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Loach ploidy identification primer based on microsatellite polymorphism and application

A microsatellite polymorphism and ploidy identification technology, which is applied in the field of loach ploidy identification primers, can solve the problems of expensive flow cytometer, inability to analyze ploidy, and small samples that cannot be sampled and analyzed. The effect of high accuracy and simple method

Active Publication Date: 2017-08-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, flow cytometers are expensive and require specialized technicians to operate
In addition, the above methods all have the following limitations: (1) only live samples can be tested, and the ploidy cannot be analyzed for long-term preserved experimental fish materials without ploidy identification; (2) the ploidy cannot be analyzed for small samples. Sampling and analysis

Method used

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  • Loach ploidy identification primer based on microsatellite polymorphism and application
  • Loach ploidy identification primer based on microsatellite polymorphism and application
  • Loach ploidy identification primer based on microsatellite polymorphism and application

Examples

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Effect test

Embodiment 1

[0032] A kind of loach ploidy identification primer based on microsatellite polymorphism, described primer specific information is as shown in table 1:

[0033] Table 1 Primers used in loach ploidy identification based on microsatellite polymorphisms

[0034]

Embodiment 2

[0036] A Ploidy Identification Method of Loach Based on Microsatellite Polymorphism

[0037] (1) Get some tissues of loach to be tested, use phenol-chloroform method to extract genomic DNA, use 1% agarose gel electrophoresis to detect the quality and integrity of DNA, detect the concentration of DNA with UV spectrophotometer (NanoDrop 2000);

[0038] (2) Take a small amount of DNA diluted to 100ng / μL working solution for later use, and store the remaining DNA in a -20°C refrigerator;

[0039] (3) carry out PCR amplification to sample DNA respectively with 5 pairs of primers (table 1) in embodiment 1, amplification system is as shown in table 2:

[0040] Table 2. Microsatellite PCR amplification reaction system

[0041]

[0042] (4) Take 5 μL of the amplified product, and electrophoresis at 150V for 3 hours in a 7% non-denaturing polyacrylamide gel (PAGE gel) (the formula is shown in Table 3);

[0043]Table 3 non-denatured polyacrylamide glue formula

[0044]

[0045] ...

Embodiment 3

[0049] Application of primers for loach chromosome ploidy identification based on microsatellite polymorphism:

[0050] (1) collect wild loach sample, after flow cytometry identification ploidy, randomly select diploid, triploid and tetraploid loach each 30 tails, be used for verifying the accuracy of technical method of the present invention;

[0051] (2) Get some tissues of loach to be tested, extract genomic DNA with phenol-chloroform method, detect DNA quality and integrity with 1% agarose gel electrophoresis, and detect DNA concentration with ultraviolet spectrophotometer (NanoDrop 2000);

[0052] (3) Take a small amount of DNA diluted to 100ng / μL working solution for later use, and store the remaining DNA in a -20°C refrigerator;

[0053] (4) Use 5 pairs of primers in Example 1 to carry out PCR amplification on the DNA of each sample to be tested respectively. The amplification system is as shown in Table 2 in Example 2. The amplification program is: 94 ° C pre-denaturat...

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Abstract

The invention belongs to the field of molecular techniques for identification of fish polyploidy, and particularly discloses a loach ploidy identification primer based on microsatellite polymorphism and application. The developed primer used for difference and polymorphism of loaches of different ploidies is high in accuracy rate and stability, a method is simple and convenient, rapid identification for the ploidies of loaches can be completed only after regular PCR amplification and polyacrylamide gel electrophoresis are conducted, the cost is low, and the primer is suitable for identification of the ploidies of various loach samples including cryopreserved samples preserved for a long time, extremely small samples and the like; a technological support is provided for distinguishing of the ploidies of the loaches, utilization of loach genetic resources based on the ploidies, polyploid breeding, genetic research and the like.

Description

technical field [0001] The invention belongs to the molecular technical field of fish polyploid identification, and in particular relates to a loach ploidy identification primer and application based on microsatellite polymorphism. Background technique [0002] Loach (Misgurnus anguillicaudatus) is known as "water ginseng" for its rich nutritional value and unique nourishing and medicinal effects, and is favored by consumers in China, Japan and South Korea. In recent years, the market demand for loach has been increasing day by day, and the market price has been rising steadily. Loach farming has become the new favorite of aquaculture farmers. [0003] Existing cytogenetic studies have found that there are not only diploids (2n=50) but also a large number of natural tetraploids (4n=100) in the wild loach population in my country. The body size of tetraploid loach is obviously larger than that of diploid, which has a significant growth advantage. Further research found that...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 周小云冯兵
Owner HUAZHONG AGRI UNIV
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