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Specificity SCAR marker for detecting puccinia striiform

A wheat stripe rust and labeling technology, applied in the fields of biotechnology and plant quarantine, can solve the problems of difficulty in defining the period and scale of initial infection, time-consuming and labor-intensive, and indistinct differences in symptoms at the seedling stage, etc.

Active Publication Date: 2014-10-29
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Not only is it time-consuming and labor-intensive, but the accuracy and reliability of the results also depend to a large extent on the technical level and experience of the surveyors, making it difficult to meet the actual needs of rapid, high-throughput diagnostic testing
The seedling symptoms of leaf rust and stripe rust are not very different, and some grassroots plant protection personnel often confuse the two and cannot accurately distinguish them
During the incubation period of wheat leaf rust, the symptoms of the host are difficult to observe, and it is difficult to determine the period and scale of initial infection

Method used

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  • Specificity SCAR marker for detecting puccinia striiform
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  • Specificity SCAR marker for detecting puccinia striiform

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1. Extraction of wheat pathogenic fungus genomic DNA (gDNA)

[0060] use and so on.

[0061] The gDNAs of the three wheat rusts and powdery mildews were extracted from the susceptible wheat leaves in the latent incubation period and the uredospores or conidia of the pathogenic fungi, and the gDNAs of other wheat pathogenic fungi were derived from the vegetative hyphae. The susceptible wheat leaves or vegetative mycelia were directly frozen and ground into powder with liquid nitrogen, and the spores of rust fungus and powdery mildew were broken by glass bead shaking, and the rest of the DNA extraction operations were carried out according to conventional methods. After digesting RNA with an appropriate amount of RNase, the quality and concentration of gDNA were determined by UV spectrophotometry.

Embodiment 2

[0062] Embodiment 2.PCR comparative analysis and the cloning and sequencing of wheat stripe rust specific nucleic acid sequence

[0063] Referring to the report of "Glass and Donaldson, 1995." on conserved genes in fungi, synthetic primers Bt2a / Bt2b, the nucleotide sequence is: Bt2a: 5'-GGTAACCAAATCGGTGCTGCTTTC-3', Bt2b: ​​5'-ACCCTCAGTGTAGTGACCCTTGGC-3'.

[0064] PCR analysis was carried out on the PTC2220 PCR instrument of MJ Research, Inc.

[0065] The PCR amplification system for gDNA of wheat stripe rust, leaf rust fungus and stem rust fungus is 25 μL, including 2 μL of 10×PCR Buffer (Mg2+Plus), 0.3 μL of dNTP Mixture (each 2.5 mmol / L), Bt2a / Bt2b (10μmol / L) 1μL, template DNA (20ng / μL) 1.0μL, TaKaRa Taq (5U / μL) 0.3μL, ddH2O 16.9μL.

[0066] The PCR reaction conditions were: 94°C for 2 min, 1 cycle; 94°C for 15 s, 55°C for 30 s, 72°C for 90 s, 30 cycles; 72°C for 10 min, 1 cycle.

[0067] The amplified products were separated by agarose gel with a mass fraction of 1.5 and ...

Embodiment 3

[0069] Example 3. Obtaining and Specificity Verification of Wheat Stripe Rust SCAR Marker

[0070]According to the specific DNA fragment sequence of wheat stripe rust obtained by PCR comparison and amplification, the specific primer TF144G / TF323G was designed and screened, and its nucleotide sequence was:

[0071] TF144G: 5'-CCTGCGATGGTGTAGACTCA-3',

[0072] TF323G: 5'-CGGCGTGTATGTTCGTGTTG-3',

[0073] Submitted to Beijing Saibaisheng Gene Technology Co., Ltd. for chemical synthesis. By further optimizing the PCR reaction system and cycle conditions, a 180bp-specific SCAR marker of wheat stripe rust was obtained.

[0074] The PCR reaction system is: 20 ng / μL template DNA 1 μL, 10 μM primer TF144G 1 μL, 10 μM primer TF323G 1 μL, 2×EasyTaq PCR SuperMix 12.5 μL, ddH 2 O9.5 μL.

[0075] The PCR reaction conditions were: pre-denaturation at 94°C for 4 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 54.5°C for 30 s, extension at 72°C for 1 min, and extension at 72°...

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Abstract

The invention relates to a Specificity SCAR marker for detecting puccinia striiform, and belongs to the technical fields of biology and plant quarantine. The length of the nucleotide sequence of the provided specificity SCAR marker is 180 bp. The SCAR marker has a high specificity on detecting puccinia striiform. The marker stably appears in the puccinia striiform standard samples from different wheat producing areas in China, and does not have polluting amplification on puccinia triticcina, puccinia graminis, or other wheat pathogenic fungi. So the SCAR marker can be applied to the diagnosis, detection, investigation, and prediction of wheat stripe rust disease.

Description

technical field [0001] The invention relates to a specific SCAR marker for detecting wheat stripe rust, belonging to the technical fields of biotechnology and plant quarantine. Background technique [0002] Wheat rusts include stripe rust (Puccinia striiformis West.f.sp.tritici Eriks.), leaf rust (Puccinia recondita Rob.ex Desm.f.sp.tritici Erikss.Et Henn.) and stem rust (Puccicinia graminis Pers.f. sp.tritici Erikss.Et Henn.) are three types of fungal diseases caused by rust fungi. This kind of disease is a kind of important disease in China and all wheat-producing countries in the world. The types, distribution and damage degree of the three rusts are different in different countries or regions in the world. [0003] Wheat stripe rust caused by Puccinia striiformis f.sp.tritici is a worldwide cereal disease. According to statistics, it can cause about 40% loss worldwide every year (Ullerup, 1991). my country is the largest wheat stripe rust epidemic area in the world (Wa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/04C12R1/645
Inventor 高利陈万权刘太国刘博
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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