Molecular marker closely linked with wheat powdery mildew resistance gene PmJ684 and application of molecular marker

Abstract

The invention discloses a molecular marker closely linked with a wheat powdery mildew resistance gene PmJ684 and application of the molecular marker, and relates to the technical field of biological genetic engineering. The molecular marker is YTULL-40, and the molecular marker is a molecular marker The nucleotide sequence of an upstream primer of the marker is as shown in SEQ ID NO: 1; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO: 2; the molecular marker is used for carrying out PCR (Polymerase Chain Reaction) amplification on to-be-detected wheat genome DNA (Deoxyribonucleic Acid) to obtain a corresponding amplification product with the molecular weight of 172bp. According to the molecular marker YTULL-40 closely linked with the wheat powdery mildew resistance gene PmJ684, provided by the invention, a PmJ684 genetic mapping group can be accurately detected, and fine positioning and cloning of the PmJ684 are facilitated. Besides, the marker is used for assisted breeding, germplasm materials with the powdery mildew resistance gene PmJ684 can be accurately selected, the breeding period is shortened, and convenience is provided for wheat breeding.

Description

Technical Field

[0001] The present invention relates to the field of biological genetic engineering technology, specifically a method for generating a wheat powdery mildew resistance gene. PmJ684 Tightly linked molecular markers and their applications. Background Art

[0002] Blumeria graminis Blumeria graminis f. sp. tritici ( Bgt ) is a major disease that causes wheat and spreads rapidly, seriously affecting wheat grain production. Wheat infection with powdery mildew generally reduces yield by 5-20%. In severe cases, it can reach 30-50%. There are many measures to prevent and control wheat powdery mildew, such as agricultural control, chemical control, biological control, planting disease-resistant varieties, etc. Among them, planting disease-resistant varieties is one of the most effective and convenient methods. By selecting and cultivating high-resistance powdery mildew varieties, the impact of powdery mildew on wheat yield can be effectively reduced. To date, more than 100 powdery mildew (Pm) resistance genes or alleles have been discovered from wheat and its closely related genera. However, with the passage of time and the continuous evolution of powdery mildew, many powdery mildew resistance genes have become ineffective, so finding new resistance genes has become a top priority in powdery mildew prevention and control.

[0003] Cultivated emmer wheat ( Triticum dicoccum , 2n = 4x = 28, AABB) is an ancient tetraploid wheat, a key intermediate species in the evolution of the genus Triticum, and plays an important role in the formation of modern hexaploid common wheat (such as bread wheat). Cultivated emmer wheat carries multiple disease resistance genes (such as powdery mildew resistance genes). Pm3h 、 Pm4a 、 Pm49 and Pm68 、 Pm71 ), has been widely used in modern wheat disease resistance breeding. Cultivated emmer wheat is not only a "living fossil" of the origin of agriculture, but also an irreplaceable genetic resource for modern wheat breeding.

[0004] Traditional breeding relies primarily on phenotypic observation and selection, a method that is not only time-consuming and labor-intensive but also susceptible to environmental factors, resulting in low selection accuracy. In contrast, marker-assisted selection (MAS) enables direct genotype detection and targeted selection by identifying molecular markers that are tightly linked or co-segregate with target genes. The advantages of this technology include: on the one hand, it can quickly and accurately identify resistance genes carried by different germplasm materials, significantly improving screening efficiency; on the other hand, it can help breeders precisely identify germplasm resources with desirable traits such as powdery mildew resistance, thereby accelerating the breeding process and promoting the dissemination of superior varieties. Therefore, MAS technology provides an efficient and precise molecular breeding method for modern crop breeding.

