Molecular marker closely linked with wheat powdery mildew resistance gene PmDR696 and detection application thereof

Abstract

The invention belongs to the technical field of agricultural biological genes, and relates to a molecular marker closely linked with a wheat powdery mildew resistance gene PmDR696, a detection primer and application. The molecular marker provided by the invention can be applied to detection and identification of the gene PmDR696, auxiliary identification of wheat powdery mildew resistance characters and molecular marker breeding. The primer provided by the invention can be used for detecting whether a wheat breeding material carries the powdery mildew resistant gene PmDR696 or not before the powdery mildew in the wheat seedling stage is attacked, a variety carrying the powdery mildew resistant gene PmDR696 is purposefully selected, and the marker not only is rapid and accurate in detection, but also saves the cost, improves the breeding efficiency and accelerates the breeding process.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural biological genes and relates to wheat genetic breeding. Background Art

[0002] Wheat is a major crop widely grown in most temperate regions of the world and one of the most important food crops in my country. Its high and stable yield is particularly important for my country's food security. Powdery mildew caused by Brucella graminis is one of the most common foliar diseases of wheat. Its conidia are spread by air currents. After infecting the wheat leaves, they compete with wheat for nutrients through haustoria, reducing the photosynthetic capacity of wheat leaves (Wu et al. Functional characterization of powderymildew resistance gene MlIW172 , a new Pm60 allele and its allelic variation in wild emmer wheat. Journal of Genetics and Genomics, 2022, 49. 787-795). Although the main prevention and control measure in production is the use of chemical pesticides, planting wheat varieties carrying effective disease resistance genes is currently the most effective and environmentally friendly management strategy.

[0003] Since 1953, the first powdery mildew resistance gene Pm1 Since it was first reported more than 70 years ago, more than 200 formally and tentatively named wheat powdery mildew resistance genes / alleles and quantitative trait loci have been identified in common wheat and its closely related species (Li et al. Dissection of a rapidly evolving wheat resistance gene clusterby long-readgenome sequencing accelerated the cloning of Pm69 . Plant Communications, 2024, 5. 1). Among them, the powdery mildew resistance gene from common wheat can be directly applied to the disease resistance improvement of wheat varieties and introduced into the target varieties through field hybridization, including Pm1a, Pm5e, Pm3b, Pm24a / b, Pm47 , Pm52 etc.; Disease resistance genes from closely related species of wheat can be used to improve wheat resistance after multiple generations of backcrossing and improving agronomic traits, including Pm2, Pm8, Pm21, Pm36, Pm37, Pm42, Pm58etc. Since the genes that confer wheat resistance are species-specific, the virulence of wheat powdery mildew strains varies rapidly, and some disease resistance genes gradually lose their resistance and become inefficient / ineffective disease resistance genes, such as Pm1, Pm2, Pm3, Pm5 and Pm8 Resistance to powdery mildew has almost been lost in most wheat-growing areas in China (Wang et al. Fighting wheat powdery mildew: from genes to fields. Theoretical and Applied Genetics, 2023, 136(9): 196.). Therefore, it is necessary to find and locate new powdery mildew resistance genes, continuously expand the powdery mildew resistance gene pool, and strategically arrange and combine these resistance genes in different wheat-growing areas.

[0004] durum wheat ( Triticum turgidum L. var. durum Desf., 2n=28, AABB) is also known as durum wheat. It is a tetraploid wheat variety with mostly spring growth habits. It is mainly planted in arid areas around the Mediterranean. Its grains are small and are mainly used to make macaroni. Durum wheat has good resistance to leaf rust, stem rust and stripe rust, and is a common variety used by breeders in wheat resistance improvement (Miedaneret al. Genetic architecture of yellow and stem rust resistance in a durum wheat diversity panel. Euphytica, 2019,215: 1-17). In durum wheat, four powdery mildew resistance genes were identified, including Mld, Pm3h, PmDR147 and Pm68 , located on 4B, 1A, 2A and 2B respectively (He et al. Characterization of Pm68, a new powderymildew resistance gene on chromosome 2BS of Greek durum wheat TRI1796.Theoretical and applied genetics, 2021, 134: 53-62). Durum wheat evolved from wild emmer wheat, while common wheat evolved from the hybridization of emmer wheat and Aegilops truncatula after a long period of evolution. Therefore, the relationship between the two provides the basis for their sexual hybridization (Mastrangelo et al. What makes bread and durum wheat different. Trends in plant science,2021, 26.(7): 677-684). Studies have shown that the hybrid offspring of durum wheat and common wheat have no significant difference in quality compared with the hybrid offspring of common wheat, except that the thousand-grain weight and number of grains per ear are slightly lower than the latter. Therefore, discovering new powdery mildew resistance genes from durum wheat, a closely related species of wheat, is of great significance for the rational use of resistance sources, accelerating gene aggregation breeding, and broadening the genetic basis of wheat. Summary of the invention

[0005] The present invention proposes a wheat powdery mildew resistance gene PmDR696 Tightly linked molecular markers and their detection applications provide guidance for breeding new wheat varieties resistant to powdery mildew.

