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Construction method for DNA fingerprint database of high flux cotton variety

A DNA fingerprinting and cotton variety technology, applied in the field of molecular biology, to achieve the effect of improving detection efficiency, ensuring accuracy and reliability, and high degree of automation

Inactive Publication Date: 2012-10-17
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Description
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  • Application Information

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Problems solved by technology

However, there is no relevant report on the method of constructing the DNA fingerprint library of cotton varieties in my country based on the multi-color fluorescent labeling detection technology of cotton SSR.

Method used

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  • Construction method for DNA fingerprint database of high flux cotton variety
  • Construction method for DNA fingerprint database of high flux cotton variety
  • Construction method for DNA fingerprint database of high flux cotton variety

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0035] Example 1 Constructing the DNA Fingerprint Library of 96 Cotton Varieties

[0036] The 96 cotton varieties in this example come from Hebei Zhongxin Seed Industry Technology Co., Ltd., Hebei Genesis Technology Co., Ltd., Hebei Nonghua Seed Industry Co., Ltd., Hejian Guoxin Rural Technical Service Association, etc. The names of 96 cotton varieties are shown in Table 4.

[0037] Table 4

[0038]

[0039] 1. DNA extraction

[0040] (1) Shell a single cotton seed, thoroughly pulverize it and transfer it to a 2ml centrifuge tube.

[0041] (2) Add 800ul DNA extraction solution (1%SDS, 0.01mol / L EDTA 8.0, 0.7mol / LNaCl, 0.05mol / L Tris-HCl, 0.5% sorbitol, 1%PVP, 1%β-mercaptoethanol), Vortex until fully mixed, then bathe in water at 65°C for 30 minutes, and shake gently at intervals of about 10 minutes.

[0042] (3) After the water bath is over, add an equal volume of 800 μl of a mixture of phenol, chloroform and isoamyl alcohol (the volume ratio is 25:24:1 in sequence), m...

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Abstract

The invention provides a construction method for a DNA fingerprint database of a high flux cotton variety. The method comprises the following steps: extracting DNAs of the cotton variety, carrying out SSR-PCR amplification by using 40 pairs of primers (with nucleotide sequences as represented by SEQ ID No. 1-80), carrying out fluorescence multiple PCR combination on products obtained after amplification, carrying out capillary five-color fluorescence electrophoresis detection with a DNA analyzer and constructing the DNA fingerprint database of the cotton variety according to a fluorescence spectrum. Compared to detection efficiency of conventional polyacrylamide gel electrophoresis, detection efficiency of the method provided in the invention is increased by more than 10 times; in addition, since the size of fragments of the amplification products is obtained through automatic comparison of internal standard of molecular weight, human errors in plate reading is reduced, and accuracy and reliability of data results are utmostly guaranteed at the same time.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for constructing a high-throughput cotton variety DNA fingerprint library. Background technique [0002] Cotton is the main economic crop in our country. The cultivation and popularization of fine new varieties is of great significance to the development of the national economy and the improvement of people's living standards. According to statistics, from 2000 to 2009, more than 700 cotton varieties passed national and provincial approvals in my country. As of February 29, 2012, 335 cotton varieties have applied for plant variety protection. However, due to the centralized use of backbone parents and the large-scale application of transgenic technology in cotton breeding, the genetic differences among cotton varieties are getting smaller and smaller, making it more and more difficult to identify varieties completely relying on traditional morphological traits. At the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12N15/10C12N15/11C12Q1/68
Inventor 匡猛杨伟华许红霞王延琴周大云冯新爱方丹
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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