Molecular marker closely linked with wheat powdery mildew resistance gene PmL424 and application of molecular marker

Abstract

The invention discloses a molecular marker closely linked with a wheat powdery mildew resistance gene PmL424. The molecular marker is YTU024-L10; the nucleotide sequence of an upstream primer of the molecular marker is as shown in SEQ ID NO: 1; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO: 2; a marker primer of the molecular marker YTU024-L10 is used for carrying out PCR (Polymerase Chain Reaction) amplification on wheat genome DNA (Deoxyribose Nucleic Acid) to be detected to obtain a corresponding amplification product with the molecular weight of 238 bp, namely the molecular marker closely linked with the wheat powdery mildew resistance gene PmL424. The molecular marker provided by the invention can more accurately and efficiently detect a genetic mapping population of PmL424, and map-based cloning and fine positioning of PmL424 are facilitated; and secondly, the marker is used for molecular marker-assisted selection of PmL424, so that the wheat breeding cycle can be shortened, and the breeding efficiency can be improved.

Description

Technical Field

[0001] The present invention relates to the field of biological genetic engineering, and particularly relates to a molecular marker closely linked to the wheat powdery mildew resistance gene PmL424 and its application. Background Art

[0002] Wheat powdery mildew is a major disease that seriously threatens wheat production, and its pathogen is Blumeria graminis Blumeria graminis f. sp. tritici ( Bgt )). Wheat powdery mildew can cause a significant reduction in the yield of affected wheat fields, generally reducing production by 5 - 10%. In severely affected areas, the yield reduction can even reach 30 - 50%. Studying the powdery mildew resistance genes carried in different wheats and then breeding excellent disease-resistant varieties is an important way to control wheat powdery mildew. So far, more than 140 powdery mildew resistance genes have been reported for different wheat types. However, with the continuous evolution of pathogen races, many resistance genes have lost their resistance. Therefore, to reduce the occurrence of powdery mildew and improve wheat yield and quality, it is necessary to continuously explore new sources of resistance. Ryegrass ( Secale cereale L , 2n = 2x = 14, RR), Agropyron cristatum ( Leynus secalinus , 2n =2x = 14, PP), Dasypyrum villosum ( Dasypyrum villosum , 2n = 2x = 14, VV) and other wheat related species, as well as Aegilops tauschii ( Aegilops tauschii , 2n = 2x = 14, DD), Triticum urartu ( Triticum urartu , 2n = 2x= 14, AA), Triticum monococcum ( Triticum monococcum L, 2n = 2x = 14, AA), Triticum dicoccoides ( Triticum turgidum , 2n = 4x = 28, AABB), Triticum timopheevii ( Triticum timopheevii , 2n =4x = 28, AAGG) and other ancestor species of common wheat are all precious resource treasuries containing disease-resistant characteristics. Among the recorded powdery mildew resistance genes, more than half are derived from the above species resources.

[0003] As a tetraploid species, Triticum dicoccoides ( Triticum dicoccum , 2n = 4x = 28, AABB) shares the A and B genomes with common wheat and is generally regarded as the ancestor of common wheat in the academic community. This species has rich excellent traits, especially showing significant advantages in coping with biotic and abiotic stresses. Taking the powdery mildew resistance gene resources as an example, important genes such as Pm4a, Pm49, Pm50, and Pm71 are all derived from Triticum dicoccoides.

[0004] In recent years, wheat breeding techniques have been continuously innovated. Molecular marker-assisted selection breeding has gradually become the core method for wheat powdery mildew resistance breeding. Having efficient, accurate, and easy-to-use molecular markers is the basis for carrying out molecular marker-assisted selection of target genes. Tracking and detecting target genes through molecular markers can significantly shorten the breeding cycle and greatly improve breeding efficiency.

