Primer group, kit and method for detecting verticillium dahliae pathotype
By identifying the VdR2e and VdR3e genes of Mycobacteria, designing primer sets and kits, high accuracy detection of physiological species No. 2 and 3 was achieved, solving the problem of identification difficulties in the prior art, and improving the ability to prevent and control Verticillium wort.
Patent Information
- Application Number
- CN202411374880.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-29
- Publication Date
- 2025-02-25
AI Technical Summary
The prior art is difficult to identify the non-toxic genes of physiological species No. 2 and 3 of Dali's Rhizoma, which leads to difficulties in monitoring the molecular epidemic of physiological species and digging of disease-resistant genes, which in turn affects the comprehensive prevention and control of verticillin wort.
By identifying the VdR2e gene and VdR3e gene of Mycetes Darien, designing corresponding primer sets and kits, and using PCR detection methods, it can accurately identify the physiological species No. 2 and No. 3 of Mycetes Darien.
It has achieved high accuracy detection of physiological species of Dali's elixir, provided effective gene resources for disease-resistant gene mining, and improved the comprehensive prevention and control capabilities of Verticillium wort.
Smart Images
Figure CN119506452A_ABST
Abstract
Description
Technical Field
[0001] The present disclosure relates to the field of biotechnology, and in particular, to a primer set, a kit and a method for detecting pathotypes of Verticillium dahliae. Background Art
[0002] Verticillium wilt caused by Verticillium dahliae poses a serious threat to global crop production safety and is extremely difficult to control. The "gene-for-gene" theory of crop resistant variety selection and resistance genetic improvement has always been an important strategy for crop Verticillium wilt prevention and control. At present, tomatoes are divided into three physiological races based on their resistance to Verticillium dahliae. The non-toxic genes of physiological races 1 and 2 and the disease-resistant genes of tomatoes to physiological race 1 have been identified, but their application in production is very limited. The main reasons are: the non-toxic genes of physiological race 3 have not yet been identified, making it difficult to implement molecular epidemic monitoring of physiological races; the disease-resistant genes to physiological races 2 and 3 have not yet been discovered, and there is a lack of effective gene resources for genetic improvement of crop resistance. Verticillium wilt is particularly serious, and physiological races 2 and 3 are the dominant populations, making it difficult to implement the prevention and control strategy of "physiological race monitoring" to guide the selection of resistant varieties and genetic improvement of resistance.
[0003] Therefore, there is an urgent need to identify the specific genes of the physiological races 2 and 3 of Verticillium dahliae and apply them to the detection of pathogenic physiological races and the comprehensive prevention and control of Verticillium wilt. Summary of the invention
[0004] The purpose of the present disclosure is to provide a method for detecting the pathotype of Verticillium dahliae, which can detect the physiological race of Verticillium dahliae through the VdR2e gene and / or the VdR3e gene.
[0005] The present disclosure also provides a primer set and a kit for detecting Verticillium dahliae, which can be used for detecting physiological races of Verticillium dahliae and have a high detection accuracy.
[0006] In order to achieve the above-mentioned object, the first aspect of the present disclosure provides a primer set for detecting Verticillium dahliae, the primer set comprising: a VdR2e gene primer set and a VdR3e gene primer set; the target gene of the VdR2e gene primer set is the Verticillium dahliae VdR2e gene; the target gene of the VdR3e gene primer set is the Verticillium dahliae VdR3e gene; Wherein, the VdR2e gene primer set includes a VdR2e gene upstream primer as shown in SEQ ID NO.3 and a VdR2e gene downstream primer as shown in SEQ ID NO.4; The VdR3e gene primer set includes a VdR3e gene upstream primer as shown in SEQ ID NO.5 and a VdR3e gene downstream primer as shown in SEQ ID NO.6.
[0007] A second aspect of the present disclosure provides a kit for detecting Verticillium dahliae, the kit comprising the VdR2e gene primer set and / or the VdR3e gene primer set according to the first aspect, and a PCR reagent.
