Repulsion-phase molecular markers, primers, screening methods and applications thereof for tobacco against bacterial wilt

Abstract

The present invention discloses a repulsion-phase molecular marker, a primer, and a screening method and application thereof for tobacco resistant to bacterial wilt, belonging to the technical field of screening tobacco resistant to bacterial wilt. The present invention provides two repulsion-phase molecular markers for tobacco resistant to bacterial wilt, namely, the repulsion-phase molecular markers of tobacco gene 4370 and tobacco gene 37372; and corresponding UTR primer pairs and SSR primer pairs are provided for these two molecular markers. The present invention also provides a method for detecting or screening tobacco breeding materials or varieties resistant to bacterial wilt. The genes of the tobacco variety to be tested are subjected to PCR amplification using the above primers, the markers are detected by electrophoresis separation technology, and it is judged whether the tobacco breeding materials or varieties to be tested are resistant to bacterial wilt according to whether bands are shown. The above method significantly improves the accuracy and discrimination efficiency of breeding tobacco varieties resistant to bacterial wilt, and overcomes the difficulties in phenotypic identification of traits and the low efficiency of conventional molecular markers in selecting quantitative traits.

Description

Technical Field

[0001] The present invention belongs to the technical field of screening of tobacco resistant to bacterial wilt, in particular to a repulsion phase molecular marker method for screening breeding materials of tobacco resistant to bacterial wilt. Background Art

[0002] Tobacco is the main raw material crop for cigarettes and also an important cash crop in China. Variety is the basis of tobacco leaf production. In recent years, due to the extension of continuous cropping years, the harm of bacterial wilt to tobacco has become increasingly serious, seriously reducing the yield and quality of tobacco, and has become an important restricting factor for tobacco leaf production. Although methods such as comprehensive agricultural control and chemical control have a certain effect on controlling bacterial wilt, practice has proved that planting varieties resistant to bacterial wilt is the most economical and effective method to control the harm of bacterial wilt.

[0003] Tobacco bacterial wilt is a bacterial soil-borne disease caused by the parasitism of Ralstonia solanacearum, which is a devastating disease threatening world tobacco production. The antigen variety used in breeding tobacco resistant to bacterial wilt is mainly TI448A. However, the resistance of TI448A and its derived tobacco varieties to bacterial wilt is controlled by multiple pairs of additive genes, showing the genetic characteristics of quantitative traits. The traditional individual selection method directly selects agronomic traits that meet the breeding goals, such as high yield, high quality, and disease resistance, that is, it selects the individual phenotype rather than the genotype. Generally speaking, this method is effective for the selection of qualitative traits; but for the selection of quantitative traits, due to the effects of pleiotropy, multiple genes with one effect, regulatory genes, and modifying genes, there is a large difference between the individual phenotype and genotype. Therefore, the accuracy of individual selection through field phenotypic traits is poor. Although traditional breeding methods have achieved many results in the breeding of varieties resistant to bacterial wilt, due to the complexity of the bacterial wilt disease and its great susceptibility to the environment, the breeding progress of disease-resistant varieties has been slow.

[0004] According to a large number of reports, the molecular marker-assisted selection (MAS) technology established based on the genetic law of disease resistance traits can reduce the influence of gene-gene, environment, and genotype interactions, improve the screening efficiency, and accelerate the breeding process. For example, Chinese Patent Application CN112410463A provides a molecular marker for tomato resistance to bacterial wilt and its application, which discloses an SNP molecular marker for identifying the genotype of tomato resistance to bacterial wilt or identifying tomato varieties resistant to bacterial wilt. However, although tomatoes and tobacco belong to the Solanaceae family, their specific varieties are different, and there will be obvious differences in the genotypes that confer resistance to bacterial wilt. Another example is that CN113278729B provides an SNP molecular marker for tobacco bacterial wilt, its obtaining method and application. However, this SNP molecular marker (a genetic marker formed by a single nucleotide variation on the gene) must be digested by enzymes before electrophoresis to convert the SNP molecular marker into an applicable PCR marker, and it has high requirements for the electrophoresis gel, generally PAGE gel is required, the operation is cumbersome, the stability is poor, and the cost is high. In addition, this marker needs to distinguish resistant homozygous plants, resistant heterozygous plants, and susceptible homozygous plants by the number of bands.

[0005] It can be seen that although a small number of common types of molecular markers related to resistance to bacterial wilt have been developed at home and abroad, unfortunately, due to the quantitative inheritance of tobacco resistance to bacterial wilt, these molecular markers are far from meeting the requirements of breeding assistant selection and utilization; so far, only a very small number of molecular markers have been found that can be directly applied to the screening of tobacco varieties resistant to bacterial wilt. Therefore, developing molecular markers that can be used for breeding tobacco varieties resistant to bacterial wilt is a very urgent task. Summary of the Invention

[0006] Aiming at the above deficiencies of the prior art, the present invention provides a repulsion-phase molecular marker and primer for tobacco resistance to bacterial wilt, and a method for screening tobacco breeding materials or varieties with resistance to bacterial wilt by using this repulsion-phase molecule or primer. By using this method, both resistant and susceptible varieties to bacterial wilt can be identified, and all susceptible plants in the hybrid offspring population can be eliminated, and the remaining plants are resistant plants, and then the remaining plants can be cultivated into varieties resistant to bacterial wilt. Specifically, it is achieved through the following technologies.

[0007] The repulsion-phase molecular marker for tobacco resistance to bacterial wilt is the repulsion-phase molecular marker of tobacco gene 4370 or the repulsion-phase molecular marker of tobacco gene 37372;

[0008] The nucleotide sequence amplified by the primer of the repulsion-phase molecular marker of tobacco gene 4370 is as shown in SEQ ID NO.1 in the sequence listing; the nucleotide sequence amplified by the primer of the repulsion-phase molecular marker of tobacco gene 37372 is as shown in SEQ ID NO.2 in the sequence listing.

[0009] Compared with the prior art CN113278729B, the repulsion-phase molecular marker of the present invention is directly designed based on the gene sequence, which is convenient to detect. After PCR amplification, it can be directly detected by electrophoresis without cumbersome steps such as enzyme digestion, with convenient and simple operation and good stability.

[0010] The CDS sequence (coding sequence) of the above-mentioned tobacco gene 4370 is shown in Sequence Listing SEQ ID NO.7; the DNA sequence of the above-mentioned tobacco gene 4370 is shown in Sequence Listing SEQ ID NO.8; the CDS sequence (coding sequence) of the above-mentioned tobacco gene 37372 is shown in Sequence Listing SEQ ID NO.9; the DNA sequence of the above-mentioned tobacco gene 37372 is shown in Sequence Listing SEQ ID NO.10.

