A molecular marker linked to wheat- agropyron hybrid line anti-powdery mildew gene PmPB74-2 and application thereof

Abstract

The present application relates to the field of agricultural biotechnology, and particularly relates to a wheat-ice grass derivative line resistant to powdery mildew gene PmPB74-2 Molecular markers and their applications. The molecular markers are detected by PCR amplification using the primer pair shown in SEQ ID NO. 1 and SEQ ID NO. 2; Pu Bing 74 amplifies 319 bp allelic bands at the site, Mingxian 169 amplifies 309 bp allelic bands, and hybrid materials simultaneously amplify the two allelic bands. In the genetic population or breeding population constructed by the donor parent PmPB74-2 The molecular markers can be used for PmPB74-2 Genetic mapping, fine mapping and recombinant screening. The molecular marker detection is stable and has good repeatability, can be used for genotype detection at the seedling stage, reduces the workload of disease-resistant phenotype identification, improves the efficiency of molecular marker assisted selection, and has good breeding application value.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a gene for powdery mildew resistance in wheat-ice grass-derived lines. PmPB74-2 Linked molecular markers and their applications. Background Technology

[0002] Wheat powdery mildew is caused by the grass family powdery mildew fungus (Phyllostachys edulis). Blumeria graminis f. sp. tritici Powdery mildew (P. brevicornu) is a fungal disease that seriously threatens wheat production. Breeding and planting resistant varieties is the most economical, effective, and sustainable strategy for controlling this disease. However, with the continuous variation of physiological races of powdery mildew, some reported wheat powdery mildew resistance genes have gradually weakened or even been lost during production and utilization. Therefore, continuously discovering new powdery mildew resistance genes and developing molecular markers linked to them is of great significance for wheat disease resistance breeding.

[0003] Ice grass ( Agropyron cristatum (L.) Gaertn., P genome) is an important wild relative of wheat, containing abundant gene resources related to excellent disease resistance, stress resistance, and agronomic traits, and is an important exogenous gene pool for wheat genetic improvement. Introducing superior disease-resistant genes from *L.) Gaertn. into common wheat through distant hybridization is an important approach to creating new sources of wheat resistance and improving wheat disease resistance.

[0004] Field identification results over many years and in various environments have shown that the wheat-ice grass derivative line Pubing 74 possesses stable powdery mildew resistance throughout its entire growth period. Lu et al. used Pubing 74 to conduct genetic analysis of powdery mildew resistance, locating a dominant powdery mildew resistance gene on the wheat 5DS chromosome. PmPB74 The gene is located at a molecular marker. Xcfd81 and HRM02 The genetic distances between them were 2.5 cM and 1.7 cM, respectively (Lu Y, Yao M, Zhang J, et al. Genetic analysis of a novel broad-spectrum powdery mildew resistance gene from the wheat- Agropyron cristatum Introgression line Pubing 74. Planta, 2016, 244(3): 713-723. DOI:10.1007 / s00425-016-2538-y). The above studies show that Pubing 74 has important value in disease resistance breeding. However, distant hybridization-derived materials usually have a more complex genetic composition, and the same material may contain multiple disease resistance genes. Existing studies mainly focus on the 5DS line. PmPB74The location has been determined, but further research is needed to determine whether other powdery mildew resistance genes exist in Pubing 74.

[0005] Currently, the screening of disease-resistant wheat materials mainly relies on phenotypic identification methods such as natural disease identification in the field and artificial inoculation identification. These methods typically require specific pathogen physiological races and suitable environmental conditions, resulting in long identification cycles, heavy workloads, and low screening efficiency in early generations. Especially in the large-scale identification of wheat-wheat grass-derived progeny, relying solely on phenotypic identification is insufficient for efficiently screening genotypes of linked regions of target disease-resistant genes.

[0006] Marker-assisted selection (MAG) technology enables rapid detection of target gene-linked sites at the DNA level, and can be used for locating target resistance genes, screening recombinants, selecting genetic backgrounds, and screening breeding progeny. Developing molecular markers that are linked to target genes, stable in detection, and easy to operate is beneficial for improving the efficiency of screening resistant materials and shortening the breeding cycle. For other powdery mildew resistance genes in Pubing 74 that require further analysis and utilization, stable molecular markers are currently lacking for continuous tracking, fine mapping, and marker-assisted selection of their linked regions. Therefore, it is necessary to further analyze the genetic basis of powdery mildew resistance in Pubing 74 and develop corresponding linkage molecular markers to provide technical means for fine mapping of resistance genes, screening recombinants, continuous tracking of linked chromosomal fragments, and utilization in powdery mildew resistance breeding. Summary of the Invention

[0007] The purpose of this invention is to provide a wheat-ice grass-derived line with a powdery mildew resistance gene. PmPB74-2 Linked codominant insertion-deletion (InDel) molecular markers Xcaaspb-21 To amplify primer pairs for this molecular marker and their applications, in order to address the lack of suitable primers in existing technologies. PmPB74-2 Problems with stable linked molecular markers in genetic mapping, fine mapping, recombinant screening, and marker-assisted selection.

