Molecular marker closely linked to wheat powdery mildew resistance gene PmDR696 and its detection applications

Abstract

The application belongs to the field of agricultural biological gene technology, and relates to a wheat powdery mildew resistance gene PmDR696 The application provides a closely linked molecular marker, a detection primer and an application. PmDR696 The molecular marker can be applied to detection, identification and auxiliary identification of a wheat powdery mildew resistance gene. PmDR696 The primer can be used for detecting whether a wheat breeding material carries the wheat powdery mildew resistance gene before the wheat powdery mildew is not developed at a wheat seedling stage. PmDR696 The marker is not only rapid, accurate, cost-saving, efficient and fast in breeding.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology and relates to wheat genetics and breeding. Background Technology

[0002] Wheat is a major crop widely cultivated in most temperate regions of the world, and it is one of my country's most important food crops. Its high and stable yields are crucial to my country's food security. Powdery mildew, caused by *Brucella brucellosa*, is one of the most common foliar diseases of wheat. Its conidia are spread by air currents and, after infecting wheat leaves, compete with the wheat for nutrients through haustoria, reducing the photosynthetic capacity of the 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 control measure in production is the use of chemical agents, planting wheat varieties carrying effective disease-resistant genes is currently the most effective and environmentally friendly control strategy.

[0003] Since the first powdery mildew resistance gene in 1953 Pm1 Since its discovery over 70 years ago, more than 200 officially and provisionally 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 cluster by long-read genome sequencing accelerated the cloning of Pm69 Plant Communications, 2024, 5. 1). Among these, powdery mildew resistance genes derived from common wheat can be directly applied to improve the disease resistance of wheat varieties, introduced into target varieties through field hybridization, including... Pm1a, Pm5e, Pm3b, Pm24a / b, Pm47 , Pm52 Disease resistance genes derived from closely related wheat species, after multiple generations of backcrossing and agronomic trait improvement, can be used for wheat resistance improvement, including... Pm2, Pm8, Pm21, Pm36, Pm37, Pm42, Pm58Etc. Because the genes conferring resistance in wheat are race-specific, the virulence of wheat powdery mildew strains varies rapidly. Some resistance genes gradually lose their resistance, becoming ineffective / ineffective resistance genes, such as... Pm1, Pm2, Pm3, Pm5 and Pm8 In most wheat-growing areas of China, resistance to powdery mildew has been almost completely lost (Wang et al. Fighting wheat powdery mildew: from genes to fields. Theoretical and Applied Genetics, 2023, 136(9): 196.). Therefore, it is essential to find and locate new powdery mildew resistance genes, continuously expand the powdery mildew resistance gene pool, and strategically deploy and combine these resistance genes in different wheat-growing areas.

[0004] durum wheat ( Triticum turgidum L. var. durum Durum wheat (Desf., 2n=28, AABB), also known as durum wheat, is a tetraploid wheat variety. It is predominantly spring-growing and mainly cultivated in arid regions around the Mediterranean. Its grains are relatively small and primarily used for pasta production. Durum wheat exhibits good resistance to leaf rust, stem rust, and stripe rust, making it a common variety for breeders in improving wheat resistance (Miedane et al. Genetic architecture of yellow and stem rust resistance in a durum wheat diversity panel. Euphytica, 2019, 215: 1-17). Four powdery mildew resistance genes have been identified in durum wheat, including... Mld, Pm3h, PmDR147 and Pm68 They were located at 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 is evolved from wild Triticum dicoccoides, while common wheat is evolved from Triticum dicoccum and Aegilops tauschii for a long time, so the genetic relationship between them provides the basis for 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 has no obvious difference in quality compared with the hybrid offspring of common wheat, except that the thousand-grain weight and spike grain number are slightly lower than the latter. Therefore, it is of great significance to mine new powdery mildew resistance genes from durum wheat, a close relative of common wheat, to reasonably use resistance sources, accelerate gene aggregation breeding, and broaden the genetic basis of wheat. SUMMARY

[0005] The present application proposes a molecular marker closely linked to the wheat powdery mildew resistance gene PmDR696 and its detection application, which provides a guidance basis for breeding new wheat varieties resistant to powdery mildew.

[0006] The technical solution of the present application is realized as follows:

[0007] The present application provides a molecular marker HENU622 of a wheat powdery mildew resistance gene PmDR696 , which is only 1.8 cM away from the gene PmDR696 and is located on the 2AL chromosome of wheat.

