KASP primer set for detecting sites significantly associated with wheat scab resistance and its application

Abstract

This invention discloses a KASP primer set for detecting loci significantly associated with wheat scab resistance and its application. Genome-wide association analysis (GWAS) was used to mine QTL loci associated with wheat scab resistance, and a QTL locus significantly associated with and having a large effect on wheat scab resistance was selected. Qfhb_3B.3 Based on this site, a KASP primer set for genotyping was developed. The KASP primer set includes primers with nucleotide sequences as shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, and can be used for... Qfhb_3B.3 Rapid and accurate detection of superior allelic variants at specific loci. Artificial inoculation results showed that wheat materials carrying the GG allelic variant at the target locus had a lower average disease spikelet rate and relatively stronger resistance to Fusarium head blight than wheat materials carrying the AA allelic variant, with the difference reaching a statistically significant level. Using the KASP primer set and its application method provided in this invention, rapid identification of wheat Fusarium head blight resistance-related genotypes and precise screening of resistant materials can be achieved.

Description

Technical Field

[0001] This invention relates to wheat genetics and breeding, and more specifically, to a KASP primer set for detecting sites significantly associated with wheat scab resistance and its application. Background Technology

[0002] Wheat scab, often called the "cancer" of wheat production, is one of the most destructive diseases affecting wheat yield and quality. It is primarily caused by the Fusarium graminearum complex (…). Fusarium graminearum This pathogen is caused by infection with the species complex (FGSC). It can infect multiple tissues of wheat, including roots, stems, leaves, and ears, and can occur throughout the entire growth period, leading to symptoms such as seedling blight, ear rot, and grain shriveling. In normal years, it can cause a yield loss of 5% to 10%, and in epidemic years, it can even lead to complete crop failure. More seriously, the pathogen can produce toxins such as deoxynivalenol (DON) during the infection process, which can contaminate grains and their processed products, thereby causing poisoning reactions such as vomiting and diarrhea in humans and animals, posing a serious food safety risk (Ma Hongxiang, Wang Yonggang, et al. (2022) Review and prospect of wheat breeding resistant to Fusarium head blight. Chinese Agricultural Science, 55(05):837-855).

[0003] The warm and humid climate of the middle and lower reaches of the Yangtze River in my country makes it a traditional high-incidence and severe area for wheat scab. In recent years, influenced by factors such as global climate change and adjustments in cropping systems, the incidence of scab has gradually expanded to major wheat-producing areas such as the Huang-Huai wheat region, with both the affected area and the severity of the disease showing an upward trend. Due to the complex epidemic process and the difficulty in controlling the disease, among various control measures, the breeding and application of scab-resistant varieties is considered the most economical, green, and sustainable control strategy.

[0004] In breeding practice, accurate phenotypic identification and the discovery and utilization of key resistance genes / locuses are crucial foundations for variety improvement. However, Fusarium head blight resistance exhibits significant multi-type characteristics and is greatly influenced by the environment, making phenotypic identification challenging. Conventional artificial inoculation and field-induced identification require strict temperature and humidity conditions and are susceptible to phenotypic biases caused by extreme weather, even leading to identification failures. This limits the efficiency of stable screening and evaluation in large-scale, multi-generational breeding populations. Furthermore, wheat Fusarium head blight resistance is a typical quantitative trait, controlled by multiple genes, resulting in a complex genetic basis. Although several QTLs related to Fusarium head blight resistance have been resolved, covering different types such as resistance to infection and spread, most QTLs have small genetic effects and insufficient stability. Fhb1While the currently cloned major resistance gene exhibits strong resistance effects, its expression varies under different genetic backgrounds. Wheat varieties carrying this gene alone may not provide sufficient resistance in years with widespread disease outbreaks, and it usually needs to be aggregated with other resistance loci for utilization (Wang Y, Yang J, et al. Development and application of a cost-effective multiplex Kompetitive Allele-Specific polymerase chain reaction assay for pyramiding resistant genes of fusarium head blight and powdery mildew in wheat. BMC Plant Biology, 2025, 25(1): 963). Therefore, the currently available stable resistance gene resources are still relatively limited, and there is a lack of efficient molecular detection tools that can be directly applied to breeding practices. This limits the effective transfer and utilization efficiency of resistance loci to breeding materials, and has become a key issue restricting the genetic improvement and molecular breeding of wheat scab resistance. Summary of the Invention