[0011] The present invention also provides a primer pair for amplifying the above-mentioned repulsion-phase molecular marker. In the UTR primer pair for amplifying the repulsion-phase molecular marker of tobacco gene 4370, the forward primer and the reverse primer have a length of 18 - 25 bp, a GC content of 30 - 60%, a annealing temperature of 56 - 62 °C, and the length of the PCR product is 100 - 350 bp;

[0012] In the SSR primer pair for amplifying the repulsion-phase molecular marker of tobacco gene 37372, the forward primer and the reverse primer have a length of 18 - 25 bp, a GC content of 30 - 60%, a annealing temperature of 56 - 62 °C, and the length of the PCR product is 100 - 350 bp.

[0013] The primer of the present invention is a repulsion-phase marker, which can exclude the interference of heterozygous genes. If there is a marker, it indicates that the breeding material or variety is not resistant to bacterial wilt, and both homozygous and heterozygous non-resistant genotypes can be eliminated at the same time; if there is no marker, it indicates that the breeding material or variety is resistant to bacterial wilt, and homozygous resistant genotypes can be screened. Therefore, the present invention only needs to distinguish resistant and susceptible plants according to the presence or absence of bands, which is very simple.

[0014] Preferably, in the UTR primer pair for amplifying the repulsion-phase molecular marker of tobacco gene 4370, the nucleotide sequence of the forward primer is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4; that is:

[0015] Forward primer: 5′-GCATGAGATACATTAAGCCAAAATC-3′;

[0016] Reverse primer: 5′-CCTGGAGCCATCACTGGG-3′;

[0017] Among the SSR primer pairs for the repulsion-phase molecular marker amplifying tobacco gene 37372, the nucleotide sequence of the forward primer is as shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.6; that is:

[0018] Forward primer: 5′-GTACTCTCGTAACTGCAGCATTAAA-3′;

[0019] Reverse primer: 5′-TGAGCCATCACAGGAAAGAAG-3′.

[0020] The applicant of the present invention used tobacco varieties resistant and susceptible to Ralstonia solanacearum as research materials, and analyzed the differential gene expression under the condition of Ralstonia solanacearum infection. Through KEGG analysis of the transcriptome data, it was found that there were significant differences in the phenylpropanoid metabolic pathway at 24 h after infection between resistant and susceptible varieties. That is, compared with the susceptible variety, the branch scopoletin glucoside metabolism in the phenylpropanoid metabolic pathway of the resistant variety showed a significant up-regulation, and scopoletin glucoside has an important relationship with resistance to Ralstonia solanacearum.

[0021] The applicant thus obtained some valuable gene fragments, verified these gene fragments by CRISPR / Cas9 technology, and identified the Ralstonia solanacearum inoculation of the T1 generation with a higher editing rate. From this, genes closely related to tobacco resistance to Ralstonia solanacearum were found: ID: gene_4370 (scopoletinglucosyltransferaselike) and ID: gene_37372 (scopoletinglucosyltransferase). The applicant further designed different molecular marker primers for these two genes. Through PCR amplification, it was found that the molecular markers developed for the two genes were both repulsion-phase molecular markers, that is, molecular markers linked to the target trait in repulsion. If there is a molecular marker, the plant does not show the target trait; if there is no molecular marker, the plant shows the target trait. The applicant verified through repeated experiments that all susceptible Ralstonia solanacearum varieties showed electrophoretic bands, while all resistant Ralstonia solanacearum varieties did not show electrophoretic bands.

[0022] Therefore, the applicant confirmed that these two repulsion-phase molecular markers can be used for the assisted selection breeding of tobacco varieties resistant to Ralstonia solanacearum. By using the above-mentioned selection of repulsion-phase molecular markers, all susceptible Ralstonia solanacearum plants containing the markers in the population can be eliminated, leaving only marker-free resistant plants, effectively overcoming the problem of low efficiency of conventional molecular markers in the selection of quantitative traits.

[0023] The present invention also provides a method for detecting or screening tobacco breeding materials or varieties resistant to Ralstonia solanacearum, which can detect or screen tobacco breeding materials or varieties resistant to Ralstonia solanacearum for the above-mentioned repulsion-phase molecular markers, or directly use the primer pairs of the above-mentioned repulsion-phase molecular markers.

[0024] Preferably, the application method of the above-mentioned repulsive phase molecule labeling comprises the following steps:

[0025] S1, extracting and using the DNA of the tobacco breeding material or variety to be tested as an amplification template, and using a primer pair for amplifying the repulsive molecular markers of tobacco gene 37372, or a primer pair for amplifying the repulsive molecular markers of tobacco gene 4370 to perform PCR amplification;

[0026] S2. Read the differential bands using electrophoresis separation technology; if bands are shown on the electrophoresis gel, the tobacco breeding material or variety to be tested is not resistant to bacterial wilt; if no bands are shown, the tobacco breeding material or variety to be tested is resistant to bacterial wilt.

[0027] Preferably, in step S1, the PCR amplification reaction system is: 16.1 μL of ddH 2 O, 2 μL 10× buffer, 0.3 μL dNTP, 0.2 μL forward primer, 0.2 μL reverse primer, 0.2 μL Easytaq PCR, 1 μL cDNA, the reaction volume was 20 μL;

[0028] The reaction conditions were as follows: pre-denaturation at 94°C for 5 min, 94°C for 30 s, annealing at 56°C for 30 s, 72°C for 30 s, 34 cycles, and extension at 72°C for 7 min.

[0029] Preferably, in the above application method, the electrophoresis separation technology in step S2 adopts a fully automatic capillary electrophoresis system or gel electrophoresis.

[0030] More preferably, in step S2, when the electrophoresis separation technology adopts a fully automatic capillary electrophoresis system, the system is operated according to the instructions of the system, using FA dsDNA Gel models 800 and 900, and selecting the Gel PrimerOuly electrophoresis program carried by the system for electrophoresis, with an electrophoresis time of 60 minutes. After the electrophoresis, the system's own software is used to analyze the electrophoresis results and read the differential bands.

