CN108950049B - Tomato bacterial spot disease closely-linked SNP (single nucleotide polymorphism) marker and application thereof - Google Patents
Tomato bacterial spot disease closely-linked SNP (single nucleotide polymorphism) marker and application thereof Download PDFInfo
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Abstract
The invention discloses a tomato bacterial spot disease close linkage SNP marker and application thereof. The invention provides 1 SNP marker located in tomato bacterial spot disease resistance gene Pto, and the genotype of the SNP site is detected by an AS-PCR method, so AS to judge the resistance of the material to the bacterial spot disease. SNP663 is located at 282-base of Pto gene, and can be used for molecular marker-assisted selection of tomato bacterial spot disease resistance. The SNP marker is located in the gene, the linkage is good, and the result is accurate.
Description
Technical Field
The invention relates to a tomato bacterial spot disease closely-linked SNP marker and application thereof.
Background
The tomato bacterial spot disease (bacterial spot of tomato) is also called tomato bacterial spot and rash disease, the pathogenic bacteria of the tomato bacterial spot disease is Pseudomonas syringae tomato pathogenic variety (Pseudomonas syringae. tomato), can infect a plurality of organs such as leaves, petioles, buds and fruits, and is one of important diseases damaging tomato production. The disease can be prevented and controlled by methods of scientific crop rotation, efficient field management, chemical agents and the like, but the methods have the problems of high requirements on planting personnel, high cost, pesticide residue and the like. Therefore, breeding disease-resistant varieties is the most economic and effective measure for preventing and treating bacterial spot diseases of tomatoes. In breeding, the disease resistance of the material needs to be judged by a method of seedling stage inoculation identification or adult stage inoculation identification on the material, and the genotype of the next generation in a family is usually presumed by using the phenotype of the next generation, which is time-consuming and labor-consuming; and due to the influence of environmental conditions, the identification result is often inaccurate, so that false positive is caused, and great difficulty is caused to disease-resistant breeding.
Molecular Marker-assisted Selection (MAS) is a novel breeding mode combining modern molecular biology and traditional genetic breeding, and can select target plants from DNA level at any stage of plant development by using molecular markers. The development of molecular marker technology goes through three important stages, namely, first generation molecular markers represented by RAPD; a second generation molecular marker represented by SSR, and a third generation molecular marker represented by SNP. RAPD technology is a technology based on PCR (polymerase chain reaction) developed in the last 90 th century, and can perform polymorphism analysis on unknown sequences. SSR (Simple Sequence Repeat, SSR) markers are developed based on DNA Sequence microsatellite Sequence polymorphism, have the characteristics of stable amplification, rich polymorphism, co-dominant inheritance and the like, and are widely applied to gene localization and variety identification; SNP markers have been developed based on Single Nucleotide Polymorphisms (SNPs), mainly DNA sequence polymorphisms caused by single nucleotide variations at the genomic level. The SNP markers have more quantity and wider distribution, are the most potential molecular markers for development at present, and play more and more roles in the research fields of crop genetic breeding, functional genomics and the like by virtue of the advantages of bimorphity, high density, high representativeness, high genetic stability and the like.
Disclosure of Invention
The invention aims to provide a tomato bacterial spot disease closely-linked SNP marker and application thereof.
The invention firstly protects the specific primer combination; consists of a primer pair I and a primer pair II;
the primer pair I consists of a primer SNP663-AF and a primer SNP 663-R;
the primer pair II consists of a primer SNP663-GF and a primer SNP 663-R;
the primer SNP663-AF is (a1) or (a2) as follows:
(a1) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(a2) DNA molecule which is obtained by substituting one or more nucleotides in the sequence 2 and has the same function as the sequence 2;
the primer SNP663-GF is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown in sequence 3 of the sequence table;
(a4) DNA molecules which are obtained by substituting the sequence 3 by one or more nucleotides and have the same functions as the sequence 3;
the primer SNP663-R is as follows (a5) or (a 6):
(a3) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;
(a4) and (b) the DNA molecule which is obtained by substituting the sequence 4 by one or more nucleotides and has the same function as the sequence 4.
