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Molecular marker closely linked with celery cabbage PHYB gene promoter allelic variation type phyB1/B2 and application of molecular marker

A technology of molecular markers and allelic variation, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as differences in flowering time and large differences in bolting and flowering traits, and achieve accurate results and easy operation. Convenience and improvement of identification efficiency

Active Publication Date: 2015-04-22
VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] There are germplasm resources of Chinese cabbage suitable for cultivation in different seasons in my country, and their bolting and flowering traits are quite different. Among them, there are many materials with significant differences in flowering time under long and short day conditions, but so far no correlation between photoreceptor mutations and flowering time has been found. report

Method used

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  • Molecular marker closely linked with celery cabbage PHYB gene promoter allelic variation type phyB1/B2 and application of molecular marker
  • Molecular marker closely linked with celery cabbage PHYB gene promoter allelic variation type phyB1/B2 and application of molecular marker
  • Molecular marker closely linked with celery cabbage PHYB gene promoter allelic variation type phyB1/B2 and application of molecular marker

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1, the cloning of PHYB gene promoter sequence in different Chinese cabbage inbred line materials

[0036] 1.1 Chinese cabbage genomic DNA extraction

[0037] (1) Put the leaves of Chinese cabbage seedlings into a liquid nitrogen pre-cooled mortar, and grind them into powder in liquid nitrogen;

[0038] (2) After the liquid nitrogen evaporates to dryness, transfer it to a 2ml centrifuge tube immediately, add about 0.6ml of CTAB extract preheated to 65°C for every 100mg of material, after melting, vigorously shake and mix the sample, place it in a 65°C water bath for 40- 60 minutes to lyse the cells;

[0039] (3) After the lysis is complete, take out the sample and let it cool down to room temperature completely. Add an equal volume of chloroform (chloroform), gently invert to mix, and place at room temperature for 10 minutes;

[0040] (4) Centrifuge at 12000 rpm for 15 minutes at room temperature;

[0041] (5) Use a pipette to carefully suck out the upper ...

Embodiment 2

[0061] Example 2 Development of co-dominant ASM markers

[0062] 2.1 Primer design

[0063] Carefully compare the genome sequences of the two allelic variants phyB1 and phyB2 of the PHYB gene. The main difference is a large deletion of 445 bp at the 5' end (such as image 3 shown). As the forward primer, the primer PF for amplifying the promoter was used, and a reverse primer PR1 was designed in the conserved region downstream (3' end) of the missing large fragment. The sequence Seq ID No.4 was commissioned to be synthesized by BGI.

[0064] 2.2 Acquisition of ASM mark

[0065] (1) Using the genomic DNA of He102 and 06-247 as templates, a PCR amplification was carried out with a common taq enzyme. Extend at 72°C for 40 seconds, 35-38 cycles, and finally extend at 72°C for 5 minutes.

[0066] (2) The amplified product was detected by electrophoresis on a 1.5% agarose gel, and photographed after EB staining. The results are as follows: Figure 5 shown. As can be seen from ...

Embodiment 3

[0067] Example 3 Detection of ASM markers on the F2 population constructed by He102×06-247.

[0068] (1) Genomic DNA extraction from different individual plants of the F2 population was as described in 1.1.

[0069] (2) PCR amplification: The preparation and amplification conditions of the PCR reaction solution are as described in Section 2.2 (1).

[0070] (3) Detection of PCR products is as described in Section 2.2 (2). Test results such as Image 6 Shown: P1 and P2 are the parents He102 and 06-247 respectively, 1-16 are 16 F2 individual plants. Individual plants 2, 3, 4, 7, and 15 in the figure are allelic variation types phyB2 in parent P1, namely He102, and individual plants 5, 9, 11, 12, 13, and 16 are allelic variation types in parent P2, namely 06-247 phyB1, the rest of the individual plants are heterozygous. M is the DNA molecular weight standard DL2000. From the amplification results, it can be seen that the pair of primers can identify homozygous wild type and h...

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Abstract

The invention discloses a molecular marker closely linked with celery cabbage PHYB gene promoter allelic variation type phyB1 / B2. The marker related to phyB1 detection is named as ASM-phyB1, the fragment size of the marker is 762bp, and the marker is shown in SEQ ID No.1; the marker related to phyB2 detection is named as ASM-phyB2, the fragment size of the marker is 317bp, and the marker is shown in SEQ ID No.2. The invention further discloses a primer for detecting the molecular marker. The primer is shown in SEQ ID No.3 and SEQ ID No.4. The invention further discloses application of the molecular marker and the primer in selection of celery cabbage seed material or transformed offspring containing the mutation type ASM-phyB1 or ASB-phyB2; by the application, screening measures can be simplified greatly, transformation period can be reduced, and blindness in selection of the conventional breeding method is avoided.

Description

technical field [0001] The invention relates to a pair of molecular markers closely linked to the bolting time of Chinese cabbage, in particular to a pair of molecular markers based on the mutation of the photoreceptor gene PHYB promoter region and the application thereof. Materials and new varieties provide tools for auxiliary selection, which can improve selection efficiency, and belong to the field of biotechnology. Background technique [0002] Chinese cabbage (Brassica rapa L. ssp pekinensis) is an important vegetable crop of the cruciferous family. It is native to China and is widely planted all over the world, especially in Southeast Asia. At present, new varieties of Chinese cabbage suitable for cultivation in spring, summer and autumn have been bred, which has enabled the annual supply of Chinese cabbage, which has played a positive role in ensuring the supply of vegetables in the market and stabilizing prices. [0003] Among the Chinese cabbage varieties cultivate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 刘栓桃赵智中张晓燕张志刚李巧云王淑芬卢金东徐文玲刘贤娴付卫民
Owner VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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