A molecular marker closely linked to wheat powdery mildew resistance gene PmL251 and application thereof
By developing the molecular marker YTUL-5, which is closely linked to the wheat powdery mildew resistance gene PmL251, the problems of long breeding cycles and low efficiency in wheat breeding have been solved. This has enabled efficient and accurate gene mapping and breeding-assisted selection, thereby improving the efficiency of powdery mildew resistant wheat breeding.
Patent Information
- Application Number
- CN202610833516.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-10
- Publication Date
- 2026-07-14
AI Technical Summary
Existing technologies make it difficult to efficiently and accurately utilize the powdery mildew resistance gene PmL251 in Sperto wheat breeding, resulting in long breeding cycles and low efficiency.
We developed the INDEL marker YTUL-5, a molecular marker closely linked to the wheat powdery mildew resistance gene PmL251, and used PCR amplification and electrophoresis detection techniques for gene localization and breeding-assisted selection.
Shorten the breeding cycle, improve breeding efficiency, achieve accurate detection and efficient introduction of PmL251, and enhance the accuracy and efficiency of powdery mildew resistant wheat breeding.
Smart Images
Figure CN122382250A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering technology, specifically to a molecular marker closely linked to the wheat powdery mildew resistance gene PmL251 and its application. Background Technology
[0002] Wheat powdery mildew is a fungal disease caused by *Erysiphe brucellae*. The disease primarily affects wheat leaves and leaf sheaths, forming white, moldy powdery spots on the surface. In severe cases, the infection can spread to the stems, glumes, and even the ears of wheat. With the infestation of numerous conidia, a white, powdery mold layer forms on the leaf surface, gradually covering the entire leaf. This significantly weakens wheat photosynthesis, increases respiration, and accelerates metabolism, ultimately leading to reduced seed setting rate, fewer grains, and decreased yield. In severe cases, it can cause plant death. Currently, the core measure for controlling powdery mildew is planting resistant varieties. However, the rapid evolution of highly virulent powdery mildew strains often breaks through species-specific resistance genes. Therefore, compared to conventional control methods such as chemical agents and biological agents, continuously discovering and utilizing novel resistance genes from untapped genetic resources is the most economical and effective means of controlling wheat powdery mildew and reducing its production damage. This is an important task in wheat breeding.
[0003] In the discovery of wheat powdery mildew resistance genes, ancestral and wild wheat species are important gene pools. Spelto wheat (Triticum spelta L.) is an ancient cultivated wheat variety with the same genome composition (AABBDD) as common wheat. Compared to cultivated wheat, Spelto wheat generally exhibits stronger resistance to biotic stresses and has evolved a large number of disease resistance and stress tolerance genes, which can be used for wheat disease resistance breeding. Studies have identified several effective wheat disease resistance genes in Spelto wheat, including Pm1d, MlHubel, Yr5 / Yr7 / YrSp, Lr44, Lr65, and Lr71. These findings indicate that Spelto wheat possesses many more undiscovered disease resistance genes that can be used for wheat disease resistance breeding.
[0004] In the history of wheat breeding, traditional breeding has been centered on experience-based breeding. Breeders used their long-term breeding experience to screen for phenotypes of target traits. This method is easily influenced by environmental and genotype interactions, resulting in low selection efficiency and a lengthy breeding cycle. Marker-assisted selection (MAS), on the other hand, utilizes molecular markers closely linked to or co-segregating with target genes to directly identify and target genotypes, significantly improving selection accuracy and genetic improvement efficiency. Currently, this technology is widely used in wheat disease resistance breeding.
[0005] Spertau wheat variety L251 has demonstrated stable, high-level resistance in the field after years of natural induction and artificial inoculation at multiple locations, making it an excellent germplasm resource resistant to powdery mildew. Seedling-stage genetic analysis combined with molecular marker genotyping revealed that resistance of L251 to wheat powdery mildew F17 is controlled by a single dominant gene, PmL251, on chromosome 2BL. Therefore, developing and validating molecular markers closely linked to or co-segregating with PmL251 is of great significance for breeding wheat varieties resistant to powdery mildew. Summary of the Invention
[0006] The purpose of this invention is to provide a molecular marker closely linked to the wheat powdery mildew resistance gene PmL251 and its application, so as to locate and detect the wheat powdery mildew resistance gene PmL251 using this molecular marker. Using this marker for marker-assisted selection of PmL251 can shorten the breeding cycle, improve breeding efficiency, and be better applied to wheat disease resistance breeding.
