A detectable molecular marker for tracking anti- powdery mildew gene PmDR144 and its application in disease-resistant breeding

Abstract

The present invention belongs to the field of genetic breeding and relates to the cultivation of powdery mildew resistant wheat. PmDR144 A detectable molecular marker and its application in disease resistance breeding, the molecular marker includes an upstream primer HENU624-F and a downstream primer HENU624-R; the nucleotide sequence of the upstream primer HENU624-F is shown in SEQ ID No. 1, and the nucleotide sequence of the downstream primer HENU624-R is shown in SEQ ID No. 2. The primers provided by the present invention can be used to detect whether wheat breeding materials carry powdery mildew resistance genes before powdery mildew occurs in wheat seedlings. PmDR144 Conduct testing and purposefully select for genes that carry resistance to powdery mildew PmDR144 For varieties, this marker not only detects quickly and accurately, but also saves costs, improves breeding efficiency and accelerates the breeding process.

Description

Technical Field

[0001] The invention belongs to the field of genetic breeding and relates to the cultivation of powdery mildew-resistant wheat. Background Art

[0002] wheat( Triticum aestivum L., 2n=42, AABBDD) is a major food crop in the world. It contains a large amount of nutrients such as starch, protein and trace elements, and plays an irreplaceable role in human activities. Therefore, breeding highly resistant and widely adaptable wheat varieties is a key topic for breeders (Gorissenet al. Ingestion of wheat protein increases in vivo muscle protein synthesis rates in healthy older men in a randomized trial. The Journal of nutrition. 2016, 146.(9):1651-1659). Wheat powdery mildew, caused by the powdery fungus Blumeria graminis, is a global fungal disease of wheat. After infection, it robs wheat plants of nutrients and hinders photosynthesis, making it difficult for plants to accumulate and transport carbohydrates, ultimately seriously affecting wheat yield. Currently, breeding and utilizing disease-resistant wheat varieties is the most economical, effective and environmentally friendly method for controlling powdery mildew.

[0003] Currently, 64 officially named disease resistance gene loci have been identified in primary and secondary gene pools of common wheat and related species ( Pm1-Pm69, Pm8=Pm17, Pm18=Pm1c, Pm22=Pm1e, Pm23=Pm4c, Pm31 =Pm21 ) and dozens of temporarily named wheat powdery mildew resistance genes. Some resistance genes have also been identified from tertiary gene pools such as Secale, Elysia, and Triticum and transferred to common wheat. However, wheat powdery mildew is caused by an obligate parasitic fungus with a fast mutation rate. Resistance genes for specific subspecies often become ineffective in a short period of time. In most wheat-growing areas in my country, Pm8 The resistance to wheat has been almost completely lost; in wheat-producing areas such as Hebei, Anhui and Jiangsu, Pm2 and Pm4b resistance is also gradually lost (Zou et al. Pm52-Effectiveness of the gene conferring resistance to powdery mildew in wheat cultivar Liangxing99. Acta Agronomica Sinica, 2017, 43(3): 332-342). Therefore, researchers have proposed to deploy diversified resistance genes to solve the dilemma of resistance loss caused by large-scale and long-term planting of a single disease-resistant variety. This approach requires the search for new powdery mildew resistance genes (McIntosh et al. Catalogue of gene symbols for wheat. Proceedings of the 12th international wheat genetics symposium. Yokohama Japan, 2013.).

[0004] durum wheat ( Triticum turgidum L. var. durum Desf., simply T. durum , 2n=28,AABB) is a secondary resource library of common wheat. Compared with common wheat, it is more resistant to stripe rust, leaf rust and bunt, and has the characteristics of drought resistance, barrenness resistance, high protein content and high nutritional value. Durum wheat is often used in artificial wheat synthesis, and its genetic background is richer than that of common wheat. The offspring obtained by directly hybridizing durum wheat with common wheat have not been found to have obvious reduction in agronomic traits. It is one of the important resources for genetic improvement of common wheat. At present, only 4 powdery mildew resistance genes have been identified in durum wheat, including 1 recessive disease resistance gene Mld and three dominant disease resistance genes Pm3h, PmDR147 and Pm68 , are located on chromosomes 4B, 1A, 2A and 2B respectively (He et al. Characterization of Pm68 Discovering new powdery mildew resistance genes from durum wheat and introducing them into common wheat not only enriches wheat's powdery mildew resistance resources but also introduces other desirable traits, increasing the genetic diversity of common wheat and benefiting the development of powdery mildew resistance breeding in my country. Summary of the Invention

[0005] The present invention provides a method for tracking powdery mildew resistance genes PmDR144 Detectable molecular markers and their application in disease resistance breeding, using primers to amplify molecular markers for wheat powdery mildew resistance genes PmDR144 Positioning and detection are carried out to purposefully select its parents in wheat breeding, providing guidance for breeding new wheat varieties resistant to powdery mildew.