[0008] Optionally, the VdR2e gene primer set is used at a concentration of 8-12 μM; and the VdR3e gene primer set is used at a concentration of 8-12 μM.
[0009] Optionally, the PCR reagents include a PCR reaction buffer, a positive control standard and a negative control standard.
[0010] A third aspect of the present disclosure provides a method for detecting the pathotype of Verticillium dahliae, the method comprising the following steps: S1, preparing a first sample to be tested containing the whole genome DNA of Verticillium dahliae; S2, performing PCR amplification using the first test sample of the whole genome DNA in step S1 as a template, and detecting whether the amplified product contains the VdR2e gene shown in SEQ ID NO.1 and / or the VdR3e gene shown in SEQ ID NO.2; If the amplified product contains the VdR2e gene as shown in SEQ ID NO.1, it indicates that the strain of Verticillium dahliae is physiological race 2 of Verticillium dahliae; if the amplified product contains the VdR3e gene as shown in SEQ ID NO.2, it indicates that the strain of Verticillium dahliae is physiological race 3 of Verticillium dahliae.
[0011] Optionally, the first sample to be tested is derived from a plant.
[0012] Optionally, the first sample to be tested is derived from at least one of a tomato plant, a cotton plant, a lettuce plant and an eggplant plant.
[0013] Through the above technical scheme, the present disclosure identifies the specific gene VdR2e gene of the physiological race 2 of Verticillium dahliae and the specific gene VdR3e gene of the physiological race 3 of Verticillium dahliae. The VdR2e gene and the VdR3e gene can be used as target genes to detect the physiological races of Verticillium dahliae. The present disclosure also designs a primer set and a kit based on the VdR2e gene and the VdR3e gene. The primer set and the kit of the present disclosure are used to realize PCR detection and identification of the physiological races of Verticillium dahliae, and the accuracy rate can reach 100%.
[0014] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings: Figure 1 This is an analysis of the characteristics of secreted proteins encoded in the lineage-specific fragments (LSRs) of three physiological subspecies of Verticillium dahliae in manipulating host immunity.
[0016] Figure 2 It is the specific fragment identification of Verticillium dahliae No. 2 and No. 3.
[0017] Figure 3 It is the potential non-toxic gene structure characteristic of Verticillium dahliae 2 and 3.
[0018] Figure 4 It is a PCR molecular detection of potential non-toxic genes of Verticillium dahliae 2 and 3. DETAILED DESCRIPTION
[0019] The specific implementation of the present disclosure is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.
[0020] The first aspect of the present disclosure provides a primer set for detecting Verticillium dahliae, the primer set comprising: a VdR2e gene primer set and a VdR3e gene primer set; the target gene of the VdR2e gene primer set is the Verticillium dahliae VdR2e gene; the target gene of the VdR3e gene primer set is the Verticillium dahliae VdR3e gene; Wherein, the VdR2e gene primer set includes a VdR2e gene upstream primer as shown in SEQ ID NO.3 and a VdR2e gene downstream primer as shown in SEQ ID NO.4; The VdR3e gene primer set includes a VdR3e gene upstream primer as shown in SEQ ID NO.5 and a VdR3e gene downstream primer as shown in SEQ ID NO.6.
[0021] The inventors of the present disclosure unexpectedly discovered that the VdR2e gene is a specific gene for the physiological race 2 of Verticillium dahliae, and the VdR3e gene is a specific gene for the physiological race 3 of Verticillium dahliae; the VdR2e gene and the VdR3e gene can be used for the detection and identification of the physiological races of Verticillium dahliae; the method for detecting the pathotype of Verticillium dahliae disclosed in the present disclosure uses the VdR2e gene and / or the VdR3e gene as target genes to detect and identify the physiological races of Verticillium dahliae, and has the advantage of high accuracy.
