A primer for amplifying a molecular marker closely linked to a wheat powdery mildew resistance gene PmCG15-009 and application thereof

Abstract

The application discloses a primer of a molecular marker closely linked to a wheat powdery mildew resistance gene PmCG15-009, and the molecular marker is YTU103-101. The marker primer of the molecular marker YTU103-101 comprises an upstream primer YTU103-101-F and a downstream primer YTU103-101-R. The marker primer of the molecular marker YTU103-101 is used for carrying out PCR amplification on the wheat genomic DNA to be detected, and a corresponding amplification product with a molecular weight of 435 bp is obtained, so that the application can be applied to the detection of the wheat powdery mildew resistance gene PmCG15-009, map-based cloning and molecular marker assisted breeding, and the breeding efficiency can be greatly improved, the breeding period can be shortened, and the application serves the wheat disease resistance breeding.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to primers for amplifying a tightly linked molecular marker for the wheat powdery mildew resistance gene PmCG15-009 and their applications. Background Technology

[0002] Wheat (Triticum aestivum L., 2n=6x=42, AABBDD) is an important food crop in my country, and its high and stable yields are crucial to national food security. Powdery mildew, caused by Brucella powdery mildew, seriously threatens wheat production. Infection with powdery mildew generally results in a 10-15% yield loss, with severe years seeing losses exceeding 40%. (Jia et al., 2018. Jia AL, Ren R, Gao FM, et al. Mapping and validation of a new QTL for adult-plant resistance to powdery mildew in Chinese elite breadwheat line Zhou8425B[J]. Theoretical and Applied Genetics, 2018, 131: 1063-1071.). Therefore, the prevention and control of powdery mildew is urgent. Compared with agricultural and chemical control, the rational use of disease-resistant genes and the cultivation of disease-resistant varieties are among the most effective measures for controlling wheat powdery mildew.

[0003] To date, 68 formally named wheat powdery mildew resistance genes (Pm1-Pm68, Pm8=Pm17, Pm18=Pm1c, Pm22=Pm1e, Pm23=Pm4c, Pm31=Pm21) and more than 30 provisionally named wheat powdery mildew resistance genes have been identified at 63 loci (He et al., 2021; McIntosh et al., 2019. He, HG, Liu, RK, Ma, PT, et al. Characterization of Pm68, a new powdery mildew resistance gene on chromosome 2BS of Greek durum wheat TRI 1796[J]. Theoretical and Applied Genetics, 2021, 134: 53-62. McIntosh RA, Dubcovsky J, Rogers WJ, et al. “Catalogue 2 of gene symbols for wheat: 2019 supplement” in Annual Wheat Newsletter [J]. Ed. WJ Raupp (Manhattan, NY: The Wheat Genetic and Genomic Resources at Kansas State University), 2019, 98-113.). Among them, Pm1, Pm2, Pm3, Pm4, Pm5, Pm24, and Pm60 are multiple allele loci. The vast majority of these disease resistance genes are race-specific resistances, which are easily lost with large-scale production and the evolution of pathogens. Studies have shown that due to the monopolization and overuse of resistance genes in production, powdery mildew resistance genes such as Pm3a, Pm3b, Pm3f, Pm4a, Pm6, Pm8, and Pm17 have lost their resistance in some parts of the United States and even throughout the country (Cowger et al., 2018. Cowger C, Mehra L, Arelano C, et al. Virulence differences in Blumeria graminis f.sp.tritici from the central and eastern United States[J]. Phytopathology, 2018, 108: 402-411.).Pm1, Pm3, Pm4, Pm5, Pm8, and Pm17 have partially or completely lost their resistance in my country (Zeller et al., 2002. Zeller FJ, Kong L, Hartl L, et al. Chromosomallocation of genes for resistance to powdery mildew in common wheat (Triticumaestivum L.em Thell.) 7. Gene Pm29 in line Pova[J]. Euphaitica, 2002, 123(2): 187-194.). Therefore, it is urgent to find new resistance sources to improve wheat resistance to powdery mildew in order to continuously address the challenges posed by wheat powdery mildew.