[0005] The cultivated emmer wheat AABB684 has outstanding overall performance in the field. Continuous multi-site trials have shown that it has excellent agronomic traits and strong resistance to powdery mildew, making it a disease-resistant donor material with great application potential. We hybridized the cultivated emmer wheat AABB684 with AABB620 to construct a genetic segregation population. After infection with the powdery mildew epidemic strain E09, we found that the resistance ratio of the secondary segregation population was 3:1, which is consistent with Mendel's law of inheritance. Therefore, we believe that the resistance of AABB684 is caused by a dominant gene. PmJ684 Control, this gene belongs to the newly discovered wheat powdery mildew resistance gene / allele. PmJ684 The closely linked molecular marker YTULL-40 is not only PmJ684 It lays the foundation for positional cloning and can also be used to screen high-quality disease-resistant wheat resources, accelerate the breeding cycle and improve breeding efficiency. Summary of the Invention

[0006] The purpose of the present invention is to provide a wheat powdery mildew resistance gene PmJ684 Tightly linked molecular markers and their application for identifying wheat powdery mildew resistance genes using the molecular markers PmJ684 Use this marker to locate and detect genes. PmJ684 Molecular marker-assisted selection can shorten the breeding cycle, improve breeding efficiency, and facilitate wheat disease-resistant breeding.

[0007] The present invention is achieved by the following method: a wheat powdery mildew resistance gene PmJ684 A tightly linked molecular marker, the molecular marker is the codominant InDel marker YTULL-40; The upstream primer of the molecular marker YTULL-40 is YTULL-40-F, and its nucleotide sequence is: 5'-ACACCAGGGGATGAAATGAAAC-3', as shown in SEQ ID NO: 1; The downstream primer of the molecular marker YTULL-40 is YTULL-40-R, and its nucleotide sequence is: 5'-CGGGTTAGATGCGAAAACTGC-3', as shown in SEQ ID NO: 2; The PCR amplification of the wheat genomic DNA was performed using the marker primers of the molecular marker YTULL-40, and the corresponding amplified product band was 172 bp, which is the wheat powdery mildew resistance gene. PmJ684 Tightly linked molecular markers.

[0008] The PCR amplification system suitable for 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 procedure for this molecular marker is as follows: pre-denaturation at 95°C for 2 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 40 s; extension at 72°C for 10 min; and storage at 4°C.

[0010] The electrophoresis separation procedure for the amplified product suitable for this molecular marker is as follows: use 8% non-denaturing polyacrylamide gel for electrophoresis, mix the amplified product with 2.5 μL 10× Loading Buffer, take 1.2 μL of the mixture for spotting, electrophoresis at a constant voltage of 140 V for 1.5-2 h, and take silver nitrate staining and photographing.

[0011] The wheat powdery mildew resistance gene provided by the present invention PmJ684 Tightly linked molecular markers in wheat powdery mildew resistance genes PmJ684 Gene mapping and its application in molecular marker-assisted selection breeding.

[0012] The application of the present invention is to detect whether the tested variety carries the wheat powdery mildew resistance gene PmJ684 The main steps include: (1) Extracting genomic DNA from fresh leaves of the wheat sample to be tested; (2) PCR amplification of the extracted wheat genomic DNA was performed using primers of the molecular marker YTULL-40 to obtain an amplified product; (3) If a specific band of 172 bp can be amplified, it indicates that the powdery mildew resistance gene exists in the wheat to be tested. PmJ684 Otherwise, the wheat powdery mildew resistance gene does not exist in the tested wheat PmJ684 .

[0013] In the application, the primers for the molecular marker YTULL-40 in step (2) include an upstream primer YTULL-40-F and a downstream primer YTULL-40-R, the nucleotide sequence of the upstream primer YTULL-40-F is shown in SEQ ID NO: 1, i.e., YTULL-40-F: 5'-ACACCAGGGGATGAAATGAAAC-3'; the nucleotide sequence of the downstream primer YTULL-40-R is shown in SEQ ID NO: 2, i.e., YTULL-40-R: 5'-CGGGTTAGATGCGAAAACTGC-3'.

[0014] The application of this molecular marker is that 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.