[0006] The technical solution of the present invention is achieved in this way: The invention provides a wheat powdery mildew resistance gene PmDR696 The molecular marker HENU622 is related to the gene PmDR696 The genetic distance is only 1.8 cM and is located on wheat chromosome 2AL.

[0007] A method for amplifying a powdery mildew resistance gene in durum wheat is also provided. PmDR696 Primers for tightly linked molecular markers, wherein the molecular marker is a co-dominant SSR marker HENU622, and the primers include an upstream primer HENU622-F and a downstream primer HENU622-R; The upstream primer of the molecular marker HENU622 is HENU622-F, and its nucleotide sequence is: 5'-TTTACGGGCCGCCGTTAGAG-3', as shown in SEQ ID No. 1; The downstream primer of the molecular marker HENU622 is HENU622-R, and its nucleotide sequence is: 5'-AATGTTTACGAGAGAGGTGGT-3', as shown in SEQ ID No. 2; Amplified durum wheat powdery mildew resistance gene provided by the present invention PmDR696 Primers for tightly linked molecular markers are located in the gene PmDR696 Detection, identification and assisted identification of wheat powdery mildew resistance and its application in molecular marker-assisted breeding.

[0008] Wheat powdery mildew resistance gene PmDR696 It was located on wheat chromosome 2AL (780,433,862-784,186,371bp).

[0009] The present invention also provides a method for detecting whether a wheat sample carries a durum wheat powdery mildew resistance gene. PmDR696 The method comprises the following steps: (1) Extracting genomic DNA from the wheat sample to be tested; (2) using molecular marker primers to perform PCR amplification on the genomic DNA of the wheat sample to be tested to obtain an amplification product; the molecular marker primers include an upstream primer HENU622-F and a downstream primer HENU622-R; The upstream primer of the molecular marker HENU622 is HENU622-F, and its nucleotide sequence is: 5'-TTTACGGGCCGCCGTTAGAG-3', as shown in SEQ ID No. 1; The downstream primer of the molecular marker HENU622 is HENU622-R, and its nucleotide sequence is: 5'-AATGTTTACGAGAGAGGTGGT-3', as shown in SEQ ID No. 2; (3) The amplified product is subjected to electrophoresis and detection. If a 214 bp closely linked specific band can be amplified, it indicates that the wheat sample to be tested carries the durum wheat powdery mildew resistance gene. PmDR696 Otherwise, the wheat sample to be tested does not carry the durum wheat powdery mildew resistance gene PmDR696 .

[0010] The system for PCR amplification in step (2) of the method described above is 10 μL, including: 1.0 μL of 20 ng / μL wheat genomic DNA, 5 μL of PCR Master Mix, 0.4 μL of 5 μM upstream primer, 0.4 μL of 5 μM downstream primer, and 3.2 μL of sterile deionized water.

[0011] The procedure of the PCR amplification in step (2) of the method is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 15 s, annealing at 60°C for 20 s, extension for 40 s, 34 cycles; extension at 72°C for 10 min; and storage at 12°C.

[0012] The electrophoresis procedure of the amplified product in step (3) of the method is as follows: electrophoresis is performed on a non-denaturing polyacrylamide gel with a mass volume percentage concentration of 8%, the amplified product is mixed with 2 μL of 6× loading buffer, 1.3 μL of the mixture is spotted, electrophoresis is performed at a constant voltage of 180 V for 1.5-2 h, and photography is performed after silver nitrate staining.

[0013] The present invention has the following beneficial effects: 1. The durum wheat JingDR696 used in the present invention is immune to powdery mildew at both the seedling and adult stages. Genetic analysis of seedling resistance to powdery mildew and molecular marker detection showed that the resistance of durum wheat JingDR696 to different toxic wheat powdery mildew at the seedling stage is mediated by a pair of dominant genes. PmDR696 Control, located on wheat chromosome 2AL, is a new wheat powdery mildew resistance gene / allele. The wheat powdery mildew resistance gene provided by the present invention PmDR696 The molecular marker HENU622 was tested in genetic segregation populations and was related to the gene PmDR696 The genetic distance is only 1.8 cM, which is similar to the gene PmDR696 Tightly linked, allowing accurate detection of genes PmDR696 Genetic mapping of large populations, applied to genes PmDR696 Fine positioning and positional cloning.