[0008] The molecular markers are used to detect wheat materials in Xcaaspb-21 The allelic type of a locus can be used to evaluate whether candidate materials and Pubing 74 exhibit polymorphism at that locus, providing a basis for hybrid combination configuration; in the breeding process, it can also be used to continuously track and... PmPB74-2 Linked cytoplasmic fragments derived from Pubing 74 are... PmPB74-2 It provides molecular tools for genetic mapping, fine mapping, recombinant screening, and marker-assisted selection.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a gene for powdery mildew resistance in wheat-ice grass-derived lines. PmPB74-2Linked co-dominant InDel molecular markers Xcaaspb-21 .

[0010] The present invention also provides a method for amplifying the molecular marker. Xcaaspb-21 Primer pairs, wherein the primer pairs include: upstream primer Xcaaspb-21 F, whose nucleotide sequence is shown in SEQ ID NO.1: 5'-TCAGAATCTAACATGCAACACCT-3'; Downstream primer Xcaaspb-21 R, whose nucleotide sequence is shown in SEQ ID NO.2: 5'-AGAGTGTCCTGAGTTCCCA-3'; The primers were used to perform PCR amplification of the wheat genomic DNA to be tested, and Pubing 74 was found to be effective. Xcaaspb-21 Amplification of the site yielded a 319 bp allele band, while amplification of Mingxian 169 yielded a 309 bp allele band. Simultaneous amplification of the hybrid material yielded both 319 bp and 309 bp allele bands. Xcaaspb-21 As a codominant InDel molecular marker, it can be used to distinguish... Xcaaspb-21 The locus was derived from homozygous and heterozygous types of Pubing 74 and homozygous types of Mingxian 169.

[0011] This invention also provides the primer pair for the powdery mildew resistance gene in wheat-ice grass-derived lines. PmPB74-2 Applications in genetic mapping, fine mapping, and recombinant screening.

[0012] This invention also provides the primer pair for detecting wheat materials Xcaaspb-21 Application in locus allelic typing. The markers can be used to evaluate the comparison of candidate materials with Plubing 74. Xcaaspb-21 The presence of polymorphism at a locus provides a basis for configuring hybrid combinations; in the breeding process, it can also be used to track and differentiate between different loci. PmPB74-2 Linked 74-derived chromosome fragments enable continuous tracking of target linked segments.

[0013] The detection method includes the following steps: (1) Extract genomic DNA from the wheat material to be tested; (2) Using the genomic DNA as a template, utilize Xcaaspb-21 Primer pairs were used for PCR amplification; (3) Determine the amplified bands of the test material. Xcaaspb-21 Allelic types of loci: When a 319 bp allelic band is obtained during amplification, the test material is determined to be in... Xcaaspb-21The locus carries the same allele as PUB 74 (i.e., the homozygous type derived from PUB 74). When a 309 bp allelic band is obtained during amplification, the test material is determined to be in... Xcaaspb-21 The locus carries the same allele type as Mingxian 169 (that is, the homozygous type from Mingxian 169). When both 319 bp and 309 bp allelic bands are obtained simultaneously by amplification, it is determined that the test material is in a state of flux. Xcaaspb-21 The site is heterozygous.

[0014] The total volume of the PCR amplification system is 9.5-10.5 μL, comprising: 49-51 ng / μL wheat genomic DNA; 0.95-1.05 μL; PCR Master Mix 4.9-5.1 μL; 0.95-1.05 μL of 9.5-10.5 μM upstream primer; 0.95-1.05 μL of 9.5-10.5 μM downstream primer; 1.9-2.1 μL of sterile deionized water.

[0015] As a preferred embodiment, the present invention provides a PCR amplification system with a total volume of 10 μL, comprising: 1 μL of 50 ng / μL wheat genomic DNA, 5 μL of PCR Master Mix, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, and 2 μL of sterile deionized water.

