Primer of a molecular marker closely linked with wheat powdery mildew resistance gene PmCW and application thereof

Abstract

The application discloses a primer of a molecular marker closely linked to a wheat powdery mildew resistance gene PmCWI77849, the molecular marker is YTUSSR-86, the nucleotide sequence of the upstream primer of the molecular marker is shown as SEQ ID NO:1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO:2, the primer of the molecular marker YTUSSR-86 is used for carrying out PCR amplification on wheat genomic DNA to be measured, and the obtained amplification product has a molecular weight of 151 bp, that is, the molecular marker closely linked to the wheat powdery mildew resistance gene PmCWI77849. The molecular marker primer closely linked to the wheat powdery mildew resistance gene PmCWI77849 provided by the application can be used for rapidly and accurately detecting whether wheat breeding materials carry the disease resistance gene PmCWI77849 at the seedling stage, and is helpful to shortening a breeding cycle, improving breeding efficiency and accelerating a 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, developing a molecular marker closely linked to PmCWI77849 and applying it to the molecular marker-assisted selection breeding of the gene has 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:

[0008] A molecular marker closely linked to the wheat powdery mildew resistance gene PmCWI77849, which is the co-dominant SSR marker YTUSSR-86;

[0009] The upstream primer of the molecular marker YTUSSR-86 is YTUSSR-86-F, and the nucleotide sequence thereof is:

[0010] 5'-ACGTCCATTTGAGTGACACG-3', as shown in SEQ ID NO: 1;

[0011] The downstream primer of the molecular marker YTUSSR-86 is YTUSSR-86-R, and the nucleotide sequence thereof is:

[0012] 5'-TACAAACAGTCGTGCGGATT-3', as shown in SEQ ID NO: 2;

[0013] The marker primer of the molecular marker YTUSSR-86 is used for PCR amplification of the wheat genomic DNA to be detected, 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.

[0014] 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 sterile deionized water.

[0015] The PCR amplification procedure suitable for the molecular 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; and 4℃ preservation.

[0016] The electrophoretic separation procedure suitable for the amplification product of the molecular marker is: electrophoresis is performed by using 8% non-denaturing polyacrylamide gel, 1.2 μL of the mixture of the amplification product and 2.5 μL 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.

[0017] The application further provides application of the molecular marker closely linked to the wheat powdery mildew resistance gene PmCWI77849 in genetic mapping, map-based cloning and marker-assisted selection breeding of the wheat powdery mildew resistance gene PmCWI77849.

[0018] The application of the application for detecting whether the wheat powdery mildew resistance gene PmCWI77849 is carried in a to-be-tested variety mainly includes the following steps:

[0019] (1) extracting fresh leaf genomic DNA of a to-be-tested wheat sample;

[0020] (2) using primers of the molecular marker YTUSSR-86 to perform PCR amplification on the extracted wheat genomic DNA to obtain an amplification product;

[0021] (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.

[0022] ​The application, the primers of 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, namely: YTUSSR-86-F: 5'-ACGTCCATTTGAGTGACACG-3'; the nucleotide sequence of the downstream primer YTUSSR-86-R is shown in SEQ ID NO:2, namely: YTUSSR-86-R: 5'-TACAAACAGTCGTGCGGATT-3'.

[0023] 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.

[0024] 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.

[0025] Detection of the PCR amplification product: electrophoresis is carried out by using 8% non-denaturing polyacrylamide gel, 1.2 μL of the mixture is taken for spotting after the amplification product is mixed with 2.5 μL of 10×Loading Buffer, electrophoresis is carried out under the condition of 220 V constant voltage for 1.5 h, silver nitrate staining is carried out after electrophoresis, and photographic treatment is carried out. The result is judged according to electrophoresis, 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.

[0026] 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.

[0027] 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

[0028] 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.

[0029] 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

[0030] 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 in the examples below were carried out in accordance with conventional methods. The experimental materials, reagents, etc. used in the examples were obtained from commercial sources unless otherwise indicated.

[0031] Example 1 Development of molecular marker YTUSSR-86 primer for wheat powdery mildew resistance gene PmCW77849

[0032] 1. Materials

[0033] The resistant parent was the cultivated two-grained wheat CWI77849, and the susceptible parent was the hard-grained wheat LDN. CWI77849 and LDN were hybridized, and the obtained F1 was selfed to obtain an F2 population.

[0034] 2. Extraction of wheat genomic DNA

[0035] The CTAB method was used to extract the wheat genomic DNA, and the process was as follows:

[0036] (1) Fresh young leaves of the wheat to be tested were taken, rapidly ground into powder after quick freezing in liquid nitrogen, and loaded into a 2 mL EP tube;

[0037] (2) 600-800 μL of CTAB extraction solution was added, and the mixture was incubated at 65°C for 1 h, with inversion and mixing every ten minutes;

[0038] (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;

[0039] (4) The mixture was centrifuged at 8000 rpm / min at room temperature for 10 min, and 400 μL of supernatant was transferred to a 1.5 mL EP tube, 3 volumes of pre-cooled 95% ethanol was added, mixed, and precipitated at -20°C for 0.5 h;

[0040] (5) The mixture was centrifuged at 12000 rpm / min at room temperature for 10 min, and the supernatant was discarded. 800 μL of 75% ethanol was added and washed 3 times;

[0041] (6) The precipitate was air-dried, and 50 μL of 1×TE buffer (100 mM Tris-Hcl, 10 mM EDTA, pH =8.0) or ddH2O was added for dissolution.