[0006] The technical solution of the present invention is achieved as follows:

[0007] The present invention found that the resistance of durum wheat JingDR144 to different toxicity wheat powdery mildew at seedling stage is caused by a pair of dominant genes through genetic analysis and molecular marker detection. PmDR144 control, located within a physical interval of 7.31 Mb (774,583,083-781,893,037 bp) on wheat chromosome 2AL, is a new wheat powdery mildew resistance gene / allele.

[0008] Wheat powdery mildew resistance gene provided by the present invention PmDR144 The molecular marker HENU624 was tested in genetic segregation populations and was PmDR144 The genetic distance is only 2.7 cM, and the PmDR144 Tightly linked, allowing accurate gene detection PmDR144 Large genetic mapping populations, applied to genes PmDR144 Fine positioning and positional cloning.

[0009] The present invention provides a method for amplifying a powdery mildew resistance gene in durum wheat. PmDR144 Primers for tightly linked molecular markers, wherein the molecular marker is the co-dominant SSR marker HENU624, and the primers include an upstream primer HENU624-F and a downstream primer HENU624-R;

[0010] The upstream primer of the molecular marker HENU624 is HENU624-F, and its nucleotide sequence is:

[0011] 5'-GACTTTGGGTAAACACTTCC-3', as shown in SEQ ID No. 1;

[0012] The downstream primer of the molecular marker HENU624 is HENU624-R, and its nucleotide sequence is:

[0013] 5'-TCATTGCTTGGTCCCTCCTC-3', as shown in SEQ ID No. 2.

[0014] Amplified durum wheat powdery mildew resistance gene provided by the present invention PmDR144 Primers for tightly linked molecular markers are located in the gene PmDR144 Detection, identification and assisted identification of wheat powdery mildew resistance traits and its application in molecular marker-assisted breeding.

[0015] The present invention also provides a method for detecting whether a wheat sample carries a durum wheat powdery mildew resistance gene. PmDR144 The method comprises the following steps:

[0016] (1) Extracting genomic DNA from the wheat sample to be tested;

[0017] (2) PCR amplification of the genomic DNA of the wheat sample to be tested is performed using molecular marker primers to obtain an amplified product; the molecular marker primers include an upstream primer HENU624-F and a downstream primer HENU624-R;

[0018] The upstream primer of the molecular marker HENU624 is HENU624-F, and its nucleotide sequence is:

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

[0020] The downstream primer of the molecular marker HENU624 is HENU624-R, and its nucleotide sequence is:

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

[0022] (3) The amplified product is subjected to electrophoresis and detection. If two closely linked specific bands of 157 and 186 bp are amplified, it indicates that the wheat sample to be tested carries the durum wheat powdery mildew resistance gene. PmDR144 Otherwise, the wheat sample to be tested does not carry the durum wheat powdery mildew resistance gene PmDR144 .

[0023] The PCR amplification system in step (2) of the above method 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.

[0024] The PCR amplification procedure in step (2) of the above method is as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 15 s, annealing at 58°C for 20 s, extension for 40 s, 34 cycles; extension at 72°C for 10 min; and storage at 12°C.

[0025] The electrophoresis procedure of the amplified product in step (3) of the above 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, and electrophoresis is performed at a constant voltage of 180 V for 1.5-2 h, and the sample is photographed after silver nitrate staining.

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

[0027] The present invention found that the resistance of durum wheat JingDR144 to different toxicity wheat powdery mildew at seedling stage is caused by a pair of dominant genes. PmDR144 control, located on wheat chromosome 2AL, is a novel wheat powdery mildew resistance gene / allele.

[0028] The present invention provides a method for tracking powdery mildew resistance genes PmDR144 The detectable molecular markers and their application in disease-resistant breeding 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 efficiency and quality of selecting high-quality powdery mildew-resistant wheat varieties or lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[0030] Figure 1 The results of molecular marker HENU624 primers detection of segregating populations of Durum wheat JingDR144 and Durum wheat Langdon and their hybrid progeny; in the figure: M: DL2000; 1: Durum wheat JingDR144 (powdery mildew resistant variety, carrying PmDR144 ); 2: Durum wheat Langdon (powdery mildew susceptible variety); 3-22: F hybrid formed by the hybridization of Durum wheat Jing DR144 and Durum wheat Langdon 2:3 Families, among which 3-7: homozygous disease-resistant F 2:3 Family, 8-17: F with resistance and segregation 2:3 Family, 18-22: homozygous susceptible F 2:3 Family; white arrows indicate traceable genes PmDR144 Specific bands.

[0031] Figure 2 The resistant parent JingDR144, the susceptible parent Langdon and some F 2:3 Family single plant seedling stage BgtResistance performance of physiological race E09. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all 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.

