Multiple KASP primer group for simultaneously detecting Fhb7 gene and Pm21 gene of wheat and application of multiple KASP primer group

Abstract

The invention discloses a multiple KASP primer group capable of simultaneously detecting a wheat gibberellic disease-resistant gene Fhb7 and a wheat powdery mildew-resistant gene Pm21 and application of the multiple KASP primer group. The primer group is Multi-KASP AssayFhb7 and Pm21, and the primer group comprises primers of which the nucleotide sequences are as shown in SEQ ID NO. 1 to SEQ ID NO. 4. The invention further discloses a kit for detecting the content of the Pm21 in the KASP AssayFhb7 in the KASP AssayFhb7. According to the primer group system, efficient joint detection of two important disease-resistant genes, namely, the wheat scab-resistant gene Fhb7 and the wheat powdery mildew-resistant gene Pm21, in single-tube and single-time reaction is realized for the first time, and the primer group system has the advantages of simplicity and convenience in operation, high flux, strong specificity, visual result and the like. The primer group is suitable for rapid and accurate identification of excellent genotypes in a large-scale genetic group in a wheat disease-resistant molecule pyramiding breeding process, improves the synergistic improvement efficiency of wheat scab and powdery mildew, can also assist in precise and efficient integration of the excellent disease-resistant genotypes and other excellent characters or target genes, shortens the breeding period, and has a good application prospect in the field of wheat disease-resistant molecule pyramiding breeding. The breeding of a new wheat variety with strong resistance and excellent comprehensive characters is accelerated.

Description

Technical Field

[0001] The present invention relates to the field of wheat genetic breeding, and specifically, to a multiplex KASP primer set capable of simultaneously detecting wheat genes resistant to Fusarium head blight Fhb7 and genes resistant to powdery mildew Pm21 and its application in disease-resistant pyramiding breeding. Background Art

[0002] Wheat ( Triticum aestivum L.) is one of the most important food crops globally, providing approximately 20% of the daily calorie and protein requirements for humans. Ensuring the safe production of wheat is crucial for maintaining national food security.

[0003] However, with the intensification of climate change and the continuous evolution of pathogenic bacteria, the threat of diseases faced by wheat is becoming increasingly severe, seriously affecting its yield and quality. Among them, Fusarium head blight caused by Fusarium graminearum Fusarium graminearum Blumeria graminis f. sp. triticiPowdery mildew caused by it is one of the two typical and devastating fungal diseases globally. Taking the wheat-growing areas in the middle and lower reaches of the Yangtze River in China as an example, the high-temperature and high-humidity climate conditions provide a favorable environment for the outbreak of these two diseases. Coupled with the complex population structure and rich variation of the pathogen, the prevention and control are extremely difficult (Yang M, Smit S, de Ridder D, Feng J, Liu T, Xu J, van der Lee TAJ, Zhang H, Chen W. Adaptation of Fusarium head blight pathogens to changes in agricultural practices and human migration. Adv Sci. 2024. 11(36): 2401899; Rana V, Batheja A, Sharma R, Rana A, Priyanka. Powdery mildew of wheat: research progress, opportunities, and challenges. In: Kashyap PL, Gupta V, Gupta OP, Sendhil R, Gopalareddy K, Jasrotia P, Singh GP, editors. New horizons in wheat and barley research: crop protection and resource management. Springer: Singapore; 2022. p. 133-178).

[0004] Although chemical control can effectively inhibit the spread of diseases in the short term, the resulting environmental pollution and high costs cannot be ignored. Currently, breeding and promoting disease-resistant varieties has become the core strategy for promoting the development of green agriculture and achieving agricultural sustainability. The realization of this goal largely depends on the effective discovery and rational utilization of excellent disease-resistant genes. However, in the actual breeding process, successfully applying disease-resistant genes to variety improvement still faces many challenges, mainly manifested as: (1) The resistance effects of widely used disease-resistant genes are limited. For example, Fhb1 As the first major gene cloned and widely used in Fusarium head blight resistance breeding, however, its resistance performance has a significant genetic background dependence. Some materials carrying Fhb1 failed to show the expected resistance and even showed susceptibility to Fusarium head blight, indicating that its resistance mechanism may be complexly regulated by modifying factors or environmental conditions. Similarly, although there are already such as Pm2a 、Pm4a Multiple powdery mildew resistance genes, etc., have been identified and applied, but most of them are race-specific and are easily overcome by the variation of pathogen populations, limiting their long-term and large-scale application. (2) Materials carrying a single disease resistance gene have a narrow resistance spectrum and are difficult to cope with the combined stress of multiple diseases. In actual production, wheat often faces the common threat of multiple diseases. Especially in areas with complex pathogen population structures and high genetic diversity, materials carrying only a certain disease resistance gene often cannot provide effective protection against other diseases. For example, some varieties carrying only the Fusarium head blight resistance gene show certain resistance to Fusarium head blight but still lack effective defense against powdery mildew, resulting in insufficient overall disease resistance.

[0005] Therefore, the current breeding work urgently needs to integrate excellent disease resistance genes with clear functions, broad-spectrum and stable resistance to breed new wheat varieties with multi-disease complex resistance, so as to effectively improve their comprehensive defense ability in complex disease environments.