[0031] More preferably, in step S2, when the electrophoretic separation technique is gel electrophoresis, a silver staining technique is used to develop and read the differential bands, and the reagents of the silver staining technique include a fixing solution, a staining solution and a color developing solution;

[0032] The fixing solution is prepared by adding glacial acetic acid to ddH2O, and the concentration of glacial acetic acid is 10%; the staining solution is prepared by adding AgNO 3 and formaldehyde were added to ddH2O, and AgNO 3 The concentration is 1g / L, the formaldehyde concentration is 0.056%; the color developing solution is Na 2 CO 3 , formaldehyde, Na2 S 2 O 3 ·5H2O was added to ddH2O to prepare it, and the concentration of Na 2 CO 3 was 30 g / L, the concentration of formaldehyde was 0.056%, and the concentration of Na 2 S 2 O 3 was 2 mg / L.

[0033] Further preferably, in step S2, the staining process of the silver staining technique is as follows:

[0034] S21. Place the electrophoresis gel in the fixing solution, drop in the xylene cyanol indicator, shake for 30 min until the solution becomes colorless; take out the electrophoresis gel, rinse it with ddH2O, drain it, and repeat the treatment 2 times;

[0035] S22. Put the electrophoresis gel into the staining solution and shake for 30 min; take out the electrophoresis gel and rinse it with ddH2O for 5 - 10 s;

[0036] S23. Put the electrophoresis gel into the developing solution, quickly shake it for 10 s, and pour out the developing solution; pour in the developing solution again and continue developing until it develops color; after developing, take out the electrophoresis gel and put it into the fixing solution to terminate the development, gently shake it for 5 min, and then rinse it clean with tap water.

[0037] Compared with the prior art, the advantages of the present invention are as follows:

[0038] 1. The present invention for the first time discloses the application of the tobacco scopoletin glucosyltransferase - like gene ID: gene_4370 and scopoletin glucosyltransferase gene ID: gene_37372 in regulating the resistance of tobacco to bacterial wilt by using the method of reverse genetics;

[0039] 2. The present invention for the first time internationally obtains two repulsion - phase molecular markers of genes ID: gene_4370 and ID: gene_37372 which are closely related to tobacco resistance to bacterial wilt; they can be directly applied to the identification of tobacco breeding materials or varieties resistant and susceptible to bacterial wilt, can simultaneously eliminate homozygous and heterozygous non - disease - resistant genotypes, and screen out homozygous disease - resistant genotypes, significantly improving the identification accuracy and efficiency of tobacco breeding materials or varieties resistant to bacterial wilt; they can also be used as an auxiliary in tobacco breeding, effectively overcoming the difficulties of phenotypic trait identification, and also overcoming the low efficiency problem of conventional molecular markers in the selection of quantitative traits, and avoiding the disadvantages such as inaccurate genotype speculation from phenotype.

[0040] 3. Since the population currently used for QTI and mapping is small, the ability to detect QTL epistasis is weak, which may underestimate the QTL epistasis effect, thereby affecting the efficiency of molecular marker-assisted selection using conventional molecular markers. By using the repulsive molecular markers obtained by the present invention, all plants susceptible to bacterial wilt in the population can be eliminated, and all the remaining plants are disease-resistant, thereby improving the efficiency of molecular marker-assisted selection and better playing the important role of molecular marker-assisted selection in crop breeding;

[0041] 4. The method of the present invention can be used to complete the screening of tobacco breeding materials or varieties resistant to bacterial wilt at the seedling stage, without being affected by the environment and phenotypic identification, significantly reducing the size of the field planting population, saving manpower, material and financial resources, greatly reducing the workload, and accelerating the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Insertion site of the UTR primer pair of the repulsive phase marker of gene ID: gene_4370 on the sequence of SEQ ID NO.8;

[0043] Figure 2 The insertion site of the SSR primer pair of the repulsive molecular marker of gene ID: gene_37372 on the sequence of SEQ ID NO.10;

[0044] Figure 3 This is the molecular marker gel map of gene ID: gene_4370. In the figure, 1: Yanyan 97; 2: DB101; 3: Corker176; 4: Anti-Emperor No. 3; 5: Xi3; 6: Bina No. 1; 7: RG17; 8: NC95; 9: HT05; 10: TI448A; 11: Honghua Dajinyuan; 12: Changbohuang; 13: Cuibi No. 1; 14: Zhongyan 90; 15: Zhongyan 100; 16: NC89; 17: 6388; 18: Yunyan 100; 19: Yunyan 85; 20: B0851; R: resistant to bacterial wilt; S: susceptible to bacterial wilt;

[0045] Figure 4 This is a molecular marker gel map of gene ID: gene_37372. In the figure, 1: Yanyan 97; 2: Honghua Dajinyuan; R: resistant to bacterial wilt; S: susceptible to bacterial wilt. DETAILED DESCRIPTION

[0046] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Example 1: Discovery and Functional Verification of Genes ID: gene_4370 and ID: gene_37372 Closely Related to Tobacco Resistance to Ralstonia solanacearum

[0048] The susceptible variety to Ralstonia solanacearum "Honghuadajinyuan" and the highly resistant variety to Ralstonia solanacearum "Yanyan 97" were used as experimental materials. After disinfecting the seeds of the two materials, they were sown in MS solid medium (purchased from Coolaber Company); after the seedlings grew out, they were separately transplanted into the dispensed MS medium for light culture. When the tobacco seedlings grew to 5-6 true leaves, a batch of tobacco seedlings with similar growth vigor was selected, and the strain material was the Ralstonia solanacearum race LC3-6 with strong pathogenicity in Hubei (provided by the Tobacco Pest Research Center of Hubei Academy of Tobacco Sciences), and the pathogen was inoculated by the stem injection method.

[0049] Samples were taken at 0h, 12h, 24h, 36h, 48h, 3d, and 5d after inoculation. At each time point, two seedlings from the experiment and the control were mixed for sampling, with a total of 78 samples (including 3 biological replicates), and total RNA was extracted using the Tiangen kit (purchased from Tiangen Biotech Co., Ltd.).

[0050] Through KEGG analysis of the transcriptome data, it was found that there were significant differences in the phenylpropanoid metabolic pathway at 24h after infection between the resistant and susceptible varieties. That is, compared with the susceptible variety, the metabolism of scopolioside, a branch of the phenylpropanoid metabolic pathway in the resistant variety, showed a significant up-regulation. The CRISPR / Cas9 technology was used for further result verification. Some genes with particularly significant up-regulation were selected from the scopolioside metabolic branch, and were constructed into the CRISPR expression vector containing the U6-7 promoter (provided by the cotton research group of Huazhong Agricultural University) using overlap extension PCR and infusion ligation reaction. After the CRISPR vector was constructed, the variety "Yanyan 97" was used as the transformation material, and tobacco was transformed by the Agrobacterium-mediated leaf disc method, and 19 transgenic tobaccos with specific gene knockout were obtained, and the seeds of the T0 generation were obtained.

