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Method for massively and efficiently developing molecular markers on basis of Indel and SSR (simple sequence repeat) site techniques

A molecular labeling and large-scale technology, applied in the fields of molecular biology and bioinformatics, can solve the problems of low effectiveness of SSR labeling and low efficiency of SSR molecular labeling, and achieve the effects of short time, low cost and low data volume

Active Publication Date: 2016-06-22
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for developing molecular markers in large quantities and efficiently based on Indel and SSR site technology for the problem of low efficiency when screening SSR molecular markers for species
It is used to solve the problem of low effectiveness of SSR marker development after high-throughput sequencing. This method can greatly improve the efficiency of molecular marker development, and the developed SSR molecular markers can be efficiently used in genetics, proliferation and release assessment, etc. research, saving a lot of time and money

Method used

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  • Method for massively and efficiently developing molecular markers on basis of Indel and SSR (simple sequence repeat) site techniques
  • Method for massively and efficiently developing molecular markers on basis of Indel and SSR (simple sequence repeat) site techniques
  • Method for massively and efficiently developing molecular markers on basis of Indel and SSR (simple sequence repeat) site techniques

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Embodiment Construction

[0025] The present invention will be described in detail below in conjunction with specific implementation methods.

[0026] Such as figure 1 As shown, taking the perch as an example, a large-scale and efficient method for developing molecular markers for perch based on Indel and SSR locus technology includes the following steps:

[0027] (1) Extraction of genomic DNA from sea bass muscle samples

[0028] Select 3 wild perch muscle tissue samples, use the Magen animal tissue DNA extraction kit, process 20-50mg tissue samples into as small fragments as possible, and transfer them to a 1.5mL centrifuge tube. Add 550 μL BufferMTL and 20 μL Proteinase K, and vortex to mix. Incubate with shaking at 55°C for 3h or overnight to digest the samples. Add 5 μL RNaseSolution to the digestion solution, mix well by inverting. Stand at room temperature for 30-60min to digest RNA. Centrifuge at 13000xg for 3 min. Transfer the supernatant to a new 2.0 mL centrifuge tube. Add 500 μL Buff...

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Abstract

The invention discloses a method for massively and efficiently developing molecular markers on the basis of Indel and SSR (simple sequence repeat) site techniques. The method comprises the following steps: (1) selecting at least 3 samples to be developed, and respectively extracting DNAs (deoxyribonucleic acids) of the samples to be developed; (2) carrying out enzyme digestion on the DNA samples of the samples to be developed, establishing a sequencing library, and carrying out sequencing; (3) mixing the genomes of all the samples to be developed, and assembling to obtain Contigs; (4) comparing the Contigs with the sequences of the sample individuals to be developed, and acquiring SSR sites with Indel inside according to the Indel and SSR site information as a candidate polymorphism SSR site; (5) designing primers according to the obtained candidate polymorphism SSR site, carrying out PCR (polymerase chain reaction) amplification and sequencing, and selecting a ribbon-shaped stable clear strip as molecular marker primers to be verified; and (6) carrying out PCR amplification on different samples to be developed by using the obtained molecular marker primers, and selecting the molecular markers with diversity, thereby obtaining the molecular markers. The method enhances the molecular marker development efficiency; and the developed SSR molecular markers can be efficiently applied to research in the aspects of genetics, multiplication release evaluation and the like.

Description

technical field [0001] The invention relates to the fields of molecular biology and bioinformatics, in particular to a method for developing molecular markers in large quantities and efficiently based on Indel and SSR site technologies. Background technique [0002] Molecular markers are genetic markers based on the detection of differences in the nucleotide sequence variation in the genetic material of individual organisms, and are a direct reflection of genetic polymorphism at the DNA level. Molecular markers are consistent in organisms at different developmental stages and in different tissues, and the greater the number of molecular markers, the higher the polymorphism, the more stable heredity, and will not be affected by the environment. Compared with several traditional genetic markers, DNA molecular markers have many advantages. With the rapid development of molecular biology, there are dozens of DNA molecular markers, which are widely used in genetic breeding, iden...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/10
CPCC12N15/1003C12Q1/6827C12Q1/6874C12Q1/6888C12Q2600/156C12Q2525/151C12Q2535/122
Inventor 张殿昌朱克诚刘田田江世贵张楠郭华阳
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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