Detectable molecular marker for tracking powdery mildew-resistant gene PmDM8 and application of detectable molecular marker in disease-resistant breeding

Abstract

The invention belongs to the field of genetic breeding, and relates to cultivation of powdery mildew resistant wheat. The invention provides a detectable molecular marker for tracking a powdery mildew-resistant gene PmDM8 and application of the detectable molecular marker in disease-resistant breeding. The molecular marker comprises an upstream primer HENU580-F and a downstream primer HENU580-R, the nucleotide sequence of the upstream primer HENU580-F is as shown in SEQ ID No. 1, and the nucleotide sequence of the downstream primer HENU580-R is as shown in SEQ ID No. 2. The primer provided by the invention can be used for detecting whether a wheat breeding material carries the powdery mildew resistant gene PmDM8 or not before the powdery mildew in the wheat seedling stage is attacked, a variety carrying the powdery mildew resistant gene PmDM8 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 present invention belongs to the field of genetic breeding and relates to the cultivation of powdery mildew-resistant wheat. Background Art

[0002] Common wheat ( Triticum aestivum L., AABBDD, 2n = 6x = 42) is one of the most important staple crops in the world, providing nearly 18% of the daily dietary calories for humans worldwide (Li et al. Wheat breeding in northern China: Achievements and technical advances. The Crop Journal, 2019, 7: 718 - 729). With the rapid growth of the global population and the reduction of available arable land area, how to improve the wheat yield potential has become the research focus of global breeders. However, powdery mildew caused by Blumeria graminis f. sp. tritici , Bgt is a highly destructive foliar disease. The incidence of the disease in production will cause a 10 - 15% reduction in wheat yield, and even a 50% reduction in severe cases (Jakobson et al. Fine mapping, phenotypic characterization and validation of non-race-specific resistance to powdery mildew in a wheat- Triticum militinae introgression line. Theoretical and Applied Genetics, 2012, 125: 609 - 623). The main control methods include the breeding of disease-resistant varieties, chemical control, and agricultural management measures. Among them, the breeding of disease-resistant varieties is the most effective and environmentally friendly method to control or delay the epidemic of powdery mildew.

[0003] So far, more than 140 powdery mildew-resistant genes or alleles have been identified and officially named, distributed at 65 different loci. The cloning of wheat powdery mildew genes mostly uses the classical map-based cloning method. The genes cloned by using this method include Pm2b, Pm5e, Pm21, Pm24, Pm38, Pm41, Pm55, Pm60 and Pm6sl (Li et al. Dissection of a rapidly evolving wheat resistance gene cluster by long-read genome sequencing accelerated the cloning of Pm69. Theoretical and Applied Genetics, Plant Communications, 2023, 100646). In the past decade, the rapid development of sequencing technology and the continuous improvement of the wheat reference genome have significantly accelerated the cloning of powdery mildew resistance genes, and some emerging methods have been reported one after another, such as gene cloning based on mutant sequencing (Mutant Sequencing, Mut-Seq). Recently reported Pm1a, Pm2, Pm4b, Pm57 and Pm69 both used new technologies for cloning (Sanchez-Martin et al. Rapid gene isolation in barley and wheat by mutant chromosome sequencing. Genome Biology, 2016, 17: 221). Although a variety of powdery mildew resistance genes have been discovered, during the breeding and promotion of wheat resistant to powdery mildew, the interaction between wheat powdery mildew strains and resistance genes will lead to continuous variation of both, causing most resistant varieties to gradually lose their resistance. However, due to large-scale promotion and long-term utilization of broad-spectrum resistant varieties, there is a trend of singleization of resistance resources. This selection pressure prompts stronger variation in wheat powdery mildew strains and accelerates their pathogenic differentiation. Currently, the powdery mildew resistance genes Pm1, Pm2 and Pm8 have been reported to have lost / almost lost their resistance in most wheat-growing regions of China (Wang et al. Fighting wheat powdery mildew: from genes to fields. Theoretical and Applied Genetics, 2023, 136: 196). Therefore, a great deal of effort needs to be invested in wheat breeding to optimize the coordinated combination of different genes and traits and explore the best coupling method to achieve the goal of coordinated improvement of complex traits.

