Method for constructing wheat SSR (single sequence repeat) fingerprint

A fingerprint and construction method technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as heavy workload, and achieve the effects of convenient operation, less environmental impact, and improved test efficiency.

Active Publication Date: 2013-08-14
CROP RES INST SHANDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for constructing wheat SSR fingerprints, and use the constructed SSR fingerprints to identify varieties i

Method used

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  • Method for constructing wheat SSR (single sequence repeat) fingerprint
  • Method for constructing wheat SSR (single sequence repeat) fingerprint
  • Method for constructing wheat SSR (single sequence repeat) fingerprint

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1) DNA extraction

[0022] Table 192 Wheat Varieties

[0023]

[0024]

[0025]

[0026] The DNA of the sample of the tested variety was extracted by CTAB method: take 0.2 g of the young tissue mixture, put it into a 1.5 mL centrifuge tube, and grind it in liquid nitrogen. Or grind the seeds and put them in a 1.5mL centrifuge tube. Preheat the DNA extraction solution to 65°C, add 400 μL to each tube, and mix the sample evenly. Place the centrifuge tube in a metal bath or water bath at 65°C, keep it warm for 30 minutes, and then remove it. Add 400 μL of 24:1 chloroform-isoamyl alcohol (V / V) to the centrifuge tube and shake to mix. Centrifuge at 10000g for 10min. Transfer 200 μL of the supernatant to another 1.5 mL centrifuge tube, add 400 μL of -20°C pre-cooled absolute ethanol to precipitate the DNA. Centrifuge at 10,000 g for 1 min, discard the supernatant, add 500 μL ethanol-ammonium acetate solution, and centrifuge at 6,000 g for 5 min to collect the pr...

Embodiment 2

[0044] Using 25 pairs of core primers (see Table 2, Table 3) to detect 92 wheat varieties (see Table 1) on denatured polyacrylamide gel. The specific operation steps of the test are as follows

[0045] 1) DNA extraction: Same as Example 1.

[0046] 2) PCR amplification: same as Example 1.

[0047] 3) Denaturing PAGE electrophoresis

[0048] a. Pre-electrophoresis

[0049] Install the gel plate on the electrophoresis tank, add about 800mL of 0.25×TBE buffer preheated to 65°C to the upper tank to make it about 3cm above the top of the gel, and add 800mL of 1×TBE buffer to the lower tank. Under 60W constant power, pre-electrophoresis for 10-20min.

[0050] b. Sample preparation

[0051] Add 10 μL of loading buffer to 25 μL of the PCR sample and mix well. Denature in a water bath or a PCR instrument at 95°C for 5 minutes, take it out, and quickly place it on crushed ice.

[0052] c. Electrophoresis

[0053] Rinse the top of the gel several times with the buffer drawn into ...

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Abstract

The invention discloses a method for constructing a wheat SSR fingerprint, which belongs to the field of agricultural wheat breeding and application technologies. The method comprises the following steps: extracting DNA (deoxyribonucleic acid) of a wheat variety to be tested; carrying out PCR (polymerase chain reaction) amplification to the DNA Of the wheat variety to be tested by using 13 pairs of basic core SSR fluorescent dye primers with the ID No.1 to 26 and shown in a sequence table SEQ and 12 pairs of extended core SSR fluorescent dye primers with the ID No.27 to 50 and shown in the sequence table SEQ respectively; and detecting PCR amplification products by denaturing polyacrylamide gel electrophoresis or five-color fluorescent capillary electrophoresis, and constructing the SSR fingerprint of the wheat variety. The method provided by the invention can identify the wheat variety in a laboratory by using technologies of the PCR amplification, the gel electrophoresis and capillary electrophoresis according to the polymorphism of SSR sites, can greatly improve the test efficiency in time, and has the advantages of high speed, high accuracy, convenience for operation and the like; and the identification result is not liable to be affected by the environment.

Description

technical field [0001] The invention relates to a method for constructing a wheat SSR fingerprint, belonging to the technical field of wheat breeding and application in agriculture. Background technique [0002] Wheat belongs to the Triticum genus of the grass family, and it is one of the earliest cultivated crops in the world. After long-term development, it has become one of the food crops with the widest distribution, the largest area, the highest total output, the largest trade volume and the highest nutritional value in the world. There are 43 countries in the world, and 35%-40% of the population takes wheat as the main food. Wheat grains are rich in starch, more protein, a small amount of fat, and a variety of mineral elements and vitamin B. It is a commercial grain with rich nutrition and high economic value. Therefore, the identification of commercial seed varieties is particularly important. [0003] At present, the main method of identifying wheat varieties in m...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 李汝玉张晗段丽丽王东建孙加梅郑永胜王雪梅李华
Owner CROP RES INST SHANDONG ACAD OF AGRI SCI
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