Primer for amplifying wild emmer wheat powdery mildew resistance gene PmLF540 molecular marker and its application

Abstract

The invention discloses a method for amplifying a wild emmer wheat powdery mildew resistance gene PmLF540 Molecular marker primers, the molecular marker primers include an upstream primer YTU540-006-F and a downstream primer YTU540-006-R; the nucleotide sequence of the upstream primer YTU540-006-F is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer YTU540-006-R is shown in SEQ ID NO: 2. The molecular marker primers provided by the present invention can be used in gene PmLF540 The primers provided by the present invention are used in the detection, identification and auxiliary identification of wheat powdery mildew resistance traits and molecular marker-assisted breeding. PmLF540 It is not only unaffected by the environment, but also has a clear selection target, fast and accurate detection, and saves costs, greatly improving the breeding efficiency of powdery mildew-resistant wheat varieties.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biological gene technology, and in particular to a method for amplifying a wild emmer wheat powdery mildew resistance gene. PmLF540 Primers for molecular markers and their applications. Background Art

[0002] Wheat is one of the important food crops in the world, and its high and stable yields are particularly important for food security. Wheat powdery mildew caused by infection with Brucella powdery mildew causes wheat production reduction, which seriously threatens the safe production of wheat. The disease is characterized by diverse physiological species and rapid mutation. It mainly infects wheat leaves and stems, affects leaf photosynthesis, and seriously affects wheat yield and quality. The measures for preventing and controlling wheat powdery mildew mainly include agricultural prevention and control, chemical control, and the selection and promotion of disease-resistant varieties. Agricultural prevention and control consumes a lot of manpower and material resources, chemical control pollutes the environment, and the purchase cost of chemical agents is high. It cannot eradicate the occurrence of wheat powdery mildew from the source. Production practice shows that introducing disease-resistant genes into wheat resources and cultivating and promoting disease-resistant varieties are the most economical, environmentally friendly and effective measures to prevent and control wheat powdery mildew.

[0003] At present, more than 110 powdery mildew resistance genes and their alleles have been identified in common wheat and its closely related species. Among them, the powdery mildew resistance genes from common wheat can be directly introduced into the target variety and can be directly used for disease resistance breeding, including Pm1a , Pm5e , Pm3b , Pm24a / b , Pm38 / Lr34 / Yr18 / Sr57 , Pm46 / Yr46 / Lr67 / Sr55 etc.; Disease resistance genes from wheat-related genera can be introduced into common wheat through continuous multi-generation backcrossing to improve wheat resistance, including Pm2 , Pm8 , Pm21 , Pm58 Although many resistance genes have been found in wheat and its relatives, new powdery mildew strains continue to emerge, and the powdery mildew resistance genes that have been found may lose resistance. Therefore, researchers need to continuously search for and locate new powdery mildew resistance genes and apply them to wheat powdery mildew resistance breeding to enrich the wheat powdery mildew resistance gene resources.

[0004] Bulked segregant exome capture sequencing is a new technology based on Bulked segregant Analysis (BSA) and exome capture sequencing. It can quickly and accurately capture and enrich the DNA in the exon region of a species' entire genome, and then perform high-throughput sequencing, detect and annotate SNPs (Single Nucleotide Polymorphisms) sites related to target traits, and then achieve the isolation of candidate genes for target traits and the development of molecular markers.

[0005] wild emmer wheat ( Triticum turgidum subsp. Dicoccoides, 2n=28, AABB) is the ancestor of cultivated emmer wheat and hexaploid wheat, and is the secondary gene resource library of common wheat. It not only has good resistance to various leaf diseases of wheat such as powdery mildew, leaf rust, and stripe rust, but also has high protein content and rich trace elements such as zinc and selenium that common wheat does not have. Its excellent genes can be directly transferred to common wheat through distant hybridization. It is a natural donor of exogenous excellent genes for common wheat and an important resource for genetic improvement of common wheat. At present, multiple powdery mildew resistance genes have been reported in wild emmer wheat, including the one located on chromosome 5BS. Pm16 and Pm30 ; 2BS chromosome Pm26 and Pm42 ; 5BL, 3BL, 2BL and 6BL Pm36 , Pm41 , Pm64 and Pm69 In addition, there are MlZec1 , MlIW72 , Ml3D232 , PmG16 , PmG3M , MlIW170 and PmAs846 Continuously exploring and studying wild emmer wheat and the powdery mildew resistance genes it carries, developing its molecular markers and applying them to molecular marker-assisted selection breeding are of great significance for the selection and breeding of powdery mildew resistant wheat varieties and the effective prevention and control of wheat powdery mildew. Summary of the invention

[0006] The purpose of the present invention is to provide a method for amplifying the powdery mildew resistance gene of wild emmer wheat. PmLF540 Molecular marker primers and their application, using primers to amplify molecular markers for wheat powdery mildew resistance genes PmLF540Through positioning and detection, parents can be selected purposefully in wheat breeding, providing guidance for breeding new wheat varieties resistant to powdery mildew.

