Plant tissue genome DNA extraction method and kit

Abstract

The invention discloses a plant tissue genome DNA extraction method and a kit. The method for extracting the genome DNA from the tomato plant tissue comprises the following steps: A1) freezing the tomato plant tissue with liquid nitrogen, and grinding to obtain powder; a2) cracking the powder with a TE cracking solution to obtain a cracking solution; a3) extracting genome DNA of tomato plant tissues from the lysate; the TE lysate is prepared from the following components: 10mmol / L of EDTA (Ethylene Diamine Tetraacetic Acid) and 150mmol The method for extracting the tomato tissue DNA is small in sample dosage, simple to operate, short in time consumption, high in efficiency, capable of realizing automatic and high-throughput extraction and safe to use; the extracted DNA is good in integrity, high in stability and good in PCR amplification effect, and can be widely applied to tomato molecular identification, molecular marker-assisted selection of breeding materials and the like.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and relates to a method for extracting genomic DNA from plant tissues and a kit, specifically to a high-throughput method for extracting genomic DNA from tomato plant tissues and a kit. Background Art

[0002] Tomato is an important vegetable crop in China, and is loved by people for its unique flavor, with huge market demand. However, with the continuous expansion of production scale, the incidence of its diseases and pests is also increasing continuously, which has a certain impact on tomato yield and quality. During the tomato production process, the invasion of diseases and pests will affect the yield and quality of tomatoes and cause economic losses to farmers. Simply relying on chemical control not only pollutes the environment, but also affects tomato yield and quality. To promote the sustainable and healthy development of the tomato industry, a more environmentally friendly and sustainable solution of green prevention and control technology should be adopted. And molecular marker technology is playing an increasingly important role in cultivating new tomato disease-resistant varieties and variety protection.

[0003] Plant DNA extraction is a basic technology in the fields of molecular biology, genetics, genomics, etc., and is widely used in fields such as species identification, genetic diversity analysis, gene function research, transgenic detection, and breeding. With the rapid development of biotechnology, the requirements for plant DNA extraction methods are also getting higher and higher, especially in terms of extraction efficiency, purity, operational simplicity, and cost control. As a rapid and simple DNA extraction method, the crude extraction method is particularly suitable for large-scale sample screening or application scenarios with low requirements for DNA purity due to its simple operation and low cost.

[0004] Traditional plant DNA extraction methods usually include steps such as cell disruption, protein removal, and DNA purification. Commonly used methods include the CTAB method, SDS method, phenol-chloroform extraction method, etc. Although these methods can obtain DNA with high purity, their operation steps are cumbersome, time-consuming, and require the use of toxic reagents (such as phenol, chloroform, etc.), which limits their application in some scenarios. In contrast, the crude extraction method can obtain a sufficient amount of DNA in a shorter time by simplifying the steps. Although its purity may not be as high as that of the fine extraction method, it can meet the requirements in many applications.

[0005] The core idea of ​​crude extraction of plant DNA is to quickly lyse cells and release DNA while minimizing the interference of impurities. Commonly used crude extraction methods include boiling method, alkaline lysis method and extraction method based on simple buffer. These methods usually have the following characteristics: (1) Simple operation: Crude extraction usually only requires a few steps and does not require complicated purification steps. It is suitable for use under limited laboratory conditions or time constraints. (2) Fast and efficient: Crude extraction can complete DNA extraction within a few minutes to tens of minutes, which is suitable for high-throughput sample processing or rapid detection. (3) Low cost: Crude extraction usually uses common chemical reagents (such as SDS, NaOH, etc.), does not require expensive purification columns or special equipment, and reduces experimental costs.

