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A multiplex PCR method for the identification of Kui clam pedigrees

A kind of multi-layer technology, applied in the field of aquatic animal family identification, can solve the problems of amplification failure, limit the application of marking, increase the cost of marking application, etc., and achieve the effect of high accuracy, time and cost saving.

Active Publication Date: 2017-02-22
OCEAN UNIV OF CHINA
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Problems solved by technology

On the other hand, in the multiplex PCR system developed at present, the fluorescent label is directly added to the 5' end of the primer. This method will not only increase the cost of label application, but also in the case of low template concentration For shellfish larvae samples, it will cause amplification failure, these factors limit the practical application of markers

Method used

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  • A multiplex PCR method for the identification of Kui clam pedigrees
  • A multiplex PCR method for the identification of Kui clam pedigrees
  • A multiplex PCR method for the identification of Kui clam pedigrees

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Embodiment 1

[0024] (1) Select 3-year-old cockles with mature gonads for artificial induction, establish a mixed-bred family consisting of 30 parents through artificial insemination, and collect all parents (30) and some offspring of the family Individuals (300).

[0025] (2) DNA samples from each parent were extracted using the phenol-chloroform method, and larval DNA was extracted using the chelating resin method.

[0026] (3) Synthesize primers according to the primer sequence information provided in Table 1.

[0027] (4) Perform multiplex PCR amplification using the primer combinations in Table 2, and the reaction system for multiplex PCR is shown in Table 3.

[0028] The reaction program of PCR is: 94°C for 3min; 94°C denaturation for 45s, 54°C annealing for 75s, 72°C extension for 90s, 35 cycles; 94°C denaturation for 45s, 53°C annealing for 75s, 72°C extension for 90s, 8 cycles; 72°C Extend for 10 min and store at 4°C.

[0029] Table 1 Sequences of microsatellite marker primers u...

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Abstract

The invention discloses a multiplex-PCR method for the parentage assignment of scapharca broughtonii. The method comprises the following steps: extracting parental and filial DNA; carrying out multiplex PCR amplification on extracted DNA samples by using eight microsatellite loci and an M13 (-21) universal primer with a fluorescent marker; detecting fluorescence signals of amplification products so as to obtain parental and filial genotype data; and carrying out genetic relationship assignment on parents and offsprings. The method disclosed by the invention is suitable for carrying out genetic relationship assignment on parents and offsprings of large-scale reproduction and releasing groups, and carrying out large-scale artificial prevalent variety cultivation experiments, and has the characteristics of rapidness, accuracy, and high throughput.

Description

technical field [0001] The invention relates to a family identification method of aquatic animals, in particular to a method for identification of clam family by adopting multiple PCR. Background technique [0002] Family identification (Parentage identification), also known as pedigree analysis, is a judgment analysis of the relationship between individuals. Understanding the lineage characteristics of a species not only has important theoretical significance in the fields of population genetics, conservation genetics, and ecology, but also has important application value in fields such as breeding of improved varieties, protection of germplasm resources, and marking of artificially released seedlings . [0003] Microsatellite markers are co-dominant molecular markers with a high degree of variation, widely distributed in the genome, consistent with Mendelian inheritance, and easy to PCR amplification. They are the most commonly used molecular markers in family identificat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q1/6888C12Q2600/16C12Q2563/107C12Q2537/143
Inventor 李琪陈辰孙楠李宁
Owner OCEAN UNIV OF CHINA
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