[0042] (7) The DNA storage solution was diluted with sterile deionized water to 50 ng / μL as a working solution for standby use.

[0043] 3. Identification of wheat seedling powdery mildew resistance and genetic analysis of resistance

[0044] Wheat seedling resistance to powdery mildew was identified in a greenhouse. The resistant parent CWI77849, the susceptible parent LDN, the F1 hybrid, and the F2 population were planted in 128-hole plug trays (3.2 x 3.2 x 4.2 cm). The parents and F1 were identified with at least 20 seeds each, and the F2 population was identified with at least 25 seeds each. The susceptible control TN18 was randomly sown and labeled. The greenhouse conditions were controlled at 18-20°C, 80% relative humidity, and a photoperiod of 14 h light / 10 h darkness. At the one-leaf stage, the plants were inoculated with the powdery mildew strain E09 using the sweeping method. After 10-14 days, when the susceptible control TN18 was fully diseased, the phenotype was investigated. The infection type (IT) was recorded according to the 0-4 scale. The resistance grade was divided as follows: 0-2 grade for resistant type, and 3-4 grade for susceptible type.

[0045] The results showed that CWI77849 was highly resistant to powdery mildew strain E09 (IT=0), LDN was highly susceptible (IT=4), and the F1 plants were all resistant (IT=0-1), indicating that CWI77849 carried a dominant resistance gene. The F2 population of this combination was identified for resistance, and the results showed that the resistant to susceptible segregation ratio was 139:53, which was consistent with the segregation ratio of 3:1 (χ 2 =0.69, P =0.405) of a single dominant gene. In summary, the resistance of CWI77849 to powdery mildew strain E09 is controlled by a single dominant gene, which is named PmCWI77849.

[0046] 4. Fine mapping of PmCWI77849

[0047] 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 throughout the genome, polymorphism detection was performed on the cultivated emmer wheat CWI77849, the durum wheat LDN, and the resistant and susceptible pools. Seventeen markers exhibited consistent polymorphism in the resistant and susceptible parents and pools. These markers were then 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 wheat chromosome 4AL.

[0048] 5. Development of molecular markers closely linked to PmCWI77849

[0049] 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.

[0050] The primers of the molecular marker YTUSSR-86 include an upstream primer and a downstream primer:

[0051] The nucleotide sequence of the upstream primer YTUSSR-86-F is 5'-ACGTCCATTTGAGTGACACG-3';

[0052] The nucleotide sequence of the downstream primer YTUSSR-86-R is 5'-TACAAACAGTCGTGCGGATT-3'.

[0053] 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.

[0054] 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.

[0055] 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 under the condition of 220 V constant voltage for 1.5 h, taking silver nitrate staining and photographing. 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.

[0056] 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.

[0057] The wheat powdery mildew resistance gene PmCWI77849 is derived from a Chinese wheat material cultivated diploid 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 improving 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 resistance mechanism.

[0058] 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. The application of a primer pair of YTUSSR-86, a molecular marker closely linked to the wheat powdery mildew resistance gene PmCWI77849, in the localization, map-based cloning, and marker-assisted breeding of the wheat powdery mildew resistance gene PmCWI77849, characterized in that... The upstream primer nucleotide sequence of the primer pair is shown in SEQ ID NO:1; the downstream primer nucleotide sequence is shown in SEQ ID NO:2; and the primer pair of the molecular marker YTUSSR-86 is used to perform PCR amplification of the wheat genomic DNA to be tested, and the corresponding amplification product has a molecular weight of 151 bp, which is a molecular marker closely linked to the wheat powdery mildew resistance gene PmCWI77849.

2. The application according to claim 1, detecting whether the wheat variety to be tested carries the wheat powdery mildew resistance gene PmCWI77849 mainly includes the following steps: (1) Extract genomic DNA from the wheat sample to be tested; (2) The extracted wheat genomic DNA was amplified by PCR using primers with the molecular marker YTUSSR-86 to obtain the amplification product; (3) Perform electrophoresis on the amplified products and judge based on the electrophoresis results. If a specific band of 151 bp can be amplified, it indicates that the powdery mildew resistance gene PmCWI77849 exists in the wheat to be tested; otherwise, the powdery mildew resistance gene PmCWI77849 does not exist in the wheat to be tested.

3. According to the application of claim 2, 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.

4. The application according to claim 2, wherein the PCR amplification system suitable for the label 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.

5. According to claim 2, the applicable PCR amplification program for the label is as follows: 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; storage at 4℃.

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