A total of 198 wheat recombinant inbred lines (RILs) of the ‘Yanzhan1×Neixiang188’ mapping population (obtained from Chinese Academy of Agricultural Sciences, CAAS) was used for linkage analysis. The mapping population for this study was planted at the experimental base of Chengdu Institute of Biology, Chinese Academy of Sciences (Shifang and Maerkang) during the seasons of 2015 to 2018, according to local legislation in Sichuan Province. Twenty seeds of each accession were planted in a row, and susceptible wheat strains ‘‘Minxian169’’ (obtained from Chengdu Institute of Biology, Chinese Academy of Sciences) as control was inserted after every 9 rows.
Lesion mimic phenotype evaluation and agronomic traits
198 RILs and 2 parents (Yanzhan1 and Neixiang188) were tested for lesion mimic (LM) phenotype at Shifang and Maerkang in Sichuan Province in 2015 to 2018. Lesion mimic phenotype were subdivided into 0-4 classification system based on flag leaf symptoms according to Yao et al.  with some modification. No visible lesions (specks) were recorded as 0, the parental phenotype; few speck (<25%) were recorded as 1; some specks (25%-50%) were recorded as 2; wheat plants with large specks (50%-75%) were recorded as 3; have a large number of specks (>75%) were recorded as 4.
Agronomic traits of RILs were investigated by our team in Chengdu Institute of Biology, Chinese Academy of Sciences at the Shifang cropping seasons. Which including plant height (PH), spikelet numbers (SPI), numbers of sterile spikelet per spike (SSNS), grain numbers per spike (GNS) and 1000-grain weight (TGW), spike length (SL). Three to five plants of each wheat lines were measured and their means were used for the analysis.
Evaluation of stripe rust resistance
All 198 RIL lines and 2 parents were evaluated for stripe rust reaction at Shifang and Maerkang in 2016 to 2018. Mixed Pst spores of races Pst-CYR32, Pst-CYR33, Pst-SU11, Pst-Hybrid46 and Pst-G22 (provided by SAAS) were suspended in 0.05% Tween 20 and were sprayed on four-leaf stage wheat seedlings.
In the adult stage, stripe rust response types (ITs) were identified, and each environment was identified at least twice, mainly on the 20 weeks to 23 weeks after sowing. Stripe rust infection types (IT) were evaluated based on a typical 0-4 classification systems .
Lesion mimic gene mapping
Seedlings leaves of the 198 RIL lines and two parents (Yanzhan1, Neixiang188) were collected, and genomic DNA was extracted from each sample using the CTAB method . Quality and quantity of DNA were identified using 1.0% agarose gel electrophoresis and a spectrophotometer (NanoDrop ND-1000, Thermo Scientific, Wilmington, DE). Polymorphic 248 SSR markers covering 21 wheat chromosomes were used to genotype the mapping population to confirm the chromosomal location. The information for SSR markers was available on the Grain Genes website (http://wheat.pw.usda.gov). The SSR markers that on 2DS were found to be associated with lesion mimic.
Based on phenotypic evaluations, 10 wheat lines with LM score 0 and 10 RILs with LM score 4 were used to constituted two extreme traits. The DNA along with parental lines were genotyped by the 660K SNP arrays at China Golden Marker Corporation (Beijing; http://www.cgmb.com.cn). The diversity SNP markers located on 2DS associated with lesion mimic were identified from the SNP typing results. Searching for whole wheat genome sequences by SNP-tagged probes sequences (https://www.ncbi.nlm.nih.gov/), or according to the possible physical intervals on 2DS obtained from the SNP analysis search the Chinese spring genomic intervals (https://urgi.versailles.inra.fr/jbrowseiwgsc/gmod_jbrowse/?data=myData%2FIWGSC_RefSeq_v1.0&loc=chr2D%3A1..651852609&tracks=DNA&highlight=). A matched scaffold sequence was obtained, and DNA repeat analysis was performed using the SSR Hunter 1.3 program (Li Qiang and Wan Jianmin 2005). The DNA sequences from both ends of the repeat are obtained, and primers are designed using primer premier 6.0 software (Canada). These primers (Table S1-1) were used for PCR and electrophoresis analysis, and primers suitable for polymorphism were selected as molecular markers to obtain genotypes in the genetic population (Table S1-2).
PCR reaction was conducted in a total volume of 20μl containing 200ng DNA template, 10μl 2× Es Taq MasterMix (Kangwei century, China), 0.6μl 10μM forward primer and 0.6μl 10μM reverse primer. The amplification procedure was as follows: 94°C for 5 min; denaturation at 94°C for 30 s, 45-60°C (adjusted according to the primers) for 30 s, 72°C for 45s, 35 cycles; 72°C for a total extension of 10 minutes. The separation of the PCR products was carried out by 1% agarose gel electrophoresis or 8% non-denaturing polyacrylamide gel electrophoresis. Agarose gel electrophoresis was stained with ethidium bromide (EB) and polyacrylamide gel electrophoresis was stained with silver nitrate .
All data of phenotypes were recorded in Microsoft office excel 2013 for statistical analysis. One-way analysis of variance (ANOVA) was conducted to evaluate the variance and significance between groups by using SPSS 20.0 and GraphPad prism 5.0. The genetic segregation ratio of normal (LM0-1) and lesion mimic phenotypes (LM2-4) was tested for goodness of fit to the expected ratio by chi-square test. Linkage map construction and QTL mapping were performed using QTL IciMapping V 4.1 software .