A broad-spectrum molecular marker for powdery mildew pathogens of crops, primers thereof and applications thereof

Abstract

The invention discloses a broad-spectrum molecular marker for powdery mildew pathogens of crops, primers and applications thereof, wherein the molecular marker comprises OG2127, and the primers of OG2127 comprise an upstream primer and a downstream primer, wherein the upstream primer is Og2127F, and its nucleotide sequence is: 5'-ATY GKT GGM CCK TWY GC-3', and the downstream primer is Og2127R, and its nucleotide sequence is: 5'-CTT TCR CCR CCR TCA AT-3'. The molecular marker gene of powdery mildew of the present application has a marking efficiency of 100% for powdery mildew pathogens under the class of Hammerglossum; the powdery mildew pathogens are detected by the molecular marker primers of the present invention, and the detection results are comprehensive and accurate; it is only necessary to detect the characteristic bands of PCR products to determine whether powdery mildew exists in the crop cultivation environment, which is conducive to timely taking management measures for the prevention and control of crop powdery mildew.

Description

Technical Field

[0001] The invention relates to the field of gene technology, and in particular to a broad-spectrum molecular marker of powdery mildew pathogens of crops, a primer thereof and an application thereof. Background Art

[0002] Powdery mildew is a typical soil-borne disease caused by fungi. It is common in many important crops and causes huge economic losses in agricultural production. The powdery mildew pathogen belongs to the order Erysiphelineales of the subphylum Ascomycota. It has high parasitic specialization and strong infectivity. It can be spread through air currents and infect plants through the stomata of leaves. The pathogen also has strong environmental resistance. Physical and chemical control measures cannot completely kill it in the crop cultivation environment. Good disease epidemic prediction is still an effective measure for powdery mildew prevention and control.

[0003] The traditional monitoring method for powdery mildew is mainly based on leaf spot judgment. This method is highly dependent on subjective and empirical evaluation, and there is a high lag between the monitoring results and the actual epidemic period of the disease, especially when the crop is slightly infected, thus missing the best time for disease prevention and control. Therefore, it is necessary to develop a highly sensitive detection method that can detect powdery mildew pathogens in the early stage of crop infection, before symptoms appear, and in the cultivation environment.

[0004] With the development of molecular biology technology, technologies based on polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (RT-PCR) methods have been successfully and widely used in pathogen detection. Although PCR and RT-PCR have outstanding advantages in specificity and sensitivity, PCR and RT-PCR have been successfully applied in the detection of pathogens such as black shank and root rot of tobacco crops. This mainly depends on the specific primers designed based on the genomic marker genes of pathogenic crops. However, the powdery mildew pathogen population is highly diverse and can infect a wide range of hosts. Specific primers designed based on the genome of a single pathogen may cause inaccurate and incomplete detection results.

[0005] Comparative genomics is based on comparative analysis of a large number of genomes and their genes and the construction of phylogenetic maps, which can reveal the diversity, differences and molecular evolutionary mechanisms of homologous genes in similar species. The key to determining broad-spectrum molecular markers for detecting powdery mildew pathogens and designing specific primers is to mine marker genes for powdery mildew pathogens by performing comparative genomics or pan-genomic analysis on a large number of powdery mildew pathogen genomes in public databases. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a broad-spectrum molecular marker for powdery mildew pathogens of crops, primers and applications thereof, which can realize pathogen detection in the cultivation environment and the early stage of crop infection, bringing great improvement to disease prevention and control.

[0007] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0008] A broad-spectrum molecular marker for powdery mildew pathogens of crops, the molecular marker comprising OG2127, the primers of OG2127 comprising an upstream primer Og2127F and a downstream primer Og2127R, the sequences of the primers of the molecular marker are as follows:

[0009] The upstream primer Og2127F has a nucleotide sequence of: 5'-ATY GKT GGM CCK TWY GC-3',

[0010] The nucleotide sequence of the downstream primer Og2127R is: 5'-CTT TCR CCR CCR TCA AT-3'.

[0011] A method for identifying broad-spectrum molecular markers of powdery mildew pathogens of crops comprises the following steps:

[0012] (1) Extracting DNA from the microorganisms in the leaf tissue of the plant to be tested;

[0013] (2) using the extracted microbial DNA as a template, and performing PCR amplification using the molecular marker primers Og2127F and Og2127R;

[0014] (3) Detect the PCR amplification product using gel electrophoresis technology; if a characteristic band of 112 bp is obtained, it means that the microorganisms in the plant leaf tissue have detected the powdery mildew pathogen; if not, it means that the microorganisms in the plant leaf tissue have not detected the powdery mildew pathogen.