[0005] The cultivated emmer wheat L424 has excellent agronomic traits. In multi-year and multi-location field appraisals, it also shows good resistance to powdery mildew and is an extremely excellent wheat germplasm resource for powdery mildew resistance. Through genetic analysis of powdery mildew resistance at the seedling stage and molecular marker detection, it was found that at the seedling stage, the resistance of L424 to the powdery mildew prevalent strain E09 is controlled by a pair of dominant genes PmL424 located on wheat chromosome 2BS. This gene belongs to a newly discovered wheat powdery mildew resistance gene / allele. Developing molecular markers tightly linked to the gene PmL424 and applying them to molecular marker-assisted selection breeding of the wheat powdery mildew resistance gene PmL424 is of crucial significance for breeding wheat varieties resistant to powdery mildew and achieving effective prevention and control of powdery mildew. Summary of the Invention

[0006] The purpose of the present invention is to provide a molecular marker tightly linked to the wheat powdery mildew resistance gene PmL424 and its application, using this molecular marker to conduct gene mapping and detection of the wheat powdery mildew resistance gene PmL424. Using this marker for molecular marker-assisted selection of PmL424 can shorten the breeding cycle and improve breeding efficiency.

[0007] The present invention is achieved by the following methods: A molecular marker tightly linked to the wheat powdery mildew resistance gene PmL424, which is the co-dominant InDel marker YTU024-L10; The upstream primer of the molecular marker YTU024-L10 is YTU024-L10-F, and its nucleotide sequence is: 5'-GCCTGGCTTCATATGCTGTG-3', as shown in SEQ ID NO: 1; The downstream primer of the molecular marker YTU024-L10 is YTU024-L10-R, and its nucleotide sequence is: 5'-TTCTCGGATCAGTAGGCCCT-3', as shown in SEQ ID NO: 2; Using the marker primers of the molecular marker YTU024-L10 to perform PCR amplification on the genomic DNA of the wheat to be tested, and obtaining the corresponding amplified product with a molecular weight of 238 bp, which is the molecular marker tightly linked to the wheat powdery mildew resistance gene PmL424.

[0008] The PCR amplification system applicable to this molecular 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.

[0009] The PCR amplification program applicable to this molecular marker is: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 40 s, for 35 cycles; extension at 72°C for 10 min; storage at 4°C.

[0010] The electrophoresis separation program for the amplification products applicable to this molecular marker is: electrophoresis is carried out using an 8% non-denaturing polyacrylamide gel. After mixing the amplification products with 2.5 μL of 10× Loading Buffer, 1.2 μL of this mixture is taken for sample loading, and electrophoresis is carried out under a constant voltage of 220 V for 1.5 - 2 h. After silver nitrate staining, photographing is carried out.

[0011] The application of the molecular marker provided by the present invention that is tightly linked to the wheat powdery mildew resistance gene PmL424 in gene mapping, map-based cloning, and molecular marker-assisted selection breeding of the wheat powdery mildew resistance gene PmL424.

[0012] For the application described in the present invention, detecting whether the tested variety carries the wheat powdery mildew resistance gene PmL424 mainly includes the following steps: (1) Extract the genomic DNA of fresh leaves of the tested wheat sample; (2) Use the primer pair of the molecular marker YTU024-L10 to perform PCR amplification on the extracted wheat genomic DNA to obtain amplification products; (3) If a specific band of 238 bp can be amplified, it indicates that the tested wheat contains the powdery mildew resistance gene PmL424; otherwise, the tested wheat does not contain the wheat powdery mildew resistance gene PmL424.

[0013] For the application described above, the primers of the molecular marker YTU024-L10 in step (2) include the upstream primer YTU024-L10-F and the downstream primer YTU024-L10-R. The nucleotide sequence of the upstream primer YTU024-L10-F is as shown in SEQ ID NO:1, that is: YTU024-L10-F: 5'-GCCTGGCTTCATATGCTGTG-3'; the nucleotide sequence of the downstream primer YTU024-L10-R is as shown in SEQ ID NO:2, that is: YTU024-L10-R: 5'-TTCTCGGATCAGTAGGCCCT-3'.

[0014] Application of the molecular marker. 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.

[0015] The applicable PCR amplification program for this marker is: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 40 s, for 35 cycles; extension at 72°C for 10 min; preservation at 4°C.

[0016] Detection of PCR amplification products: Electrophoresis is carried out using an 8% non-denaturing polyacrylamide gel. After mixing the amplification products with 2.5 μL of 10× Loading Buffer, 1.2 μL of this mixture is taken for spotting. Electrophoresis is carried out at a constant voltage of 220 V for 1.5 - 2 h, followed by silver nitrate staining and photographing. Judgments are made based on the electrophoresis results. If a specific band of 238 bp can be amplified, it indicates the presence of the powdery mildew resistance gene PmL424 in the tested wheat germplasm; otherwise, the wheat powdery mildew resistance gene PmL424 is absent in the tested wheat germplasm.

