Group of molecular marker primers for screening early bolting of non-heading Chinese cabbages and application of molecular marker primers

Abstract

The invention discloses a group of molecular marker primers for screening early bolting of non-heading Chinese cabbages and application of the molecular marker primers, and belongs to the technical field of molecular biology. According to the molecular marker primer FTinsert-1F / 1R provided by the invention, the target fragment is amplified by using the genome DNA of a sample to be identified as a template through a PCR (Polymerase Chain Reaction) technology, and the early bolting of the non-heading Chinese cabbage can be identified according to the length of an amplified band. According to the molecular marker provided by the invention, early bolting varieties or materials of the non-heading Chinese cabbages can be more intuitively and quickly detected, and the non-heading Chinese cabbages can be bred in advance by using the group of molecular markers, so that time and land resources are greatly saved.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a set of molecular marker primers for screening early bolting of non-heading Chinese cabbage and their applications. Background Technology

[0002] Non-heading cabbage ( Brassica campestris (syn.) Brassica rapa )ssp chinensis Brassica oleracea, belonging to the Brassicaceae family, is an important leafy green vegetable. It is popular with consumers due to its short growth cycle, strong adaptability, and rich nutritional content. Non-heading cabbage is primarily consumed for its leaves, but bolting and flowering signify a shift from vegetative to reproductive growth, reducing its edibility and marketability, ultimately leading to significant economic losses.

[0003] Therefore, it is of great significance to design a method specifically for the rapid identification of early bolting, non-heading Chinese cabbage varieties or materials. Summary of the Invention

[0004] One objective of this invention is to provide the application of primer pair FTinsert-1F / 1R in the identification / screening of early bolting, non-heading Chinese cabbage, the sequence of which is: FTinsert-1F: 5'-GTAGACGCAAACAGCGCCTC-3', FTinsert-1R: 5'-CACGTCGCTACGGAAGGTCG-3'.

[0005] The genomic DNA of the non-heading Chinese cabbage to be identified was amplified using the primer pair FTinsert-1F / 1R. If a 732 bp fragment was obtained, it indicates that the non-heading Chinese cabbage to be identified is an early bolting non-heading Chinese cabbage.

[0006] The second objective of this invention is to provide a molecular marker method for identifying / screening early bolting, non-heading Chinese cabbage, comprising the following steps: Step 1: Extract genomic DNA from the non-heading Chinese cabbage to be identified; Step 2: Using the genomic DNA extracted in Step 1 as a template, amplify it using primer pair FTinsert-1F / 1R; The sequence of the primer pair FTinsert-1F / 1R is as follows: FTinsert-1F: 5'-GTAGACGCAAACAGCGCCTC-3', FTinsert-1R: 5'-CACGTCGCTACGGAAGGTCG-3'; Step 3: Use agarose gel electrophoresis to separate the amplification products from Step 2 and observe the band size. If a 732 bp fragment is obtained, it indicates that the non-heading Chinese cabbage to be identified is early bolting non-heading Chinese cabbage.

[0007] Further, in step 2, the PCR amplification is as follows: 0.5 μL each of 10 μM FTinsert-1F / 1R primers, 1 μL of 60 ng / μL genomic DNA, and 5 μL of 2× PrimeSTAR MAX DNA Polymerase, followed by the addition of 3 μL of deionized water to a final volume of 10 μL; the PCR reaction program is as follows: pre-deformation at 95℃ for 2 min, denaturation at 98℃ for 30 s, annealing at 56.5℃ for 30 s, extension of the target fragment at 72℃ for 1 min, and final extension at 72℃ for 5 min, for a total of 28 cycles. Beneficial effects

[0008] 1. This invention uses molecular marker primers FTinsert-1F / 1R to amplify target fragments in samples to be identified, which can accurately and rapidly screen early bolting, non-heading Chinese cabbage.