[0006] In the field of modern molecular biology, DNA extraction and identification are very important technologies. Therefore, developing an efficient, rapid, low-cost crude DNA extraction method that is applicable to a variety of plant materials has important practical significance and application value. Summary of the invention

[0007] The technical problem to be solved by the present invention is how to extract high-quality genomic DNA from tomato plant tissues in a simple, rapid, efficient, safe, high-throughput and / or low-cost manner, which can be used for molecular marker-assisted selection of tomatoes or identification of tomato varieties. The technical problem to be solved is not limited to the technical subject matter described, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0008] In order to solve the above technical problems, the present invention first provides a method for extracting genomic DNA from tomato plant tissues, the method comprising the following steps:

[0009] A1) freezing tomato plant tissue with liquid nitrogen and then grinding it to obtain powder;

[0010] A2) cracking the powder with TE cracking solution to obtain a cracking solution;

[0011] A3) extracting genomic DNA of tomato plant tissue from the lysate;

[0012] The composition of the TE lysate may be: 10 mmol / L EDTA, 150-200 mmol / L Tris-HCl.

[0013] In the above method, the composition of the TE lysis solution may be: 10mmol / L EDTA, 150mmol / L Tris-HCl.

[0014] In the above method, the lysis conditions in A2) can be: after the powder and the TE lysis solution are fully mixed, they are heated at 90° C. for 15-30 min.

[0015] In the above method, the step A3) may include the following steps: centrifuging the lysate obtained in step A2) at 4000 rpm for 3 min, harvesting the supernatant, freezing the supernatant at -20 °C for 30 - 60 min, then thawing it at room temperature, and centrifuging the thawed supernatant at 4000 rpm for 10 min. The harvested supernatant is the genomic DNA of tomato plant tissue.

[0016] In the above method, the tomato plant tissue may be tomato leaves.

[0017] Further, the method may include the following steps:

[0018] (1) Put the young leaves of tomato into a 2 mL centrifuge tube or a 1.2 mL 96-well deep well plate;

[0019] (2) Add a steel bead, freeze it in liquid nitrogen, grind it with a high-throughput grinder for 1 min until the leaves are fully ground, and then centrifuge;

[0020] (3) Add 300 μL of TE lysate and mix well by shaking;

[0021] (4) Place it in an oven at 90 °C for 15 - 30 min;

[0022] (5) Cool it to room temperature, centrifuge at 4000 rpm for 3 min, aspirate 150 μL of the supernatant, and place it in an environment at -20 °C for 30 - 60 min;

[0023] (6) Let it stand at room temperature until it is completely thawed, centrifuge at 4000 rpm for 10 min, and the harvested supernatant is the extracted genomic DNA (genomic DNA of tomato plant tissue).

[0024] Dilute the supernatant obtained in step (6) by 20 - 30 times, and then PCR amplification or KASP amplification can be carried out.

[0025] Specifically, in an embodiment of the present invention, the method includes the following steps:

[0026] (1) Place about 1 cm 2 young tomato leaves in a well plate, then add 1 steel bead with a diameter of 4 mm, freeze it in liquid nitrogen until there are no obvious bubbles in the liquid nitrogen, and then put it into a high-throughput grinder for grinding until the leaves are fully ground;

[0027] (2) Add 300 μL of TE lysate to the sample powder obtained after grinding;

[0028] (3) After thoroughly mixing the sample powder and the TE lysate, place it in an oven at 90 °C and heat it for 15 - 30 min;

[0029] (4) Remove the orifice plate from the oven. After cooling to room temperature, centrifuge at 4000 rpm for 3 min, aspirate 150 μl of the supernatant, and place it at -20 °C for freezing for 30 - 60 min;

[0030] (5) After the freezing is completed, completely melt the frozen supernatant at room temperature;

[0031] (6) Centrifuge the melted supernatant at 4000 rpm for 10 min, and the harvested supernatant is the extracted genomic DNA (genomic DNA of tomato plant tissue).

[0032] The composition of the TE lysis buffer described herein may be: 10 mmol / L EDTA, 150 - 200 mmol / L Tris-HCl.

[0033] Further, the composition of the TE lysis buffer may be: 10 mmol / L EDTA, 150 mmol / L Tris-HCl.

[0034] The present invention also provides a kit for extracting genomic DNA from tomato plant tissue, and the kit contains the TE lysis buffer described herein.

[0035] Further, the kit may also contain reagents for detecting tomato KASP molecular markers.

[0036] Further, the reagents for detecting tomato KASP molecular markers may include primers for detecting tomato KASP molecular markers and reagents for PCR amplification thereof.

