Wheat powdery mildew resistance main effect QTL, KASP molecular marker closely linked with wheat powdery mildew resistance main effect QTL and application

Abstract

The invention belongs to the technical field of molecular genetics, and particularly relates to a wheat powdery mildew resistance main effect QTL, a KASP molecular marker closely linked with the wheat powdery mildew resistance main effect QTL and application of the wheat powdery mildew resistance main effect QTL. The invention provides a wheat powdery mildew resistance major QTL, the major QTL locus is located at the end part of a wheat 1A chromosome short arm and is located in a 4.4 cM genetic interval between flanking KASP markers Chr1A6275522K2 and Chr1A11524652K9, nine KASP molecules are developed, auxiliary selective breeding and screening of powdery mildew resistance wheat strains can be carried out, and a new available molecular marker is provided for screening of wheat powdery mildew resistance materials.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics technology, and specifically relates to a major QTL for wheat resistance to powdery mildew and its closely linked KASP molecular marker and its application. Background Technology

[0002] Wheat is one of the most important food crops for humankind, and it is also the most widely cultivated and highest-yielding staple crop globally, holding significant strategic importance for world food security and social stability. Wheat growth is frequently harmed by various external abiotic and biotic stresses. Among the many diseases, those caused by the obligate parasitic grass species *Brucea fulvidraco* (powdery mildew) are particularly prevalent. Blumeria graminis f.sp. tritici , Bgt Powdery mildew, caused by wheat, is one of the world's major diseases and ranks among the leading factors contributing to crop yield reduction globally. It is widespread, spreads rapidly, and causes immense damage, seriously threatening global food security.

[0003] Kompetitive allele-specific PCR (KASP) is a novel genotyping technique based on single nucleotide polymorphisms. KASP technology can accurately determine biallelic alleles for SNPs and InDels at specific sites in a wide range of genomic DNA samples, with high accuracy and reproducibility, making it suitable for identification and screening of large populations.

[0004] To date, more than 130 powdery mildew resistance genes have been discovered in wheat and its closely related species, distributed across more than 70 loci. Among them, 69 are wheat powdery mildew resistance genes. Pm1 - Pm69 It was officially named. Among them... Pm1 , Pm2 , Pm3 , Pm4 , Pm5 , Pm8 and Pm24 It contains multiple resistance sites, that is Pm1 ( Pm1a - Pm1e ), Pm2 ( Pm2a , Pm2b ), Pm3 ( Pm3a - Pm3k ), Pm4 ( Pm4a - Pm4d ), Pm5 ( Pm5a - Pm5e ), Pm8 ( Pm8- Pm17 )and Pm24 ( Pm24a , Pm24b Furthermore, in the officially named wheat powdery mildew resistance genes... Pm8 = Pm17 , Pm18 = Pm1c , Pm22 = Pm1e , Pm23 = Pm4c , Pm31 = Pm21 Although many wheat powdery mildew resistance genes have been discovered and located, only 17 genes have been cloned to date, namely... Pm1 , Pm2 , Pm3 , Pm4 , Pm5 , Pm6Sl , Pm8 , Pm13a , Pm17 , Pm21 , Pm24 , Pm26 , Pm36 , Pm38 , Pm41 , Pm46 , Pm57 , Pm60 and PmAeu1 . Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a QTL with major efficacy against wheat powdery mildew.

[0006] This invention further provides a KASP molecular marker that is closely linked to the major QTL for wheat resistance to powdery mildew.

[0007] Another objective of this invention is to provide an application of the aforementioned KASP molecular marker, which can be used for assisted selection breeding to screen wheat varieties resistant to powdery mildew.

[0008] This invention identified powdery mildew resistance in 55 imported American wheat accessions and detected molecular markers of known resistance genes. Screening revealed that PI601806 exhibited high resistance to powdery mildew at both the seedling and mature stages, demonstrating broad-spectrum resistance, and may contain an unknown powdery mildew resistance gene. By constructing a genetic population with Jimai 22 and conducting powdery mildew resistance identification, it was determined that PI601806 carries a dominant single resistance gene, tentatively named... Pm601806 Cluster segregation analysis combined with wheat exon capture sequencing (BSE-seq) was used to infer powdery mildew resistance genes. Pm601806It is likely located on the wheat 1AS chromosome. This study aims to build upon this by developing markers using genetically segregating populations, BSE-seq sequencing results, and the newly released wheat reference genome sequence to further investigate its location. Pm601806 Genes are located.