[0015] Preferably, in step (3), the reaction system is 10uL in total, and the 10uL PCR reaction system includes:

[0016] 2ng plant DNA, 1× Taq enzyme buffer; 2mmol·L -1 MgCl2, 0.2mmol·L -1 dNTPs, upstream and downstream primers were 2.5 pmol·L -1 and 1U Taq DNA polymerase, and make up to 10uL with ddH2O.

[0017] Preferably, in step (3), the PCR reaction conditions are set as follows:

[0018] Pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 50-55°C for 30 s, extension at 72°C for 30 s, 35 cycles; final extension at 72°C for 10 min.

[0019] Preferably, the powdery mildew pathogen of crops originates from hydroponic tobacco.

[0020] The invention discloses an application of a molecular marker in screening powdery mildew resistance genes of crops or in breeding of powdery mildew resistance of crops.

[0021] The invention discloses an application of a molecular marker primer in screening powdery mildew genes of crops or breeding of powdery mildew resistance of crops.

[0022] The above technical solution of the present invention has the following beneficial effects:

[0023] The molecular marker gene of powdery mildew of the present application has a labeling efficiency of 100% for Erysiphales under Leotiomycetes. The molecular marker primers of the present invention are used to detect powdery mildew pathogens, and the detection results are comprehensive and accurate. Only by detecting the characteristic bands of the PCR product, it is possible to determine whether powdery mildew exists in the crop cultivation environment, which is conducive to timely taking management measures for the prevention and control of crop powdery mildew. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0025] Figure 1 It is the conserved region of the gene family OG2127 unique to the Powderymilales. The fragment in the black box on the left is the template for the design of the 5' primer, and the fragment in the black box on the right is the template for the design of the 3' primer.

[0026] Figure 2 The PCR amplification products of endophytes in tobacco leaves using powdery mildew-specific primers Og2127F / Og2127R, where M refers to leaves infected with powdery mildew and H refers to healthy leaves without the disease. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0028] The present application is aimed at the problems of poor specificity, low sensitivity, incomplete and inaccurate detection results of existing powdery mildew pathogen detection methods, and the characteristics of powdery mildew pathogen populations with high diversity and a wide host range. The present invention uses bioinformatics analysis methods to conduct comparative genomics analysis of powdery mildew pathogens from various crops, determines the marker genes of powdery mildew pathogens and specific primers for polymerase chain reaction, and provides molecular markers and primers with broad-spectrum powdery mildew pathogen detection.

[0029] Through comparative genomics analysis, the present invention determined that the OG2127 homologous gene is a molecular marker gene of powdery mildew; specific primers for the polymerase chain reaction of the gene were designed, including an upstream primer Og2127F and a downstream primer Og2127R, which can PCR amplify a characteristic band with a length of 112bp in the DNA of plant tissue with powdery mildew lesions.

[0030] Example 1: Identification of Powdery Mildew Molecular Markers

[0031] Based on the 38 Erysiphales genomes available at NCBI (GCA_000151065.3, GCA_000208805.1, GCA_000214055.1, GCA_000401615.1, GCA_000401675.1, GCA_000417025.1, GCA_000417865.1, GCA_000418435.1, GCA_000441875.1, GCA_000798715.1, GCA_000798735.1, GCA_00079875 5.1, GCA_000798775.1, GCA_000798795.1, GCA_002918395.1, GCA_003610855.1, GCA_003611195.1, GCA_003611215.1, GCA_0036112 35.1, GCA_003957845.1, GCA_006912115.1, GCA_010015925.1, GCA_013170925.1, GCA_014884795.1, GCA_016906895.1, GCA_018398 735.1, GCA_019455505.1, GCA_019455665.1, GCA_022627015.2, GCA_023065745.1, GCA_024363405.1, GCA_024703715.1, GCA_900237765.1, GCA_900239735.1, GCA_900519115.1, GCA_900638725.1, GCA_905067625.1, GCA_927323605.2) and their Leotiomycetes The other 8 genomes of 8 orders were analyzed, and 2 homologous gene families specific to Powdery mildew were identified through pan-genome analysis. The above-mentioned Powdery mildew-specific genes were further compared with the NCBI non-redundant fungal gene database. The results showed that the similarity of the two gene families based on the best matching gene in the database was less than 50%. Through repeated verification of 38 powdery mildew genomes (known), the primers of 2 molecular markers were determined to be able to indicate the presence of powdery mildew, OG2127 was one of the molecular markers, and 18 gene sequences were obtained after de-redundancy of the gene family. The conserved regions of the 18 gene sequences (such as Figure 1As shown in the figure, degenerate primers were designed using HYDEN software, including the upstream primer Og2127F (whose nucleotide sequence is 5'-ATY GKT GGM CCKTWY GC-3') and the downstream primer Og2127R (whose nucleotide sequence is 5'-CTT TCR CCR CCR TCA AT-3'). The primers can be used to amplify a characteristic band with a length of 112 bp by PCR.