[0037] The present invention also provides the application of the method for extracting genomic DNA or the kit described in any one of the above in any one of the following:

[0038] B1) Application in extracting genomic DNA of tomato plant tissue;

[0039] B2) Application in molecular marker-assisted selection of tomatoes or variety identification of tomatoes;

[0040] B3) Application in detecting tomato genetic markers or detecting tomato genetic markers based on the KASP method.

[0041] The tomato genetic marker may be a molecular marker capable of identifying and tracking specific DNA sequences in the tomato genome.

[0042] Further, the tomato genetic marker may be a tomato KASP molecular marker.

[0043] The tomato KASP molecular markers include, but are not limited to: KASP molecular markers for sterile genes (such as KASP molecular markers for the tomato sterile gene DYT1), KASP molecular markers for disease resistance-related genes (such as KASP molecular markers for the late blight resistance gene Ph-3, KASP molecular markers for the yellow leaf curl virus disease resistance genes Ty-1 and Ty-3, KASP molecular markers for the root-knot nematode resistance gene Mi-1, etc.), KASP molecular markers for tomato variety purity detection, KASP molecular markers related to tomato quality traits, etc.

[0044] The tomato KASP molecular marker described herein can be the KASP molecular marker for the tomato sterile gene DYT1.

[0045] Furthermore, the KASP molecular marker for the tomato sterile gene DYT1 consists of the following two DNA molecules:

[0046] DNA molecule 1: A DNA molecule amplified using the tomato sterile gene DYT1 as a template, with the forward primer 1 (5’-GAAGGTGACCAAGTTCATGCT GCGAAAGGCTTCTTCAATTACG-3’, SEQ ID NO:1) and the downstream primer (5’-CATTGCATTTGTTATGTTTGGGA CC-3’, SEQ ID NO:3).

[0047] DNA molecule 2: A DNA molecule amplified using the tomato sterile gene DYT1 as a template, with the forward primer 2 (5’-GAAGGTCGGAGTCAACGGATT GCGAAAGGCTTCTTCAATTACA-3’, SEQ ID NO:2) and the downstream primer (5’-CATTGCATTTGTTATGTTTGGGA CC-3’, SEQ ID NO:3).

[0048] Among them, the nucleotide sequence of the tomato sterile gene DYT1 is positions 144 - 797 of GenBank Accession No. XM_026029418 (Update Date 24 - OCT - 2024).

[0049] The present invention also provides a method for detecting tomato KASP molecular markers. The method may include the following steps:

[0050] C1) Using the genomic DNA of tomato plant tissues obtained by any of the methods for extracting genomic DNA described herein as a template, performing KASP amplification using the detection primers for tomato KASP molecular markers.

[0051] C2) Determine whether the tomato KASP molecular marker exists based on the amplification product, and further determine the genotype of the tomato.

[0052] Furthermore, the tomato KASP molecular marker can be the KASP molecular marker of the tomato sterility gene DYT1.

[0053] Furthermore, the detection primers of the tomato KASP molecular marker in C1) can be a primer composition. The primer composition can consist of forward primer 1 (SEQ ID NO: 1), forward primer 2 (SEQ ID NO: 2), and a downstream primer (SEQ ID NO: 3).

[0054] When performing KASP amplification with the above primer composition, if the amplification product contains DNA molecule 1, it is determined that the genotype of the tomato to be tested is S (wild); if the amplification product contains DNA molecule 2, it is determined that the genotype of the tomato to be tested is R (sterile); in the case of containing both DNA molecule 1 and DNA molecule 2, it is determined to be heterozygous.

[0055] Compared with the prior art, the TE method for extracting tomato genomic DNA of the present invention has the following beneficial effects:

[0056] (1) The extraction method is simple and time-consuming.

[0057] (2) It can achieve automated and high-throughput extraction, which saves both labor costs and time costs.

[0058] (3) The operation is safe, and toxic and harmful reagents such as chloroform and benzene commonly used in traditional methods are not used, which is both environmentally friendly and can reduce the harm of reagents to the bodies of experimental personnel.

