Detectable molecular marker for tracking powdery mildew-resistant gene PmDR144 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 PmDR144 and application of the detectable molecular marker in disease-resistant breeding. The molecular marker comprises an upstream primer HENU624-F and a downstream primer HENU624-R, the nucleotide sequence of the upstream primer HENU624-F is as shown in SEQ ID No. 1, and the nucleotide sequence of the downstream primer HENU624-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 PmDR144 or not before the powdery mildew in the wheat seedling stage is attacked, a variety carrying the powdery mildew resistant gene PmDR144 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 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. However, due to the frequent occurrence of extreme climate and urban development in recent years, the reduction of cultivated land and the continuous increase in pests and diseases have seriously threatened the yield and quality of wheat in my country. Therefore, breeding highly resistant and widely adaptable wheat varieties is currently 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 Bacillus graminis of the Poaceae family is a global fungal disease of wheat. After infection, it will plunder the nutrition of wheat plants and hinder the photosynthesis of plants, making it difficult for plants to accumulate and transport carbohydrates, which will eventually seriously affect wheat yield. At present, breeding and using disease-resistant wheat varieties is the most economical, effective and environmentally friendly method to prevent and control powdery mildew.

[0003] At present, 64 officially named disease resistance gene loci have been identified in primary and secondary gene pools including common wheat and closely 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, Elymus, 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 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 proposed to deploy diversified resistance genes to solve the dilemma of loss of resistance caused by planting a single disease-resistant variety over a large area for a long time. This method 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 found 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 , a new powdery mildew resistance gene on chromosome2BS of Greek durum wheat TRI 1796.Theoretical and applied genetics, 2021,134: 53-62). Discovering new powdery mildew resistance genes from durum wheat and introducing them into common wheat can not only enrich the sources of powdery mildew resistance in wheat, but also introduce other excellent traits carried by it, increase the genetic diversity of common wheat, and be beneficial to the development of wheat powdery mildew resistance breeding in my country. Summary of the invention

[0005] The invention provides a method for tracking powdery mildew resistance gene PmDR144 Detectable molecular markers and their application in disease resistance breeding, using primers to amplify molecular markers for wheat powdery mildew resistance genes PmDR144 Through positioning and detection, parents can be selected purposefully in wheat breeding, providing guidance for breeding new wheat varieties resistant to powdery mildew.

[0006] The technical solution of the present invention is achieved in this way: The present invention found through genetic analysis of powdery mildew resistance in durum wheat seedling stage and molecular marker detection that the resistance of durum wheat JingDR144 to different toxic wheat powdery mildew in seedling stage is caused by a pair of dominant genes. PmDR144 control, which was 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.

[0007] 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, which is similar to the gene PmDR144 Tightly linked, allowing accurate detection of genes PmDR144 Genetic mapping of large populations, applied to genes PmDR144 Fine positioning and positional cloning.

[0008] The invention provides a method for amplifying a powdery mildew resistance gene of durum wheat. PmDR144 Primers for tightly linked molecular markers, wherein the molecular marker is a co-dominant SSR marker HENU624, and the primers include an upstream primer HENU624-F and a downstream primer HENU624-R; The upstream primer of the molecular marker HENU624 is HENU624-F, and its nucleotide sequence is: 5'-GACTTTGGGTAAACACTTCC-3', as shown in SEQ ID No. 1; The downstream primer of the molecular marker HENU624 is HENU624-R, and its nucleotide sequence is: 5'-TCATTGCTTGGTCCCTCCTC-3', as shown in SEQ ID No.2.

[0009] 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 and its application in molecular marker-assisted breeding.

[0010] 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: (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 HENU624-F and a downstream primer HENU624-R; The upstream primer of the molecular marker HENU624 is HENU624-F, and its nucleotide sequence is: 5'-GACTTTGGGTAAACACTTCC-3', as shown in SEQ ID No. 1; The downstream primer of the molecular marker HENU624 is HENU624-R, and its nucleotide sequence is: 5'-TCATTGCTTGGTCCCTCCTC-3', as shown in SEQ ID No. 2; (3) The amplified product is subjected to electrophoresis and detection. If two closely linked specific bands of 157 bp 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 .

