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Molecular markers for distinguishing two new haplotypes of Brassica rapaL. ssp pekinensis eIF4E.c and application thereof

An eif4e.c, haplotype technology, applied in the determination/inspection of microorganisms, 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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  • Molecular markers for distinguishing two new haplotypes of Brassica rapaL. ssp pekinensis eIF4E.c and application thereof
  • Molecular markers for distinguishing two new haplotypes of Brassica rapaL. ssp pekinensis eIF4E.c and application thereof
  • Molecular markers for distinguishing two new haplotypes of Brassica rapaL. ssp pekinensis eIF4E.c and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Cloning of eIF4E.c in different Chinese cabbage inbred lines

[0034] 1.1 Chinese cabbage genomic DNA extraction

[0035] (1) Put the leaves of Chinese cabbage seedlings into a liquid nitrogen pre-cooled mortar, and grind them into powder in liquid nitrogen;

[0036] (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;

[0037] (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;

[0038] (4) Centrifuge at 12000 rpm for 15 minutes at room temperature;

[0039] (5) Use a pipette to carefully suck out the upper aqueous phase, add it to a new 1.5ml...

Embodiment 2

[0057] Example 2 Development of co-dominant ASM markers

[0058] 2.1 Primer design

[0059] Careful comparison of the two haplotypes of eIF4E.c Hap r and Hap s The difference in the genome sequence is mainly in the InDels in the intron region. In view of this difference, two sets of primers Fd1 and Rd1, Fd2 and Rd2 were designed in the conserved region upstream of the deletion region, and they were commissioned by Huada Gene Co., Ltd. to synthesize them.

[0060] 2.2 Acquisition of ASM mark

[0061] The differential region was amplified by nested PCR technique:

[0062] (1) Use the outer primer combination Fd1 and Rd1 to amplify He102 and 06-247 genomic DNA, and cycle 20 times; use the first PCR product as a template, use the inner primer combination Fd2 and Rd2 to perform the second PCR amplification, and the reaction solution The preparation and amplification conditions are as described in Section 1.2 (2).

[0063] (2) The amplification results were detected by electrop...

Embodiment 3

[0065] Example 3 Identification of Individual Plants in Callback Population (He102×06-247)×06-247 by ASM Markers

[0066] (1) The genomic DNA of different individual plants of the callback population was extracted as described in 1.1.

[0067] (2) PCR amplification: The preparation and amplification conditions of the PCR reaction solution are as described in item (2) of 1.2.

[0068] (3) Detection of PCR products is as described in item (3) of 1.2. Test results such as Figure 4 As shown, P1 and P2 are the parents 06-247 and He102, respectively, and 1-23 are 23 Chinese cabbage materials. In the figure, individual plants 11, 12, 21, and 22 are the haplotypes in the parent P1, namely 06-247, and individual plants 2, 4, 5, 6, 14, and 18 are the haplotypes in the parent P2, namely He102. strains are heterozygous.

[0069]

[0070]

[0071]

[0072]

[0073]

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Abstract

The invention relates to molecular markers for distinguishing two new haplotypes of Brassica rapaL. ssp pekinensis eIF4E.c and application thereof. A marker related to detection of eIF4E.c haplotype Hap<r> in a resistant material is named as ASM-R, has a fragment size of 187bp and contains a sequence shown as a SEQ ID No.1; and a marker related to detection of eIF4E.c haplotype Hap<s> in a high sensitive material is named as ASM-S, has fragment size of 193bp and contains a sequence shown as a SEQ ID No.2. The invention employs homology-based cloning technology to explore two new haplotypes of the eIF4E.c locus, and develops and identifies molecular markers for the two. With the markers, genotype of a backcross generation can be accurately chosen. At the same time, the markers can also be used for the screening of genetic resources of Brassica rapaL. ssp pekinensis, so as to find mutant materials with more abundant genetic background.

Description

technical field [0001] The present invention relates to the development of two different haplotypes of a gene and its related site-specific molecular markers, in particular to the two haplotype mutations of a Chinese cabbage eukaryotic translation initiation factor 4E.c and its site Spot-specific molecular marker development and application. The two haplotypes can provide a source of variation for the selection of virus-resistant Chinese cabbage germplasm containing this site, and site-specific molecular markers can be directly applied to molecular marker-assisted breeding to select Chinese cabbage materials containing this haplotype site The method for improving selection efficiency belongs to the field of biotechnology. Background technique [0002] Chinese cabbage (Brassica rapaL.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 perennial supply of vegetables ...

Claims

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Application Information

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