[0004] The sources of wheat powdery mildew resistance genes discovered so far mainly include common wheat and its closely related genera and species. For example, Pm21 (He et al., 2018. He HG, Zhu SY, Zhao RH, et al. Pm21, Encoding a typical CC-NBS-LRR protein, confers broad-spectrum resistance to wheat powdery mildew disease[J]. Molecular Plant, 2018, 11(6): 879-882.) is derived from Dasypyrum villosum, and Pm36 (Blanco et al., 2008. Blanco A, Gadaleta A, Cenci A, et al. Molecular mapping of the novel powdery mildew resistance gene Pm36 introgressed from Triticum turgidum var. dicoccoides indurum wheat[J]. Theoretical and Applied Genetics, 2008, 117:135-142.) and Pm60 from the ancestor of wheat A genome, T. urartu (Zou et al., 2018.Zou SH,Wang H,LiYW, et al.The NB-LRR gene Pm60 confers powdery mildew resistance in wheat[J].New Phytologist, 2018, 218(1):298-309.), etc. These genes are mostly linked to unfavorable traits. Materials carrying these disease resistance genes cannot be directly used as parents for wheat disease resistance breeding. They need to be backcrossed and self-crossed with the main wheat varieties for multiple generations to break the linkage burden before they can be applied to wheat disease resistance breeding.Of the powdery mildew resistance genes that have been discovered, nearly half are derived from common wheat. For example, the powdery mildew resistance gene Pm65 is derived from the Chinese wheat cultivar Xinmai 208 (Li et al., 2019.Li GQ,Cowger C,Wang XW, et al.Characterization of Pm65,a new powdery mildew resistance gene on chromosome 2AL of a facultative wheat cultivar[J].Theoretical and Applied Genetics,2019,132:2625-2632.), and Pm5e is derived from the Chinese local variety Fuzhuang 30 (Huang et al., 2003.Huang XQ,Wang LX,Xu MX, et al.Microsatellite mappingof the powdery mildew resistance gene Pm5e in common wheat(Triticum aestivum L.)[J].Theoretical and Applied Genetics,2003,106:858-865.). Pm59 originates from the Afghan local variety PI181356 (Tan et al., 2018. Tan CC, Li GQ, Cowger C, et al. Characterization of Pm59, a novel powdery mildew resistance gene in Afghanistan wheat landrace PI181356[J]. Theoretical and Applied Genetics, 2018a, 131: 1145-1152.). These disease-resistant genes from common wheat are easier to utilize and can be directly applied to disease-resistant breeding through traditional hybridization and backcrossing techniques (Li et al., 2019; Li GQ, Cowger C, Wang XW, et al. Characterization of Pm65, a new powdery mildew resistance gene on chromosome 2AL of a facultative wheat cultivar[J]. Theoretical and Applied Genetics, 2019, 132: 2625-2632.). Therefore, it is of great significance to discover and utilize new powdery mildew resistance gene resources from common wheat.

[0005] Once a gene exhibiting superior resistance is discovered, its efficient application in wheat disease resistance breeding is crucial. In recent years, with the continuous development of wheat breeding technology, molecular marker-assisted selection (MMR) breeding has become a primary method for breeding wheat resistant to powdery mildew. Efficient, accurate, and user-friendly molecular markers are prerequisites for MMR-assisted selection of target genes. Utilizing molecular markers to track and detect target genes can significantly shorten the breeding process and improve breeding efficiency.

[0006] Shimai CG15-009 is a provincial-level regional trial wheat line with excellent agronomic traits. In multi-year, multi-location field trials, it has demonstrated good resistance to powdery mildew, making it an excellent wheat germplasm resource resistant to powdery mildew. Genetic analysis and molecular marker detection of powdery mildew resistance at the seedling stage revealed that the resistance of Shimai CG15-009 to the prevalent powdery mildew strain E09 at the seedling stage is controlled by a pair of dominant genes, PmCG15-009, located on the wheat 2BL chromosome. This represents a novel wheat powdery mildew resistance gene / allele. Therefore, developing molecular markers tightly linked to the PmCG15-009 gene and applying them to marker-assisted selection breeding for the PmCG15-009 powdery mildew resistance gene is of great significance for breeding powdery mildew-resistant wheat varieties and effectively controlling wheat powdery mildew. Summary of the Invention

[0007] The purpose of this invention is to provide primers for amplifying a tightly linked molecular marker of the wheat powdery mildew resistance gene PmCG15-009 and its application, so as to locate and detect the wheat powdery mildew resistance gene PmCG15-009 by amplifying the molecular marker using the primers, and to make targeted selection of its parents in wheat breeding, thus providing guidance for breeding new wheat varieties resistant to powdery mildew.