[0009] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a major QTL for wheat resistance to powdery mildew, wherein the major QTL is located at the end of the short arm of wheat chromosome 1A, and is a flanking KASP marker. Chr1A_6275522_K2 and Chr1A_11524652_K9 Within the 4.4 cM genetic interval.

[0010] This invention provides a KASP molecular marker closely linked to the above-mentioned major QTL for wheat powdery mildew resistance, wherein the KASP molecular marker is KASP-2, KASP-4, KASP-5, KASP-9, KASP-12, KASP-17, KASP-22, KASP-24, or KASP-25; Two KASP forward primers were modified by adding fluorescent tag sequences to their 5' ends: FAM 5'-GAAGGTGACCAAGTTCATGCT-3; HEX 5'-GAAGGTCGGAGTCAACGGATT-3'.

[0011] Preferably, the specific sequence of KASP-2, KASP-4, KASP-5, KASP-9, KASP-12, KASP-17, KASP-22, KASP-24, and KASP-25 is as follows: ①KASP-2 FAM primers: GAAGGTGACCAAGTTCATGCTCCTCAACATGTGTCACCCG, as shown in SEQ ID NO.1; HEX primers: GAAGGTCGGAGTCAACGGATTCCTCAACATGTGTCACCCA, as shown in SEQ ID NO.2; Reverse primer: CCTTACATTCCTGGCATGCA, as shown in SEQ ID NO.3; ②KASP-4 FAM primers: GAAGGTGACCAAGTTCATGCTACAACAATTTTTTTCATAGTGCC, as shown in SEQ ID NO.4; HEX primers: GAAGGTCGGAGTCAACGGATTACAACAATTTTTTTCATAGTGCT, as shown in SEQ ID NO.5; Reverse primer: TCCGTCAACAAATCTCAAGC, as shown in SEQ ID NO.6; ③KASP-5 FAM primers: GAAGGTGACCAAGTTCATGCTCTACACCCGGTTTTTTCTCAA, as shown in SEQ ID NO.7; HEX primers: GAAGGTCGGAGTCAACGGATTCTACACCCGGTTTTTTCTCAG, as shown in SEQ ID NO.8; Reverse primer: GCTTACACTCCTCGTTGATGTC, as shown in SEQ ID NO.9; ④KASP-9 FAM primers: GAAGGTGACCAAGTTCATGCTTAGGAAATGCCCCGTGAACA, as shown in SEQ ID NO.10; HEX primer: GAAGGTCGGAGTCAACGGATTTAGGAAATGCCCCGTGAACG, as shown in SEQ ID NO.11; reverse primer: ATCGGATGCTACCACTGGAC, as shown in SEQ ID NO.12; ⑤KASP-12 FAM primers: GAAGGTGACCAAGTTCATGCTCTATGGACAAGAAGAACAACATTCA, as shown in SEQ ID NO.13; HEX primers: GAAGGTCGGAGTCAACGGATTCTATGGACAAGAAGAACAACATTCG, as shown in SEQ ID NO.14; Reverse primer: TCCGTCAACAAATCTCAAGC, as shown in SEQ ID NO.15; ⑥KASP-17 FAM primers: GAAGGTGACCAAGTTCATGCTCTGACCCACAGGCAATTGC, as shown in SEQ ID NO.16; HEX primers: GAAGGTCGGAGTCAACGGATTCTGACCCACAGGCAATTGT, as shown in SEQ ID NO.17; Reverse primer: AGTGTGGTGCAGCAAATCAG, as shown in SEQ ID NO.18; ⑦KASP-22 FAM primers: GAAGGTGACCAAGTTCATGCTGCCGCGTTCCTTGATCTCG, as shown in SEQ ID NO.19; HEX primers: GAAGGTCGGAGTCAACGGATTGCCGCGTTCCTTGATCTCA, as shown in SEQ ID NO.20; Reverse primer: GGAAGATGGAAGAACACAACAATTT, as shown in SEQ ID NO.21; ⑧KASP-24 FAM primers: GAAGGTGACCAAGTTCATGCTGCCGCGTTCCTTGATCTCG, as shown in SEQ ID NO.22; HEX primers: GAAGGTCGGAGTCAACGGATTGCCGCGTTCCTTGATCTCA, as shown in SEQ ID NO.23; Reverse primer: TGGGAAGATGGAAGAACACAAC, as shown in SEQ ID NO.24; ⑨KASP-25 FAM primers: GAAGGTGACCAAGTTCATGCTGCCGCGTTCCTTGATCTCG, as shown in SEQ ID NO.25; HEX primers: GAAGGTCGGAGTCAACGGATTGCCGCGTTCCTTGATCTCA, as shown in SEQ ID NO.26; Reverse primer: TCATTTCCTGGGAAGATGGAAGAA, as shown in SEQ ID NO.27.

