A dCAPS marker primer for identifying tomato stigma color and its identification method

Abstract

The present invention provides a dCAPS marker primer for identifying the color of tomato stigma, comprising a dCAPS upstream primer and a dCAPS downstream primer, and the nucleotide sequence is shown in SEQ ID NO: 1-2. A method for identifying the color of tomato stigma using the marker primer is also provided, wherein the dCAPS upstream primer and the dCAPS downstream primer are used to perform a PCR amplification reaction on the genomic DNA of the extracted sample to be tested, to obtain a PCR product, and then the PCR product is digested by endonuclease BseXI after 1.5% agarose gel electrophoresis detection, and the digested product is subjected to 12% polyacrylamide gel electrophoresis, and the digestion detection result is observed by silver staining and development; if the digestion detection result is a single band of 105bp, it is a yellow stigma tomato plant; if the digestion detection result is two bands of 68bp and 37bp, it is a homozygous green stigma tomato plant; if the digestion detection result is three bands of 105bp, 68bp and 37bp, it is a heterozygous green stigma tomato plant. The present invention provides a new basis for the genetic evaluation of tomato appearance morphology germplasm resources.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and particularly relates to a dCAPS marker primer for identifying tomato stigma color and an identification method thereof. Background Art

[0002] Tomato is the most widely cultivated and consumed vegetable crop in the world. It has obvious hybrid vigor and its stigma is generally green. The stigma is an important part of the pistil. It receives pollen and is of great significance to the pollination and fertilization of tomatoes and the development of seeds and fruits. Rapid screening of stigma color can greatly save manpower and material resources in the hybrid breeding process. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a dCAPS marker primer for identifying tomato stigma color and an identification method thereof in view of the deficiencies of the above-mentioned prior art. The dCAPS marker primer is used as an excellent marker trait in tomato breeding and is used for the selection and breeding of hybrid progeny. The application of this trait can greatly save manpower and material resources in the hybrid breeding process.

[0004] To solve the above technical problems, the technical solution adopted by the present invention is: a dCAPS marker primer for identifying the stigma color of tomato, the dCAPS marker primer comprises a dCAPS upstream primer and a dCAPS downstream primer, the nucleotide sequence of the dCAPS upstream primer is shown in SEQ ID NO:1; the nucleotide sequence of the dCAPS downstream primer is shown in SEQ ID NO:2.

[0005] The present invention also provides a method for identifying the color of a tomato stigma using the dCAPS marker primers for identifying the color of a tomato stigma, the method comprising:

[0006] S1, extracting genomic DNA of the tomato to be tested;

[0007] S2, using dCAPS upstream primer and dCAPS downstream primer to perform PCR amplification reaction on the genomic DNA of the sample to be tested extracted in S1 to obtain a PCR product;

[0008] The PCR amplification system is as follows: 1 µL of genomic DNA of the tomato sample to be tested, 0.5 µL of dCAPS upstream primer, 0.5 µL of dCAPS downstream primer, 5 µL of 2×Taq Master Mix, ddH 2 Make up to 10 µL with O;

[0009] The reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 45 s, annealing at 55°C for 30 s; extension at 72°C for 30 s, 30 cycles; extension at 72°C for 7 min; storage at 4°C;

[0010] S3, take 3 μL of the PCR product obtained in S2 and perform 1.5% agarose gel electrophoresis detection, then use BseXI endonuclease to digest the PCR product obtained in S2 at 65°C for 30 minutes to obtain the digestion product;

[0011] The enzyme digestion reaction system is: PCR product 7µL, 10×BseXI buffer 1µL, BseXI enzyme 0.2 µL, ddH 2 O to make up to 10 µL;

[0012] S4. The enzyme digestion product obtained in S3 is subjected to 12% polyacrylamide gel electrophoresis at a power of 20 W for 1.5 h. After the electrophoresis, silver staining and development are performed to observe the enzyme digestion detection results; Result judgment:

[0013] If the result of enzyme digestion test is a single band of 105 bp, it is a yellow stigma tomato plant;

[0014] If the result of enzyme digestion test is two bands of 68 bp and 37 bp, it is a homozygous green stigma tomato plant;

[0015] The results of enzyme digestion detection are three bands of 105bp, 68bp and 37bp, which means it is a heterozygous green stigma tomato plant.

