Rhizoctonia solani kuha SSR mark, as well as preparation method and application thereof

A corn sheath blight, marker technology, applied in the direction of biochemical equipment and methods, microorganism-based methods, DNA/RNA fragments, etc., can solve the limited, less genetic research of corn sheath blight, affecting corn yield And other issues

Active Publication Date: 2017-02-15
CHINA NAT RICE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, maize sheath blight is a soil-borne disease widely occurring in maize producing areas, which seriously affects the yield of maize and causes huge economic losses. Its pathogenic bacteria is Rhizoctonia solani ( Rhizoctonia solani Kühn), the host range of this fungus is very wide, it can infect 263 species of plants in 43 families such as rice, corn, soybean, wheat, etc., causing sheath blight, standing blight and other diseases
However, there are few genetic studies on Rhizoctonia solani, especially at the DNA molecular level.
The research foundation of the genetic diversity of Rhizoctonia solani has been relatively weak, and the sequence information available in public databases is relatively limited

Method used

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  • Rhizoctonia solani kuha SSR mark, as well as preparation method and application thereof
  • Rhizoctonia solani kuha SSR mark, as well as preparation method and application thereof
  • Rhizoctonia solani kuha SSR mark, as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0090] Example 1: A preparation method for screening SSR markers of corn sheath blight by magnetic bead enrichment method

[0091] 1. Test materials: 60 corn sheath blight strains.

[0092] 2. Use water agar method to isolate corn sheath blight; for specific methods refer to Zhou Erxun, Yang Mei. A fast and simple technique for isolating Rhizoctonia solani from plant disease tissues. Journal of South China Agricultural University, 1998, 19(1): 125-126.

[0093] 3, get the mycelia of corn sheath blight, adopt the CTAB method to extract the total DNA of the genome; pick the mycelia of corn sheath blight and inoculate it in potato dextrose liquid medium (PDB), shake culture at constant temperature (28 ℃, 120 r / min) for 6 days, the mycelium was collected. For the specific method of extracting total genome DNA by CTAB method, refer to Huang Peitang et al., Molecular Cloning Experiment Guide (Third Edition) (Chinese translation), 2002. Take 5 μL of DNA samples for agarose gel ele...

Embodiment 2

[0142] Example 2: Application of a magnetic bead enrichment method for screening SSR markers of corn sheath blight in analyzing the genetic diversity of corn sheath blight

[0143] (1) Amplify 60 corn sheath blight materials with the designed SSR primers;

[0144] The test materials were all collected from the Fuyang Experimental Base of the China Rice Research Institute in Hangzhou, Zhejiang Province. Total DNA was extracted from 60 mycelia of Rhizoctonia solani and amplified by PCR. The total volume of PCR reaction is 10μL (DNA: 10ng; 10pmol / L upstream and downstream primers: 0.5μL each; 10×Taq Buffer: 1μL; 25mmol / L MgCl 2 : 1μL; 10mmol / L dNTPs: 1μL; 5U / μL Taq enzyme: 0.1μL). The PCR amplification reaction program was: pre-denaturation at 94°C for 5 min; then, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, and 30 cycles; finally, extension at 72°C for 10 min. The PCR products were separated by molecular weight by 8% polyacrylamide g...

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Abstract

The invention discloses a rhizoctonia solani kuha SSR mark, as well as a preparation method and application thereof, and belongs to the technical field of molecular organism. The rhizoctonia solani kuha SSR mark comprises 8 groups of primer pairs, and the preparation method comprises the following steps: firstly, extracting genome DNA of rhizoctonia solani kuha; secondly, obtaining a DNA fragment through restriction enzyme digestion total NDA; thirdly, ensuring that a biotin labeled probe is cross-fertilized with a target fragment; fourthly, enriching a DNA fragment containing an SSR sequence through a magnetic bead; fifthly, amplifying and purifying the DNA fragment containing the SSR sequence; sixthly, performing clone sequencing on the DNA fragment; seventhly, designing an SSR primer through an SSR flanking sequence, which is used for amplifying a microsatellite fragment of a locus. The rhizoctonia solani kuha SSR mark is high in primer specificity, stable in amplification, simple in operation, short in period and low in cost, meanwhile has the advantages of rich codominant inheritance and polymorphism, and can be applied to analysis of the genetic diversity of rhizoctonia solani kuha.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a corn sheath blight SSR marker, a preparation method and an application thereof. Background technique [0002] Microsatellite DNA, also known as simple sequence repeat (SSR), is a repetitive sequence consisting of 2-6 nucleotides evenly distributed in the genome, and is widely distributed in various organisms. Microsatellites have the advantages of high polymorphism, co-dominant inheritance, abundant quantity, high resolution, wide genome coverage distance and easy detection, making SSR markers one of the widely used molecular markers. There are many methods for isolating microsatellite loci, such as database query method, related species screening method, genomic library screening method, enrichment library method, and EST-SSR method. Since the magnetic bead enrichment method has the function of efficiently enriching microsatellite DNA and does not requir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q2600/156
Inventor 王玲黄世文刘连盟
Owner CHINA NAT RICE RES INST
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