[0008] A primer for amplifying a molecular marker closely linked to a powdery mildew resistance gene PmDR696 of durum wheat is also provided, and the molecular marker is the co-dominant SSR marker HENU622, and the primer includes an upstream primer HENU622-F and a downstream primer HENU622-R.

[0009] The upstream primer of the molecular marker HENU622 is HENU622-F, and the nucleotide sequence thereof is:

[0010] 5'-TTTACGGGCCGCCGTTAGAG-3', as shown in SEQ ID No. 1;

[0011] The downstream primer of the molecular marker HENU622 is HENU622-R, and the nucleotide sequence is:

[0012] 5'-AATGTTTACGAGAGAGGTGGT-3', as shown in SEQ ID No. 2;

[0013] The application provides an amplified hard wheat powdery mildew resistance gene PmDR696 The primer of the closely linked molecular marker is used for detecting, identifying and assisting in identifying the wheat powdery mildew resistance trait and application in molecular marker assisted breeding. PmDR696

[0014] The wheat powdery mildew resistance gene PmDR696 is located on the wheat 2AL chromosome (780,433,862-784,186,371 bp).

[0015] The application further provides a method for detecting whether a wheat sample carries the hard wheat powdery mildew resistance gene PmDR696 , comprising the following steps:

[0016] (1) extracting genomic DNA of the wheat sample to be tested;

[0017] (2) performing PCR amplification on the genomic DNA of the wheat sample to be tested by using a molecular marker primer, to obtain an amplification product; the molecular marker primer comprises an upstream primer HENU622-F and a downstream primer HENU622-R;

[0018] The upstream primer of the molecular marker HENU622 is HENU622-F, and the nucleotide sequence is:

[0019] 5'-TTTACGGGCCGCCGTTAGAG-3', as shown in SEQ ID No. 1;

[0020] The downstream primer of the molecular marker HENU622 is HENU622-R, and the nucleotide sequence is:

[0021] 5'-AATGTTTACGAGAGAGGTGGT-3', as shown in SEQ ID No. 2;

[0022] (3) performing electrophoresis and detection on the amplification product, if a specific band of 214 bp closely linked can be amplified, it indicates that the wheat sample to be tested carries the hard wheat powdery mildew resistance gene PmDR696 ; otherwise, the wheat sample to be tested does not carry the hard wheat powdery mildew resistance gene PmDR696 .

[0023] ​The PCR amplification system in step (2) of the method is 10 μL, including: 20 ng / μL wheat genomic DNA 1.0 μL, 5 μL PCR Master Mix, 5 μM upstream primer 0.4 μL, 5 μM downstream primer 0.4 μL, and 3.2 μL of sterile deionized water.

[0024] The PCR amplification program in step (2) of the method is: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 60℃ annealing for 20 s, extension for 40 s, 34 cycles; 72℃ extension for 10 min; 12℃ storage.

[0025] The electrophoresis program of the amplification product in step (3) of the method is: electrophoresis is carried out on a non-denaturing polyacrylamide gel with a mass percentage concentration of 8%, the amplification product is mixed with 2 μL of 6x loading buffer, 1.3 μL of the mixture is taken for sample loading, electrophoresis is carried out at a constant voltage of 180 V for 1.5-2 h, and silver nitrate staining is carried out before taking a photo.

[0026] The present application has the following beneficial effects:

[0027] 1、The hard wheat Jing DR696 used in the present application is immune to powdery mildew at seedling stage and adult stage, and through seedling stage powdery mildew resistance genetic analysis and molecular marker detection, it is shown that the resistance of the hard wheat Jing DR696 to different virulence wheat powdery mildew at seedling stage is controlled by a pair of dominant genes PmDR696 , which is located on the wheat 2AL chromosome and is a new wheat powdery mildew resistance gene / allele. PmDR696 The molecular marker HENU622 of the wheat powdery mildew resistance gene PmDR696 provided in the present application is detected through a genetic separation population, and the genetic distance with the gene PmDR696 is only 1.8 cM, and the gene PmDR696 is closely linked, so that the genetic mapping large population of the gene PmDR696 can be accurately detected, and the fine mapping and map-based cloning of the gene can be applied.