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

[0016] Detection of PCR amplification products: Use 8% non-denaturing polyacrylamide gel electrophoresis. Mix the amplified product with 2.5 μL of 10× Loading Buffer, take 1.2 μL of this mixture for spotting, and run electrophoresis at 140 V for 1.5-2 hours. Stain with silver nitrate and take a picture. Based on the electrophoresis results, if a specific band of 172 bp is amplified, it indicates that the powdery mildew resistance gene is present in the wheat germplasm to be tested. PmJ684 Otherwise, the wheat powdery mildew resistance gene does not exist in the tested wheat germplasm. PmJ684 .

[0017] The present invention shows that the resistance of cultivated emmer wheat AABB684 to the powdery mildew epidemic strain E09 at the seedling stage is controlled by a single dominant gene through genetic analysis of powdery mildew resistance at the seedling stage and molecular marker detection. PmJ684 Using 300 pairs of molecular markers uniformly distributed throughout the genome, we identified the pathogenicity of cultivated emmer wheat AABB684, susceptible wheat AABB620, and the F-type hybrid of AABB684×AABB620. 2:3 Polymorphisms were detected in the disease-resistant pool and the disease-susceptible pool, which consisted of 10 homozygous disease-resistant and 10 homozygous disease-susceptible families in the family. 14 pairs of markers showed consistent polymorphisms in the resistant and susceptible parents and the resistant and susceptible pool. These markers were then used to genotype 600 F clones of AABB684×AABB620.2:3 The genotype of the family was PmJ684 It was initially located in the interval of 16.28-17.01Mb on wheat chromosome 2BS. Based on the sequence of durum wheat reference genome in this interval, primer5.0 software was used to design and screen the genes. PmJ684 Tightly linked Insertion-Deletion (InDel) marker YTULL-40. The wheat powdery mildew resistance gene provided by the present invention PmJ684 The molecular marker YTULL-40 was tested in genetic segregation populations and was PmJ684 The genetic distance between PmJ684 Tight linkage enables more accurate and efficient detection PmJ684 Genetic mapping populations are beneficial for PmJ684 Positional cloning and fine positioning.

[0018] The present invention provides a wheat powdery mildew resistance gene PmJ684 Tightly linked molecular markers, when used in powdery mildew-resistant wheat breeding, can not only greatly save costs and reduce breeding cycles, but are also more accurate and efficient, and can be better applied in powdery mildew-resistant wheat breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results show that the marker YTULL-40 was partially amplified in 600 F2:3 families derived from AABB684 × AABB620.

[0020] M in the figure: pUC19 Msp I; 1: AABB684 (resistant parent); 2: AABB620 (susceptible parent); 3-17: F of AABB684×AABB620 2:3 Families, 3-7: homozygous disease-resistant families, 8-12: resistance-susceptibility segregation families, 13-17: homozygous disease-susceptibility families; white arrows indicate PmJ684 Specific bands. DETAILED DESCRIPTION

[0021] The following examples are provided to facilitate understanding and application of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials and reagents used in the examples, unless otherwise specified, are commercially available.

[0022] Example 1 Wheat Powdery Mildew Resistance Gene PmJ684 Development of the molecular marker YTULL-40 Material The resistant parent is cultivated emmer wheat AABB684, and the susceptible parent is cultivated emmer wheat AABB620. AABB684 and AABB620 were crossed, and the resulting F1 was self-pollinated to obtain F 2:3 Family lineage.

[0023] 2. Extraction of wheat genomic DNA Wheat genomic DNA was extracted using the CTAB method, and the process is as follows: 1) Take young fresh leaves of wheat to be tested, freeze them in liquid nitrogen, grind them into powder, and put them into 2 mL EP tubes; 2) Add 600-800 μL of CTAB extract and incubate at 65°C in a water bath for 1 h, mixing by inversion every 10 minutes. 3) Add an equal volume of chloroform and mix on a shaker for 30 min; 4) Centrifuge at 8000 rpm for 10 min at room temperature. Pipette 400 μL of the supernatant into a 1.5 mL EP tube. Add 3 volumes of pre-chilled 95% ethanol, mix well, and incubate at -20°C for 0.5 h. 5) Centrifuge at 12,000 rpm for 10 min at room temperature, 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 stock solution with sterile deionized water to 50 ng / μL as working solution.

