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Vegetable soy bean DNA finger print map and its construction method and use

A DNA fingerprinting and fingerprinting technology, applied in the biological field, can solve the problems of complex genetic analysis, production and commercial application limitations, and influence on RAPD amplification results.

Inactive Publication Date: 2007-04-11
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current RAPD technology still has the following two problems: (1) The repeatability and stability are poor. There are many factors that will affect the amplification results of RAPD. Before performing RAPD analysis, it is necessary to repeatedly optimize the RAPD analysis conditions
(2) Too sensitive to complicate genetic analysis, because some RAPD markers do not follow the expected genetic pattern
Since AFLP is patented, its production and commercial applications are limited

Method used

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  • Vegetable soy bean DNA finger print map and its construction method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of vegetable soybean fingerprint map:

[0044] 1. Extraction of DNA

[0045] Take ground samples of fresh leaves of various varieties / lines, extract their DNA by CTAB method, and measure the concentration of the resulting DNA stock solution by EB method, and then dilute it into a DNA dilution of about 10ng / ul.

[0046] 2. PCR amplification

[0047] The PCR reaction was carried out on the PCR amplification machine model PTC-225. The reaction system is 10ul, which contains:

[0048] Template genomic DNA solution 4.5ul (10ng / ul)

[0049] 10xbuffer(100mmol / l tris-Hclph8.0 1.36ul

[0050] 500mmol / lkcl 0.1% gelatin)

[0051] dNTP(10mM) 0.24ul

[0052] Taq enzyme (5u / ul) 0.1ul

[0053] Mg2+(25Mm) 0.8ul

[0054] Primer (1ppm) 3.0ul

[0055] Attached PCR reaction program:

[0056] (1) 94 degree pre-denaturation 3min

[0057] (2) 95 degree denaturation 1min

[0058] (3)55 degree annealing for 110s

[0059] (4) 72 degree extension 60s

[0060] (5) 72 degree heat...

Embodiment 2

[0082] Use the vegetable soybean DNA fingerprinting pattern of the present invention for the authenticity detection of a certain vegetable soybean variety:

[0083] After the vegetable soybean standard DNA fingerprint is established, when a certain variety (line) is to be identified, the currently widely used CTAB method is used to extract the DNA, and the extracted DNA is used as a template, and the 13 primers selected are used for PCR Amplify and plot the presence or absence of specific bands on the same SSR site in the electrophoresis pattern of the DNA sample of the vegetable soybean variety (line) based on the primers (see Table 2 for primer sequences). By comparing the DNA fingerprint of the seed to be tested with the standard DNA fingerprint, you can know which of the 28 vegetable soybean varieties (lines) used to construct the DNA fingerprint.

Embodiment 3

[0085] Use the vegetable soybean DNA fingerprinting pattern of the present invention for the authenticity detection of a certain vegetable soybean variety:

[0086] For example, the "Qing Su No. 2" vegetable soybean variety in the test materials is an excellent variety that has been widely promoted and applied in production. The existing batch of seeds cannot be determined whether they are genuine "Qing Su No. 2" seeds. The invented method establishes the DNA fingerprint map of this batch of seeds, and then compares and identifies it against the standard map.

[0087] Identification result: If its DNA fingerprint is consistent with the fingerprint of "Qing Su No. 2" in the standard DNA fingerprinting pattern, this batch of seeds is the real "Qing Su No. 2". If the DNA fingerprint of the sample to be tested is not consistent with the fingerprint of "Qing Su No. 2" in the standard DNA fingerprint, then this batch of seeds is not the real "Qing Su No. 2".

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Abstract

The present invention discloses vegetable soybean DNA fingerprint map and its constituting method and application. The vegetable soybean DNA fingerprint map is constituted with 28 vegetable soybean varieties as the test material and by means of DNA extraction, PCR amplification, PCR produce identification and data analysis. The DNA fingerprint map may be used in identification of soybean varieties and purity.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a plant DNA fingerprint map and its construction method and its application for plant species identification. Background technique [0002] Vegetable soybeans are one of the important characteristic vegetables that have been developed in recent years and are welcomed by the international market, especially the traditional characteristic vegetables that the residents of southern my country like to eat. The annual cultivation area is more than 5 million mu, and there is a trend of continuous expansion. [0003] The selection and breeding of new vegetable soybean varieties has always been the main goal of soybean breeders in Japan and Taiwan Province of my country. Breeders in Taiwan Province of my country have been studying vegetable soybean breeding since the 1950s, and have successively introduced many excellent varieties such as Taiwan 290, Taiwan 292, Taiwan 75-1, 9308, KVS844 and KVS86...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12Q1/68
Inventor 顾卫红麻浩王丽群马坤宋荣浩高文瑞杨红娟
Owner SHANGHAI ACAD OF AGRI SCI
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