SSR (Simple Sequence Repeat) molecular marking method for identifying variety authenticity and/ or variety purity of high-quality transgenic hybrid cotton CCRI (Chinese cotton research institute) 70

A technology of variety purity and labeling method, which is applied in the field of biotechnology applications, can solve the problems of large environmental impact on morphological identification, heavy identification workload, and seasonal restrictions, and achieve stable and reliable detection results, stable genetics, and simple and convenient operation.

Active Publication Date: 2012-07-18
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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AI Technical Summary

Problems solved by technology

[0003] At present, the identification of the authenticity and / or variety purity of cotton seeds in my country still mainly relies on the identification method of planting morphology in field plots, but the identification of morphology is greatly affected by the environment, the identification workload is large, the cost is high, it takes a long time, and it is limited by seasons (Kuang Meng etc. Research progress of molecular marker technology in identification of cotton varieties. Acta Cotton Sinica, 2009,21(4):330-334)

Method used

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  • SSR (Simple Sequence Repeat) molecular marking method for identifying variety authenticity and/ or variety purity of high-quality transgenic hybrid cotton CCRI (Chinese cotton research institute) 70
  • SSR (Simple Sequence Repeat) molecular marking method for identifying variety authenticity and/ or variety purity of high-quality transgenic hybrid cotton CCRI (Chinese cotton research institute) 70

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Methods: Seed DNA was extracted, PCR was amplified using SSR molecular markers, the amplified products were subjected to gel electrophoresis, the results were counted and characteristic primers were screened.

[0027] 1. Extract cotton genomic DNA.

[0028] The experimental material in this example is the high-quality insect-resistant hybrid cotton "Zhongmian 70" commercial seeds (provided by Hunan Longping Hi-Tech Yahua Cotton Oil Seed Industry Co., Ltd.).

[0029] (1) Remove the outer hard shell of a single cotton seed, thoroughly crush it, transfer it to a 2 mL centrifuge tube, add 800 μL of SDS extract (composition: 1% SDS, 0.01mol / L EDTA, 0.7 mol / L NaCl, 0.05 mol / L Tris, 0.5% sorbitol, 1% PVP, 1% β-mercaptoethanol), after fully mixing.

[0030] (2) Put in a water bath at 65°C for 30 minutes, and shake gently every 10 minutes or so.

[0031] (3)) Add an equal volume of 800 μL phenol:chloroform:isoamyl alcohol (25:24:1), mix up and down until there is no layer...

Embodiment 2

[0054] The four characteristic primers screened out in Example 1 were used to identify the genetic purity of the seeds.

[0055] The experimental material in this example is the high-quality insect-resistant hybrid cotton "Zhongmian 70" commercial seeds (provided by Hunan Longping Hi-Tech Yahua Cotton Oil Seed Industry Co., Ltd.). Generally, 100 seeds are extracted for detection and genetic purity is calculated.

[0056] 1. Cotton genomic DNA was extracted, and the extraction method was the same as in Example 1.

[0057] 2. Use CGR5390, BNL1552, CGR5111 and CIR24 primers, and use the genomic DNA extracted in the first step above as a template for PCR amplification.

[0058] The PCR amplification reaction system is 10 μl, including 6.40 μl ultrapure water, 1.0 μl 10×Buffer (containing Mg 2+ / 20mM), 10mM dNTPs 0.50μ1, 10μM forward primer 0.50μ1, 10μM reverse primer 0.50μ1, 30ng / μ1 template DNA 1.0μ1, 5U / μ1 Taq DNA polymerase 0.10μ1. The PCR reaction program was: pre-denatur...

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Abstract

The invention discloses an SSR (Simple Sequence Repeat) molecular marking method for identifying variety authenticity and/ or variety purity of high-quality transgenic hybrid cotton CCRI (Chinese cotton research institute) 70, which comprises the following steps: extracting genomic DNA (Deoxyribose Nucleic Acid) from a cotton hybrid 'CCRI 70' and the patient seed or the young seedling leaves thereof; taking the genomic DNA extracted in step one as a template, and carrying out PCR (Polymerase Chain Reaction) amplification by an SSR primer; carrying out gel electrophoresis on an amplified product; and analyzing an electrophoresis result, wherein a seed or a signal plant is identified as a real hybrid if the seed or the signal plant has special bands of parents, and is identified as a false hybrid if any one band lacks. The SSR molecular marking method has the advantages of good repeatability, high polymorphism, stable hereditary, stable and reliable detection result and low cost, is free from the influence of environmental condition, is quick and accurate, is simple and convenient to operate and is favorable for quickly detecting on a large scale.

Description

technical field [0001] The invention relates to an SSR marking method for identifying the authenticity and / or variety purity of high-quality insect-resistant hybrid cotton "Zhongmian 70" seeds. These specific primers are used to identify high-quality cotton hybrid F 1 The authenticity identification and / or identification of the variety purity of the seeds of the variety "Zhongmiansuo 70" belongs to the application field of biotechnology. Background technique [0002] Cotton is an important economic crop in our country and plays an important role in the development of our national economy and society. In recent years, the successful breeding and promotion of insect-resistant hybrid cotton has promoted the rapid development of hybrid cotton production in my country, and has become one of the two countries in the world where hybrid cotton is planted on a large scale (Qiu Xinping. Progress in research and utilization of cotton heterosis [J]. China Cotton, 2000, 27 (10): 4-7). I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 袁友禄石玉真李俊文商海红刘爱英龚举武张金凤葛瑞华龚万奎王涛
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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