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Primer combination and kit for identifying flue-cured tobacco Zhongyan 100, application and detection method

A technology of primer combinations and kits, which is applied in biochemical equipment and methods, microbial measurement/inspection, recombinant DNA technology, etc., can solve the problems of complex operation and high detection cost, and achieve flexible experimental design, high cost performance, and typing The effect of high accuracy of results

Active Publication Date: 2017-11-17
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method is complicated to operate and the detection cost is very high

Method used

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  • Primer combination and kit for identifying flue-cured tobacco Zhongyan 100, application and detection method
  • Primer combination and kit for identifying flue-cured tobacco Zhongyan 100, application and detection method
  • Primer combination and kit for identifying flue-cured tobacco Zhongyan 100, application and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The primer combination used to identify Zhongyan 100 in flue-cured tobacco is designed for the specific SNP site marker of Zhongyan 100 in flue-cured tobacco, including:

[0046] 1. Genome-wide SNP typing detection of main tobacco varieties.

[0047] The 420K Tobacco SNP array (Affymetrix) was used to scan the whole genome of the main tobacco varieties in my country in recent years, and the SNP typing of the sample DNA was performed relying on the Gene Titan chip platform (Affymetrix) of the National Tobacco Gene Research Center. After whole genome amplification of tobacco sample DNA, the product was randomly fragmented into fragments between 25 and 125 bp. After purification, the fragments were resuspended and hybridized with the 420K Tobacco SNP array. Each SNP is differentially linked by a two-color ligation reaction that occurs on the surface of the chip. Stringent washes are performed after the hybridization process to remove non-specific binding. After the liga...

Embodiment 2

[0054] Flue-cured tobacco Zhongyan 100 identification kit: including 5 mL of the amplification primer mixture (1 μM) in Example 1, 5 mL of 10×PCR buffer, 25 mM MgCl 2 4mL, 25mM dNTPs 2mL, 5U / μL PCR Enzyme 2mL, 1.7U / μL SAP 3mL, 10×SAP buffer 3mL, 10×iPLEX buffer 2mL, 10×iPLEX termination mix 2mL, 33U / μL iPLEXEnzyme 1mL, Example 1 Medium extension primer mixture (1 μM) 5mL, water 200mL.

Embodiment 3

[0056] The identification method of Tobacco 100 in flue-cured tobacco comprises the following steps:

[0057] 1. DNA extraction of the sample to be tested: collect the fresh leaf tissue of the sample, use the Gene Pure Neway PlantGenomic DNA Kit kit (Gene Answer) to extract the genomic DNA of the sample; use the nucleic acid protein analyzer NanoDrop ND-2000 (ThermoFisher Scientific) to detect the DNA concentration, and DNA was diluted to 10ng / μL for use.

[0058] 2. MassARRAY detection: the operation method is carried out according to the requirements of the MassARRAY system platform (Agena), and the reaction is carried out using the iPLEX Gold Reagent Kit (Agena), specifically:

[0059] 1) SNP site multiplex PCR amplification reaction:

[0060] Using the genomic DNA of the sample to be tested as a template, the amplification primers in Example 1 are used to perform multiple PCR amplification reactions to obtain PCR products;

[0061] The reaction system is: PCR buffer (10×...

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Abstract

The invention relates to a primer combination and a kit for identifying flue-cured tobacco Zhongyan 100, application and a detection method, and belongs to the technical field of tobacco variety identification. According to the primer combination and the kit for identifying the flue-cured tobacco Zhongyan 100, application and the detection method, a 420K Tobacco SNP array is utilized to conduct whole-genome SNP typing on the mainly-planted varieties of tobacco in our country in recent years, a set of specific SNP markers suitable for identifying the flue-cured tobacco Zhongyan 100 are obtained through screening according to intervarietal polymorphic SNP sites, wherein the number of the specific SNP markers is thirteen; thirteen SNP site flanking sequences obtained from a reference genome of cultivated tobacco are aligned, the sequences are as shown in SEQ ID NO: 1-13, and primers are designed based on the sequences; and the sites are subjected to typing detection by utilizing the designed primers and adopting a matrix-assisted laser desorption-ionization time of flight mass spectrometry technology. An SNP typing result is obtained according to a detection result, and whether a sample is the Zhongyan 100 or not is identified. By adopting the detection method, the needed detection sample amount is small in the detecting process, the detection throughput is high, and the identification result is accurate.

Description

technical field [0001] The invention relates to a primer combination, a kit, an application and a detection method for identifying flue-cured tobacco Zhongyan 100, and belongs to the technical field of tobacco variety identification. Background technique [0002] Tobacco is an important economic crop and the main raw material for cigarette production. The flue-cured tobacco variety Zhongyan 100 is a high-quality variety bred by the China Tobacco Genetics and Breeding Research (Northern) Center using the high-quality variety NC82 as the central parent and the multi-resistant complementary parent 9201, and then backcrossing NC82 for 5 generations. The new variety of multi-resistant flue-cured tobacco was approved by the National Tobacco Variety Approval Committee in December 2002. The plant is cylindrical, with about 24 leaves, 20-22 leaves can be collected, the leaf shape is oval, the leaf surface is slightly wrinkled, the leaf tip is acuminate, the leaf color is green, the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/156C12Q2600/16
Inventor 张剑锋杨军王燃魏攀吴寿明李锋武明珠王中罗朝鹏
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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