[0005] To address the issues of wheat scab phenotypic identification being greatly affected by the environment, the cumbersome process of resistance evaluation through artificial inoculation, and the limited number of discovered resistance loci with insufficient application in breeding, this invention provides a KASP primer set for detecting loci significantly associated with wheat scab resistance and its application. This primer set enables rapid detection and genotyping of target resistance loci, providing technical support for wheat scab resistance identification and molecular marker-assisted breeding.

[0006] To achieve the above objectives, this invention uses core wheat germplasm resources from the middle and lower reaches of the Yangtze River as research materials. Combining multi-environment Fusarium head blight resistance phenotypic identification and genome-wide association analysis (GWAS), multiple genetic loci associated with wheat Fusarium head blight resistance were identified. Furthermore, a QTL locus significantly associated with Fusarium head blight resistance and exhibiting a large genetic effect was identified through screening. Qfhb_3B.3 Based on this, a matching KASP molecular marker was developed for this site to promote its application in wheat scab resistance breeding.

[0007] The first aspect of this invention provides a site for detecting sites significantly associated with wheat scab resistance. Qfhb_3B.3 The KASP primer set, the Qfhb_3B.3Located on wheat chromosome 3B, with a physical region of 796,734,063–802,734,063 bp; the KASP primer set includes a first competitive forward primer with a nucleotide sequence as shown in SEQ ID NO.3, a second competitive forward primer with a nucleotide sequence as shown in SEQ ID NO.4, and a universal reverse primer with a nucleotide sequence as shown in SEQ ID NO.5.

[0008] A second aspect of the present invention provides a site for detecting sites significantly associated with wheat scab resistance. Qfhb_3B.3 The kit includes the aforementioned KASP primer set.

[0009] A third aspect of this invention provides the application of the above-described KASP primer set or kit in any of the following: 1) Positioning Qfhb_3B.3 Related genes; 2) Testing wheat resistance to Fusarium head blight; 3) Develop wheat varieties with high resistance to Fusarium head blight.

[0010] A fourth aspect of the present invention provides a method for detecting wheat scab resistance, comprising the following steps: (1) Genomic DNA was extracted from the wheat material to be tested as an amplification template; (2) Fluorescent PCR amplification reaction was performed using the above-mentioned KASP primer set; (3) Fluorescence signal acquisition and cluster analysis were performed on the amplification products, and the wheat material to be tested was determined based on the typing results. Qfhb_3B.3 Genotype of loci: If a sample clusters near the Y-axis in the genotyping clustering diagram and shows a blue FAM fluorescence signal, it is identified as the AA genotype; if a sample clusters near the X-axis in the genotyping clustering diagram and shows a red HEX fluorescence signal, it is identified as the GG genotype; among them, wheat materials carrying the GG allele showed significantly higher resistance to Fusarium head blight than wheat materials carrying the AA allele.

[0011] Specifically, the total volume of the fluorescence PCR amplification reaction system is 10 μL, including: 2.5 μL of the test sample DNA at a concentration of 120 ng / μL, 5 μL of 2×KASP Master Mix, 0.14 μL of KASP Assay Mix, and 2.36 μL of ddH2O; The preparation method for each 100 μL KASP Assay Mix is ​​as follows: 12 μL each of two 100 μM positive competitive primers (SEQ ID NO.3 and SEQ ID NO.4), 30 μL of a 100 μM reverse universal primer (SEQ ID NO.5), and 46 μL of ddH2O.

[0012] The fluorescent PCR amplification program included: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, annealing / extension at 61℃~55℃ for 1 min, for a total of 10 touch-down cycles, with a decrease of 0.6℃ per cycle; followed by 94℃ denaturation for 20 s, annealing / extension at 55℃ for 1 min, for 28 cycles; and incubation at 30℃ after amplification.

