Molecular marker primer closely linked with wheat powdery mildew resistance gene PmCWI77849 and application of molecular marker primer

Abstract

The invention discloses a primer of a molecular marker in close linkage with a wheat powdery mildew resistance gene PmCWI77849. The molecular marker is YTUSSR-86; the nucleotide sequence of an upstream primer of the molecular marker is as shown in SEQ ID NO: 1; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO: 2; a primer of the molecular marker YTUSSR-86 is used for carrying out PCR (Polymerase Chain Reaction) amplification on to-be-detected wheat genome DNA (Deoxyribose Nucleic Acid), and the molecular weight of an obtained amplification product is 151bp, namely the molecular marker closely linked with the wheat powdery mildew resistance gene PmCWI77849. The molecular marker primer closely linked with the wheat powdery mildew-resistant gene PmCWI77849 provided by the invention can be used for rapidly and accurately detecting whether a wheat breeding material carries the anti-disease gene PmCWI77849 or not in a seedling stage, and is beneficial to shortening the breeding period, improving the breeding efficiency and accelerating the breeding process.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural bioengineering, in particular to a primer of a molecular marker closely linked to a wheat powdery mildew resistance gene PmCW178849 and application thereof. BACKGROUND

[0002] Wheat is a major food crop for human survival, and Blumeria graminis f. sp. tritici Wheat powdery mildew caused by Bgt is a pandemic disease that poses a serious threat to global wheat production. Powdery mildew causes significant yield reduction in wheat, with a general range of 5-10%, and severe losses of up to 30-50%. Exploring wheat powdery mildew resistance genes and breeding disease-resistant varieties is a key strategy to reduce pesticide dependence in agricultural production. So far, more than 140 powdery mildew resistance genes and alleles have been identified in different germplasm resources of wheat. However, the continuous variation of pathogen races has led to the failure of many original resistance genes. Therefore, in order to resist the occurrence of powdery mildew, it is urgent to continuously explore, locate and clone new resistance sources from various types of wheat germplasm resources.

[0003] Wheat's close relatives and ancestral species contain rich genetic variation and are an important gene source for wheat genetic improvement. More than half of the reported powdery mildew resistance genes are derived from these species. Cultivated emmer ( Triticum dicoccum , 2n=4x=28, AABB) is a tetraploid and is widely considered to be the ancestor of common wheat due to its shared A and B genomes. This species contains rich superior traits, particularly in resisting biotic and abiotic stresses. In terms of powdery mildew resistance genes, key genes such as Pm4a, Pm49, Pm50, Pm71 all originate from cultivated emmer.

[0004] After exploring superior resistance genes, how to efficiently integrate them into wheat breeding is crucial. In the past decade, wheat breeding technology has continued to develop, and molecular marker-assisted selection (MAS) has become a core means of powdery mildew resistance breeding. Developing efficient, precise and easy-to-use molecular markers is the basis for achieving MAS of target genes. Using molecular markers to track target genes can significantly shorten the breeding cycle and improve efficiency.

[0005] The cultivated Triticum dicoccum CWI77849 has excellent agronomic traits, and shows good resistance to powdery mildew in multi-year and multi-point field identification, and is a valuable resistant powdery mildew germplasm resource. The genetic analysis of seedling resistance and molecular marker detection confirm that the resistance of CWI77849 to the prevalent powdery mildew strain E09 is controlled by a pair of dominant genes located on the 4AL chromosome of wheat, which is named as PmCWI77849 and belongs to a new wheat powdery mildew resistance gene that has not been reported. Therefore, the development of a molecular marker closely linked to PmCWI77849 and its application in the molecular marker assisted selection breeding of the gene have extremely important significance for breeding new wheat varieties resistant to powdery mildew and realizing effective prevention and control of the disease. SUMMARY

[0006] The purpose of the present application is to provide a molecular marker closely linked to the wheat powdery mildew resistance gene PmCWI77849 and its application, so as to locate and detect the wheat powdery mildew resistance gene PmCWI77849 by using the molecular marker.