[0011] 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.

[0012] The PCR amplification procedure in step (2) of the above method is: 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.

[0013] 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, electrophoresis is performed at a constant voltage of 180 V for 1.5-2 h, and photography is performed after silver nitrate staining.

[0014] The present invention has the following beneficial effects: The present invention found that the resistance of durum wheat JingDR144 to different toxicity wheat powdery mildew at the 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.

[0015] The 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 selection efficiency and quality of high-quality powdery mildew-resistant wheat varieties or lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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.

[0017] Figure 1 The results of molecular marker HENU624 primers detecting segregation 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 of Durum wheat JingDR144 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 PmDR144 Specific bands.

[0018] Figure 2 The resistant parent JingDR144, the susceptible parent Langdon and some F 2:3 Family single plant seedling stage Bgt Resistance performance of physiological race E09. DETAILED DESCRIPTION

[0019] 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.

[0020] 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.

[0021] Example 1: Wheat powdery mildew resistance gene PmDR144 Development of primers for molecular marker HENU624 1 Materials 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 .

[0022] Table 1 Powdery mildew resistance phenotypes of plant materials used Table 2 Beijing DR144 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 with liquid nitrogen. Take about 0.5 g and place it in a 2.0 mL centrifuge tube.

[0023] (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.

[0024] (3) Add 600 μL of chloroform-isoamyl alcohol (24:1, v / v) and gently shake on a shaker for 15 min.

[0025] (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 to precipitate for 60 min.

[0026] (5) Pick out the flocculent DNA precipitate and wash it twice with 75% pre-cooled ethanol.

[0027] (6) Pick out the DNA precipitate, place it in a 1.5 mL centrifuge tube, and air-dry it indoors.

[0028] (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.

[0029] (8) Dilute the DNA storage solution with ultrapure water to 20-30 ng / μL as working solution.

[0030] 3 Identification of resistance to powdery mildew in wheat seedlings and genetic analysis of resistance 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 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 for the family, and the susceptible control Zhoumai 18 was randomly sown and labeled for distinction. After sowing, the growth conditions were controlled to a 14 h light / 10 h dark cycle, a temperature of 20°C, and a relative humidity of 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 to be: dark, a temperature of 20°C, and a relative humidity of 100%, and then the conditions were controlled to a 14 h light / 10 h dark cycle, a temperature of 18-22°C, and a relative humidity of 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.

[0031] 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 the 151 F2 plants, 116 were resistant and 35 were susceptible, which was consistent with the 3:1 ratio of resistance to sensitivity. 2:3 There were 32 homozygous resistance, 84 heterozygous, and 35 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 JingDR144 to E09 strain was controlled by a single dominant gene, named gene PmDR144 .

[0032] 4 Preliminary localization of molecular markers for wheat powdery mildew resistance gene JingDR144 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 JingDR144, 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 resistant and 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 gene PmDR144 The gene was initially located at the end of wheat chromosome 2AL.

[0033] 5 and genes PmDR144 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 JingDR144×durum wheat Langdon. 2:3 The pedigree was genotyped and the gene PmDR144 The closely linked SSR marker HENU624 had a genetic distance of only 2.7 cM.

[0034] The primers for the molecular marker HENU624 include an upstream primer and a downstream primer: The nucleotide sequence of the upstream primer HENU624-F: 5′-GACTTTGGGTAAACACTTCC-3′ (as shown in SEQ ID No. 1); The nucleotide sequence of the downstream primer HENU624-R is: 5'-TCATTGCTTGGTCCCTCCTC-3' (as shown in SEQ ID No. 2).

[0035] 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.

[0036] The PCR amplification program 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.

[0037] 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.