[0032] Example 2: Verification of Powdery Mildew Molecular Marker Primers

[0033] The microbial DNA of three hydroponic tobacco powdery mildew diseased leaf tissues was extracted, and each DNA sample was amplified by PCR using molecular labeled primers (Og2127F / Og2127R) for powdery mildew detection. The 10uL PCR reaction system includes: 2ngDNA, 1×Taq enzyme buffer; 2mmol·L -1 MgCl2, 0.2mmol·L -1 dNTPs, upstream and downstream primers were 2.5 pmol·L -1 and 1U Taq DNA polymerase, ddH2O to 10uL. Reaction procedure: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 52℃ annealing for 30s, 72℃ extension for 30s, 32 cycles; finally 72℃ extension for 10min. PCR products were separated by 1.2% polyacrylamide gel electrophoresis, SYBR Green fluorescent dye was blue, and the characteristic bands of PCR amplified DNA were observed by gel imaging.

[0034] The results showed that the identification efficiency of this marker was 100%.

[0035] Example 3: Verification of Powdery Mildew Molecular Marker Primers

[0036] Extract three microbial DNA from healthy leaf tissues of hydroponic tobacco, and use molecular marker primers (Og2127F / Og2127R) for PCR amplification of each DNA sample for powdery mildew detection. The PCR reaction system is as in Example 2. Separate the PCR products by 1.2% polyacrylamide gel electrophoresis, SYBR Green fluorescent dye blue, and observe the characteristic bands of PCR amplified DNA by gel imaging. The results show that no characteristic bands are found in the microbial DNA of healthy leaf tissues of tobacco, and the identification efficiency of the marker for negative controls is 100%.

[0037] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. A method for identifying broad-spectrum molecular markers of powdery mildew pathogens of crops, characterized in that: The following steps are involved: (1) Extracting microbial DNA from leaf tissues of the plants to be tested; (2) Using the above-extracted microbial DNA as a template, PCR amplification was performed using molecular marker primers Og2127F and Og2127R; The sequences of the primers for the molecular markers are as follows: The upstream primer Og2127F has a nucleotide sequence of: 5'-ATY GKT GGM CCK TWY GC-3', The downstream primer Og2127R had the following nucleotide sequence: 5′-CTT TCR CCR CCR TCA AT-3′; (3) Detect the PCR amplification product using gel electrophoresis technology; if a characteristic band of 112 bp is obtained, it means that the microorganisms in the plant leaf tissue have detected the powdery mildew pathogen; if not, it means that the microorganisms in the plant leaf tissue have not detected the powdery mildew pathogen.

2. The method for identifying broad-spectrum molecular markers of powdery mildew pathogens of crops according to claim 1, characterized in that: In step (3), the reaction system is 10uL in total, and the 10uL PCR reaction system includes: 2 ng plant DNA, 1 × Taq enzyme buffer; 2 mmol·L -1 MgCl2, 0.2 mmol·L -1 dNTPs, upstream and downstream primers were 2.5 pmol·L -1 and 1U Taq DNA polymerase, and make up to 10uL with ddH2O.

3. The method for identifying broad-spectrum molecular markers of powdery mildew pathogens of crops according to claim 2, characterized in that: In step (3), the PCR reaction conditions are set as follows: Pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 50-55°C for 30 s, extension at 72°C for 30 s, 35 cycles; final extension at 72°C for 10 min.

4. The method for identifying broad-spectrum molecular markers of powdery mildew pathogens of crops according to claim 2, characterized in that: The powdery mildew pathogen of crops originates from hydroponic tobacco.

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