[0012] The present invention also provides a genotyping method for the above-mentioned major QTL for wheat resistance to powdery mildew, comprising the following steps: extracting wheat genomic DNA, and performing PCR amplification on the extracted genomic DNA using primer sequences labeled with KASP-2, KASP-4, KASP-5, KASP-9, KASP-12, KASP-17, KASP-22, KASP-24, and KASP-25.

[0013] Preferably, the PCR amplification method is as follows: the PCR amplification reaction system (total volume 4 μL) consists of: 2 μL 2x HiGeno reagent, 0.056 μL primer mixture, and 1 μL DNA template (100 ng).

[0014] Preferably, the PCR amplification reaction program is as follows: pre-denaturation at 94℃ for 15 min; denaturation at 94℃ for 5 min; annealing at 62-55℃ (Touch down PCR, decreasing by 0.8℃ per cycle) for 30 s, 10 cycles; extension at 72℃, annealing at 57℃ for 30 s; extension at 72℃ for 10 min; after the reaction, the fluorescence value is read on the Phestar instrument, and the genotyping results are analyzed using KLUSTER software; the amplification product with the FAM adapter primer emits blue fluorescence, the amplification product with the HEX adapter primer emits red fluorescence, and the product corresponding to the heterozygote emits green fluorescence.

[0015] This invention also provides the application of the KASP molecular marker, which is tightly linked to the major QTL for wheat powdery mildew resistance, in wheat disease resistance breeding.

[0016] The beneficial effects of this invention are as follows: This invention provides a major QTL for wheat resistance to powdery mildew, the major QTL locus being located at the end of the short arm of wheat chromosome 1A, flanked by the KASP marker. Chr1A_6275522_K2 and Chr1A_11524652_K9 Within the 4.4 cM genetic interval, nine KASP molecules were developed, which can be used for assisted selection breeding to screen wheat lines resistant to powdery mildew, providing new and usable molecular markers for screening wheat materials resistant to powdery mildew. Attached Figure Description

[0017] Figure 1 Identification of powdery mildew resistance in seedlings of PI601806; A, Identification of the resistance spectrum of PI601806, JM229, Jimai 229, JM22, and Jimai 22; B, Identification of powdery mildew resistance in seedlings of resistant parent PI601806, susceptible parent Jimai 22, F1 and some F2 plants, with Jimai 229 as a susceptible control; Figure 2 Using the Euclidean distance (ED) algorithm to Pm601806 Initial localization is performed, where the upper figure shows the fitted curve and the lower figure shows the original values, with each point representing the ED4 value; Figure 3 Enrichment distribution of differential SNPs in the resistant / susceptible mixed pool of PI601806×Jimai 22 hybrid F2; differential SNP density at the screening thresholds of parental heterozygosity <0.4 and genotype frequency difference >0.67, each vertical line represents the number of SNPs within 1 Mb; the redder the color, the higher the density; Figure 4KASP marker genotyping results of the F2 population; the three clusters correspond to the homozygous type of parent 1 (PI601806), the homozygous type of parent 2 (Jimai 22), and the heterozygous type of F1, respectively, with NTC as the blank control; Figure 5 Pm601806 Genetic linkage map and its physical regions on chromosome 1AS; linkage map of chromosome 1A constructed based on the F2 population, Pm601806 Located at the mark Chr1A_6275522_K2 and Chr1A_11524652_K9 The physical interval is approximately 5.2 Mb (CS RefSeq v2.1); the right side shows the annotated genes within this 5.2 Mb interval. Detailed Implementation

[0018] The technical solution of the present invention will be further explained and described below through specific embodiments. Unless otherwise specified, all raw materials used in the present invention are commercially available.