[0051] Through Hi-TOM target detection of the T0 generation plants, it was found that there were various editing types in the positive plants, and there were more than 5 plants with each gene completely edited, which could meet the subsequent research. Therefore, 3 lines were randomly selected for each gene, 6 plants were planted in each line, and Hi-TOM target detection was carried out on each plant.

[0052] It was found that deletions and insertions of varying degrees occurred at the predicted editing sites, and the editing rate of the target sites with higher editing rates could reach 60-70%. Further, plants with higher editing rates were detected in 3 lines of each gene and verified for disease resistance by inoculating with the pathogen. The disease incidence of different genes and different lines was counted, and it was found that two genes, Gene ID: gene_4370 and ID: gene_37372, were closely related to the resistance of tobacco to bacterial wilt.

[0053] DNA of the susceptible bacterial wilt variety Honghuadajinyuan and the highly resistant bacterial wilt variety Yanyan 97 was extracted using the plant genomic DNA extraction kit from TIANGEN company. Further, different molecular marker primers were designed for these two candidate genes, and the DNA of Honghuadajinyuan and Yanyan 97 was subjected to PCR amplification. The molecular markers were detected by electrophoresis technology, and different band patterns were obtained in both resistant and susceptible materials, confirming that these two genes were indeed closely related to the resistance of tobacco to bacterial wilt.

[0054] The molecular marker gel diagrams of Gene ID: gene_4370 and ID: gene_37372 are as Figure 3 、 4 shown. Figure 3 In [diagram], well 1 is the disease-resistant variety Yanyan 97, and well 11 is the disease-susceptible variety Honghuadajinyuan; Figure 4 In [diagram], R is the disease-resistant variety Yanyan 97 and S is the disease-susceptible variety Honghuadajinyuan.

[0055] By sequencing, the CDS sequence of Gene ID: gene_4370 (NCBI ID: 107800062) is shown in SEQ ID NO.7, and the DNA sequence is shown in Sequence Listing SEQ ID NO.8. The CDS sequence of Gene ID: gene_37372 (NCBI ID) is shown in SEQ ID NO.9, and the DNA sequence is shown in Sequence Listing SEQ ID NO.10.

[0056] Example 2: Development and verification of the repulsion-phase molecular marker of Gene ID: gene_4370 (NCBI ID: 107800062)

[0057] First, the software Batchprimer3 (https: / / wheat.pw.usda.gov / demos / BatchPrimer3 / ) was used to develop corresponding UTR primer pairs based on the repulsion-phase molecular marker sequence (UTR sequence) of Gene ID: gene_4370, where:

[0058] Forward primer: 5′-GCATGAGATACATTAAGCCAAAATC-3′;

[0059] Reverse primer: 5′-CCTGGAGCCATCACTGGG-3′;

[0060] Insertion site Figure 1 shown. Figure 1 In the method, the UTR molecular marker site is the site G of the fifth base from the 5' end of the sequence shown in SEQ ID NO.8, and the material of the 254 bp band generated by the electrophoresis is a susceptible variety.

[0061] Then, the genomic DNA of the plants of Yanyan 97, DB101, Corker176, Fandi No. 3, Xi 3, Bina No. 1, RG17, NC95, HT05, TI448A, which are highly resistant to bacterial wilt, and Honghua Dajinyuan, Changbohuang, Cuibi No. 1, Zhongyan 90, Zhongyan 100, NC89, 6388, Yunyan 100, Yunyan 85, B0851, which are highly susceptible to bacterial wilt, were extracted using the plant genomic DNA extraction kit of TIANGEN Company.

[0062] The highly resistant varieties Yanyan 97, DB101, Fandi No. 3, Xi3, RG17, and TI448A, and the highly susceptible variety Cuibi No. 1 were provided by the Fujian Tobacco Agricultural Science Research Institute;

[0063] The highly resistant varieties Corker176 and NC95, and the highly susceptible varieties Honghua Dajinyuan and NC89 were provided by the Tobacco Research Institute of Anhui Academy of Agricultural Sciences;

[0064] The highly resistant variety Bina No. 1 was provided by Bijie City Branch of Guizhou Provincial Tobacco Company;

[0065] HT05, a highly resistant variety to bacterial wilt, provided by Yongzhou Branch of Hunan Tobacco Company

[0066] The highly susceptible varieties to bacterial wilt, Longbohuang and 6388, were provided by the Tobacco Research Institute of Henan Academy of Agricultural Sciences;

[0067] The highly susceptible varieties Zhongyan 90 and Zhongyan 100 were provided by the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences;

[0068] The highly susceptible varieties Yunyan 100 and Yunyan 85 were provided by Yunnan Tobacco Agricultural Science Research Institute;

[0069] The highly susceptible variety B0851 is a variety bred by the Hubei Tobacco Science Research Institute.

[0070] Next, the designed UTR primers were used to perform PCR amplification on the extracted genomic DNA of the 20 tobacco varieties. The PCR amplification reaction volume was 20 μL, including 16.1 μL of ddH 20 μL, 2 μL of 10× buffer, 0.3 μL of dNTP, 0.2 μL of forward primer, 0.2 μL of reverse primer, 0.2 μL of Easytaq PCR, 1 μL of cDNA.

[0071] The amplification reaction conditions were pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, for 34 cycles, and final extension at 72°C for 7 min.

[0072] Finally, electrophoresis separation was performed using a capillary electrophoresis instrument, Fragment Analyzer (FA) and Zero Agarose Gel (ZAG), according to the instrument operation manual, and the differential bands were read.

[0073] The method for preparing electrophoresis reagents is as follows.

[0074] (1) Gel preparation: Use FA dsDNA Gel with model numbers 800 and 900, and prepare the gel according to the capillary gel preparation amounts listed in the following table. The gel preparation operation is as follows: Measure the gel with a clean graduated cylinder, then transfer it to a 250 mL conical bottom flask. Use a pipette to aspirate the fluorescent reagent (Intercalation Dye - Profect from Light). Slowly add it below the gel surface, and then tilt the conical bottom flask and slowly rotate it to mix evenly. The capillary gel preparation amounts are shown in Table 1 below.

[0075] Table 1 Capillary gel preparation amounts

[0076]

[0077] Reagent 5×930 dsDNA Inlet Buffer: Dilute 5 times, add to a 96 - well deep - well plate, 1.1 ml per well.