[0013] Through the above technical solution, the present invention achieves the following beneficial effects: 1. This invention utilizes genome-wide association analysis of wheat germplasm resources in the middle and lower reaches of the Yangtze River to identify QTL loci significantly associated with wheat scab resistance. Qfhb_3B.3 The wheat-growing areas in the middle and lower reaches of the Yangtze River are regions in my country with a high incidence and susceptibility to wheat scab. Long-term disease stress has created a rich genetic variation base for disease resistance. Therefore, the loci discovered have good breeding application value and can directly serve the improvement of regional disease-resistant varieties and the creation of disease-resistant germplasm.

[0014] 2. The KASP molecular marker developed in this invention is based on the principle of allelic variant-specific amplification and can be used for... Qfhb_3B.3 The locus enables stable and rapid genotyping, with advantages such as high genotyping accuracy, good repeatability, and suitability for high-throughput detection, thereby improving the reliability of target resistance locus detection and the efficiency of breeding application and transformation.

[0015] 3. Compared with traditional artificial inoculation or field natural induction identification methods, the markers and their applications described in this invention are not affected by environmental conditions or identification period, and can realize the rapid identification and screening of superior Fusarium head blight resistance genotypes in large-scale breeding populations, meet the needs of early selection of resistant materials and evaluation of resistance potential, thereby improving the efficiency of wheat Fusarium head blight resistance breeding. Attached Figure Description

[0016] Figure 1 The results of a genome-wide association study on wheat scab resistance; Figure 2 This diagram illustrates the genotyping effect of the KASP molecular marker of the present invention and the genotyping results in the validation population. In the diagram, a represents the genotyping and clustering results of the KASP molecular marker in the test samples, and b represents the genotyping results of the KASP molecular marker in high-generation wheat breeding lines. The horizontal and vertical axes in the diagram represent the fluorescence signal intensities of HEX and FAM, respectively. The black dots represent the negative control (NTC, ddH2O), and the blue and red dots represent different genotypes. Figure 3 The figure shows the comparison of the average disease spikelet rate of two different genotypes of the tested wheat high-generation breeding lines, detected by KASP molecular markers. Detailed Implementation

[0017] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0018] Genome-wide association study (GWAS) can identify genetic loci significantly associated with complex quantitative traits based on genotypic and phenotypic variation at the population level, and has become an important technique for genetic analysis and gene mapping of wheat disease resistance traits. In breeding methods, marker-assisted selection (MAS) and multi-resistance locus / gene aggregation breeding have become important strategies for the genetic improvement of wheat disease resistance. By aggregating multiple Fusarium head blight resistance loci with markers, the efficiency of resistance improvement can be effectively improved, and strong resistant varieties such as Yanghong 617 and Yangmai 53 have been successfully bred. KASP (kompetitive allele specific PCR) technology, based on the principle of allele-specific primer amplification, can achieve high-throughput and accurate genotyping of biallelic alleles, with advantages such as high detection throughput, simple operation, and stable results. Therefore, by identifying efficient resistance QTLs based on GWAS and developing KASP molecular markers for breeding, rapid genotyping and stable detection of target loci can be achieved, improving the utilization efficiency of resistance loci in breeding and promoting the efficient development of molecular breeding for wheat scab resistance.

[0019] This invention uses core wheat germplasm from the middle and lower reaches of the Yangtze River as materials to conduct multi-environment Fusarium head blight resistance phenotypic identification and, combined with genome-wide association analysis, to identify genetic loci significantly associated with Fusarium head blight resistance. Based on this, corresponding KASP molecular markers are further developed for rapid detection of target allelic variations and screening of superior germplasm. This provides molecular tools and application methods for evaluating wheat germplasm resources resistant to Fusarium head blight and for marker-assisted breeding, accelerating the breeding process of new disease-resistant wheat varieties.

[0020] Unless otherwise specified, the reagents, equipment, etc. used in the following examples are all commercially available.

[0021] The wheat materials used in this invention are all germplasm resources preserved in the Jiangsu Provincial Crop Germplasm Resource Bank (Crop, Yangzhou University Bank), which can be obtained and used by technical and research personnel in this field.