[0007] The present invention is achieved by the following technical scheme: a method for amplifying the powdery mildew resistance gene of wild two-grain wheat PmLF540 Molecular marker primers, the molecular marker primers include an upstream primer YTU540-006 and a downstream primer YTU540-006-R; the nucleotide sequence of the upstream primer YTU540-006-F is shown in SEQ ID NO:1, and the nucleotide sequence of the downstream primer YTU540-006-R is shown in SEQ ID NO:2.

[0008] Amplification of wild emmer wheat powdery mildew resistance gene provided by the present invention PmLF540 Molecular marker primers in gene PmLF540 Detection, identification and assisted identification of wheat powdery mildew resistance and its application in molecular marker-assisted breeding.

[0009] The present invention also provides a method for detecting whether a wheat sample carries a wild emmer wheat powdery mildew resistance gene PmLF540 The method comprises the following steps:

[0010] (1) Extracting genomic DNA from the wheat sample to be tested;

[0011] (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 YTU540-006 and a downstream primer YTU540-006-R; the nucleotide sequence of the upstream primer YTU540-006 is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer YTU540-006-R is shown in SEQ ID NO: 2;

[0012] (3) The amplified product is subjected to electrophoresis and detection. If a specific band of 160 bp can be amplified, it indicates that the wild emmer wheat powdery mildew resistance gene is carried in the wheat sample to be tested. PmLF540 Otherwise, the wheat sample to be tested does not carry the wild emmer powdery mildew resistance gene PmLF540 .

[0013] The PCR amplification system in the method of the present invention is 10 μL, including: 1.0 μL of 50 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.

[0014] The PCR amplification procedure in the method of the present invention is: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 15 seconds, annealing at 55°C for 20 seconds, extension for 40 seconds, 30 cycles; extension at 72°C for 10 minutes; and storage at 4°C.

[0015] The electrophoretic separation procedure of the amplified product in the method of the present invention 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, 2 μL of the mixture is spotted, electrophoresis is performed at a constant voltage of 180 V for 1-1.5 h, and photography is performed after silver nitrate staining.

[0016] A new dominant powdery mildew resistance gene was found in wild emmer wheat LF540 PmLF540 , and developed a molecular marker YTU540-006 that is closely linked to the disease resistance gene. Through molecular marker linkage analysis and genetic segregation population detection, PmLF540 The genetic distance is only 2.2 cM, which is similar to the gene PmLF540 Tightly linked, can be applied to genes PmLF540 Detection, identification and auxiliary identification of wheat powdery mildew resistance traits and molecular marker-assisted selection breeding; it can also accurately detect genes PmLF540 Genetic mapping of large populations, and genes PmLF540 The primers are used in wheat breeding for powdery mildew resistance, which can not only quickly and accurately screen for powdery mildew resistance genes, but also can be used for the precise positioning and map-based cloning of powdery mildew resistance genes. PmLF540 It is not affected by the environment, has a clear selection target, saves production costs, and greatly improves the selection efficiency and quality of high-quality powdery mildew-resistant wheat varieties or lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 :The results of using the primers of molecular marker YTU540-006 to detect the segregating populations of disease-resistant wild emmer wheat LF540, susceptible durum wheat Langdon (LDN) and their hybrid progeny.

[0018] In the figure: M: pUC19 / MspI; 1: wild emmer wheat LF540 (disease-resistant emmer wheat, carrying the gene PmLF540 ); 2: Durum wheat Langdon (susceptible emmer wheat, LDN); 3-17: F hybrid formed by the hybridization of wild emmer wheat LF540 and durum wheat Langdon 2:3 Families, among which, 3-7: homozygous disease-resistant F 2:3 Family, 8-12: homozygous susceptible F 2:3 Family, 13-17: F resistant to infection separation 2:3 Pedigree; arrows indicate genes that can be traced PmLF540 Specific bands. DETAILED DESCRIPTION

[0019] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials, reagents, etc. used in the examples are all commercially available unless otherwise specified.

[0020] Example 1 Wheat powdery mildew resistance gene PmLF540 Development of Primers for Molecular Marker YTU540-006

[0021] 1. Materials

[0022] Wild emmer wheat LF540 and durum wheat Langdon (LDN) are resistant and susceptible to wheat powdery mildew, respectively. Wild emmer wheat LF540 and durum wheat Langdon were crossed, and the resulting F1 was self-pollinated to obtain F 2 Groups and corresponding F 2:3 Family lineage.

[0023] 2. Extraction of wheat genomic DNA

[0024] Wheat genomic DNA extraction CTAB method, the process is as follows:

[0025] (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.