[0059] (4) Compared with other crude extraction methods (such as the alkali boiling method), the DNA integrity is better, and it can maintain the stability of DNA under the conditions of -20°C or 4°C.

[0060] In summary, the present invention provides a simple and efficient method for extracting genomic DNA from tomato plant tissues. Using the method of the present invention, high-quality tomato genomic DNA can be extracted in trace amounts, efficiently, and rapidly. The extracted tomato genomic DNA can meet the requirements of PCR amplification and KASP genotyping detection. When combined with the KASP detection method, tomato materials carrying the target trait genotypes can be quickly screened out, improving the efficiency of tomato breeding. Verified by experiments, the DNA extracted using the method of the present invention for KASP genotyping detection has accurate results, achieving the same genotyping effect as the SDS method. However, compared with the SDS method, the present invention has fewer steps, simpler operation, lower cost, and does not require harmful reagents such as phenol / chloroform and isopentyl alcohol, making it safer. The DNA extraction method of the present invention for DNA extraction from tomato tissues not only realizes high-quality and high-throughput DNA extraction but also greatly improves the efficiency of the entire DNA extraction process, significantly reducing the DNA extraction cost compared with conventional extraction schemes. The entire process of DNA extraction from lysis, purification to product collection can be completed by instruments, with a high degree of automation, and the reagents used for extraction have high safety, which can avoid harm to the human body to the greatest extent. This method has a small sample amount, simple and economical operation, short time, high efficiency, is easy to operate in large batches for extraction, is safe to use, the extracted DNA has good integrity and high stability, and has good effects for PCR amplification. It can be widely applied to tomato molecular identification and molecular marker-assisted selection of breeding materials, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 It is a genotyping detection diagram of tomato DNA extracted by the SDS method.

[0062] Figure 2 It is a genotyping detection diagram of tomato DNA extracted by the TE method.

[0063] Figure 3 It is a partial electrophoresis diagram of tomato DNA marker detection extracted by the TE method. Among them, lane 1 is Marker, and lanes 2 - 12 are PCR products of the molecular marker SCAR1 of 11 tomato materials.

[0064] Figure 4 It is a genotyping detection diagram of tomato DNA stored at 4°C for two weeks extracted by the TE method.

[0065] Figure 5 It is a partial electrophoresis diagram of tomato DNA marker detection stored at 4°C for two weeks extracted by the TE method. Among them, lane 1 is Marker, and lanes 2 - 12 are PCR products of the molecular marker SCAR1 of 11 tomato materials. DETAILED DESCRIPTION OF THE INVENTION

[0066] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0067] In the experimental methods in the following embodiments, unless otherwise specified, they are all conventional methods and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0068] EDTA (purchased from Beijing Solarbio Science & Technology Co., Ltd., product number E8030) in the following embodiments is disodium ethylenediaminetetraacetate dihydrate (Na2EDTA·2H2O), with the molecular formula C 10 H 14 N2O8Na2·2H2O, molecular weight 390.24, purity > 99%, analytical pure.

[0069] NaOH in the following embodiments is sodium hydroxide, with the molecular formula NaOH, analytical pure, purchased from Sinopharm Chemical Reagent Co., Ltd., product number: 1310-73-2.

[0070] HCl in the following embodiments is hydrochloric acid, with the molecular formula HCl, analytical pure, purchased from Yantai Far East Fine Chemical Co., Ltd.

[0071] Tris in the following embodiments is tris(hydroxymethyl)aminomethane, with the molecular formula C4H 11 NO3, molecular weight 121.14, purity > 99%, analytical pure, purchased from Beijing Solarbio Science & Technology Co., Ltd., product number T8060.

[0072] The primer preparation and gene sequencing work involved in the following embodiments were provided and completed by Sangon Biotech (Shanghai) Co., Ltd.

[0073] In the following embodiments, 2×Taq Plus Master MixⅡ for PCR amplification was purchased from Nanjing Novoprotein Scientific Inc. (product number P213-03); 2×PACE Master Mix for KASP amplification was purchased from Chengdu HanChen Guangyi Technology Co., Ltd. (product number 102001000080), and the restriction endonuclease was purchased from New England Biolabs (product number R0149S).