[0009] 2. This invention is low-cost and resource-saving. Conventional breeding methods are time-consuming and labor-intensive. This invention can screen early-bolting, non-heading Chinese cabbage materials or varieties using molecular markers during the seedling stage, greatly shortening the selection time, saving a lot of manpower and land resources, and accelerating the breeding speed. Attached Figure Description

[0010] Figure 1-3 This is an agarose gel electrophoresis image of molecular marker primers FTinsert-1F / 1R amplified in various non-heading Chinese cabbage genomic DNA samples, where M is the DL2000 Marker. Figure 1 Lanes 1-21, 23, and 24 in the middle swimming lane are early bolting, non-heading Chinese cabbage; lanes 22, 25-27 are late bolting, non-heading Chinese cabbage. Figure 2 Lanes 1-16, 18-22, 24, 26, and 29-32 in the middle swimming lanes are early bolting, non-heading Chinese cabbage; lanes 17, 23, 25, 27, and 28 are late bolting, non-heading Chinese cabbage. Figure 3 Lanes 1-16 in the middle section are all early bolting, non-heading Chinese cabbage. Detailed Implementation

[0011] This invention provides a pair of molecular marker primers, FTinsert-1F / 1R, for screening early-bolting, non-heading Chinese cabbage. Genomic DNA is extracted from various early and late-bolting non-heading Chinese cabbage varieties or materials to be tested, and the fragment is amplified by PCR. Based on the length of the amplified band, early-bolting non-heading Chinese cabbage varieties or materials can be screened. The molecular markers provided by this invention allow for more intuitive and rapid detection of early-bolting non-heading Chinese cabbage varieties or materials. Using this set of molecular markers allows for early selection and breeding of non-heading Chinese cabbage, greatly saving time and land resources.

[0012] The present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used are commercially available, and techniques not described in detail are performed according to standard methods well known to those skilled in the art. The reagents mentioned in this application are commercially available or otherwise publicly available; they are merely examples and not the only ones applicable to the present invention. Other suitable tools or biological materials can be substituted. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Example 1

[0013] This embodiment provides a molecular marker screening method for early bolting and non-heading Chinese cabbage, comprising the following steps: Step 1: Extract 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 Biotech Co., Ltd.), as follows: 1) Take 100mg of leaf tissue from the non-heading Chinese cabbage material or variety to be tested, and then grind it in liquid nitrogen; 2) Preheat buffer GP1 in a 65℃ water bath, add mercaptoethanol to a final concentration of 0.1%, then add GP1 to the ground sample to be tested, and immediately mix with a vortex mixer. After mixing, place the sample to be tested in a 65℃ water bath for 20 minutes. 3) Add 700µL of chloroform to the above sample, mix thoroughly on a vortex mixer, and centrifuge at 12,000rpm for 5min. 4) Transfer the supernatant after centrifugation to a new centrifuge tube, then add 700µL of GP2 buffer and mix well; 5) Transfer the above liquid to a CB3 adsorption column, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid; 6) Add 500µL of GD buffer to the CB3 adsorption column, centrifuge at 12,000rpm for 30s, and discard the waste liquid; 7) Add 600µL of PW washing solution to the CB3 adsorption column, centrifuge at 12,000rpm for 30s, and discard the waste liquid; 8) Repeat step 7 above; 9) Place the CB3 adsorption column in a new collection tube, centrifuge at 12,000 rpm for 2 min, and discard the tube. Place the CB3 adsorption column at 25℃ for 5 min to completely remove any residual washing solution from the material; 10) Transfer the CB3 adsorption column into a new 1.5 mL centrifuge tube, add 30 μL of elution buffer to the CB3 adsorption membrane, then incubate at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min to collect the solution; 11) Detect the genomic DNA concentration of non-heading Chinese cabbage and place it in a -20℃ refrigerator.

[0014] Step 2: Amplify the genomic DNA of non-heading Chinese cabbage using molecular marker primers FTinsert-1F / 1R. 0.5 μL each of 10 μM FTinsert-1F / 1R primers, 1 μL of 60 ng / μL genomic DNA, and 5 μL of 2× PrimeSTARMAX DNA Polymerase were added, followed by 3 μL of deionized water to a final volume of 10 μL. FTinsert-1F / 1R labeled primers: FTinsert-1F: 5'-GTAGACGCAAACAGCGCCTC-3' FTinsert-1R: 5'-CACGTCGCTACGGAAGGTCG-3'; The PCR reaction program was as follows: pre-deformation at 95℃ for 2 min, denaturation at 98℃ for 30 s, annealing at 56.5℃ for 30 s, extension of the target fragment at 72℃ for 1 min, and final extension at 72℃ for 5 min, for a total of 28 cycles. The sample was stored at 5℃ and then placed on ice for later use.

