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Specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei and application thereof

A technology of microsatellite markers and specific primers, which is applied in the field of DNA molecular genetic markers in molecular biology, achieving scientific, time-saving and wide-ranging effects

Active Publication Date: 2011-01-12
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A large number of methods for screening polymorphic microsatellite loci from EST libraries of Litopenaeus vannamei have not been reported so far

Method used

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  • Specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei and application thereof
  • Specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei and application thereof
  • Specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Screening of microsatellite sites and determination of polymorphic markers thereof

[0035] The Litopenaeus vannamei used in the experiment came from the Litopenaeus vannamei purchased randomly in the market, the WSSV-resistant Litopenaeus vannamei "Zhongxing No. 1" family selected at the Zhanjiang 863 base, and the Litopenaeus vannamei imported from the Hawaii Oceanographic Research Institute. .

[0036] 1. Source of microsatellite loci and screening of microsatellite sequences

[0037] Existing Litopenaeus vannamei ( Litopenaeus vannamei ) EST sequence, use the batch sequence assembly tool CAP3 software to splice and cluster the ESTs sequence in FASTA format, and then use the microsatellite site search software SSRhunter to search for the microsatellite sequence, for 2~6 base repeat units Microsatellite fragments with a number greater than 5 were screened and separated to obtain the original EST sequences LV_GL_RA09G18f, LV_HC_RA071F01f, LV_HC_RA074D14f, ...

Embodiment 2

[0054] Example 2: Application example - application of microsatellite markers in family identification

[0055] 1. Extraction of the genome of Litopenaeus vannamei

[0056] (1) Put 30 mg of Litopenaeus vannamei muscle into a 2.0 ml centrifuge tube, cut it into pieces, and add 600 μl of STE lysis buffer (100 mmol / L NaCl, 10 mmol / L Tris-Cl, pH 8.0; 1 mmol / L L EDTA, pH 8.0), 50 μl SDS and 5 μl proteinase K (20 mg / ml), mix well;

[0057] (2) Digest at 56 ℃ until the lysate is clear, centrifuge at 6000 rpm for 3 min;

[0058] (3) Take the supernatant, add an equal volume of saturated phenol (250 μl), chloroform / isoamyl alcohol (24:1) (250 μl), mix gently up and down for a few minutes, extract to remove protein, and centrifuge at 10,000 rpm for 10 min ;

[0059] (4) Take the supernatant and repeat step 3 until there is no protein layer between the aqueous phase and the organic phase;

[0060] (5) Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (500 μl), ...

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Abstract

The invention discloses specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei. The specific primers are five pairs together, and the nucleotide sequences are shown as SEQIDNO:1-10. The invention also discloses application of the specific primers in screening the microsatellite markers of the EST sequences of the Litopenaeus vannamei, performing diversity analysis of germplasm resources of the Litopenaeus vannamei, performing genetic diversity analysis, identifying families, studying molecular population genetics, constructing genetic maps, positioning important economic traits, studying functional genes, and assisting the molecular genetic breeding or culture of the Litopenaeus vannamei. Compared with the prior art, the specific primers for the microsatellite markers have the characteristics of high efficiency, simpleness, convenience, and quickness in screening required microsatellite markers.

Description

technical field [0001] The invention belongs to DNA molecular genetic marker technology of molecular biology, in particular to marine economic animal Penaeus vannamei (Litopenaeus vannamei) Litopenaeus vannamei ) EST (Express Sequence Tag, expressed sequence tag) specific primers for microsatellite markers and their application. Background technique [0002] Litopenaeus vannamei ( Litopenaeus vannamei ), commonly known as vannamei ( Penaeus vannamei ), belonging to the phylum Arthropoda, Crustacea, Decapoda, Natantia, Penaeidae, and the genus Penaeus ( Penaeus ), Lito-penaeus The subgenus is wide-temperature and wide-saline tropical shrimp. It is the shrimp species with the highest single yield among the three major cultured prawns (Penaeus sinensis, Penaeus monodon, and Litopenaeus vannamei) in the world. important position. [0003] In July 1988, Litopenaeus vannamei was introduced into my country from Hawaii, USA by the Institute of Oceanology, Chinese Academy of ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C12Q1/68
Inventor 李朝政吕玲张付霞翁少萍何建国
Owner SUN YAT SEN UNIV
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