Method for detecting DNA fingerprint purity in cotton crossbreeds

A technology of purity detection and hybridization, which is applied in the field of molecular biology, can solve the problems of lack of systematic research on variety identification, insufficient identification ability, and high experimental cost, and achieve stable and reliable detection results, which is conducive to standardization and simple and convenient operation. Effect

Inactive Publication Date: 2009-10-21
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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Problems solved by technology

However, there is a lack of systematic research on species identification using the existing molecular marker technology, and there are problems such as insufficient identification ability, many operation steps, long time-consuming, high experimental cost, etc., and cannot fully meet the needs of practical testing.

Method used

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  • Method for detecting DNA fingerprint purity in cotton crossbreeds
  • Method for detecting DNA fingerprint purity in cotton crossbreeds
  • Method for detecting DNA fingerprint purity in cotton crossbreeds

Examples

Experimental program
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Effect test

Embodiment 1

[0030] The purity detection of the 48 seed samples of China Cotton Research Institute includes the following steps. Among them, the primer information used in this method is shown in Table 1; the reagent preparation used is shown in Table 2.

[0031] 1. Single grain cotton DNA extraction

[0032] (1) Shell a single cotton seed, thoroughly pulverize it and transfer it to a 2ml centrifuge tube.

[0033] (2) Add 800ul DNA extraction solution (1% SDS, 0.01M EDTA 8.0, 0.7M NaCl, 0.05M Tris-HCl, 0.5% sorbitol, 1% PVP, 1% β-mercaptoethanol), vortex until fully mixed , in a 65°C water bath for 30 minutes, and shake gently at intervals of about 10 minutes.

[0034] (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 2-5:24:1 in turn), mix up and down until there is no layering, and centrifuge at 10,000 rpm for 10 min.

[0035](4) Transfer the supernatant to another 2ml centrifuge tube, add 1uL RNa...

Embodiment 2

[0069] According to the steps of Example 1, the purity of the 48 seed samples of China Cotton Research Institute was tested using the characteristic primer BNL1705. The results of data statistics and analysis are as follows:

[0070] figure 2 Shown is the purity test result of the characteristic primer BNL1705 on 20 samples of 48 seeds of China National Cotton Research Institute, and the control figure 1 The male parent, female parent and hybrid F of Zhongmian Institute 48 shown in 1 The standard electrophoresis bands of the standard electrophoresis bands show that: ①Paternal band type: sample No. 16; ②Maternal band type: sample No. 7 and No. 20; ③Other hybrid band types: sample No. 13; ④True hybrid band type: except the above Except for the banding patterns of the three hybrids, the other 16 samples were all banding patterns of true hybrids. Therefore, the purity of these 20 Zhongmian Institute 48 seed samples is 80%; the rate of hybrid plants is 20% (wherein the rate of ...

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Abstract

The invention relates to a method for detecting CRI48 DNA fingerprint purity in cotton crossbreeds, which uses an SSR marking technique and uses one or more characteristic primers selected from NAU1190, CIR246, CIR105, BNL1705, NL4030 and NAU1322 to perform detection. The method comprises the following steps: extracting a DNA; and using one or more characteristic primers selected from the NAU1190, the CIR246, the CIR105, the BNL1705, the BNL4030 and the NAU1322 to perform SSR-PCR amplification, electrophoresis and silver staining detection. The method is simple, quick, accurate and stable, and only needs 2 days to finish the purity detection of a submitted sample (100 granules).

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for detecting the purity of the cotton hybrid 48 DNA fingerprint of the cotton hybrid. Background technique [0002] China Cotton Institute 48 is an insect-resistant hybrid cotton variety bred by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences. This hybrid has the characteristics of large bolls and high quality. It has been planted in major cotton areas in my country for many years and has become the main cotton variety in my country. With the increasing number of cotton varieties and the increasingly narrow genetic basis, it is becoming more and more difficult to distinguish cotton varieties based on morphological traits, which brings different degrees of difficulties to various links such as seed production, operation, management, and rights protection. Such as the phenomenon of many varieties, chaos and miscellaneous. In recent years, t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 匡猛杨伟华许红霞王延琴周大云冯新爱
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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