General microsatellite multiplex fluorescent PCR method for hypophthalmichthys molitrix and hypophthalmichthys nobilis

A technology for microsatellites and silver carp and bighead carp is applied in the field of general microsatellite fluorescence multiplex PCR for silver carp and bighead carp, and achieves the effects of being beneficial to large-scale and batch experimental operations, improving work efficiency, and avoiding overlap and confusion.

Inactive Publication Date: 2018-11-06
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

In the past few decades, studies on the genetic diversity of wild resources of silver carp and bighead carp populations in China have also been carried out. Basic reference; there are also population genetic studies using microsatellite markers, but most of the detection techniques use polyacrylamide gel electrophoresis, and there is room for improvement and improvement in resolution and accu

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  • General microsatellite multiplex fluorescent PCR method for hypophthalmichthys molitrix and hypophthalmichthys nobilis
  • General microsatellite multiplex fluorescent PCR method for hypophthalmichthys molitrix and hypophthalmichthys nobilis
  • General microsatellite multiplex fluorescent PCR method for hypophthalmichthys molitrix and hypophthalmichthys nobilis

Examples

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Embodiment 1

[0034] Example 1 is used for silver carp and bighead carp based on microsatellite marker population genetic variation analysis

[0035] (1) Two populations of silver carp and bighead carp were collected in the lower reaches of the Yangtze River, with 45 samples per population, a total of 180 individuals from 4 populations, and the genomic DNA was extracted by the phenol-chloroform method, and detected by 1% agarose gel electrophoresis Integrity, use a UV spectrophotometer for concentration detection, and dilute to 50ng / μL, and store at 0°C.

[0036] (2) Based on the DNA of a total of 180 individuals from 4 silver carp and bighead carp populations, PCR amplification was performed using the 4-fold PCR system modified by 3 sets of fluorescence according to the present invention.

[0037] (3) Random sampling of PCR amplification products, after passing the 2.5% agarose gel electrophoresis, using ABI3730XL automatic DNA sequencer for capillary electrophoresis detection, based on GS...

Embodiment 2

[0042] Example 2 is used for the parentage analysis of the artificial breeding parents and offspring of bighead carp

[0043] (1) Using 12 male and 12 female mature individuals of bighead carp, carry out artificial breeding according to the partial factormating design (Partial factorating design, Gjedrem 2005), divide them into 4 mating groups, each group has 3 pairs of parents, and there is 1 female fish in the group (or male) and 2 males (or females) for artificial insemination. The fertilized eggs are mixed for hatching, and routine fry cultivation is carried out.

[0044] (2) Sample collection of parents and progeny, cut 24 parental fin ray samples during the artificial breeding process, and use absolute ethanol for fixed preservation; and collect 192 whole fishes of progeny summer flower fry (~30 days old) , and were fixed and preserved with absolute ethanol.

[0045] (3) Utilize phenol-chloroform method to carry out genomic DNA extraction to above-mentioned 24 tail par...

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Abstract

The invention relates to a general microsatellite multiplex fluorescent PCR method for hypophthalmichthys molitrix and hypophthalmichthys nobilis and belongs to the technical field of aquatic animal germplasm identification in the aquaculture field. The method comprises the steps: firstly, collecting hypophthalmichthys molitrix and hypophthalmichthys nobilis pterygiophore samples; utilizing a traditional phenol-chloroform method to extract genome DNA; according to primer pairs referred to by a target amplified fragment length difference requirement, choosing 3 groups of quadruple PCR systems with clear amplified bands; totally referring to 12 microsatellite markers; finally, performing fluorescence decoration and amplification verification on the primer. The multiplex PCR method disclosedby the invention has the characteristics of quickness in operation, convenience in implementation, low cost and the like and can provide a novel molecular marker assistive technology for researches ofhypophthalmichthys molitrix and hypophthalmichthys nobilis germplasm genetic evaluation, family breeding and the like.

Description

technical field [0001] The invention relates to a general-purpose microsatellite fluorescent multiplex PCR method for silver carp and bighead carp, which is suitable for research on genetic variation and parentage identification of silver carp and bighead carp populations, and belongs to the technical field of germplasm identification of aquatic animals in the field of aquaculture. Background technique [0002] Silver carp (Hypophthalmichthys molitrix) and bighead carp (Hypophthalmichthys nobilis) are closely related species in Cyprinidae. As important indigenous fishes in China, they are widely distributed in major rivers and lakes. Both are carnivorous fish, important biological resources of water purification fishery, and have important ecological value in current water environment protection and governance. At the same time, as the traditional bulk freshwater fish farming species in China, the two have a long history of farming and large breeding volume, occupying an ext...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/156C12Q2563/107C12Q2537/143C12Q2525/151C12Q2565/125
Inventor 傅建军董在杰朱文彬方敏
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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