[0022] Example 1: Genome-wide association analysis of wheat scab resistance (1) This embodiment uses 335 wheat varieties selected from the wheat-growing areas of the middle and lower reaches of the Yangtze River as the research object. Field planting and related trait data collection were carried out at the Yangzhou Experimental Base (YZ) (32.34 °N, 119.40 °E), Zhenjiang Experimental Base (ZJ) (32.34 °N, 119.40 °E), and Nanjing Experimental Base (NJ) (32.48 °N, 118.63 °E) from 2022 to 2025. Among them, the identification of wheat scab resistance phenotype was carried out at the Yangzhou Experimental Base. The experiment adopted a randomized block design with two replicates. Each replicate was planted in 4 rows with a row length of 1.5 m and a row spacing of 0.3 m. The wheat cultivation and management methods published in "100 Questions on Key Practical Technologies of Wheat Industry" (Ma Hongxiang et al., 2021) were used for consistent field management.

[0023] (2) During the wheat flowering stage, following the method described in (Chang Lei et al. (2018) Identification and evaluation of resistance to Fusarium head blight in new wheat varieties in Jiangsu Province. Jiangsu Agricultural Sciences, 46(16):87-91), artificial inoculation identification of Fusarium head blight was conducted using the "single flower drip method". Sumai 3 and Anong 8455 were used as resistant and susceptible controls, respectively. Twenty ears were selected from each line, and 10 μL of conidial solution was inoculated at one floret in the middle of each ear. The concentration of the conidial solution was 5×10⁻⁶. 5 The number of spikelets / mL was sprayed after inoculation and covered with a plastic bag to keep it moist for 3 days. The disease incidence was investigated 21 days after inoculation. The number of diseased spikelets per ear and the total number of spikelets were counted, and the diseased spikelet rate (DIS) was calculated. The average value was used for analysis.

[0024] (3) Subsequently, combined with the previous GBS genotype data, genome-wide association analysis (GWAS) was conducted using the Fast3VmrMLM software package (Wang J, et al. Fast3VmrMLM: A fast algorithm that integrates genome-wide scanning with machine learning to accelerate gene mining and breeding by design for polygenic traits in large-scale GWAS datasets. Plant Communications, 2025, 6(7)). In the analysis, P-value = 1.0 × 10⁻⁶. -3 The probability threshold for whole-genome scanning was used, and the likelihood ratio test statistic LOD=3 was further used to identify significantly associated sites. The visualization of the correlation analysis results was performed in the R language environment.

[0025] (4) Wheat Fusarium head blight resistance-related loci andQfhb_3B.3 Screening: The study detected a total of 42 significantly associated markers ( Figure 1 Using a population-average linkage disequilibrium distance of 3 Mb as a reference, significant loci located on the same chromosome with overlapping LD intervals were merged into a single QTL, ultimately yielding 39 QTLs significantly associated with Fusarium head blight resistance, distributed across 18 chromosomes: 1B, 2A, 2B, 2D, 3A, 3B, 4A, 4B, 4D, 5A, 5B, 5D, 6A, 6B, 6D, 7A, 7B, and 7D. Among these, the QTL located on chromosome 3B... Qfhb_3B.3 With major antiviral site Fhb1 The overlapping physical locations of the regions further validate the reliability of the association analysis results in this study (Su Z, et al. A deletion mutation in TaHRC confers Fhb1 Resistance to Fusarium head blight in wheat. Nature genetics, 2019, 51(7): 1099-1105). The remaining QTLs further enriched the genetic resources related to wheat scab resistance, providing new candidate loci for the discovery of resistance genes and genetic improvement.

[0026] Of the above loci, the one located on chromosome 3B is... Qfhb_3B.3 The association was highly significant and the genetic effect was large (r 2 =3.15%), with a physical interval of 796,734,063–802,734,063 bp, a peak label of rs-799,734,063, and a LOD of 28.01. Considering factors such as association strength, genetic effects, and localization accuracy, Qfhb_3B.3 It has significant potential for breeding applications. To promote the application of this locus in wheat scab resistance breeding, this application further developed [a technology / mechanism]. Qfhb_3B.3 The corresponding KASP molecular markers are used for rapid detection of superior allelic variations, screening of disease-resistant materials, and marker-assisted selection breeding.