[0014] 2. The present invention provides a gene for wheat resistance to powdery mildew PmDR696 Primers of tightly linked molecular markers and their applications not only allow for rapid and accurate screening of target varieties without being affected by the environment and with clear selection targets, but also save production costs and greatly improve the selection efficiency and quality of high-quality powdery mildew-resistant wheat varieties or lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 The results of molecular marker HENU622 detection of segregation populations of durum wheat JingDR696 and durum wheat Langdon and their hybrid progeny. MspI;1: Durum wheat JingDR696 (powdery mildew resistant variety, carrying powdery mildew resistance gene PmDR696 ); 2: Durum wheat Langdon (powdery mildew susceptible variety); 3-22: F hybrid of Durum wheat JingDR696 and Durum wheat Langdon 2:3 Families, among which, 3-7: homozygous disease-resistant F 2:3 Family, 8-17: F with resistance and sensitivity 2:3 Family, 18-22: homozygous susceptible F 2:3 Family tree; white arrows indicate traceable genes PmDR696 Specific bands.

[0017] Figure 2 The responses of the parents Jing DR696, Langdon and the hybrid F1 progeny to the E09 strain. DETAILED DESCRIPTION

[0018] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0020] Example 1: Wheat powdery mildew resistance gene PmDR696 Development of primers for molecular marker HENU622 1 Materials Durum wheat JingDR696 and durum wheat Langdon are resistant and susceptible to wheat powdery mildew, respectively. Durum wheat JingDR696 and durum wheat Langdon were crossed, and the resulting F1 was self-pollinated to obtain the F2 population and the corresponding F 2:3 The pedigrees are shown in Tables 1 and 2.

[0021] Table 1 Powdery mildew resistance phenotypes of plant materials Table 2 Beijing DR696 and Langdon and their F2 and F 2:3 Seedling inoculation Bgt Separation ratio of physiological race E09 2. Extraction of wheat genomic DNA Wheat genomic DNA extraction CTAB method, the process is as follows: (1) Cut fresh wheat leaves and grind them in liquid nitrogen. Take about 0.5 g and place it in a 2.0 mL centrifuge tube.

[0022] (2) Add 600 μL of CTAB extraction solution and incubate in a 65°C water bath for 40 min, mixing up and down every 10 min.

[0023] (3) Add 600 μL of chloroform-isoamyl alcohol (24:1, v / v) and gently shake on a shaker for 15 min.

[0024] (4) Centrifuge at 12,000 rpm for 15 min. Take the supernatant and place it in another 2 mL centrifuge tube. Add 3 times the volume of pre-cooled anhydrous ethanol and place it in a -20℃ refrigerator to precipitate for 60 min.

[0025] (5) Pick out the flocculent DNA precipitate and wash it twice with pre-cooled 75% ethanol.

[0026] (6) Pick out the DNA precipitate, place it in a 1.5 mL centrifuge tube, and air-dry it indoors.

[0027] (7) Add 60 μL TE buffer (100 mM Tris-HCl, 10 mM EDTA, pH = 8.0) to dissolve the DNA precipitate to prepare a DNA storage solution.

[0028] (8) Dilute the DNA storage solution with ultrapure water to 20-30 ng / μL as working solution.

[0029] 3 Identification of powdery mildew resistance in wheat seedlings and genetic analysis of disease resistance The disease-resistant parent durum wheat JingDR696, the disease-susceptible parent durum wheat Langdon, the F1 hybrid, the F2 population and the F 2:3 The families were planted in a 128-hole tray (2 × 2 cm), with 5 seeds sown in each hole. 20 seeds were identified for both the parents and the F1, and 20 seeds were sown for each F1. 2:3 Twenty seeds were sown in the family, and the susceptible control Zhoumai 18 was randomly sown and labeled. After sowing, the growth conditions were controlled to 14 h light / 10 h dark cycle, temperature 20℃, and relative humidity 30-40%. When the seedlings grew to the one-leaf stage, they were inoculated with the wheat powdery mildew strain E09. The conditions for the first 24 h after inoculation were controlled as: dark, temperature 20℃, and relative humidity 100%, and then the conditions were controlled as 14 h light / 10 h dark cycle, temperature 18-22℃, and relative humidity 100%. When the first leaf of the susceptible control Zhoumai 18 was fully diseased, the investigation phenotype was recorded according to the 0-4 standard, of which 0-2 was considered as the disease-resistant level and 3-4 was the disease-susceptible level.

