Molecular marker method for screening late bolting and non-heading Chinese cabbage materials in seedling stage

Abstract

The invention discloses a molecular marker method for screening late bolting and non-heading Chinese cabbage materials in the seedling stage, and belongs to the field of molecular marker assisted breeding. According to the method, genome DNA of the non-heading Chinese cabbage is taken as a template, a specific primer A06-33293887-indel-F / R is adopted to amplify a target fragment through a polymerase chain reaction (PCR), polyacrylamide gel electrophoresis detection is combined, and early / late bolting materials and varieties of the non-heading Chinese cabbage can be rapidly identified and distinguished according to the size of a target band. According to the molecular marker detection method provided by the invention, early bolting or late bolting materials can be quickly screened in the seedling stage of the non-heading Chinese cabbages, the breeding selection efficiency is remarkably improved, the breeding period is shortened, and a technical support is provided for genetic improvement of flowering time regulation of the non-heading Chinese cabbages.

Description

Technical Field

[0001] The invention belongs to the field of molecular marker-assisted breeding, and particularly relates to a molecular marker method for screening late-bolting non-heading Chinese cabbage materials at the seedling stage. Background Art

[0002] Non-heading cabbage ( Brassica campestris (syn. Brassica rapa )ssp . chinensis ) originates in my country and belongs to the Brassica subspecies of the Brassicaceae family. It is an important leafy vegetable, nutritious and highly adaptable. The bolting timing of non-heading cabbage varies depending on the variety or material. Premature bolting can lead to leaf fibrosis and a sharp drop in yield, seriously affecting its commercial value and economic value. Therefore, studying the bolting timing of non-heading cabbage is crucial for improving its economic value and accelerating breeding.

[0003] Based on this, the present invention designs a molecular marker method for screening late-bolting non-heading Chinese cabbage materials at the seedling stage. Summary of the Invention

[0004] One of the objectives of the present invention is to provide a primer set A06-33293887-indel-F / R for use in identifying early / late bolting materials or varieties of non-heading Chinese cabbage at the seedling stage; The sequence of the primer set A06-33293887-indel-F / R is as follows: A06-33293887-indel-F: 5'-TGATTTAGGGTTTAGGGATTAGGA-3' (SEQ ID NO.1) A06-33293887-indel-R: 5'-ATAATAGTTAGAGAAAAACGAGTT-3' (SEQ ID NO. 2).

[0005] A second object of the present invention is to provide a kit for identifying early / late bolting materials or varieties of non-heading Chinese cabbage at the seedling stage, comprising a primer set A06-33293887-indel-F / R. The sequence of the primer set A06-33293887-indel-F / R is as follows: A06-33293887-indel-F:5'-TGATTTAGGGTTTAGGGATTAGGA-3' A06-33293887-indel-R:5'-ATAATAGTTAGAGAAAAACGAGTT-3'.

[0006] A third object of the present invention is to provide a method for identifying early / late bolting materials or varieties of non-heading Chinese cabbage at the seedling stage, comprising the following steps: Step 1: Take tissue of the material or variety to be identified and extract genomic DNA; Step 2: Using the genomic DNA extracted in step 1 as a template, PCR amplification was performed using specific primers A06-33293887-indel-F / R; The sequence of the specific primer A06-33293887-indel-F / R is as follows: A06-33293887-indel-F:5'-TGATTTAGGGTTTAGGGATTAGGA-3' A06-33293887-indel-R:5'-ATAATAGTTAGAGAAAAACGAGTT-3'; Step 3, performing polyacrylamide gel electrophoresis separation on the amplified products of step 2 to obtain the band pattern of the material or variety to be identified. If the fragment size is 597 bp, it indicates that the material or variety has an early bolting trait; if the fragment size is 568 bp, it indicates that the material or variety has a late bolting trait.

[0007] Furthermore, in step 2, the PCR reaction system is: in a 10 μL reaction system, 2 μL deionized water, 1 μL each of 10 μM A06-33293887-indel-F / R primers, 1 μL 80 ng / μL genomic DNA, and 5 μL 2× Rapid Taq MasterMix.

[0008] Furthermore, in step 2, the PCR amplification reaction program is: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 32 cycles, and storage at 12°C.

[0009] Furthermore, in step 3, the configuration system of polyacrylamide gel electrophoresis is: in a 60 ml gel system, 10×TBE 6 mL, 40% PA 12 mL, TEMED 600 μL, 10% APS 60 μL, and deionized water 42 mL.

[0010] Furthermore, in step 3, the silver staining and development system of the polyacrylamide gel is: 2 g AgNO3 / 1000 mLH2O staining solution, 3 g NaOH / 200 mL H2O-2 mL formaldehyde stock solution developer.

[0011] Furthermore, in step 3, the silver staining and development reaction procedures of the polyacrylamide gel are as follows: staining with a nitrate solution for 5 minutes, rinsing with distilled water three times for 1 minute each time, and after rinsing, placing in a sodium hydroxide-formaldehyde color development solution and gently shaking on a horizontal shaker for 3-5 minutes. Beneficial effects

[0012] 1. The present invention uses the marker primer A06-33293887-indel-F / R to quickly distinguish early / late bolting varieties and materials of non-heading Chinese cabbage at the seedling stage.