[0017] Genetic analysis of powdery mildew resistance at the seedling stage and molecular marker detection in this invention show that the resistance of cultivated emmer wheat L424 to the powdery mildew prevalent strain E09 at the seedling stage is controlled by a single dominant gene, which is named PmL424. Using 155 pairs of molecular markers evenly distributed across the whole genome to conduct polymorphism detection on cultivated emmer wheat L424, susceptible wheat Langdon (LDN), and the resistant pool and susceptible pool composed of 10 homozygous resistant and 10 homozygous susceptible families in the F2 population of L424×Langdon (LDN), 22 pairs of markers showed consistent polymorphisms in the resistant and susceptible parents and the resistant and susceptible pools. Subsequently, these markers were used to genotype 185 F2 populations of L424×Langdon (LDN), and PmL424 was initially mapped to the interval of 21.62 - 21.81 Mb on chromosome 2BS of wheat. Further, according to the sequence of the Chinese Spring wheat reference genome in this interval, the Insertion-Deletion (INDEL) marker YTU024-L10 tightly linked to the gene PmL424 was designed and screened using primer5.0 software. The molecular marker YTU024-L10 of the wheat powdery mildew resistance gene PmL424 provided in this invention has been tested in a genetic segregation population, and its genetic distance from the gene PmL424 is only 0.5 cM, which is tightly linked to PmL424. It can more accurately and efficiently detect the genetic mapping population of PmL424, facilitating the map-based cloning and fine mapping of PmL424.

[0018] The present invention provides molecular markers closely linked to the wheat powdery mildew resistance gene PmL424, which are applied in the breeding of powdery mildew-resistant wheat. It can not only greatly save costs and reduce the breeding cycle, but also be more accurate and efficient, and can be better applied to the breeding work of powdery mildew-resistant wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the partial amplification result of the marker YTU024-L10 in 185 F2 populations derived from L424 × Langdon (LDN).

[0020] In the figure, M: pUC19 Msp I; 1: L424 (disease-resistant parent); 2: Langdon (LDN) (disease-susceptible parent); 3-17: F2 population of L424×Langdon (LDN), among which, 3-7: homozygous disease-resistant families, 8-12: disease-resistant and disease-susceptible segregating families, 13-17: homozygous disease-susceptible families; the white arrow is the specific band of PmL424. DETAILED IMPLEMENTATION MANNER

[0021] The following examples are used to better understand and use the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials, reagents, etc. used in the examples can be obtained from commercial channels unless otherwise specified.

[0022] Example 1 Development of the molecular marker YTU024-L10 of the wheat powdery mildew resistance gene PmL424.

[0023] 1. Materials The disease-resistant parent is the cultivated emmer wheat L424, and the disease-susceptible parent is the durum wheat Langdon (LDN). L424 and Langdon (LDN) are crossed, and the obtained F1 is self-crossed to obtain an F2 population.

[0024] 2. Extraction of wheat genomic DNA The CTAB method is used for the extraction of wheat genomic DNA, and the process is as follows: 1) Take the young and fresh leaves of the wheat to be tested, quickly grind them into powder after being frozen in liquid nitrogen, and load them into a 2 mL EP tube; 2) Add 600 - 800 μL of CTAB extraction solution, water bath at 65°C for 1 h, and invert and mix every ten minutes during this period; 3) Add an equal volume of chloroform, and mix well on a shaker for 30 min; 4) Centrifuge at 8000 rpm for 10 min at room temperature, aspirate 400 μL of the supernatant into a 1.5 mL EP tube, add 3 volumes of pre-cooled 95% ethanol in advance, mix well, and precipitate at -20°C for 0.5 h; 5) Centrifuge at 12000 rpm for 10 min at room temperature, discard the supernatant, and wash 3 times with 800 μL of 75% ethanol; 6) Air-dry the precipitate and dissolve it with 50 μL of 1×TE or ddH2O; 7) Dilute the DNA stock solution with sterile deionized water to 50 ng / μL as the working solution for standby.