[0022] The inventors of the present disclosure designed a primer set with high specificity based on the obtained CDS sequences of the VdR2e gene and the VdR3e gene using primer design software, and compared them in the NCBI database to verify the specificity of the designed primers. Specifically, the upstream primer and downstream primer for specific detection of the physiological subspecies 2 of Verticillium dahliae were designed based on the sequence of the VdR2e gene; the upstream primer and downstream primer for specific detection of the physiological subspecies 3 of Verticillium dahliae were designed based on the sequence of the VdR3e gene. The primer set disclosed in the present disclosure can realize the rapid detection and identification of Verticillium dahliae, and has the advantages of fast detection speed and high accuracy, and the accuracy rate can be as high as 100%.
[0023] A second aspect of the present disclosure provides a kit for detecting Verticillium dahliae, the kit comprising the VdR2e gene primer set and / or the VdR3e gene primer set according to the first aspect, and a PCR reagent.
[0024] In one embodiment of the present disclosure, the VdR2e gene primer set is used at a concentration of 8-12 μM; the VdR3e gene primer set is used at a concentration of 8-12 μM.
[0025] In one embodiment of the present disclosure, the PCR reagents include a PCR reaction buffer, a positive control standard, and a negative control standard.
[0026] A third aspect of the present disclosure provides a method for detecting the pathotype of Verticillium dahliae, the method comprising the following steps: S1, preparing a first sample to be tested containing the whole genome DNA of Verticillium dahliae; S2, performing PCR amplification using the first test sample of the whole genome DNA in step S1 as a template, and detecting whether the amplified product contains the VdR2e gene shown in SEQ ID NO.1 and / or the VdR3e gene shown in SEQ ID NO.2; If the amplified product contains the VdR2e gene as shown in SEQ ID NO.1, it indicates that the strain of Verticillium dahliae is physiological race 2 of Verticillium dahliae; if the amplified product contains the VdR3e gene as shown in SEQ ID NO.2, it indicates that the strain of Verticillium dahliae is physiological race 3 of Verticillium dahliae.
[0027] In the present disclosure, the step of preparing the first sample to be tested can be carried out according to the conventional method of preparing samples for nucleic acid detection in the art, and the present disclosure has no special requirements. For example, tissues can be collected from plant plants and cultured on a culture medium containing resistance, and then colonies of Verticillium dahliae can be screened, identified and separated, and then the whole genome DNA of Verticillium dahliae can be extracted using a fungal DNA extraction kit to obtain the first sample to be tested.
[0028] Wherein, the PCR amplification reaction comprises the following steps: (a) 95°C, 3 min; (b) 95°C, 30 s; (c) 58°C, 30 s; (d) 72°C, 30 s; (e) 72°C, 5 min; (b)-(d) were cycled 35 times.
[0029] According to the present disclosure, the first sample to be tested may be derived from a plant; preferably, the first sample to be tested may be derived from at least one of a tomato plant, a cotton plant, a lettuce plant and an eggplant plant. Specifically, the first sample to be tested may be derived from a root or stem of a plant.
[0030] In the present disclosure, the second sample to be tested may be derived from a plant; specifically, the second sample to be tested may be derived from at least one of a tomato plant, a cotton plant, a lettuce plant, and an eggplant plant.
[0031] The present disclosure is further illustrated below by way of examples, but the present disclosure is not limited thereby.
[0032] DNAsecure new plant genomic DNA extraction kit (Tiangen): used to extract DNA from samples to be tested; TransScript Ⅱ One-Step gDNA Removal and cDNA Synthesis SuperMix (Quanshi Gold): used for reverse transcription to obtain cDNA as the test sample to amplify VdR2e and VdR3e; Phanta Max Super-Fidelity DNA Polymerase (Nuoweizan): used for PCR amplification; 5 min TA / Blunt-Zero Cloning Kit (Novozymes): The amplified VdR2e and VdR3e target sequences were ligated to this vector for sequencing and analysis of sequence information.
[0033] The primers were synthesized by Bioengineering Corporation.
[0034] Example 1 This example is used to illustrate the identification of VdR2e gene and VdR3e gene.