[0078] Reagent 5×Capillary Conditioning Buffer: Dilute 5 times, transfer to a 250 ml conical bottom flask.

[0079] Storage Solution: Aliquot with a PCR plate, 100 μL per well.

[0080] Reagent Maker: Aliquot with a PCR plate, 33 μL per well.

[0081] Dilution of PCR amplification products: Aspirate 2 μL of the PCR amplification product and transfer it to a new PCR plate. Add 22 μL of Dilution Buffer 1×TE to each well for dilution. The total volume per well is 24 μL. Add 10 μL of DNA Ladder and 14 μL of Dilution Buffer 1×TE to the last well (H12 well) of the PCR plate.

[0082] The electrophoresis process is as follows:

[0083] (1) Turn on the computer and the instrument;

[0084] (2) Put the prepared reagents into the corresponding positions according to the instrument operation manual;

[0085] (3) After the reagents are placed, successively open the control software "Utilities", "Solution Levels", and input the volume of the prepared reagents;

[0086] (4) Select the Gel Primer Ouly electrophoresis program for electrophoresis, with 6 - 10 plates of PCR as one round;

[0087] (5) The electrophoresis time is about 60 min;

[0088] (6) After electrophoresis, use the "PROsize2.0" software to analyze the electrophoresis results.

[0089] The obtained molecular marker gel diagram is shown in Figure 3. Figure 3 Among them, the first 10 wells are for the high-resistant bacterial wilt varieties Yanyan 97, DB101, Corker176, Anti-Imperial No. 3, Line 3, Bina 1, RG17, NC95, HT05, TI448A; the last 10 wells are for the high-susceptible bacterial wilt varieties Honghuadajinyuan, Changbohuang, Cuibi 1, Zhongyan 90, Zhongyan 100, NC89, 6388, Yunyan 100, Yunyan 85, B0851.

[0090] Figure 3 The results of the molecular marker gel diagram show that all high-susceptible bacterial wilt varieties show bands, while all high-resistant bacterial wilt varieties do not show bands. This indicates that the above UTR primers developed based on the UTR sequence of gene ID: gene_4370 are repulsion-phase molecular markers, which can be used for the identification of tobacco bacterial wilt-resistant varieties and molecular marker-assisted selection breeding. In practical applications, tobacco bacterial wilt-resistant breeding materials or varieties can be screened according to whether bands are shown on the molecular marker polyacrylamide gel. If there are bands, it indicates that the breeding material or variety is not resistant to bacterial wilt and should be excluded; if there are no bands, it indicates that the breeding material or variety is resistant to bacterial wilt and should be retained.

[0091] Example 3: Development and verification of repulsion-phase molecular markers for gene ID: gene_37372 (NCBI ID: 107826982)

[0092] First, based on the nucleotide sequence of gene ID: gene_37372, use SSRHunter (v1.3) to scan and search for SSR sites, and use PrimerPrimer5 software to design SSR primer pairs, where:

[0093] Forward primer: 5′-GTACTCTCGTAACTGCAGCATTAAA-3′;

[0094] Reverse primer: 5′-TGAGCCATCACAGGAAAGAAG-3′;

[0095] The insertion site is as Figure 2 shown. Figure 2 Among them, the SSR molecular marker locus is the locus G of the 11th base from the 5′ end of the sequence shown in SEQ ID NO.10, and the material of the 181bp band generated by electrophoresis is the disease-susceptible variety.

[0096] Then, use the plant genomic DNA extraction kit of TIANGEN Company to extract the genomic DNA of the plants of the highly resistant bacterial wilt variety Yanyan 97 and the highly susceptible bacterial wilt variety Honghuadajinyuan, respectively.

[0097] Next, use the above-designed SSR primers to perform PCR amplification on the genomic DNA of the above two tobacco varieties extracted. The reaction volume and reaction conditions of PCR amplification are the same as those in Example 2.

[0098] Finally, use 8% non-denaturing polyacrylamide gel (SSCP) for electrophoresis separation, and use silver staining technology to develop color and read the differential bands after electrophoresis.

[0099] The reagent preparation for silver staining technology is as follows:

[0100] Fixing solution (10% glacial acetic acid): Add 150 mL of glacial acetic acid to 1350 mL of ddH 2 O.

[0101] Staining solution (1 g / L AgNO 3 , 0.056% formaldehyde): Add 2 g of AgNO 3 and 3.0 mL of 37% formaldehyde to 2 L of ddH 2 O.

[0102] Developing solution (30 g / L Na 2 CO 3 , 0.056% formaldehyde, 2 mg / L Na 2 S 2 O 3 ·5H 2 O): Dissolve 75 g of Na 2 CO 3 in 2.5 L of ddH 2 O, and after loading the samples, place it at -20 °C for pre-cooling. A few minutes before use, add 6 mL of 37% formaldehyde and 400 μL of sodium thiosulfate with a concentration of 10 mg / mL.

[0103] The staining process is as follows:

[0104] (1) Fixation: Place the long glass plate with the adhesion glue in the fixing solution and place it on a shaker for 30 minutes until all the xylene cyanol disappears;

[0105] (2) Rinsing: Place the glass plate in ddH 2 O and rinse for 3 minutes. Take out the glass plate, drain it slightly, and repeat the treatment 2 times;

[0106] (3) Staining: Place the glass plate in a prepared plastic box containing the staining solution and place it on a shaker for 30 minutes;

[0107] (4) Rinsing: Take out the glass plate from the staining solution and quickly rinse it once in the cleaning box, with the time strictly controlled within 5 - 10 seconds;

[0108] (5) Development: Place the glass plate in 1 L of the developing solution, quickly shake it for 10 seconds, pour out the developing solution, and pour in the remaining developing solution to continue development. After development, place it in the fixing solution to terminate the development reaction. Gently shake it for 5 minutes, and then rinse the glass plate thoroughly with tap water.