[0025] 3. Identification of powdery mildew resistance and genetic analysis of resistance at the wheat seedling stage The identification of powdery mildew resistance at the wheat seedling stage was completed in the greenhouse. The resistant parent L424, the susceptible parent Langdon (LDN), the F1 hybrid, and the F2 population were planted in 128-cell trays (3.2×3.2×4.2 cm). At least 20 seeds of the parents and F1 were identified, and at least 25 seeds of each F2 population were identified. The susceptible control TN18 was randomly sown and labeled for distinction. The greenhouse conditions were controlled at 18 - 20°C, relative humidity 80%, and photoperiod 14 h light / 10 h dark. At the one-leaf stage, the powdery mildew strain E09 was inoculated by the brushing method. After 10 - 14 days, when the susceptible control TN18 was fully diseased, the phenotypes were investigated. The reaction type (Infection type, IT) was recorded according to the 0 - 4 grade standard. The resistance level was divided as follows: 0 - 2 grades were the resistant type, and 3 - 4 grades were the susceptible type.

[0026] The investigation results showed that: L424 showed high resistance (IT = 0) to the powdery mildew strain E09, Langdon (LDN) showed high susceptibility (IT = 4), and all F1 plants showed resistance (IT was 0 - 1), indicating that L424 carried a dominant resistance gene; the resistance of the F2 population of this combination was identified, and the results showed that the segregation ratio of resistant to susceptible was 127:58, and the chi-square test conformed to the segregation ratio of a single dominant gene of 3:1 (χ 2 = 3.98, P = 0.046). In summary, the resistance of L424 to the powdery mildew strain E09 was controlled by a single dominant gene, and this powdery mildew resistance gene was named PmL424.

[0027] 4. Fine mapping of molecular markers for PmL424 According to the phenotypic identification results, 10 homozygous disease-resistant families and 10 homozygous disease-susceptible families were respectively selected to construct a disease-resistant pool and a disease-susceptible pool. 155 pairs of molecular markers evenly distributed across the whole genome were used to detect polymorphisms in cultivated emmer wheat L424, durum wheat Langdon (LDN), the disease-resistant pool, and the disease-susceptible pool. 22 pairs of markers showed consistent polymorphisms in the resistant and susceptible parents and the resistant and susceptible pools. Subsequently, these markers were used to genotype an F2 population of 185 L424×Langdon (LDN), and PmL424 was initially mapped to the interval of 21.62 - 21.81 Mb on chromosome 2BS of wheat.

[0028] 5. Development of molecular markers tightly linked to PmL424 Based on the sequence information of the Chinese Spring wheat reference genome in the candidate interval of 21.62 - 21.81 Mb, Insertion-Deletion (INDEL) markers were designed using primer5.0 software and used to genotype the F2 population of L424×Langdon (LDN) to obtain the INDEL marker YTU024-L10 tightly linked to the gene PmL424, with a genetic distance of only 0.5 cM.

[0029] The primers of the molecular marker YTU024-L10 include an upstream primer and a downstream primer: The nucleotide sequence of the upstream primer YTU024-L10-F: 5'-GCCTGGCTTCATATGCTGTG-3'; The nucleotide sequence of the downstream primer YTU024-L10-R: 5'-TTCTCGGATCAGTAGGCCCT-3'.

[0030] The PCR amplification system suitable 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 PCR amplification program suitable for this marker is: pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 30 s; annealing at 55℃ for 30 s; extension at 72℃ for 40 s, for 35 cycles; extension at 72℃ for 10 min; and storage at 4℃.

[0032] The electrophoresis separation procedure of the amplification product is as follows: Electrophoresis is carried out using an 8% non-denaturing polyacrylamide gel. After mixing the amplification product with 2.5 μL of 10× Loading Buffer, 1.2 μL of this mixture is taken for spotting, and electrophoresis is carried out under a constant voltage of 220 V for 1.5 - 2 h. After silver nitrate staining, photography is performed. If a specific band of 238 bp can be amplified, it indicates that the wheat germplasm to be tested contains the powdery mildew resistance gene PmL424. Otherwise, the wheat germplasm to be tested does not contain the wheat powdery mildew resistance gene PmL424.

