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Genetic detection method of powdery mildew-resistant near-isogenic lines Brock/Jing411<7>

A powdery mildew resistance gene and base sequence technology, which is applied in the field of molecular detection and application of genetic background of gene lines, can solve the problems of long cycle, low selection efficiency, developmental and environmental conditions, etc.

Inactive Publication Date: 2012-05-30
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cultivation and selection of NIL takes 7-8 years, which is a long cycle, and the evaluation index of selection efficiency is backcross algebra and appearance shape. This method is easily affected by development and environmental conditions, and the selection efficiency is low.

Method used

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  • Genetic detection method of powdery mildew-resistant near-isogenic lines Brock/Jing411&lt;7&gt;
  • Genetic detection method of powdery mildew-resistant near-isogenic lines Brock/Jing411&lt;7&gt;
  • Genetic detection method of powdery mildew-resistant near-isogenic lines Brock/Jing411&lt;7&gt;

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the preparation of near isogenic line:

[0037] The preparation of the near-isogenic line of the present invention: the parent of the disease-resistant gene donor is from the British common wheat variety Brock, and it is crossed with the parent Jing 411, which has excellent agronomic traits but is susceptible to the disease, and the hybrid combination is "Brock / Jing 411", and the obtained F 1 They are all resistant to powdery mildew. Backcross with Beijing 411, under the stress of Physiological race No. 15 of powdery mildew that is prevalent in North China, select a single disease-resistant plant, and after 7 generations, self-cross again to obtain the powdery mildew resistant near-isogenic line Brock / Beijing 411 7 . figure 1 It is Jing 411, near-isogenic line Brock / 015 / / Jing 411 7 , near isogenic line Brock / Jing 411 7 Compared with Brock leaves infected with bacteria for 2 weeks, it was found that 411 was seriously infected with bacteria, and the hyp...

Embodiment 2

[0038] Embodiment 2, the separation of genomic DNA:

[0039] Take 0.2g of etiolated seedling leaves and grind them in liquid nitrogen until they are as powder as possible, quickly add 2ml of extraction buffer (50mM Tris-HCl, pH 8.0; 20mM EDTA; 50mM NaCl), mix gently, and incubate at 65°C for 15min, during which Mix by inversion 2-3 times. Ice bath at 0°C for 10 minutes, centrifuge at 0-4°C at 3000 rpm for 15 minutes, remove the supernatant, add it to a new tube, add an equal volume of Tris-HCl (pH 8.0) saturated phenol, and mix gently. Centrifuge at 0-4°C at 3000 rpm for 10 minutes, take the supernatant, add an equal volume of phenol, chloroform-isoamyl alcohol (25:24:1) for extraction, and mix gently. Centrifuge at 0-4°C at 3000 rpm for 10 minutes, take the supernatant, add an equal volume of chloroform-isoamyl alcohol (25:24:1) for extraction, and mix gently. 0-4°C, 3000 rpm, centrifuge for 10 minutes, take the supernatant, add 2 times the volume of cold ethanol, and preci...

Embodiment 3

[0040] Embodiment 3, genetic background detection:

[0041] AFLP analysis refers to Vos (Vos, P., R.Hogers, M.Bleeker, M.Reijans, T.van de Lee, M.Hornes, A.Frijters, J.Pot, J.Peleman, M.Kuiper, M. Zabeau. AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res, 1995, 21: 4407-4414) and other methods. The adapters and primers used are listed in Table 1.

[0042] 1) Genomic DNA double digestion and adapter ligation: 100×BSA 0.2μl, NEB buffer 4.0μl, Pst I (20U / μl) 0.25μl, Mse I (10U / μl) 0.5μl, Pst I linker (5pM) 1.0μl , Mse I adapter (50pM) 1.0μl, T4 DNAligase 0.4μl, 10×T4buffer 2.0μl, DNA (100ng / μl) 5.0μl, ddH 2 O make up 40 μl. Incubate at 37°C for 8 hours.

[0043] 2) Preamplification: The reaction system is 25 μL, containing 1 μL of ligation product, 0.6 pmol / L primer, 2.5 μL of 10×PCR buffer, 0.2 mmol / L dNTPs, and 1 U Taq polymerase. The PCR amplification program was 94°C for 2min; 94°C for 35S, 56°C for 35S, 72°C for 60S, 30 cycles; 72°C for 5min. The pre-ampl...

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Abstract

The invention discloses a molecular detection method of a mating system and a genetic background of powdery mildew-resistant near-isogenic lines Brock / Jing411<7>, and a nucleotide sequence of a molecular marker P15 / M14-160 closely linked with a powdery mildew-resistant gene, which belong to the field of gene engineering of agricultural biology. The near-isogenic lines are obtained by hybridizing common powdery mildew-resistant wheat Brock serving as a disease-resistant gene donor parent with cultivated wheat Jing411 which has high agronomical characters, does not resist powdery mildew and serves as a recurrent parent, wherein the first generation (F1) is continuously backcrossed with the Jing411 for seven generations and inbred for one generation to breed the near-isogenic lines Brock / Jing411<7>. Disease-resistant single plants are selected under the stress of the powdery mildew during every hybridization and backcross. Four AFLP spectral patterns are disclosed, namely parental ditype, partial disease-resistant donor Brock type, partial recurrent parent Jing411 type and crossover type, and can reflect the biparental genetic background in the near-isogenic lines.

Description

technical field [0001] The invention belongs to agricultural biotechnical engineering, and relates to the preparation of powdery mildew-resistant near-isogenic lines, in particular to the molecular detection and application of the genetic background of wheat Brock powdery mildew-resistant near-isogenic lines. technical background [0002] Near-isogenic lines (Near-isogenic Lines, NIL) were proposed by Yong et al., which refers to a group of strains with similar genetic background and different target genes (Young N D, Zamir D G, Tanksley S D. Use of isogenic lines and simultaneoue probing to identify DNA markers tightly linked to the TM-Za gene in tomato.Genetics.1988, 120:579-585), which can be used in gene pleiotropic analysis, target gene isolation, gene fine-mapping and cloning, etc. (Tian Qingzhen, Zhou Ronghua, Jia Jizeng. Molecular detection of genetic background of wheat powdery mildew resistant near-isogenic lines. Acta Crops, 2004, 30(3): 205-209; Zhang Yi, Li Yunf...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 王振英彭永康刘晓颖陈欧陈宏
Owner TIANJIN NORMAL UNIVERSITY
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