[0019] Example 1 1. Materials and Methods The F1 generation was produced by crossing the powdery mildew-resistant American wheat variety PI601806 with the powdery mildew-susceptible wheat variety Jimai 22. All F1 plants were harvested and then planted individually to produce the F2 population (134 plants). The harvested F2 plants were then planted individually to obtain the F1 generation. 2:3 The family lineages (105) were used as the target group.

[0020] (1) Seedling resistance identification and genetic analysis: Wheat seedling resistance identification was conducted in the artificial climate chamber of Shandong Academy of Agricultural Sciences. After disinfecting the seeds of PI601806, Jimai 22 and F1 with 75% alcohol solution, they were sown in 24-well seedling trays (54×28×4.2 cm), with 5 seeds per well. Every 4 wells, one highly susceptible powdery mildew variety, Jimai 229 (susceptible control), was sown to provide the inoculum source for the experimental groups to ensure sufficient disease development in the experimental materials for seedling identification. F2 generation seeds were sown in 128-well (3.2×3.2×4.2 cm) plug trays (54×28×4.2 cm), with 1 seed per well. A total of 3 surveys were conducted (resistant single plants were denoted as R, and susceptible single plants were denoted as S).

[0021] In F 2:3To ensure the accuracy of phenotypic responses in F2 generation plants, 20 to 35 seeds from each family were selected for identification during pedigree testing. After sowing, seedling trays were placed in an artificial climate chamber (relative humidity approximately 70%, light intensity 600 μmol m⁻² s⁻¹, photoperiod 12 h, temperature controlled at 22 ℃ under light and 16 ℃ under no light). At the one-leaf stage, wheat materials were inoculated with powdery mildew strains by spraying the leaves with water and sweeping. 10-14 days later, when spores were fully developed on the first leaf of the susceptible control Jimai 229, a 0-4 graded system was used to grade the resistant parent PI601806, the susceptible parent Jimai 22, and its F1, F2, and F3 populations. 2:3 The infection type of the family was investigated. Infection type (IT) was investigated and recorded plant by plant according to a 0-4 grade standard. Resistance levels were classified as follows: 0-2 for resistant, 3-4 for susceptible. All identification tests required three replicates to ensure reliability. The survey data were entered into an Excel spreadsheet, and the F2 and F3 pedigrees were analyzed. 2:3 The phenotypic segregation ratio of family groups was analyzed using a chi-square test to determine whether it conformed to Mendel's laws of inheritance.

[0022] (2) BSE-seq analysis Based on the phenotypic identification results of F2, 40 resistant and 40 susceptible leaves were selected from each of the 134 F2 populations. Equal amounts of DNA from the resistant and susceptible plants were mixed to construct resistant and susceptible pools. Simultaneously, leaves from 10 resistant and 10 susceptible parents were taken and mixed in equal amounts to construct resistant-susceptible parent pools. Exon capture sequencing was performed on the F2 single-plant resistant-susceptible mixed pools and parent pools using the Illumina platform. Exon sequencing was performed by Qingdao Ouyi Biotechnology Co., Ltd. Adapters were removed and low-quality raw sequencing data (raw reads) were filtered out using FastP v0.12.4 software. Data that passed quality control were aligned to the Chinese spring reference genome (IWSGC Ref v2.1) using BWA software. The resulting SAM files were converted to BAM format using SAMtools, indexed, and sorted. Duplicate reads generated during library preparation were removed, and non-specifically aligned reads were filtered out. SNP calling analysis was performed using Bcftools with the following parameters: "theminimum-mapping-quality: 30; the minimum-depth: 5". The Euclidean distance (ED) algorithm was used to calculate the BAM file of the F2 mixed pool to identify segments associated with powdery mildew resistance genes.