[0007] The present invention is achieved by the following method: a molecular marker closely linked to the wheat powdery mildew resistance gene PmL251 and its application, wherein the molecular marker is the INDEL marker YTUL-5 closely linked to PmL251; The upstream primer for the molecular marker YTUL-5 is YTUL-5-F, and its nucleotide sequence is as follows: 5'-TGCAGACCAAAAACAACTGTGA-3', as shown in SEQ ID NO: 1; The downstream primer for the molecular marker YTUL-5 is YTUL-5-R, and its nucleotide sequence is as follows: 5'-TGCAGGTTTCATGTTGTGGC-3', as shown in SEQ ID NO: 2; The marker primers for the molecular marker YTUL-5 were used to amplify the wheat genomic DNA to be tested by PCR. The corresponding amplification product had a molecular weight of 359 bp, which is a molecular marker closely linked to the wheat powdery mildew resistance gene PmL251.
[0008] The appropriate PCR amplification system for this molecular marker is 10 μL, comprising: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.25 μL of 5 μM upstream primer, 0.25 μL of 5 μM downstream primer, and 4.5 μL of sterile deionized water.
[0009] The applicable PCR amplification program for this molecular marker is as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; store at 4℃.
[0010] The electrophoretic separation procedure for the amplification products applicable to this molecular marker is as follows: electrophoresis is performed using an 8% non-denaturing polyacrylamide gel. The amplification product is mixed with 2.0 μL of 10× Loading Buffer, and 1.0 μL of the mixture is loaded onto the gel. Electrophoresis is performed at a constant voltage of 170 V for 1.5-2 h. The gel is then stained with silver nitrate and photographed.
[0011] The application of the primers provided in this invention, which are closely linked to the wheat powdery mildew resistance gene PmL251, in gene localization, map-based cloning, and marker-assisted selection breeding of the wheat powdery mildew resistance gene PmL251.
[0012] The application described in this invention, which detects whether a wheat variety carries the powdery mildew resistance gene PmL251, mainly includes the following steps: (1) Extract genomic DNA from fresh leaves of the wheat sample to be tested; (2) The extracted wheat genomic DNA was amplified by PCR using primers with the molecular marker YTUL-5 to obtain the amplification product; (3) If a specific band of 359 bp can be amplified, it indicates that the powdery mildew resistance gene PmL251 exists in the wheat to be tested; otherwise, the powdery mildew resistance gene PmL251 does not exist in the wheat to be tested.
[0013] In the application described above, the primers for the molecular marker YTUL-5 in step (2) include an upstream primer YTUL-5-F and a downstream primer YTUL-5-R. The nucleotide sequence of the upstream primer YTUL-5-F is shown in SEQ ID NO:1, namely: YTUL-5-F: 5'-TGCAGACCAAAAACAACTGTGA-3'; the nucleotide sequence of the downstream primer YTUL-5-R is shown in SEQ ID NO:2, namely: YTUL-5-R: 5'-TGCAGGTTTCATGTTGTGGC-3'.
[0014] The application of this molecular marker is based on a 10 μL PCR amplification system, which includes: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.25 μL of 5 μM upstream primer, 0.25 μL of 5 μM downstream primer, and 4.5 μL of sterile deionized water.
[0015] The applicable PCR amplification program for this marker is as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; store at 4℃.
[0016] Detection of PCR amplification products: Electrophoresis was performed using an 8% non-denaturing polyacrylamide gel. The amplification product was mixed with 2.0 μL of 10× Loading Buffer, and 1.0 μL of this mixture was loaded onto the gel. Electrophoresis was carried out at a constant voltage of 170 V for 1.5–2 h. The gel was then stained with silver nitrate and photographed. The electrophoresis results were used to determine the presence of the powdery mildew resistance gene PmL251 in the tested wheat germplasm. Otherwise, the powdery mildew resistance gene PmL251 was not present in the tested wheat germplasm.