[0028] 2、The present application provides primers of a molecular marker closely linked to the wheat powdery mildew resistance gene Figure 1 and application thereof, which not only quickly and accurately screen target varieties, are not affected by the environment, have clear selection targets, save production costs, and greatly improve the selection efficiency and quality of high-quality powdery mildew-resistant wheat varieties or lines. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only need to be some embodiments of the present application, and other drawings can be obtained by those of ordinary skill in the art without any creative effort.

[0030] Msp Results of detecting hard wheat Jing DR696 and hard wheat Langdon and their hybrid offspring segregation population by molecular marker HENU622. In the figure, M: pUC19 DNA PmDR696 I; 1: hard wheat Jing DR696 (resistant to powdery mildew variety, carrying powdery mildew resistance gene PmDR696 ) 2: hard wheat Langdon (powdery mildew susceptible variety); 3-22: F 2:3 family formed by hybridization of hard wheat Jing DR696 and hard wheat Langdon, wherein 3-7: homozygous resistant F 2:3 family, 8-17: resistant and susceptible segregation F 2:3 family, 18-22: homozygous susceptible F 2:3 family; white arrow is the specific band that can track the gene Figure 2 .

[0031] PmDR696 Reaction of parents Jing DR696, Langdon and hybrid F1 offspring to E09 strain. DETAILED DESCRIPTION

[0032] The technical solutions of the present application will be described clearly and completely in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without any creative effort are within the scope of protection of the present application.

[0033] The experimental methods used in the following experimental examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained commercially unless otherwise specified.

[0034] Example 1: Wheat powdery mildew resistance gene Bgt Development of molecular marker HENU622 primer

[0035] 1 Material

[0036] Hard red winter wheat Jing DR696 and hard red winter wheat Langdon showed resistant and susceptible to wheat powdery mildew, respectively, hard red winter wheat Jing DR696 and hard red winter wheat Langdon were crossed, the obtained F1 was selfed, F2 population and corresponding F 2:3 Families, as shown in Table 1 and Table 2.

[0037] Table 1 relates to the phenotype of plant materials against powdery mildew

[0038]

[0039] Table 2 Jing DR696 and Langdon and their F2 and F 2:3 Seedling inoculation Figure 2 Isolation ratio of physiological race E09

[0040]

[0041] 2 Extraction of wheat genomic DNA

[0042] The CTAB method for extracting wheat genomic DNA is as follows:

[0043] (1) Fresh wheat leaves were cut and ground in liquid nitrogen, and about 0.5 g was taken and placed in a 2.0 mL centrifuge tube.

[0044] (2) Add 600 μL CTAB extraction solution, 65℃ water bath for 40 min, and mix well every 10 min.

[0045] (3) Add 600 μL chloroform-isoamyl alcohol (24:1, v / v), and shake gently on a shaker for 15 min.

[0046] (4) Centrifuge at 12,000 rpm for 15 min, take the supernatant into another 2 mL centrifuge tube, add 3 times the volume of pre-cooled anhydrous ethanol, and precipitate in a-20℃ refrigerator for 60 min.

[0047] (5) Take out the flocculent DNA precipitate and wash it with pre-cooled 75% ethanol for 2 times.

[0048] (6) Take out the DNA precipitate and place it in a 1.5 mL centrifuge tube, and dry it naturally in the room.

[0049] (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.

[0050] (8) Dilute the DNA storage solution with ultrapure water to 20-30 ng / μL as working solution for standby.

[0051] 3. Identification of powdery mildew resistance in wheat seedlings and genetic analysis of disease resistance

[0052] The disease-resistant parent durum wheat Jing DR696, the disease-susceptible parent durum wheat Langdon, F1 hybrids, F2 populations, and F1 hybrids were compared. 2:3 The families were planted in 128-cell trays (2×2cm), with 5 seeds sown in each cell. Both the parents and F1 strains had 20 seeds each. 2:3 Twenty seeds were sown from each family, while the susceptible control Zhoumai 18 was randomly sown and tagged for identification. After sowing, growth conditions were controlled as a 14-hour light / 10-hour dark cycle, temperature 20℃, and relative humidity 30-40%. When seedlings reached the one-leaf stage, they were inoculated with wheat powdery mildew strain E09. For the first 24 hours after inoculation, conditions were controlled as darkness, temperature 20℃, and relative humidity 100%. Afterward, conditions were controlled as a 14-hour light / 10-hour dark cycle, temperature 18-22℃, and relative humidity 100%. When the first leaf of the susceptible control Zhoumai 18 showed full disease, phenotypes were recorded according to a 0-4 scale, with 0-2 considered resistance and 3-4 considered susceptibility.