[0027] Table 1. Genome-wide associated QTL loci for wheat scab resistance

[0028] Example 2: Development, screening and validation of KASP molecular markers (1) According to Qfhb_3B.3 The physical location and SNP variation information of peak markers rs-799,734,063 were obtained, and KASP markers suitable for high-throughput genotyping were developed.

[0029] Specifically, genomic flanking sequences of 150 bp upstream and downstream of the peak SNP were obtained as templates for primer design. Based on the allelic differences of the target SNP locus, multiple candidate primer combinations were designed using Primer Premier 5. Each candidate primer combination consisted of two allelic-specific forward primers and one shared reverse primer. The 3' ends of the two forward primers were matched with different alleles of the target SNP locus to achieve specific identification of different allelic variations. To ensure primer amplification efficiency, DNAMAN was used to analyze parameters such as secondary structure, complementarity, and annealing temperature of candidate primers, and the optimal primer combination was determined by manual screening. The following conditions were mainly considered during screening: 1) No consecutive 5 or more complementary bases are formed within a single primer; 2) No consecutive 4 or more bases at the 3' end of the primer are complementary to its internal sequence; 3) No consecutive 6 or more complementary bases are formed between the forward and reverse primers; the primer Tm value was controlled between 59℃ and 65℃; 4) The primer length was not less than 19 bp, and the amplified fragment length was preferably not more than 150 bp.

[0030] In addition, to ensure the accuracy of genotyping and eliminate the risk of non-specific amplification, candidate primer sequences were submitted to the Ensembl Plants database for whole-genome alignment to screen primer combinations that can specifically identify and amplify target sites.

[0031] Finally, after determining the candidate primer combinations, FAM and HEX fluorescent tag sequences were ligated to the 5' ends of the two allele-specific forward primers to form KASP competitive primers; the shared reverse primer was not ligated with a fluorescent tag. The resulting KASP primer set includes three sequences: a FAM-tagged forward competitive primer, a HEX-tagged forward competitive primer, and a shared reverse primer. Among them: The FAM tag sequence is 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO.1); The HEX tag sequence is 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO.2).

[0032] The final KASP primer sequences are shown in Table 2. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0033] Table 2 KASP primer design

[0034] (2) Validation of KASP marker genotyping effect: To evaluate the effectiveness of the developed KASP primer pair Qfhb_3B.3To assess the detection accuracy and genotyping stability of site-specific SNPs, 50 randomly selected wheat samples were used for KASP genotyping verification.

[0035] Specifically, refer to Stein et al. (2001) (Stein N, Herren G, and Keller B (2001) A new DNA extraction method for high-throughput marker analysis in a large-genomespecies such as Triticum aestivum The method disclosed in Plant Breed 120:354-356 uses the CTAB method to extract genomic DNA from the test materials, and the DNA concentration is uniformly adjusted to about 120 ng / μL with sterile ultrapure water.

[0036] Subsequently, fluorescent PCR amplification was performed using the KASP primer set listed in Table 2. The total reaction volume was 10 μL, including 2.5 μL of sample DNA (120 ng / μL), 5 μL of 2×KASP Master Mix, 0.14 μL of KASP Assay Mix, and 2.36 μL of ddH2O. The KASP Assay Mix was prepared as follows for every 100 μL: 12 μL each of two 100 μM forward competing primers (SEQ ID NO. 3 and SEQ ID NO. 4), 30 μL of a 100 μM reverse universal primer (SEQ ID NO. 5), and 46 μL of ddH2O.

[0037] The fluorescent PCR amplification program included: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, annealing / extension at 61℃~55℃ for 1 min, for a total of 10 touch-down cycles, with a decrease of 0.6℃ per cycle; followed by 94℃ denaturation for 20 s, annealing / extension at 55℃ for 1 min, for 28 cycles; and incubation at 30℃ after amplification.