[0109] The obtained molecular marker gel diagram is shown in Figure 4. Figure 4 Among them, the resistant variety Yanyan 97 is on the left and the susceptible variety Honghuadajinyuan is on the right. Figure 4 The results of the molecular marker gel diagram show that the highly susceptible bacterial wilt variety Honghuadajinyuan shows bands, while the highly resistant bacterial wilt variety Yanyan 97 does not show bands. This indicates that the above SSR primers developed based on the nucleotide sequence of Gene ID: gene_37372 are repulsion-phase molecular markers and can be used for the identification of tobacco bacterial wilt-resistant varieties and molecular marker-assisted selection breeding. In practical applications, tobacco bacterial wilt-resistant breeding materials or varieties can be screened according to whether bands are shown on the molecular marker polyacrylamide gel. If there are bands, it indicates that the breeding material or variety is not resistant to bacterial wilt and should be excluded; if there are no bands, it indicates that the breeding material or variety is resistant to bacterial wilt and should be retained. Sequence Listing <110> Hubei Academy of Tobacco Sciences <120> Repulsion-phase Molecular Marker, Primer and Their Screening Method and Application for Tobacco Bacterial Wilt Resistance <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 254 <212> DNA <213> tobacco <400> 1 gcatgagata cattaagcca aaatcagaac aaacttttct taaaccttga acccttacaa 60 atcctcgtta gtctataaaa ataggatgag ctaaggctag aaatacaact actcatatct 120 tctttcaact cctgttctct ttattctttt ctccctcatc agctggtaaa gcattgtttt 180 acttatacct tagctacaat gaataaaaga actgatcaac ttcatgcata ctttttccca 240 gtgatggctc cagg 254 <210> 2 <211> 181 <212> DNA <213> Tobacco <400> 2 gtactctcgt aactgcagca ttaaacactc atgcaatcat acaagaactg aaaacaacca 60 cacagcttta tatttctttc tattttctgc tactaaacta ggagtacatc tttctttctt 120 tctttcaagc attttcacaa atgggtcagc tccatatttt cttctttcct gtgatggctc 180 a 181 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <400> 3 gcatgagata cattaagcca aaatc 25 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <400> 4 cctggagcca tcactggg 18 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <400> 5 gtactctcgt aactgcagca ttaaa 25 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <400> 6 tgagccatca caggaaagaa g 21 <210> 7 <211> 1458 <212> DNA <213> tobacco <400> 7 atgaataaaa gaactgatca acttcatgca tactttttcc cagtgatggc tccaggccac 60 atgataccac tagtggacat ggccaggcaa tttgctcggc gtggtgtgaa ggcaaccctt 120 gtcaccactc ctctcaacgc acctaaattc tccaaaacaa tccaaagaga aagagagttg 180 ggcagtgata ttagcatccg aacaatcgag ttcccttgca aagaagctgg attgccagat 240 ggctgtgaaa acttagcttc tactactagt acagaaatga cccagaaatt catcaaagcc 300 ctgtacttgt ttcaacaacc aattgaacaa ttcctagaag aagatcatcc agattgtcta 360 atagcaagca ctctcttccc ttggactgtt gacgttgcag ccaagctggg aattccaaga 420 ctagttttca gtggcaccgg tttacttcct atttgtgctt atcacagttt gatggaatac 480 aaacctcact tgaaggtcga atctgacaca gaagagttca tcatccctgg ccttcctcac 540 acaataaaga tgtcaagaca acagctctct gaccatataa aggatgaaac agaaaaccca 600 atgactgaaa taattaaaga tgttatgaga gcagaaatga ctagttatgg agctattgta 660 aacagctttt atgaggtgga accaaattat gtaaaacatt acagggaagt ggtagggaaa 720 aaagcatggc acattggtcc agtttcactc tgcaataagg acaatgaaga taaagctcaa 780 agaggacaag aaatctgctt ttctgagcat cagtgtttag attggctcaa ttccaagaaa 840 ccaaaatcgg tcgtttacat ctgtttcggc agcatgtcgt tcttttcatc ggctcagttg 900 cttgagatag caatggctct tgaagcttca gaccagcaat ttatttgggt ggtgaagcaa 960 aatacaaaaa atgaagagca gaatgagtgg atgccagaag gatttgagga aaagctgaag 1020 ggacgaggtc tgataataaa aggatgggca ccgcaggtgc tgatccttga tcatgaagca 1080 attggaggtt ttgtcactca ctgcggatgg aactcgtcgc tggaaggagt aactgctgga 1140 gttccaatgg ttacgtggcc actgtcagct gagcaatttt ttaatgaaaa gctgctcgta 1200 gagattttaa agataggagt tccagtaggt gctcaggcct ggtcgcaaag aacagacagc 1260 agagtcccca taaacaggga aaacatacag agagcaatga ccaaactgat ggttggttcg 1320 gaagctgagg aaatgcgaag ccgtgcagct gccttaggaa aattggctaa aatggctgtg 1380 gagaaaggtg gatcctctga caacaacttg gtttccttgc tagaagaatt gaggaagaag 1440 caaagcaact ccaactga 1458 <210> 8 <211> 1715 <212> DNA <213> tobacco <400> 8 tgctgcatga gatacattaa gccaaaatca gaacaaactt ttcttaaacc ttgaaccctt 60 acaaatcctc gttagtctat aaaaatagga tgagctaagg ctagaaatac aactactcat 120 atcttctttc aactcctgtt ctctttattc ttttctccct catcagctgg taaagcattg 180 ttttacttat accttagcta caatgaataa aagaactgat caacttcatg catacttttt 240 cccagtgatg gctccaggcc acatgatacc actagtggac atggccaggc aatttgctcg 300 gcgtggtgtg aaggcaaccc ttgtcaccac tcctctcaac gcacctaaat tctccaaaac 360 aatccaaaga gaaagagagt tgggcagtga tattagcatc cgaacaatcg agttcccttg 420 caaagaagct ggattgccag atggctgtga aaacttagct tctactacta gtacagaaat 480 gacccagaaa ttcatcaaag ccctgtactt gtttcaacaa ccaattgaac aattcctaga 540 agaagatcat ccagattgtc taatagcaag cactctcttc ccttggactg ttgacgttgc 600 agccaagctg ggaattccaa gactagtttt cagtggcacc ggtttacttc ctatttgtgc 660 ttatcacagt ttgatggaat acaaacctca cttgaaggtc gaatctgaca cagaagagtt 720 catcatccct ggccttcctc acacaataaa gatgtcaaga caacagctct ctgaccatat 780 aaaggatgaa acagaaaacc caatgactga aataattaaa gatgttatga gagcagaaat 840 gactagttat ggagctattg taaacagctt ttatgaggtg gaaccaaatt atgtaaaaca 900 ttacagggaa gtggtaggga aaaaagcatg gcacattggt ccagtttcac tctgcaataa 960 ggacaatgaa gataaagctc aaagaggaca agaaatctgc ttttctgagc atcagtgttt 1020 agattggctc aattccaaga aaccaaaatc ggtcgtttac atctgtttcg gcagcatgtc 