Primer and identifying method for identifying different genetic collateral series of aphelinid
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A technology of solar bees and branches, applied in the field of agricultural biology, can solve problems such as high chance of polymorphism
Inactive Publication Date: 2013-05-08
QINGDAO AGRI UNIV
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In addition, since many restriction endonucleases can digest DNA amplification fragments, there is a greater chance of detecting polymorphisms
[0005] At present, there is no report on the method of using PCR-RFLP technology to construct and distinguish the genetic lineages of Sunbee
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Embodiment 3
[0035] The Sunbee described in Example 3 was collected in Changzhi City, Shanxi Province in 2007; the above Sunbee was tested, and the Sunbee collected in Changzhi City, Shanxi Province was genetic branch 1 and genetic branch 2.
Embodiment 4
[0036] The Sunbee described in Example 4 was collected in Dalian City, Liaoning Province in 2007; the above Sunbee was tested, and the Sunbee collected in Dalian was evenly divided into genetic lineage 2.
[0037] The Alu I endonuclease, Tris-HCl, ethylenediaminetetraacetic acid, and sodium lauryl sulfate described in the examples were all purchased from Shanghai Bioengineering Company, and other reagents were all commercially available products.
Embodiment 1
[0039] (i) Extraction of Sunbee Genomic DNA
[0040] Place a single sunbee in a 0.2ml centrifuge tube containing 60 μl of alkaline lysate: 50 mmol L -1 Tris-HCl (pH8.0), 20mmol·L -1 NaCl, 1mmol L -1 EDTA (ethylenediaminetetraacetic acid) and 1% SDS (sodium dodecyl sulfate) are fully ground and homogenized with a sealed gun head, placed in a water bath at 65°C for 15 minutes, and then placed in a water bath at 95°C for 10 minutes to obtain Sunbee Genomic DNA Solution.
[0041] (ii) PCR amplification of COI gene of sunbee
[0042] Carry out PCR amplification with the solar bee genomic DNA solution as a template to obtain PCR amplification products;
[0046]Sense primer Clade-F: 5'-TCTCATATAATTTGTAATGAAAG-3';
[0047] Antisense primer Clade...
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Abstract
The invention relates to a primer and an identifying method for identifying different genetic collateral series of aphelinid. The identifying method comprises the following steps: (1), extracting aphelinid genome DNA (Deoxyribose Nucleic Acid); (2) carrying out PCR (Polymerase Chain Reaction) augmentation on a mitochondria COI (Cytochrome Oxidase Subunit) gene by adopting the aphelinid genome DNA as a template; (3), digesting the prepared PCR product by restriction enzyme Alu I to obtain enzyme-digested products; and (4), carrying out sepharose gel electrophoretic analysis on the prepared enzyme-digested products. The restriction enzyme is normal restriction enzyme, so that simple and stable enzyme digestion marks are provided for selecting the aphelinid with two genetic collateral series; and meanwhile, an identifying technology for the aphelinid with two genetic collateral series is established, so that the foundation is laid for population dynamics identification for the aphelinid with two genetic collateral series, the biology as well as the research of a mechanism of transmission.
Description
technical field [0001] The invention relates to a primer and a method for identifying genetic branches of sunbees based on PCR-RFLP technology, and belongs to the field of agricultural biotechnology. Background technique [0002] The apple aphid Eriosoma lanigerum (Hausmann) is an important pest of the genus Malus, and is also an important quarantine object at home and abroad. In recent years, the harm in my country has become increasingly serious, and there is a tendency to further expand and spread. Apple cotton aphid mainly parasitizes as wingless viviparous aphids on the wounds of pests, pruning cuts, cracks in old skin, axils of new shoots, short fruit, stem depressions, calyx depressions, fruit stalks and roots of fruit trees. harm. The cell division of the cortical tissue at the damaged part gradually forms tumor-like protrusions, which seriously affects the yield and quality of fruit trees, and even leads to failure and death of the whole fruit tree. The parasitic ...
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