CAPS molecular marker related to botrytis cinerea resistance and application thereof

Abstract

The invention provides a CAPS molecular marker related to botrytis cinerea resistance. The nucleotide sequence of the CAPS molecular marker is as shown in SEQ ID NO. 1; the 125th basic group of the sequence shown as SEQ ID NO.1 in the disease-resistant material is C, and the 125th basic group of the sequence shown as SEQ ID NO.1 in the susceptible material is T. Furthermore, a pair of detection primers is designed according to the molecular marker and can be used for detecting the botrytis cinerea resistance, a feasible method is provided for subsequent breeding of the tomato with high botrytis cinerea resistance, and waste of resources such as manpower and material resources in the early stage caused by a phenotype distinguishing method is avoided.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular biology, and specifically relates to tomato Adi3 Application of genes in regulating resistance to gray mold. Background Art

[0002] tomato( Solanum lycopersicum ), also known as tomato, tomato, etc., is an annual herbaceous plant of the genus Tomato in the Solanaceae family. It is rich in vitamins and lycopene, nutritious, and has antioxidant function. It is widely cultivated and is one of the main economic vegetable crops in my country and even in other countries in the world. my country's tomato industry has developed rapidly in recent years with the advancement of agricultural science and technology and the improvement of planting technology. However, in the process of tomato planting, gray mold seriously affects crop health and yield.

[0003] Gray mold is a disease caused by the necrotrophic Botrytis cinerea ( Botrytis cinerea ) is a global fungal disease that infects different parts of tomatoes, such as leaves, stems, flowers and fruits, and produces a large number of gray hyphae and spores. It spreads extremely quickly and reproduces rapidly under humid conditions, causing serious economic losses to the tomato industry.

[0004] To increase economic value, breeding tomato varieties with high resistance to gray mold is crucial for cultivation. Resistance is typically determined by phenotyping after the plants are infected with gray mold during growth. This method significantly wastes resources, labor, and other early-stage input. Therefore, there is an urgent need to develop a method that can rapidly identify tomato resistance to gray mold at the seedling stage. Summary of the Invention

[0005] In view of this, the present invention provides a CAPS molecular marker related to tomato gray mold resistance, and the acquisition of the CAPS molecular marker is based on Adi3 The result shows that there are differences in single nucleotide polymorphisms (SNPs) in the coding regions of genes in different resistant and susceptible materials.

[0006] One of the purposes of the present invention is to provide a CAPS molecular marker associated with resistance to tomato gray mold, wherein the nucleotide sequence of the CAPS molecular marker is shown in SEQ ID NO.1; in disease-resistant materials, the 125th base of the sequence shown in SEQ ID NO.1 is C, and in susceptible materials, the 125th base of the sequence shown in SEQ ID NO.1 is T; SEQ ID NO.1 is as follows:

[0007] A second object of the present invention is to provide an application of the above-mentioned CAPS molecular marker in identifying resistance to tomato gray mold.

[0008] A third object of the present invention is to provide a primer set for detecting the above-mentioned CAPS molecular marker, wherein the primer set is as follows: CAPS-FW:ATGCTTGGTGTAGTGTAC; CAPS-RV:TGGTGTACTGCAACGTAT.

[0009] A fourth object of the present invention is to provide an application of the above primer set in detecting resistance to tomato gray mold.

[0010] A fifth object of the present invention is to provide a kit for detecting resistance to tomato mildew, the kit comprising the primer set according to claim 3.

[0011] A sixth object of the present invention is to provide a method for detecting mildew resistance in tomatoes, comprising the following steps: S1, extracting DNA from the tomato to be tested; S2. Performing PCR amplification on the DNA obtained in S1 using the primer set described in claim 3; S3, digesting the PCR amplification product in S2 with Eco0109I and then performing electrophoresis to obtain the digestion product; S4. If the enzyme cleavage product shows three electrophoretic bands, it is a disease-resistant material; if the enzyme cleavage product shows only one electrophoretic band, it is a disease-susceptible material.