[0038] If two closely linked specific bands of 157 and 186 bp can be amplified, the tested wheat carries the powdery mildew resistance gene. PmDR144 ; Two closely linked specific bands of 157 and 186 bp were not amplified, indicating that the wheat to be tested does not carry the wheat powdery mildew resistance gene PmDR144 .

[0039] Example 2: Wheat powdery mildew resistance gene PmDR144 Application of primers for molecular marker HENU624 Utilizing wheat powdery mildew resistance genes PmDR144 Detection of durum wheat JingDR144, durum wheat Langdon and the hybrid of the two by amplification of HENU624 primers 2:3 The DNA extraction method for the materials in the family was the same as in Example 1.

[0040] Samples to be tested: Durum wheat Jing DR144, Durum wheat Langdon and their hybrid progeny F 2:3 Family lineage.

[0041] The genomic DNA of the above materials was extracted as a PCR amplification template, and the primers of the molecular marker HENU624 developed by the present invention were used for amplification: Nucleotide sequence of upstream primer HENU624-F: 5'-GACTTTGGGTAAACACTTCC-3' (as shown in SEQ ID No. 1); Nucleotide sequence of downstream primer HENU624-R: 5'-TCATTGCTTGGTCCCTCCTC-3' (as shown in SEQ ID No. 2).

[0042] 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.

[0043] The PCR amplification program 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.

[0044] 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 180 V for 1.5-2 h, and photography is performed after silver nitrate staining.

[0045] The results of molecular marker detection are shown in Figure 1 . Figure 1 HENU624 was labeled in the F strain derived from Durum wheat DR144 × Durum wheat Langdon. 2:3 Some amplification results in the family. In the figure: M: DL2000; 1: Durum wheat JingDR144 (powdery mildew resistant variety, carrying powdery mildew resistance gene PmDR144 ); 2: Durum wheat Langdon (powdery mildew susceptible variety); 3-22: F hybrid of Durum wheat JingDR144 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 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 did not amplify the target band in the susceptible parent durum wheat Langdon and the susceptible family.

[0046] 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 gene PmDR144 Fine positioning and map-based cloning of genes PmDR144 The efficient transformation and in-depth analysis of disease resistance mechanisms are of great significance.

[0047] 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. Powdery mildew resistance gene PmDR144 Application in breeding powdery mildew resistant wheat.

2. The use according to claim 1, characterized in that: Powdery mildew resistance gene PmDR144 It was located in a physical interval of 7.31 Mb between 774,583,083-781,893,037 bp on wheat chromosome 2AL.

3. Used to detect powdery mildew resistance genes PmDR144 A molecular marker characterized by: Powdery Mildew Resistance Gene PmDR144 Tightly linked, with a genetic distance of only 2.7 cM.

4. Used to detect powdery mildew resistance genes PmDR144 A primer characterized in that: The primers are a primer pair for detecting the molecular marker according to claim 3.

5. The method for detecting powdery mildew resistance gene according to claim 4. PmDR144 A primer characterized in that: The sequences of the primer pair are shown in SEQ ID No.1 and SEQ ID No.

2.

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

7. The use according to claim 6, characterized in that: The application is to identify whether wheat has a powdery mildew resistance gene PmDR144 achieved.

8. Use of the molecular marker according to claim 3 or the primer according to any one of claims 4 to 5 in assisting the breeding of powdery mildew resistant wheat.

9. A method for detecting whether wheat is a powdery mildew resistant variety, characterized in that: The method is to detect whether wheat contains a powdery mildew resistance gene PmDR144 achieved.

10. The method for detecting whether wheat is a powdery mildew resistant variety according to claim 9, characterized in that: The steps are: using the primers described in claim 4 or 5 to amplify the genome of the wheat sample to be tested, if two closely linked specific bands of 157 bp and 186 bp can be amplified, it means that the wheat sample to be tested carries the powdery mildew resistance gene PmDR144 , which is a powdery mildew resistant variety; otherwise, the wheat sample to be tested does not carry it, which is a powdery mildew resistant variety.

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