Method of screening pacific oyster EST micro-satellite mark

A technology of microsatellite markers and Pacific oysters, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc.

Inactive Publication Date: 2007-10-24
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Claims
  • Application Information

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Problems solved by technology

There is no research report on EST microsatellite markers in Pacific oyster

Method used

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  • Method of screening pacific oyster EST micro-satellite mark
  • Method of screening pacific oyster EST micro-satellite mark

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Embodiment 1

[0014] Example 1: Screening of microsatellite sites and determination of polymorphic markers. The Pacific oysters used in the experiment were all from Fujian farming populations.

[0015] 1. Source of microsatellite loci and screening of microsatellite sequences

[0016] Collect and download (in FASTA format) existing EST sequences from the NCBI (http: / / www.ncbi.nlm.nih.gov) database, use SSRhunter software to search and analyze the 3390 sequences obtained one by one, for 2-6 bases Microsatellite DNA sequences with basic repeating units and repeats greater than 5 were isolated. The ESTs containing microsatellites were clustered and analyzed using SeqMan in the software DNAstar, and the ESTs clustered in different contigs were selected to design primers.

[0017] 2. Design of microsatellite marker primers

[0018] Use primer design software Primer Premier 5.0 and Primerselect in DNAstar to design primers at the flanking sequences of microsatellite repeats; primers are requir...

Embodiment 2

[0025] Embodiment 2: application example

[0026] 1. DNA extraction from Pacific oysters

[0027] The Pacific oyster material used in this experiment was randomly extracted from 13 DNA samples, and DNA was extracted from frozen muscle tissue. Cut 100mg of muscle tissue, put it into 0.7ml extraction buffer (6M Urea (urea), 10mM Tris-HCl.125mM NaCl (sodium chloride), 1% SDS (sodium lauryl sarcosulphate), 10 mM EDTA (ethylenediaminetetraacetic acid), pH=7.5), proteinase K (20 mg / ml) at a final concentration of 0.1 mg / ml was added, and digested overnight at 37°C. The reactant was extracted three times with phenol:chloroform (1:1), and the extracted supernatant was extracted once with an equal volume of chloroform. The supernatant was precipitated with isopropanol, and then dissolved in 5001 μl×TE (10 mM Tris.HCl, 1 mM EDTA, pH=8.0). Treat the DNA sample with RNase (20 μg / ml) at 37°C for 1 hour, then perform DNA purification with phenol / chloroform extraction solution, extract on...

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Abstract

The invention discloses a screening method of Pacific oyster EST micro satellite label, which comprises the following steps: finding micro satellite site with Pacific oyster EST sequence announced by GeneBank and micro satellite searching software SSRhunter; screening and separating 2-6 base repeat unit micro satellite fragment (repeat times >5); getting repeat ESTs sequence with micro satellite; designing primer at two sides flank sequence of micro satellite repeat sequence; detecting optimization primer as the micro satellite label further; proceeding overall merit for micro satellite repeatability, stability, polymorphism; getting micro satellite label. The invention can be used for Pacific oyster species resource and genetic heterogeneity, molecular population genetics, species resource determination, genetic atlas construct, functional gene research, Pacific oyster breeding and cultivation.

Description

Technical field: [0001] The invention belongs to the field of DNA marker technology and application of molecular biology, and in particular relates to a screening method and application of EST (Express Sequence Tag, expressed sequence tag) microsatellite marker of marine economic shellfish Pacific oyster (Crassostrea gigas). technical background: [0002] Pacific oysters are originally produced in Japan, South Korea, China and other places. They are the marine shellfish with the widest range of cultivation and the highest yield in the world, and they are also one of the important species of marine culture in my country. In my country, Pacific oyster farming is mainly distributed in coastal areas such as Liaoning, Shandong, Fujian, and Guangdong. With the continuous deepening of the research on the genetic diversity of Pacific oyster, genetic breeding and improvement, and the construction of genetic maps, the demand for molecular markers of Pacific oyster is also increasing. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
Inventor 喻子牛王艳红
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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