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Specific molecular marker of Brassica rapa L.ssp pekinensis eIF (iso) 4E.a locus large fragment deletion mutation and application thereof

A technology of asm-iso4e.a, deletion mutation, applied in the direction of DNA / RNA fragments, recombinant DNA technology, etc., to achieve the effect of improving screening efficiency, stable results, and easy operation

Inactive Publication Date: 2013-05-15
VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Up to now, there have been no reports at home and abroad on the mutation of Chinese cabbage at the above two gene loci and the development of markers for the detection of related mutants.

Method used

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  • Specific molecular marker of Brassica rapa L.ssp pekinensis eIF (iso) 4E.a locus large fragment deletion mutation and application thereof
  • Specific molecular marker of Brassica rapa L.ssp pekinensis eIF (iso) 4E.a locus large fragment deletion mutation and application thereof
  • Specific molecular marker of Brassica rapa L.ssp pekinensis eIF (iso) 4E.a locus large fragment deletion mutation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Cloning of eIF4E.a in different Chinese cabbage inbred lines

[0030] 1.1 Genomic DNA extraction from Chinese cabbage

[0031] (1) Put the leaves of Chinese cabbage seedlings into a liquid nitrogen pre-cooled mortar, and grind them into powder in liquid nitrogen;

[0032] (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;

[0033] (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;

[0034] (4) Centrifuge at 12000 rpm for 15 minutes at room temperature;

[0035] (5) Use a pipette to carefully suck out the upper aqueous phase, add it to a new ...

Embodiment 2

[0053] Example 2 Development of co-dominant ASM markers

[0054] 2.1 Primer design

[0055] Carefully compare the genome sequences of BraeIF(iso)4E.a (wild type) and BraeIF(iso)4e.a (mutant), the difference is mainly the deletion of the 3' end of the latter. For such differences, the upstream of the deletion region is conserved A forward primer (as shown in SEQ ID NO.3) is designed for the region, and a reverse primer Rd is used as a primer for genome sequence amplification (as shown in SEQ ID NO.4). The primers were synthesized by Jinan Boshang Biotechnology Co., Ltd.

[0056] 2.2 Acquisition of ASM mark:

[0057] (1) Amplification in He102 and 06-247 by combination of forward and reverse primers: The preparation of PCR reaction solution and amplification conditions are as described in item (2) of 1.2.

[0058] (2) The detection of the amplification result is as described in item (3) of 1.2.

[0059] (3) Amplification results such as image 3 As shown, the two primers ca...

Embodiment 3

[0060] Example 3 Identification of Individual Plants in Backcross Population (He102×06-247)×He102 by ASM Markers

[0061] (1) Genomic DNA extraction from different individual plants of the backcross population was as described in 1.1.

[0062] (2) PCR amplification: The preparation and amplification conditions of the PCR reaction solution are as described in item (2) of 1.2.

[0063] (3) Detection of PCR products is as described in item (3) of 1.2. Test results such as Figure 4 As shown, 1-21 is 21 parts of Chinese cabbage materials, M is DNA molecular weight standard DL2000. As can be seen from the figure, Figure 4 1-4 in A are heterozygous, 5-10 are mutants, Figure 4 11-21 in B is wild type.

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Abstract

The invention discloses a locus specific co-dominant ASM (allele specific marker) directly related to identification of Brassica rapa L.ssp pekinensis eIF (iso) 4E.a wild type and mutant. The marker related to wild type locus detection is named as ASM-iso4E.a, has fragment size of 1636bp, and is shown as a SEQ ID No.1. The corresponding mutation locus detection related marker is named as ASM-iso4e.a, has fragment size of 640bp, and is shown as a SEQ ID No.2. In the invention, by making use of a homology based cloning technology, a mutant of the eIF (iso) 4E.a locus is discovered, and the molecular marker for identifying the locus is developed. The marker can be utilized for accurate selection of the genotype of backcrossing transformation progenies. Meanwhile, the marker also can be used for screening Brassica rapa L.ssp pekinensis germplasm resources so as to seek mutant materials with more abundant genetic backgrounds.

Description

technical field [0001] The present invention relates to the development of a mutant of a gene and its related site-specific molecular markers, in particular to the mutation of a Chinese cabbage eukaryotic translation initiation factor 4E isomer eIF(iso)4E.a and its site Spot-specific molecular marker development and application. The mutant can provide a source of variation for the selection of virus-resistant Chinese cabbage germplasm containing the mutation site, and the site-specific molecular markers can be directly applied to molecular marker-assisted breeding to select Chinese cabbage materials containing the mutation site and improve the eIF( The invention discloses selection efficiency of iso)4E mutant, and belongs to the field of biotechnology. Background technique [0002] Chinese cabbage (Brassica rapa L.ssp pekinensis) is an important vegetable crop of the Brassicaceae family, which originated in China and is currently grown all over the world. Especially in the...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 刘栓桃赵智中张志刚李巧云卢金东
Owner VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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