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Multiplex-polymerase chain reaction (PCR) kit for detecting wheat disease-resistant gene and application of kit

A technology related to disease resistance genes and kits, applied in the fields of application, plant gene improvement, recombinant DNA technology, etc., to achieve short time-consuming, strong specificity, and reliable results

Inactive Publication Date: 2012-07-25
CROP RES INST SHANDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2003, Helguera et al. established a set of PCR analysis methods for effectively screening the accumulation of resistance gene Yr17-Lr37-Sr38 in wheat breeding programs, including PCR-based markers, CAPS and TaqMan systems. This dominant marker is associated with rust resistance. The sex genes Yr17, Lr37 and Sr38 are fully linked; Groenewald et al. , 83-85.) also developed the corresponding STS molecular markers (XustSSR2001-7DL) of the Pch1 gene. These good marker systems have played an important role in molecular assisted breeding (MAS); but they all detect a single gene alone, for Simultaneous detection of Yr17-Lr37-Sr38 and Pch1 genes has not been reported

Method used

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  • Multiplex-polymerase chain reaction (PCR) kit for detecting wheat disease-resistant gene and application of kit
  • Multiplex-polymerase chain reaction (PCR) kit for detecting wheat disease-resistant gene and application of kit
  • Multiplex-polymerase chain reaction (PCR) kit for detecting wheat disease-resistant gene and application of kit

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1, multiple PCR kit for amplifying wheat disease resistance-related genes

[0039] The wheat disease resistance-related genes of the present invention are Yr17-Lr37-Sr38 gene cluster and Pch1 gene.

[0040] The multiplex PCR kit for amplifying wheat disease resistance-related genes provided by the present invention consists of the following components:

[0041] 1) 2×Taq Plus PCR MasterMix (Taq enzyme, dNTPs mixture, Tris-HCl, KCl and MgCl 2 Solution): Tiangen Biochemical (Beijing) Technology Co., Ltd., 2×Taq Plus PCRMasterMix (with dye, blue) catalog number: KT205-01.

[0042] 2) Positive control substance (CK1) of the Yr17-Lr37-Sr38 gene cluster: a wheat genome DNA template solution containing the Yr17-Lr37-Sr38 gene cluster;

[0043] 3) Pch1 gene homozygous control substance (CK2): a wheat genome DNA template solution containing the Pch1 gene;

[0044] 4) Control substance (CK3) without Pch1 gene: wheat genome DNA template solution without the Pch1 gene ...

Embodiment 2

[0053] Embodiment 2, multiple PCR amplification of known genotype wheat disease resistance-related genes

[0054] 1. Preparation of wheat genome

[0055] Genomic DNA was extracted from 5 wheat varieties with known genotypes by SDS method, and the genomic DNAs corresponding to 5 wheat varieties with known genotypes were obtained, which were used as templates for multiplex PCR amplification of wheat disease resistance-related genes. Among them, 5 wheat varieties with known genotypes were VPM1, Madsen, Lankao 906, H-93-70 and Jimai 2462. In the following three references, molecular marker detection was performed on the Yr17-Lr37-Sr38 gene cluster or the genotype of the Pch1 gene of the above-mentioned wheat varieties, and the results are shown in Table 1:

[0056] 1) Santra.D.K., Watt.C, Little L.Comparison of a modified assay method for the endopeptidase marker Ep-D1b the Sequence Tag Site marker XustSSR2001-7DL for strawbreaker foot rot resistance in wheat, Plant Breeding, 200...

Embodiment 3

[0074] Embodiment 3, multiple PCR amplification of unknown genotype wheat disease resistance-related genes

[0075] 1. Preparation of wheat genome

[0076] Genomic DNA was extracted from 12 wheat varieties with genotypes to be tested by SDS method, and the genomic DNAs corresponding to 12 wheat varieties with genotypes to be tested were obtained, which were used as templates for multiplex PCR amplification of wheat disease resistance-related genes. Among them, 12 offspring of wheat varieties VPM1 and Jimai 22 to be tested for genotype: Jimai 2462-1-2, Jimai 2462-1-4, Jimai 2462-1-9, Jimai 2463-1-5 , Jimai 2463-1-6, Jimai 2463-2-2, Jimai 2464-1-1, Jimai 2464-1-2, Jimai 2464-1-6, Jimai 2464-1-7, Jimai Mai 2464-2-4, Jimai 2464-3-7. .

[0077] 2. Multiplex PCR amplification of wheat disease resistance-related genes

[0078] Using the genomic DNAs of the 12 wheat varieties of the genotype to be tested prepared in the above step 1 as templates, the multiplex PCR kit of Example 1...

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Abstract

The invention discloses a multiplex-polymerase chain reaction (PCR) kit for detecting a wheat disease-resistant gene and application of the kit. The PCR kit provided by the invention comprises a multiplex-PCR primer pair group used for identifying or auxiliarily identifying the wheat disease-resistant gene, wherein the primer pair group consists of a primer pair specific for a Yr17-Lr37-Sr38 gene cluster and a primer pair specific for a Pch1 gene; the primer pair specific for the Yr17-Lr37-Sr38 gene cluster is the primer pair 1 consisting of two single stranded deoxyribonucleic acids (DNA) shown as the sequence 1 and the sequence 2 of a sequence list; and the primer pair specific for the Pch1 gene is the primer pair 2 consisting of two single stranded deoxyribonucleic acids (DNA) shown as the sequence 3 and the sequence 4 of the sequence list. Inhibition and mispairing do not exist among the primers of the primer pair group, a group of genes is identified through PCR at one time, the kit is reliable in result, high in repeatability and specificity, low in cost and short in time, and a reference basis for breeding wheat disease-resistant varieties is provided.

Description

technical field [0001] The invention relates to a PCR kit for identifying or assisting identification of wheat disease resistance genes and its application, in particular to a pair of multiplex PCR primers for identifying or assisting identification of Yr17-Lr37-Sr38 and Pch1 genes of wheat to be tested Groups, kits and their applications. Background technique [0002] At present, the main diseases in my country's wheat production include wheat stripe rust, powdery mildew, head blight, root rot and take-all. The stripe rust fungus usually invades wheat plants in winter or early spring. As the temperature rises, the stripe rust fungus accelerates its reproduction, often causing a pandemic of stripe rust after wheat heading. It is caused by the wheat stripe rust fungus (Puccinia striiformis f.sp.tritici) Wheat stripe rust is the number one disease of wheat in the country, and in major occurrence years, the yield of susceptible varieties can be greatly reduced or even grain fa...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11A01H1/00
Inventor 曹新有刘建军程敦公宋健民李豪圣刘爱峰赵振东
Owner CROP RES INST SHANDONG ACAD OF AGRI SCI