[0004] The related species of common wheat are considered to have important application value in breeding due to their rich genetic diversity. These wild resources contain a large number of excellent disease-resistant, stress-resistant and high-yield alleles, and are important gene pools for improving the agronomic traits of common wheat. Currently, more than half of the reported powdery mildew-resistant genes come from the related species of common wheat, including diploid, tetraploid cultivated varieties and wild relatives from the A, B and D genomes. The diploid relatives include wild emmer wheat (AA), cultivated emmer wheat (AA), Aegilops speltoides (BB) and Aegilops tauschii (DD); the tetraploid relatives (AABB) include wild emmer wheat, cultivated emmer wheat, durum wheat and Persian wheat (Nyine et al. The haplotype-based analysis of Aegilops tauschii introgression into hard red winter wheatand its impact on productivity traits. Frontiers in Plant Science, 2023, 12:716955). The genomes of these wild relatives are derived from the ancestral species of common wheat and can undergo homologous recombination with the common wheat genome. This genetic compatibility provides the possibility for the introduction of powdery mildew-resistant genes, thus promoting the transfer of disease-resistant genes and the genetic improvement of excellent wheat varieties, and can improve the disease resistance and agronomic traits of common wheat.

[0005] Cultivated emmer wheat ( Triticum dicoccum, AABB, 2n = 4x = 28) belongs to the secondary gene pool of common wheat, shares the A and B genomes with common wheat, and is an important source of excellent powdery mildew-resistant genes in wheat breeding. The reported Pm4a, Pm5a, Pm49 and Pm50 were both discovered from cultivated emmer wheat materials. In addition, cultivated emmer wheat has excellent resistance to various diseases such as stem rust and leaf rust, can also resist insect pests, and has excellent stress-resistant traits such as strong drought tolerance, poor soil tolerance and salt tolerance (Lhamo et al. Genome-wide association analyses of leaf rustresistance in cultivatedemmer wheat. Theoretical and Applied Genetics, 2023,136: 20). Therefore, mining new powdery mildew-resistant genes from cultivated emmer wheat and introducing them into common wheat can not only expand the powdery mildew resistance gene pool of wheat, but also simultaneously introduce the excellent agronomic traits it carries, and enhance the genetic diversity of common wheat. Summary of the Invention

[0006] The present invention provides a detectable molecular marker for tracking powdery mildew resistance genes PmDM8 and its application in disease-resistant breeding. By using primer amplification of molecular markers, the powdery mildew resistance gene PmDM8 in wheat is located and detected, and its parents are purposefully selected in wheat breeding, providing a guiding basis for breeding new wheat varieties resistant to powdery mildew.

[0007] The technical solution of the present invention is realized as follows: Through genetic analysis of powdery mildew resistance and molecular marker detection in the seedling stage of cultivated emmer wheat, it is found that the resistance of cultivated emmer wheat Jing DM8 to different virulent powdery mildew fungi in the seedling stage is controlled by a pair of dominant genes PmDM8 and is located in a physical interval of 560.78 kb (693,556,693 - 694,117,473 bp) on chromosome 6BL of wheat, which is a new wheat powdery mildew resistance gene / allele.

[0008] The molecular marker HENU580 of the wheat powdery mildew resistance gene PmDM8 provided by the present invention, after being detected in a genetic segregation population, has a genetic distance of only 0.03 cM from the gene PmDM8 and is tightly linked to the gene PmDM8 , and can accurately detect the large genetic mapping population of the gene PmDM8 and be applied to the fine mapping and map-based cloning of the gene PmDM8 .

[0009] The present invention provides a primer for amplifying a molecular marker tightly linked to the powdery mildew resistance gene PmDM8 in cultivated emmer wheat. The molecular marker is a co-dominant SSR marker HENU580, and its primers include an upstream primer HENU580-F and a downstream primer HENU580-R; The upstream primer of the molecular marker HENU580 is HENU580-F, and its nucleotide sequence is: 5'-CATCGACGCATCCCACATTT-3', as shown in SEQ ID No.1; The downstream primer of the molecular marker HENU580 is HENU580-R, and its nucleotide sequence is: 5'-AGTTTGGTTGTGTAATCTCGCA-3', as shown in SEQ ID No.2.

[0010] The primer for amplifying a molecular marker tightly linked to the powdery mildew resistance gene PmDM8 in cultivated emmer wheat provided by the present invention is used in the detection and identification of the gene PmDM8 , the auxiliary identification of wheat powdery mildew resistance traits, and molecular marker-assisted breeding.