[0030] The survey results are as follows Figure 2The results showed that Durum wheat JingDR696 was immune to E09 strain, while Durum wheat Langdon (LDN) was highly susceptible to E09 strain. When Durum wheat JingDR696 was crossed with Durum wheat Langdon, the F1 showed resistance; among the 166 F2 plants, 125 were resistant and 41 were susceptible, which was consistent with the 3:1 ratio of resistance to sensitivity; 2:3 There were 36 homozygous resistance, 89 heterozygous, and 41 homozygous susceptible families, and the ratio of homozygous resistance: heterozygous: homozygous susceptible was consistent with the segregation ratio of 1:2:1. Therefore, it was speculated that the resistance of durum wheat JingDR696 to E09 strain was controlled by a single dominant gene, named gene PmDR696 .

[0031] 4 Preliminary localization of molecular markers for wheat powdery mildew resistance gene JingDR696 in durum wheat According to the results of phenotypic identification, 20 homozygous resistant families and 20 homozygous susceptible families were selected to construct disease-resistant pools and disease-susceptible pools, respectively. Polymorphisms of durum wheat JingDR696, durum wheat Langdon, and disease-resistant pools and disease-susceptible pools were detected using molecular markers evenly distributed throughout the genome. Ten pairs of markers showed consistent polymorphisms in resistant and susceptible parents and the resistant and susceptible pools. These markers were then used to identify 166 durum wheat JingDR696×durum wheat Langdon F pairs. 2:3 The pedigree was genotyped and the gene PmDR696 The gene was initially located on wheat chromosome 2AL.

[0032] 5. Genes PmDR696 Development of tightly linked molecular markers Based on the sequence information of the hexaploid wheat reference genome SY Mattis v1.0, Simple Sequence Repeat (SSR) markers were designed using primer5.0 software to identify the F2999 durum wheat JingDR696×durum wheat Langdon. 2:3 The pedigree was genotyped and the gene PmDR696 The tightly linked SSR marker HENU622 had a genetic distance of only 1.8 cM.

[0033] The primers for the molecular marker HENU622 include an upstream primer and a downstream primer: The nucleotide sequence of the upstream primer HENU622-F: 5′-TTTACGGGCCGCCGTTAGAG-3′ (as shown in SEQ ID No. 1); The nucleotide sequence of the downstream primer HENU622-R is: 5'-AATGTTTACGAGAGAGGTGGT-3' (as shown in SEQ ID No. 2).

[0034] The PCR amplification system is 10 μL, including: 1.0 μL of 20 ng / μL wheat genomic DNA, 5 μL of PCR Master Mix, 0.4 μL of 5 μM upstream primer, 0.4 μL of 5 μM downstream primer, and 3.2 μL of sterile deionized water.

[0035] The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 15 s, annealing at 60°C for 20 s, extension for 40 s, 34 cycles; extension at 72°C for 10 min; and storage at 12°C.

[0036] The electrophoretic separation procedure of the amplified product is as follows: electrophoresis is performed on a non-denaturing polyacrylamide gel with a mass volume percentage concentration of 8%, the amplified product is mixed with 2 μL of 6× loading buffer, 1.3 μL of the mixture is spotted, electrophoresis is performed at a constant voltage of 180V for 1.5-2 h, and photography is performed after silver nitrate staining.

[0037] A 214 bp tightly linked specific band was amplified, indicating that the wheat to be tested carries the powdery mildew resistance gene PmDR696 ; No 214 bp closely linked specific band was amplified, indicating that the tested wheat does not carry the wheat powdery mildew resistance gene PmDR696 .

[0038] Example 2: Wheat powdery mildew resistance gene PmDR696 Application of primers for molecular marker HENU622 Utilizing wheat powdery mildew resistance genes PmDR696 Detection of durum wheat JingDR696, durum wheat Langdon and the hybrid of the two by amplification with HENU622 primers 2:3 The DNA extraction method for the materials in the family was the same as in Example 1.

[0039] Samples to be tested: Durum wheat Jing DR696, Durum wheat Langdon and their hybrid F 2:3 Family lineage.

[0040] The genomic DNA of the above materials was extracted as a PCR amplification template, and the primers of the molecular marker HENU622 developed by the present invention were used for amplification: Nucleotide sequence of upstream primer HENU622-F: 5'-TTTACGGGCCGCCGTTAGAG-3' (as shown in SEQ ID No. 1); Nucleotide sequence of downstream primer HENU622-R: 5'-AATGTTTACGAGAGAGGTGGT-3' (as shown in SEQ ID No. 2).