[0025] 3. Identification of powdery mildew resistance and genetic analysis of resistance in wheat seedlings The identification of wheat powdery mildew resistance at seedling stage was completed in a greenhouse. 2:3 The families were planted in a 128-hole tray (3.2×3.2×4.2 cm). At least 20 seeds were identified from each parent and F1. 2:3 At least 25 seeds were identified from each family, and the susceptible control, Tainong 18, was randomly sown and labeled for differentiation. Greenhouse conditions were maintained at 18-20°C, 80% relative humidity, and a photoperiod of 14 hours light / 10 hours dark. Powdery mildew strain E09 was inoculated at the one-leaf stage using the sweeping method. Phenotypic analysis was performed 10-14 days later, when the susceptible control, Tainong 18, had fully developed disease. The infection type (IT) was recorded on a scale of 0-4. Disease resistance was categorized as 0-2 for resistant and 3-4 for susceptible.

[0026] The results showed that AABB684 was highly resistant to powdery mildew strain E09 (IT=0), while AABB620 was highly susceptible (IT=4). All F1 plants were resistant (IT=0-1), indicating that AABB684 carried a dominant resistance gene. Resistance identification of the F2 plants of this combination showed a resistant-susceptible segregation ratio of 188:60, which was consistent with the segregation ratio of a single dominant gene (χ2 test). 2 =0.086, P =0.769). Further, the derived F 2:3 The results showed that the segregation ratio of homozygous resistant families: resistant and susceptible families: homozygous susceptible families was 68:120:60 (χ 2 1:2:1 =0.77, P =0.68). In summary, the resistance of AABB684 to powdery mildew strain E09 is controlled by a single dominant gene, and this powdery mildew resistance gene is named PmJ684 .

[0027] 4. PmJ684 Fine mapping of molecular markers Based on the phenotypic identification results, 10 homozygous resistant families and 10 homozygous susceptible families were selected to construct disease-resistant and disease-susceptible pools, respectively. Using 300 pairs of molecular markers uniformly distributed throughout the genome, polymorphisms were detected in cultivated emmer wheat AABB684, cultivated emmer wheat AABB620, and the disease-resistant and disease-susceptible pools. Fourteen pairs of markers showed consistent polymorphisms in the resistant and susceptible parents and the disease-susceptible pools. These markers were then used to identify 600 F pairs of AABB684×AABB620. 2:3 The genotype of the family was PmJ684 It was initially located in the interval of 16.28-17.01Mb on wheat chromosome 2BS.

[0028] 5. With PmJ684 Development of tightly linked molecular markers According to the sequence information of the durum wheat reference genome within the candidate interval of 16.28-17.01 Mb, Insertion-Deletion (InDel) markers were designed using primer5.0 software to identify the F of AABB684×AABB620. 2:3 The pedigree was genotyped and the gene PmJ684 The tightly linked InDel marker YTULL-40 has a genetic distance of only 0.4 cM.

[0029] The primers for the molecular marker YTULL-40 include an upstream primer and a downstream primer: Nucleotide sequence of the upstream primer YTULL-40-F: 5′-ACACCAGGGGATGAAATGAAAC-3′; The nucleotide sequence of the downstream primer YTULL-40-R is: 5'-CGGGTTAGATGCGAAAACTGC-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 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 for this marker is as follows: 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, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0032] The electrophoresis separation procedure for the amplified product is as follows: The electrophoresis separation procedure for the amplified product suitable for this molecular marker is as follows: Use 8% non-denaturing polyacrylamide gel electrophoresis, mix the amplified product with 2.5 μL 10× Loading Buffer, take 1.2 μL of this mixture for spotting, electrophoresis at a constant voltage of 140 V for 1.5-2 hours, take silver nitrate staining and take pictures. If a specific band of 172 bp can be amplified, it indicates that the powdery mildew resistance gene is present in the wheat germplasm to be tested. PmJ684 Otherwise, the wheat powdery mildew resistance gene does not exist in the tested wheat germplasm. PmJ684 .

