A primer for amplifying a molecular marker of wild two-grain wheat powdery mildew resistance gene PmAB543 and application thereof

Abstract

The present application relates to the field of agricultural bioengineering technology, and particularly relates to a primer for amplifying a wild two-grain wheat powdery mildew resistance gene PmAB543 molecular marker and application thereof.The molecular marker is YTU543-L103; the nucleotide sequence of the upstream primer of the molecular marker is shown as SEQ ID NO:1; the nucleotide sequence of the downstream primer is shown as SEQ ID NO:2; the primer of the molecular marker YTU543-L103 is used to carry out PCR amplification on the wheat genomic DNA to be tested, and a corresponding amplification product with a molecular weight of 503 bp is obtained, which is a molecular marker closely linked to the wheat powdery mildew resistance gene PmAB543.The molecular marker primer closely linked to the wheat powdery mildew resistance gene PmAB543 provided in the present application can be used to quickly and efficiently detect whether the wheat breeding material carries the disease resistance gene PmAB543 at the seedling stage, and is helpful to improve the breeding efficiency.

Description

Technical Field

[0001] This invention relates to the field of agricultural bioengineering technology, specifically to a primer for amplifying the molecular marker of the wild emmer wheat powdery mildew resistance gene PmAB543 and its application. Background Technology

[0002] Wheat powdery mildew is a serious and prevalent fungal disease that severely damages wheat production. Its pathogen is *Blumeria graminis* f. sp. tritici (Bgt), a species of grass. This disease occurs in all major wheat-producing regions worldwide. Powdery mildew inhibits host photosynthesis, reduces effective tillering, and hinders grain filling, leading to a significant decrease in yield and severely degrading grain processing quality, posing a major threat to safe wheat production. Currently, utilizing disease-resistant genes and breeding new resistant varieties is the most economical, effective, and sustainable strategy for controlling wheat powdery mildew. Continuously exploring disease-resistant genes and enriching the reserve of resistance sources are urgent needs in current wheat powdery mildew resistance breeding efforts.

[0003] Wild emmer wheat (Triticum turgidum var. dicoccoides, AABB, 2n=4x=28), as a tetraploid ancestor of hexaploid common wheat, contains rich genetic variation and is an excellent genetic resource for improving wheat powdery mildew resistance. Wild emmer wheat AB543 is a wheat germplasm resource with excellent comprehensive traits, exhibiting stable powdery mildew resistance in field identification at multiple locations over many years. Genetic analysis and molecular marker detection of seedling powdery mildew resistance in this material showed that seedling resistance to the prevalent powdery mildew strain E09 is controlled by a single dominant gene located on chromosome 4AL. This gene, named PmAB543, is a novel wheat powdery mildew resistance gene that has not been previously reported, and no closely linked molecular markers have been reported, making this invention highly innovative. Therefore, developing molecular markers closely linked to PmAB543 and applying them to marker-assisted selection breeding of this gene has significant theoretical and practical value for breeding new disease-resistant wheat varieties and effectively controlling wheat powdery mildew. Summary of the Invention

[0004] The purpose of this invention is to provide a primer for amplifying the molecular marker of the powdery mildew resistance gene PmAB543 in wild emmer wheat and its application, so as to use the molecular marker to locate and detect the powdery mildew resistance gene PmAB543 in wheat.

[0005] This invention is achieved through the following method: A primer for amplifying the molecular marker PmAB543, a gene for resistance to powdery mildew in wild emmer wheat, and its application. The molecular marker is the codominant insertion-deletion (InDel) marker YTU543-L103. The upstream primer for the molecular marker YTU543-L103 is YTU543-L103-F, and its nucleotide sequence is as follows: 5'- TATTGCTGCTAGGCTGTC-3', as shown in SEQ ID NO: 1; The downstream primer for the molecular marker YTU543-L103 is YTU543-L103-R, and its nucleotide sequence is as follows: 5'- GCTATCCTCACGCTCAAC-3', as shown in SEQ ID NO: 2; The primers for the molecular marker YTU543-L103 were used to amplify the wheat genomic DNA to be tested by PCR. The corresponding amplification product had a molecular weight of 503 bp, which is a molecular marker closely linked to the wheat powdery mildew resistance gene PmAB543.

[0006] The appropriate PCR amplification system for this molecular marker is 10 μL, comprising: 1.5 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR Master Mix, 0.25 μL of 10 μM upstream primer, 0.25 μL of 10 μM downstream primer, and 4 μL of sterile deionized water.

[0007] The applicable PCR amplification program for this molecular marker is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 48℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; store at 4℃.

[0008] 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 2.5 μL of 10× Loading Buffer, and 0.5 μL of the mixture is loaded onto the gel. Electrophoresis is performed at a constant voltage of 220 V for 1.5-2 h. The gel is then stained with silver nitrate and photographed.