[0007] The present application is realized by the following method: A molecular marker closely linked to the wheat powdery mildew resistance gene PmCWI77849, which is the co-dominant SSR marker YTUSSR-86; The upstream primer of the molecular marker YTUSSR-86 is YTUSSR-86-F, and the nucleotide sequence thereof is: 5'-ACGTCCATTTGAGTGACACG-3', as shown in SEQ ID NO: 1; The downstream primer of the molecular marker YTUSSR-86 is YTUSSR-86-R, and the nucleotide sequence thereof is: 5'-TACAAACAGTCGTGCGGATT-3', as shown in SEQ ID NO: 2; The molecular marker YTUSSR-86 is used for PCR amplification of the wheat genomic DNA to be detected by using the marker primer, and the corresponding amplification product has a molecular weight of 151 bp, which is the molecular marker closely linked to the wheat powdery mildew resistance gene PmCWI77849.

[0008] The PCR amplification system suitable for the molecular marker is 10 μL, including: 50 ng / μL wheat genomic DNA 1.5 μL, 4 μL PCR Master Mix, 10 μM upstream primer 0.25 μL, 10 μM downstream primer 0.25 μL, and 4 μL of sterile deionized water.

[0009] The PCR amplification procedure suitable for the molecular marker is as follows: pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 40 s, 35 cycles, extension at 72℃ for 10 min, and preservation at 4℃.

[0010] The electrophoretic separation procedure suitable for the amplification product is as follows: electrophoresis is performed on an 8% non-denaturing polyacrylamide gel, 1.2 μL of the mixture of the amplification product and 2.5 μL of 10×Loading Buffer is taken for spotting, electrophoresis is performed at a constant voltage of 220 V for 1.5 h, silver nitrate staining is performed, and photographing is performed.

[0011] The application further provides application of the molecular marker which is closely linked to the wheat powdery mildew resistance gene PmCWI77849 in gene location, map-based cloning and marker-assisted selection breeding of the wheat powdery mildew resistance gene PmCWI77849.

[0012] The application of the application for detecting whether the wheat powdery mildew resistance gene PmCWI77849 is carried in a to-be-tested variety mainly comprises the following steps: (1) extracting fresh leaf genome DNA of a to-be-tested wheat sample; (2) performing PCR amplification on the extracted wheat genome DNA by using primers of the molecular marker YTUSSR-86 to obtain an amplification product; (3) performing electrophoresis on the amplification product, and judging according to the electrophoretic result; if a specific band of 151 bp can be amplified, it is indicated that the wheat powdery mildew resistance gene PmCWI77849 exists in the to-be-tested wheat; otherwise, the wheat powdery mildew resistance gene PmCWI77849 does not exist in the to-be-tested wheat.

[0013] The primers of the molecular marker YTUSSR-86 comprise an upstream primer YTUSSR-86-F and a downstream primer YTUSSR-86-R, the nucleotide sequence of the upstream primer YTUSSR-86-F is shown in SEQ ID NO: 1, namely: YTUSSR-86-F: 5'-ACGTCCATTTGAGTGACACG-3'; and the nucleotide sequence of the downstream primer YTUSSR-86-R is shown in SEQ ID NO: 2, namely: YTUSSR-86-R: 5'-TACAAACAGTCGTGCGGATT-3'.

[0014] ​The application of the molecular marker, the PCR amplification system suitable for the marker is 10 μL, including: 50 ng / μL wheat genomic DNA 1.5 μL, 4 μL PCR Master Mix, 10 μM upstream primer 0.25 μL, 10 μM downstream primer 0.25 μL, and 4 μL of sterile deionized water.

[0015] The PCR amplification procedure suitable for the marker is: 95 ℃ pre-denaturation for 5 min; 95 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 40 s, 35 cycles; 72 ℃ extension for 10 min; 4 ℃ preservation.

[0016] Detection of the PCR amplification product: electrophoresis is performed by using 8% non-denaturing polyacrylamide gel, 1.2 μL of the mixture of the amplification product and 2.5 μL of 10×Loading Buffer is taken for spotting, electrophoresis is performed under the condition of 220 V constant voltage for 1.5 h, silver nitrate staining is performed after electrophoresis, and photographing is performed. According to the electrophoresis result, if a specific band of 151 bp can be amplified, it is indicated that the wheat germplasm to be tested contains the wheat powdery mildew resistance gene PmCWI77849, otherwise, the wheat germplasm to be tested does not contain the wheat powdery mildew resistance gene PmCWI77849.