[0026] (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.

[0027] (3) Add 600 μL of chloroform-isoamyl alcohol (24:1, v / v) and gently shake on a shaker for 15 min.

[0028] (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 ºC refrigerator to precipitate for 60 min.

[0029] (5) Pick out the flocculent DNA precipitate and wash it twice with 75% pre-cooled ethanol.

[0030] (6) Pick out the DNA precipitate, place it in a 1.5 mL centrifuge tube, and air-dry it indoors.

[0031] (7) Add 60 μL TE buffer (100 mM Tris-HCl, 10 mM EDTA, pH = 8.0) to dissolve the DNA precipitate and prepare a DNA storage solution.

[0032] (8) Dilute the DNA storage solution with ultrapure water to 20-30 ng / μL as working solution.

[0033] 3. Identification of resistance to powdery mildew in wheat seedlings and genetic analysis of resistance

[0034] The identification of wheat powdery mildew resistance at the seedling stage was completed in an artificial climate chamber. The disease-resistant parent, wild emmer wheat LF540, the disease-susceptible parent, durum wheat Langdon, and F 1 Hybrid, F 2 Group and F 2:3 The families were planted in a 128-hole tray (2 × 2 cm), with 5 seeds sown in each hole. 1 Twenty seeds were identified for each F 2:3 Twenty seeds were sown in the family, and the susceptible control Tainong 18 was randomly sown and labeled for distinction. After sowing, the growth conditions were controlled to a light 14h / dark 10h 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 24h after inoculation were controlled as follows: darkness, temperature of 20°C, and relative humidity of 100%, and then the conditions were controlled as a light 14h / dark 10h cycle, temperature of 18-22°C, and relative humidity of 100%. When the first leaf of the susceptible control wheat variety Tainong 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.

[0035] The disease-resistant wild emmer wheat LF540 and the susceptible durum wheat Langdon were used to construct a segregating population. The hybrid F 1 Representative is necrosis (IT: 0;), F 2 Of the 149 individuals in the first generation, 114 were resistant (IT: 0 or 0;), and 35 were susceptible (IT: 3 or 4). 2 The test was consistent with a separation ratio of 3:1 (χ 2 =0.18, P=0.67). Among the 149 resistant strains F 2:3 In the family, there are 36 F 2:3 The family showed homozygous disease resistance, 76 F 2:3 The family showed resistance and sensitivity separation. 2 37 susceptible strains F 2:3 All families showed susceptible disease, and the segregation ratio of resistance was consistent with the segregation ratio of 1:2:1 (χ 2 =0.07, P=0.96). Combined with F 1 、F 2 and F 2:3The results of the identification of the disease-resistant parent, wild emmer wheat LF540, to the powdery mildew strain E09 were controlled by a single dominant gene, named gene PmLF540 .

[0036] 4. With PmLF540 Development of tightly linked molecular markers

[0037] In LF540×Langdon's F 2:3 In the family, 30 homozygous highly resistant and 30 homozygous highly susceptible powdery mildew plants were randomly selected to construct disease-resistant and disease-susceptible pools. BSE-Seq sequencing analysis was performed by Chengdu Tiancheng Future Biotechnology Co., Ltd. Data analysis found that the whole genome was only significantly enriched on chromosome 4A. Further, based on the difference information in the candidate interval and combined with the Chinese spring wheat reference genome sequence, primers for the molecular marker YTU540-006 were designed using primer5.0 software to genotype the F of LF540×Langdon. 2:3 The pedigree was genotyped and the gene PmLF540 The closely linked SSR marker YTU540-006 had a genetic distance of only 2.2 cM.

[0038] The primers for molecular marker YTU540-006 include an upstream primer and a downstream primer:

[0039] The nucleotide sequence of the upstream primer YTU540-006-F is: 5'- CAACCTTATTGGAGGACTT-3' (SEQ ID NO: 1);

[0040] The nucleotide sequence of the downstream primer YTU540-006-R is: 5′-GACGGAGAAATGATATGTTAG-3′ (SEQ ID NO: 2).

[0041] The PCR amplification system is 10 μL, including: 1.0 μL of 50 ng / μL wheat genomic DNA, 5 μL of PCR Master Mix, 0.4 μL of 5 μM upstream primer, 0.4 μL of 5 μM downstream primer, and 3.2 μL of sterile deionized water.

[0042] The PCR amplification procedure is: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 15 seconds, annealing at 55°C for 20 seconds, extension for 40 seconds, 30 cycles; extension at 72°C for 10 minutes; and storage at 4°C.

[0043] The electrophoretic separation procedure of the amplified product is as follows: electrophoresis is performed on a non-denaturing polyacrylamide gel with a mass volume percentage concentration of 8%, the amplified product is mixed with 2 μL of 6× loading buffer, 2 μL of the mixture is spotted, electrophoresis is performed at a constant voltage of 180 V for 1-1.5 h, and photography is performed after silver nitrate staining.

