Method for establishing DNA (deoxyribonucleic acid) fingerprint of green vegetable hybrid and application thereof

A technology of DNA fingerprinting and establishing method, which is applied in the field of establishing DNA fingerprinting of green cabbage hybrids, can solve the problems that the quality of seeds cannot be supervised in advance, the information of seed purity cannot be provided in time, and additional land is occupied, so as to protect legitimate rights and interests, Ease of reading and analysis

Inactive Publication Date: 2011-06-29
SHANGHAI ACAD OF AGRI SCI +1
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  • Abstract
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AI Technical Summary

Problems solved by technology

The traditional field identification method uses the color, shape and other agronomic traits of seeds, seedlings, leaves, flowers, fruits, pollen and other tissues and organs as the observation indicators for identification. Although it is a relatively feasible method in production practice, field identification requires Additional land occupation, long period, high cost, not only limited by seasons, but also the performance of many traits is affected by cultivation measures and environmental factors, and the observation experience of the appraiser also restricts the accuracy of appraisal
The biggest defect of Daejeon identification is that it cannot provide timely information on the purity of seeds, and cannot provide information on the quality of seeds before a large number of seeds are traded, and cannot play a role in pre-supervising the quality of seeds
Biochemical identification methods include protein electrophoresis and isoenzyme electrophoresis, which have been increasingly widely used, but they cannot fully display the polymorphisms between varieties, and it is difficult to effectively distinguish varieties with close relatives

Method used

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  • Method for establishing DNA (deoxyribonucleic acid) fingerprint of green vegetable hybrid and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0021] In this example, the SDS method was used to extract the genomic DNA of the vegetable variety "Xinlu" and its parents, and its DNA fingerprint was constructed by RAPD, ISSR, and SRAP molecular markers for identification of seed purity. The specific operation is as follows:

[0022] 1. Selected test materials:

[0023] The materials are green vegetable hybrid "Xinlu" and its parents.

[0024] 2. Extraction and purification of vegetable DNA:

[0025] Extract the genomic DNA of each green vegetable material according to the following procedures:

[0026] Take 2g of fresh tissue, grind it into powder in liquid nitrogen, put it in a 50ml centrifuge tube, add 8ml of extraction solution (100mM Tris pH 8.0, 50mM EDTA, 500mM NaCl, 1.5% SDS), keep it at 65°C for 30 minutes, and mix it upside down from time to time Mix well, add 2.5ml 5M potassium acetate in ice bath for 10 minutes, add 0.5ml chloroform, mix upside down, centrifuge at 10000rmp for 8 minutes, take the supernatant...

Embodiment 2

[0036] DNA fingerprint of the present invention is used for the identification of " new green " seed purity:

[0037] Randomly select 50-80 individual plants from the "new green" hybrids produced in the seed production base, extract the DNA of each individual plant sample by micro-DNA extraction method, use the extracted DNA as a template, and perform PCR amplification on it with corresponding primers Incremental and electrophoretic analysis. For example, if the DNA fingerprint of a single plant detected by UBC812 primer analysis is "11010111111111", it is the "New Green" true hybrid strain, and the DNA fingerprint is "10101111111011" or "11010111111110", which is the "New Green" parental inbred strain, that is, the pseudo hybrid strain . For another example, the DNA fingerprint of a single plant detected by M1-E4 primer analysis is "11110111111101111011011", which is the "new green" true hybrid strain, and the DNA fingerprint is "111001011111011101101111" or "111111011110011...

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Abstract

The invention discloses a method for establishing a DNA (deoxyribonucleic acid) fingerprint of green vegetable hybrid and parents thereof, and application thereof. The method comprises the following steps: 1) extracting and purifying a green vegetable DNA; 2) carrying out RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeat) and SRAP (sequence-related amplified polymorphism) analysis by using the acquired high-purity green vegetable DNA as a template; and 3) choosing a typical polymorphism amplification strip to establish the DNA fingerprint of tested material, wherein both the green vegetable hybrid and the parents thereof in the DNA fingerprint have specific DNA fingerprints which can be distinguished. The DNA fingerprint disclosed by the invention is presented in a graphic mode, so the DNA fingerprint is visualized and can be easily understood. The graphic mode of the DNA fingerprint can be converted into a digital code presenting mode, thereby being conveniently read and analyzed by a computer. By using the method disclosed by the invention, the seed purity of the green vegetable hybrid can be identified from the heredity nature, the result is accurate and reliable, and the detection can be quickly performed.

Description

technical field [0001] The invention relates to identification technology of cabbage seeds, in particular to a method for establishing DNA fingerprints of hybrid cabbage and its application. Background technique [0002] At present, many green vegetables (Brassic campestris L. ssp. Chinensis (L.) Makino) varieties popularized and applied in my country are the first generation of hybrids. The first generation of hybrids can synthesize many excellent characters of the parents, and the economic benefits are better. [0003] However, in the process of seed production, due to improper management measures and the defects of self-incompatibility itself, self-seeds, ie false hybrids, are often produced, resulting in a decrease in seed purity. Therefore, improving the purity of hybrids is an important topic in the field of heterosis utilization research. At present, there are roughly three methods for variety identification and seed purity analysis, namely field morphology, biochem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 薄天岳朱玉英马金骏陈锦秀缪体云
Owner SHANGHAI ACAD OF AGRI SCI
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