[0074] Example 1. Method for Extracting Genomic DNA from Tomato Plant Tissues

[0075] 1. Extract genomic DNA by the TE method

[0076] Through extensive and in - depth research, the inventors of this application have developed a TE method for extracting genomic DNA from tomato plant tissues. This method is based on Tris - EDTA lysis buffer (TE lysis buffer) to quickly and efficiently extract genomic DNA from plant tissues. In this example, this method is used to extract genomic DNA from tomato leaves, and the specific steps are as follows:

[0077] (1) Place 1 cm 2 young and tender tomato leaves in a 1.2 ml 96 - well deep - well plate. After sampling, add 1 steel bead with a diameter of 4 mm to each well, cover with a silica gel cover plate, and place in liquid nitrogen until there are no obvious bubbles in the liquid nitrogen. Then place the 96 - well plate in a high - throughput grinder for grinding. If the sample is an ordinary plant leaf or seedling, the grinding time is 1 min until the leaf is fully ground;

[0078] (2) Place the ground 96 - well deep - well plate in a centrifuge and centrifuge for 2 min to centrifuge the sample powder on the silica gel cover plate to the inside of the tube. Remove the silica gel cover plate, and add 300 μL of TE lysis buffer to each well using a liquid - separating platform or a multi - channel pipette gun. After covering the silica gel cover plate, shake with the grinder for 3 seconds (check and exclude the situation that the silica gel cover of individual wells is not properly covered);

[0079] (3) After the sample and TE lysis buffer are fully mixed, place it in an oven at 90 °C for 15 - 30 min (to release DNA and inactivate nucleases at the same time);

[0080] (4) Take out the deep - well plate from the oven and let it stand at room temperature for 5 - 10 min (cool to room temperature), then centrifuge at 4000 rpm for 3 min. Use a 96 - channel pipetting platform to aspirate 150 μL of the supernatant from each well and place it at - 20 °C for 30 - 60 min;

[0081] (5) Take out the deep - well plate from the - 20 °C refrigerator and let it stand at room temperature until it completely melts;

[0082] (6) Centrifuge the deep - well plate at 4000 rpm for 10 min to precipitate impurities, and the harvested supernatant is the extracted genomic DNA.

[0083] Aspirate the supernatant and dilute it 20 - 30 times, then PCR amplification can be carried out.

[0084] In the above steps, the composition of the TE lysis buffer is: 10 mmol / L EDTA, 150 mmol / L Tris - HCl, and the pH value is 8.0.

[0085] The preparation method of the TE lysis buffer is as follows:

[0086] 1) Solution preparation

[0087] Preparation of 0.5M EDTA (pH 8.0) solution: Weigh 186.1 g of Na2EDTA·2H2O, add 800 mL of distilled water. During stirring, slowly add NaOH to adjust the pH to 8.0. After the dissolution of Na2EDTA·2H2O, transfer it to a volumetric flask and make up the volume to 1000 mL with distilled water. Autoclave at 121 °C for 20 min.

[0088] Preparation of 1M Tris-HCl (pH 8.0) solution: Weigh 60.6 g of Tris, add 400 mL of distilled water. After the dissolution of Tris, add HCl to adjust the pH value to 8.0, and make up the volume to 500 mL with distilled water. Autoclave at 121 °C for 20 min.

[0089] 2) Preparation of TE lysis buffer

[0090] Measure 150 mL of 1M Tris-HCl (pH 8.0) and 20 mL of 0.5M EDTA (pH 8.0) with a graduated cylinder and transfer them to a volumetric flask. Make up the volume to 1000 mL with distilled water. The final formula of the TE lysis buffer is: 150 mM Tris-HCl and 10 mM EDTA in the final concentration, which is used for DNA lysis.