[0006] Fhb7 and Pm21 genes, as important disease resistance resources derived from wild relatives of wheat, have become the focus of current molecular breeding research on disease resistance due to their excellent resistance performance. Among them, Fhb7 derived from Thinopyrum elongatum, its product is glutathione S-transferase (GST), which can open the epoxy structure of the Fusarium head blight toxin DON and catalyze its formation of a complex with glutathione (GSH) (DON-GSH), thereby achieving the detoxification and clearance of the toxin (Wang H, Sun S, Ge W, Zhao L, Hou B, Wang K, Lyu Z, Chen L, Xu S, Guo J, et al. Horizontal gene transfer of Fhb7 from fungus underlies Fusarium head blight resistance in wheat. Science. 2020, 368(6493):eaba5435); Pm21 genes are derived from Haynaldia villosa and belong to typical CC-NBS-LRR class disease resistance genes, which can provide broad-spectrum and durable resistance to a variety of wheat powdery mildew strains (He H, Zhu S, Zhao R, Jiang Z, Ji Y, Ji J, Qiu D, Li H, Bie T. Pm21, encoding a typical CC-NBS-LRR protein, confers broad-spectrum resistance to wheat powdery mildew disease. Mol Plant. 2018, 11(6):879-882). Currently, in the Yangtze River Basin and the southwestern wheat regions of China, more than 20 wheat varieties resistant to powdery mildew carrying Pm21 genes have been successfully bred. Compared with varieties carrying other Pm resistance genes, these varieties show better performance in terms of resistance breadth and stability (Bie T, Zhao R, Zhu S, Chen S, Cen B, Zhang B, Gao D, Jiang Z, Chen T, Wang L, et al. Development and characterization of marker MBH1 simultaneously tagging genes Pm21 and PmV conferring resistance to powdery mildew in wheat. Mol Breed. 2015, 35:1-8).

[0007] Therefore, pyramiding these two key genes with clear functions and broad-spectrum resistance, Fhb7 and Pm21 is of great practical significance for breeding excellent wheat germplasms with the ability to resist both Fusarium head blight and powdery mildew. Given that wheat is a hexaploid crop with a complex genome, multi-gene pyramiding breeding faces many challenges in practice. In this process, efficient and precise marker-assisted selection (MAS) technology has become a key tool for improving breeding efficiency and achieving targeted pyramiding of target genes. However, the effective implementation of MAS highly depends on the practicality and accuracy of the molecular markers themselves. Currently, although a variety of molecular markers for Fhb7 and Pm21 have been developed, most of them are still traditional gel electrophoresis-based markers (such as SSR, RAPD, SCAR, etc.), which generally have limitations such as low specificity, poor resolution, and cumbersome operation. With Fhb7 and Pm21Based on the successful cloning, SNP-based KASP markers have been gradually applied to the detection and selection of these two disease-resistant genes, showing certain advantages in simplifying the detection process and controlling costs. However, conventional KASP markers generally only support the detection of a single gene, making it difficult to achieve parallel identification and efficient detection of multiple loci and multiple genes, and it is difficult to meet the application requirements of parallel, efficient selection and molecular aggregation of multiple target genes in large-scale breeding practices.

[0008] Therefore, it is urgent to construct a new multi-gene detection system that can simultaneously detect Fhb7 and Pm21 and has the characteristics of high throughput, low cost, high specificity and good stability, so as to break through the technical bottlenecks of existing methods and markers in multi-gene identification and selection, further improve the breeding utilization efficiency of key disease-resistant genes, and thus more powerfully support the molecular improvement of wheat disease resistance and the precise cultivation of new multi-resistant wheat germplasms. SUMMARY OF THE INVENTION

[0009] Aiming at the problem that conventional KASP markers only support the detection of a single gene and it is difficult to achieve Fhb7 and Pm21 parallel identification and efficient detection, the present invention provides a multiplex KASP (Multi-KASP) primer set - Multi-KASP Assay Fhb7 for simultaneously detecting the Fusarium head blight resistance gene Pm21 and powdery mildew resistance gene Fhb7 and Pm21 in wheat, and its application. This primer set can achieve simple, accurate, high-throughput detection and assisted selection of double-resistant excellent genotype materials in a single tube and single reaction system, and has important practical value in wheat disease-resistant molecular breeding.

[0010] Specifically, the technical solutions provided by the present invention are as follows: First of all, the present invention provides a Multi-KASP primer set Multi-KASP Assay Fhb7 and Pm21 that can simultaneously detect important resistance genes of Fusarium head blight and powdery mildew in wheat Fhb7 and Pm21 Multi-KASP Assay Fhb7 and Pm21 consists of 4 primers, including: the Fhb7 gene forward specific marker primer Multi-KASP Assay Fhb7 and Pm21 -F1 with the nucleotide sequence shown in SEQ ID NO.1, the Fhb7 gene reverse specific primer Multi-KASP Assay Fhb7 and Pm21 -R1 with the nucleotide sequence shown in SEQ ID NO.2, and the Pm21Forward specific marker primer for gene Multi-KASP Assay Fhb7 and Pm21 -F2, whose nucleotide sequence is as shown in SEQ ID NO.4 Pm21 Reverse specific primer for gene Multi-KASP Assay Fhb7 and Pm21 -R2

[0011] The above forward specific marker primer for gene Multi-KASP Assay Fhb7 and Pm21 -F1 and Multi-KASP Assay Fhb7 and Pm21 -F2 are respectively connected with different fluorescent probe tags at their 5' ends. Among them, Multi-KASP Assay Fhb7 and Pm21 -F1 is connected with the HEX fluorescent probe tag as shown in SEQ ID NO.5 at its 5' end; Multi-KASP Assay Fhb7 and Pm21 -F2 is connected with the FAM fluorescent probe tag as shown in SEQ ID NO.6 at its 5' end.

