MY17,BLUP | 3.13–5.82 | 10.54–20.26 | |
| 1138233 | 3B | 92.78 | FDS | MY17,BLUP | 3.09–4.73 | 9.97–16.2 | |
| | | | AUDPC | MY17,BLUP | 3.56–4.94 | 11.47–16.93 | |
| 1107260 | 3B | 93.62 | FDS | MY17,BLUP | 3.08–3.65 | 9.95–12.28 | |
| | | | AUDPC | CZ16,MY17,BLUP | 3.04–4.1 | 10.07–13.87 | |
| 3940970 | 3B | 92.68 | FDS | MY17,BLUP | 3.63–5.97 | 11.83–20.92 | |
| | | | AUDPC | CZ17,MY17,BLUP | 3.09–5.66 | 10.39–19.64 | |
| 4439724 | 3B | 92.68 | FDS | MY17,BLUP | 4.16–5.19 | 13.69–17.91 | |
| | | | AUDPC | MY17,BLUP | 4.34–5.43 | 14.17–18.8 | |
QYrsicau-4A | 1231042 | 4A | 83.92 | IT | CZ16,CZ17,BLUP | 3.13–3.36 | 10.41–11.31 | [64] |
QYrsicau-5B.1 | 3944166 | 5B | 50.14 | FDS | CZ17,MY17,BLUP | 3.99–5.47 | 13.08–18.86 | [30, 73] |
| | | | AUDPC | CZ17,MY16,MY17,BLUP | 3.99–6.07 | 13.28–21.23 | |
Table 4 continued
QTL Name | Marker | Chromosome | Position (cM) | Trait | Environment | −log 10 (P) | Marker R2 (%) | References |
QYrsicau-5B.2 | 3022447 | 5B | 55.6 | FDS | CZ17,MY16,MY17,BLUP | 3.1–4.82 | 10.25–16.53 | [71, 74] |
| | | | AUDPC | MY16,MY17,BLUP | 4.87–6.63 | 16.03–23.41 | |
| 1103656 | 5B | 55.6 | FDS | CZ17,MY16,MY17,BLUP | 3.4–5.75 | 11.27–19.88 | |
| | | | AUDPC | CZ17,MY16,MY17,BLUP | 4.27–5.99 | 14.31–20.92 | |
| 3936865 | 5B | 55.6 | FDS | CZ17 | 4.05 | 13.61 | |
| | | | AUDPC | CZ17,MY17,BLUP | 3.11–5.32 | 9.94–18.57 | |
| 3024339 | 5B | 55.71 | FDS | MY16,MY17,BLUP | 3.47–4.97 | 11.53–17.12 | |
| | | | AUDPC | MY16,MY17,BLUP | 5.13–6.72 | 16.95–23.77 | |
| 2276711 | 5B | 57.24 | FDS | CZ17,MY17,BLUP | 3.98–5.47 | 13.06–18.85 | |
| | | | AUDPC | CZ17,MY16,MY17,BLUP | 4.01–5.99 | 13.36–20.91 | |
| 3956366 | 5B | 59.68 | FDS | CZ17,MY17 | 3.07–4.17 | 10.23–14.06 | |
| | | | AUDPC | CZ17,MY17 | 3.92–4.25 | 13.35–14.42 | |
QYrsicau-5B.3 | 1108002 | 5B | 64.83 | FDS | CZ17,MY17,BLUP | 4.18–5.75 | 13.75–20.05 | [31, 48, 57] |
| | | | AUDPC | CZ17,MY16,MY17,BLUP | 4.3–6.6 | 14.39–23.3 | |
| 1223817 | 5B | 66.35 | FDS | CZ17,MY17,BLUP | 4.26–5.8 | 14.04–20.08 | |
| | | | AUDPC | CZ17,MY16,MY17,BLUP | 4.31–6.09 | 14.44–21.31 | |
QYrsicau-6A | 3021470 | 6A | 78.71 | FDS | CZ17 | 3.44 | 11.44 | |
| | | | AUDPC | CZ16,CZ17,BLUP | 3.06–4.2 | 9.76–14.38 | |
Favorable Allele Analyses
Fifteen QTLs were significantly associated with stripe rust in at least two environments in the field. Of these QTLs, four favorable alleles were identified based on the high PVE, namely QYrsicau-3B.3, QYrsicau-5B.1, QYrsicau-5B.2 and QYrsicau-5B.3, for which the PVE were as high as 20.92%, 21.23%, 23.77% and 23.3%, respectively. Among the 143 accessions, QYrsicau-3B.3, QYrsicau-5B.1, QYrsicau-5B.2 and QYrsicau-5B.3 showed the higher frequencies of 83.22%, 86.01%, 90.21% and 84.62% among the favorable resistance-associated alleles. Examination of the subpopulation distribution of favorable resistance-associated alleles revealed that these four favorable alleles showed almost the same frequencies in Gp1 and Gp2. Accessions exhibited higher resistance to stripe rust when the favorable alleles were present. In contrast, when the favorable alleles were absent, the accession showed higher susceptibility (Fig. 4).