[0011] The present invention also provides a method for detecting whether a wheat sample carries the powdery mildew resistance gene of Triticum dicoccoides PmDM8 , comprising the following steps: (1) Extracting the genomic DNA of the wheat sample to be tested; (2) Performing PCR amplification on the genomic DNA of the wheat sample to be tested using molecular marker primers to obtain an amplification product; the molecular marker primers include an upstream primer HENU580-F and a downstream primer HENU580-R; The upstream primer of the molecular marker HENU580 is HENU580-F, and its nucleotide sequence is: 5'-CATCGACGCATCCCACATTT-3', as shown in SEQ ID No.1; The downstream primer of the molecular marker HENU580 is HENU580-R, and its nucleotide sequence is: 5'-AGTTTGGTTGTGTAATCTCGCA-3', as shown in SEQ ID No.2; (3) Performing electrophoresis and detection on the amplification product. If a specific band of 273 bp tightly linked can be amplified, it indicates that the wheat sample to be tested carries the powdery mildew resistance gene of Triticum dicoccoides PmDM8 ; otherwise, the wheat sample to be tested does not carry the powdery mildew resistance gene of Triticum dicoccoides PmDM8 .

[0012] In the above method, the PCR amplification system in step (2) 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.

[0013] In the above method, the PCR amplification program in step (2) is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 15 s, annealing at 56°C for 20 s, extension for 30 s, 34 cycles; extension at 72°C for 10 min; preservation at 12°C.

[0014] In the above method, the electrophoresis program of the amplification product in step (3) is: performing electrophoresis on a non-denaturing polyacrylamide gel with a mass-volume percentage concentration of 8%. After mixing the amplification product and 2 μL of 6× loading buffer, taking 1.3 μL of the mixture for sample loading, and performing electrophoresis at a constant voltage of 180 V for 1.5 - 2 h, followed by silver nitrate staining and photographing.

[0015] The present invention has the following beneficial effects: The present invention discovers that the resistance of cultivated emmer wheat Jing DM8 at the seedling stage to different virulent powdery mildew fungi of wheat is controlled by a pair of dominant genes PmDM8 and is located on chromosome 6BL of wheat, which is a new wheat powdery mildew resistance gene / allele.

[0016] The present invention provides a detectable molecular marker for tracking the powdery mildew resistance gene PmDM8 and its application in disease-resistant breeding. It can not only screen target varieties quickly and accurately, 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 wheat varieties or lines resistant to powdery mildew. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 Results of primer detection of molecular marker HENU580 for cultivated emmer wheat Jing DM8, durum wheat Langdon and their hybrid offspring segregation population; in the figure: M: DL2000; 1: cultivated emmer wheat Jing DM8 (powdery mildew-resistant variety, carrying PmDM8 ); 2: durum wheat Langdon (powdery mildew-susceptible variety); 3 - 22: F 2:3 families formed by the hybridization of cultivated emmer wheat Jing DM8 and durum wheat Langdon, among which, 3 - 7: homozygous disease-resistant F 2:3 families, 8 - 17: F 2:3 families with disease-resistant and disease-susceptible segregation, 18 - 22: homozygous disease-susceptible F 2:3 families; white arrows are specific bands that can track the gene PmDM8 .

[0019] Figure 2 Results of primer detection of molecular marker HENU580 for cultivated emmer wheat Jing DM8, durum wheat Langdon and main powdery mildew-susceptible cultivated wheat varieties in wheat-growing regions of China; in the figure: M: DL2000; 1: cultivated emmer wheat Jing DM8 (powdery mildew-resistant variety, carrying PmDM8 ); 2: durum wheat Langdon (powdery mildew-susceptible variety); 3 - 17: Yumai 16, Zhengzhou 5, Zhengzhou 24, Yangmai 1, Yangmai 158, Jinan 8, Jinan 9, Lumai 7, Lumai 12, Lumai 14, Lumai 15, Zhongyou 206, Zhongyou 9507, Pinchun 16, Shiyou 17;; white arrows are specific bands that can track the gene PmDM8 .

[0020] Figure 3 For the disease-resistant parent Jing DM8, the disease-susceptible parent Langdon, and some F 2:3 Single plants of the family at the seedling stage were tested for Bgt Resistance performance against physiological race E09. Specific implementation manner

[0021] The technical solutions 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 a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0023] Example 1: Development of primers for wheat powdery mildew resistance gene PmDM8 HENU580 1 Materials The cultivated emmer wheat Jing DM8 and the durum wheat Langdon showed disease resistance and disease susceptibility to wheat powdery mildew respectively. The cultivated emmer wheat Jing DM8 and the durum wheat Langdon were crossed, and the obtained F1 was self-crossed to obtain the F2 population and the corresponding F 2:3 Family as shown in Table 1 and Table 2.