[0012] This Multi-KASP primer set provides a molecular marker tool with simple operation, high throughput, accurate and intuitive results for the integrated and efficient detection of wheat scab resistance gene Fhb7 and powdery mildew resistance gene Pm21 . It is applicable to the efficient identification of excellent disease-resistant genotypes in large-scale genetic populations and breeding materials during disease-resistant breeding, meets the requirements of early screening of target disease-resistant traits and evaluation of resistance potential, and significantly improves the collaborative improvement efficiency of wheat multiple resistances.

[0013] Secondly, the present invention also provides the application of the above Multi-KASP primer set in wheat disease-resistant pyramiding breeding, and the application includes any one or more of the following: (1) The above Multi-KASP primer set is used for the synchronous joint detection of scab resistance gene Fhb7 and powdery mildew resistance gene Pm21 in wheat; (2) The above Multi-KASP primer set is used for breeding and creating excellent wheat new germplasms or varieties with both scab and powdery mildew resistances; (3) The above Multi-KASP primer set is used for promoting the pyramiding breeding of double-gene polymerization materials simultaneously carrying Fhb7 and Pm21 with other excellent traits or functional genes / loci.

[0014] Specifically, the detection method includes the following steps: (1) Using the genomic DNA of the wheat to be tested as a template, perform fluorescence quantitative PCR amplification with the above Multi-KASP primer set to obtain amplification products; (2) Use a fluorescence quantitative PCR instrument to scan the fluorescence signals of the amplification products, and perform genotyping and clustering on the wheat materials to be tested according to the fluorescence signals: If the fluorescence signal of the amplification product appears red in the obtained genotyping and clustering map (the fluorescence signal values are aggregated close to the X-axis), it is considered that the wheat material to be tested contains the Fusarium head blight resistance gene Fhb7 and does not contain the powdery mildew resistance gene Pm21 , denoted as Fhb7 + Pm21 - genotype; if the fluorescence signal of the amplification product appears blue in the obtained genotyping and clustering map (the fluorescence signal values are aggregated close to the Y-axis), it is considered that the wheat material to be tested does not contain the Fusarium head blight resistance gene Fhb7 and contains the powdery mildew resistance gene Pm21 , denoted as Fhb7 - Pm21 + genotype; if the fluorescence signal of the amplification product appears green in the obtained genotyping and clustering map (the fluorescence signal values are aggregated on the diagonal, that is, close to the middle of the X-axis and the Y-axis), it is considered that the wheat material to be tested contains both the Fusarium head blight resistance gene Fhb7 and the powdery mildew resistance gene Pm21 , denoted as Fhb7 + Pm21 + genotype; if the fluorescence signal of the amplification product appears black in the obtained genotyping and clustering map (the fluorescence signal values are aggregated in the region close to the origin, that is, clustered with the negative control (NTC)), it is considered that the wheat material to be tested contains neither the Fusarium head blight resistance gene Fhb7 nor the powdery mildew resistance gene Pm21 , denoted as Fhb7 - Pm21 - genotype.

[0015] Fhb7 + Pm21 + genotype and Fhb7 + Pm21 - The tested wheat materials with Fhb7 - Pm21 + genotype and Fhb7 -Pm21 - Genotyped wheat materials for testing. Fhb7 + Pm21 + Genotype and Fhb7 - Pm21 + The genotyped wheat materials for testing are superior in powdery mildew resistance to Fhb7 + Pm21 - Genotype and Fhb7 - Pm21 - the genotyped wheat materials; Fhb7 + Pm21 + The genotype is an excellent double-gene pyramiding material that simultaneously carries the Fusarium head blight resistance gene Fhb7 and the powdery mildew resistance gene Pm21 and exhibits synergistic resistance to Fusarium head blight and powdery mildew.

[0016] Specifically, in step (1), the amplification product is obtained by the following method: The total volume of the PCR amplification reaction system is 10 μL, and its specific components include: 2 μL of genomic DNA with a concentration of 250 ng / μL, 5 μL of 2× HiGeno Probe Mix A (JasonGen, Beijing, China), 0.15 μL of Multi-KASP Assay Mix, and 2.85 μL of ddH2O; Among them, the preparation method of every 100 μL of Multi-KASP Assay Mix is: 12 μL of the forward specific marker primer Multi-KASP Assay Fhb7 -F1 of the gene with a concentration of 100 μM, 15 μL of the reverse specific primer Multi-KASP Assay Fhb7 and Pm21 -R1 of the gene with a concentration of 100 μM, 12 μL of the forward specific marker primer Multi-KASP Assay Fhb7 -F2 of the gene with a concentration of 100 μM, 15 μL of the reverse specific primer Multi-KASP Assay Fhb7 and Pm21 -R2 of the gene with a concentration of 100 μM, and 46 μL of ddH2O; Pm21 -F2 of the gene with a concentration of 100 μM, 15 μL of the reverse specific primer Multi-KASP Assay Fhb7 and Pm21 -F2 of the gene with a concentration of 100 μM, 15 μL of the reverse specific primer Multi-KASP Assay Pm21 -R2 of the gene with a concentration of 100 μM, and 46 μL of ddH2O; Fhb7 and Pm21 -R2 of the gene with a concentration of 100 μM, and 46 μL of ddH2O; The PCR amplification reaction procedure was as follows: In the first step, heat activation was performed at 94°C for 15 min; in the second step, denaturation was carried out at 94°C for 20 s, annealing was performed at 61 - 55°C for 60 s, and 10 touch-down cycles were carried out, with a decrease of 0.6°C in each cycle; in the third step, denaturation was carried out at 94°C for 20 s, renaturation was performed at 55°C for 1 min, and 30 cycles were carried out; finally, the amplified product was incubated at 30°C.

