Haplotype primer for identifying Q-shaped bemisia tabaci and identification method

A Bemisia tabaci, haplotype technology, applied in the field of agricultural biology

Inactive Publication Date: 2012-09-19
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method of using PCR-RFLP technology to distinguish the haplotypes of Q-type Bemisia tabaci has not been reported yet.

Method used

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  • Haplotype primer for identifying Q-shaped bemisia tabaci and identification method
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  • Haplotype primer for identifying Q-shaped bemisia tabaci and identification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 3

[0031] The Q-type Bemisia tabaci described in Example 3 was collected in Shouguang City, Shandong Province in 2011; 6(10):e25579.doi:10.1371 / journal.pone.0025579.) to detect the above Q-type Bemisia tabaci, and the Q-type Bemisia tabaci collected in Shouguang City, Shandong Province were divided into haplotype 1 and haplotype 2.

Embodiment 4

[0032]The Q-type Bemisia tabaci described in Example 4 was collected in many regions of China in 2011: A: Liaocheng City, Shandong Province, B: Dezhou City, Shandong Province, C: Zaozhuang City, Shandong Province, D: Linyi City, Shandong Province, E : Beijing, F: Shanghai, G: Nanjing, Jiangsu, H: Nanchang, Jiangxi; according to (De Barro P, Ahmed MZ, 2011. Genetic Networking of the Bemisia tabaci Cryptic Species Complex Reveals Pattern of Biological Invasions.PLoS ONE, 6(10):e25579.doi:10.1371 / journal.pone.0025579.) to detect the above Q-type Bemisia tabaci, and the Q-type Bemisia tabaci collected in the above cities were divided into haplotype 1 and Haplotype 2.

[0033] The Hin1II endonuclease described in the examples was purchased from Fermentas Company, Tris-HCl, ethylenediaminetetraacetic acid, and sodium lauryl sulfate were all purchased from Shanghai Bioengineering Company, and other reagents were all commercially available products.

Embodiment 1

[0035] (1) Extraction of Q-type Bemisia tabaci genomic DNA

[0036] Place a single Q-type Bemisia tabaci 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 Genomic DNA solution of type Q Bemisia tabaci.

[0037] (2) PCR amplification of COI gene of Q-type Bemisia tabaci

[0038] Using the Q-type Bemisia tabaci genomic DNA solution and the Q-type Bemisia tabaci haplotype 2 genome DNA solution as templates for PCR amplification to obtain PCR amplification products;

[0039] The PCR amplification system is:

[0040] Type Q Bemisia tabaci 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;

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Abstract

The invention relates to a haplotype primer for identifying Q-shaped bemisia tabaci and an identification method. The method comprises the following steps of: (1) extracting Q-shaped bemisia tabaci genomic DNA; (2) carrying out PCR (Polymerase Chain Reaction) amplification on mitochondrial COI (cytochrome oxidase subunit I) genes of the Q-shaped bemisia tabaci genomic DNA by taking the Q-shaped bemisia tabaci genomic DNA as a template; (3) digesting a PCR product prepared in step (2) by a restriction enzyme Hin1II (N1aIII) to obtain a digestion product; and (4) carrying out agarose gel electrophoresis analysis on the digestion product prepared in step (3). The restriction enzyme is a common restriction enzyme, a simple and stable digestion mark is provided for screening two types of haplotype Q-shaped bemisia tabaci, at the same time an identification technology for the two types of haplotype Q-shaped bemisia tabaci is developed and established, and a foundation is established for future population dynamic identification and biology and invasion mechanism researches of the two types of haplotype Q-shaped bemisia tabaci.

Description

technical field [0001] The invention relates to a primer and an identification method for identifying Q-type Bemisia tabaci haplotypes based on PCR-RFLP technology, and belongs to the field of agricultural biotechnology. Background technique [0002] The whitefly Bemisia tabaci (Gennadius) is a worldwide agricultural pest. Due to its wide host spectrum, strong explosiveness and great harm, it is listed as one of the 100 most harmful invasive species in the world by the International Union for Conservation of Nature (IUCN). At the same time, it is also the only insect that has been dubbed the title of "super pest" in the history of the international scientific and technological circles. In recent years, with the spread of the geminivirus Tomato Yellow Leaf Curl Virus (TYLCV) transmitted by Bemisia tabaci, the harm of Bemisia tabaci has become more and more serious, and even led to the extinction of tomato production in some areas such as Jiangsu, Henan, and Shandong. Bemisi...

Claims

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Application Information

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