[0091] 2. Extraction of genomic DNA by SDS method

[0092] Take the SDS method as a control to extract the genomic DNA of tomato leaves. The steps are as follows:

[0093] (1) Put the sample (1 cm 2 young and tender tomato leaves) into a 96-well deep-well plate, add steel beads, freeze in liquid nitrogen for 3 min, and then put the 96-well plate into a high-throughput grinder for grinding.

[0094] (2) After grinding, add 400 uL of SDS lysis buffer to each well, mix well, and place it in an oven at 65 °C for 30 min.

[0095] (3) Add 300 ul of isopropanol to each well into a new 1.2 mL 96-well deep-well plate, pre-cool it in a -20 °C refrigerator to obtain a pre-cooled 96-well deep-well plate containing isopropanol.

[0096] (4) Use a liquid separation platform to add 200 uL of 6M ammonium acetate to each well of the 96-well plate cooled after heating in step (2), and mix the samples well.

[0097] (5) Place the 96-well plate after mixing in step (4) in a 4 °C refrigerator for 15 min for pre-cooling.

[0098] (6) Centrifuge the 96-well plate after pre-cooling in step (5) at 4000 rpm for 15 min.

[0099] (7) After centrifugation in step (6), 300 μL of the supernatant was aspirated from each well and placed into the pre-cooled 96-well deep well plate containing isopropanol described in step (3). The plate was inverted up and down to mix evenly, and then placed in a -20 °C refrigerator for 15 min.

[0100] (8) The 96-well deep well plate frozen in step (7) was centrifuged at 4000 rpm for 10 min. The supernatant was discarded.

[0101] (9) 300 μL of 75% ethanol was added to each well of the 96-well deep well plate in step (8) for washing. After 5 min, it was centrifuged at 4000 rpm for 1 min, and the 75% ethanol was discarded.

[0102] (10) The sample in step (9) was placed in an oven at 65 °C for 15 min to volatilize the alcohol.

[0103] (11) 200 μL of ddH2O was added to each well to dissolve the DNA, and it was placed in a 4 °C refrigerator for standby.

[0104] The composition of the SDS lysis solution is: 0.1 M Tris-HCl (pH 7.5), 0.5 M EDTA (pH = 8.0), 10% SDS, 6 M ammonium acetate solution.

[0105] Example 2. Verification of DNA quality by PCR

[0106] In this example, the quality and application of the genomic DNA extracted from tomato leaves in Example 1 were verified by PCR amplification and KASP detection of tomato fertility markers.

[0107] The verification steps are as follows:

[0108] 1. Using the DNA extracted by the TE method, after 20-fold dilution, it was used as a template for PCR experiments:

[0109] The PCR amplification reaction system is shown in Table 1:

[0110] Table 1. PCR amplification system

[0111] Reagent Added volume (ul) DNA template 2 2×Taq Plus Master MixⅡ 5 <![CDATA[Forward primer (10 μmol·L -1 )]]> 0.2 <![CDATA[Downstream primer (10 μmol·L -1 )]]> 0.2 <![CDATA[ddH2O]]> 2.6 Total volume 10

[0112] The primers in Table 1 were used to amplify the SCAR1 molecular marker of the tomato disease-resistant gene CF9. Among them, the upstream primer is SCAR1F: 5’-CAAACTGGTTGGGATACCTATTTC-3’. The downstream primer is SCAR1R: 5’-ATCCAACAAGATGATTGTGAGAGA-3’.

[0113] The DNA templates in Table 1 were extracted from tomato materials such as AC-1-1 to AC-1-10, AC-2-1 to AC-2-10, AC-3-1 to AC-3-10, AC-4-1 to AC-4-10, AC-5-1 to AC-5-4, etc. according to the TE method (i.e., the method for extracting genomic DNA of the present invention) in Step 1 of Example 1.

[0114] The reaction program for PCR amplification was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s; annealing at 55°C for 40 s; extension at 72°C for 50 s; 32 cycles; extension at 72°C for 10 min; end the reaction, and store the amplification product at 4°C in the refrigerator. The amplification product was identified by 2% agarose gel. When performing gel detection, the electrophoresis buffer was 1×TAE buffer, the voltage was 200 V, the current was 300 mA, and electrophoresis was carried out for 20 min.