[0035] The whole genome sequencing of 4 strains of No. 3 physiological subspecies (HoMCLT, Gf-Cb5, GF1300 and GF1192), 2 strains of No. 2 physiological subspecies (TO22 and Ud1-4-1) and 1 strain of No. 1 physiological subspecies (VdLs.16) was completed (No. 1 physiological subspecies JR2 was used as the reference genome), and the genomes at the near chromosome assembly level were obtained. HoMCLT, TO22 and JR2 were used as representatives of No. 3, No. 2 and No. 1 physiological subspecies strains, respectively. The genome structure comparison and colinearity analysis showed that each physiological subspecies strain contained a subspecies-specific genome fragment (LSRs), No. 3 physiological subspecies HoMCLT contained 28 LSRs, No. 2 physiological subspecies TO22 contained 56 LSRs, and No. 1 physiological subspecies JR2 contained 45 LSRs; the genes encoded in the LSRs of the three physiological subspecies and the secretion characteristics of the encoded genes were predicted. In order to further study the functions of LSRs in different physiological races, the functions of 28 LSRs in race 3, 156 LSRs in race 2, and 45 LSRs in race 1 were predicted. A small number of specific LSRs and gene expansions occurred in race 3, and most LSRs overlapped in the three physiological races. Further analysis of the secretory protein manipulation immune characteristics in LSRs revealed a large number of secretory proteins that stimulate / suppress immunity. The secretory protein genes encoded in LSRs of races 2 and 3 were analyzed one by one, and it was found that the gene TO22_V02745 (VdR2e) encoding secretory proteins in LSR-52 of race 2 only existed in strains of race 2; the gene HoMCLT_V04112 (VdR3e) encoding secretory proteins in LSR-10 of race 3 only existed in strains of race 3, and was completely missing in races 1 and 2.
[0036] Figure 1 This is an analysis of the properties of secreted proteins encoded in three lineage-specific fragments (LSRs) of Verticillium dahliae that manipulate host immunity. Figure 1 It can be seen that three physiological subspecies-specific gene fragments (LSRs) were identified, and the secretory proteins encoded by most LSRs have the function of inhibiting the host immune response, which is beneficial to promoting pathogen infection.
[0037] Depend on Figure 2 It can be seen that the secretory protein genes encoded in the No. 2 and No. 3 LSRs were analyzed one by one, and it was found that the gene TO22_V02745 (VdR2e) encoding the secretory protein in the No. 2 physiological race LSR-52 only exists in the No. 2 physiological race strain; the gene HoMCLT_V04112 (VdR3e) encoding the secretory protein in the No. 3 physiological race LSR-10 only exists in the No. 3 physiological race strain, and is completely missing in the No. 1 and No. 2 races.
[0038] Depend on Figure 3It can be seen that the sequence characteristics of the proteins encoded by the VdR2e and VdR3e genes were preliminarily analyzed. Among them, VdR2e encodes 297 amino acids with a signal peptide, which is a small molecule cysteine-rich protein (containing 6 cysteines and predicted to form 3 disulfide bonds). This gene is located in Scaffold 3 of the genome of the No. 2 physiological subspecies strain. VdR3e encodes 114 amino acids with a signal peptide, which is a small molecule cysteine-rich protein (containing 4 cysteines and predicted to form 2 disulfide bonds); this gene is located in Scaffold 11 of the VdR3e genome of the No. 3 physiological subspecies strain, and the flanking sequences are rich in transposons and tandem repeats.
[0039] Depend on Figure 4 It can be seen that the gene detection markers were developed and PCR verification was performed using the physiological subspecies 2 and 3 of Verticillium dahliae, proving the specificity of the two gene markers. VdR2e only exists in the physiological subspecies 2 strain; VdR3e only exists in the physiological subspecies 3 strain, and is completely absent in the physiological subspecies 2 population. Preliminary results show that VdR2e is a secreted protein unique to physiological subspecies 2; VdR3e is a secreted protein unique to physiological subspecies 3.
[0040] Example 2 This example is used to illustrate the detection and identification of the pathotype of Verticillium dahliae in a sample to be tested.