[0015] Step 3, Agarose gel electrophoresis detection After the PCR reaction was completed, agarose gel electrophoresis was performed. 10 μL of sample was loaded, and the electrophoresis buffer was 1×TAEBuffer. After electrophoresis for 25 min, the results were viewed in a gel imaging system.

[0016] Figure 1-3 This is an agarose gel electrophoresis image of molecular marker primers FTinsert-1F / 1R amplified in various non-heading Chinese cabbage genomic DNA samples, where M is the DL2000 Marker. Figure 1 Lanes 1-21, 23, and 24 in the middle swimming lane are early bolting, non-heading Chinese cabbage; lanes 22, 25-27 are late bolting, non-heading Chinese cabbage. Figure 2Lanes 1-16, 18-22, 24, 26, and 29-32 in the middle swimming lanes are early bolting, non-heading Chinese cabbage; lanes 17, 23, 25, 27, and 28 are late bolting, non-heading Chinese cabbage. Figure 3 Lanes 1-16 in the middle section are all early bolting, non-heading Chinese cabbage.

[0017] Electrophoresis results showed that if a 732bp band could be amplified in the genomic DNA of non-heading Chinese cabbage using molecular marker primers FTinsert-1F / 1R, then the material or variety is an early bolting non-heading Chinese cabbage.

Claims

1. The application of primer pair FTinsert-1F / 1R in the identification / screening of early bolting, non-heading Chinese cabbage, characterized in that, The sequence of the primer pair FTinsert-1F / 1R is as follows: FTinsert-1F: 5'-GTAGACGCAAACAGCGCCTC-3', FTinsert-1R: 5'-CACGTCGCTACGGAAGGTCG-3'.

2. The application according to claim 1, characterized in that, The genomic DNA of the non-heading Chinese cabbage to be identified / screened was amplified using the primer pair FTinsert-1F / 1R. If a 732 bp fragment was obtained, it indicates that the non-heading Chinese cabbage to be identified / screened is an early bolting non-heading Chinese cabbage.

3. A molecular marker method for identifying / screening early bolting, non-heading Chinese cabbage, characterized in that, Includes the following steps: Step 1: Extract genomic DNA from non-heading Chinese cabbage plants to be identified / screened; Step 2: Using the genomic DNA extracted in Step 1 as a template, amplify it using primer pair FTinsert-1F / 1R; The sequence of the primer pair FTinsert-1F / 1R is as follows: FTinsert-1F: 5'-GTAGACGCAAACAGCGCCTC-3', FTinsert-1R: 5'-CACGTCGCTACGGAAGGTCG-3'; Step 3: Use agarose gel electrophoresis to separate the amplification products from Step 2 and observe the band size. If a 732 bp fragment is obtained, it indicates that the non-heading Chinese cabbage to be identified / screened is early bolting non-heading Chinese cabbage.

4. The molecular marker method according to claim 3, characterized in that, In step 2, the PCR amplification is as follows: 0.5 μL each of 10 μM FTinsert-1F / 1R primers, 1 μL of 60 ng / μL genomic DNA, 5 μL of 2× PrimeSTAR MAX DNA Polymerase, and then 3 μL of deionized water is added to a final volume of 10 μL.

5. The molecular marker method according to claim 3, characterized in that, In step 2, the PCR reaction program is as follows: pre-deformation at 95℃ for 2 min, denaturation at 98℃ for 30 s, annealing at 56.5℃ for 30 s, extension of the target fragment at 72℃ for 1 min, and post-extension at 72℃ for 5 min, for a total of 28 cycles.

6. A reagent kit for identifying / screening early bolting, non-heading Chinese cabbage, characterized in that, This includes the primer pair FTinsert-1F / 1R, the sequence of which is: FTinsert-1F: 5'-GTAGACGCAAACAGCGCCTC-3', FTinsert-1R: 5'-CACGTCGCTACGGAAGGTCG-3'.

Images