[0053] The survey results are as follows PmDR696 The results showed that durum wheat DR696 was immune to strain E09, while durum wheat Langdon (LDN) was highly susceptible to strain E09. A cross between durum wheat DR696 and durum wheat Langdon resulted in F1 plants that were resistant; of the 166 F2 plants, 125 were resistant and 41 were susceptible, consistent with a 3:1 segregation ratio of resistant to susceptible. 2:3 In the family, 36 strains were homozygous resistant, 89 were heterozygous, and 41 were homozygous susceptible. The ratio of homozygous resistant to heterozygous to homozygous susceptible conformed to a segregation ratio of 1:2:1. Therefore, it is speculated that the resistance of durum wheat Jing DR696 to strain E09 is controlled by a single dominant gene, named gene [gene name missing]. PmDR696 .

[0054] 4. Preliminary molecular marker localization of the powdery mildew resistance gene in durum wheat Jing DR696

[0055] Based on phenotypic identification results, 20 homozygous resistant families and 20 homozygous susceptible families were selected to construct resistant and susceptible pools, respectively. Polymorphism detection was performed on durum wheat DR696 and Langdon, as well as the resistant and susceptible pools, using genome-wide uniformly distributed molecular markers. Ten pairs of markers showed consistent polymorphism in the resistant and susceptible parents and the resistant-susceptible pools. Subsequently, these markers were used to detect F1 mutations in 166 durum wheat DR696 × Langdon durum wheat lines. 2:3 Genotyping of families, gene PmDR696 Preliminary localization to wheat chromosome 2AL.

[0056] 5 with the gene PmDR696 Development of closely linked molecular markers

[0057] According to the sequence information of the hexaploid wheat reference genome SY Mattis v1.0, a Simple Sequence Repeat (SSR) marker was designed by using primer 5.0 software, and the F 2:3 family of hard wheat Jing DR696 x hard wheat Langdon was genotyped to obtain the SSR marker HENU622 closely linked to the gene PmDR696 with a genetic distance of only 1.8 cM.

[0058] The primer of the molecular marker HENU622 comprises an upstream primer and a downstream primer.

[0059] The nucleotide sequence of the upstream primer HENU622-F is 5'-TTTACGGGCCGCCGTTAGAG-3' (as shown in SEQ ID No. 1);

[0060] The nucleotide sequence of the downstream primer HENU622-R is 5'-AATGTTTACGAGAGAGGTGGT-3' (as shown in SEQ ID No. 2).

[0061] The PCR amplification system is 10 μL, including: 20 ng / μL wheat genomic DNA 1.0 μL, 5 μL PCR Master Mix, 5 μM upstream primer 0.4 μL, 5 μM downstream primer 0.4 μL, and 3.2 μL of sterile deionized water.

[0062] The PCR amplification program is: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 60℃ annealing for 20 s, extension for 40 s, 34 cycles; 72℃ extension for 10 min; 12℃ storage.

[0063] The electrophoresis separation program of the amplification product is: electrophoresis on a non-denaturing polyacrylamide gel with a mass percentage concentration of 8%, mix the amplification product and 2 μL 6x loading buffer, take 1.3 μL of the mixture for sample loading, electrophoresis at 180V constant voltage for 1.5-2 h, and take a photo after silver nitrate staining.

[0064] A specific band of 214 bp closely linked to the gene PmDR696 was amplified; and a specific band of 214 bp closely linked to the gene PmDR696 was not amplified.

[0065] Example 2: Wheat powdery mildew resistance genePmDR696 Application of the molecular marker HENU622 primers

[0066] Using wheat powdery mildew resistance genes Figure 1 The molecular marker HENU622 primer amplification was used to detect durum wheat DR696, durum wheat Langdon, and the F1 hybrid formed by their cross. 2:3 The DNA extraction method for materials from the family lineage was the same as in Example 1.

[0067] Samples to be tested: durum wheat DR696, durum wheat Langdon, and their hybrid F1 strain. 2:3 Family lineage.

[0068] Genomic DNA was extracted from the above materials and used as a PCR amplification template. Amplification was performed using primers for the molecular marker HENU622 developed in this invention.