[0017] Through the above technical solutions, the present invention achieved the following beneficial effects: 1. The Multi-KASP primer set provided by the present invention realized for the first time the single-tube and single-system high-efficiency combined detection of wheat scab resistance genes Fhb7 and powdery mildew resistance genes Pm21 , having the advantages of simple operation, high throughput, intuitive results, and strong specificity, and meeting the actual needs of high-efficiency detection of target disease-resistant genotypes and resistance-assisted prediction in large-scale genetic generations and breeding materials in wheat disease-resistant breeding.

[0018] 2. Using the Multi-KASP primer set provided by the present invention can provide strong technical support for the disease-resistant gene pyramiding breeding strategy, solve the problems of timeliness and accuracy in the coordinated improvement of wheat scab and powdery mildew resistance; it can also assist in the efficient integration of Fhb7 and Pm21 excellent disease-resistant genotypes with other excellent traits or genes / loci, shorten the breeding cycle, and accelerate the breeding of new wheat varieties with outstanding resistance and excellent comprehensive traits.

[0019] 3. The present invention expands the types and application methods of molecular markers in wheat disease-resistant breeding, provides an efficient and practical technical means for the breeding of multi-resistant and excellent wheat new varieties, and has important breeding utilization value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For Fhb7 and Pm21 the design of gene-specific primers and the genomic regions they anchor; wherein, (A) is Fhb7 a partial sequence of the open reading frame (ORF), (B) is Pm21 a partial sequence of the third exon; the numbers represent the nucleotide positions relative to Fhb7 the start site of the ORF and Pm21 the start site of the third exon; single underlines and double underlines respectively represent the anchoring positions of the forward primer and the reverse primer; Figure 2 is the genotyping effect of the Multi-KASP Assay Fhb7 and Pm21 primer set provided by the present invention and the genotype detection result diagram of the verification materials; Figure 3The Multi-KASP Assay provided by the present invention Fhb7 and Pm21 Application and verification of the primer set in MAS breeding for wheat resistance to Fusarium head blight and powdery mildew; wherein, (A) is a schematic diagram of the genotyping results of the primer set in the BC2F2 population; (B) and (C) are respectively MBH1 and GST (26102F / R) Markers were used for gel electrophoresis verification of four genotype samples identified by Multi-KASP; a1–a4 are schematic diagrams of the amplification results using MBH1 ; b1–b4 are schematic diagrams of the amplification results using GST (26102F / R) ; a1 / b1, a2 / b2, a3 / b3, and a4 / b4 respectively represent the amplification results of BC2F2 materials carrying only Pm21 , only carrying Fhb7 , carrying both Pm21 and Fhb7 , and BC2F2 materials without either gene; M is the DNA molecular weight standard of DL 1000; Figure 4 are representative results of the resistance of different genotype materials identified by the Multi-KASP Assay Fhb7 and Pm21 primer set to powdery mildew and Fusarium head blight; "+" indicates the presence of the gene, and "–" indicates the absence of the gene. Specific embodiments

[0021] The following examples are used to illustrate the specific embodiments of the present invention in detail. It should be understood that the specific embodiments described herein are only for the purpose of illustration and explanation of the present invention, and are not intended to limit the present invention.

[0022] The reagents, equipment, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0023] The wheat materials used in the present invention are all germplasm resource materials preserved in the Jiangsu Provincial Crop Germplasm Resource Bank (crops, Yangzhou University Bank), and can be obtained and used by those skilled in the art and researchers.

[0024] Example 1 Multi-KASP Assay Fhb7 and Pm21 Design and verification of genotyping results of the primer set (1) Primer set design: Extract the cloned Pm21 gene sequence (GenBank: MF370199.1) (He H, ZhuS, Zhao R, Jiang Z, Ji Y, Ji J, Qiu D, Li H, Bie T. Pm21, encoding a typical CC-NBS-LRR protein, confers broad-spectrum resistance to wheat powdery mildew disease. Mol Plant. 2018;11(6):879-882) and Fhb7 The open reading frame (ORF) sequence of the gene (Wang H, Sun S, Ge W, Zhao L, Hou B, Wang K, Lyu Z, Chen L, Xu S, Guo J, et al. Horizontal gene transfer of Fhb7 from fungus underlies Fusarium head blight resistance in wheat. Science. 2020, 368(6493):eaba5435), using the "Chinese Spring" wheat reference genome (IWGSC Reference Sequence v1.0) as a control, was aligned with the wheat A, B, and D subgenomes respectively to screen out conserved regions or specific SNPs with significant differences and no homologous sequence interference in these three genomes as the anchoring regions of the Multi-KASP primer set.