[0009] The present invention also provides the application of the molecular markers closely linked to the wheat powdery mildew resistance gene PmAB543 in the localization of the wheat powdery mildew resistance gene PmAB543 and in marker-assisted selection breeding.

[0010] The application described in this invention, which detects whether a wheat variety carries the powdery mildew resistance gene PmAB543, 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 YTU543-L103 to obtain the amplification product; (3) Perform electrophoresis on the amplified products and judge based on the electrophoresis results. If a specific band of 503 bp can be amplified, it indicates that the powdery mildew resistance gene PmAB543 exists in the wheat to be tested; otherwise, the powdery mildew resistance gene PmAB543 does not exist in the wheat to be tested.

[0011] In the application described above, the primers for the molecular marker YTU543-L103 in step (2) include the upstream primer YTU543-L103-F and the downstream primer YTU543-L103-R. The nucleotide sequence of the upstream primer YTU543-L103-F is shown in SEQ ID NO:1, namely: YTU543-L103-F: 5'-TATTGCTGCTAGGCTGTC-3'; the nucleotide sequence of the downstream primer YTU543-L103-R is shown in SEQ ID NO:2, namely: YTU543-L103-R: 5'-GCTATCCTCACGCTCAAC-3'.

[0012] The application of this molecular marker is based on a 10 μL PCR amplification system, which includes: 1.5 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR Master Mix, 0.25 μL of 10 μM upstream primer, 0.25 μL of 10 μM downstream primer, and 4 μL of sterile deionized water.

[0013] The applicable PCR amplification program for this marker is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 48℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; store at 4℃.

[0014] Detection of PCR amplification products: Electrophoresis was performed using an 8% non-denaturing polyacrylamide gel. The amplification product was mixed with 2.5 μL of 10× Loading Buffer, and 0.5 μL of this mixture was loaded onto the gel. Electrophoresis was carried out at a constant voltage of 220 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 PmAB543 in the tested wheat germplasm. Otherwise, the powdery mildew resistance gene PmAB543 was not present in the tested wheat germplasm.

[0015] This invention, through genetic analysis and molecular marker detection of seedling powdery mildew resistance, demonstrates that the resistance of wild erin wheat AB543 seedlings to the prevalent powdery mildew strain E09 is controlled by a single dominant gene, named PmAB543. Polymorphism was detected in 40 homozygous resistant and 40 homozygous susceptible families from 160 pairs of molecular markers evenly distributed throughout the genome in resistant wild erin wheat AB543, susceptible wild erin wheat AB647, and their hybrid F2 populations. 24 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 180 AB543×AB647 F2 populations and 1376 AB543×AB647 F2 populations. 2：3 Genotyping was performed on the population, and PmAB543 was located within the interval 712.37-712.79 Mb on wheat chromosome 4AL. Further, based on the sequence of wild emmer wheat within this interval, the InDel marker YTU543-L103, closely linked to the PmAB543 gene, was designed and screened using Primer 5.0 software. The molecular marker YTU543-L103 for the wheat powdery mildew resistance gene PmAB543 provided by this invention, after genetic segregation population testing, showed a genetic distance of only 1.0 cM from the gene YTU543-L103, indicating close linkage with PmAB543. This allows for more accurate and efficient detection of the genetic mapping population of PmAB543, facilitating fine mapping and map-based cloning of PmAB543.

[0016] The molecular marker provided by this invention, which is closely linked to the wheat powdery mildew resistance gene PmAB543, can be applied to powdery mildew resistant wheat breeding. It can not only quickly and accurately screen materials containing PmAB345, but also greatly save costs, shorten the breeding cycle, and improve breeding efficiency. Attached Figure Description

[0017] Figure 1 The results of primer detection of molecular marker YTU543-L103 on disease-resistant wild emmer wheat AB543 and disease-susceptible wild emmer wheat AB647 and their hybrid offspring segregating populations.

[0018] In the diagram, M: pUC19 Msp I; 1: Wild emmer wheat AB543 (disease-resistant parent); 2: Wild emmer wheat AB647 (disease-susceptible parent); 3-17: F1 generation of AB543 × AB647 2:3 The populations are divided into groups, where 3-7 are homozygous resistant families, 8-12 are homozygous susceptible families, and 13-17 are resistant-susceptible segregating families; the white arrows represent specific bands of PmAB543. Detailed Implementation

[0019] 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.