[0012] In some specific embodiments, preferably, it is characterized in that the reaction system for PCR amplification in S2 is: 25µL of KOD, 1.5µL of each of the forward and reverse primers with a concentration of 10nmol / L, 1µL of sample cDNA, and 21µL of ddH2O.

[0013] The PCR reaction program was as follows: 94°C for 2 min, 98°C for 10 s, 56°C for 10 s, 68°C for 1 min, for a total of 45 cycles, 68°C for 5 min, and 4°C for 10 min.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention has been found through preliminary research Adi3 The gene plays a role in regulating tomato gray mold; further, on this basis, a CAPS molecular marker for identifying tomato gray mold resistance was developed. The CAPS molecular marker developed by the present invention can accurately distinguish the resistance of tomatoes to gray mold. This result provides a feasible method for the subsequent breeding of tomatoes with high gray mold resistance, avoiding the waste of early human, material and other resources such as relying on phenotypic differentiation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 TS9 and TS286 in Example 1 of the present invention Adi3 cDNA sequence alignment diagram.

[0016] Figure 2 This is a comparison diagram of the nucleic acid sequences of different anti-susceptibility materials in Example 1 of the present invention.

[0017] Figure 3 For different anti-sensing materials in Example 1 of the present invention Adi3 Gene amplification electrophoresis.

[0018] Figure 4This is an electrophoresis diagram of the amplified products of different anti-susceptibility materials in Example 1 of the present invention after being digested by restriction endonucleases. DETAILED DESCRIPTION

[0019] The present invention will be further described in detail below in conjunction with specific examples so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0020] Example 1 This example provides the development of CAPS molecular markers related to identification of tomato gray mold resistance and the acquisition of primers. The specific steps are as follows: In the early stage, more than 300 tomato materials were inoculated with gray mold multiple times, and the inoculation results were correlated with the whole genome sequencing results. Adi3 The gene (nucleotide sequence shown in SEQ ID NO.2) has single nucleotide polymorphism (SNP) differences in the coding region of different resistant and susceptible materials. Furthermore, the resistant varieties have a specific restriction enzyme site. Based on this, a corresponding CAPS marker was developed. The CAPS marker contains the Eco0109I restriction enzyme recognition site (see Figure 1 ), with the potential to differentiate between resistant and susceptible varieties.

[0021]

[0022] Based on this finding, specific primers were designed within the 1500 bp region upstream and downstream of the SNP. Genomic DNA was extracted from each resistant and susceptible accession and then amplified by PCR. The amplified products from the resistant accessions TS107, TS57, TS29, and TS286 (TGRC, Tomato Genetic Resource Center, USA) and the susceptible accessions TS100, TS98, TS112, and TS9 (TGRC, Tomato Genetic Resource Center, USA) were sequenced to verify the marker and design primers.

[0023] 1.1 Extraction of genomic DNA from resistant and susceptible materials Weigh 0.2 g of fresh tomato leaves from the susceptible strain into a 2 mL nuclease-free centrifuge tube, snap-freeze in liquid nitrogen, and grind into a powder using a tissue grinder. Pipette 800 µL of preheated CTAB into the tube and incubate in a 65°C water bath for 1-2 hours, inverting every 10 minutes. After cooling to room temperature, add 800 µL of chloroform-isoamyl alcohol (24V:1V) and gently invert 100 times to mix thoroughly. Allow to stand at room temperature for 10 minutes and centrifuge at 12,000 rpm for 10 minutes. Transfer approximately 500 µL of the supernatant to a new tube and gently mix with an equal volume of pre-chilled isopropanol. Allow to stand at 4°C for at least 30 minutes and centrifuge at 12,000 rpm for 10 minutes. Discard the supernatant. Wash the pellet by pipetting with 1 mL of 75% ethanol, centrifuge at 12,000 rpm for 5 minutes, and discard the supernatant. Wash twice. Aspirate with a 20µL pipette and blow dry in a fume hood. Dissolve the DNA precipitate with 100µL ddH2O. After passing the ultraviolet-visible spectrophotometer test, store at -20°C for future use.