[0115] The enzyme digestion reaction system is shown in Table 2.

[0116] Table 2. Enzyme digestion reaction system

[0117] Reagent Added volume (ul) PCR product 5 10×buffer 0.15 TaqI restriction endonuclease 0.5 <![CDATA[ddH2O]]> 4.35 Total volume 10

[0118] Enzyme digestion conditions: The optimal reaction temperature of the restriction endonuclease was 65°C, and the reaction was carried out for 4 h.

[0119] Perform 2% agarose gel electrophoresis to detect the enzyme digestion products. After electrophoresis, place the gel block in a gel imager to view the electrophoresis results. It was found that after the DNA extracted by the TE method was amplified with the upstream primer SCAR1F and the downstream primer SCAR1R, the gel electrophoresis bands were clear, the separation effect was significant, and it was easy to accurately genotype ( Figure 3 ).

[0120] 2. Using the DNA extracted by the TE method and the DNA extracted by the SDS method in Example 1 respectively, after 20-fold dilution, they were used as templates for the KASP reaction: The KASP reaction system was 3 μl (see Table 3), and the DNA was dried in an oven at 65°C (about 20 min).

[0121] Table 3. KASP reaction system

[0122] Reagent Added amount DNA template 2μg (dried) 2×PACE Master Mix 1.5ul <![CDATA[Forward primer 1 (10 μmol·L -1 )]]> 0.048ul <![CDATA[Forward primer 2 (10 μmol·L -1 )]]> 0.048ul <![CDATA[Downstream primer (10 μmol·L -1 )]]> 0.12ul <![CDATA[ddH2O (containing 1 mmol / L MgCl2)]]> 1.284ul Total volume 3ul

[0123] The primers in Table 3 were used to detect tomato genetic markers (KASP molecular markers for tomato sterility gene DYT1). The primers in Table 3 were: forward primer 1 (5’-GAAGGTGACCAAGTTCATGCTGCGAAAGGCTTCTTCAATTACG-3’, SEQ ID NO:1) for amplifying the KASP molecular marker of tomato sterility gene DYT1, forward primer 2 (5’-GAAGGTCGGAGTCAACGGATTGCGAAAGGCTTCTTCAATTACA-3’, SEQ ID NO:2), and the downstream primer (5’-CATTGCATTTGTTATGTTTGGGACC-3’, SEQ ID NO:3).

[0124] The DNA templates in Table 3 were the DNAs extracted from tomato materials (see Table 4) by the TE method and SDS method in Example 1 respectively:

[0125] The KASP reaction procedure was: 94°C: 15 minutes; 94°C: 20 seconds, 65°C (decreasing 0.8°C per cycle): 1 minute, for 10 cycles; 94°C: 20 seconds, 57°C: 1 minute, for 30 cycles. After the reaction ended, the PCR products were put into a microplate reader to read the experimental data, and the experimental data was analyzed using the analysis website (http: / / www.snpway.com:8339 / ).

[0126] Result determination:

[0127] The KASP molecular marker of tomato sterility gene DYT1 consisted of the following two DNA molecules:

[0128] DNA molecule 1: A DNA molecule amplified using tomato sterility gene DYT1 as the template, forward primer 1 (5’-GAAGGTGACCAAGTTCATGCTGCGAAAGGCTTCTTCAATTACG-3’, SEQ ID NO:1), and the downstream primer (5’-CATTGCATTTGTTATGTTTGGGACC-3’, SEQ ID NO:3).

[0129] DNA molecule 2: A DNA molecule amplified using tomato sterility gene DYT1 as the template, forward primer 2 (5’-GAAGGTCGGAGTCAACGGATTGCGAAAGGCTTCTTCAATTACA-3’, SEQ ID NO:2), and the downstream primer (5’-CATTGCATTTGTTATGTTTGGGACC-3’, SEQ ID NO:3).

[0130] Among them, the nucleotide sequence of the tomato sterility gene DYT1 is from positions 144 to 797 of GenBank Accession No. XM_026029418 (Update Date 24 - OCT - 2024).