[0008] The present invention is achieved by the following method: a primer for amplifying a tightly linked molecular marker of the wheat powdery mildew resistance gene PmCG15-009 and its application, wherein the molecular marker is the codominant SSR marker YTU103-101;

[0009] The upstream primer for the molecular marker YTU103-101 is YTU103-101-F, and its nucleotide sequence is as follows:

[0010] 5'-GGGAGAGCCGTCAAAGAACA-3', as shown in SEQ ID NO:1;

[0011] The downstream primer for the molecular marker YTU103-101 is YTU103-101-R, and its nucleotide sequence is as follows:

[0012] 5'-CTTCTCATTTTCTCCGCGCG-3', as shown in SEQ ID NO:2;

[0013] The marker primers of the molecular marker YTU103-101 were used to amplify the wheat genomic DNA to be tested by PCR. The corresponding amplification product had a molecular weight of 435 bp, which is a molecular marker closely linked to the wheat powdery mildew resistance gene PmCG15-009.

[0014] The PCR amplification system is 10 μL, comprising: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.5 μL of 5 μM upstream primer, 0.5 μL of 5 μM downstream primer, and 4 μL of sterile deionized water.

[0015] The present invention relates to the application of the molecular markers closely linked to the wheat powdery mildew resistance gene PmCG15-009 in the localization, map-based cloning, and marker-assisted selection breeding of the wheat powdery mildew resistance gene PmCG15-009.

[0016] The application described in this invention, which detects whether a wheat variety carries the powdery mildew resistance gene PmCG15-009, mainly includes the following steps:

[0017] (1) Genomic DNA was extracted from fresh leaves of the wheat sample to be tested;

[0018] (2) The wheat genomic DNA was amplified by PCR using primers with molecular marker YTU103-101 to obtain the amplification product;

[0019] (3) If a specific band of 435bp can be amplified, it indicates that the powdery mildew resistance gene PmCG15-009 exists in the wheat to be tested; otherwise, the powdery mildew resistance gene PmCG15-009 does not exist in the wheat to be tested.

[0020] In the application described above, the primers for the molecular marker YTU103-101 in step (2) include an upstream primer YTU103-101-F and a downstream primer YTU103-101-R. The nucleotide sequence of the upstream primer YTU103-101-F is shown in SEQ ID NO:1, namely: YTU103-101-F: 5'-GGGAGAGCCGTCAAAGAACA-3'; the downstream primer of the molecular marker YTU103-101 is YTU103-101-R, and its nucleotide sequence is shown below; the nucleotide sequence of the downstream primer YTU103-101-R is shown in SEQ ID NO:2, YTU103-101-R: 5'-CTTCTCATTTTCTCCGCGCG-3'.

[0021] In the aforementioned application, the PCR amplification system is 10 μL, comprising: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.5 μL of 5 μM upstream primer, 0.5 μL of 5 μM downstream primer, and 4 μL of sterile deionized water.

[0022] The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 55℃ annealing for 15 s, extension for 40 s, 30 cycles; 72℃ extension for 10 min; storage at 4℃.

[0023] PCR amplification product detection: Electrophoresis was performed on an 8% (w / v) non-denaturing polyacrylamide gel. The amplification product was mixed with 2 μL of 6× loading buffer, and 2 μL of the mixture was loaded onto the gel. Electrophoresis was performed at a constant voltage of 180V for 2.5-3 h. After silver nitrate staining, the sample was photographed. If a specific band of 435 bp was amplified, it indicates that the powdery mildew resistance gene PmCG15-009 is present in the wheat germplasm being tested. Otherwise, the powdery mildew resistance gene PmCG15-009 is not present in the wheat germplasm being tested.

