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GmDGK7 gene molecular marker significantly related with soybean oil content and application of GmDGK7 gene molecular marker

A technology of molecular markers and soybean oil, which is applied in the direction of recombinant DNA technology, microbial measurement/testing, DNA/RNA fragments, etc., can solve problems such as difficult typing, restricted breeding units, lack of functional molecular markers in soybean high-oil molecular breeding, etc. , to speed up the breeding process and improve the breeding efficiency

Active Publication Date: 2015-07-08
NANJING AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

A few SNP markers related to fatty acid metabolism genes (such as FAD2 gene) have been used in molecular breeding for high oleic acid in soybean, but there is still a lack of functional molecular markers for molecular breeding for high oleic acid in soybean
In addition, SNP marker typing generally requires special instruments, and it is difficult to type in conventional laboratories, which limits its wide promotion to breeding units

Method used

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  • GmDGK7 gene molecular marker significantly related with soybean oil content and application of GmDGK7 gene molecular marker
  • GmDGK7 gene molecular marker significantly related with soybean oil content and application of GmDGK7 gene molecular marker
  • GmDGK7 gene molecular marker significantly related with soybean oil content and application of GmDGK7 gene molecular marker

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Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1: the development of the primer pair of the SNP-dCAPS molecular marker of amplifying GmDGK7 gene

[0031] According to the sequence of the molecular marker (as shown in the sequence table Seq ID No.1) in phytozome (http: / / www.phytozome.net / ), the soybean genome sequence with a sequence identity rate equal to 100% was obtained by using the BLAST method. The primer design software PRIMER5.0 was used to design a primer pair that specifically amplifies the sequence Seq ID No.1 containing all or part of the molecular marker. In phytozome (http: / / www.phytozome.net / ), BLAST was performed on the primer pair to detect that the unique product amplified by the primer pair in soybean was the molecular marker sequence shown.

[0032] Use Premier 5.0 software to design primers upstream and downstream of the SNP site:

[0033] (1) The length of the primer is generally 15-30bp;

[0034] (2) The primer sequence has a high similarity within the template, especially the 3' e...

Embodiment 2

[0042] Embodiment 2: the preparation of the SNP-dCAPS molecular marker of GmDGK7 gene

[0043] (1) Using the CTAB method to extract soybean genomic DNA, the specific steps are as follows:

[0044] Step 1: Take 2g of fresh young leaves, grind them into fine powder with liquid nitrogen, add 2×CTAB extract (2% CTAB; 1.4M NaCl, 0.1M Tris-HCl, pH 8.0, 0.1M EDTA) preheated to 65°C , pH 8.0) 15ml, mix well.

[0045] Step 2: Take a water bath at 65°C for 30-45 minutes, and shake gently to mix well. After cooling to room temperature, add an equal volume of chloroform:isoamyl alcohol (24:1), mix gently until the supernatant becomes milky, and centrifuge at 4000rpm for 10min.

[0046] Step 3: Take the supernatant, add an equal volume of isopropanol, and place in an ice bath to precipitate DNA.

[0047] Step 4: Mark out the DNA, wash twice with 70% ethanol, once with absolute ethanol, air-dry the DNA, and dissolve in an appropriate amount of 1×TE solution with pH 8.0. Add RNase to a f...

Embodiment 3

[0056] Example 3: Genotype analysis of the SNP-dCAPS molecular marker of the GmDGK7 gene in representative low-oil and high-oil soybean varieties

[0057] The assay method of grease content: gas chromatograph is American Thermo-Trace GC, and chromatographic column is Agilent capillary column 122-3232DB-FFAP (30m * 0.25mm * 0.25 μ m); Detector is hydrogen flame ion detector (FID), detects The temperature of the instrument is constant at 250°C; the temperature of the injection port is 220°C; the temperature of the column is 200°C; the injection volume is 1 μL, the split injection method is used, and the split ratio is 25:1; the carrier gas is nitrogen, and the nitrogen flow rate is 30.0ml·min- 1. The flow rate of hydrogen is 35.0ml·min-1, and the flow rate of air is 350.0ml·min-1; the heating program is: keep at 200°C for 1min, then rise to 210°C at a rate of 8°C·min-1 for 5min, and then Rise to 220°C at a rate of 5°C·min-1 and keep for 5 minutes. Use the fatty acid standard as...

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Abstract

The invention discloses a GmDGK7 gene molecular marker significantly related with the soybean oil content and application of the GmDGK7 gene molecular marker to solve the problem that soybean high-oil breeding is short of a molecular marker closely linked with the oil content. The nucleotide sequence of the molecular marker is shown by a sequence table Seq ID No.1, a G154-A154 basic group at the 154th site mutates to cause SNP-dCAPs polymorphism. Moreover, the invention also relates to a primer pair shown by sequence tables Seq ID No.2 and Seq ID No.3 for amplifying the molecular marker, and application of the molecular marker and the primers in screening of high-oil and low-oil soybeans and assistant selection of a soybean oil content molecular marker.

Description

technical field [0001] The invention relates to soybean molecular marker assisted breeding, belongs to the field of soybean genetic breeding, in particular to a GmDGK7 gene molecular marker significantly related to soybean oil content and application thereof. Background technique [0002] Soybean is the most important oil crop. Soybean oil accounts for 28% of global vegetable oil production, and fatty acids account for about 20% of soybean seed mass. Fatty acids play an important role in the prevention and treatment of several diseases, including cancer, heart disease and myopia. Soybean oil can also be used as an important raw material for biodiesel. Therefore, increasing the oil content of soybean seeds not only plays an important role in human health, but also has great significance for my country's economic and energy issues. [0003] Soybean oil content is a complex quantitative trait controlled by multiple genes and influenced by the environment. At present, there h...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 李艳陆亮盖钧镒
Owner NANJING AGRICULTURAL UNIVERSITY
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