[0017] This invention, through genetic analysis and molecular marker detection of seedling powdery mildew resistance, demonstrates that the resistance of Sperto wheat L251 seedlings to the prevalent powdery mildew strain F17 is controlled by a single dominant gene, named PmL251. Using 173 pairs of molecular markers evenly distributed throughout the genome, resistance was investigated in Sperto wheat L251, susceptible wheat L211, and the F17 strain derived from L251×L211. 2:3 Polymorphism was detected in disease-resistant and disease-susceptible pools composed of 10 homozygous resistant and 10 homozygous susceptible families. Seventeen pairs of markers showed consistent polymorphism in both the resistant and susceptible parents and the resistant-susceptible pools. These markers were then used to analyze the F1 generation of 219 L251×L211 pedigrees. 2:3 Genotyping of the family pedigree initially located PmL251 within the region of chromosome 701.81-704.33 Mb on wheat 2BL. Further, based on the sequence of the Chinese spring wheat reference genome within this region, the INDEL marker YTUL-5, closely linked to the PmL251 gene, was designed and screened using Primer 5.0 software. The molecular marker YTUL-5 for the wheat powdery mildew resistance gene PmL251 provided by this invention, after genetic segregation population testing, showed a genetic distance of only 2.2 cM from the PmL251 gene, indicating close linkage. This allows for more accurate and efficient detection of the genetic mapping population of PmL251, facilitating map-based cloning and localization of PmL251.
[0018] This invention provides a molecular marker closely linked to the wheat powdery mildew resistance gene PmL251 and its application. When applied to powdery mildew resistant wheat breeding, it can not only greatly save costs and shorten the breeding cycle, but also be more accurate and efficient, and can be better applied to powdery mildew resistant wheat breeding work. Attached Figure Description
[0019] Figure 1 To label YTUL-5 in 219 L251×L211 derived F 2:3 Partial amplification results from the family pedigree.
[0020] In the diagram, M: pUC19 Msp I; 1: L251 (disease-resistant parent); 2: L211 (susceptible parent); 3-17: F1 generation of L251 × L211. 2:3 The families are shown in the following order: 3-7: homozygous resistant families; 8-12: homozygous susceptible families; 13-17: resistant-susceptible segregating families; the arrows indicate specific bands of PmL251. Detailed Implementation
[0021] The following examples are provided to better understand and use 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.
[0022] Example 1: Development of the molecular marker YTUL-5 for the wheat powdery mildew resistance gene PmL251 1. Materials The disease-resistant parent is Spertoh wheat L251, and the disease-susceptible parent is Spertoh wheat L211. L251 and L211 are crossed, and the resulting F1 generation is self-crossed to obtain the F2 population.
[0023] 2. Extraction of wheat genomic DNA Wheat genomic DNA was extracted using the CTAB method, and the procedure is as follows: 1) Take tender fresh leaves of the wheat to be tested, freeze them quickly with liquid nitrogen, grind them into powder, and put them into 2 mL EP tubes; 2) Add 600-800 μL of CTAB extraction solution and incubate in a 65°C water bath for 1 h, inverting the container every ten minutes during the incubation period; 3) Add an equal volume of chloroform and mix on a shaker for 30 minutes; 4) Centrifuge at 8000 rpm at room temperature for 10 min, aspirate 400 μL of supernatant into a 1.5 mL EP tube, add 3 times the volume of pre-cooled 95% ethanol, mix well, and allow to settle at -20°C for 0.5 h. 5) Centrifuge at 12000 rpm at room temperature for 10 min, discard the supernatant, and wash three times with 800 μL of 75% ethanol; 6) Air dry the precipitate and dissolve it in 50 μL of 1×TE or ddH2O.
[0024] 7) Dilute the DNA storage solution with sterile deionized water to 50 ng / μL as a working solution for later use.