[0024] This invention, through genetic analysis and molecular marker detection of seedling powdery mildew resistance, demonstrates that the resistance of Shimai CG15-009 seedlings to the prevalent powdery mildew strain E09 is controlled by a single dominant gene, named PmCG15-009. Using 321 pairs of molecular markers evenly distributed throughout the genome, the resistance of Shimai CG15-009, the susceptible wheat variety Yannong 21, and the F1 generation of Shimai CG15-009 × Yannong 21 were compared. 2:3 Polymorphism was detected in a disease-resistant pool and a disease-susceptible pool, consisting of 20 homozygous resistant families and 20 homozygous susceptible families. Ten pairs of markers showed consistent polymorphism in both the resistant and susceptible parents and the resistant-susceptible pool. These markers were then used to detect F15 strains of 115 Shimai CG15-009 × Yannong 21. 2:3 Genotyping of families revealed that the PmCG15-009 gene was initially located within the 688.67-723.48 Mb region of wheat chromosome 2BL. Further, based on the sequence of the Chinese spring wheat reference genome within this region, the Simple Sequence Repeat (SSR) marker YTU103-101, closely linked to the PmCG15-009 gene, was designed and screened using Primer 5.0 software. The molecular marker YTU103-101 for the wheat powdery mildew resistance gene PmCG15-009 provided by this invention, after genetic segregation population testing, showed a genetic distance of only 1.2 cM from the PmCG15-009 gene, indicating close linkage. This marker can efficiently and accurately detect large genetic mapping populations of the PmCG15-009 gene and can be applied to the fine mapping and map-based cloning of the PmCG15-009 gene.

[0025] This invention provides a molecular marker closely linked to the wheat powdery mildew resistance gene PmCG15-009 for use in powdery mildew resistant wheat breeding. This not only allows for rapid and accurate screening, unaffected by the environment, and with clear selection targets, but also saves production costs and greatly improves the selection efficiency and quality of high-quality powdery mildew resistant wheat varieties or lines. Attached Figure Description

[0026] Figure 1 To mark YTU103-101 in 115 stone wheat CG15-009× tobacco farmer 21 derived F 2:3 Partial amplification results from the family pedigree.

[0027] In the diagram, M: pUC18 Msp I; 1: Shimai CG15-009 (disease-resistant parent); 2: Yannong 21 (susceptible parent); 3-17: F1 generation of Shimai CG15-009 × Yannong 21 2:3 The families are 3-7: homozygous disease-resistant families, 8-12: disease-resistant segregating families, and 13-17: homozygous disease-susceptible families; the white arrows are specific bands of PmCG15-009.

[0028] Figure 2 This is a partial amplification result of YTU103-101 in 40 susceptible wheat varieties.

[0029] In the diagram, M: pUC18 Msp I; 1: Shimai CG15-009 (disease-resistant parent); 2: Yannong 21 (susceptible parent); 3-17: Shannong 1538, Hanmai 13, Huaimai 0226, Zhoumai 27, Yanmai 1212, Xinong 979, Lumai 185, Zhongyu 1311, Jimai 268, Tainong 1014, Jimai 229, Jimai 21, Jimai 20, Daimai 2173, and Zhongmai 1751. The white arrows indicate the specific bands of PmCG15-009. Detailed Implementation

[0030] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials and reagents used in the examples are commercially available.

[0031] Example 1: Development of the molecular marker YTU103-101 for the wheat powdery mildew resistance gene PmCG15-009

[0032] 1. Materials

[0033] The resistant parent, Shimai CG15-009, is a provincial-level experimental wheat line, while the susceptible parent, Yannong 21, is a nationally approved wheat variety. Shimai CG15-009 and Yannong 21 were crossed, and the resulting F1 generation was self-crossed to obtain the F2 population and corresponding F3 generation. 2:3 Family lineage.

[0034] 2. Extraction of wheat genomic DNA

[0035] The CTAB method for wheat genomic DNA extraction is as follows:

[0036] 1) Take tender leaves of wheat material, quick-freeze them in liquid nitrogen, grind them into powder, and put them into 2mL EP tubes;

[0037] 2) Add 600-800 μL of CTAB extraction solution and incubate in a water bath at 65°C for 1 hour, inverting the container several times during the incubation period.

[0038] 3) Add an equal volume of chloroform and mix on a shaker for 15 minutes;

[0039] 4) Centrifuge at 12000 rpm at room temperature for 10 min, take 400 μL of supernatant into a 1.5 mL EP tube, add 3 times the volume of pre-cooled anhydrous ethanol, mix well, and let it settle at -20℃ for 2 h.

[0040] 5) Centrifuge at 12000 rpm at room temperature for 10 min, discard the supernatant, and wash three times with 800 μL of 70% ethanol.

[0041] 6) Air dry the precipitate and dissolve it in 50 μL of 1×TE or ddH2O.

