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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

Inactive Publication Date: 2013-08-21
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the construction of a method for distinguishing Western flower thrips using EST microsatellite markers

Method used

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  • Primer and method for authenticating frankliniella occidentalis by using expressed sequence tag (EST) microsatellite markers
  • Primer and method for authenticating frankliniella occidentalis by using expressed sequence tag (EST) microsatellite markers
  • Primer and method for authenticating frankliniella occidentalis by using expressed sequence tag (EST) microsatellite markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 3

[0028] The thrips described in Example 3 were collected in Qingdao, Shandong Province in 2011; according to (PC Brunner et al., 2002. A molecular identification key for economically important thrips species (Thysanoptera: Thripidae) using direct sequenciong and a PCR-RFLP -based approach. Agricultural and Forest Entomology, 4(2): 127-136.) to detect the above-mentioned thrips. The thrips collected in Qingdao City, Shandong Province are respectively flower thrips, yellow-breasted thrips, Thrips palm, thrips western flower.

[0029] Tris-HCl, ethylenediaminetetraacetic acid, and sodium lauryl sulfate described in the examples were purchased from Shanghai Bioengineering Company, and other reagents were common commercially available products.

Embodiment 1

[0031] (1) Extraction of thrips genomic DNA

[0032] Put the single-head sample to be identified in a 0.2ml centrifuge tube containing 60μl of alkaline lysis solution, the alkaline lysis solution is: 50mmol·L -1 Tris-HCl (pH8.0), 20mmol·L -1 NaCl, 1 mmol·L -1 EDTA (ethylenediaminetetraacetic acid), 1% SDS (sodium dodecyl sulfate), fully ground and homogenized with a sealed pipette tip, placed in a water bath at 65°C for 15min, and then in a 95°C water bath for 10min to obtain Genomic DNA solution.

[0033] (2) PCR amplification of thrips COI gene

[0034] PCR amplification is performed using the genomic DNA solution prepared in step (1) as a template 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...

Embodiment 2

[0043] A method for identifying Western flower thrips, the steps are as follows:

[0044] (1) The single-headed thrips individuals collected in Jinan City, Shandong Province were placed in a 0.2 ml centrifuge tube containing 60 μl of alkaline lysis solution: 50 mmol·L -1 Tris-HCl (pH8.0), 20mmol·L -1 NaCl, 1 mmol·L -1 EDTA, 1% SDS, fully grind the homogenate with a sealed pipette tip, place it in a water bath at 65°C for 15 minutes, and then in a 95°C water bath 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 25μl;

[0048] The primer sequences are as follows:

[0049] Sense primer EST-F: 5'-CTGCCTTTTCCCTTGAT-3'; SEQ ID NO.1

[0...

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Abstract

The invention relates to a primer and a method for authenticating frankliniella occidentalis by using expressed sequence tag (EST) microsatellite markers. The method comprises the following steps of: (1) extracting the genomic DNA of thrips; (2) performing polymerase chain reaction (PCR) on the genomic DNA of thrips, which serves as a template; and (3) performing sepharose gel electrophoretic analysis on an enzyme-digested product prepared in the step (2). By the authentication method, simple and stable molecule markers are supplied to screening of frankliniella occidentalis and other three types of thrips and a foundation is laid for the future research on the population dynamic authentication, the biology and an invasive mechanism of frankliniella occidentalis.

Description

technical field [0001] The invention relates to a primer and a method for identifying Western flower thrips by using EST microsatellite markers, and belongs to the field of agricultural biotechnology. Background technique [0002] The western flower thrip (Frankliniella occidentalis) is a worldwide invasive pest. In addition to causing harm to plants, it also spreads a variety of plant viruses, causing serious losses to flowers and crops. Western flower thrips have been widely distributed in more than 60 countries and regions such as the United States, the Netherlands, and Japan. In 2000, my country first intercepted the worm on the Burmese bonsai exhibited at Kunming International Flower Festival in Yunnan Province. Western flower thrips occur mixed with other thrips, and the nymphal stage is similar in shape and difficult to distinguish. Effective differentiation of Western flower thrips is the early stage and basis for the control of this insect, and it has important t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 褚栋陶云荔国栋
Owner QINGDAO AGRI UNIV
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