[0025] 3. Identification of powdery mildew resistance in wheat seedlings and genetic analysis of resistance Wheat seedling powdery mildew resistance identification was conducted in a greenhouse. The resistant parent L251, the susceptible parent L211, the F1 hybrid, and the F2 population were planted in 128-cell trays (3.2 × 3.2 × 4.2 cm). At least 20 seeds from each parent and F1 were identified. 2:3 Each family was identified with at least 20 seeds. The susceptible control TN18 was randomly sown and tagged for identification. Greenhouse conditions were controlled at 18-20°C, 80% relative humidity, and a photoperiod of 17 h light / 10 h dark. Powdery mildew strain F17 was inoculated using the sweeping method at the one-leaf stage. Phenotypic assessment was conducted 10-17 days later, when the susceptible control TN18 showed full disease development. Response types were 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.
[0026] The results showed that L251 was highly resistant to powdery mildew strain F17, while L211 was highly susceptible. All F1 plants were resistant, indicating that L251 carries a dominant resistance gene. Resistance identification of the F2 population of this combination showed a resistance-susceptibility segregation ratio of 162:57, which, according to the chi-square test, conformed to a 3:1 segregation ratio for a single dominant gene (χ²). 2 =0.12, P =0.73), for F 2:3 Further pedigree analysis revealed a segregation ratio of 53:109:57 between homozygous resistant, susceptible-resistant, and homozygous susceptible families. This ratio, as determined by the chi-square test, conformed to a 3:1 segregation ratio for a single dominant gene (χ²). 2 =0.15, P =0.93). In summary, the resistance of L251 to powdery mildew strain F17 is controlled by a single dominant gene, which is named PmL251.
[0027] 4. Molecular marker localization of PmL251 Based on the phenotypic identification results, 10 homozygous resistant families and 10 homozygous susceptible families were selected to construct resistant and susceptible pools, respectively. Polymorphism detection was performed on Spertoh wheat L251, Spertoh wheat L211, and the resistant and susceptible pools using 38 pairs of molecular markers evenly distributed throughout the genome. Twelve pairs of markers showed consistent polymorphism in both the resistant and susceptible parents and the resistant-susceptible pools. Subsequently, these markers were used to genotype 219 F2 populations of L251×L211, locating PmL251 within the interval 701.81-704.33 Mb on wheat chromosome 2BL.
[0028] 5. Development of molecular markers closely linked to PmL251 Based on the sequence information of the Chinese spring wheat reference genome within the candidate region of 701.81-704.33 Mb, INDEL markers were designed using Primer 5.0 software, and the F251×L21 ... 2:3 Genotyping of the family revealed the INDEL marker YTUL-5, which is closely linked to gene PmL251, with a genetic distance of 2.2 cM, indicating close linkage.
[0029] The primers for the molecular marker YTUL-5 consist of one upstream primer and one downstream primer: The nucleotide sequence of the upstream primer YTUL-5-F is: 5'-TGCAGACCAAAAACAACTGTGA-3'; The nucleotide sequence of the downstream primer YTUL-5-R is: 5'-TGCAGGTTTCATGTTGTGGC-3'.
[0030] The applicable PCR amplification system for this marker is 10 μL, including: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.25 μL of 5 μM upstream primer, 0.25 μL of 5 μM downstream primer, and 4.5 μL of sterile deionized water.
[0031] The applicable PCR amplification program for this marker is as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s; 55℃ annealing for 30 s; 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; storage at 4℃.
[0032] The electrophoretic separation procedure for the amplified products is as follows: Electrophoresis is performed using an 8% non-denaturing polyacrylamide gel. The amplified product is mixed with 2.0 μL of 10× Loading Buffer, and 1.0 μL of this mixture is loaded onto the gel. Electrophoresis is carried out at a constant voltage of 170 V for 1.5–2 h. After silver nitrate staining, the sample is photographed. If a specific band of 359 bp is amplified, it indicates the presence of the powdery mildew resistance gene PmL251 in the tested wheat germplasm; otherwise, the powdery mildew resistance gene PmL251 is not present in the tested wheat germplasm.