The invention also protects the application of the specific primer combination, which is at least one of the following (b1) - (b 8):
(b1) identifying or assisting in identifying tomato bacterial spot disease resistance;
(b2) screening or auxiliary screening of bacterial spot disease tomato;
(b3) screening or auxiliary screening of disease-resistant tomatoes with bacterial spot diseases;
(b4) screening or auxiliary screening of bacterial spot disease resistant tomatoes;
(b5) preparing a kit for identifying or assisting in identifying the resistance to bacterial spot disease of tomato;
(b6) preparing a kit for screening or auxiliary screening of the bacterial spot disease tomato;
(b7) preparing a kit for screening or assisting in screening the tomato with bacterial spot disease resistance;
(b8) preparing a kit for screening or assisting in screening the bacterial spot disease resistant tomatoes.
The invention also protects a kit containing the specific primer combination; the application of the kit is at least one of the following (c1) - (c 4):
(c1) identifying or assisting in identifying tomato bacterial spot disease resistance;
(c2) screening or auxiliary screening of bacterial spot disease tomato;
(c3) screening or auxiliary screening of disease-resistant tomatoes with bacterial spot diseases;
(c4) screening or auxiliary screening of tomato with bacterial spot disease resistance.
The invention also provides a preparation method of the kit, which comprises the step of independently packaging each primer in the specific primer combination.
The invention also protects a method for identifying or assisting in identifying resistance to bacterial spot disease in tomato (method A), comprising the following steps: the method comprises the steps of taking genomic DNA of tomatoes to be detected as templates, respectively carrying out PCR amplification on the primer pair I and the primer pair II, obtaining specific strips if the primer pair I is used for amplifying the templates, obtaining the specific strips if the primer pair II is used for amplifying the templates, obtaining the bacterial spot disease-resistant tomatoes if the primers are used for amplifying the templates, obtaining the specific strips if the primers are used for amplifying the templates and obtaining the bacterial spot disease-resistant tomatoes if the primers are used for amplifying the templates.
The size of the specific band is 303 bp.
The invention also protects a method for identifying or assisting in identifying resistance to bacterial spot disease in tomato (method B), comprising the steps of:
detecting whether the genotype based on the SNP663 locus in the genomic DNA of the tomato to be detected is an AA genotype, a GG genotype or an AG genotype;
the AA genotype tomato is bacterial spot disease tomato;
the GG genotype tomato is a disease-resistant tomato with bacterial spot disease;
the AG genotype tomato is bacterial spot disease resistant tomato;
the SNP663 site is the 60 th nucleotide from the 5' end of the sequence 1 in the sequence table.
The invention also provides a method for screening or assisting in screening the tomato with the bacterial spot disease (method C), which comprises the following steps:
(d1) identifying tomato bacterial spot disease resistance according to method a or method b;
(d2) screening or aiding in the screening of bacterial spot disease tomatoes based on the results of step (d 1).
The invention also provides a method for screening or assisting in screening the tomato with bacterial spot disease resistance (method D), which comprises the following steps:
(d1) identifying tomato bacterial spot disease resistance according to method a or method b;
(d2) screening or assisting in screening tomato resistant to bacterial spot disease based on the results of step (d 1).
The invention also provides a method for screening or assisting in screening the disease-resistant tomatoes with the bacterial spot diseases (method E), which comprises the following steps:
(d1) identifying tomato bacterial spot disease resistance according to method a or method b;
(d2) screening or aiding in the screening of bacterial spot disease resistant tomatoes based on the results of step (d 1).
The invention also protects the application of the specific primer combination, or the SNP663 locus, or the method in tomato breeding.
The tomato with any bacterial spot disease can be specifically tomato with disease index of more than 40.1 through disease resistance detection. The tomato with any bacterial spot disease resistance can be specifically tomato with disease index of 0.1-20.0 through disease resistance detection. The tomato with bacterial spot disease resistance can be tomato with disease index of 20.1-40.0 by disease resistance detection. The disease resistance detection method comprises the following steps: and (3) seedling the tomato material to be detected by using a nutrition pot, transplanting when the seedling grows to 2-3 true leaves, and transplanting into the nutrition pot, wherein 9 plants are planted in each material, and three plants are one repeat and are three repeats in total. And (4) placing the greenhouse in a greenhouse at 15-35 ℃ for management, and strictly preventing plant diseases and insect pests. The length of the variety to be detected is 7-8Spray inoculation is carried out when the leaves are true. Before inoculation, the strain PST27 (purchased from China microbial strains inquiry network, http:// www.biobw.org/, strain platform number: bio-80067) was transferred to KB medium, cultured at 28 ℃ for two days, and then prepared into 1 × 10 with sterile water82L of cfu/mL bacterial suspension was used for inoculation. The inoculation is based on spraying the leaves with water. After inoculation, the materials are placed in a moisturizing rack with the average temperature of 28 ℃, normal management is carried out after the materials are moisturized for 48 hours, grading investigation is carried out on the sixth day after inoculation, and the disease index is calculated. The disease grading criteria are as follows: level 0: the plant has no disease spots; level 1: the diseased leaves of the plants are less than 25 percent; and 2, stage: the diseased leaves of the plants are more than 25 percent and less than 50 percent; and 3, level: the diseased leaves of the plants are more than 50 percent and less than 75 percent; 4, level: the diseased leaves of the plants exceed 75 percent. Disease index calculation formula: the disease index ∑ (number of diseased plants at each stage × the disease grade value)/(total number of investigated plants × highest grade value) × 100%.
