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Method for establishing microsatellite molecular marker of scylla paramamosain

A technology of molecular markers for Scylla simulans is applied in the field of construction of microsatellite molecular markers of Scylla simulans, which can solve the problems of a small number of microsatellite markers, limiting the development of genetics research and the like.

Inactive Publication Date: 2010-11-17
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

At present, the number of microsatellite markers available in Scylla cryptae is very small, which severely limits the development of related genetic research.

Method used

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  • Method for establishing microsatellite molecular marker of scylla paramamosain

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Embodiment 1

[0219] 1. Extraction of Genomic DNA of Scylla pseudocaveus

[0220] Cut about 100-150 mg of a small amount of muscle tissue from Scylla pseudocarpus, and place it in 500 μl of tissue extract (10 mmol / L Tris-CI, pH8.0; 100 mmol / L EDTA, pH8.0; 100 mmol / L NaCl; 0.5% SDS) Cut into pieces, add proteinase K (final concentration 20mg / ml) and RNaseA (final concentration 100μg / ml), mix thoroughly, digest at 55°C for 2-3 hours until clear; mix with phenol: chloroform: isoamyl alcohol solution (volume ratio 25:24:1) twice; then precipitate DNA with 2 volumes of absolute ethanol, wash with 70% ethanol and dry naturally, then dissolve in 50 μL TE (10 mmol / L Tris-HCl, pH8.0; 10mmol / L EDTA, pH8.0); Finally, the DNA concentration was diluted to 100ng / μl and stored at -20°C for later use;

[0221] 2. Enzyme digestion and ligation of the genomic DNA of Scylla pseudomae

[0222] Use the restriction endonuclease MseI to digest the genomic DNA of Scylla syringae. The total volume of the digestio...

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Abstract

The invention relates to a method for establishing a microsatellite molecular marker of scylla paramamosain. The method comprises the following steps of: extracting genome DNA of the scylla paramamosain; performing enzyme digestion on the genome DNA, jointing and pre-amplifying; hybridizing an amplification product, a probe and a magnetic bead and performing secondary PCR amplification; cloning and sequencing the amplification product; analyzing the sequence and designing a microsatellite specific primer; performing PCR amplification on the genome DNA of different geographic groups or individuals in the group; and performing 6 percent modified polyacrylamide gel electrophoresis to detect the PCR amplification product so as to obtain a polymorphic map of genetic variation of the scylla paramamosain. The method has a good application prospect in the fields such as genetic variation analysis of the scylla paramamosain, germplasm resource protection and management, genetic linkage map establishment, marker-assisted breeding and the like.

Description

technical field [0001] The invention belongs to the technical field of DNA molecular genetic markers of aquatic organisms, and in particular relates to a method for constructing microsatellite molecular markers of Scylla pseudocavei. Background technique [0002] Microsatellite DNA (Microsatellite DNA) exists in almost all eukaryotic genomes. It is a simple sequence repeat (Simple Sequence Repeats, SSR) composed of 1-6 nucleotides. It is a new type of molecule developed in the past ten years. Marking technology. Microsatellites have the advantages of large number, random distribution, high polymorphism, strong repeatability, co-dominant inheritance, and simple operation, and are widely used in population genetic diversity analysis, germplasm resource protection and management, and genetic linkage maps. Construction and QTL mapping and other fields. [0003] Scholars at home and abroad have developed microsatellite markers in most important aquaculture animals. For example...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 马凌波马洪雨马春艳
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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