Method for identifying petal color of chinese cabbage in seedling stage

Abstract

The application discloses a method for identifying the petal color of Chinese cabbage in the seedling stage. The method uses the DNA of Chinese cabbage varieties as a template, adopts a primer group wF35 to amplify the fragment through polymerase chain reaction (PCR), and according to the size of the target band after electrophoresis, the petal color of Chinese cabbage varieties can be preliminarily inferred before bolting and flowering, and corresponding petal color varieties are screened. The method provided by the application can quickly detect the petal color of Chinese cabbage varieties, realize early and accurate prediction of the petal color, greatly save time and resources, greatly improve the selection efficiency, thereby accelerate the breeding process, and has an important role in production practice.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to a method for identifying flower petal color of Chinese cabbage at seedling stage. BACKGROUND

[0002] Chinese cabbage belongs to Brassica rapa L. ssp. Pekinensis (Lour.) Olsson. Brassica campestris ssp . chinensis Makino, belongs to Brassicaceae and Brassica rapa L. ssp. Pekinensis. Common flower color of Brassica rapa L. is yellow, and there are also white, orange and other variant colors. The flower color trait is stably inherited and easy to observe, and can be used as a marker trait to identify hybrid plants, and has an important role in production practice. Chinese cabbage is widely planted in China, and its yield and quality are extremely concerned characteristics. Flowers are important reproductive organs of Chinese cabbage, and preliminary identification of flower colors of different varieties helps to accelerate the breeding process of flower color traits of Chinese cabbage and cultivate more Chinese cabbage varieties with rich flower colors.

[0003] Therefore, it is extremely important to design a screening and identification method specifically for Chinese cabbage yellow petal / white petal varieties or materials. SUMMARY

[0004] The purpose of the application is to provide a method for identifying yellow petal / white petal of Chinese cabbage at seedling stage, which can quickly identify the petal color of the variety or material to be tested by means of primer set wF35, and accurately predict the flower color before bolting and flowering, thereby accelerating the breeding process.

[0005] In order to achieve the above purpose, the application adopts the following technical scheme:

[0006] A kit for identifying yellow petal / white petal of Chinese cabbage, comprising a primer set wF35, the sequence of the primer set wF35 being:

[0007] wF35F: 5'-CGAAACGAAGAAGAAGAGTCC-3' (SEQ ID NO. 1),

[0008] wF35R: 5'-GGGTGGGAACAAAAGAGCTAC-3' (SEQ ID NO. 2).

[0009] The above kit is applied to identifying yellow petal or white petal of Chinese cabbage.

[0010] A method for identifying flower petal color of Chinese cabbage at seedling stage, comprising the following steps:

[0011] Step 1, extracting genomic DNA of the variety or material to be identified;

[0012] Step 2, using the DNA extracted in step 1 as a template, PCR amplification is carried out on the template by using the primer set wF35 in the kit;

[0013] Step 3, the PCR amplification product in step 2 is subjected to polyacrylamide gel electrophoresis separation to obtain the band type of the variety or material to be identified, if a 115bp fragment is obtained, it indicates that the petals of the variety or material to be identified are yellow; if a 93bp fragment is obtained, it indicates that the petals of the variety or material to be identified are white.

[0014] Further, in step 2, the PCR amplification system is as follows: in a 10μL reaction system, 50ng / μL DNA template 1μL, 10μM wF35 primer 0.5μL each, 2× Primer Star 5μL, deionized water 3μL; the PCR amplification program is as follows: 95℃ pre-denaturation for 2min, 98℃ denaturation for 30s, 55.7℃ annealing for 30s, 72℃ extension for 30s, 72℃ post-extension for 10min, 35 cycles, 4℃ storage.

[0015] Beneficial effects:

[0016] 1. The present application can quickly and efficiently identify the petal color of Brassica chinensis var. rosularis variety or material before bolting and flowering by using the labeled primer set wF35.

[0017] 2. The present application is convenient for assisted selection and saves cost. If the flower color of Brassica chinensis var. rosularis is screened during the flowering period, the early stage consumes a long period, has high cost, and consumes a lot of manpower and material resources. With the aid of the present application, seedling selection can be realized, the workload is reduced, and the selection efficiency is improved.

[0018] 3. Flowers are not only important reproductive organs of Brassica chinensis var. rosularis but also have certain ornamental value. Early identification of flower color helps to speed up the breeding process of Brassica chinensis var. rosularis, and thus more Brassica chinensis var. rosularis varieties with rich flower colors can be quickly bred, which has more profound ornamental significance. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is an electrophoresis result diagram after PCR amplification of the primer set wF35 in Brassica chinensis var. rosularis with different flower colors, wherein: M is DL500 Marker, lanes 1, 2, 5, 6, 8, 10, 13, 15, 17, 19 are yellow petal varieties or materials, and lanes 3, 4, 7, 9, 11, 12, 14, 16, 18, 20 are white petal varieties or materials. DETAILED DESCRIPTION

[0020] The application will be further described in connection with specific examples. It should be noted that the experimental methods and materials reagents used in the following examples, if not specifically stated, are all conventional methods, and can be obtained commercially; at the same time, these methods or reagents are only used as an example, and are not the only way to achieve the application.

[0021] The main technical implementation route of the application is: extracting the genomic DNA of the non-heading Chinese cabbage variety or material to be tested as a template, using primer group wF35 to amplify the fragment by polymerase chain reaction (PCR), and after polyacrylamide gel electrophoresis, analyzing the size of the band, so as to identify the petal color of the non-heading Chinese cabbage variety to be tested.