Any one of the tomatoes to be tested may specifically be the tomatoes shown in table 1.
The invention develops 1 SNP marker located in tomato bacterial spot disease resistance gene Pto, and detects the genotype of SNP locus by AS-PCR method, so AS to judge the resistance of the material to bacterial spot disease. SNP663 is located at 282-base of Pto gene, and can be used for molecular marker-assisted selection of tomato bacterial spot disease resistance. The SNP marker is located in the gene, the linkage is good, and the result is accurate.
Drawings
FIG. 1 shows the results of the detection in step 2 of example 3.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 molecular marker development for the assisted identification of bacterial Spot disease resistance in tomato
A large number of sequence analyses, alignments and preliminary experiments are carried out, and a molecular marker SNP663 for assisting in identifying the resistance of tomato bacterial spot disease is found and is positioned at 282-bit base of Pto gene, and the polymorphic site of SNP663 is positioned at 60-bit nucleotide of sequence 1. Designing a primer aiming at the SNP663 polymorphic site, which is shown as follows:
SNP663-AF (sequence 2 of sequence Listing): 5'-CCTATCAGTGAAACCAGATGTGtA-3', respectively;
SNP663-GF (sequence 3 of sequence Listing): 5'-CCTATCAGTGAAACCAGATGTGtG-3', respectively;
SNP663-R (sequence 4 of sequence Listing): 5'-GGGAACCAACTATTCCAAGCCAACAA-3' are provided.
Example 2 detection method of tomato Pto genotype based on SNP663
1. And extracting the genomic DNA of the tomato to be detected.
2. And (2) performing PCR amplification by using the genome DNA obtained in the step (1) as a template and adopting a primer pair consisting of the primer SNP663-AF and the primer SNP663-R prepared in the example 1 and a primer pair consisting of the primer SNP663-GF and the primer SNP663-R to obtain a PCR amplification product.
Reaction system of PCR amplification: ES Taq Master mix (CW 0690, Congwort Biotechnology Co., Ltd.) 5. mu.l, primer SNP663-AF/SNP663-GF 0.5. mu.l, primer SNP 663-R0.5. mu.l, template DNA 3. mu.l, sterile H2O 1μl。
Each primer is added into a reaction system in a primer solution form, and the initial concentration of each primer in the primer solution is 10 mu M; the template DNA was added to the reaction system in the form of a DNA solution, and the initial concentration of the template DNA in the DNA solution was 50 ng/. mu.L.
Reaction procedure for PCR amplification: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; keeping the temperature at 72 ℃ for 5min, and keeping the temperature at 16 ℃ for forever.
3. And (3) carrying out agarose gel electrophoresis on the PCR amplification product obtained in the step (2). When loading, loading the SNP663-AF/SNP663-R amplification product, and after three minutes, loading the SNP663-GF/SNP663-R amplification product to the same loading hole.
4. The electrophoresis results of step 3 were analyzed as follows:
when the electrophoresis result shows that only the SNP663-AF/SNP663-R amplification product has a strip, the genotype of the material to be detected is A/A homozygous, and the genome of the tomato material to be detected does not contain the gene Pto for resisting bacterial spot disease; when the electrophoresis result shows that only the SNP663-GF/SNP663-R amplification product has a strip, the genotype of the material to be detected is G/G homozygous, and the genome of the tomato material to be detected contains an anti-bacterial spot disease gene Pto; when the electrophoresis result shows that the amplification products of the SNP663-AF/SNP663-R and the SNP663-GF/SNP663-R have strips, the genotype of the material to be detected is A/G heterozygous, and the genome of the tomato material to be detected contains the gene Pto (heterozygous type) for resisting bacterial spot disease.
The size of the band is 303 bp.
The sequencing confirms that the detection result of the SNP663 is consistent with the sequencing result, and the method is proved to be correct.