[0017] The present application shows that the resistance of the cultivated emmer wheat CWI77849 to the epidemic strain E09 of powdery mildew in the seedling stage is controlled by a single dominant gene, which is named as PmCWI77849 through genetic analysis of the resistance to powdery mildew in the seedling stage and molecular marker detection. The resistance pool and the susceptible pool composed of 10 homozygous resistant families and 10 homozygous susceptible families in the F2 population of the hybrid of the cultivated emmer wheat CWI77849, the susceptible hard wheat Langdon (LDN) and the F2 population of the hybrid of the two are detected for polymorphism by using 184 molecular markers uniformly distributed in the whole genome, 17 markers exhibit consistent polymorphism in the resistant and susceptible parents and the resistant and susceptible pools, and then the genotypes of 192 F2 populations of the hybrid of CWI77849 and LDN are typed by using the markers, so that the PmCWI77849 is preliminarily located in the interval of 708.71-709.63 Mb of the wheat 4AL chromosome. Further, according to the sequence in the interval of the hard wheat reference genome, the Wekits software is used to design and screen the SSR marker YTUSSR-86 closely linked to the gene PmCWI77849. The molecular marker YTUSSR-86 of the wheat powdery mildew resistance gene PmCWI77849 provided in the present application is detected by using the genetic segregation population, and the genetic distance with the gene PmCWI77849 is only 0.3 cM, which is closely linked to PmCWI77849, so that the PmCWI77849 can be detected more accurately and efficiently, and the fine mapping, map-based cloning and marker-assisted backcross of PmCWI77849 are greatly promoted.

[0018] The molecular marker closely linked to the wheat powdery mildew resistance gene PmCWI77849 provided in the present application is applied to the breeding of the wheat resistant to powdery mildew, which can not only rapidly and accurately screen the material containing the disease resistance gene PmCWI77849, but also greatly save the cost, shorten the breeding period and improve the breeding efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : Detection results of the primer of the molecular marker YTUSSR-86 on the resistant cultivated emmer wheat CWI77849, the susceptible hard wheat LDN and the segregation population of the hybrid offspring of the two.

[0020] M: pUC19 in the figure Msp I; 1: CWI77849 (resistant parent); 2: LDN (susceptible parent); 3-17: F2 population of the hybrid of CWI77849 and LDN, wherein, 3-7: homozygous resistant family, 8-12: resistant and susceptible segregation family, 13-17: homozygous susceptible family; white arrow is the specific band of PmCWI77849. DETAILED DESCRIPTION

[0021] The following examples are intended to better illustrate and enable the practice of the present application and are not intended to limit its scope. Unless otherwise indicated, the experimental methods used in the examples below were carried out in accordance with conventional methods. The test materials, reagents and the like used in the examples were obtained from commercial sources unless otherwise noted.

[0022] Example 1 Development of molecular marker YTUSSR-86 primer for wheat powdery mildew resistance gene PmCW77849 1. Materials The resistant parent was the cultivated bread wheat CWI77849, and the susceptible parent was the Durum wheat LDN. The F2 population was obtained by selfing the F1 derived from the cross between CWI77849 and LDN.

[0023] 2. Extraction of wheat genomic DNA The CTAB method was used to extract the wheat genomic DNA, and the procedure was as follows: (1) The young fresh leaves of the wheat to be tested were quickly ground into powder after quick freezing in liquid nitrogen, and were loaded into 2 mL EP tubes; (2) 600-800 μL of CTAB extraction solution was added, and the mixture was incubated at 65°C for 1 h, and was inverted and mixed every ten minutes during the incubation; (3) An equal volume of chloroform-isoamyl alcohol (24:1, v / v) was added, and the mixture was mixed on a shaker for 30 min; (4) The mixture was centrifuged at 8000 rpm / min for 10 min at room temperature, and 400 μL of supernatant was transferred to a 1.5 mL EP tube, and 3 volumes of pre-cooled 95% ethanol was added, mixed, and precipitated at -20°C for 0.5 h; (5) The mixture was centrifuged at 12000 rpm / min for 10 min at room temperature, and the supernatant was discarded, and 800 μL of 75% ethanol was added for washing 3 times; (6) The precipitate was air-dried, and 50 μL of 1x TE buffer (100 mM Tris-Hcl, 10 mM EDTA, pH = 8.0) or ddH2O was added for dissolving.

[0024] (7) The DNA storage solution was diluted with sterile deionized water to 50 ng / μL as a working solution for standby.