[0024] 1.2 PCR amplification The designed primers were used to amplify the genomic DNA of the tomato samples. The PCR amplification reaction system was: 25µL KOD, 1.5µL each of forward and reverse primers (10nmol / L), 1µL sample cDNA, and 21µL ddH2O.

[0025] The reaction program was as follows: 94°C for 2 min, 98°C for 10 s, 56°C for 10 s, 68°C for 1 min, for a total of 45 cycles, 68°C for 5 min, and 4°C for 10 min.

[0026] The amplification primers are as follows: Adi3 (CAPS-684bp)-FW:ATGCTTGGTGTAGTGTAC; Adi3 (CAPS-684bp)-RV:TGGTGTACTGCAACGTAT.

[0027] 1.3 Sequencing and enzyme digestion identification Sequence comparison and analysis of the above PCR amplification products showed that the sequences of TS107, TS57, TS29, and TS286 of the disease-resistant materials were identical, and the sequences of TS100, TS98, TS112, and TS9 of the disease-susceptible materials were also completely identical, but there was a single base difference between the sequences of the two types of materials. Based on this, it was determined that the disease-resistant materials TS107, TS57, TS29, and TS286 and the disease-susceptible materials TS100, TS98, TS112, and TS9 had the same SNP difference on the Adi3 gene. The results are shown in the figure. Figure 2 .

[0028] The PCR amplification products were cleaved using Eco0109I restriction endonuclease (see Figure 3 、 4 Electrophoresis results showed that the PCR products of the resistant varieties (TS107, TS57, TS29, and TS286) displayed three bands after enzyme digestion, indicating that they contained the EcoO1O9I restriction site. However, the PCR products of the susceptible varieties (TS100, TS98, TS112, and TS9) were not digested and displayed only a single band. This result demonstrates that the developed CAPS marker can accurately distinguish resistant from susceptible varieties.

[0029] In summary, the CAPS polymorphic marker can well distinguish eight different types of tomato varieties, verifying the effectiveness and feasibility of the developed CAPS marker in the identification of tomato disease resistance.

[0030] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0031] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A CAPS molecular marker associated with tomato gray mold resistance, characterized in that: The CAPS molecular marker nucleotide sequence is shown in SEQ ID NO.1; in the disease-resistant material, the 125th base of the sequence shown in SEQ ID NO.1 is C, and in the disease-susceptible material, the 125th base of the sequence shown in SEQ ID NO.1 is T.

2. Use of the CAPS molecular marker according to claim 1 in identifying resistance to tomato gray mold.

3. A primer set for detecting the CAPS molecular marker according to claim 1, characterized in that: The primer set is specifically as follows: CAPS-FW:ATGCTTGGTGTAGTGTAC; CAPS-RV:TGGTGTACTGCAACGTAT.

4. Use of the primer set according to claim 3 in detecting resistance to tomato gray mold.

5. A kit for detecting mildew resistance in tomatoes, characterized in that: The kit comprises the primer set according to claim 3.

6. A method for detecting mildew resistance in tomatoes, characterized in that: The following steps are involved: S1, extracting DNA from the tomato to be tested; S2. Performing PCR amplification on the DNA obtained in S1 using the primer set described in claim 3; S3, digesting the PCR amplification product in S2 with Eco0109I and then performing electrophoresis to obtain the digestion product; S4. If the enzyme cleavage product shows three electrophoretic bands, it is a disease-resistant material; if the enzyme cleavage product shows only one electrophoretic band, it is a disease-susceptible material.

7. The method according to claim 6, characterized in that The reaction system for PCR amplification in S2 was as follows: KOD 25 μL, 1.5 μL each of forward and reverse primers at a concentration of 10 nmol / L, 1 μL of sample cDNA, and 21 μL of ddH O . The PCR reaction program was as follows: 94°C for 2 min, 98°C for 10 s, 56°C for 10 s, 68°C for 1 min, for a total of 45 cycles, 68°C for 5 min, and 4°C for 10 min.

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