[0022] The method provides a set of molecular markers for yellow / white petal of non-heading Chinese cabbage, which can quickly detect the flower color of non-heading Chinese cabbage, greatly save time and resources, improve selection efficiency, help the development of China's vegetable industry, especially ornamental vegetables, and also has an important role in production practice.

[0023] Example 1

[0024] The method for identifying the flower color of non-heading Chinese cabbage at the seedling stage provided by the embodiment comprises the following steps:

[0025] Step 1, extraction of non-heading Chinese cabbage genomic DNA

[0026] According to the instructions of the plant genomic DNA rapid extraction kit (purchased from Beijing Tiangen Bioscience Co., Ltd.), the DNA is extracted as follows:

[0027] 1) Take about 100 mg of fresh plant tissue and add liquid nitrogen to grind thoroughly;

[0028] 2) Quickly transfer the ground powder to a centrifuge tube preloaded with 700 µL of buffer GP1 preheated at 65°C (add mercaptoethanol to the preheated GP1 before the experiment to make the final concentration 0.1%), mix well by inverting quickly, and then place the centrifuge tube in a 65°C water bath for 20 min;

[0029] 3) Add 700 µL of chloroform and mix thoroughly, then centrifuge at 12,000 rpm for 5 min;

[0030] 4) Carefully transfer the upper aqueous phase obtained in the previous step to a new centrifuge tube, add 700 µL of buffer GP2, and mix thoroughly;

[0031] 5) Transfer the mixed liquid to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid;

[0032] 6) Add 500 μL Buffer GD (check if anhydrous ethanol has been added before use) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in the collection tube;

[0033] 7) Add 600 μL of the washing liquid PW (check if anhydrous ethanol has been added before use) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in the collection tube;

[0034] 8) Repeat operation step 7;

[0035] 9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to completely dry the residual washing liquid in the adsorption material;

[0036] 10) Transfer the adsorption column CB3 into a clean centrifuge tube, add 100 μL of the elution buffer TE to the middle part of the adsorption membrane, and place it at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min, and collect the solution into the centrifuge tube;

[0037] 11) After the DNA is detected in terms of purity and concentration, dilute it to 50 ng / μL, and store it in a refrigerator at -20℃ for standby use.

[0038] Step 2, label primer wF35 amplification in Brassica campestris L. genome

[0039] In a 10 μL reaction system, 50 ng / μL DNA template 1 μL, 10 uM labeled primer wF35 0.5 μL each, 2×PrimerStar 5 μL, deionized water 3 μL.

[0040] wF35 labeled primer:

[0041] wF35F: 5'-CGAAACGAAGAAGAAGAGTCC-3',

[0042] wF35R: 5'-GGGTGGGAACAAAAGAGCTAC-3'.

[0043] The PCR amplification program is as follows: 95℃ pre-denaturation for 2 min, 98℃ denaturation for 30 s, 55.7℃ annealing for 30 s, 72℃ extension for 30 s, 72℃ post-extension for 10 min, 35 cycles, and 4℃ storage.

[0044] Step 3, polyacrylamide gel electrophoresis detection

[0045] After the PCR amplification, polyacrylamide gel electrophoresis (PAGE gel electrophoresis) was used, the electrophoresis buffer was 1xTBE, 1 μL was loaded, 150V electrophoresis for 56 min, then staining, observation and photography.

[0046] The electrophoresis results are as shown in Figure 1. Figure 1 It can be seen that the yellow petal materials in lanes 1, 2, 5, 6, 8, 10, 13, 15, 17, 19 can all amplify 115bp fragments, and the white petal materials in lanes 3, 4, 7, 9, 11, 12, 14, 16, 18, 20 can all amplify 93bp fragments.

[0047] From the above results, it can be seen that the yellow flower material / white flower material of non-heading Chinese cabbage is amplified by using the labeled primer wF35, if 115bp fragments are obtained, it indicates that the petals of the to-be-identified variety or material are yellow; if 93bp fragments are obtained, it indicates that the petals of the to-be-identified variety or material are white.

Claims

1. The application of the reagent kit in identifying the flower color of non-heading Chinese cabbage petals during the seedling stage, characterized in that... The kit includes primer set wF35, the sequence of which is: wF35F: 5'-CGAAACGAAGAAGAAGAGTCC-3', wF35R: 5'-GGGTGGGAACAAAAGAGCTAC-3'; The genomic DNA of the non-heading Chinese cabbage variety or material to be identified was amplified using primer set wF35. If a 115bp fragment was obtained, it indicates that the petals of the variety or material to be identified are yellow, and if a 93bp fragment was obtained, it indicates that the petals of the variety or material to be identified are white.

2. A method for identifying the flower petal color of non-heading Chinese cabbage during the seedling stage, characterized in that, Includes the following steps: Step 1: Extract genomic DNA from the variety or material to be identified; Step 2: Using the DNA extracted in Step 1 as a template, perform PCR amplification using primer set wF35; The sequence of the primer set wF35 is as follows: wF35F: 5'-CGAAACGAAGAAGAAGAGTCC-3', wF35R: 5'-GGGTGGGAACAAAAGAGCTAC-3'; Step 3: Perform polyacrylamide electrophoresis to separate the PCR amplification products from Step 2 to obtain the band pattern of the variety or material to be identified. If a 115bp fragment is obtained, it indicates that the petals of the variety or material to be identified are yellow; if a 93bp fragment is obtained, it indicates that the petals of the variety or material to be identified are white.

3. The method according to claim 2, characterized in that: In step 2, the PCR amplification system is as follows: in a 10 μL reaction system, there is 1 μL of 50 ng / μL DNA template, 0.5 μL each of 10 μM wF35 primers, 5 μL of 2× Primer Star, and 3 μL of deionized water.

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