[0023] (3) KASP marker development and genotyping Based on the enrichment regions of polymorphic SNPs on chromosomes in the BSA results, polymorphic SNPs associated with disease resistance traits were screened for subsequent KASP marker development. Primers were designed using the WheatOmics 1.0 database (http: / / wheatomics.sdau.edu.cn / snprimer / ) to develop KASP primers for population validation. Fluorescent tag sequences were added to the 5' ends of two KASP forward primers (FAM 5'-GAAGGTGACCAAGTTCATGCT-3; HEX 5'-GAAGGTCGGAGTCAACGGATT-3'), and these were synthesized by Qingke Biotechnology Co., Ltd.

[0024] Table 1. KASP marker sequences used for genotyping Remark: Forward primer Allele 1: Add a FAM fluorescent adapter sequence (5'-GAAGGTGACCAAGTTCATGCT-3') to the 5' end; #Forward primer Allele 2: Add a HEX fluorescent adapter sequence (5'-GAAGGTCGGAGTCAACGGATT-3') to the 5' end.

[0025] The developed molecular markers were first validated for effectiveness in parental materials, followed by KASP marker genotyping in F2 plants to locate the target disease resistance gene. The specific procedures were as follows: First, a mixture of KASP molecular marker primers was prepared: FAM, HEX, and reverse primers were diluted to 100 μM with ddH2O. 12 μL each of primers A and B, 30 μL of primer C, and 46 μL of ddH2O were added and vortexed to mix. The PCR reaction was performed on a CFX96 Real-Time PCR instrument according to the PCR 384 plate reaction system and procedure. The reaction system (total volume 4 μL) consisted of: 2 μL 2x HiGeno reagent, 0.056 μL primer mixture, and 1 μL DNA template (100 ng). The reaction program was as follows: pre-denaturation at 94℃ for 15 min; denaturation at 94℃ for 5 min; annealing at 62-55℃ (Touchdown PCR, decreasing by 0.8℃ per cycle) for 30 s, for 10 cycles; extension at 72℃, followed by annealing at 57℃ for 30 s; and extension at 72℃ for 10 min. After the reaction, fluorescence values ​​were read on a Phester instrument, and the genotyping results were analyzed using KLUSTER software. Amplification products with FAM adapter primers emitted blue fluorescence, those with HEX adapter primers emitted red fluorescence, and the products corresponding to heterozygotes emitted green fluorescence.

[0026] 2. Results (1) Genetic analysis of powdery mildew resistance gene in PI601806 Powdery mildew resistance was assessed using mixed races of powdery mildew against the parents of PI601806 and Jimai 22 and their hybrids. When the susceptible control variety Jimai 229 was fully infected, PI601806 showed no obvious symptoms on the first leaf and was therefore identified as immune (IT=0). Figure 1 B). Compared to the susceptible control, Jimai 22 had abundant spores on its first leaf, and was therefore rated as highly susceptible (IT=4). There was a significant genetic difference in the response to powdery mildew between the two. Figure 1 B). The F1 population obtained from the cross between PI601806 × JM22 and the F2 population obtained from continuous self-pollination, and the F3 population. 2:3 A seedling resistance phenotype investigation was conducted on the pedigrees. The results showed that all 10 F1 seedlings exhibited a resistant phenotype (IT=0), indicating that the resistance of PI601806 to powdery mildew is controlled by a dominant resistance gene. In the tested F2 population of 134 plants, 101 were resistant and 35 were susceptible, meeting the Mendelian single-gene segregation ratio of 3:1 as determined by chi-square test (χ²=0.03, P=0.86) (Table 2). Furthermore, in 105 F2... 2:3 Family identification for powdery mildew showed that the number of homozygous resistant lines, resistant-susceptible segregating lines, and homozygous susceptible lines were 27, 54, and 20 respectively. Chi-square test confirmed that these ratios conformed to a 1:2:1 segregation ratio of a single dominant gene (χ²=1.55, P=0.46) (Table 2). In summary, the genetic segregation population survey results indicate that powdery mildew resistance in PI601806 is controlled by a single dominant gene. This key powdery mildew resistance gene has been tentatively named... Pm601806 Combined with the anti-spectral results ( Figure 1 A), Pm601806 It exhibits broad-spectrum resistance to powdery mildew strains.