[0025] The Multi-KASP primer set includes two sets of specifically designed forward and reverse primers, each targeting the target gene Fhb7 and Pm21 to ensure the accurate identification and efficient amplification of the two genes in the same reaction system. Therefore, to ensure the amplification efficiency, sensitivity, and resolution of the primers, the quality of the designed primers was evaluated using DNAMAN. At the same time, the primers must be screened again manually for melting temperature matching analysis and dimer structure checking.

[0026] Finally, the 130-150th and 190-207th base intervals of the ORF of the Fhb7 gene were selected as the anchoring regions of the forward and reverse specific primers respectively; the 1522-1543rd and 1561-1580th base intervals of the third exon of the Fhb7 gene were selected as the anchoring regions of the forward and reverse specific primers respectively ( Pm21 ). Finally, different fluorescent probe tags were introduced at the 5' end of the two forward specific primers, among which the Pm21 primer was incorporated with the HEX fluorescent probe tag, Figure 1 Fhb7 The Pm21The forward primer is incorporated with a FAM fluorescent probe label, and the reverse primer is unmodified to simplify the primer system and maintain the amplification efficiency. The applicant self-named this Multi-KASP primer set as Multi-KASP Assay Fhb7 and Pm21 , including 4 primers: 1) The forward specific marker primer Multi-KASPAssay Fhb7 for the gene with the nucleotide sequence shown in SEQ ID NO.1 Fhb7 and Pm21 -F1; 2) The reverse specific primer Multi-KASPAssay Fhb7 for the gene with the nucleotide sequence shown in SEQ ID NO.2 Fhb7 and Pm21 -R1; 3) The forward specific marker primer Multi-KASPAssay Pm21 for the gene with the nucleotide sequence shown in SEQ ID NO.3 Fhb7 and Pm21 -F2; 4) The reverse specific primer Multi-KASPAssay Pm21 for the gene with the nucleotide sequence shown in SEQ ID NO.4 Fhb7 and Pm21 -R2.

[0027] Among them, the nucleotide sequences of the HEX and FAM fluorescent probe labels are as follows: HEX label sequence (SEQ ID NO.5): 5’-GAAGGTCGGAGTCAACGGATT-3’; FAM label sequence (SEQ ID NO.6): 5’-GAAGGTGACCAAGTTCATGCT-3’.

[0028] All primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and the specific information is shown in Table 1.

[0029] Table 1 Multi-KASP Assay Fhb7 and Pm21 Sequence information

[0030] Note: The bold sequences represent the fluorescent probe labels HEX (SEQ ID NO.5) and FAM (SEQ IDNO.6) introduced into the sequences.

[0031] (2) Genotype identification and verification: In order to further test the high resolution and accuracy of Multi-KASP Assay Fhb7 and Pm21 in the synchronous detection of two target genes to prove the successful development of the marker, it is necessary to verify its genotyping effect. The specific method is as follows: 1) Selection of experimental materials: Yangnongmai 158 (YNM158), a wheat-Thinopyrum elongatum Th. elongatum ) translocation line, carrying Fhb7 , showing good resistance to Fusarium head blight (Dai Y, Fei W, Chen S, Shi J, Ma H, Li H,Li J, Wang Y, Gao Y, Zhu J, et al. Using transcriptomics to determine themechanism for the resistance to Fusarium head blight of a wheat- Th. elongatum translocation line. Int J Mol Sci. 2024, 25(17):9452); Yangmai 30, a wheat variety with excellent agronomic traits in the middle and lower reaches of the Yangtze River, without Fhb7 , but carrying Pm21 , highly resistant to powdery mildew; Other materials include: plasmid vectors carrying Fhb7 (V- Fhb7 ); plasmid vectors carrying Pm21 (V- Pm21 ); wheat varieties known to carry Pm21 (Yangmai 18, Yangmai 21, Yangmai 29, Yangfumai 20, Zhenmai 9 and Yangmai 30 (YM30)); wheat varieties known not to carry Pm21 and Fhb7 (Yangmai 16, Yangmai 19, Yangmai 23, Yangmai 25 and Yangmai 28); 5 DNA samples equally mixed by D. villosum and Th. Elongatum .

[0032] 2) DNA extraction: Take young leaves, refer to Stein et al. (2001) (Stein N, Herren G, and KellerB (2001) A new DNA extraction method for high-throughput marker analysis in alarge-genome species such as Triticum aestivum . Plant Breed 120:354-356), and extract genomic DNA from them using the CTAB method. Then, uniformly dilute the DNA with sterilized ultrapure water to a concentration of about 250 ng / μL.

[0033] 3) Genotype detection and result verification: Use the Multi-KASP primer set Multi-KASP Assay obtained in Example 1 Fhb7 and Pm21 (Table 1) to perform fluorescence quantitative PCR amplification. The reaction system, amplification program, and genotype typing method are as follows: ① Reaction system: The total volume is 10 μL, including 2 μL of sample DNA (250 ng / μL), 5 μL of 2× HiGenoProbe Mix A (JasonGen, Beijing, China), 0.15 μL of Multi-KASP Assay Mix, and 2.85 μL of ddH2O.