Example 3 detection of resistance to tomato bacterial Spot disease
Tomato material to be tested: see table 1 for details.
1. Phenotypic identification of tomato resistance to bacterial spot disease
And (3) seedling the tomato materials to be detected in the table 1 by using a nutrition pot, transplanting when the seedlings grow to 2-3 true leaves, transplanting into the nutrition pot, and planting 9 plants in each material, wherein three plants are one repeat and three repeats are arranged. And (4) placing the greenhouse in a greenhouse at 15-35 ℃ for management, and strictly preventing plant diseases and insect pests. And carrying out spray inoculation when the variety to be detected grows to 7-8 true leaves. Before inoculation, the strain PST27 (purchased from China microbial strains inquiry network, http:// www.biobw.org/, strain platform number: bio-80067) was transferred to KB medium, cultured at 28 ℃ for two days, and then prepared into 1 × 10 with sterile water82L of cfu/mL bacterial suspension was used for inoculation. The inoculation is based on spraying the leaves with water. After inoculation, the materials are placed in a moisturizing rack with the average temperature of 28 ℃, normal management is carried out after the materials are moisturized for 48 hours, grading investigation is carried out on the sixth day after inoculation, and the disease index is calculated.
The disease grading criteria are as follows:
level 0: the plant has no disease spots;
level 1: the diseased leaves of the plants are less than 25 percent;
and 2, stage: the diseased leaves of the plants are more than 25 percent and less than 50 percent;
and 3, level: the diseased leaves of the plants are more than 50 percent and less than 75 percent;
4, level: the diseased leaves of the plants exceed 75 percent.
Reactive type division standard:
immunization: the disease index is 0;
disease resistance: disease index is 0.1-20.0;
disease resistance: disease index is 20.1-40.0;
the infection: the disease index is above 40.1.
Disease index calculation formula: the disease index ∑ (number of diseased plants at each stage × the disease grade value)/(total number of investigated plants × highest grade value) × 100%. The statistical results are shown in Table 1.
2. The method in example 2 is adopted to detect the genotype of each tomato material to be detected based on the SNP663, and the result is shown in Table 1 and figure 1. In FIG. 1, the SNP663-AF/SNP663-R combination band corresponds to Marker1(M1), and the SNP663-GF/SNP663-R combination band corresponds to Marker2 (M2). In FIG. 1, the numbers 1-30 sequentially correspond to the materials of the numbers 1801 and 1830 in Table 1.
3. Analyzing the relevance of the SNP663 locus of the material to be detected and the bacterial spot disease resistance of tomato
Comparing the result of the phenotype identification with the result of the SNP663 marking identification, wherein the genotype of the SNP663 locus is A/A material, the phenotype identification result is all bacterial-sensing spot disease, the genotype of the SNP663 is G/G, the phenotype is all bacterial-resistant spot disease, the genotype of the SNP663 is A/G, and the phenotype is all bacterial-resistant spot disease.
The results show that the molecular marker and the specific primer pair thereof provided by the invention can be used for identifying the resistance of tomato bacterial spot disease.
TABLE 1 tomato variety and test results
In the disease-resistant gene list, R represents that the disease-resistant gene is contained, S represents that the disease-resistant gene is not contained, and H represents a heterozygous genotype.
Sequence listing
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Claims (10)
1. Specific primer combination; consists of a primer pair I and a primer pair II;
the primer pair I consists of a primer SNP663-AF and a primer SNP 663-R;
the primer pair II consists of a primer SNP663-GF and a primer SNP 663-R;
the primer SNP663-AF is a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
the primer SNP663-GF is a single-stranded DNA molecule shown in a sequence 3 of a sequence table;
the primer SNP663-R is a single-stranded DNA molecule shown in a sequence 4 of a sequence table.
2. The use of the specific primer combination of claim 1, which is at least one of the following (b1) - (b 8):
(b1) identifying or assisting in identifying tomato bacterial spot disease resistance;
(b2) screening or auxiliary screening of bacterial spot disease tomato;
(b3) screening or auxiliary screening of disease-resistant tomatoes with bacterial spot diseases;
(b4) screening or auxiliary screening of bacterial spot disease resistant tomatoes;
(b5) preparing a kit for identifying or assisting in identifying the resistance to bacterial spot disease of tomato;
(b6) preparing a kit for screening or auxiliary screening of the bacterial spot disease tomato;
(b7) preparing a kit for screening or assisting in screening the tomato with bacterial spot disease resistance;
(b8) preparing a kit for screening or assisting in screening the bacterial spot disease resistant tomatoes.