1080 gttcttttca tcggctcagt tgcttgagat agcaatggct cttgaagctt cagaccagca 1140 atttatttgg gtggtgaagc aaaatacaaa aaatgaagag cagaatgagt ggatgccaga 1200 aggatttgag gaaaagctga agggacgagg tctgataata aaaggatggg caccgcaggt 1260 gctgatcctt gatcatgaag caattggagg ttttgtcact cactgcggat ggaactcgtc 1320 gctggaagga gtaactgctg gagttccaat ggttacgtgg ccactgtcag ctgagcaatt 1380 ttttaatgaa aagctgctcg tagagatttt aaagatagga gttccagtag gtgctcaggc 1440 ctggtcgcaa agaacagaca gcagagtccc cataaacagg gaaaacatac agagagcaat 1500 gaccaaactg atggttggtt cggaagctga ggaaatgcga agccgtgcag ctgccttagg 1560 aaaattggct aaaatggctg tggagaaagg tggatcctct gacaacaact tggtttcctt 1620 gctagaagaa ttgaggaaga agcaaagcaa ctccaactga tgggaatttg tccatatgtg 1680 atcaaactag actgctaata aaaatgagta ggtgt 1715 <210> 9 <211> 1431 <212> DNA <213> Tobacco <400> 9 atgggtcagc tccatatttt cttctttcct gtgatggctc atggccacat gattcctaca 60 ctagacatgg cgaagctctt tgcttcacgt ggtgttaagg ccactataat cacaacccca 120 ctcaatgaat tcgttttctc caaagctatt caaagaaaca agcatttggg tatcgaaatc 180 gaaatccgtt tgatcaaatt cccagctgtt gaaaacggct tacctgaaga atgcgaacgc 240 ctcgatcaaa tcccttcaga tgagaagctc ccaaactttt tcaaagctgt agctatgatg 300 caagaaccac tagaacagct tattgaagaa tgtcgccccg attgtcttat ttcagatatg 360 ttccttcctt ggactactga tactgcagca aaatttaaca ttccaagaat agtctttcat 420 ggcacaagct tctttgctct ttgtgttgag aatagcgtca ggctaaataa gcctttcaag 480 aatgtgtcct cagattctga aacttttgtt gtaccggatt tgcctcacga aattaagctg 540 accagaaccc aggtgtctcc gtttgagcga tctggggaag agacggctat gacccggatg 600 accagaaccc aggtgtctcc gtttgagcga tctggggaag agacggctat gacccggatg 600 ataaaaacag tcagggaatc agattcaaag agctatggag ttgttttcaa cagtttctat 660 ataaaaacag tcagggaatc agattcaaag agctatggag ttgttttcaa cagtttctat 660 gagcttgaaa cagattatgt tgagcattat actaaggtgc tgggtagaag agcttgggct 720 gagcttgaaa cagattatgt tgagcattat actaaggtgc tgggtagaag agcttgggct 720 attggccctc tatcgatgtg caacagggac attgaagata aagctgaaag aggaaagaaa 780 attggccctc tatcgatgtg caacagggac attgaagata aagctgaaag aggaaagaaa 780 tcctctattg ataaacacga gtgcttgaaa tggcttgatt cgaagaaacc aagttccgtc 840 tcctctattg ataaacacga gtgcttgaaa tggcttgatt cgaagaaacc aagttccgtc 840 gtttacattt gttttggaag cgtagcgaat ttcactgcat cacaactgca cgaacttgct 900 gtttacattt gttttggaag cgtagcgaat ttcactgcat cacaactgca cgaacttgct 900 atgggagttg aagcttccgg acaagaattc atttgggttg ttagaacaga actagacaac 960 atgggagttg aagcttccgg acaagaattc atttgggttg ttagaacaga actagacaac 960 gaagattggt tgcctgaagg attcgaggaa agaacgaaag agaaaggttt aataataaga 1020 gaagattggt tgcctgaagg attcgaggaa agaacgaaag agaaaggttt aataataaga 1020 ggatgggcac cccaagtact aattcttgat cacgaatctg tgggagcttt tgttacacat 1080 ggatgggcac cccaagtact aattcttgat cacgaatctg tgggagcttt tgttacacat 1080 tgtggttgga attcaacact agaaggagtt tcaggagggg ttccaatggt aacatggcct 1140 tgtggttgga attcaacact agaaggagtt tcaggagggg ttccaatggt aacatggcct 1140 gtatttgctg agcaattttt caatgagaag ttagtgactg aggttttgaa aactggagct 1200 gtatttgctg agcaattttt caatgagaag ttagtgactg aggttttgaa aactggagct 1200 ggtgttggtt cgatacaatg gaagagatca gctagtgaag gagtgaaaag agaagcaata 1260 ggtgttggtt cgatacaatg gaagagatca gctagtgaag gagtgaaaag agaagcaata 1260 gctaaggcaa taaagagagt aatggtgagt gaagaagcag atggattcag aaacagagct 1320 aaagcgtata aggagatggc aagaaaggct attgaagaag gagggtcatc ttacactgga 1380 ttgactactt tgttggaaga tataagtaca tatagttcca ctggtcatta a 1431 <210> 10 <211> 1735 <212> DNA <213> tobacco <400> 10 taaagtgaga gtactctcgt aactgcagca ttaaacactc atgcaatcat acaagaactg 60 aaaacaacca cacagcttta tatttctttc tattttctgc tactaaacta ggagtacatc 120 tttctttctt tctttcaagc attttcacaa atgggtcagc tccatatttt cttctttcct 180 gtgatggctc atggccacat gattcctaca ctagacatgg cgaagctctt tgcttcacgt 240 ggtgttaagg ccactataat cacaacccca ctcaatgaat tcgttttctc caaagctatt 300 caaagaaaca agcatttggg tatcgaaatc gaaatccgtt tgatcaaatt cccagctgtt 360 gaaaacggct tacctgaaga atgcgaacgc ctcgatcaaa tcccttcaga tgagaagctc 420 ccaaactttt tcaaagctgt agctatgatg caagaaccac tagaacagct tattgaagaa 480 tgtcgccccg attgtcttat ttcagatatg ttccttcctt ggactactga tactgcagca 540 aaatttaaca ttccaagaat agtctttcat ggcacaagct tctttgctct ttgtgttgag 600 aatagcgtca ggctaaataa gcctttcaag aatgtgtcct cagattctga aacttttgtt 660 gtaccggatt tgcctcacga aattaagctg accagaaccc aggtgtctcc gtttgagcga 720 tctggggaag agacggctat gacccggatg ataaaaacag tcagggaatc agattcaaag 780 agctatggag ttgttttcaa cagtttctat gagcttgaaa cagattatgt tgagcattat 840 actaaggtgc tgggtagaag agcttgggct attggccctc tatcgatgtg caacagggac 900 attgaagata aagctgaaag aggaaagaaa tcctctattg ataaacacga gtgcttgaaa 960 tggcttgatt cgaagaaacc aagttccgtc gtttacattt gttttggaag cgtagcgaat 1020 ttcactgcat cacaactgca cgaacttgct atgggagttg aagcttccgg acaagaattc 1080 atttgggttg ttagaacaga actagacaac gaagattggt tgcctgaagg attcgaggaa 1140 agaacgaaag agaaaggttt aataataaga ggatgggcac cccaagtact aattcttgat 1200 cacgaatctg tgggagcttt tgttacacat tgtggttgga attcaacact agaaggagtt 1260 tcaggagggg ttccaatggt aacatggcct gtatttgctg agcaattttt caatgagaag 1320 ttagtgactg aggttttgaa aactggagct ggtgttggtt cgatacaatg gaagagatca 1380 gctagtgaag gagtgaaaag agaagcaata gctaaggcaa taaagagagt aatggtgagt 1440 gaagaagcag atggattcag aaacagagct aaagcgtata aggagatggc aagaaaggct 1500 attgaagaag gagggtcatc ttacactgga ttgactactt tgttggaaga tataagtaca 1560 tatagttcca ctggtcatta agttatgaat agcaacaaaa aaaaatgtag tactccgtac 1620 ttggtattat ttctgtactg tttttgtgct tttcctgtat gtgctaattt aaacatttct 1680 ttttgtcact ttttataatt gcaatgatga tatgtggaag aaccccttgc ctcca 1735