[0020] Example 1: Development of the YTU543-L103 primer, a molecular marker for the wheat powdery mildew resistance gene PmAB543. 1. Materials The resistant parent was wild emmer wheat AB543, and the susceptible parent was wild emmer wheat AB647. These materials were donated by Associate Professor Xu Hongxing of Henan University (85 Minglun Street, Kaifeng City, Henan Province) in September 2022. AB543 and AB647 were crossed, and the resulting F1 generation was self-crossed to obtain the F2 population. The F2 generation was then self-crossed to obtain the F1 generation. 2：3 group.

[0021] 2. Extraction of wheat genomic DNA The CTAB method for extracting wheat genomic DNA is as follows: (1) Take tender fresh leaves of wheat to be tested, freeze them with liquid nitrogen, grind them into powder, and put them into 2 mL EP tubes; (2) Add 600-800 μL of CTAB extract and incubate in a 65°C water bath for 1 h, inverting and mixing every ten minutes during the incubation period; (3) Add an equal volume of chloroform and mix on a shaker for 30 min; (4) Centrifuge at 8000 rpm / min 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 settle at -20°C for 0.5 h; (5) Centrifuge at 12000 rpm / min at room temperature for 10 min, discard the supernatant, and wash 3 times with 800 μL of 75% ethanol; (6) Air dry the precipitate and dissolve it in 50 μL of 1×TE or ddH2O.

[0022] (7) Dilute the DNA storage solution with sterile deionized water to 50 ng / μL as a working solution for later use.

[0023] 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 AB543, the susceptible parent AB647, F1 hybrids, F2 populations, and F4 seedlings were used. 2：3Populations were planted in 128-cell trays (3.2×3.2×4.2 cm). At least 20 seeds were identified in each parent and F1 population, and at least 25 seeds were identified in each F2 population. The susceptible control, Tai Nong 18, was randomly sown and tagged for identification. Greenhouse conditions were controlled at 18-20°C, 80% relative humidity, and a photoperiod of 14 h light / 10 h 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, Tai Nong 18, showed full disease development. 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.

[0024] The survey results showed that wild emmer wheat AB543 exhibited high resistance to powdery mildew strain E09 (IT=0), while wild emmer wheat AB647 showed high susceptibility (IT=4). All F1 plants showed resistance (IT = 0-1), indicating that wild emmer wheat AB543 carries a dominant resistance gene. Resistance identification of the F2 population of this combination showed a resistance-susceptibility segregation ratio of 136:44, which, according to the chi-square test, conformed to a segregation ratio of 3:1 for a single dominant gene (χ²=0). 2 =0.03, P=0.86), for F 2:3 Population identification revealed a segregation ratio of 38:75:30 for homozygous resistant families, resistant-susceptible families, and homozygous susceptible families. Chi-square test confirmed a segregation ratio of 1:2:1 for a single dominant gene. 2 =1.24, P=0.54). In conclusion, the resistance of wild emmer wheat AB543 to powdery mildew strain E09 is controlled by a single dominant gene, which is named PmAB543.

[0025] 4. Fine localization of molecular markers for PmAB543 Based on phenotypic identification results, 40 homozygous resistant families and 40 homozygous susceptible families were selected to construct resistant and susceptible populations, respectively. Polymorphism detection was performed on wild emmer wheat AB543 and AB647, as well as the resistant and susceptible populations, using 160 pairs of molecular markers evenly distributed throughout the genome. 24 pairs of markers showed consistent polymorphism in both the resistant and susceptible parents and the resistant-susceptible populations. Subsequently, these markers were used to analyze 180 AB543×AB647 F2 populations and 1376 F2 populations. 2：3 Genotyping of the population revealed that PmAB543 was located in the region of 712.37-712.79 Mb on the wheat 4AL chromosome.

[0026] 5. Development of molecular markers closely linked to PmAB543 Based on the sequence information of the wild emmer wheat reference genome within the candidate region of 712.37-712.79 Mb, InDel markers were designed using Primer 5.0 software. These markers were then used to target the F2 population of AB543×AB647 and the F... 2：3 Genotyping of the population yielded the InDel marker YTU543-L103, which is closely linked to the gene PmAB543, with a genetic distance of only 1.0 cM, indicating close linkage.

[0027] The primers for the molecular marker YTU543-L103 include one upstream primer and one downstream primer: The nucleotide sequence of the upstream primer YTU543-L103-F is: 5'-TATTGCTGCTAGGCTGTC-3'; The nucleotide sequence of the downstream primer YTU543-L103-R is: 5'-GCTATCCTCACGCTCAAC-3'.

[0028] The applicable PCR amplification system for this marker is 10 μL, including: 1.5 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR Master Mix, 0.25 μL of 10 μM upstream primer, 0.25 μL of 10 μM downstream primer, and 4 μL of sterile deionized water.

[0029] The applicable PCR amplification program for this marker is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 48℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; store at 4℃.