[0027] Table 2 PI601806×Jimai 22 F1, F2 and F 2:3 Genetic analysis of powdery mildew resistance in families (2) Pm601806 Positioning DNA from 33 resistant and 32 susceptible F2 plants was pooled to create two pools (Pool-R and Pool-S), along with DNA from the parents PI601806 and Jimai 22, for a total of four samples used for exon sequencing. After quality control, the cleanreads were aligned to the Chinese Spring reference genome (RefSeq v2.1). The alignment results from the two F2 pools were analyzed using the ED algorithm, identifying four DNA sequences related to the F2 strain. Pm601806 The relevant genomic segments are located on chromosomes 1A, 3A, 5A, and 7A, respectively. Figure 2 Using GATK software, a region with SNP enrichment was found in the 0-100 Mb interval of 1A from these reads. Figure 3 The SNP density on chromosome 1A was significantly higher than on other chromosomes. A total of 232 SNPs were detected on chromosome 1A (heterozygosity was 0.4 in both resistant and susceptible pools; genotype frequency difference was 0.67), of which 113 (48.7%) were located in the 0-16 Mb region on chromosome 1AS, indicating that… Pm601806 It may be located within this 16Mb interval. Further, using SNPs within the 1Mb-14Mb interval on the short arm of chromosome 1A, nine pairs of valid KASP markers were developed. Figure 4 ),Will Pm601806 Positioning at KASP mark Chr1A_ 6275522_K2 and Chr1A_11524652_K9 The physical distance between them, corresponding to the Chinese Spring reference genome (IWGSC RefSeq v2.1), is 5.2 Mb. Chr1A_6275522_K2 and Pm601806 The genetic distance is 4.4 cM ( Figure 5 ).

Claims

1. A major QTL for wheat resistance to powdery mildew, characterized in that, The major-effect QTL is located at the end of the short arm of wheat chromosome 1A, flanked by the KASP marker. Chr1A_6275522_K2 and Chr1A_11524652_K9 Within the 4.4 cM genetic interval.

2. A KASP molecular marker closely linked to the major QTL for wheat powdery mildew resistance as described in claim 1, characterized in that, The KASP molecular markers are KASP-2, KASP-4, KASP-5, KASP-9, KASP-12, KASP-17, KASP-22, KASP-24, and KASP-25; Two KASP forward primers were modified by adding fluorescent tag sequences to their 5' ends: FAM 5'-GAAGGTGACCAAGTTCATGCT-3; HEX 5'-GAAGGTCGGAGTCAACGGATT-3'.