Claims

1. A method for detecting or screening tobacco breeding materials or varieties resistant to bacterial wilt, It is characterized in that Using primer pairs of mutually exclusive molecular markers for PCR amplification of tobacco gene 4370 to detect or screen tobacco breeding materials or varieties resistant to bacterial wilt; After PCR amplification using the primer pair, the differential bands are read using electrophoresis separation technology; if bands are shown on the electrophoresis gel, the tested tobacco breeding material or variety is not resistant to bacterial wilt; if no bands are shown, the tested tobacco breeding material or variety is resistant to bacterial wilt; The nucleotide sequence amplified by the primer pair for PCR amplification of the repulsive phase molecular marker of tobacco gene 4370 is shown in the sequence table SEQ ID NO.1, the nucleotide sequence of the forward primer of the primer pair is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

4.

2. The method for detecting or screening tobacco breeding materials or varieties resistant to bacterial wilt according to claim 1, It is characterized in that The following steps are involved: S1, extracting and using the DNA of the tobacco breeding material or variety to be tested as an amplification template, and using a primer pair of mutually exclusive molecular markers for amplifying tobacco gene 4370 for PCR amplification; S2. Read the differential bands using electrophoresis separation technology; if bands are shown on the electrophoresis gel, the tobacco breeding material or variety to be tested is not resistant to bacterial wilt; if no bands are shown, the tobacco breeding material or variety to be tested is resistant to bacterial wilt.

3. The method for detecting or screening tobacco breeding materials or varieties resistant to bacterial wilt according to claim 2, It is characterized in that In step S1, the PCR amplification reaction system is as follows: 16.1 μL of ddH 2 O, 2 μL of 10× buffer, 0.3 μL of dNTP, 0.2 μL of forward primer, 0.2 μL of reverse primer, 0.2 μL of Easytaq PCR, 1 μL of cDNA, and the reaction volume is 20 μL; The reaction conditions were as follows: pre-denaturation at 94°C for 5 min, 94°C for 30 s, annealing at 56°C for 30 s, 72°C for 30 s, 34 cycles, and extension at 72°C for 7 min.

4. The method for detecting or screening tobacco breeding materials or varieties resistant to bacterial wilt according to claim 2, It is characterized in that In step S2, the electrophoresis separation technique uses a fully automatic capillary electrophoresis system or gel electrophoresis.

5. The method for detecting or screening tobacco breeding materials or varieties resistant to bacterial wilt according to claim 4, It is characterized in that In step S2, the electrophoresis separation technology uses a fully automatic capillary electrophoresis system to operate according to the system's instructions, using FA dsDNA Gels of models 800 and 900, and selecting the Gel Primer Ouly electrophoresis program carried by the system for electrophoresis. The electrophoresis time is 60 minutes. After the electrophoresis is completed, the system's own software is used to analyze the electrophoresis results and read the differential bands.

6. The method for detecting or screening tobacco breeding materials or varieties resistant to bacterial wilt according to claim 5, It is characterized in that In step S2, the electrophoretic separation technique uses gel electrophoresis, and the silver staining technique is used to develop and read the differential bands. The reagents of the silver staining technique include a fixing solution, a staining solution, and a color developing solution; The fixing solution is prepared by adding glacial acetic acid to ddH 2 O, and the concentration of glacial acetic acid is 10%; the staining solution is prepared by adding AgNO 3 and formaldehyde to ddH 2 O, and the concentration of AgNO 3 is 1 g / L and the concentration of formaldehyde is 0.056%; the developing solution is prepared by adding Na 2 CO 3 , formaldehyde, and Na 2 S 2 O 3 ·5H 2 O to ddH 2 O, and the concentration of Na 2 CO 3 is 30 g / L, the concentration of formaldehyde is 0.056%, and the concentration of Na 2 S 2 O 3 is 2 mg / L.

7. The method for detecting or screening tobacco breeding materials or varieties resistant to bacterial wilt according to claim 6, It is characterized in that In step S2, the silver staining process is as follows: S21. Place the electrophoresis gel in the fixing solution, add xylene cyanol indicator dropwise, and shake for 30 min until the solution becomes colorless; take out the electrophoresis gel and rinse it with ddH 2 O, drain it, and repeat the treatment twice; S22. Place the electrophoresis gel in the staining solution and shake for 30 min; take out the electrophoresis gel and rinse it with ddH 2 O for 5 - 10 s; S23. Place the electrophoresis gel into the developing solution, quickly shake it for 10 s, and pour out the developing solution; pour in the developing solution again and continue developing until the development is complete; after development, take out the electrophoresis gel and put it into the fixing solution to terminate the development. After gently shaking for 5 min, rinse it thoroughly with tap water.

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