[0030] 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 2.5 μL of 10× Loading Buffer, and 0.5 μL of this mixture is loaded onto the gel. Electrophoresis is carried out at a constant voltage of 220 V for 1.5–2 h. After silver nitrate staining, the sample is photographed. If a specific band of 503 bp is amplified, it indicates the presence of the powdery mildew resistance gene PmAB543 in the tested wheat germplasm; otherwise, the powdery mildew resistance gene PmAB543 is not present in the tested wheat germplasm.

[0031] Molecular marker detection results are shown below Figure 1 .in Figure 1The results of primer detection for the molecular marker YTU543-L103 were obtained from the segregation of resistant wild emmer wheat AB543, susceptible wild emmer wheat AB647, and their hybrid progeny. In the figure, M: pUC19Msp I; 1: wild emmer wheat AB543 (resistant parent); 2: wild emmer wheat AB647 (susceptible parent); 3-17: F1 generation of AB543 × AB647. 2:3 Populations were defined as follows: 3-7: homozygous resistant families; 8-12: homozygous susceptible families; 13-17: resistant-susceptible segregating families; white arrows indicate specific bands of PmAB543. Amplification results showed that the marker YTU543-L103 amplified a specific band of 503 bp in the resistant parent AB543 and resistant families, but not in the susceptible parent AB647 and susceptible families.

[0032] The wheat powdery mildew resistance gene PmAB543, derived from wild emmer wheat AB543, is a novel gene exhibiting excellent resistance. Currently, there are no reports on its localization, map-based cloning, or application in molecular breeding. Using the molecular marker YTU543-L103 provided by this invention to detect large-scale genetically mapped populations is beneficial for achieving precise localization and map-based cloning of the PmAB543 gene. Introducing PmAB543 into major wheat varieties susceptible to powdery mildew, using the primer pair of the molecular marker YTU543-L103 from this invention, allows for efficient and accurate detection of large breeding populations, significantly improving the efficiency and accuracy of transferring the disease-resistant gene PmAB543. This is of paramount importance for the efficient transfer of the PmAB543 gene and for in-depth analysis of the disease resistance mechanism.

[0033] 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. Primers for amplifying the molecular marker PmAB543, a powdery mildew resistance gene in wild emmer wheat, wherein the molecular marker is YTU543-L103, and the nucleotide sequence of the upstream primer is shown in SEQ ID NO:1; the nucleotide sequence of the downstream primer is shown in SEQ ID NO:2; PCR amplification of the wheat genomic DNA to be tested using the primers of the molecular marker YTU543-L103 yields an amplification product with a molecular weight of 503 bp, which is a molecular marker closely linked to the wheat powdery mildew resistance gene PmAB543.

2. The molecular marker tightly linked to the wheat powdery mildew resistance gene PmAB543 according to claim 1, wherein the applicable PCR amplification system for the marker is 10 μL, comprising: 1.5 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR MasterMix, 0.25 μL of 10 μM upstream primer, 0.25 μL of 10 μM downstream primer, and 4 μL of sterile deionized water.

3. The molecular marker closely linked to the wheat powdery mildew resistance gene PmAB543 according to claim 1, wherein the applicable PCR amplification program for the marker is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 48℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; storage at 4℃.

4. The application of the molecular marker as described in claim 1, which is closely linked to the wheat powdery mildew resistance gene PmAB543, in the gene localization of the wheat powdery mildew resistance gene PmAB543 and in marker-assisted breeding.

5. According to the application described in claim 4, detecting whether a wheat variety carries the powdery mildew resistance gene PmAB543 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 molecular marker YTU543-L103 to obtain the amplification product; (3) Perform electrophoresis on the amplified products and judge based on the electrophoresis results. If a specific band of 503 bp can be amplified, it indicates that the powdery mildew resistance gene PmAB543 exists in the wheat to be tested; otherwise, the powdery mildew resistance gene PmAB543 does not exist in the wheat to be tested.

6. In the application according to claim 5, the primers for the molecular marker YTU543-L103 in step (2) include an upstream primer YTU543-L103-F and a downstream primer YTU543-L103-R, wherein the nucleotide sequence of the upstream primer YTU543-L103-F is shown in SEQ ID NO:1 and the nucleotide sequence of the downstream primer YTU543-L103-R is shown in SEQ ID NO:

2.

7. According to the application of claim 5, the PCR amplification system suitable for the labeling in step (2) is 10 μL, comprising: 1.5 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR Master Mix, 0.25 μL of 10 μM upstream primer, 0.25 μL of 10 μM downstream primer, and 4 μL of sterile deionized water.

8. According to the application of claim 5, the PCR amplification program applicable to the label in step (2) is as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 48℃ annealing for 30 s, 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; storage at 4℃.

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