[0033] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0034] Example 1: Wheat powdery mildew resistance gene PmDR144 Development of primers for molecular marker HENU624

[0035] 1. Materials

[0036] Durum wheat JingDR144 and Durum wheat Langdon are resistant and susceptible to wheat powdery mildew, respectively. Durum wheat JingDR144 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.

[0037] Table 1 Powdery mildew resistance phenotypes of plant materials used

[0038]

[0039] Table 2 Beijing DR144 and Langdon and their F2 and F 2:3 Seedling inoculation Bgt Separation ratio of physiological race E09

[0040]

[0041] 2. Extraction of wheat genomic DNA

[0042] Wheat genomic DNA was extracted using the CTAB method. The process is as follows:

[0043] (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.

[0044] (2) Add 600 μL of CTAB extract and incubate in a 65°C water bath for 40 min, mixing up and down every 10 min.

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

[0046] (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°C refrigerator for precipitation for 60 min.

[0047] (5) Pick out the flocculent DNA precipitate and wash it twice with 75% pre-cooled ethanol.

[0048] (6) Pick out the DNA precipitate, place it in a 1.5 mL centrifuge tube, and air-dry it indoors.

[0049] (7) Add 60 μL of TE buffer (100 mM Tris-HCl, 10 mM EDTA, pH = 8.0) to dissolve the DNA precipitate to prepare DNA storage solution.

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

[0051] 3 Identification of powdery mildew resistance and genetic analysis of wheat seedling resistance

[0052] The disease-resistant parent Durum wheat JingDR144, the disease-susceptible parent Durum wheat Langdon, the F1 hybrid, the F2 population and the F 2:3 The family was 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 from each family, and the susceptible control, Zhoumai 18, was randomly sown and labeled for identification. After sowing, the seedlings were grown under a 14-hour light / 10-hour dark cycle, a temperature of 20°C, and a relative humidity of 30-40%. When the seedlings reached the one-leaf stage, they were inoculated with the wheat powdery mildew strain E09. For the first 24 hours after inoculation, the seedlings were kept in darkness, a temperature of 20°C, and a relative humidity of 100%. Thereafter, the seedlings were kept under a 14-hour light / 10-hour dark cycle, a temperature of 18-22°C, and a relative humidity of 100%. After the first leaf of the susceptible control, Zhoumai 18, was fully diseased, the phenotype was recorded on a scale of 0-4, with 0-2 considered resistant and 3-4 considered susceptible.

[0053] The survey results are as follows Figure 2 The results showed that Durum wheat JingDR144 was highly resistant to E09 strain, while Durum wheat Langdon (LDN) was highly susceptible to E09 strain. When Durum wheat JingDR144 was crossed with Durum wheat Langdon, the F1 showed resistance; among 151 F2 plants, 116 were resistant and 35 were susceptible, which was consistent with the resistant to susceptible ratio of 3:1. 2:3There were 32 homozygous resistant, 84 heterozygous, and 35 homozygous susceptible families in the family. The ratio of homozygous resistant: heterozygous: homozygous susceptible was consistent with the segregation ratio of 1:2:1. Therefore, it was speculated that the resistance of durum wheat JingDR144 to E09 strain was controlled by a single dominant gene, named gene PmDR144 .

[0054] 4 Preliminary Molecular Marker Mapping of the Powdery Mildew Resistance Gene in Durum Wheat JingDR144

[0055] Based on the phenotypic identification results, 20 homozygous resistant families and 20 homozygous susceptible families were selected to construct disease-resistant and disease-susceptible pools, respectively. Polymorphisms of durum wheat JingDR144, durum wheat Langdon, and disease-resistant and disease-susceptible pools were detected using molecular markers uniformly distributed throughout the genome. Ten pairs of markers showed consistent polymorphisms in the resistant and susceptible parents and the disease-susceptible pools. These markers were then used to identify 151 F pairs of durum wheat JingDR144×durum wheat Langdon. 2:3 The pedigree was genotyped and the genes PmDR144 It was initially located at the end of wheat chromosome 2AL.

[0056] 5 and genes PmDR144 Development of tightly linked molecular markers

[0057] 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 F1 of durum wheat JingDR144×durum wheat Langdon. 2:3 The pedigree was genotyped and the gene PmDR144 The tightly linked SSR marker HENU624 has a genetic distance of only 2.7 cM.

[0058] The primers for the molecular marker HENU624 include an upstream primer and a downstream primer:

[0059] The nucleotide sequence of the upstream primer HENU624-F is: 5′-GACTTTGGGTAAACACTTCC-3′ (as shown in SEQ ID No. 1);

[0060] The nucleotide sequence of the downstream primer HENU624-R is: 5'-TCATTGCTTGGTCCCTCCTC-3' (as shown in SEQ ID No. 2).

[0061] 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.

