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Primer combination and kit for identifying flue-cured tobacco Bina No. 1, application and detection method

A primer combination and kit technology, used in biochemical equipment and methods, microbial determination/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. Results with high accuracy

Active Publication Date: 2019-12-27
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 Bina No. 1, application and detection method
  • Primer combination and kit for identifying flue-cured tobacco Bina No. 1, application and detection method
  • Primer combination and kit for identifying flue-cured tobacco Bina No. 1, application and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The primer combination used to identify flue-cured tobacco Bina No. 1 is designed for the specific SNP site marker of flue-cured tobacco Bina No. 1, including:

[0047] 1. Genome-wide SNP typing detection of main tobacco varieties.

[0048] 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 ligation react...

Embodiment 2

[0055] Flue-cured tobacco Bina No. 1 identification kit: including 5 mL of 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, in Example 1 Extension primer mixture (1μM) 5mL, water 200mL.

Embodiment 3

[0057] The identification method of flue-cured tobacco Bina No. 1 comprises the following steps:

[0058] 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.

[0059] 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:

[0060] 1) SNP site multiplex PCR amplification reaction:

[0061] 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;

[0062] The reaction system is: PCR buffer (10×) 0....

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Abstract

The invention relates to a primer combination and a kit for identifying flue-cured tobacco Bina No.1, 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 Bina No.1, 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 Bina No.1 are obtained through screening according to intervarietal polymorphic SNP sites, wherein the number of the specific SNP markers is fifteen; fifteen SNP site flanking sequences obtained from a reference genome of cultivated tobacco are aligned, the sequences are as shown in SEQ ID NO: 1-15, 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 Bina No.1 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 Bina No. 1, 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 Bina No. 1 is a new flue-cured tobacco variety selected from the natural variant strain of the flue-cured tobacco variety Yunyan 85 by the Bijie City Company of Guizhou Tobacco Company. It passed the National Tobacco Variety Approval Committee Guizhou Province in October 2012 Approved by the Tobacco Variety Approval Group. The plant type is tower-shaped, and it is tube-shaped after topping. The natural plant height is about 210cm, and the topping plant height is about 150cm. The number of natural leaves is about 32, and the number of harvestable leaves is 24-26. cm, the waist leaf is 78.2cm long, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6895C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2521/525C12Q2533/101C12Q2565/627
Inventor 张剑锋金静静吴寿明何声宝金鑫罗朝鹏谢小东王中杨军
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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