[0034] Among them, the preparation method of Multi-KASP Assay Mix is as follows: Every 100 μL of Multi-KASP Assay Mix contains 12 μL of Multi-KASP Assay Fhb7 and Pm21 -F1 with a concentration of 100 μM, 15 μL of Multi-KASPAssay Fhb7 and Pm21 -R1 with a concentration of 100 μM, 12 μL of Multi-KASP Assay Fhb7 and Pm21 -F2 with a concentration of 100 μM, 15 μL of Multi-KASP Assay Fhb7 and Pm21 -R2 with a concentration of 100 μM, and 46 μL of ddH2O.

[0035] ② Reaction program: First step, heat activation at 94°C for 15 min; Second step, denaturation at 94°C for 20 s, annealing at 61 - 55°C for 60 s, 10 touch-down cycles, with a decrease of 0.6°C per cycle; Third step, denaturation at 94°C for 20 s, renaturation at 55°C for 1 min, 30 cycles; Finally, keep the amplified product at 30°C.

[0036] ③ Fluorescence signal scanning and genotype detection: Use the fluorescence quantitative PCR instrument Applied Biosystems ABIViia7 Real Time PCR System (Thermo Scientific, USA) to scan the fluorescence signal and perform genotype typing clustering. The results are as follows: a. Samples with red fluorescence signals in the obtained typing clustering map (fluorescence signal values aggregated close to the X-axis) represent samples bound to the HEX fluorophore, which contain the scab resistance gene Fhb7 and do not contain the powdery mildew resistance gene Pm21 , and are denoted as Fhb7 + Pm21- Genotype; b. Samples with blue fluorescence signals in the obtained genotyping clustering map (fluorescence signal values aggregated close to the Y-axis) represent samples conjugated with the FAM fluorophore and do not contain the gene for resistance to Fusarium head blight Fhb7 but contain the gene for resistance to powdery mildew Pm21 and are denoted as Fhb7 - Pm21 + Genotype; c. Samples with green fluorescence signals in the obtained genotyping clustering map (fluorescence signal values aggregated on the diagonal, i.e., close to the middle of the X-axis and Y-axis) represent samples conjugated with both HEX and FAM fluorophores and contain both the gene for resistance to Fusarium head blight Fhb7 and the gene for resistance to powdery mildew Pm21 and are denoted as Fhb7 + Pm21 + Genotype; d. Samples with black fluorescence signals in the obtained genotyping clustering map (fluorescence signal values aggregated in the region close to the origin, i.e., clustered with the negative control (NTC)) represent samples not conjugated with HEX and FAM fluorophores and contain neither the gene for resistance to Fusarium head blight Fhb7 nor the gene for resistance to powdery mildew Pm21 and are denoted as Fhb7 - Pm21 - Genotype.

[0037] As Figure 2 shown by the genotyping results in Th. Elongatum : The red samples aggregated close to the X-axis included Fhb7 V- Fhb7 + Pm21 - and Yangnongmai 158 and were D. villosum V- Pm21 and wheat varieties Yangmai 18, Yangmai 21, Yangmai 29, Yangfumai 20, Zhenmai 9, and Yangmai 30, and were Fhb7 - Pm21 + The green samples aggregated on the diagonal, i.e., close to the middle of the X-axis and Y-axis, were all from the five independent pooled DNA samples of D. villosum and Th. elongatum and were Fhb7 + Pm21+ Genotype; Samples aggregated in the region near the origin, that is, samples clustered with the negative control (NTC) (marked with black crosses) included wheat varieties Yangmai 16, Yangmai 19, Yangmai 23, Yangmai 25, and Yangmai 28 that did not carry Pm21 and Fhb7 and were Fhb7 - Pm21 - genotypes.

[0038] The above four genotypes showed good separation effects and clear clustering characteristics in the genotyping clustering map, and the results were completely consistent with the known genotypes, indicating the accuracy and reliability of the Multi-KASP primer set Multi-KASP Assay provided by the present invention Fhb7 and Pm21 in the simultaneous detection of different disease-resistant genotypes.

[0039] Example 2 Multi-KASP Assay Fhb7 and Pm21 Application of the primer set in the pyramiding breeding of wheat scab and powdery mildew resistance genes (1) Test materials: Using the test material Yangmai 30 described in Example 1 as the female parent and recurrent parent, and Yangnongmai 158 as the male parent, a BC2F2 population containing 615 lines was constructed through two rounds of backcrossing and self-crossing for genotyping verification of the Multi-KASPAssay Fhb7 and Pm21 primer set in the population materials, as well as high-throughput screening and identification of breeding materials simultaneously integrating two resistance genes in MAS. The DNA preparation method of the test materials was the same as that in Example 1.

[0040] (2) Genotype identification of the Multi-KASP Assay Fhb7 and Pm21 primer set in the breeding population Using the Multi-KASP primer set obtained in Example 1, Multi-KASP Assay Fhb7 and Pm21 (Table 1) to detect the genotypes of the above BC2F2 population. The fluorescence quantitative PCR amplification reaction system, amplification program, and genotype typing method were the same as those in Example 1.