3. The KASP molecular marker according to claim 1 or 2, characterized in that, The specific sequences of KASP-2, KASP-4, KASP-5, KASP-9, KASP-12, KASP-17, KASP-22, KASP-24, and KASP-25 are as follows: ①KASP-2 FAM primers: GAAGGTGACCAAGTTCATGCTCCTCAACATGTGTCACCCG, as shown in SEQ ID NO.1; HEX primers: GAAGGTCGGAGTCAACGGATTCCTCAACATGTGTCACCCA, as shown in SEQ ID NO.2; Reverse primer: CCTTACATTCCTGGCATGCA, as shown in SEQ ID NO.3; ②KASP-4 FAM primers: GAAGGTGACCAAGTTCATGCTACAACAATTTTTTTCATAGTGCC, as shown in SEQ ID NO.4; HEX primers: GAAGGTCGGAGTCAACGGATTACAACAATTTTTTTCATAGTGCT, as shown in SEQ ID NO.5; Reverse primer: TCCGTCAACAAATCTCAAGC, as shown in SEQ ID NO.6; ③KASP-5 FAM primers: GAAGGTGACCAAGTTCATGCTCTACACCCGGTTTTTTCTCAA, as shown in SEQ ID NO.7; HEX primers: GAAGGTCGGAGTCAACGGATTCTACACCCGGTTTTTTCTCAG, as shown in SEQ ID NO.8; Reverse primer: GCTTACACTCCTCGTTGATGTC, as shown in SEQ ID NO.9; ④KASP-9 FAM primers: GAAGGTGACCAAGTTCATGCTTAGGAAATGCCCCGTGAACA, as shown in SEQ ID NO.10; HEX primers: GAAGGTCGGAGTCAACGGATTTAGGAAATGCCCCGTGAACG, as shown in SEQ ID NO.11; Reverse primer: ATCGGATGCTACCACTGGAC, as shown in SEQ ID NO.12; ⑤KASP-12 FAM primers: GAAGGTGACCAAGTTCATGCTCTATGGACAAGAAGAACAACATTCA, as shown in SEQ ID NO.13; HEX primers: GAAGGTCGGAGTCAACGGATTCTATGGACAAGAAGAACAACATTCG, as shown in SEQ ID NO.14; Reverse primer: TCCGTCAACAAATCTCAAGC, as shown in SEQ ID NO.15; ⑥KASP-17 FAM primers: GAAGGTGACCAAGTTCATGCTCTGACCCACAGGCAATTGC, as shown in SEQ ID NO.16; HEX primers: GAAGGTCGGAGTCAACGGATTCTGACCCACAGGCAATTGT, as shown in SEQ ID NO.17; Reverse primer: AGTGTGGTGCAGCAAATCAG, as shown in SEQ ID NO.18; ⑦KASP-22 FAM primers: GAAGGTGACCAAGTTCATGCTGCCGCGTTCCTTGATCTCG, as shown in SEQ ID NO.19; HEX primers: GAAGGTCGGAGTCAACGGATTGCCGCGTTCCTTGATCTCA, as shown in SEQ ID NO.20; Reverse primer: GGAAGATGGAAGAACACAACAATTT, as shown in SEQ ID NO.21; ⑧KASP-24 FAM primers: GAAGGTGACCAAGTTCATGCTGCCGCGTTCCTTGATCTCG, as shown in SEQ ID NO.22; HEX primers: GAAGGTCGGAGTCAACGGATTGCCGCGTTCCTTGATCTCA, as shown in SEQ ID NO.23; Reverse primer: TGGGAAGATGGAAGAACACAAC, as shown in SEQ ID NO.24; ⑨KASP-25 FAM primers: GAAGGTGACCAAGTTCATGCTGCCGCGTTCCTTGATCTCG, as shown in SEQ ID NO.25; HEX primers: GAAGGTCGGAGTCAACGGATTGCCGCGTTCCTTGATCTCA, as shown in SEQ ID NO.26; Reverse primer: TCATTTCCTGGGAAGATGGAAGAA, as shown in SEQ ID NO.

27.

4. A genotyping method for a major QTL for wheat powdery mildew resistance as described in claim 1, characterized in that, Includes the following steps: Wheat genomic DNA was extracted and amplified by PCR using primer sequences labeled with KASP-2, KASP-4, KASP-5, KASP-9, KASP-12, KASP-17, KASP-22, KASP-24, and KASP-25.

5. The genotyping method according to claim 4, characterized in that, The PCR amplification method is as follows: The PCR amplification reaction system (total volume 4 μL) consists of: 2 μL 2x HiGeno reagent, 0.056 μL primer mixture, and 1 μL DNA template (100 ng).

6. The genotyping method according to claim 4 or 5, characterized in that, The PCR amplification reaction program was as follows: pre-denaturation at 94℃ for 15 min; denaturation at 94℃ for 5 min; annealing at 62-55℃ (Touch down PCR, decreasing by 0.8℃ per cycle) for 30 s, for 10 cycles; extension at 72℃, annealing at 57℃ for 30 s; extension at 72℃ for 10 min; after the reaction, the fluorescence values ​​were read on the Phester instrument, and the genotyping results were analyzed using KLUSTER software; the amplification products with FAM adapter primers emitted blue fluorescence, the amplification products with HEX adapter primers emitted red fluorescence, and the products corresponding to heterozygotes emitted green fluorescence.

7. The application of a KASP molecular marker tightly linked to a major QTL for wheat powdery mildew resistance as described in any one of claims 1-3 in wheat disease resistance breeding.

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