[0044] A specific band of 160 bp can be amplified, indicating that the wheat to be tested carries a powdery mildew resistance gene PmLF540 ; No specific band of 160 bp was amplified, indicating that the wheat tested does not carry the powdery mildew resistance gene PmLF540 .

[0045] Example 2 Wheat powdery mildew resistance gene PmLF540 Application of molecular marker YTU540-006

[0046] The primers of the molecular marker YTU540-006 obtained in Example 1 were used to detect the disease-resistant parent wild emmer wheat LF540, the disease-susceptible parent durum wheat Langdon (LDN), and the F hybrid formed by the hybridization thereof. 2:3 The extraction method was the same as in Example 1.

[0047] Samples to be tested: resistant wild emmer wheat LF540, susceptible durum wheat Langdon, and their hybrid F 2:3 Family lineage.

[0048] The genomic DNA of the above materials was extracted as a PCR amplification template, and the primers of the molecular marker YTU540-006 developed by the present invention were used for amplification:

[0049] Nucleotide sequence of the upstream primer:

[0050] YTU540-006-F: 5'- CAACCTTATTGGAGGACTT-3' (SEQ ID NO: 1);

[0051] Nucleotide sequence of downstream primer:

[0052] YTU540-006-R: 5'-GACGGAGAAATGATATGTTAG-3' (SEQ ID NO: 2).

[0053] The PCR amplification system is 10 μL, including: 1.0 μL of 50 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.

[0054] The PCR amplification procedure is: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 15 seconds, annealing at 55°C for 20 seconds, extension for 40 seconds, 30 cycles; extension at 72°C for 10 minutes; and storage at 4°C.

[0055] 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, 2 μL of the mixture is spotted, electrophoresis is performed at a constant voltage of 180 V for 1-1.5 h, and photography is performed after silver nitrate staining.

[0056] If a specific band of 160 bp can be amplified, it means that the powdery mildew resistance gene exists in the wheat to be tested. PmLF540 Otherwise, the wheat powdery mildew resistance gene does not exist in the tested wheat PmLF540 .

[0057] The results of molecular marker detection are shown in Figure 1 The amplification results showed that the primers of the molecular marker YTU540-006 amplified a specific band of 160 bp in the disease-resistant parent wild emmer wheat LF540 and the disease-resistant family, but did not amplify the target band in the susceptible parent durum wheat Langdon and the susceptible family.

[0058] The test results show that the gene PmLF540 Originated from wild emmer wheat variety LF540, the gene has not yet been located and cloned. The primers of the molecular marker YTU540-006 provided by the present invention are used to detect genetic mapping of large populations, which is helpful for gene PmLF540 Fine positioning and map-based cloning of genes PmLF540 The efficient transformation and in-depth analysis of disease resistance mechanisms are of great significance.

[0059] The above embodiments are optimized implementation schemes of the present invention, which are only used to illustrate the present invention but not to limit the present invention. Any modification or equivalent substitution made by those skilled in the art without departing from the purpose and principle of the implementation scheme of the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for amplifying the powdery mildew resistance gene of wild emmer wheat PmLF540 The primer for molecular labeling is characterized in that The molecular marker primers include an upstream primer YTU540-006-F and a downstream primer YTU540-006-R; the nucleotide sequence of the upstream primer YTU540-006-F is shown in SEQ ID NO:1, and the nucleotide sequence of the downstream primer YTU540-006-R is shown in SEQ ID NO:

2.

2. An amplified wild emmer wheat powdery mildew resistance gene according to claim 1 PmLF540 Molecular marker primers in gene PmLF540 Detection, identification and assisted identification of wheat powdery mildew resistance and its application in molecular marker-assisted breeding.

3. A method for detecting whether wheat samples carry wild emmer wheat powdery mildew resistance genes PmLF540 The method is characterized in that The following steps are involved: (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 YTU540-006 and a downstream primer YTU540-006-R; the nucleotide sequence of the upstream primer YTU540-006 is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer YTU540-006-R is shown in SEQ ID NO: 2; (3) The amplified product is subjected to electrophoresis and detection. If a specific band of 160 bp can be amplified, it indicates that the wild emmer wheat powdery mildew resistance gene is carried in the wheat sample to be tested. PmLF540 Otherwise, the wheat sample to be tested does not carry the wild emmer powdery mildew resistance gene PmLF540 .

4. The method according to claim 3, characterized in that The PCR amplification system is 10 μL, including: 1.0 μL of 50 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.

5. The method according to claim 3 or 4, characterized in that: The PCR amplification procedure is: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 15 seconds, annealing at 55°C for 20 seconds, extension for 40 seconds, 30 cycles; extension at 72°C for 10 minutes; and storage at 4°C.

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