[0025] 3. Identification of wheat seedling resistance to powdery mildew and genetic analysis of resistance Wheat seedling resistance to powdery mildew was identified in greenhouse. The resistant parent CWI77849, the susceptible parent LDN, the F1 hybrid, the F2 population were planted in 128-hole plug trays (3.2 x 3.2 x 4.2 cm), and at least 20 seeds of the parents and F1 were identified, and at least 25 seeds of each F2 population were identified, and the susceptible control TN18 was randomly sown and labeled. The greenhouse conditions were controlled at 18-20°C, relative humidity 80%, and photoperiod 14 h light / 10 h dark. At the one-leaf stage, the powdery mildew strain E09 was inoculated by sweeping method. After 10-14 days, when the susceptible control TN18 was fully diseased, the phenotype was investigated. The reaction type (Infection type, IT) was recorded according to the 0-4 level standard. The resistance grade was divided: 0-2 level was resistant type, and 3-4 level was susceptible type.

[0026] The investigation results showed that CWI77849 showed high resistance to powdery mildew strain E09 (IT=0), LDN showed high susceptibility (IT=4), and the F1 plants showed resistance (IT was 0-1), indicating that CWI77849 carried a dominant resistance gene; the F2 population of the combination was identified for resistance, and the results showed that the resistance-susceptibility segregation ratio was 139:53, and the chi-square test was consistent with the segregation ratio of single dominant gene 3:1 (χ 2 =0.69, P =0.405). In summary, the resistance of CWI77849 to powdery mildew strain E09 is controlled by a single dominant gene, and the powdery mildew resistance gene is named PmCWI77849.

[0027] 4. Fine mapping of PmCWI77849 According to the phenotypic identification results, 10 pure resistant families and 10 pure susceptible families were selected to construct the resistant pool and the susceptible pool. Using 184 pairs of molecular markers evenly distributed in the whole genome, polymorphism detection was performed on the cultivated emmer wheat CWI77849, the hard wheat LDN, and the resistant pool and the susceptible pool. Seventeen markers exhibited consistent polymorphism in the resistant and susceptible parents and pools. Subsequently, these markers were used to genotype the 192 F2 populations of CWI77849 x LDN, and PmCWI77849 was preliminarily mapped to the interval of 708.71-709.63 Mb on the wheat 4AL chromosome.

[0028] 5. Development of molecular markers closely linked to PmCWI77849 According to the sequence information of the hard wheat reference genome in the candidate interval 708.71-709.63 Mb, the Wekits software was used to design a SSR marker, and the F2 population of CWI77849xLDN was genotyped, and the INDEL marker YTUSSR-86 closely linked to the gene PmCWI77849 was obtained, with a genetic distance of only 0.3 cM.

[0029] The primers of the molecular marker YTUSSR-86 include an upstream primer and a downstream primer: The nucleotide sequence of the upstream primer YTUSSR-86-F is 5'-ACGTCCATTTGAGTGACACG-3'; The nucleotide sequence of the downstream primer YTUSSR-86-R is 5'-TACAAACAGTCGTGCGGATT-3'.

[0030] The suitable PCR amplification system for the marker is 10 μL, including: 50 ng / μL wheat genomic DNA 1.5 μL, 4 μL PCR Master Mix, 10 μM upstream primer 0.25 μL, 10 μM downstream primer 0.25 μL, and 4 μL sterile deionized water.

[0031] The suitable PCR amplification procedure for the marker is: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 55℃ annealing for 30 s; 72℃ extension for 40 s, 35 cycles; 72℃ extension for 10 min; 4℃ storage.

[0032] The electrophoretic separation procedure of the amplification product is: the suitable electrophoretic separation procedure of the amplification product of the molecular marker is: using 8% non-denaturing polyacrylamide gel for electrophoresis, mixing the amplification product and 2.5 μL 10xLoading Buffer, taking 1.2 μL of the mixture for spotting, and electrophoresis at 220 V constant voltage for 1.5 h, taking silver nitrate staining and taking pictures. If a specific band of 151 bp can be amplified, it indicates that the wheat germplasm to be tested contains the wheat powdery mildew resistance gene PmCWI77849, otherwise, the wheat germplasm to be tested does not contain the wheat powdery mildew resistance gene PmCWI77849.