[0016] The PCR amplification procedure is as follows: Pre-denaturation at 94-96 ℃ for 4.5-5.5 min; Denaturation at 94-96 ℃ for 28-32 s, annealing at 57-59 ℃ for 28-32 s, and extension at 71-73 ℃ for 28-32 s, for a total of 31-33 cycles; Extend the time to 71-73 ℃ for 4.5-5.5 min; Store at 3-5°C.

[0017] As a preferred embodiment, the present invention provides a PCR amplification program: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 58 °C annealing for 30 s, 72 °C extension for 30 s, for a total of 32 cycles; 72 °C extension for 5 min; storage at 4 °C.

[0018] Preferably, the PCR amplification products are detected by 8% non-denaturing polyacrylamide gel electrophoresis.

[0019] This invention also provides a method for marker-assisted selection of wheat using the primer pair, comprising extracting genomic DNA from the wheat material to be tested, and using... Xcaaspb-21 Primer pairs were used for PCR amplification to detect the test material in Xcaaspb- 21 Allelic types at loci were determined, with priority given to materials carrying the 319 bp allelic band consistent with Pubing 74; if necessary, disease resistance phenotype identification and genetic background analysis were combined to obtain carriers. PmPB74-2 Target material for linked chromosomal segments.

[0020] The present invention has the following beneficial effects: (1) This invention provides a gene for resistance to powdery mildew in wheat-ice grass-derived lines. PmPB74-2 Linked co-dominant InDel molecular markers Xcaaspb-21 It can be used for detection Xcaaspb-21 Allelic type of the locus, and track its relationship with PmPB74-2 Linked 74-derived chromosome fragments.

[0021] (2) This molecular marker can distinguish between homozygous and heterozygous genotypes from Pubing 74 and homozygous genotypes from Mingxian 169, and can be used for genotyping of genetically mapped populations. PmPB74-2 Genetic mapping, fine mapping, and recombinant screening.

[0022] (3) The molecular marker can be detected by conventional PCR amplification and polyacrylamide gel electrophoresis. The detection method is simple, the amplification band is clear, and the repeatability is good.

[0023] (4) This molecular marker can be used for genotyping during the seedling stage and is independent of powdery mildew inoculation conditions. It can also be used for continuous tracking and monitoring during the breeding process. PmPB74-2 Linked cytokine fragments derived from Pubing 74 reduce the workload of subsequent disease resistance phenotype identification and improve the efficiency of marker-assisted selection, thus providing a basis for... PmPB74-2 It provides technical support for molecular marker-assisted breeding. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 The molecular markers provided in the embodiments of the present invention Xcaaspb-21The primer detection results of the disease-resistant wheat-Isodon sibiricum introgression line and the disease-susceptible common wheat variety Mingxian 169 and the segregating populations of their hybrid offspring were shown in the figure. M: 50 bp DNA Ladder; 1: Wheat-Isodon sibiricum derivative Pubing 74 (disease-resistant parent); 2: Common wheat variety Mingxian 169 (susceptible parent); 3-17: F2 populations of Pubing 74 × Mingxian 169, where 3-7: homozygous disease-resistant families, 8-12: segregating families of resistance and susceptibility, and 13-17: homozygous susceptible families; white arrows indicate... Xcaaspb-21 319 bp allele band at the locus.

[0026] Figure 2 The results of detecting the molecular marker Xcaaspb-21 provided in this embodiment of the invention in wheat materials with different genetic backgrounds are shown in the figure. M: pBR322 / MspI; 1: Pubing 74; 2: Mingxian 169; 3: Jimai 44; 4: Shixin 6171; 5: 1650594; 6: Shimai 25; 7: 16-4316; 8: Zhoumai 36. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0028] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0029] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0030] Example 1: Wheat powdery mildew resistance gene PmPB74-2 Location and linkage molecular markers Xcaaspb-21 development Previous studies have located the powdery mildew resistance gene on chromosome 5DS in Pubing 74 wheat. PmPB74 To further investigate whether Pubing 74 carries other powdery mildew resistance genes, a hybridization was conducted using the wheat-ice grass derivative Pubing 74 as the resistant parent and the common wheat variety Mingxian 169 as the susceptible parent to obtain F1 plants. F1 plants were then self-pollinated to obtain the F2 population. Individual F2 plants were then self-pollinated to obtain the corresponding F1 generation. 2:3 Family lineage.