[0042] 7) Dilute the DNA storage solution with sterile deionized water to 50 ng / μL as a working solution for later use.

[0043] 3. Identification of powdery mildew resistance in wheat seedlings and genetic analysis of resistance

[0044] Wheat seedling powdery mildew resistance identification was completed in a greenhouse. The resistant parent Shimai CG15-009, the susceptible parent Yannong 21, F1 hybrids, F2 populations, and F4 populations were used. 2:3 The families were planted in 128-cell seed trays (3.2×3.2×4.2cm). Both parents and F1 strains were identified as having at least 20 seeds per cell. 2:3Each family was identified with at least 25 seeds. The susceptible control, Mingxian 169, was randomly sown and tagged for identification. Greenhouse conditions were controlled at 18-20℃, relative humidity 80%, and a photoperiod of 14h light / 10h dark. Powdery mildew strain E09 was inoculated using the sweeping method at the one-leaf stage. Phenotypic analysis was conducted 10-14 days later, when the susceptible control, Mingxian 169, was fully infected. Infection type (IT) was recorded according to a 0-4 grade standard. Disease resistance grades were classified as follows: 0-2 for resistant types, and 3-4 for susceptible types (Si Quanmin et al., 1987).

[0045] The survey results showed that Shimai CG15-009 exhibited high resistance to powdery mildew strain E09 (IT=0), while Yannong 21 showed high susceptibility (IT=4). All F1 plants showed resistance (IT=0-1), indicating that Shimai CG15-009 carries a dominant resistance gene. Resistance identification of the F2 population of this combination showed a resistance-susceptibility segregation ratio of 79:36, which, according to the chi-square test, conformed to a segregation ratio of 3:1 for a single dominant gene (χ²=0). 2 =2.11, P=0.15), further F 2:3 The pedigree analysis results showed that the segregation ratio of homozygous resistant families: resistant-susceptible families: homozygous susceptible families was 22:57:36, which, according to the chi-square test, was consistent with a segregation ratio of 1:2:1 for a single dominant gene. 2 =3.42, P=0.18). In conclusion, the resistance of Shimai CG15-009 to powdery mildew strain E09 is controlled by a single dominant gene, which is named PmCG15-009.

[0046] 4. Initial localization of the molecular marker PmCG15-009

[0047] Based on phenotypic identification results, 20 homozygous resistant families and 20 homozygous susceptible families were selected to construct resistant and susceptible pools, respectively. Polymorphism detection was performed on Shimai CG15-009, the susceptible wheat variety Yannong 21, and the resistant and susceptible pools using 321 pairs of molecular markers evenly distributed throughout the genome. Ten pairs of markers showed consistent polymorphism in the resistant and susceptible parents and the resistant-susceptible pools. Subsequently, these markers were used to analyze the F1 generation of 115 Shimai CG15-009 × Yannong 21 pairs. 2:3 Genotyping of the family revealed that PmCG15-009 was preliminarily located within the range of 688.67-723.48 Mb on the wheat 2BL chromosome.

[0048] 5. Development of molecular markers closely linked to PmCG15-009

[0049] Based on the sequence information of the Chinese spring wheat reference genome within the candidate region of 688.67-723.48 Mb, primers marked with Simple Sequence Repeat (SSR) were designed using Primer 5.0 software to target the F1 generation of *Strombus haemocarpa* CG15-009 × *Strombus spp.* var. *yannong* 21. 2:3 Genotyping of the family revealed the SSR marker YTU103-101, which is closely linked to the gene PmCG15-009, with a genetic distance of only 1.2 cM.

[0050] The primers for the molecular marker YTU103-101 include one upstream primer and one downstream primer:

[0051] The nucleotide sequence of the upstream primer YTU103-101-F is: 5'-GGGAGAGCCGTCAAAGAACA-3';

[0052] The nucleotide sequence of the downstream primer YTU103-101-R is: 5'-CTTCTCATTTTCTCCGCGCG-3'.

[0053] The PCR amplification system is 10 μL, comprising: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.5 μL of 5 μM upstream primer, 0.5 μL of 5 μM downstream primer, and 4 μL of sterile deionized water.

[0054] The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 55℃ annealing for 15 s, extension for 40 s, 30 cycles; 72℃ extension for 10 min; storage at 4℃.