[0041] The results showed that each genotype showed good differentiation and clear clustering distribution in the genotyping clustering map ( Figure 3 A). Among them, Fhb7 + Pm21 + genotype materials were aggregated in the middle and were double-gene heterozygous; those carrying Fhb7 or Pm21Homozygous samples form clusters along the X-axis and Y-axis directions and are consistent with the clustering patterns of their original donors ( Th. elongatum and D. villosum ), as well as the transgenic vectors carrying the corresponding genes; materials without Fhb7 and Pm21 and the negative control (NTC) samples are clustered near the origin, indicating no amplification signal.

[0042] (3) Comparative verification of genotype typing results To further verify the accuracy of the Multi-KASP Assay Fhb7 and Pm21 in genotype typing, 40 representative samples were randomly selected from the BC2F2 population, covering the above 4 typical genotypes ( Fhb7 - Pm21 + , Fhb7 + Pm21 - , Fhb7 + Pm21 + and Fhb7 - Pm21 - , with 10 samples for each genotype), and the typing results were repeatedly verified using two reported and applied molecular markers, among which: MBH1 Marker detection Pm21 gene (Bie T, Zhao R, Zhu S, Chen S, Cen B, Zhang B, Gao D, Jiang Z, Chen T, Wang L, et al. Development and characterization of marker MBH1 simultaneously tagging genes Pm21 and PmV conferring resistance to powdery mildew in wheat. Mol Breed. 2015;35:1-8.), GST (26102F / R) Marker detection Fhb7 gene (Guo X, Wang M, Kang H, Zhou Y, Han F. Distribution, polymorphism and function characteristics of the GST-encoding Fhb7in Triticeae. Plants. 2022;11:2074.). The results showed ( Figure 3 B): MBH1 The marker could amplify a specific fragment of 341 bp (6V#4S-341), which corresponded to the 6V#2S chromosome carrying the Pm21 gene. This fragment could be amplified in the parental Yangmai 30, Pm21 homozygous and Fhb7 / Pm21 heterozygous BC2F2 samples, but not in Yangnongmai 158 and BC2F2 samples without Pm21 ; GST (26102F / R) The primer could amplify a specific fragment of about 1,000 bp, which could be detected in Yangnongmai 158, Fhb7 homozygous and Fhb7 homozygous and Fhb7 / Pm21 heterozygous samples, but not in Yangmai 30 and other samples without Fhb7 . The above results were completely consistent with the genotyping results of the Multi-KASP Assay Fhb7 and Pm21 , further verifying the accuracy and stability of the Multi-KASP Assay Fhb7 and Pm21 primer set provided by the present invention in high-throughput genotype identification.

[0043] (4) Genotype-phenotype comparison and verification To further verify the practical application value of the Multi-KASP Assay Fhb7 and Pm21 genotyping system provided by the present invention in MAS breeding for resistance to Fusarium head blight and powdery mildew, references were made to Troch et al. (2013) (Troch V, Audenaert K, Vanheule A, Bekaert B, Höfte M, Haesaert G. Evaluation of resistance to powdery mildew in triticale seedlings and adult plants. Plant Dis. 2013, 97(3):410-417) and He et al. (2020) (He Y, Wu L, Liu X, Jiang P, Yu L, Qiu J, Wang G, Zhang X, Ma H. TaUGT6, a novel UDP - glycosyltransferase gene enhances the resistance to FHB and DON accumulation in wheat. Front Plant Sci. 2020;11:574775), the evaluation methods for powdery mildew and Fusarium head blight resistance, and further phenotypic identification of Fusarium head blight and powdery mildew resistance was carried out on 40 BC2F2 population materials. The results showed ( Figure 4 ): Materials carrying the Pm21 gene ( Fhb7 + Pm21 + 、 Fhb7 - Pm21 + )showed strong resistance to powdery mildew, and materials carrying the Fhb7 gene ( Fhb7 + Pm21 + 、 Fhb7 + Pm21 - )showed significantly reduced symptoms of Fusarium head blight; Detection of BC2F2 materials carrying both Pm21 and Fhb7 genes ( Fhb7 + Pm21 + )showed significant dual resistance to powdery mildew and Fusarium head blight in terms of resistance performance. The above results verified that the Multi - KASP Assay Fhb7 and Pm21 genotyping system provided by the present invention has good stability and practicability in the high - throughput and accurate identification of disease - resistant genotypes, and has high application value in the molecular pyramiding breeding of wheat disease resistance.

[0044] In summary of the above results, the present invention for the first time provides a novel multiplex primer set - Multi - KASP Assay Fhb7 that can simultaneously detect the important Fusarium head blight - resistant gene Pm21 and powdery mildew - resistant gene Fhb7 and Pm21 in wheat in a single reaction, with the remarkable advantages of high - throughput, low - cost, strong specificity and excellent stability, and has been successfully applied to the molecular pyramiding breeding of wheat disease resistance. This primer set and its application have broken through the technical bottlenecks of existing molecular markers and detection methods in the simultaneous identification of multiple genes and the precise and efficient screening of genotypes, significantly improving the efficiency and accuracy of the coordinated improvement of wheat resistance to Fusarium head blight and powdery mildew, and providing strong technical support for the molecular improvement of disease - resistant traits and the efficient breeding of new multi - resistant wheat germplasms.