[0016] Preferably, among the bands indicated by the 1.5% agarose gel electrophoresis detection results of the PCR products in S2, the nucleotide sequence of the band corresponding to the PCR results of the homozygous green stigma tomato plant is shown as SEQ ID NO:3; the nucleotide sequence of the band corresponding to the PCR results of the yellow stigma tomato plant is shown as SEQ ID NO:4.

[0017] Preferably, the nucleotide sequence of the 37 bp band in the enzyme digestion detection result in S4 is shown as SEQ ID NO:5; the nucleotide sequence of the 68 bp band is shown as SEQ ID NO:6; and the nucleotide sequence of the 105 bp band is shown as SEQ ID NO:7.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The dCAPS molecular marker primers of the present invention are suitable for genetic evaluation, germplasm identification and molecular marker-assisted breeding research of tomato stigma color germplasm resources, and have good repeatability and high reliability. An effective identification method is provided for cultivating new tomato varieties with new stigma colors, thereby increasing tomato genetic diversity and morphological markers that are beneficial to identification of tomato hybrid progeny. At the same time, the present invention provides a new basis for genetic evaluation of tomato appearance morphology germplasm resources.

[0020] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 2 is a diagram of green and yellow stigmas of tomatoes according to Example 1 of the present invention.

[0022] Figure 2 This is a diagram showing the detection results of the dCAPS molecular marker of Example 1 of the present invention in the tomato material to be detected. DETAILED DESCRIPTION

[0023] Example 1

[0024] The dCAPS marker primers used to identify the tomato stigma color in this embodiment include a dCAPS upstream primer and a dCAPS downstream primer. The nucleotide sequence of the dCAPS upstream primer is shown in SEQ ID NO:1; the nucleotide sequence of the dCAPS downstream primer is shown in SEQ ID NO:2.

[0025] This embodiment also provides a method for identifying the color of tomato stigma using the above-mentioned dCAPS marker primers for identifying the color of tomato stigma, the method being:

[0026] S1, extracting genomic DNA of the tomato to be tested;

[0027] S2, using dCAPS upstream primer and dCAPS downstream primer to perform PCR amplification reaction on the genomic DNA of the sample to be tested extracted in S1 to obtain a PCR product;

[0028] The PCR amplification system is as follows: 1 µL of genomic DNA of the tomato sample to be tested, 0.5 µL of dCAPS upstream primer, 0.5 µL of dCAPS downstream primer, 5 µL of 2×Taq Master Mix, ddH 2 Make up to 10 µL with O;

[0029] The reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 45 s, annealing at 55°C for 30 s; extension at 72°C for 30 s, 30 cycles; extension at 72°C for 7 min; storage at 4°C;

[0030] Among the bands indicated by the 1.5% agarose gel electrophoresis detection results of the PCR products, the nucleotide sequence of the band corresponding to the PCR result of the homozygous green stigma tomato plant is shown in SEQ ID NO:3; the nucleotide sequence of the band corresponding to the PCR result of the yellow stigma tomato plant is shown in SEQ ID NO:4;

[0031] S3, take 3 μL of the PCR product obtained in S2 and perform 1.5% agarose gel electrophoresis detection, then use the endonuclease BseXI to digest the PCR product obtained in S2 at 65°C for 30 minutes to obtain the digestion product;

[0032] The enzyme digestion reaction system is: PCR product 7µL, 10×BseXI buffer 1µL, BseXI enzyme 0.2 µL, ddH 2 O to make up to 10 µL;

[0033] S4. The enzyme digestion product obtained in S3 is subjected to 12% polyacrylamide gel electrophoresis at a power of 20 W for 1.5 h. After the electrophoresis, silver staining and development are performed to observe the enzyme digestion detection results; Result judgment:

[0034] If the result of enzyme digestion detection is a single band of 105 bp, it is a yellow stigma tomato plant;

[0035] If the result of enzyme digestion test is two bands of 68 bp and 37 bp, it is a homozygous green stigma tomato plant;

[0036] If the result of enzyme digestion is three bands of 105bp, 68bp and 37bp, it is a heterozygous green stigma tomato plant;

[0037] The nucleotide sequence of the 37 bp band is shown in SEQ ID NO:5;

[0038] The nucleotide sequence of the 68 bp band is shown in SEQ ID NO: 6;

[0039] The nucleotide sequence of the 105 bp band is shown in SEQ ID NO:7.