[0038] Finally, fluorescence signals were acquired and genotype clustering analysis was performed using an ABI ViiA 7 Real-Time PCR System (Thermo Scientific, USA). In the genotyping results, samples clustered near the Y-axis and showing blue FAM fluorescence signals were identified as AA genotype; samples clustered near the X-axis and showing red HEX fluorescence signals were identified as GG genotype; and black signals clustered near the origin represented a blank control without DNA.

[0039] The results are as follows Figure 2As shown in Figure a, the developed KASP marker amplification effect is good, with clear clustering and typing, and can effectively distinguish between different clusters. Qfhb_3B.3 The different allelic types at the locus, and the complete consistency between the genotyping results and the gene chip detection results, indicate that this KASP marker has good detection accuracy and genotyping stability. The applicant named this KASP primer set GWAS- fhb3B _799734063, the primer set includes Fhb3B _F1 (SEQ ID NO.3) Fhb3B _F2 (SEQ ID NO.4) and Fhb3B The three primer sequences _Com (SEQ ID NO.5) can be used for wheat scab resistance-related loci. Qfhb_3B.3 Molecular detection, screening for superior allelic variations, and molecular marker-assisted breeding.

[0040] Example 3: KASP molecular marker primer combination GWAS- fhb3B Application of _799734063 To further validate the KASP primer set GWAS- fhb3B The genotyping and application effects of _799734063 in wheat breeding materials were investigated in this embodiment. 286 wheat materials were randomly selected from high-generation ratio and identification materials in our unit's breeding experiments for joint verification of genotype and disease resistance phenotype. These materials were derived from wheat varieties from the Yangtze River mid-lower reaches wheat region as parents and were planted at the Yangzhou University Yangzijiang Campus experimental base in 2025-2026, with 40 seeds sown in a single row at a row length of 1.5 m. Field management methods and Fusarium head blight inoculation and identification methods were the same as described in Example 1.

[0041] Genomic DNA was extracted from each material according to the method described in Example 2, and GWAS- was performed using KASP primer set. fhb3B _799734063 Qfhb_3B.3 Fluorescent PCR amplification and genotype detection were performed at the loci. DNA extraction, KASP reaction system, amplification procedure, and genotype determination method were all as described in Example 2. Fluorescent signal detection and genotype clustering results are as follows: Figure 2 As shown in b.

[0042] The results showed that the KASP primer set had good amplification effect, clear genotyping and clustering, and could effectively distinguish different breeding materials in... Qfhb_3B.3 The allelic variants carried by the locus indicate that the marker can stably amplify and accurately genotype breeding materials. Specifically, samples near the Y-axis and exhibiting blue FAM fluorescence signals in the genotyping clustering diagram were identified as AA genotypes, while samples near the X-axis and exhibiting red HEX fluorescence signals were identified as GG genotypes. The genotyping results are shown in Table 3.

[0043] Table 3. High-generation wheat breeding lines Qfhb_3B.3 Allelic variation and mean diseased spikelet rate

[0044] The resistance of wheat materials of different genotypes was identified by artificial inoculation with Fusarium head blight, and the average diseased spikelet percentage was used as the evaluation index for Fusarium head blight resistance. The higher the average diseased spikelet percentage, the more severe the disease susceptibility of the material; the lower the average diseased spikelet percentage, the stronger the Fusarium head blight resistance of the material.

[0045] SPSS 19.0 software was used to statistically analyze the average disease incidence rate of breeding materials with different genotypes. The results showed that ( Figure 3 The average diseased spikelet rate of wheat materials carrying the AA genotype was 38.18%, while that of wheat materials carrying the GG genotype was 27.19%. Compared with the AA genotype, the average diseased spikelet rate of the GG genotype materials decreased by 10.99 percentage points, a decrease of 28.78%, indicating that wheat materials carrying the GG genotype showed relatively stronger resistance to Fusarium head blight, while materials carrying the AA genotype were relatively more susceptible to the disease (Table 4).

[0046] Table 4. Average disease spikelet rate of high-generation wheat breeding materials of different genotypes

[0047] Note: ** indicates the significance level. P <0.01.