[0024] Table 1 Phenotypes of plant materials used for powdery mildew resistance Germplasm number Variety name E09 Beijing DM8 Cultivated emmer wheat 0 Langdon Durum wheat 4 Table 2 Jing DM8 and Langdon and their F2 and F 2:3 Inoculation at the seedling stage Bgt Segregation ratio of physiological race E09 2 Extraction of wheat genomic DNA The CTAB method for extracting wheat genomic DNA has the following procedure: (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.

[0025] (2) Add 600 μL of CTAB extraction solution, and incubate in a water bath at 65 °C for 40 min, mixing up and down every 10 min during this period.

[0026] (3) Add 600 μL of chloroform-isoamyl alcohol (24:1, v / v), and gently shake it on a shaker for 15 min.

[0027] (4) Centrifuge at 12,000 rpm for 15 min. Transfer the supernatant to another 2 mL centrifuge tube, add 3 volumes of pre-cooled absolute ethanol, and place it in a -20°C refrigerator for 60 min for precipitation.

[0028] (5) Pick out the flocculent DNA precipitate and wash it twice with 75% pre-cooled ethanol.

[0029] (6) Pick out the DNA precipitate, place it in a 1.5 mL centrifuge tube, and air-dry it naturally indoors.

[0030] (7) Add 60 μL of TE buffer (100 mM Tris-Hcl, 10 mM EDTA, pH = 8.0) to dissolve the DNA precipitate to prepare a DNA storage solution.

[0031] (8) Dilute the DNA storage solution with ultrapure water to 20 - 30 ng / μL as a working solution for standby.

[0032] 3 Identification of Powdery Mildew Resistance and Genetic Analysis of Disease Resistance at the Seedling Stage of Wheat Plant the disease-resistant parent Triticum dicoccoides Jing DM8, the susceptible parent Triticum durum Langdon, the F1 hybrid, the F2 population, and the F 2:3 families in 128-hole trays (2×2 cm). Sow 5 seeds in each hole. Identify 20 seeds for the parents and F1, and sow 20 seeds for each F 2:3 family. Randomly sow the susceptible control Zhoumai 18 and insert labels for distinction. After sowing, control the growth conditions as a 14 h light / 10 h dark cycle, temperature 20°C, and relative humidity 30 - 40%. When the seedlings grow to the one-leaf stage, inoculate with the wheat powdery mildew strain E09. The conditions for the first 24 h after inoculation are: dark, temperature 20°C, relative humidity 100%. After that, control the conditions as a 14 h light / 10 h dark cycle, temperature 18 - 22°C, and relative humidity 100%. When the first leaf of the susceptible control Zhoumai 18 is fully diseased, record the phenotypes according to the 0 - 4 grade standard. Among them, grades 0 - 2 are regarded as the disease-resistant grades, and grades 3 - 4 are the susceptible grades.

[0033] The survey results show that Triticum dicoccoides Jing DM8 is immune to the E09 strain, and Triticum durum Langdon (LDN) is highly susceptible to the E09 strain. Cross Triticum dicoccoides Jing DM8 with Triticum durum Langdon, and the F1 shows disease resistance; among 229 F2 plants, 174 are disease-resistant and 55 are susceptible, conforming to the 3:1 resistant-to-susceptible segregation ratio; 197 F 2:3Among the families, there are 44 homozygous resistant ones, 107 heterozygous ones, and 46 homozygous susceptible ones. The ratio of homozygous resistant:heterozygous:homozygous susceptible conforms to the segregation ratio of 1:2:1. Therefore, it is speculated that the resistance of cultivated emmer wheat Jing DM8 to strain E09 is controlled by a single dominant gene, named gene PmDM8 .

[0034] 4 Fine mapping of the powdery mildew resistance gene in cultivated emmer wheat Jing DM8 According to the phenotypic identification results, 15 homozygous resistant families and 15 homozygous susceptible families were respectively selected to construct a resistant pool and a susceptible pool. Molecular markers evenly distributed across the whole genome were used to detect polymorphisms in cultivated emmer wheat Jing DM8, durum wheat Langdon, the resistant pool, and the susceptible pool. 14 pairs of markers showed consistent polymorphisms in the resistant and susceptible parents and pools. Subsequently, these markers were used to genotype 197 F 2:3 families of cultivated emmer wheat Jing DM8 × durum wheat Langdon, and the gene PmDM8 was preliminarily mapped to the end of chromosome 6BL of wheat.

