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
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[0030] Example 1: Development of a primer pair for amplifying the SNP-dCAPS molecular marker of GmDGK7 gene
[0031] According to the sequence of molecular markers (as shown in the sequence table Seq ID No. 1) in phytozome (http: / / www.phytozome.net / ), using the BLAST method, a soybean genome sequence with a sequence identity rate equal to 100% is obtained. The primer design software PRIMER5.0 is 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 is performed on the primer pair, and the unique product amplified by the detection primer pair in soybean is the indicated molecular marker sequence.
[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 high similarity in the template, especially the 3'end, otherwise it is easy to ca...
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[0042] Example 2: Preparation of SNP-dCAPS molecular marker of GmDGK7 gene
[0043] (1) Using 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℃ , PH 8.0) 15ml, mix well.
[0045] Step 2: Water bath at 65°C for 30-45min, during which shaking gently to mix. After cooling to room temperature, add an equal volume of chloroform: isoamyl alcohol (24:1), gently mix until the supernatant is milky, and centrifuge at 4000 rpm for 10 min.
[0046] Step 3: Take the supernatant, add an equal volume of isopropanol, and place it in an ice bath to precipitate DNA.
[0047] Step 4: Tick off the DNA, wash twice with 70% alcohol, and wash once with anhydrous ethanol to air dry the DNA, and dissolve it in an appropriate amount of 1×TE solution with pH 8.0. Add RNase to a final concentratio...
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[0056] Example 3: Genotype analysis of SNP-dCAPS molecular markers of GmDGK7 gene in representative low-oil and high-oil soybean varieties
[0057] Determination method of oil content: The gas chromatograph is Thermo-Trace GC, the chromatographic column is Agilent capillary column 122-3232DB-FFAP (30m×0.25mm×0.25μm); the detector is a hydrogen flame ion detector (FID) for detection The temperature of the device is constant at 250°C; the temperature of the injection port is 220°C; the column temperature is 200°C; the sample volume is 1μL, split injection mode, split ratio 25:1; carrier gas is nitrogen, nitrogen flow rate is 30.0ml·min- 1. The hydrogen flow rate is 35.0ml·min-1, the air flow rate is 350.0ml·min-1; the heating program is: 200°C for 1 min, then at a rate of 8°C·min-1 to 210°C for 5 minutes, and then The rate of 5℃·min-1 was increased to 220℃ and kept for 5min. Using fatty acid standards as reference materials, prepare a series of concentration gradient standard solu...
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