[0055] The electrophoretic separation procedure for the amplified products is as follows: electrophoresis is performed on an 8% (w / v) non-denaturing polyacrylamide gel. The amplified products are mixed with 2 μL of 6× loading buffer, and 2 μL of the mixture is loaded onto the gel. Electrophoresis is performed at a constant voltage of 180V for 2.5-3 hours, followed by silver nitrate staining and photographing. If a specific band of 435 bp is amplified, it indicates that the powdery mildew resistance gene PmCG15-009 is present in the tested wheat germplasm; otherwise, the powdery mildew resistance gene PmCG15-009 is not present in the tested wheat germplasm.

[0056] Example 2: Application of the YTU103-101 primer, a molecular marker for the wheat powdery mildew resistance gene PmCG15-009.

[0057] The samples to be tested included the resistant parent Shimai CG15-009, the susceptible parent Yannong 21, and 115 F1 strains derived from Shimai CG15-009 × Yannong 21. 2:3Family lineages and 40 susceptible wheat varieties that do not carry the resistance gene PmCG15-009 (Shannong 1538, Hanmai 13, Huaimai 0226, Zhoumai 27, Yan 1212, Xinong 979, Lumai 185, Zhongyu 1311, Jimai 268, Tainong 1014, Jimai 229, Jimai 21, Jimai 20, Daimai 2173, Zhongmai 1751, Jinan 17, Zhongmai 9398, Womai 8). The following wheat varieties were tested: Liangxing 619, Shimai 15, Xinluo 4, Qingmai 6, Zhengmai 0856, Wunong 6, Huixian Hong, Pumai 28, Zhongxinmai 77, Yannong 15, Yannong 17, Yannong 23, Yannong 24, Yannong 161, Yannong 301, Yannong 390, Yannong 745, Yannong 836, Yannong 5158, Yannong 999, Yannong 215, and Yannong 199. The method for extracting DNA from the wheat was the same as in Example 1.

[0058] Using the genomic DNA of the above materials as a PCR amplification template, amplification was performed using primers for the molecular marker YTU103-101 developed in this invention:

[0059] The nucleotide sequence of the upstream primer YTU103-101-F is: 5'-GGGAGAGCCGTCAAAGAACA-3';

[0060] The nucleotide sequence of the downstream primer YTU103-101-R is: 5'-CTTCTCATTTTCTCCGCGCG-3'.

[0061] The PCR amplification system consisted of 10 μL, including: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.5 μL of 5 μM upstream primer, 0.5 μL of 5 μM downstream primer, and 4 μL of sterile deionized water.

[0062] The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 55℃ annealing for 15 s, extension for 40 s, 30 cycles; 72℃ extension for 10 min; storage at 4℃.

[0063] The electrophoretic separation procedure for the amplified products is as follows: electrophoresis is performed on a non-denaturing polyacrylamide gel with a mass-volume percentage concentration of 8%. The amplified products are mixed with 2 μL of 6× loading buffer, and 2 μL of the mixture is loaded onto the gel. Electrophoresis is performed at a constant voltage of 180V for 2.5-3 hours. The gel is then stained with silver nitrate and photographed.

[0064] Molecular marker detection results are shown below Figure 1 and Figure 2 .in Figure 1 To mark YTU103-101 in 115 stone wheat CG15-009× tobacco farmer 21 derived F 2:3Partial amplification results in the family. In the figure, M: pUC18 Msp I; 1: Shimai CG15-009 (disease-resistant parent); 2: Yannong 21 (disease-susceptible parent); 3-17: F1 generation of Shimai CG15-009 × Yannong 21. 2:3 The families are shown in the diagram: 3-7: homozygous disease-resistant families; 8-12: segregating resistant and susceptible families; 13-17: homozygous susceptible families. The white arrows indicate the specific band of gene PmCG15-009. Amplification results showed that marker YTU103-101 amplified a specific 435 bp band in the disease-resistant parent *Shimai* CG15-009 and in disease-resistant families, but did not amplify this target band in the susceptible parent *Yannong 21* and in susceptible families.

