Rapid detection method for purity of cucumber seeds

A detection method and seed technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of human factors and environmental factors, low accuracy rate, laborious and other problems, and achieve manpower saving and high accuracy , determine the effect of simple

Inactive Publication Date: 2013-04-03
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The object of the present invention is to provide a method suitable for cucumber variety 'Zhexiu 1, aiming at the disadvantages of human factors and environmental factors, such a

Method used

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  • Rapid detection method for purity of cucumber seeds
  • Rapid detection method for purity of cucumber seeds
  • Rapid detection method for purity of cucumber seeds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: (Using three pairs of primers to make the standard map of cucumber variety 'Zhexiu No. 1' respectively)

[0032] (1) Extraction of DNA: Take 3 seeds of 'Zhexiu No. 1', remove the seed coat, and extract genomic DNA by CTAB method;

[0033] (2) PCR amplification: The total PCR amplification reaction system is 20μl, and the amplification reaction system components are: Zhexiu No. 1'seed genomic DNA 20ng each, 10pmol / μl primers CSⅥ-37.2-F770, CSⅥ-24.9-F757 and CSⅤ-18.5-F664 upstream primer, downstream primer each 0.4μl, 2.5mM Mg 2+ 2μl, 2mM dNTP 1μl, 5U / μl Taq DNA polymerase 0.1μl, 10×buffer 2μl, sterile distilled water to make up to 20μl;

[0034]The amplification program of the PCR reaction is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 sec, annealing at 55°C for 1 min, extension at 72°C for 1 min, 35 cycles, extension at 72°C for 10 min, and storage at 4°C;

[0035] (3) Detection of amplified products and preparation of the standard m...

Embodiment 2

[0038] Example 2: (Detection of 'Zhexiu 1' and its parents and mixed seeds using primer CSⅥ-37.2-F770)

[0039] (1) DNA extraction: Take 20 seeds of 'Zhexiu 1', 1 seed of the male parent 'XD23', 1 seed of the female parent 'XH65' and 4 seeds of other cucumber inbred lines artificially mixed in, after removing the seed coat, Genomic DNA was extracted by CTAB method;

[0040] (2) PCR amplification: The total PCR amplification reaction system is 20 μl, and the amplification reaction system components are: Zhexiu No. 1', male parent, female parent, and samples to be tested (in this example, other cucumber inbred lines artificially mixed 20ng of seed genomic DNA, 10pmol / μl of primer CSⅥ-37.2-F770, 0.4μl of upstream primer and downstream primer of primer CSⅥ-37.2-F770, 2.5mM Mg 2+ 2μl, 2mM dNTP 1μl, 5U / μl Taq DNA polymerase 0.1μl, 10×buffer 2μl, sterile distilled water to make up to 20μl;

[0041] The amplification program of the PCR reaction is: pre-denaturation at 94°C for 5 ...

Embodiment 3

[0045] Example 3: (Rapid detection of 'Zhexiu No. 1', parents and samples to be tested using primer CSⅥ-24.9-F757)

[0046] (1) Extraction of DNA: Take 20 seeds of 'Zhexiu 1', 1 seed of the male parent 'XD23', 1 seed of the female parent 'XH65' and 4 seeds of the male parent 'XD23' (in this case, artificially mixed male parent seeds As the cucumber seeds to be tested), after removing the seed coat, the genomic DNA was extracted by the CTAB method;

[0047] (2) PCR amplification: The total PCR amplification reaction system is 20μl, and the amplification reaction system components are: Zhexiu No. 1', 20ng each of male parent, female parent and sample to be tested, 10pmol / μl primer CSⅥ -24.9-F757 upstream primer, downstream primer 0.4μl, 2.5mM Mg 2+ 2μl, 2mM dNTP 1μl, 5U / μl Taq DNA polymerase 0.1μl, 10×buffer 2μl, sterile distilled water to make up to 20μl;

[0048] The amplification program of the PCR reaction is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for...

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Abstract

The invention discloses a rapid detection method for the purity of cucumber seeds, belonging to the technical field of biological detection on vegetable seeds. The method comprises the steps of extracting the DNA (deoxyribonucleic acid) of 'Zhexiu No.1'; carrying out PCR (polymerase chain reaction) amplification on the system, compositions and programs of the DNA; screening SSR (simple sequence repeat) primers; detecting amplification products, and preparing a 'Zhexiu No.1' breed standard map; identifying the purity of sample seeds to be detected, and the like. According to the method, through selecting any one of three pairs of primers to carry out PCR amplification, according to the relative position on a gel strip and in comparison with the standard map, the purity of 'Zhexiu No.1' seeds can be rapidly and accurately detected in 4-5 hours, therefore, the method has the characteristics of convenience in operation, good repeatability, accurate results and the like. The method disclosed by the invention can be popularized and applied in the production, multiplication and selling enterprises of 'Zhexiu No.1' seeds.

Description

technical field [0001] The invention relates to the field of biotechnology for detecting the quality of vegetable crop seeds, in particular to a method for quickly detecting the seed purity of cucumber variety 'Zhexiu No. 1' by using a combination of SSR molecular markers. Background technique [0002] Seed purity is the core indicator of seed quality and the main standard for seed quality grading. At present, the method used in the identification of cucumber seed purity is mainly field phenotyping, but field phenotyping has problems such as long cycle, heavy workload, easy to be affected by environmental factors, and phenotypic characteristics will have a certain degree of deviation, which seriously affects the varieties. The efficiency and accuracy of purity identification. With the acceleration of cucumber breeding process, more and more materials need to be identified for variety purity, and field phenotypic identification has become increasingly unable and unable t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 张鹏周胜军朱育强陈新娟陈丽萍
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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