[0035] 5 Development of molecular markers tightly linked to the gene PmDM8 Based on the sequence information of the hexaploid wheat reference genome Chinese Spring v1.0, Simple Sequence Repeat (SSR) markers were designed using PrimerPrimer5.0 software and used to genotype the F 2:3 families of cultivated emmer wheat Jing DM8 × durum wheat Langdon, and an SSR marker HENU580 tightly linked to the gene PmDM8 was obtained, with a genetic distance of only 0.03 cM.

[0036] The primers of the molecular marker HENU580 include an upstream primer and a downstream primer: Nucleotide sequence of the upstream primer HENU580-F: 5'-CATCGACGCATCCCACATTT-3' (as shown in SEQ ID No.1); Nucleotide sequence of the downstream primer HENU580-R: 5'-AGTTTGGTTGTGTAATCTCGCA-3' (as shown in SEQ ID No.2).

[0037] 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. ​

[0038] The procedure for PCR amplification is as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 15 s, annealing at 56°C for 20 s, extension at 30 s, for 34 cycles; extension at 72°C for 10 min; preservation at 12°C.

[0039] The procedure for electrophoretic separation of the amplification product is as follows: electrophoresis is carried out on a non-denaturing polyacrylamide gel with a mass-volume percentage concentration of 8%. After mixing the amplification product with 2 μL of 6× loading buffer, 1.3 μL of the mixture is loaded for electrophoresis at a constant voltage of 180 V for 1.5 - 2 h, and then photographed after silver nitrate staining.

[0040] If a specific band of 273 bp in tight linkage can be amplified, the tested wheat carries the powdery mildew resistance gene. PmDM8 ; If the specific band of 273 bp in tight linkage is not amplified, the tested wheat does not carry the powdery mildew resistance gene in wheat. PmDM8 .

[0041] Example 2: Application of primers of molecular marker HENU580 for wheat powdery mildew resistance gene PmDM8 Materials in the F family formed by hybridization of cultivated emmer wheat Jing DM8, durum wheat Langdon and their hybrids were amplified and detected using primers of molecular marker HENU580 for wheat powdery mildew resistance gene; and the main susceptible wheat varieties in wheat-growing areas of China. The DNA extraction method was the same as in Example 1. Using primers of molecular marker HENU580 for wheat powdery mildew resistance gene PmDM8 to amplify and detect materials in the F family formed by hybridization of cultivated emmer wheat Jing DM8, durum wheat Langdon and their hybrids; and the main susceptible wheat varieties in wheat-growing areas of China. The DNA extraction method was the same as in Example 1. 2:3 Materials in the F family formed by hybridization of cultivated emmer wheat Jing DM8, durum wheat Langdon and their hybrids; and the main susceptible wheat varieties in wheat-growing areas of China. The DNA extraction method was the same as in Example 1.

[0042] Test samples: cultivated emmer wheat Jing DM8, durum wheat Langdon and their hybrid offspring F 2:3 family; the main susceptible wheat varieties in wheat-growing areas of China.

[0043] Genomic DNA of the above materials was extracted as the PCR amplification template, and amplified using the primers of molecular marker HENU580 developed in the present invention: Nucleotide sequence of upstream primer HENU580-F: 5'-CATCGACGCATCCCACATTT-3' (as shown in SEQ ID No.1); Nucleotide sequence of downstream primer HENU580-R: 5'-AGTTTGGTTGTGTAATCTCGCA-3' (as shown in SEQ ID No.2).

[0044] The system for PCR amplification 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.

[0045] The procedure for PCR amplification is as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 15 s, annealing at 56°C for 20 s, extension for 30 s, for 34 cycles; extension at 72°C for 10 min; preservation at 12°C.

[0046] The procedure for electrophoretic separation of the amplification products is as follows: electrophoresis is carried out on a non-denaturing polyacrylamide gel with a mass-volume percentage concentration of 8%. After mixing the amplification products with 2 μL of 6× loading buffer, 1.3 μL of the mixture is loaded for electrophoresis at a constant voltage of 180 V for 1.5 - 2 h, and then photographed after silver nitrate staining.