[0031] The disease-resistant parent Pubing 74, the disease-susceptible parent Mingxian 169, F1 hybrids, F2 populations, and F1 strains were used.2:3 Family cultivars were planted in 72-cell trays for seedling powdery mildew resistance assessment, with Heng 4399 as a susceptible control.

[0032] Powdery mildew resistance identification at the seedling stage was conducted under greenhouse conditions: temperature 18-20 ℃, relative humidity 80%, and photoperiod of 14 h light / 10 h dark. Powdery mildew strain A09 was inoculated using the sweeping method at the one-leaf stage. Phenotypic assessment was conducted 10-14 days after inoculation, when the susceptible control Heng 4399 showed full disease development. Infection type (IT) was recorded according to a 0-4 grade standard. Resistance levels were classified as follows: 0-2 for resistant types and 3-4 for susceptible types.

[0033] The results showed that the wheat-ice grass derivative line Pubing 74 was nearly immune to powdery mildew strain A09 (IT=0;), while the common wheat line Mingxian 169 was highly susceptible (IT=4). All F1 plants showed resistance (IT=0;), indicating that the resistance of the wheat-ice grass derivative line Pubing 74 to powdery mildew strain A09 is controlled by a dominant gene. Resistance identification of the F2 population of this combination showed a resistance-susceptibility segregation ratio of 203:79, which, according to the chi-square test, conformed to a segregation ratio of 3:1 for a single dominant gene (χ²). 2 =1.367, P =0.242), for the corresponding F 2:3 Family pedigree analysis revealed a segregation ratio of 75:128:79 for homozygous disease-resistant, disease-resistant, and disease-susceptible families. Chi-square tests confirmed a segregation ratio of 1:2:1 for a single dominant gene. 2 =2.511, P =0.285). The above results indicate that the seedling resistance of the wheat-ice grass derivative line Pubing 74 to powdery mildew strain A09 is controlled by a single dominant gene, which is named... PmPB74-2 .

[0034] According to F 2:3 To identify powdery mildew resistance in the population, 40 homozygous resistant families and 40 homozygous susceptible families were selected to construct resistant and susceptible populations, respectively. A mixed segregant population transcriptome sequencing analysis (BSR) was then performed. Association analysis of differentially expressed sites between the resistant and susceptible populations identified candidate regions associated with resistance traits. The results showed… PmPB74-2 It is located on chromosome 4AL.

[0035] Further utilizing resequencing data from the resistant parent Pubing 74 and the susceptible parent Mingxian 169, the study... PmPB74-2SNPs and InDel variant sites within candidate intervals were screened and sequence differential analyses were performed. Using the Chinese spring reference genome sequence as a reference, sequence sites with stable insertion / deletion differences between parents within candidate intervals were selected. Based on conserved sequences flanking these differential sites, InDel molecular marker primers were designed using Primer 5.0 software. Subsequently, using the resistant parent Pubing 74, the susceptible parent Mingxian 169, and the F2 segregating population of Pubing 74 × Mingxian 169 as materials, polymorphism screening and genotyping of the developed markers were performed. Linkage analysis was then conducted in conjunction with population data on powdery mildew resistance phenotypes to further refine the sequence. PmPB74-2 Molecular markers located on chromosome 4AL Xcaas-in65 and Xcaas-in128 Between these points, the corresponding physical region is 730.38-735.27 Mb of the Chinese Spring reference genome.

[0036] To further reduce PmPB74-2 The target region was identified, and an expanded F2 population of 2220 individuals was constructed and identified. Genotyping of the expanded F2 population was performed using flanking linkage markers. Recombinant exchange individuals within the target region were screened. Based on the resequencing data of Pubing 74 and Mingxian 169, InDel molecular markers were further developed within the target region. According to the parental InDel differential sites within the target region, marker primers were designed using Primer 5.0 software. After parental polymorphism screening, recombinant individual genotyping, and combined phenotypic analysis, markers corresponding to the target region were finally selected. PmPB74-2 Linked co-dominant InDel molecular markers Xcaaspb-21 Using molecular markers Xcaaspb-21 Detecting recombinant exchange single plants can identify the recombination type within the target range and assist in... PmPB74-2 It is located within the 730.38-733.50 Mb physical region of the Chinese spring reference genome.

[0037] Example 2 Molecular Markers Xcaaspb-21 PCR amplification and codominance verification Molecular markers Xcaaspb-21 The primers consist of one upstream primer and one downstream primer: upstream primer Xcaaspb-21 The nucleotide sequence of F is: 5'-TCAGAATCTAACATGCAACACCT-3'; Downstream primer Xcaaspb-21 The nucleotide sequence of R is: 5'-AGAGTGTCCTGAGTTCCCA-3'.

