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InDel mark relating to mutation of untranslated region of Chinese cabbage PHYB gene mRNA5'and application thereof

A Chinese cabbage and gene technology, which is applied to InDel markers related to mutations in the 5' untranslated region of Chinese cabbage PHYB gene mRNA and its application field, can solve problems such as differences in flowering time, large differences in bolting and flowering characteristics, and achieve simple operation. Effect

Inactive Publication Date: 2014-04-30
VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
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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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  • InDel mark relating to mutation of untranslated region of Chinese cabbage PHYB gene mRNA5'and application thereof
  • InDel mark relating to mutation of untranslated region of Chinese cabbage PHYB gene mRNA5'and application thereof
  • InDel mark relating to mutation of untranslated region of Chinese cabbage PHYB gene mRNA5'and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Cloning of PHYB gene promoter sequence in different Chinese cabbage inbred line materials of embodiment 1

[0034] 1.1 Chinese cabbage total RNA 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 1.5ml centrifuge tube immediately, add about 1ml Trizol reagent for every 100mg of material, shake and mix, leave at room temperature for 5 minutes, then add 200μl chloroform, shake and mix, and place at room temperature for 5 minutes;

[0037] (3) Centrifuge at 12,000 rpm for 15 minutes at 4°C;

[0038] (4) Carefully suck out the upper aqueous phase with a pipette, add to a new 1.5ml centrifuge tube, add 500μl of isopropanol (according to 1:1 volume ratio), mix well, and settle at room temperature for 10min;

[0039] (5) Centrifuge at 12000rpm for 10min at 4°C, discard the supernatant carefull...

Embodiment 2

[0055] Example 2 Development of co-dominant InDel markers

[0056] 2.1 Primer design

[0057]Carefully compare the genome sequences corresponding to the ESTs of the two allelic variants phyB3 and phyB4 of the PHYB gene. The differences are mainly in the small insertion / deletion mutations of 20 bp near the start codon (such as figure 2 shown). Design primers in the conserved regions on both sides as follows:

[0058] Forward primer PF2: 5'-AGAATCTCTCGGCTTCAATTTTC-3';

[0059] Reverse primer PR2: 5'-CCGCCGCCGACTCCGGAA-3';

[0060] The primer sequences are shown in SeqID No.3 and 4, which were synthesized by Huada Genomics Co., Ltd.

[0061] 2.2 Obtaining InDel mark

[0062] (1) Use the genomic DNA of He102 and 06-247 as templates, and perform a PCR amplification with common taq enzyme. Extend at 72°C for 40 seconds, 35 to 38 cycles, and finally extend at 72°C for 5 minutes.

[0063] (2) The amplified product was detected by electrophoresis on a 6% polyacrylamide gel, and...

Embodiment 3

[0064] Example 3 Detection of InDel markers on the F2 population constructed by He102×06-247.

[0065] (1) The CTAB method was used to extract genomic DNA from different individual plants of the F2 population.

[0066] (2) PCR amplification: The preparation and amplification conditions of the PCR reaction solution are as described in item (2) of 1.2.

[0067] (3) Detection of PCR products is as described in Section 2.2 (2). Test results such as Figure 5 Shown: P1 and P2 are the parents He102 and 06-247 respectively, 1-45 are 45 F2 individual plants. In the figure, there is no amplified band for individual plant 18, which may be due to a problem with the genome extraction. 1, 19, 22, 23, 26, 27, 33, and 44 are the allelic variation type phyB4 in parent P1, that is, He102. Individual plants 2, 3, 5, 9, 10, 12, 13, 16, 28, 35, 37, 45 are the allelic variation type phyB3 in the parent P2, that is, 06-247, and the rest of the individual plants are heterozygous. It can be seen ...

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Abstract

The invention discloses a co-dominance InDel mark relating to the mutation of a noncoding region at 5'end of Chinese cabbage PHYB gene mRNA5'. A mark relating to phyB3 detection is named as InDel-phy B3, and the fragment size is 139bp, shown in SEQIDNo.1; a mark relating to phyB4 detection is named as InDel-phy B4, and the fragment size is 121bp, shown in SEQIDNo.2. The invention further discloses a pair of primers for detecting the InDel mark: the sequence is shown in SEQIDNo.3 and No.4. The invention further discloses application of the mark and the primers in selecting germplasm materials containing InDel-phy B3 or InDel-phy B4 mutation types or in offspring breeding, an assistant selection tool for cultivating bolting resisting Chinese cabbages can be provided, the application of the invention ensures that selection means are greatly simplified, the transformation age limit is shortened, and selecting blindness in a conventional breeding method is avoided.

Description

technical field [0001] The invention relates to a pair of molecular markers related to the mutation of Chinese cabbage bolting time candidate functional genes, in particular to a pair of co-dominant InDel markers related to the mutation of the photoreceptor gene PHYB mRNA 5' untranslated region (UTR) and its application. The marker provides an auxiliary selection tool for breeding materials and new varieties containing corresponding types of bolting-resistant Chinese cabbage, can improve selection efficiency, and belongs to the field of biotechnology. Background technique [0002] Chinese cabbage (Brassicarapa L. ssppekinensis) 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 different seasons of spring, summer and autumn have been bred, which has enabled the annual supply of Chinese cabbage, which has...

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张晓燕徐文玲刘贤娴付卫民
Owner VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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