[0047] The results of detecting the cultivated emmer wheat Jing DM8, durum wheat Langdon and their hybrid progeny segregation population with the molecular marker HENU580 primer are shown in Figure 1 . Figure 1 For the partial amplification results of the marker HENU580 in the F 2:3 families derived from the cross of cultivated emmer wheat Jing DM8 × durum wheat Langdon. In the figure: M: DL2000; 1: cultivated emmer wheat Jing DM8 (powdery mildew-resistant variety, carrying the powdery mildew-resistant gene PmDM8 ); 2: durum wheat Langdon (powdery mildew-susceptible variety); 3 - 22: F 2:3 families formed by the cross of cultivated emmer wheat Jing DM8 and durum wheat Langdon. Among them, 3 - 7: homozygous resistant F 2:3 families, 8 - 17: resistant-susceptible segregating F 2:3 families, 18 - 22: homozygous susceptible F 2:3 families; the white arrow is the specific band that can trace the gene PmDM8 . The amplification results show that the marker HENU580 amplified a specific band closely linked to 273 bp in the resistant parent cultivated emmer wheat Jing DM8 and the resistant families, and did not amplify the target band in the susceptible parent durum wheat Langdon and the susceptible families.

[0048] The results of detecting the cultivated emmer wheat Jing DM8, durum wheat Langdon and the main susceptible wheat varieties in the wheat-growing areas of China with the molecular marker HENU580 primer are shown in Figure 2 . Figure 2Amplification results of marker HENU580 in cultivated emmer wheat Jing DM8, durum wheat Langdon and the main susceptible cultivated wheat varieties in wheat-growing regions of China. In the figure: DL2000; 1: cultivated emmer wheat Jing DM8 (powdery mildew-resistant variety, carrying powdery mildew resistance gene PmDM8 ); 2: durum wheat Langdon (powdery mildew-susceptible variety); 3-17: Yumai 16, Zhengzhou 5, Zhengzhou 24, Yangmai 1, Yangmai 158, Jinan 8, Jinan 9, Lumai 7, Lumai 12, Lumai 14, Lumai 15, Zhongyou 206, Zhongyou 9507, Pinchun 16, Shiyou 17;; The white arrow is the specific band that can trace the gene PmDM8 . The amplification results show that marker HENU580 amplified a specific band tightly linked to 273 bp in the powdery mildew-resistant cultivated emmer wheat Jing DM8, and this target band was not amplified in the powdery mildew-susceptible durum wheat Langdon.

[0049] Powdery mildew resistance gene PmDM8 originates from the cultivated emmer wheat Jing DM8 variety. Currently, there is no report on the mapping and map-based cloning of this gene. Detecting a large genetic mapping population through the molecular marker HENU580 provided by the present invention helps in the PmDM8 fine mapping and map-based cloning of the gene, and is of great significance for the PmDM8 efficient introgression of the gene and in-depth analysis of the disease resistance mechanism.

[0050] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of wheat powdery mildew resistance gene PmDM8 in the cultivation of powdery mildew-resistant wheat.

2. The application according to claim 1, wherein: The powdery mildew resistance gene PmDM8 is located in a 560.78 kb physical interval between 693,556,693 - 694,117,473 bp on wheat chromosome 6BL.

3. Molecular marker for detecting powdery mildew resistant gene PmDM8 which is characterized in that: The molecular marker is tightly linked to the powdery mildew resistance gene PmDM8 with a genetic distance of only 0.03 cM.

4. Primers for detecting powdery mildew resistance genes PmDM8 which are characterized in that: The primer is a primer pair for detecting the molecular marker described in claim 3.

5. The primer for detecting the powdery mildew resistance gene according to claim 4 PmDM8 is characterized in that: The sequences of the primer pair are as shown in SEQ ID No.1 and SEQ ID No.

2.

6. Use of the molecular marker described in claim 3 or the primer described in any one of claims 4-5 in assisting in identifying whether wheat has powdery mildew resistance.

7. The application according to claim 6, wherein: The application is achieved by identifying whether wheat has powdery mildew resistance genes PmDM8 ​ 8. Use of the molecular marker described in claim 3 or the primer described in any one of claims 4-5 in assisting in cultivating powdery mildew-resistant wheat.

9. A method for detecting whether a wheat variety is resistant to powdery mildew, characterized in that: The method is achieved by detecting whether wheat contains the powdery mildew resistance gene PmDM8 and is realized 10. The method for detecting whether wheat is a powdery mildew-resistant variety according to claim 9, characterized in that: The steps are as follows: Using the primer described in claim 4 or 5, amplify the genome of the wheat sample to be tested. If a specific band tightly linked with 273 bp can be amplified, it indicates that the wheat sample to be tested carries the powdery mildew resistance gene. PmDM8 , it is a powdery mildew resistant variety; on the contrary, the wheat sample to be tested does not carry it and is a powdery mildew susceptible variety.

Images