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Methods for acquiring micro-satellite sequence and polymorphic micro-satellite markers of hucho taimen and polymorphic micro-satellite markers of hucho taimen

A technology of microsatellite sequence and acquisition method, which is applied in the acquisition of jelly salmon microsatellite sequence and polymorphic microsatellite markers and the field of jelly salmon polymorphic microsatellite markers, can solve the problem of time-consuming and labor-intensive development of microsatellite markers. The risk and cost of microsatellite sequence separation, to achieve the effect of low cost and high safety

Inactive Publication Date: 2012-10-03
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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Problems solved by technology

At present, the development of microsatellite markers is time-consuming and laborious, and the main difficulties are concentrated in the acquisition of microsatellite sequences, primer design and optimization of primer PCR amplification conditions.
There are many methods for the isolation or cloning of microsatellite sequences, such as hybridization screening of small fragment genome libraries, FIASCO (Fast Isolation by AFLP of Sequences), magnetic bead enrichment method, etc., because the magnetic bead enrichment method has a positive clone rate and The microsatellite sequence has a high yield, so it is widely used at present, but this method needs to use isotope (disadvantage is radioactive) or biotin secondary hybridization (disadvantage is more toxic) technology, which increases the separation of microsatellite sequences. risk and cost, so there are limitations in use

Method used

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  • Methods for acquiring micro-satellite sequence and polymorphic micro-satellite markers of hucho taimen and polymorphic micro-satellite markers of hucho taimen
  • Methods for acquiring micro-satellite sequence and polymorphic micro-satellite markers of hucho taimen and polymorphic micro-satellite markers of hucho taimen
  • Methods for acquiring micro-satellite sequence and polymorphic micro-satellite markers of hucho taimen and polymorphic micro-satellite markers of hucho taimen

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specific Embodiment approach 1

[0020] Specific embodiment 1: In this embodiment, the microsatellite sequence of salmon salmon is obtained according to the following steps: 1. Extraction of salmon genome DNA; 2. Enzyme digestion of salmon genome DNA and construction of microsatellite enrichment library; 3. Bacteria colony PCR amplification and positive clone detection and sequencing, that is, to obtain the microsatellite sequence of jellyfish salmon.

specific Embodiment approach 2

[0021] Specific embodiment 2: The difference between this embodiment and specific embodiment 1 is that in step 2, restriction endonucleases are used to single-enzyme or double-enzyme digest the salmon genome DNA. Other steps and parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0022] Specific implementation mode three: the difference between this implementation mode and specific implementation mode two is: the steps are divided into the following steps to realize:

[0023] a. Enzyme digestion of salmon genome DNA:

[0024]The jelly salmon genomic DNA digestion system is 20 μL, which consists of 2 μL 10× digestion buffer, 0.2 μL BSA with a concentration of 10 ng / μL, 2.5 U restriction endonucleases, 5 μL jelly salmon genomic DNA with a concentration of 100 ng / μL and The remaining amount is composed of sterilized deionized water; the enzyme digestion incubation time is 4h;

[0025] b. Connection:

[0026] The ligation system was 40 μL, which consisted of 20 μL of enzyme-digested fragments in step a, 2 μL of sticky-end double-stranded adapters, 4 μL of 10×buffer, 6 Weiss units of T4 DNA ligase and the rest of sterilized deionized water, and ligated overnight in a water bath at 4°C; The double-stranded linker was prepared according to the following ste...

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Abstract

The invention discloses methods for acquiring a micro-satellite sequence and polymorphic micro-satellite markers of hucho taimen and the polymorphic micro-satellite markers of the hucho taimen, which relate to a method for acquiring the micro-satellite sequence and the polymorphic micro-satellite markers and the polymorphic micro-satellite markers. The method for acquiring the micro-satellite sequence comprises the following steps: firstly, extracting a hucho taimen genome DNA; secondly, performing enzyme cutting on the hucho taimen genome DNA and constructing a micro-satellite enrichment library; and thirdly, performing colony PCR amplification and performing positive clone detection and sequencing. The method for acquiring the polymorphic micro-satellite markers of the hucho taimen comprises the following steps: performing the first step to the third step the same as that of the method for acquiring the micro-satellite sequence; fourthly, analyzing the micro-satellite sequence of the hucho taimen and designing a primer; and fifth, performing polymorphic identification on the primer of the micro-satellite sequence. The method acquires seven pairs of the polymorphic micro-satellite markers of the hucho taimen. The biological materials obtained by the methods can be used for conservation genetics, genetic relationship analysis, linkage map construction and genetic management ofcultured populations of the hucho taimen.

Description

technical field [0001] The invention relates to a microsatellite sequence and a method for acquiring polymorphic microsatellite markers and polymorphic microsatellite markers. Background technique [0002] Zheluo salmon is a rare and precious fish native to our country, and it is the largest fish in the family Salmonidae. Due to factors such as environmental degradation and overfishing, its current resources are very scarce and endangered; therefore, in 1998, the salmon was included in the "China Red Book of Endangered Animals (Fish)", and in 2004 it was included in the "Chinese Species Red List. Zheluo salmon is also an excellent cold-water cultured fish. It has the characteristics of fast growth and strong stress resistance. At present, the artificial domestication of wild Zheluo salmon has been successfully carried out, and large-scale production has been successfully carried out on this basis. Breeding and farming. However, due to its serious self-mutilation, intolera...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12P19/34C12R1/19
Inventor 匡友谊佟广香尹家胜孙效文
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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