Claims

1. A multiplex KASP primer set capable of simultaneously detecting wheat genes resistant to Fusarium head blight Fhb7 and powdery mildew Pm21 , characterized in that The primer set is named Multi-KASP Assay Fhb7 and Pm21 , and includes the forward specific marker primer of the Fhb7 gene Multi-KASP Assay with the nucleotide sequence shown in SEQ ID NO.1 Fhb7 and Pm21 -F1, the reverse specific primer of the Fhb7 gene Multi-KASP Assay with the nucleotide sequence shown in SEQ ID NO.2 Fhb7 and Pm21 -R1, the forward specific marker primer of the Pm21 gene Multi-KASP Assay with the nucleotide sequence shown in SEQ ID NO.3 Fhb7 and Pm21 -F2, the reverse specific primer of the Pm21 gene Multi-KASP Assay with the nucleotide sequence shown in SEQ ID NO.4 Fhb7 and Pm21 -R2.

2. Use of the multiplex KASP primer set according to claim 1 in any of the following: (1)For the synchronous joint detection of the gene resistant to Fusarium head blight Fhb7 and the gene resistant to powdery mildew Pm21 in wheat disease-resistant pyramiding breeding; (2) For breeding and creating excellent new wheat germplasms or varieties with resistance to both Fusarium head blight and powdery mildew; (3) For promoting the pyramiding breeding of double-gene pyramiding materials carrying Fhb7 and Pm21 with other excellent traits or functional genes / loci.

3. The application according to claim 2, characterized in that, Fusarium head blight resistance gene Fhb7 and powdery mildew resistance gene Pm21 The synchronous joint detection method comprises the following steps: (1) Using the genomic DNA of the wheat to be tested as a template, performing fluorescence quantitative PCR amplification with the multiplex KASP primer set to obtain an amplification product; (2) Using a fluorescence quantitative PCR instrument to scan the fluorescence signal of the amplification product, and performing genotyping and clustering on the wheat material to be tested according to the fluorescence signal: If the fluorescence signal of the amplification product appears red in the obtained genotyping clustering map, the wheat material to be tested contains the gene for resistance to Fusarium head blight Fhb7 , but does not contain the gene for resistance to powdery mildew Pm21 , denoted as Fhb7 + Pm21 - genotype; if the fluorescence signal of the amplification product appears blue in the obtained genotyping clustering map, the wheat material to be tested does not contain the gene for resistance to Fusarium head blight Fhb7 , but contains the gene for resistance to powdery mildew Pm21 , denoted as Fhb7 - Pm21 + genotype; if the fluorescence signal of the amplification product appears green in the obtained genotyping clustering map, the wheat material to be tested contains both the gene for resistance to Fusarium head blight Fhb7 and the gene for resistance to powdery mildew Pm21 , denoted as Fhb7 + Pm21 + genotype; if the fluorescence signal of the amplification product appears black in the obtained genotyping clustering map, the wheat material to be tested contains neither the gene for resistance to Fusarium head blight Fhb7 , nor the gene for resistance to powdery mildew Pm21 , denoted as Fhb7 - Pm21 - genotype; Among them, Fhb7 + Pm21 + the genotypes and Fhb7 + Pm21 - the tested wheat materials with these genotypes are superior in resistance to Fusarium head blight to Fhb7 - Pm21 + the genotypes and Fhb7 - Pm21 - the tested wheat materials with these genotypes; Fhb7 + Pm21 + the genotypes and Fhb7 - Pm21 + the tested wheat materials with these genotypes are superior in resistance to powdery mildew to Fhb7 + Pm21 - the genotypes and Fhb7 - Pm21 - the tested wheat materials with these genotypes; Fhb7 + Pm21 + The genotype is an excellent double-gene pyramiding material that simultaneously carries the gene for resistance to Fusarium head blight Fhb7 and the gene for resistance to powdery mildew Pm21 and shows coordinated resistance to Fusarium head blight and powdery mildew.

4. The application according to claim 3, wherein In step (1), the fluorescence quantitative PCR amplification system is 10 μL, including: 2 μL of genomic DNA with a concentration of 250 ng / μL, 5 μL of 2× HiGeno Probe Mix A, 0.15 μL of Multi-KASP Assay Mix, and 2.85 μL of ddH2O; Among them, the preparation method of every 100 μL of Multi-KASP Assay Mix is as follows: the forward specific labeled primer of the Fhb7 gene Multi-KASP Assay Fhb7 and Pm21 -F1 12 μL, the reverse specific primer of the Fhb7 gene Multi-KASP Assay Fhb7 and Pm21 -R1 15 μL, the forward specific labeled primer of the Pm21 gene Multi-KASP Assay Fhb7 and Pm21 -F2 12 μL, the reverse specific primer of the Pm21 gene Multi-KASP Assay Fhb7 and Pm21 -R2 15 μL, ddH2O 46 μL; The PCR amplification reaction procedure is as follows: First step, heat activation at 94°C for 15 min; Second step, denaturation at 94°C for 20 s, annealing at 61 - 55°C for 60 s, 10 touch-down cycles, with a decrease of 0.6°C per cycle; Third step, denaturation at 94°C for 20 s, renaturation at 55°C for 1 min, 30 cycles; Finally, keep the amplification product at 30°C for incubation and scan the fluorescence signal.

5. A multiplex KASP kit for synchronously detecting wheat genes resistant to Fusarium head blight Fhb7 and powdery mildew resistance genes Pm21 , characterized in that Including the multiplex KASP primer set according to claim 1.

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