[0033] Molecular marker detection results are shown below Figure 1 .in Figure 1 To label YTUL-5 in 219 L251×L211 derived F 2:3 Partial amplification results from the family. In the figure, M: pUC19 Msp I; 1: L251 (resistant parent); 2: L211 (susceptible parent); 3-17: F1 generation of L251 × L211. 2:3The families are shown in the diagram: 3-7: homozygous resistant families; 8-12: homozygous susceptible families; 13-17: resistant-susceptible segregating families; the red arrows indicate the specific band for PmL251. Amplification results showed that the marker YTUL-5 amplified a specific band of 359 bp in the resistant parent L251 and the resistant families, but not in the susceptible parent L211 and the susceptible families.
[0034] The wheat powdery mildew resistance gene PmL251 originates from the Sperto wheat L251. Currently, there are no reports on the localization, map-based cloning, or molecular breeding of this gene, nor are there reports on molecular markers closely linked to it, thus highlighting its significant innovation. Using the molecular marker YTUL-5 provided in this invention to detect large-scale genetic mapping populations aids in the localization and map-based cloning of gene PmL251. After introducing PmL251 into major wheat varieties susceptible to powdery mildew, the molecular marker YTUL-5 developed in this invention can efficiently and accurately detect large-scale breeding populations, greatly improving the efficiency and accuracy of transferring the disease-resistant gene PmL251. This is of great significance for the efficient transfer of gene PmL251.
[0035] 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. A molecular marker tightly linked to the wheat powdery mildew resistance gene PmL251 and its application, wherein the upstream primer nucleotide sequence of the molecular marker YTUL-5 is shown in SEQ ID NO:1; the downstream primer nucleotide sequence is shown in SEQ ID NO:2; the marker primer of molecular marker YTUL-5 is used to perform PCR amplification on the wheat genomic DNA to be tested, and the corresponding amplification product with a molecular weight of 359 bp is obtained, which is the molecular marker tightly linked to the wheat powdery mildew resistance gene PmL251.
2. The molecular marker tightly linked to the wheat powdery mildew resistance gene PmL251 according to claim 1, wherein the applicable PCR amplification system for the marker is 10 μL, comprising: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCRMasterMix, 0.25 μL of 5 μM upstream primer, 0.25 μL of 5 μM downstream primer, and 4.5 μL of sterile deionized water.
3. The molecular marker closely linked to the wheat powdery mildew resistance gene PmL251 according to claim 1 or 2, wherein the applicable PCR amplification program for the marker is as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; storage at 4℃.
4. The application of a molecular marker closely linked to the wheat powdery mildew resistance gene PmL251 as described in any one of claims 1-3 in gene localization, map-based cloning, and marker-assisted breeding of the wheat powdery mildew resistance gene PmL251.
5. According to the application described in claim 4, detecting whether a test variety carries the wheat powdery mildew resistance gene PmL251 mainly includes the following steps: (1) Extract genomic DNA from the wheat sample to be tested; (2) The extracted wheat genomic DNA was amplified by PCR using primers with the molecular marker YTUL-5 to obtain the amplification product; (3) Perform electrophoresis on the amplified products and judge based on the electrophoresis results. If the wheat DNA can amplify a specific band of 359 bp, it indicates that the wheat to be tested contains the powdery mildew resistance gene PmL251; otherwise, the wheat to be tested does not contain the wheat powdery mildew resistance gene PmL251.
6. In the application according to claim 5, the primers for the molecular marker YTUL-5 in step (2) include an upstream primer YTUL-5-F and a downstream primer YTUL-5-R, wherein the nucleotide sequence of the upstream primer YTUL-5-F is shown in SEQ ID NO:1 and the nucleotide sequence of the downstream primer YTUL-5-R is shown in SEQ ID NO:
2.
7. The application according to claim 5, wherein the PCR amplification system for which the label is applicable is 10 μL, comprising: 1.0 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.25 μL of 5 μM upstream primer, 0.25 μL of 5 μM downstream primer, and 4.5 μL of sterile deionized water.
8. According to claim 5, the applicable PCR amplification program for the label is as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; storage at 4℃.