Primer and method for authenticating frankliniella occidentalis by using expressed sequence tag (EST) microsatellite markers
A technology of microsatellite marker, Western flower thrips, applied in the field of agricultural biology
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Embodiment 3
[0028] Thrips described in Example 3 was collected in Qingdao City, Shandong Province in 2011; based approach.Agricultural and Forest Entomology, 4(2):127-136.) to detect the above-mentioned thrips, and the thrips collected in Qingdao City, Shandong Province were flower thrips, yellow-breasted thrips, palm thistle Horse, western flower thrips.
[0029] The Tris-HCl, ethylenediaminetetraacetic acid, and sodium lauryl sulfate described in the examples were all purchased from Shanghai Bioengineering Company, and other reagents were common commercially available products.
Embodiment 1
[0031] (1) Extraction of Thrips Genomic DNA
[0032] Put a single head of the sample to be identified in a 0.2ml centrifuge tube containing 60 μl of alkaline lysate: 50mmol 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 Genomic DNA solution.
[0033] (2) PCR amplification of thrips COI gene
[0034] Using the genomic DNA solution prepared in step (1) as a template to perform PCR amplification to obtain a PCR amplification product;
[0035] The PCR amplification system is:
[0036] Genomic DNA solution: 3μl; 20μM primer: 0.5μl; 5U / μl Taq enzyme: 0.5μl; 10×Taq Buffer: 5μl; 10mM dNTP: 1μl; ddH 2 0 to 50μl;
[0037] The primer sequences are as follows:
[0038] Sense primer EST-F: 5'-CTGCCTTTTCCCTTGAT-3'; SEQ ID NO.1
[0039] A...
Embodiment 2
[0043] A method for identifying Thrips occidentalis, the steps are as follows:
[0044] (1) Place individual thrips individuals collected in Jinan City, Shandong Province 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, 1% SDS, fully grind the homogenate with a sealed pipette tip, put it in a water bath at 65°C for 15 minutes, and then put it in a water bath at 95°C for 10 minutes to obtain a genomic DNA solution;
[0045] (2) Using the genomic DNA obtained in step (1) as a template, performing PCR amplification on the EST sequence in the genomic DNA to obtain a PCR amplification product;
[0046] The PCR amplification system is:
[0047] Genomic DNA solution 2μl, 20μM primer 0.5μl, 5U / μl Taq enzyme 0.25μl, 10×Taq Buffer 2.5μl, 10mM dNTP 0.5μl, ddH 2 0 to 25 μl;
[0048] The primer sequences are as follows:
[0049] Sense primer EST-F: 5'-CTGCCTTTTCCCTTGAT-3'; SEQ ID NO.1
[0050] Antisense p...
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