A caps molecular marker related to tomato botrytis resistance and application thereof

Abstract

The application provides a CAPS molecular marker related to tomato botrytis resistance, wherein the nucleotide sequence is shown as SEQ ID NO.1; the 125th base of the sequence shown as SEQ ID NO.1 is C in the disease-resistant material, and the 125th base of the sequence shown as SEQ ID NO.1 is T in the disease-susceptible material. Further, a pair of detection primers is designed according to the above-mentioned molecular marker, which can be used for detecting the tomato botrytis resistance, and provides a feasible method for subsequent breeding of high botrytis resistance tomato, and avoids the waste of resources such as manpower and material resources caused by the phenotype distinguishing method.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, specifically relating to tomatoes. Adi3 Application of genes in regulating resistance to gray mold. Background Technology

[0002] tomato( Solanum lycopersicum Tomato, also known as foreign persimmon or tomato, is an annual herbaceous plant belonging to the genus *Solanum* of the Solanaceae family. It is rich in vitamins and lycopene, and has antioxidant properties. It is widely cultivated and is one of the major economic vegetable crops in my country and even the world. my country's tomato industry has developed rapidly. In recent years, with the progress of agricultural technology and the improvement of planting techniques, gray mold disease has seriously affected crop health and yield in the process of tomato cultivation.

[0003] Gray mold is a disease caused by the necrotrophic form of *Botrytis cinerea* (… Botrytis cinerea This fungal disease, which causes widespread damage, infects different parts of tomatoes, including leaves, stems, flowers, and fruits. It generates a large number of gray mycelia and spores, spreads very quickly, and multiplies rapidly under humid conditions, causing serious economic losses to the tomato industry.

[0004] To enhance economic value, breeding tomato varieties with high resistance to gray mold is crucial for cultivation. Currently, seedling resistance is typically determined by observing phenotypic changes after gray mold infection during plant growth. However, this method is wasteful of resources and manpower in the early stages. Therefore, there is an urgent need to develop a method that can rapidly identify tomato resistance to gray mold during the seedling stage. Summary of the Invention

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

[0006] One objective of this 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 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; The specific details of SEQ ID NO.1 are as follows:

[0007] The second objective of this invention is to provide the application of the aforementioned CAPS molecular marker in identifying tomato gray mold resistance.

[0008] The third objective of this invention is to provide a primer set for detecting the aforementioned CAPS molecular marker, the primer set being as follows: CAPS-FW:ATGCTTGGTGTAGTGTAC; CAPS-RV:TGGTGTACTGCAACGTAT.

[0009] The fourth objective of this invention is to provide the application of the above-mentioned primer set in the detection of tomato gray mold resistance.

[0010] The fifth objective of this invention is to provide a kit for detecting tomato resistance to fungal diseases, the kit comprising the primer set as described in claim 3.

[0011] The sixth objective of this invention is to provide a method for detecting tomato fungal resistance, comprising the following steps: S1. Extract DNA from the tomatoes to be tested; S2. PCR amplification of the DNA obtained in S1 using the primer set described in claim 3; S3. The PCR amplification product in S2 was digested with Eco0109I and then electrophoresed to obtain the digested product. S4. If the enzyme digestion product shows three electrophoretic bands, it is an resistant material; if the enzyme digestion product shows only one electrophoretic band, it is a susceptible material.

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

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

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discovered through preliminary research Adi3 Genes play a role in regulating tomato gray mold; furthermore, based on this, CAPS molecular markers were developed for identifying tomato gray mold resistance. The CAPS molecular markers developed in this invention can accurately distinguish the resistance of tomatoes to gray mold. This result provides a feasible method for subsequent breeding of tomatoes with high gray mold resistance, avoiding the waste of manpower and material resources in the early stage caused by relying on phenotypic differentiation methods. Attached Figure Description

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

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

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

[0018] Figure 4This is an electrophoresis diagram of the amplification products of different resistant materials in Example 1 of the present invention after being digested by restriction endonuclease. Detailed Implementation

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

[0020] Example 1 This embodiment provides the development of CAPS molecular markers for identifying resistance to tomato gray mold and the acquisition of primers. The specific steps are as follows: In the preliminary stage, more than 300 tomato samples were repeatedly inoculated with Botrytis cinerea, and the inoculation results were correlated with whole-genome sequencing results. It was found that... Adi3 The gene (nucleotide sequence shown in SEQ ID NO.2) exhibits differences in single nucleotide polymorphisms (SNPs) in the coding region of different resistant and susceptible materials. Furthermore, resistant varieties possess a specific restriction enzyme site. Based on this, a corresponding CAPS marker was developed, which contains an Eco0109I restriction enzyme recognition site (see...). Figure 1 It has the potential to distinguish between disease-resistant and disease-susceptible varieties.