[0065] Figure 2 This figure shows the partial amplification results of YTU103-101 in 40 susceptible wheat varieties. In the figure, M: pUC18MspI; 1: Shimai CG15-009 (resistant parent); 2: Yannong 21 (susceptible parent); 3-17: Shannong 1538, Hanmai 13, Huaimai 0226, Zhoumai 27, Yanmai 1212, Xinong 979, Lumai 185, Zhongyu 1311, Jimai 268, Tainong 1014, Jimai 229, Jimai 21, Jimai 20, Daimai 2173, and Zhongmai 1751. The white arrows indicate the specific bands of PmCG15-009. The amplification results showed that the marker YTU103-101 amplified a specific 435bp band only in the resistant parent *Shimai* CG15-009. This target band was not amplified in the susceptible parent *Yannong 21* or any of the susceptible wheat varieties, indicating that *Yannong 21* and the 15 mentioned wheat varieties do not contain the gene PmCG15-009 and are therefore susceptible wheat varieties. No 435bp specific band was amplified in any of the 40 susceptible wheat varieties. Fifteen materials were selected as examples for amplification illustration; the results for the remaining 25 materials were completely consistent with the 15 examples shown in the illustration.

[0066] The wheat powdery mildew resistance gene PmCG15-009 originates from the wheat variety Shimai CG15-009, which is tested in regional trials in my country. It is a novel gene exhibiting excellent resistance, but currently, there are no reports on its localization, map-based cloning, or molecular breeding applications. Using the molecular marker YTU103-101 provided by this invention to detect large genetically mapped populations helps achieve fine localization and map-based cloning of the PmCG15-009 gene. Introducing PmCG15-009 into susceptible wheat varieties, using the molecular marker YTU103-101 developed in this invention, can efficiently and accurately detect large breeding populations, greatly improving the efficiency and accuracy of transferring the disease-resistant gene PmCG15-009. This is of great significance for the efficient transfer of the PmCG15-009 gene and the in-depth analysis of its disease resistance mechanism.

[0067] The above embodiments are optimized implementations of the present invention and are used only to illustrate the present invention, not to limit it. Modifications or equivalent substitutions made by those skilled in the art without departing from the spirit and principles of the embodiments of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. An amplification gene for wheat resistance to powdery mildew PmCG15-009 Primers with tightly linked molecular markers, characterized in that, The molecular marker is YTU103-101; the marker primers for the molecular marker YTU103-101 include an upstream primer YTU103-101-F and a downstream primer YTU103-101-R. The nucleotide sequence of the upstream primer YTU103-101-F is shown in SEQ ID NO:1; the nucleotide sequence of the downstream primer YTU103-101-R is shown in SEQ ID NO:

2. PCR amplification of the wheat genomic DNA to be tested using the marker primers for the molecular marker YTU103-101 yielded an amplification product with a molecular weight of 435 bp, which corresponds to the wheat powdery mildew resistance gene. PmCG15-009 Tightly linked molecular markers.

2. The amplified wheat powdery mildew resistance gene as described in claim 1 PmCG15-009 Primers for tightly linked molecular markers in wheat powdery mildew resistance genes PmCG15-009 The detection of powdery mildew and its application in molecular marker-assisted breeding of wheat.

3. A method for detecting whether a wheat variety carries a gene for resistance to powdery mildew. PmCG15-009 The method is characterized by, Includes the following steps: (1) Extract genomic DNA from the wheat sample to be tested; (2) The genomic DNA of the wheat sample to be tested was amplified by PCR using the marker primers of molecular marker YTU103-101 to obtain the amplification product; the marker primers of molecular marker YTU103-101 include upstream primer YTU103-101-F and downstream primer YTU103-101-R, the nucleotide sequence of upstream primer YTU103-101-F is shown in SEQ ID NO:1; the nucleotide sequence of downstream primer YTU103-101-R is shown in SEQ ID NO:2; (3) The amplified product is subjected to electrophoresis and detection. If a specific band of 435 bp can be amplified, it indicates that the wheat sample to be tested contains a powdery mildew resistance gene. PmCG15-009 Otherwise, the wheat sample tested does not contain the wheat powdery mildew resistance gene. PmCG15- 009 .

4. The method according to claim 3, characterized in that, The PCR amplification system was 10 μL, comprising: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.5 μL of 5 μM upstream primer, 0.5 μL of 5 μM downstream primer, and 4 μL of sterile deionized water.

5. The method according to claim 4, characterized in that, The PCR amplification program was as follows: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 15 s, annealing at 55℃ for 15 s, extension for 40 s, 30 cycles; extension at 72℃ for 10 min; storage at 4℃.

Images