[0033] The results of molecular marker detection are shown in Figure 1 .in Figure 1 YTULL-40 was labeled at 600 AABB684 × AABB620 derived F 2:3 Some amplification results in the family. M in the figure: pUC19 Msp I; 1: AABB684 (resistant parent); 2: AABB620 (susceptible parent); 3-17: F of AABB684×AABB620 2:3 Families, 3-7: homozygous disease-resistant families, 8-12: resistance-susceptibility segregation families, 13-17: homozygous disease-susceptibility families; white arrows indicate PmJ684 The amplification results showed that the marker YTULL-40 amplified a 172 bp specific band in the resistant parent AABB684 and the resistant family, but did not amplify the target band in the susceptible parent AABB620 and the susceptible family.

[0034] The wheat material AABB684 belongs to the cultivated emmer wheat and carries a new gene for powdery mildew resistance. PmJ684 The specific marker YTULL-40 designed by the present invention can be accurately screened in genetically segregated populations, PmJ684 This laid the foundation for the fine positioning and positional cloning of PmJ684 The introduction of resistance genes into wheat resources with excellent traits can better promote the breeding of powdery mildew resistance. The use of molecular markers can significantly improve the efficiency and accuracy of disease-resistant fragments, thereby accelerating PmJ684 breeding applications.

[0035] The above embodiments are optimized implementation schemes of the present invention and are only used to illustrate the present invention rather than to 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 are all within the scope of protection claimed by the present invention.

Claims

1. A gene related to wheat powdery mildew resistance PmJ684 A closely linked molecular marker, YTULL-40, has an upstream primer nucleotide sequence as shown in SEQ ID NO: 1; a downstream primer nucleotide sequence as shown in SEQ ID NO:

2. PCR amplification of the wheat genomic DNA using the primers of the molecular marker YTULL-40 yielded a 172 bp amplified product, which is the marker for the wheat powdery mildew resistance gene. PmJ684 Tightly linked molecular markers.

2. The wheat powdery mildew resistance gene according to claim 1 PmJ684 Tightly linked molecular markers, wherein the marker is suitable for a 10 μL PCR amplification system, including: 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 wheat powdery mildew resistance gene according to claim 1 or 2. PmJ684 Tightly linked molecular markers, the markers are suitable for PCR amplification procedures as follows: 95°C pre-denaturation for 2 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 40 s, 35 cycles; 72°C extension for 10 min; and storage at 4°C.

4. A molecular marker according to any one of claims 1 to 3 in a wheat powdery mildew resistance gene PmJ684 Gene mapping and its application in molecular marker-assisted breeding.

5. The use according to claim 4, for detecting whether the tested variety carries the wheat powdery mildew resistance gene PmJ684 The main steps include: (1) extracting genomic DNA from the wheat sample to be tested; (2) using primers of the molecular marker YTULL-40 to perform PCR amplification on the wheat genomic DNA to obtain an amplified product; (3) Perform electrophoresis on the amplified product and judge based on the electrophoresis results. If the wheat DNA can amplify a specific band of 172 bp, it indicates that the powdery mildew resistance gene exists in the wheat to be tested. PmJ684 Otherwise, the wheat powdery mildew resistance gene does not exist in the tested wheat PmJ684 .

6. The use according to claim 5, wherein the primers for the molecular marker YTULL-40 in step (2) include an upstream primer YTULL-40-F and a downstream primer YTULL-40-R, the nucleotide sequence of the upstream primer YTULL-40-F is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer YTULL-40-R is shown in SEQ ID NO:

2.

7. The use according to claim 5, wherein the PCR amplification system suitable for the 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.

8. The use according to claim 5, wherein the PCR amplification program applicable to 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, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

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