Citrus Mitochondrial Indel Molecular Marker and Its Application
A molecular marker, mitochondrial technology, applied in recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problems of poor specificity and narrow application range of citrus, achieve strong stability, save identification time, distinguish high rate effect
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Embodiment 1
[0058] (1) DNA extraction: Genomic DNA of 62 varieties of Citrus, Hovenia citrus, and kumquat were extracted by CTAB method, and the variety numbers are shown in figure 1 Description of drawings.
[0059] (2) PCR amplification: Utilize primer pair HBF / HBR, G1F / G1R, ZKF / ZKR, SJGF / SJGR to carry out PCR amplification to the genomic DNA of above-mentioned 62 varieties respectively, utilize primer pair BTCF / BTCR pair No. 1-25 varieties were amplified by PCR. The reaction system for PCR amplification is: 5 μL of PCR reaction Mix, 0.5 μL of forward and reverse primers (10 mmol / L), 1 μL of DNA template (100 ng / μL) and ddH 2 O 3 μL. The PCR amplification steps are: denaturation at 94°C for 5 minutes; 35 cycles of 94°C, 30s, 55°C, 30s, 72°C, 60s; 72°C, 5min, 12°C, 30min, and storage at 4°C.
[0060] (3) Electrophoresis detection: use 2.5% agarose gel electrophoresis to detect the amplified product, the conditions of the electrophoresis solution are: 1×TAE buffer solution (0.04M Tris-...
Embodiment 2
[0063] (1) DNA extraction: Genomic DNA of cytoplasmic hybrids Huayou 2, Guoqing 1 Wenzhou mandarin and Huayou 1 were extracted by CTAB method.
[0064] (2) PCR amplification: the genomic DNA of the above-mentioned citrus varieties was amplified by using the primer pair HBF / HBR. The reaction system for PCR amplification is: 5 μL of PCR reaction Mix, 0.5 μL of forward and reverse primers (10 mmol / L), 1 μL of DNA template (100 ng / μL) and ddH 2 O 3 μL. The PCR amplification steps are: denaturation at 94°C for 5 minutes; 35 cycles of 94°C, 30s, 55°C, 30s, 72°C, 60s; 72°C, 5min, 12°C, 30min, and storage at 4°C.
[0065] (3) Electrophoresis detection: use 2.5% agarose gel electrophoresis to detect the amplified product, the conditions of the electrophoresis solution are: 1×TAE buffer solution (0.04M Tris-acetate, 0.001M EDTA, pH=8.0), voltage 60V , electrophoresis 80min. After electrophoresis, use the gel imaging system (UVP) to take pictures of the gel and save the pictures.
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Embodiment 3
[0068] (1) DNA extraction: Genomic DNA of cocktail grapefruit, Shatian pomelo, National Day No. 1 Wenzhou mandarin and rock sugar orange were extracted by CTAB method.
[0069] (2) PCR amplification and electrophoresis detection 1: Genomic DNA of cocktail grapefruit, Shatian pomelo, rock sugar orange, and National Day No. 1 Wenzhou mandarin were amplified by using the primer pair HBF / HBR. The reaction system for PCR amplification is: 5 μL of PCR reaction Mix, 0.5 μL of forward and reverse primers (10 mmol / L), 1 μL of DNA template (100 ng / μL) and ddH 2 O 3 μL. The PCR amplification steps are: denaturation at 94°C for 5 minutes; 35 cycles of 94°C, 30s, 55°C, 30s, 72°C, 60s; 72°C, 5min, 12°C, 30min, and storage at 4°C. The amplified product was detected by 2.5% agarose gel electrophoresis, 1×TAE buffer solution (0.04M Tris-acetate, 0.001M EDTA, pH=8.0), voltage 60V, electrophoresis for 80min. After electrophoresis, use the gel imaging system (UVP) to take pictures of the gel an...
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