[0040] The specific preparation of the test agent and the specific test method in this embodiment are as follows:

[0041] 1. Reagent preparation

[0042] 1. Preparation of electrophoresis reagents:

[0043] (1) Preparation of 0.5 mol / L EDTA (pH 8.0): Add 18.61 g EDTANa2 to 80 mL of distilled water, stir magnetically, adjust the solution to pH 8.0 with 10 mol / L NaOH (or solid NaOH), make up to 100 mL, sterilize under high pressure at 121°C for 25 min, and set aside.

[0044] (2) Preparation of 10×TBE: 108 g Tris, 55 g boric acid, 37.25 mL 0.5 mol / L EDTA (pH 8.0), and dilute to 1 L with water;

[0045] Preparation of 1×TBE buffer: dilute 10×TBE 10 times;

[0046] (3) 30% stock solution preparation: 290 g acrylamide, 10 g bisacrylamide, add water to 1 L, heat to dissolve, and store at 4 °C away from light;

[0047] Preparation of 12% polyacrylamide gel solution: 400 mL 30% stock solution, 100 mL 10×TBE, 500 mL ddH 2 O, stir to mix, and store at 4℃ away from light.

[0048] (4) 10% ammonium persulfate: Add water to 1 g ammonium persulfate to make up to 10 mL and store at 4°C.

[0049] 2. Preparation of silver staining reagent

[0050] Silver staining solution formula: 1 g AgNO 3 Dissolve to 1 L ddHO 2 O in;

[0051] 3. Preparation of developer

[0052] The developer solution formula is: 15 g sodium hydroxide, 10 mL formaldehyde, 2 L distilled water, mix well.

[0053] (2) Specific operations

[0054] 1. Preparation of polyacrylamide gel

[0055] (1) Gel preparation: Place 50 mL of 12% polyacrylamide gel solution, 50 µL of TEMED, and 500 µL of 10% ammonium persulfate in a gel-filling bottle and mix thoroughly.

[0056] (2) Glue filling: Slowly pour the glue into the gap between the two glass plates along the glue filling port to prevent bubbles. After the glue filling is completed, insert a comb into the glue filling port. Be careful to prevent bubbles when inserting the comb. Gel polymerization for more than 1 hour.

[0057] 2. Electrophoresis

[0058] (1) Install the gel plate in the electrophoresis tank and fill the upper and lower tanks with 1×TBE buffer. The buffer level in the lower tank should just cover the lower edge of the gel, and the buffer level in the upper tank should just cover the upper edge of the short plate.

[0059] (2) Gently pull out the comb teeth and use a syringe to blow out the bubbles in the spot holes.

[0060] (3) Take 1 μL of the amplified product after enzyme digestion and add it to the sample spotting hole. Perform electrophoresis at 20 W for 1.5 h in the Beijing Liuyi Factory double-plate sandwich vertical electrophoresis tank. The electrophoresis ends when the bromophenol blue dye reaches the bottom.

[0061] (4) Recover the electrophoresis buffer, remove the glass plate and place it flat on the table, and carefully separate the two pieces of glass.

[0062] 3. Silver staining

[0063] (1) Glue stripping: Place the long plate with glue in distilled water to separate the glue from the glass plate, and rinse twice to remove the electrophoresis buffer.

[0064] (2) Silver staining: Place the film in the silver stain solution, shake gently for about 15 minutes in a dark place, pour out the silver stain solution, and then rinse with distilled water three times.

[0065] (3) Development: Place the film in the developer and shake slowly for about 7-10 minutes until clear bands appear. Pour out the developer, rinse the film with tap water 3-5 times, and photograph the gel with a digital camera.