[0069] Nucleotide sequence of upstream primer HENU622-F:

[0070] 5'-TTTACGGGCCGCCGTTAGAG-3' (as shown in SEQ ID No. 1);

[0071] The nucleotide sequence of the downstream primer HENU622-R is as follows:

[0072] 5'-AATGTTTACGAGAGAGGTGGT-3' (as shown in SEQ ID No. 2).

[0073] The PCR amplification system was 10 μL, comprising: 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.

[0074] The PCR amplification procedure is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 15 s, 60℃ annealing for 20 s, extension for 40 s, 34 cycles; 72℃ extension for 10 min; storage at 12℃.

[0075] The electrophoretic separation procedure for the amplified products is as follows: electrophoresis is performed on a non-denaturing polyacrylamide gel with a mass-volume percentage concentration of 8%. The amplified products are mixed with 2 μL of 6× loading buffer, and 1.3 μL of the mixture is loaded. Electrophoresis is performed at a constant voltage of 180V for 1.5-2 h. The gel is then stained with silver nitrate and photographed.

[0076] Molecular marker detection results are shown below Figure 1 . MspTo mark HENU622 in the F 2:3 partially amplified results in the family derived from hard wheat Jing DR696 x hard wheat Langdon. In the figure: M: pUC19 DNA / PmDR696 I; 1: hard wheat Jing DR696 (a resistant variety to powdery mildew, carrying the resistance gene PmXu6) PmDR696 ); 2: hard wheat Langdon (a susceptible variety to powdery mildew); 3-22: F 2:3 families formed by the cross of hard wheat Jing DR696 and hard wheat Langdon, wherein 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; white arrows are specific bands that can track the gene PmDR696 The amplification results show that the marker HENU622 amplifies a 214 bp tightly linked specific band in the resistant parent hard wheat Jing DR696 and the resistant families, and does not amplify the specific band in the susceptible parent hard wheat Langdon and the susceptible families.

[0077] Wheat powdery mildew resistance gene PmDR696 derived from the hard wheat Jing DR696 variety, and the gene has not been reported to be located and map-based cloned. The detection of the genetic mapping large population by the molecular marker HENU622 provided by the application is helpful for fine mapping and map-based cloning of the gene PmDR696 , and has very important significance for efficient transformation of the gene ​ and in-depth analysis of the disease resistance mechanism.

[0078] The above only describes the preferred embodiments of the application and is not used to limit the application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. Use of primers of molecular markers tightly linked to the wheat powdery mildew resistance gene PmDR696 The use of primers of molecular markers tightly linked to the wheat powdery mildew resistance gene The primers include an upstream primer HENU622-F having a nucleotide sequence as shown in SEQ ID No. 1 and a downstream primer HENU622-R having a nucleotide sequence as shown in SEQ ID No.

2.

2. Use according to claim 1, characterized in that: The application is by identifying whether the Durum wheat has the resistance to powdery mildew gene PmDR696 achieved; The steps are: using the primer of claim 1 to amplify the genome of the wheat sample to be tested, if a 214 bp specific band tightly linked can be amplified, it indicates that the wheat sample to be tested carries the wheat powdery mildew resistance gene PmDR696 , and has the powdery mildew resistance; otherwise, the wheat sample to be tested does not carry the powdery mildew resistance gene and does not have the powdery mildew resistance.

3. A method for detecting the presence of a wheat powdery mildew resistance gene PmDR696 The use of primers for closely linked molecular markers in the assisted breeding of powdery mildew resistant Durum wheat, characterized by: The primers include an upstream primer HENU622-F having a nucleotide sequence as shown in SEQ ID No. 1 and a downstream primer HENU622-R having a nucleotide sequence as shown in SEQ ID No.

2.

4. A method of detecting whether a Durum wheat is a resistant variety to powdery mildew, characterized in that: The method is by detecting whether the Durum wheat contains the anti-mildew gene PmDR696 achieved; The steps are: using the primers described in SEQ ID No. 1-2 to amplify the genome of the wheat sample to be tested, if a 214 bp tightly linked specific band can be amplified, it indicates that the wheat sample to be tested carries the wheat powdery mildew resistance gene PmDR696 is a powdery mildew resistant variety; otherwise, the wheat sample to be tested does not carry it, which is a powdery mildew susceptible variety.

Images