[0131] If the KASP amplification product contains DNA molecule 1, the genotype of the tomato to be tested is determined as S; if the amplification product contains DNA molecule 2, the genotype of the tomato to be tested is determined as R; and it is determined as heterozygous when both DNA molecule 1 and DNA molecule 2 are present.

[0132] KASP genotyping results ( Figure 1 and Figure 2 ) showed that for the DNA extracted by the TE method, the sample clustering was clear, the population structure was distinct, and the differentiation of each genotype group was significant, making it easy to accurately genotype ( Figure 2 ). Compared with the experimental results of the DNA extracted by the SDS method, it was found that the results were exactly the same (Table 4). The time taken to extract tomato DNA by the TE method of the present invention is about 2 h, while the steps for extracting DNA by the SDS method are cumbersome and it takes about 4 h.

[0133] Table 4. Amplification results of tomato sterility gene DYT1 using DNA extracted by the TE method and the SDS method

[0134]

[0135]

[0136] Note: The tomato materials in Table 4 are experimental material AC, self - propagated in this laboratory; R represents sterile, S represents wild - type, and H represents heterozygous.

[0137] Example 3. Experiment on the storage time of DNA extracted by the TE method of the present invention

[0138] The DNA of the tomato materials extracted by the TE method in Example 2 was stored at 4°C for 2 weeks and then the above - mentioned PCR experiment and KASP genotyping experiment were carried out again. It was found that the gel result bands were clear ( Figure 5 ), and the KASP genotyping was good ( Figure 4 ), and there was no difference in the results. Therefore, the DNA extracted by this method has good integrity and maintains good stability under the conditions of - 20°C or 4°C.

[0139] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made by using conventional techniques known in the art.

Claims

1. A method for extracting genomic DNA from tomato plant tissue, characterized in that: The method comprises the following steps: A1) freezing tomato plant tissue with liquid nitrogen and then grinding it to obtain powder; A2) cracking the powder with TE cracking solution to obtain a cracking solution; A3) extracting genomic DNA of tomato plant tissue from the lysate; The composition of the TE lysis solution is: 10mmol / L EDTA, 150-200mmol / L Tris-HCl.

2. The method according to claim 1, characterized in that The composition of the TE lysis solution is: 10mmol / L EDTA, 150mmol / L Tris-HCl.

3. The method according to claim 1 or 2, characterized in that: The lysis conditions in A2) are: the powder and the TE lysis solution are fully mixed and then heated at 90° C. for 15-30 min.

4. The method according to any one of claims 1 to 3, characterized in that: The A3) comprises the following steps: centrifuging the lysate obtained in step A2) at 4000 rpm for 3 min, harvesting the supernatant, freezing the supernatant at -20°C for 30-60 min, and then thawing it at room temperature, centrifuging the thawed supernatant at 4000 rpm for 10 min, and harvesting the supernatant, which is the genomic DNA of tomato plant tissue.

5. The method according to any one of claims 1 to 4, characterized in that: The tomato plant tissue is tomato leaves.

6. A kit for extracting genomic DNA from tomato plant tissue, characterized in that: The kit comprises the TE lysate according to claim 1 or 2.

7. The kit according to claim 6, characterized in that The kit also contains reagents for detecting tomato KASP molecular markers.

8. Use of the method according to any one of claims 1 to 5 or the kit according to claim 6 or 7 in any of the following: B1) Application in extracting genomic DNA from tomato plant tissues; B2) Application in molecular marker-assisted selection of tomatoes or identification of tomato varieties; B3) Application in detecting tomato genetic markers or detecting tomato genetic markers based on the KASP method.

9. A method for detecting tomato KASP molecular markers, characterized in that: The method comprises the following steps: C1) using the tomato plant tissue genomic DNA obtained by the method according to any one of claims 1 to 5 as a template, and using the detection primers of the tomato KASP molecular marker to amplify KASP; C2) judging whether the tomato KASP molecular marker exists according to the amplified product, and then determining the genotype of the tomato.

10. The method according to claim 9, characterized in that The tomato KASP molecular marker is a KASP molecular marker used to detect the tomato sterile gene DYT1.

Images