[0038] The primer pair was used to perform PCR amplification with genomic DNA of the wheat material to be tested as a template.

[0039] The applicable PCR amplification system for this marker is 10 μL, which includes: 1 μL of 50 ng / μL wheat genomic DNA, 5 μL of PCR Master Mix, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, and 2 μL of sterile deionized water.

[0040] The applicable PCR amplification program for this marker is as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 58 °C annealing for 30 s, 72 °C extension for 30 s, 32 cycles; 72 °C extension for 5 min; store at 4 °C.

[0041] The electrophoretic separation procedure for the amplification products was as follows: electrophoresis was performed using an 8% non-denaturing polyacrylamide gel, 1.5 μL of PCR amplification products were loaded onto the gel, and electrophoresis was performed at a constant voltage of 200 V for 1.5-2 h. The gel was then stained with silver nitrate and photographed for record-keeping.

[0042] Using the wheat-ice grass derivative line Pubing 74, the common wheat variety Mingxian 169, and the segregating population of the progeny of the Pubing 74 × Mingxian 169 hybrid as materials, the above primer pairs and PCR amplification conditions were used to... Xcaaspb-21 The site was detected, and the results were as follows: Figure 1 As shown. Figure 1 In the diagram, M represents a 50 bp DNA ladder; 1 represents the wheat-wheat-ice grass derivative line Pubing 74; 2 represents the common wheat variety Mingxian 169; 3-17 represent segregating populations of the progeny of Pubing 74 × Mingxian 169, where 3-7 represent homozygous disease-resistant families, 8-12 represent resistant-susceptible segregating families, and 13-17 represent homozygous susceptible families; white arrows indicate molecular markers. Xcaaspb-21 The 319 bp band at the locus.

[0043] Amplification results showed that molecular markers Xcaaspb-21 A 319 bp allelic band was amplified in the disease-resistant parent wheat-ice grass derivative line Pubing 74, and a 309 bp allelic band was amplified in Mingxian 169. Heterozygous materials from the Pubing 74 × Mingxian 169 cross could simultaneously amplify allelic bands derived from both Pubing 74 and Mingxian 169. Materials that amplified only the 319 bp allelic band identical to that of Pubing 74 were considered... Xcaaspb-21 The locus was homozygous for Pubing 74; only materials that amplified a 309 bp allele consistent with Mingxian 169 were identified as having this locus. Xcaaspb-21 The locus is homozygous from Mingxian 169; materials that simultaneously amplify the above two allele bands are determined to have... Xcaaspb-21 The site is heterozygous.

[0044] The above results indicate that Xcaaspb-21It exhibits stable insertion / deletion polymorphism between PUB 74 and Mingxian 169, and can distinguish between homozygous types from PUB 74, homozygous types from Mingxian 169, and heterozygous types, thus serving as a codominant InDel molecular marker.

[0045] In the segregating population of the progeny of the hybridization of Pubing 74 × Mingxian 169, using Xcaaspb-21 Genotyping of the test material can be used for... PmPB74-2 Genetic mapping, fine mapping, screening of single plants through recombination and exchange, and molecular marker-assisted selection.

[0046] Example 3 Molecular Markers Xcaaspb-21 Analysis of detection and breeding applications in wheat materials with different genetic backgrounds To verify molecular markers Xcaaspb-21 To investigate the auxiliary screening effect in the screening of wheat breeding materials resistant to powdery mildew, six wheat breeding materials with different genetic backgrounds were selected as test materials, with Pubing 74 and Mingxian 169 used as controls, for a total of eight materials. Genomic DNA was extracted from each material and used... Xcaaspb-21 PCR amplification was performed using primer pairs, and the PCR amplification system and reaction procedure were carried out according to Example 2. The amplification products were detected by 8% non-denaturing polyacrylamide gel electrophoresis to analyze the different materials. Xcaaspb-21 Amplification type of the site.

[0047] Test results as follows Figure 2 As shown, all six tested materials amplified to obtain 309 bp allele bands, with clear bands and good reproducibility. The six materials in... Xcaaspb-21 The amplification types of the loci were consistent with those of Mingxian 169 (consistent with the known genetic background of the material), but different from the 319 bp allele type of Pubing 74.

