Quick molecular detection method of novel mycelium T4 injecting wheat stripe rust with AABBDDXX

A technology of wheat stripe rust and bacterial strains, applied in the direction of microbial-based methods, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of identification, long cycle time, difficult standard samples, etc., and achieve high specificity Effect

Inactive Publication Date: 2014-03-26
李强 +2
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Problems solved by technology

[0006] However, the traditional method of identifying hosts has the disadvantages of heavy workload, long cycle, difficulty in identifying a large number of standard samples, and the accuracy is easily affected by external factors such as personnel and identification conditions.
Moreover, the detection of pathogens can only be carried out by collecting leaves for identification and detection after the onset of wheat, which has a certain lag and is not suitable for early detection of pathogenic bacteria species.

Method used

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  • Quick molecular detection method of novel mycelium T4 injecting wheat stripe rust with AABBDDXX
  • Quick molecular detection method of novel mycelium T4 injecting wheat stripe rust with AABBDDXX
  • Quick molecular detection method of novel mycelium T4 injecting wheat stripe rust with AABBDDXX

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Embodiment example

[0031] 1. Sample Collection

[0032] On September 30, 2013, at the Taibai Wheat Stripe Rust Test Station of Northwest Agriculture and Forestry University, standard samples of wheat leaves inoculated with stripe rust T4, Su11-4, V26, CYR29, CYR31, CYR32, and CYR33 but without symptoms were collected. , rinse the wheat leaves with deionized water, and store them at -80°C for later use;

[0033] 2. Molecular detection of new virulent strain T4 of wheat stripe rust

[0034] (1) Using the CTAB method to extract genomic DNA from wheat leaf samples;

[0035] (2) PCR amplification: 15μL reaction system includes: 10×Reaction Buffer 1.5μL, MgCl 2 (25mmol / L) 0.9μL, dNTP S (10mmol / L ) 0.3μL, Taq DNA polymerase (5U / μL, Fermentas Company) 0.15μL, upstream and downstream primers (10ng / μL) each 1.0μL, template DNA (50ng / μL) 1.5μL, ddH 2 O 8.65 μL. Sterile deionized water was used as a negative control. The amplification reaction was carried out on a Bio-Rad S1000 PCR thermal cycler, an...

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Abstract

The invention discloses a quick molecular detection method of a novel mycelium T4 injecting wheat stripe rust with AABBDDXX. The method comprises the following steps: screening to obtain two specific RAPD (Random Amplified Polymorphic DNA) marks S1311 and S1363 of T4 by use of 189 RAPD random primers among the novel mycelium T4, epidemic strains of wheat stripe rust CYR29, CYR31, CYR32, CYR33 and Su11-4 and a novel mycelium V26 injecting an anti-stripe rust gene Yr26; recovering, purifying, cloning and sequencing the specific stripes, designing an SCAR (Sequence Characterized Amplified Regions) primer according to the sequencing result, and successfully converting the RAPD marks to stable SCAR marks T4SP8-1 / T4SP8-2 (the sequence is 5'GGGCGTGGATGAAGAG-3' / 5'-GGAGCAGAAGCAGGTTT3'); stably amplifying the specific stripe of the novel mycelium T4 at 700bp by the marks. The specific SCAR marks T4SP8-1 / T4SP8-2 of the novel mycelium T4 provided by the invention can be used for realizing quick molecular detection of the novel mycelium T4 in an early stage in a large field so as to understand the variation dynamic condition of the pathogenic bacteria, thereby providing a reliable technical support and a theoretical foundation for prophylaxis and treatment policy of the mycelium and the wheat stripe rust.

Description

technical field [0001] The present invention relates to PCR (Polymerase Chain Reaction) technology, RAPD (Random Amplified Polymorphic DNA) and SCAR (Sequence Characterized Amplified Regions) molecular marker technology in molecular biology. Screening of T4-specific molecular markers for new strains of wheat stripe rust in Si" and a new method for rapid detection of new virulent strains T4 in the early stage of wheat pathogenicity using SCAR markers; Background technique [0002] Wheat stripe rust is caused by wheat stripe rust ( Puccinia striformis f. sp. tritici ) is one of the most important diseases of wheat worldwide. my country is the hardest-hit area for wheat stripe rust, mainly distributed in winter wheat areas in Northwest, Southwest, North China, Huaibei and other places and spring wheat areas in Northwest China. In epidemic years, wheat production can be reduced by 20-30%, and in extremely epidemic years, it can be reduced by more than 50%, or even no harves...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/02C12R1/645
CPCC12Q1/6895C12Q2600/156
Inventor 李强王保通樊玉
Owner 李强
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