Method for screening scylla paramamosain microsatellite molecular marker

A technique of molecular markers of Scylla pseudosinus, which is applied in the field of screening of microsatellite molecular markers of Scylla pseudosinus, can solve the problems of the small number of microsatellite markers and the limitation of genetic research and development.

Inactive Publication Date: 2011-08-03
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 screening scylla paramamosain microsatellite molecular marker
  • Method for screening scylla paramamosain microsatellite molecular marker
  • Method for screening scylla paramamosain microsatellite molecular marker

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Embodiment 1

[0177] 1. Extraction of Genomic DNA of Scylla pseudocaveus

[0178] Cut a small amount of muscle tissue (about 100-150 mg) of mud crabs (collected in the coastal waters of Ningde City, Fujian Province) and put them in 500 μL of tissue extract (10 mmol / L Tris-Cl, pH 8.0; 100 mmol / L EDTA, pH 8.0). 0; 100mmol / L NaCl; 0.5% SDS), add proteinase K (final concentration 20mg / ml) and RNaseA (final concentration 100μg / mL), mix well, and digest at 55°C for 2-3 hours to clarification; extracted twice with phenol: chloroform: isoamyl alcohol mixture (volume ratio 25: 24: 1); then precipitated DNA with 2 times the volume of absolute ethanol, washed with 70% ethanol and dried naturally, Dissolve in 50μL TE (10mmol / L Tris-HCl, pH 8.0; 10mmol / L EDTA, pH 8.0); finally dilute the DNA concentration to 100ng / μL, and store it at -20℃ for future use.

[0179] 2. Design 5' anchor primers and PCR amplification

[0180] Design primers containing merged bases and repeated bases, where the merged base ...

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Abstract

The invention relates to a method for screening a scylla paramamosain microsatellite molecular marker. The method comprises the following steps of: extracting scylla paramamosain genome DNA (Deoxyribonucleic Acid) and diluting the DNA for later use; designing a 5' anchor primer and performing PCR (Polymerase Chain Reaction) amplification; cloning an amplification product and sequencing; performing sequence analysis and designing a microsatellite specific marker; performing PCR amplification on the genome DNA of different geographical populations of scylla paramamosain or individuals in the populations by using the primer; detecting a PCR amplification product by using 6 percent denaturing polyacrylamide gel electrophoresis; and determining the genotype of each individual according to different migration distances of the amplification product to obtain a polymorphic map of the genetic variance of scylla paramamosain. The method is easy to operate, is safe and has a high positive rate and a true and reliable result. By adopting the method, the polymorphic map of the genetic variance of scylla paramamosain can be obtained quickly. The method is mainly applied to genetic variance analysis of scylla paramamosain, protection and management of the germ plasm resource, establishment of a genetic linkage map and genetic breeding.

Description

technical field [0001] The invention belongs to the field of microsatellite molecular markers of Scylla pseudocavei, in particular to a screening method for 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: GUO et al. develope...

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Application Information

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