[0062] The PCR amplification procedure was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 15 s, annealing at 58°C for 20 s, and extension for 40 s, for 34 cycles; extension at 72°C for 10 min; and storage at 12°C.

[0063] The electrophoresis 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 hours, and photos are taken after silver nitrate staining.

[0064] If two closely linked specific bands of 157 and 186 bp can be amplified, the wheat to be tested carries the powdery mildew resistance gene. PmDR144 ; Two closely linked specific bands of 157 and 186 bp were not amplified, indicating that the wheat tested did not carry the powdery mildew resistance gene in wheat. PmDR144 .

[0065] Example 2: Wheat powdery mildew resistance gene PmDR144 Application of primers for the molecular marker HENU624

[0066] Utilizing wheat powdery mildew resistance genes PmDR144 The molecular marker HENU624 primers were used to amplify and detect the durum wheat JingDR144, durum wheat Langdon and the hybrid of the two. 2:3 The DNA extraction method for the materials in the family was the same as that in Example 1.

[0067] Samples to be tested: Durum wheat Jing DR144, Durum wheat Langdon and their hybrid progeny F 2:3 Family lineage.

[0068] The genomic DNA of the above materials was extracted and used as a template for PCR amplification, and the primers for the molecular marker HENU624 developed by the present invention were used for amplification:

[0069] Nucleotide sequence of upstream primer HENU624-F:

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

[0071] Nucleotide sequence of downstream primer HENU624-R:

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

[0073] 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.

[0074] The PCR amplification procedure was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 15 s, annealing at 58°C for 20 s, extension for 40 s, 34 cycles; extension at 72°C for 10 min; and storage at 12°C.

[0075] The electrophoresis 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 180 V for 1.5-2 h, and photography is performed after silver nitrate staining.

[0076] The results of molecular marker detection are shown in Figure 1 . Figure 1 HENU624 was a marker of the durum wheat DR144 × durum wheat Langdon derived F 2:3 Some amplification results in the family. In the figure: M: DL2000; 1: Durum wheat JingDR144 (powdery mildew resistant variety, carrying the powdery mildew resistance gene PmDR144 ); 2: Durum wheat Langdon (powdery mildew susceptible variety); 3-22: F hybrid formed by the hybridization of Durum wheat Jing DR144 and Durum wheat Langdon 2:3 Families, among which 3-7: homozygous disease-resistant F 2:3 Family, 8-17: F with resistance and segregation 2:3 Family, 18-22: homozygous susceptible F 2:3 Family; white arrows indicate traceable genes PmDR144 The amplification results showed that the marker HENU624 amplified two closely linked specific bands of 157 and 186 bp in the disease-resistant parent durum wheat JingDR144 and the disease-resistant family, but no such target bands were amplified in the disease-susceptible parent durum wheat Langdon and the disease-susceptible family.

[0077] Gene PmDR144 It is derived from the durum wheat variety JingDR144. Currently, there is no report on the location and positional cloning of this gene. The molecular marker HENU624 provided by the present invention is used to detect genetic mapping of large populations, which is helpful for the study of gene PmDR144 Fine mapping and map-based cloning of genes PmDR144 The efficient transformation and in-depth analysis of the disease resistance mechanism are of great significance.

[0078] 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 principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Used to detect powdery mildew resistance genes PmDR144 The use of a molecular marker primer pair in assisting identification of whether durum wheat has powdery mildew resistance is characterized by: The sequences of the primer pair are shown in SEQ ID No. 1 and SEQ ID No.

2.

2. The use according to claim 1, characterized in that: The application is to identify whether durum wheat has a powdery mildew resistance gene PmDR144 achieved; The steps are: using the primer pair described in claim 1 to amplify the genome of the durum wheat sample to be tested; if two closely linked specific bands of 157 bp and 186 bp can be amplified, it indicates that the wheat sample to be tested carries the powdery mildew resistance gene. PmDR144 , and has resistance to powdery mildew; on the contrary, the durum wheat sample to be tested does not carry it and has no resistance to powdery mildew.

3. Used to detect powdery mildew resistance genes PmDR144 The use of a molecular marker primer pair in assisting the cultivation of powdery mildew-resistant durum wheat is characterized by: The sequences of the primer pair are shown in SEQ ID No. 1 and SEQ ID No.

2.

4. A method for detecting whether durum wheat is a powdery mildew resistant variety, characterized in that: The method is to detect whether durum wheat contains powdery mildew resistance genes PmDR144 achieved; The steps are: using the primer pair described in claim 1 to amplify the genome of the durum wheat sample to be tested; if two closely linked specific bands of 157 bp and 186 bp can be amplified, it indicates that the wheat sample to be tested carries the powdery mildew resistance gene. PmDR144 , which is a powdery mildew-resistant variety; on the contrary, the durum wheat sample to be tested does not carry it, which is a powdery mildew-non-resistant variety.

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