[0048] Independent samples t-tests were further used to analyze the significance of the difference in average diseased spikelet rate between the AA and GG genotypes. The results showed that the average diseased spikelet rate of the GG genotype was significantly lower than that of the AA genotype, with a highly significant difference (t=4.44, P=2.42×10⁻⁶). -5 ),show Qfhb_3B.3 Different allelic variations at different loci are significantly associated with wheat scab resistance. Among them, the GG genotype is associated with strong scab resistance and can be used as a dominant disease-resistant genotype for molecular marker-assisted breeding of wheat scab resistance and improvement of superior traits.

[0049] In summary, this invention, based on wheat germplasm resources from the middle and lower reaches of the Yangtze River, combined with Fusarium head blight resistance phenotypic identification and genome-wide association analysis, identified QTL loci significantly associated with wheat Fusarium head blight resistance. Qfhb_3B.3 They also developed the KASP primer set GWAS- which is closely associated with this site. fhb3B_799734063. This primer set enables rapid and accurate typing of target allelic variations, and has the advantages of simple operation, stable typing, intuitive results, and suitability for high-throughput detection. It can be used for efficient laboratory screening of superior disease-resistant genotypes in large-scale genetic populations and breeding materials, providing technical support for early selection of wheat scab resistance, evaluation of resistance potential, and molecular marker-assisted breeding.

[0050] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0051] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0052] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for detecting loci significantly associated with wheat scab resistance. Qfhb_3B.3 The KASP primer set is characterized by, The Qfhb_3B.3 located on wheat chromosome 3B, with a physical interval of 796,734,063-802,734,063 bp; the KASP primer set comprises a first competitive forward primer with a nucleotide sequence as shown in SEQ ID NO. 3, a second competitive forward primer with a nucleotide sequence as shown in SEQ ID NO. 4, and a universal reverse primer with a nucleotide sequence as shown in SEQ ID NO.

5.

2. A method for detecting loci significantly associated with wheat scab resistance. Qfhb_3B.3 The reagent kit is characterized by, Includes the KASP primer set as described in claim 1.

3. The use of the KASP primer set of claim 1 or the kit of claim 2 in any of the following: 1) Positioning Qfhb_3B.3 Related genes; 2) Testing wheat resistance to Fusarium head blight; 3) Develop wheat varieties with high resistance to Fusarium head blight.

4. A method for detecting wheat resistance to Fusarium head blight, characterized in that, Includes the following steps: (1) Genomic DNA was extracted from the wheat material to be tested as an amplification template; (2) Fluorescent PCR amplification reaction was performed using the KASP primer set described in claim 1; (3) Fluorescence signal acquisition and cluster analysis were performed on the amplification products, and the wheat material to be tested was determined based on the typing results. Qfhb_3B.3 Genotype of loci: If a sample clusters near the Y-axis in the genotyping clustering diagram and shows a blue FAM fluorescence signal, it is identified as the AA genotype; if a sample clusters near the X-axis in the genotyping clustering diagram and shows a red HEX fluorescence signal, it is identified as the GG genotype; among them, wheat materials carrying the GG allele showed significantly higher resistance to Fusarium head blight than wheat materials carrying the AA allele.

5. The detection method according to claim 4, characterized in that, The total volume of the fluorescent PCR amplification reaction system was 10 μL, including: 2.5 μL of the test sample DNA at a concentration of 120 ng / μL, 5 μL of 2×KASP Master Mix, 0.14 μL of KASP Assay Mix, and 2.36 μL of ddH2O; The preparation method for each 100 μL KASP Assay Mix is ​​as follows: 12 μL each of two 100 μM forward competitive primers, 30 μL of 100 μM reverse universal primers, and 46 μL of ddH2O.

6. The detection method according to claim 4, characterized in that, The fluorescent PCR amplification program included: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, annealing / extension at 61℃~55℃ for 1 min, for a total of 10 touch-down cycles, with a decrease of 0.6℃ per cycle; followed by 94℃ denaturation for 20 s, annealing / extension at 55℃ for 1 min, for 28 cycles; and incubation at 30℃ after amplification.

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