Plant materials and breeding strategy
Xiaoyan22 was acquired from Researcher Zhang Li, Northwest institute of botany of Chinese Academy of Sciences. Xiaoyan22D with shorter plant height, which was discovered by ourself in population XY22, was a spontaneous mutant, and served as the recipient parent for improving the lodging ability of XY22 (Fig. S1). The other three donors as follows: (l) 91260 with gene ML91260-1 and Ml91260-2 conferring resistance to Blumeria graminis f. sp. Tritici was a gift from the Dr. Zhen Fang, Agricultural Research Center of Shaanxi  . (2): 92R137 with gene Yr26 conferring resistance to Puccinia striiformis West. f.sp. tritici Eriks et Henn Was got from Dr. Peidu Chen, Nanjing Agricultural University[18-19] . (3) ZhengNong 16 with two tightly linked Glu-D1 genes encoding high-molecular-weight (HMW) glutenin subunits, Glu-D1 “x-type” and Glu-D1 “y-type” (Glu-D1-5/Glu-D1-10 was developed by Tiwen Lei , Academy of agriculture and forestry of Zhengzhou  .These four parents were crossed in pairs to produce three single-cross F1 hybrids, then intercrossed to produce a double-cross F1 hybrid (DCHF1). The plants of DCHF1, which is genotype (Ml91260-1, Ml91260-2, Yr26) was double affirmed by SSR marker and field evaluation of resistance. The affirmed DCHF1 with genotype (Ml91260-1, Ml91260-2, Yr26) was crossed with the third single-cross F1 hybrids produce a three-cross F1 hybrid (TCHF1) for pyramiding of disease resistance genes and HMW-GS Dx5+Dy10. The plants of TCHF1 with genotype (Ml91260-1, Ml91260-2, Yr26, Dx+Dy10) was backcrossed with XY22D two times to produce plants which agronomic traits similar to those of XY22D (TCHF1BC1 to TCHF1BC2) with MAS and field disease resistance evaluation. The subsequent two generations (TCHF1BC2F1 to TCHF1BC2F2) were raised through selfing, with MAS exercised and agronomic traits investigated in each generation, eventually leading to selection of six improved groups, including eighteen lines, designated PYLs and Plants (PYL1-6; Plant 1-18, Fig. S1). The eighteen plants were multiplied individually to produce sufficient progenies for evaluation of phenotypic traits, yield and grain quality.
DNA extraction and PCR amplification
In each generation, MAS was exercised using SSR markers for tracking specific genes. The total DNA for PCR amplification of every generation of plant material was extracted according to the procedure of Sharp et al . Specific SSR primers which are adjacent to ML91260-1, ML92160-2, Yr26 and Dx5+Dy10，were adapted for PCR amplification (Table 1). Total reaction mixture was 15 μL containing: 100 ng of genomic DNA, 1X Taq DNA polymerase buffer, 10 pmol of forward and reverse primers, 2.5 mM of each dNTP and 0.15 U of Taq DNA polymerase (Takara，USA). Template DNA was initially denatured at 95℃ for 5 min, prior to 32 cycles of denaturation at 94 ℃ for 1min, annealing at 50-65℃ for 45s and extension at 72℃ for 30 s. In the final step, the reaction mixture was incubated at 72 ℃ for 10 min before completion. After amplification, 3 μL of restriction buffer (10×) was added to the PCR products, which were separated on a 1 % agarose gel.DNA extraction and PCR amplification
SDS-PAGE for analysis of HMW glutenin subunits Dx5+Dy10 in the TCHF1BC2F3
was conducted as described by Damania with minor modifications . Protein was extracted from half seeds of each plant and electrophoresis was performed at a constant voltage of 200 V with a low concentration of running buffer [16.7 mM Tris 127.9 mM glycine, 0.07 % (w/v) SDS]. Electrophoresis was continued until protein bands carrying the smallest molecules reached near the end of the gel.
Evaluation agronomic traits and disease resistance of XY22 and the PYLs
Six PYLs and control XY22 were sown at the experiment station of Northwest A&F University in ShaanXi province with three replications from 2015 to 2017．Each entry consisted of eight rows (4 m) with 161 seeds per row and a row-to-row distance of 25 cm. Seeds of XiNong2000, a variety highly susceptible to yellow rust and powdery mildew were sown at the border as a guard row. Leaves of plants in the guard row were inoculated with Bgt isolate E09 and Pst isolates TiaoZhong33 at shooting period. E-09 pathotypes caused severe powdery mildew epidemics in the wheat-growing regions of north China and TiaoZhong33 pathotypes caused severe yellow rust epidemics in the wheat-growing regions of south GanSu and central ShaanXi, China . Data were recorded on the following nine traits: (1) plant height at shooting period and maturation period (cm), (2) powdery mildew resistance, (3) yellow rust resistance, (4) grain quality, (5) kernel number of 30 spikes, (6) 1000-grain weight (g), (7) grain yield (t/ha), (8) productive tillers in 1m2, (9) days to heading and flowering. The disease symptoms were scored according to the resistance level as described by Sun  for powdery mildew and Mcintosh et al  for yellow rust. A strong wind, resulting in the lodging of XY22, occurred in May of 2017. Thus, the data was investigated only in 2015 and 2016.
Flour Quality analysis of XY22 and PYLs
For each PYL, 1.5 kg was analyzed for flour quality. Wheat gluten content was analyzed by Glutograph-E (Germany). Wheat bulk density was measured with a bulk density meter (BLH-5000, China). Dough formation time and stability time were analyzed by a wheat extensograph（Farinograph-E, Germany).
Comparison of the means of the data of all nine agronomic traits in two years were performed using the analysis of variance. The data on grain yield (kg) was converted into grain yield (t/ha) for further statistical analyses. P values less than 0.05 were considered to be statistically significant. Statistical analysis was performed using the SPSS 19.0 software (IBM Inc. Armonk, NY, USA).