[0066] like Figure 1 Shown are the appearance pictures of the green stigma (wild type, left) and yellow stigma (mutant, right) of the tomato to be tested. The stigma of the wild type is green, and the stigma of the mutant is yellow.

[0067] Electrophoresis experiments were performed on tomato green stigma material samples and tomato yellow stigma mutant samples, as well as F1 population samples constructed from green stigma material and yellow stigma mutant, and F2 population yellow stigma single plant samples. The results are as follows Figure 2 As shown, lane M is Mark, lane G is homozygous green stigma material, ys is yellow stigma mutant, F1 is the first generation of hybrids of green stigma and yellow stigma mutant (heterozygous green stigma), and lanes 1-32 are yellow stigma single plant samples of the second generation (F2) of hybrids of green stigma and yellow stigma mutant. Figure 2 It can be clearly seen that the homozygous green stigma lane G has two bands of 68bp and 37bp, while the F1 lane has three bands of 105bp, 68bp and 37bp, and the yellow stigma mutant ys and the yellow stigma tomato in F2 have a detection rate of 100% at 105bp.

[0068] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A dCAPS-labeled primer for identifying tomato stigma color, It is characterized in that The dCAPS marker primers include a dCAPS upstream primer and a dCAPS downstream primer. The nucleotide sequence of the dCAPS upstream primer is shown in SEQ ID NO:1; the nucleotide sequence of the dCAPS downstream primer is shown in SEQ ID NO:

2.

2. A method for identifying tomato stigma color using the dCAPS marker primers for identifying tomato stigma color as claimed in claim 1, It is characterized in that The method is: S1, extracting genomic DNA of the tomato to be tested; S2, using dCAPS upstream primer and dCAPS downstream primer to perform PCR amplification reaction on the genomic DNA of the sample to be tested extracted in S1 to obtain a PCR product; The PCR amplification system is as follows: 1 µL of genomic DNA of the tomato sample to be tested, 0.5 µL of dCAPS upstream primer, 0.5 µL of dCAPS downstream primer, 5 µL of 2×Taq Master Mix, ddH 2 Make up to 10 µL with O; The reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 45 s, annealing at 55°C for 30 s; extension at 72°C for 30 s, 30 cycles; extension at 72°C for 7 min; storage at 4°C; S3, take 3 μL of the PCR product obtained in S2 and perform 1.5% agarose gel electrophoresis detection, then use the endonuclease BseXI to digest the PCR product obtained in S2 at 65°C for 30 minutes to obtain the digestion product; The enzyme digestion reaction system is: PCR product 7µL, 10×BseXI buffer 1µL, BseXI enzyme 0.2 µL, ddH 2 O to make up to 10 µL; S4. The enzyme digestion product obtained in S3 is subjected to 12% polyacrylamide gel electrophoresis at a power of 20 W for 1.5 h. After the electrophoresis, silver staining and development are performed to observe the enzyme digestion detection results; Result judgment: If the result of enzyme digestion test is a single band of 105 bp, it is a yellow stigma tomato plant; If the result of enzyme digestion test is two bands of 68 bp and 37 bp, it is a homozygous green stigma tomato plant; The results of enzyme digestion detection are three bands of 105bp, 68bp and 37bp, which means it is a heterozygous green stigma tomato plant.

3. The method according to claim 2, It is characterized in that Among the bands indicated by the results of 1.5% agarose gel electrophoresis detection of the PCR product in S3, the nucleotide sequence of the band corresponding to the PCR result of the homozygous green stigma tomato plant is shown as SEQ ID NO:3; the nucleotide sequence of the band corresponding to the PCR result of the yellow stigma tomato plant is shown as SEQ ID NO:

4.

4. The method according to claim 2, It is characterized in that The nucleotide sequence of the 37 bp band in the enzyme digestion detection result described in S4 is shown in SEQ ID NO:5; the nucleotide sequence of the 68 bp band is shown in SEQ ID NO:6; and the nucleotide sequence of the 105 bp band is shown in SEQ ID NO:7.

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