3. A kit comprising the specific primer combination of claim 1; the application of the kit is at least one of the following (c1) - (c 4):
(c1) identifying or assisting in identifying tomato bacterial spot disease resistance;
(c2) screening or auxiliary screening of bacterial spot disease tomato;
(c3) screening or auxiliary screening of disease-resistant tomatoes with bacterial spot diseases;
(c4) screening or auxiliary screening of tomato with bacterial spot disease resistance.
4. A method for preparing the kit according to claim 3, comprising the step of packaging each primer in the specific primer combination according to claim 1 separately.
5. A method for identifying or aiding in the identification of resistance to bacterial leaf spot disease in tomato comprising the steps of: the method comprises the steps of taking genomic DNA of tomatoes to be detected as templates, respectively carrying out PCR amplification by using a primer pair I and a primer pair II as described in claim 1, obtaining a specific strip if the template is amplified by using the primer pair I, obtaining a specific strip if the template is amplified by using the primer pair II, obtaining the bacterial spot disease-resistant tomato if the template is amplified by using the primer pair II, obtaining the specific strip if the template is amplified by using the primer pair I, obtaining the bacterial spot disease-resistant tomato if the tomato is detected, obtaining the specific strip if the template is amplified by using the primer pair I, obtaining the specific strip if the template is amplified by using the primer pair II, and obtaining the bacterial spot disease-resistant tomato if the tomato is detected.
6. A method for identifying or aiding in the identification of resistance to bacterial leaf spot disease in tomato comprising the steps of: detecting whether the genotype based on the SNP663 locus in the genomic DNA of the tomato to be detected is an AA genotype, a GG genotype or an AG genotype;
the AA genotype tomato is bacterial spot disease tomato;
the GG genotype tomato is a disease-resistant tomato with bacterial spot disease;
the AG genotype tomato is bacterial spot disease resistant tomato;
the SNP663 site is the 60 th nucleotide from the 5' end of the sequence 1 in the sequence table.
7. A method for screening or aiding in screening for bacterial spot disease tomato comprising the steps of:
(d1) identifying resistance to bacterial leaf spot disease in tomato according to the method of claim 5 or 6;
(d2) screening or aiding in the screening of bacterial spot disease tomatoes based on the results of step (d 1).
8. A method for screening or auxiliary screening of tomato with bacterial spot disease resistance comprises the following steps:
(d1) identifying resistance to bacterial leaf spot disease in tomato according to the method of claim 5 or 6;
(d2) screening or assisting in screening tomato resistant to bacterial spot disease based on the results of step (d 1).
9. A method for screening or assisting in screening for bacterial spot disease resistant tomatoes comprising the steps of:
(d1) identifying resistance to bacterial leaf spot disease in tomato according to the method of claim 5 or 6;
(d2) screening or aiding in the screening of bacterial spot disease resistant tomatoes based on the results of step (d 1).
10. Use of the specific primer combination of claim 1 or the SNP663 locus of claim 6 or the method of any one of claims 5 to 9 in tomato breeding.
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| "Determining resistance to Pseudomonas syringae in tomato,a comparison of different molecular markers";Ilaria Orsi et al.;《Mol Breeding》;20111221;第30卷;第967-974页 * |
| "Marker-assisted Selection for Combining Resistance to Bacterial Spot and Bacterial Spot and Bacterial Speck in Tomato";Wencai Yang et al.;《J.AMER.SOC.HORT.SCI.》;20051231;第130卷(第5期);第716-721页 * |
| "Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato";SUN Wan-yu et al.;《Agricultural Sciences in China》;20110630;第10卷(第6期);第827-837页 * |
| "Use of modern tomato breeding germplasm for deciphering the genetic control of agronomical traits by Genome Wide Association study";Guillaume Bauchet et al.;《Theor Appl Genet》;20170210;第130卷;第875-889页 * |
| "加工番茄 Ve-1、 I-2、 Mi-1 和 Pto 基因关联变异";刘磊等;《园艺学报》;20180630;第45卷(第6期);第1089-1100页 * |
| "番茄抗病基因 Tm-2、 Pto、 Sw-5 和 Ve1 的 SNP标记检测";苏晓梅等;《园艺学报》;20141231;第41卷(第10期);第2012-2020页 * |
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| Publication number | Publication date |
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| CN108950049A (en) | 2018-12-07 |
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