[0013] 2. The present invention facilitates assisted selection, is low-cost, and simple to operate. Conventional methods for selecting non-heading cabbage for bolting traits are time-consuming and labor-intensive. However, the present invention allows selection to be performed at the seedling stage, significantly reducing subsequent screening costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 、 Figure 2 This is the polyacrylamide gel electrophoresis result of the marker primer A06-33293887-indel-F / R amplified in the non-heading cabbage early-bolting variety Suzhou Qing 'NHCC001', the non-heading cabbage late-bolting variety Wuyueman 'NHCC004', the F1 material obtained by hybridization of these two materials, and the F2 material obtained by self-pollination of the F1. Where: M is DL2000 Marker, Figure 1 Middle lanes 2–33, 35–36, and 37–39 represent early-bolting F2 materials, and lanes 44–52, 54–76, and 78–80 represent late-bolting F2 materials; Figure 2 The middle lanes 1-27 are all early-bolting F2 materials. DETAILED DESCRIPTION

[0015] This invention provides a set of specific molecular marker primers, A06-33293887-indel-F / R, for identifying the early-bolting trait in non-heading Chinese cabbage. Using genomic DNA from the early-bolting non-heading Chinese cabbage variety Suzhou Qing 'NHCC001' (P1), the late-bolting variety Wuyueman 'NHCC004' (P2), the F1 hybrid of these two varieties, and the F2 self-fertilized from the F1 hybrid, the target fragments are amplified by polymerase chain reaction. Early-bolting and late-bolting varieties and materials can be screened based on the size of the target band.

[0016] This study developed a novel detection method based on an insertion-deletion marker at locus 33293887 on chromosome A06. Compared with conventional phenotypic identification, this method can accurately predict bolting traits in the seedling stage, shortening the breeding cycle and reducing testing costs. This technology provides a highly efficient and reliable means for molecular marker-assisted selection of bolting traits in non-heading Chinese cabbage, and is of great significance for accelerating the breeding of new leafy vegetable varieties.

[0017] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0019] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Example

[0020] This embodiment provides a method for identifying the early / late bolting time of non-heading Chinese cabbage at the seedling stage, comprising the following steps: Step 1, extraction of genomic DNA from non-heading Chinese cabbage DNA was extracted according to the instructions of the Plant Genomic DNA Rapid Extraction Kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), as follows: 1) Take 100 mg of leaves from non-heading cabbage materials or varieties (including the early-bolting non-heading cabbage variety Suzhou Qing 'NHCC001' (P1), the late-bolting variety Wuyueman 'NHCC004' (P2), the F1 material obtained by hybridizing these two materials, and the F2 material obtained by self-pollination of the F1), place them in liquid nitrogen, and grind thoroughly; 2) Before the experiment begins, preheat a centrifuge tube containing 700 µL of GP1 in a 65°C water bath. Add mercaptoethanol to a final GP1 concentration of 0.1%. Add the GP1 to the submerged powder, mix quickly, and place in a 65°C water bath for 20 minutes. 3) Add 700 µL of chloroform to the reaction mixture, mix thoroughly, and centrifuge at 12,000 rpm for 5 minutes. 4) Transfer the supernatant from the above centrifugation to a new centrifuge tube, add 700µL of GP2 buffer and mix thoroughly; 5) Transfer the mixed liquid to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid; 6) Add 500 µL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid; 7) Add 600 µL of rinse solution PW to the adsorption column CB3 again, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube; 8) Repeat step 7; 9) Return the adsorption column CB3 to the collection tube and centrifuge at 12,000 rpm for 2 minutes. Discard the waste liquid. Leave the adsorption column CB3 uncovered at room temperature for 2 minutes to completely remove the rinse solution from the adsorption material. 10) Place the adsorption column CB3 in a new centrifuge tube, add 50 μL of elution buffer TE to the middle of the adsorption column CB3, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes to collect the genomic DNA into the centrifuge tube; 11) Determine the purity and concentration of the DNA, dilute it to 80 ng / μL, and store it in a -20°C refrigerator.

[0021] Step 2: Amplify the non-heading cabbage genomic DNA with the labeled primer A06-33293887-indel-F / R In a 10 μL reaction system, 1 μL of 80 ng / μL DNA template, 1 μL of 10 μM labeled primer A06-33293887-indel-F / R, 5 μL of 2× Rapid Taq Master Mix, and 2 μL of deionized water were added.

[0022] A06-33293887-indel-F / R marker primers: A06-33293887-indel-F:5'-TGATTTAGGGTTTAGGGATTAGGA-3' A06-33293887-indel-R: 5'-ATAATAGTTAGAGAAAAACGAGTT-3'.

[0023] The PCR amplification program was as follows: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 32 cycles, and storage at 12°C.