[0033] The detection results of the molecular marker are shown in Figure 1 . Among them Figure 1 are the detection results of the molecular marker YTUSSR-86 for the resistant landrace wheat CWI77849 and the susceptible hard wheat LDN and the segregation population of the hybrid offspring of the two. In the figure, M: pUC19 MspI; 1: CWI77849 (resistant parent); 2: LDN (susceptible parent); 3-17: F2 population of CWI77849 x LDN, wherein 3-7: homozygous resistant family, 8-12: resistant-susceptible segregating family, 13-17: homozygous susceptible family; white arrow is specific band of PmCWI77849. The amplification result shows that the marker YTUSSR-86 amplifies a specific band of 151 bp in the resistant parent CWI77849 and the resistant family, and does not amplify the specific band in the susceptible parent LDN and the susceptible family.

[0034] The wheat powdery mildew resistance gene PmCWI77849 is derived from a Chinese wheat material cultivated two-grain wheat CWI77849, and is an excellent new gene with resistance. At present, there is no related report on molecular positioning, map-based cloning and molecular breeding of the gene. With the aid of the molecular marker YTUSSR-86 provided in the present application, detection is carried out on a large genetic mapping population, which is beneficial to fine positioning and map-based cloning of the gene PmCWI77849. By introducing PmCWI77849 into a wheat main cultivar susceptible to powdery mildew, the molecular marker YTUSSR-86 of the present application can efficiently and accurately detect a breeding large population, greatly improves the efficiency and accuracy of transferring the disease-resistant gene PmCWI77849, and has extremely important significance for efficient transfer of the gene PmCWI77849 and in-depth analysis of the disease-resistant mechanism.

[0035] The above examples are optimized embodiments of the present application, and are used to illustrate the present application but not to limit the present application. Modifications or equivalent replacements made by those skilled in the art without departing from the purpose and principles of the embodiments of the present application shall fall within the scope of the present application.

Claims

1. A primer for a molecular marker tightly linked to the wheat powdery mildew resistance gene PmCWI77849, wherein the upstream primer nucleotide sequence of the molecular marker YTUSSR-86 is shown in SEQ ID NO: 1; the downstream primer nucleotide sequence is shown in SEQ ID NO:

2. PCR amplification of wheat genomic DNA using the marker primers for the molecular marker YTUSSR-86 yields an amplified product with a molecular weight of 151 bp, which is a molecular marker tightly linked to the wheat powdery mildew resistance gene PmCWI77849.

2. The molecular marker tightly linked to the wheat powdery mildew resistance gene PmCWI77849 according to claim 1, wherein the marker is suitable for a 10 μL PCR amplification system comprising: 1.5 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR Master Mix, 0.25 μL of 10 μM upstream primer, 0.25 μL of 10 μM downstream primer, and 4 μL of sterile deionized water.

3. The molecular marker tightly linked to the wheat powdery mildew resistance gene PmCWI77849 according to claim 1 or 2, wherein the PCR amplification program applicable to the marker is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 40 s, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

4. Use of the molecular marker according to any one of claims 1 to 3 in gene mapping, map-based cloning and marker-assisted breeding of the wheat powdery mildew resistance gene PmCWI77849.

5. The method according to claim 4, wherein the method comprises the following steps: (1) Extracting genomic DNA from the wheat sample to be tested; (2) Using the primers of the molecular marker YTUSSR-86, the extracted wheat genomic DNA was amplified by PCR to obtain the amplified product; (3) Perform electrophoresis on the amplified product and make judgment based on the electrophoresis results. If a specific band of 151 bp can be amplified, it means that the powdery mildew resistance gene PmCWI77849 is present in the tested wheat; otherwise, the powdery mildew resistance gene PmCWI77849 is not present in the tested wheat.

6. The use according to claim 5, wherein the primers for the molecular marker YTUSSR-86 in step (2) include an upstream primer YTUSSR-86-F and a downstream primer YTUSSR-86-R, the nucleotide sequence of the upstream primer YTUSSR-86-F is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer YTUSSR-86-R is shown in SEQ ID NO:

2.

7. The use according to claim 5, wherein the PCR amplification system suitable for the marker is 10 μL, comprising: 1.5 μL of 50 ng / μL wheat genomic DNA, 4 μL of PCR Master Mix, 0.25 μL of 10 μM upstream primer, 0.25 μL of 10 μM downstream primer, and 4 μL of sterile deionized water.

8. The use according to claim 5, wherein the PCR amplification program applicable to the marker is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 40 s, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

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