[0048] The above results indicate that Xcaaspb-21 can be stably amplified in different wheat materials tested, and can detect the allelic type at this locus. Furthermore, the tested materials exhibit polymorphism at this locus compared to Pubing 74. Using this marker, the allelic type of candidate wheat materials can be detected before hybridization breeding. Xcaaspb-21 The allelic type of the locus was determined, and materials with polymorphism at this locus were screened, providing a basis for hybrid combination configuration and marker detection in subsequent breeding populations.

[0049] In conjunction with Example 1 Xcaaspb-21 and PmPB74-2 The linkage analysis results and the co-dominance typing results of this marker in Example 2, with Pulvin 74 as the base PmPB74-2 During molecular marker-assisted breeding of donor parents, the aforementioned markers can be used to help identify those consistent with Pubing 74. Xcaaspb-21 Equivalent types, tracking and PmPB74-2The linkage of the Pubing 74 chromosome fragment prioritizes the retention of materials carrying the target linkage fragment, reducing the number of materials that need to be identified for disease resistance phenotype and improving the efficiency of molecular marker-assisted selection for wheat resistance to powdery mildew.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gene for powdery mildew resistance in wheat-ice grass-derived lines PmPB74-2 Linked co-dominant InDel molecular markers Xcaaspb-21 Its characteristics are, The molecular markers were detected by PCR amplification using a primer pair consisting of the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.

2. A 319 bp allele band was obtained from the amplification of Pubing 74, and a 309 bp allele band was obtained from the amplification of Mingxian 169. Both allele bands were obtained from the simultaneous amplification of the hybrid material.

2. A method for amplifying the molecular marker of claim 1 Xcaaspb-21 The primer pair is characterized in that, The primer pair includes: (1) The upstream primer with a nucleotide sequence as shown in SEQ ID NO.1; (2) The downstream primer with the nucleotide sequence shown in SEQ ID NO.

2.

3. The primer pair according to claim 2, characterized in that, When the primer pairs were used to amplify wheat genomic DNA by PCR, Pubing 74 amplified a 319 bp allele band, Mingxian 169 amplified a 309 bp allele band, and the heterozygous material amplified both of the above allele bands.

4. The primer pair according to claim 2 or 3 in wheat powdery mildew resistance gene PmPB74-2 Applications in genetic mapping, fine mapping, or recombinant screening.

5. The primer pair according to claim 2 or 3 in the detection of wheat materials Xcaaspb-21 Application in locus allele types.

6. The application according to claim 5, characterized in that, Includes the following steps: (1) Extract genomic DNA from the wheat material to be tested; (2) Using the genomic DNA as a template, perform PCR amplification using the primer pair described in claim 2; (3) Electrophoresis detection of PCR amplification products; (4) When only a 319 bp allelic band consistent with the disease-resistant parent Pubing 74 is detected, the material is determined to be in Xcaaspb- 21 The site is homozygous for the Pubing 74 source; when only a 309 bp allelic band consistent with Mingxian 169 is detected, the material is determined to be homozygous. Xcaaspb-21 The locus is homozygous from Mingxian 169; when both 319 bp and 309 bp allelic bands are detected simultaneously, the material is determined to be homozygous. Xcaaspb-21 The site is heterozygous.

7. The application according to claim 6, characterized in that, The applicable PCR amplification system for the primer pair is 9.5-10.5 μL, comprising: 0.95-1.05 μL of 49-51 ng / μL wheat genomic DNA, 4.9-5.1 μL of PCR Master Mix, 0.95-1.05 μL of 9.5-10.5 μM upstream primer, 0.95-1.05 μL of 9.5-10.5 μM downstream primer, and 1.9-2.1 μL of sterile deionized water. The applicable PCR amplification program for the primer pair is: 94-96 ℃ pre-denaturation for 4.5-5.5 min; 94-96 ℃ denaturation for 28-32 s, 57-59 ℃ annealing for 28-32 s, 71-73 ℃ extension for 28-32 s, 31-33 cycles; 71-73 ℃ extension for 4.5-5.5 min; and storage at 3-5 ℃.

8. A method for auxiliary selection of wheat resistance to powdery mildew using the primer pair described in claim 2 or 3, characterized in that, include: (1) Extract genomic DNA from the wheat material to be tested; (2) PCR amplification was performed using the primer pair; (3) Detect the genotype of the amplification product; (4) Materials carrying the same 319 bp allelic band as Pubing 74 were selected as candidate materials, and in conjunction with disease resistance phenotype identification, materials carrying the same 319 bp allelic band as Pubing 74 were screened. PmPB74-2 The target material for the linked Pubing 74-derived chromosome fragment.

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