[0024] Step 3, polyacrylamide gel electrophoresis detection After PCR amplification, perform polyacrylamide gel electrophoresis. Use 1× TBE as the running buffer. Add 10 μL of the PCR amplified product to an appropriate amount of bromophenol blue solution and mix thoroughly. Pipette 1 μL into the gel wells. Run the gel at 150 V and 300 W for 100–120 min (the electrophoresis time can be adjusted depending on the length of the PCR product). Then, perform silver staining and development. Place the gel in silver nitrate staining solution (2 g AgNO₃ / 1000 mL H₂O) for 5 minutes. Rinse with distilled water three times for 1 minute each. After rinsing, transfer the gel to a sodium hydroxide-formaldehyde (3 g NaOH / 200 mL H₂O-2 mL C₂O) colorimetric solution. Gently shake the gel on a horizontal shaker for 3–5 minutes until the bands are clearly visible. The gel is then visualized using a gel imager.

[0025] From the electrophoresis results, we can see that Figure 1 The early-bolting F2 materials in lanes 1-10, 12-32, 34-35, and 37-39 all amplified a 597 bp fragment, while the late-bolting F2 materials in lanes 44-52, 54-76, and 78-80 all amplified a 568 bp fragment. The specific molecular marker primers A06-33293887-indel-F / R of the present invention can effectively distinguish between early and late bolting in non-heading Chinese cabbage.

[0026] At the same time, specific molecular marker primers A06-33293887-indel-F / R were used to amplify materials of known shapes, such as Figure 2 As shown, the early-bolting F2 materials in lanes 1-7, 9, 11-12, 14-15, 17-19, 21-23, and 26 were all able to amplify a 597 bp fragment.

[0027] From the above results, it can be seen that when the molecular marker primer A06-33293887-indel-F / R is used to amplify in the genome of non-heading cabbage, if a 597 bp fragment is amplified, it indicates that the non-heading cabbage variety or material has the early bolting trait, and if a 568 bp fragment is amplified, it indicates that the non-heading cabbage variety or material has the late bolting trait.

Claims

1. Application of primer set A06-33293887-indel-F / R in identifying early / late bolting materials or varieties of non-heading Chinese cabbage at the seedling stage; The sequence of the primer set A06-33293887-indel-F / R is as follows: A06-33293887-indel-F:5'-TGATTTAGGGTTTAGGGATTAGGA-3' A06-33293887-indel-R:5'-ATAATAGTTAGAGAAAAACGAGTT-3'.

2. A kit for identifying early / late bolting materials or varieties of non-heading Chinese cabbage at the seedling stage, comprising primer set A06-33293887-indel-F / R. The sequence of primer set A06-33293887-indel-F / R is as follows: A06-33293887-indel-F:5'-TGATTTAGGGTTTAGGGATTAGGA-3' A06-33293887-indel-R:5'-ATAATAGTTAGAGAAAAACGAGTT-3'.

3. A method for identifying early / late bolting materials or varieties of non-heading Chinese cabbage at the seedling stage, characterized in that: The following steps are involved: Step 1: Take tissue of the material or variety to be identified and extract genomic DNA; Step 2: Using the genomic DNA extracted in step 1 as a template, PCR amplification was performed using specific primers A06-33293887-indel-F / R; The sequence of the specific primer A06-33293887-indel-F / R is as follows: A06-33293887-indel-F:5'-TGATTTAGGGTTTAGGGATTAGGA-3' A06-33293887-indel-R:5'-ATAATAGTTAGAGAAAAACGAGTT-3'; Step 3, performing polyacrylamide gel electrophoresis separation on the amplified products of step 2 to obtain the band pattern of the material or variety to be identified. If the fragment size is 597 bp, it indicates that the material or variety has an early bolting trait; if the fragment size is 568 bp, it indicates that the material or variety has a late bolting trait.

4. The method according to claim 3, wherein In step 2, the PCR reaction system is as follows: in a 10 μL reaction system, 2 μL deionized water, 1 μL each of 10 μM A06-33293887-indel-F / R primers, 1 μL 80 ng / μL genomic DNA, and 5 μL 2× Rapid Taq Master Mix.

5. The method according to claim 3, characterized in that In step 2, the PCR amplification reaction program was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 32 cycles, and storage at 12°C.

6. The method according to claim 3, wherein In step 3, the polyacrylamide gel electrophoresis system is as follows: in a 60 ml gel system, 10× TBE 6 mL, 40% PA 12 mL, TEMED 600 μL, 10% APS 60 μL, and deionized water 42 mL.

7. The method according to claim 3, wherein In step 3, the silver staining and development system for the polyacrylamide gel is as follows: 2 g of AgNO3 per 1000 mL of H2O staining solution, and 3 g of NaOH per 200 mL of H2O and 2 mL of formaldehyde mixed stock solution.

8. The method according to claim 3, characterized in that In step 3, the silver staining and development reaction procedures of the polyacrylamide gel are as follows: staining with nitrate solution for 5 minutes, rinsing with distilled water three times, each for 1 minute; after rinsing, placing in sodium hydroxide-formaldehyde color development solution, and gently shaking on a horizontal shaker for 3-5 minutes.

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