[0033] The results of molecular marker detection are shown in Figure 1 。 Figure 1 It is a partial amplification result of the marker YTU024-L10 in 185 L424×Langdon (LDN)-derived F2 populations. In the figure, M: pUC19 Msp I; 1: L424 (resistant parent); 2: Langdon (LDN) (susceptible parent); 3 - 17: F2 population of L424×Langdon (LDN), among which, 3 - 7: homozygous resistant families, 8 - 12: resistant-susceptible segregating families, 13 - 17: homozygous susceptible families; the white arrow is the specific band of PmL424. The amplification results show that the marker YTU024-L10 amplified a specific band of 238 bp in the resistant parent L424 and resistant families, and did not amplify the target band in the susceptible parent Langdon (LDN) and susceptible families.

[0034] The wheat powdery mildew resistance gene PmL424 is derived from the cultivated emmer wheat L424 and is a new gene with excellent resistance. Currently, there are no relevant reports on the mapping, map-based cloning, and molecular breeding of this gene. By using the molecular marker YTU024-L10 provided by the present invention to detect large genetic mapping populations, it is beneficial to achieve the fine mapping and map-based cloning of the gene PmL424. Introducing PmL424 into the main cultivated wheat varieties susceptible to powdery mildew and using the molecular marker YTU024-L10 developed by the present invention can efficiently and accurately detect large breeding populations, greatly improving the efficiency and accuracy of transferring the resistant gene PmL424, which is of great significance for the efficient introgression of the gene PmL424 and the in-depth analysis of the disease resistance mechanism.

[0035] The above embodiments are the optimized implementation schemes of the present invention, which are only used to illustrate the present invention rather than limit the present invention. Modifications or equivalent substitutions made by those skilled in the art without departing from the purpose and principle of the implementation scheme of the present invention all belong to the scope protected by the present invention.

Claims

1. A molecular marker closely linked to the wheat powdery mildew resistance gene PmL424. This molecular marker is YTU024-L10. The nucleotide sequence of its upstream primer is as shown in SEQ ID NO:1; the nucleotide sequence of its downstream primer is as shown in SEQ ID NO:

2. Using the marker primers of the molecular marker YTU024-L10 to perform PCR amplification on the genomic DNA of the wheat to be tested, the molecular weight of the corresponding amplification product obtained is 238 bp, which is the molecular marker closely linked to the wheat powdery mildew resistance gene PmL424.

2. The molecular marker closely linked to the wheat powdery mildew resistance gene PmL424 according to claim 1, wherein the applicable PCR amplification system for the marker is 10 μL and 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, 4.5 μL of sterile deionized water.

3. The molecular marker closely linked to the wheat powdery mildew resistance gene PmL424 according to claim 1 or 2. The applicable PCR amplification program for the marker is: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 40 s, for 35 cycles; extension at 72°C for 10 min; preservation at 4°C.

4. Application of the molecular marker closely linked to the wheat powdery mildew resistance gene PmL424 according to any one of claims 1-3 in gene mapping, map-based cloning and molecular marker-assisted breeding of the wheat powdery mildew resistance gene PmL424.

5. According to the application described in claim 4, detecting whether the wheat variety to be tested carries the wheat powdery mildew resistance gene PmL424 mainly includes the following steps: (1) Extract the genomic DNA of the wheat sample to be tested; (2) Use the primers of the molecular marker YTU024-L10 to perform PCR amplification on the extracted wheat genomic DNA to obtain an amplification product; (3) Electrophorese the amplification product and make a judgment according to the electrophoresis result. If the wheat DNA can amplify a specific band of 238 bp, it indicates that the wheat to be tested contains the powdery mildew resistance gene PmL424; otherwise, the wheat to be tested does not contain the wheat powdery mildew resistance gene PmL424.

6. According to the application described in claim 5, the primers of the molecular marker YTU024-L10 in step (2) include the upstream primer YTU024-L10-F and the downstream primer YTU024-L10-R. The nucleotide sequence of the upstream primer YTU024-L10-F is as shown in SEQ ID NO:1, and the nucleotide sequence of the downstream primer YTU024-L10-R is as shown in SEQ ID NO:

2.

7. The application according to claim 5, wherein the PCR amplification system applicable to the label is 10 μL and comprises: 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, 4.5 μL of sterile deionized water.

8. The PCR amplification program applicable to the label in the application according to claim 5 is: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 40 s, for 35 cycles; extension at 72°C for 10 min; storage at 4°C.

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