[0021]

[0022] Based on this, specific amplification primers were designed within a 1500bp range upstream and downstream of this SNP. Genomic DNA was extracted from each resistant and susceptible material and then amplified by PCR. Sequencing was performed on the amplification products of resistant materials TS107, TS57, TS29, TS286 (TGRC, U.S. Tomato Genetic Resource Center) and susceptible materials TS100, TS98, TS112, TS9 (TGRC, U.S. Tomato Genetic Resource Center) to verify the markers and design primers.

[0023] 1.1 Genomic DNA Extraction from Anti-Sensitive Materials Weigh 0.2 g of fresh tomato leaves (resistance material) into a 2 mL nuclease-free centrifuge tube, flash-freeze in liquid nitrogen, and grind into powder using a tissue homogenizer. Add 800 µL of preheated CTAB to the tube, incubate at 65°C for 1-2 hours, inverting the tube every 10 minutes. After cooling to room temperature, add 800 µL of chloroform-isoamyl alcohol (24V:1V), gently invert 100 times to mix, let stand at room temperature for 10 minutes, and centrifuge at 12000 rpm for 10 minutes. Transfer approximately 500 µL of the supernatant to a new tube, add an equal volume of pre-cooled isopropanol, mix gently, let stand at 4°C for at least 30 minutes, centrifuge at 12000 rpm for 10 minutes, and discard the supernatant. Add 1 mL of 75% ethanol to wash the precipitate by pipetting, centrifuge at 12000 rpm for 5 minutes, discard the supernatant, and wash twice. After aspirating 20µL of the solution with a pipette and drying it in a fume hood, dissolve the DNA precipitate with 100µL of ddH2O. Once the DNA precipitate passes the test using an ultra-micro UV-Vis spectrophotometer, store it at -20℃ for later use.

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

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

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

[0027] 1.3 Sequencing and enzyme digestion identification Sequence alignment analysis of the PCR amplification products revealed that the TS107, TS57, TS29, and TS286 sequences of the resistant material were identical, as were the TS100, TS98, TS112, and TS9 sequences of the susceptible material. However, a single base difference existed between the sequences of the two types of materials. Based on this, it was determined that the resistant materials TS107, TS57, TS29, and TS286 and the susceptible materials TS100, TS98, TS112, and TS9 share the same SNP difference in the Adi3 gene. The results are shown below. Figure 2 .

[0028] The PCR amplification products described above were cleaved using the Eco0109I restriction endonuclease (see [link to article]). Figure 3 , 4 Electrophoresis results showed that the PCR products of resistant varieties (TS107, TS57, TS29, TS286) exhibited three bands after enzyme digestion, indicating the presence of the EcoO1O9I restriction site; while the PCR products of susceptible varieties (TS100, TS98, TS112, TS9) were not cleaved, showing only one band. This result indicates that the developed CAPS marker can accurately distinguish between resistant and susceptible varieties.

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

[0030] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0031] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a reagent for detecting SNP molecular markers in identifying tomato gray mold resistance, characterized in that, The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1; in disease-resistant tomatoes, the 125th base of the sequence shown in SEQ ID NO.1 is C, and in disease-susceptible tomatoes, the 125th base of the sequence shown in SEQ ID NO.1 is T.

2. The application of the primer set for detecting the SNP molecular marker described in claim 1 in detecting tomato gray mold resistance, characterized in that, The primer set is as follows: CAPS-FW:ATGCTTGGTGTAGTGTAC; CAPS-RV:TGGTGTACTGCAACGTAT; In practical application: First, use the primer set to perform PCR amplification on the DNA of the tomato to be tested; then digest the PCR amplification product with Eco0109I enzyme and electrophoresis to obtain the enzyme digestion product; finally, if the enzyme digestion product shows three electrophoretic bands, it is a disease-resistant tomato; if the enzyme digestion product shows only one electrophoretic band, it is a disease-susceptible tomato.

3. A method for detecting resistance to gray mold in tomatoes, characterized in that, Includes the following steps: S1. Extract DNA from the tomatoes to be tested; S2. Perform PCR amplification on the DNA obtained in S1 using the primer set described in claim 2; S3. The PCR amplification product in S2 was digested with Eco0109I and then electrophoresed to obtain the digested product. S4. If the enzyme digestion product shows three electrophoretic bands, it is a disease-resistant tomato; if the enzyme digestion product shows only one electrophoretic band, it is a disease-susceptible tomato.

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