[0041] Take the stem base of the plant to be tested (tomato), remove the phloem, disinfect it with sodium hypochlorite and 75% alcohol, grow it in PDA medium, and extract fungal DNA as the sample to be tested according to the instructions of the DNAsecure new plant genomic DNA extraction kit for subsequent testing.
[0042] PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase (Novozyme) with the DNA of the sample to be tested as a template. The VdR2e gene primer set included an upstream primer of the VdR2e gene as shown in SEQ ID NO.3 and a downstream primer of the VdR2e gene as shown in SEQ ID NO.4; the VdR3e gene primer set included an upstream primer of the VdR3e gene as shown in SEQ ID NO.5 and a downstream primer of the VdR3e gene as shown in SEQ ID NO.6.
[0043] The concentration of the VdR2e gene primer set was 10 μM; the concentration of the VdR3e gene primer set was 10 μM.
[0044] The PCR reaction system was: Phanta Max Super-Fidelity DNA Polymerase 10 μL, primers 1 μL+1 μL, DNA sample 1 μL, and H2O 7 μL.
[0045] PCR amplification reaction conditions: 95°C, 3 min; 95°C, 30 s, 58°C, 30 s, 72°C, 30 s, 35 cycles; 72°C, 5 min.
[0046] Analysis of amplification products: The amplified VdR2e and VdR3e target sequences were ligated to the vector (5 min TA / Blunt-ZeroCloning Kit (Novozyme)) for sequencing to analyze the sequence information; When the VdR2e gene primer set is used for PCR reaction, if the size of the amplified product is 1121 bp, it means that the sample to be tested contains the physiological subspecies 2 of Verticillium dahliae. When the VdR3e gene primer set is used for PCR reaction, if the size of the amplified product is 345 bp, it means that the sample to be tested contains physiological race 3 of Verticillium dahliae.
[0047] The preferred embodiments of the present disclosure are described in detail above in conjunction with the accompanying drawings; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
[0048] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.
[0049] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.
Claims
1. A primer set for detecting Verticillium dahliae, characterized in that: The primer set includes: a VdR2e gene primer set and a VdR3e gene primer set; the target gene of the VdR2e gene primer set is the Verticillium dahliae VdR2e gene; the target gene of the VdR3e gene primer set is the Verticillium dahliae VdR3e gene; Wherein, the VdR2e gene primer set includes a VdR2e gene upstream primer as shown in SEQ ID NO.3 and a VdR2e gene downstream primer as shown in SEQ ID NO.4; The VdR3e gene primer set includes a VdR3e gene upstream primer as shown in SEQ ID NO.5 and a VdR3e gene downstream primer as shown in SEQ ID NO.
6.
2. A kit for detecting Verticillium dahliae, characterized in that: The kit comprises the VdR2e gene primer set and / or the VdR3e gene primer set according to claim 1, and PCR reagents.
3. The kit according to claim 2, wherein The VdR2e gene primer set is used at a concentration of 8-12 μM; The VdR3e gene primer set is used at a concentration of 8-12 μM.
4. The kit according to claim 2, wherein The PCR reagents include a PCR reaction buffer, a positive control standard and a negative control standard.
5. A method for detecting the pathotype of Verticillium dahliae, characterized in that: The method comprises the following steps: S1, preparing a first sample to be tested containing the whole genome DNA of Verticillium dahliae; S2, performing PCR amplification using the first test sample of the whole genome DNA in step S1 as a template, and detecting whether the amplified product contains the VdR2e gene shown in SEQ ID NO.1 and / or the VdR3e gene shown in SEQ ID NO.2; If the amplified product contains the VdR2e gene as shown in SEQ ID NO.1, it indicates that the strain of Verticillium dahliae is physiological race 2 of Verticillium dahliae; if the amplified product contains the VdR3e gene as shown in SEQ ID NO.2, it indicates that the strain of Verticillium dahliae is physiological race 3 of Verticillium dahliae.
6. The method according to claim 5, wherein: The first sample to be tested comes from a plant.
7. The method according to claim 6, wherein: The first sample to be tested comes from at least one of a tomato plant, a cotton plant, a lettuce plant and an eggplant plant.