[0041] The PCR amplification system is 10 μL, including: 1.0 μL of 20 ng / μL wheat genomic DNA, 5 μL of PCR Master Mix, 0.4 μL of 5 μM upstream primer, 0.4 μL of 5 μM downstream primer, and 3.2 μL of sterile deionized water.

[0042] The PCR amplification program is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 15 s, annealing at 60°C for 20 s, extension for 40 s, 34 cycles; extension at 72°C for 10 min; and storage at 12°C.

[0043] The electrophoretic separation procedure of the amplified product is as follows: electrophoresis is performed on a non-denaturing polyacrylamide gel with a mass volume percentage concentration of 8%, the amplified product is mixed with 2 μL of 6× loading buffer, 1.3 μL of the mixture is spotted, electrophoresis is performed at a constant voltage of 180V for 1.5-2 h, and photography is performed after silver nitrate staining.

[0044] The results of molecular marker detection are shown in Figure 1 . Figure 1 HENU622 was labeled in the durum wheat DR696 × durum wheat Langdon derived F 2:3 Some amplification results in the family. In the figure: M: pUC19 DNA / Msp I;1: Durum wheat JingDR696 (powdery mildew resistant variety, carrying powdery mildew resistance gene PmDR696 ); 2: Durum wheat Langdon (powdery mildew susceptible variety); 3-22: F hybrid of Durum wheat JingDR696 and Durum wheat Langdon 2:3 Families, among which, 3-7: homozygous disease-resistant F 2:3 Family, 8-17: F with resistance and sensitivity 2:3 Family, 18-22: homozygous susceptible F 2:3 Family tree; white arrows indicate traceable genes PmDR696 The amplification results showed that the marker HENU622 amplified a 214 bp tightly linked specific band in the disease-resistant parent durum wheat JingDR696 and the disease-resistant family, but did not amplify the target band in the disease-susceptible parent durum wheat Langdon and the disease-susceptible family.

[0045] Wheat powdery mildew resistance gene PmDR696 It is derived from the durum wheat variety Jing DR696. Currently, there is no report on the location and positional cloning of this gene. The molecular marker HENU622 provided by the present invention is used to detect genetic mapping of large populations, which is helpful for gene PmDR696 Fine positioning and map-based cloning of genes PmDR696The efficient transformation and in-depth analysis of disease resistance mechanisms are of great significance.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Genes related to wheat resistance to powdery mildew PmDR696 Tightly linked molecular markers characterized by: The molecular marker is located on chromosome 2AL of wheat and is closely related to the powdery mildew resistance gene PmDR696 The genetic distance is only 1.8 cM.

2. The wheat powdery mildew resistance gene according to claim 1 PmDR696 Tightly linked molecular markers characterized by: Powdery mildew resistance gene PmDR696 Located on wheat chromosome 2AL, the corresponding physical interval of the SY Mattis v1.0 reference genome is 780,433,862-784,186,371 bp.

3. The wheat powdery mildew resistance gene according to claim 2 PmDR696 Tightly linked molecular markers characterized by: The wheat is of the durum variety.

4. A primer for detecting the molecular marker according to claim 1, characterized in that: The primers include an upstream primer HENU622-F whose nucleotide sequence is shown as SEQ ID No.1 and a downstream primer HENU622-R whose nucleotide sequence is shown as SEQ ID No.

2.

5. The molecular marker according to any one of claims 1 to 3 or the primer according to claim 4 is used to detect whether wheat contains a powdery mildew resistance gene PmDR696 Application in.

6. Use of the molecular marker according to any one of claims 1 to 3 or the primer according to claim 4 in identifying or assisting in identifying whether wheat has a powdery mildew resistance trait.

7. Use of the molecular marker according to any one of claims 1 to 3 or the primer according to claim 4 in assisting the breeding of powdery mildew-resistant wheat.

8. A method to detect whether wheat carries a gene for resistance to powdery mildew PmDR696 The method is characterized in that The steps are: using the primers described in claim 2 to amplify the genome of the wheat sample to be tested; if a 214 bp closely linked specific band can be amplified, it means that the wheat sample to be tested carries the wheat powdery mildew resistance gene PmDR696 ; otherwise, the wheat sample to be tested does not carry it.

9. Powdery mildew resistance gene PmDR696 Application in breeding powdery mildew resistant wheat.

10. The use according to claim 9, characterized in that: Powdery mildew resistance gene PmDR696 Located within the physical interval of 780,433,862-784,186,371 bp on chromosome 2AL of the SYMattis v1.0 reference genome.

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