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Method for carrying out parentage identification and paternity identification by utilizing multiple haliotis discus hannai Ino fluorescence-labeled microsatellite

A technology of wrinkled plate abalone and paternity identification, which is applied in the direction of biochemical equipment and methods, microbiological measurement/inspection, etc., can solve the problems of being susceptible to subjective influence and low efficiency, and achieve the standardization and standardization of genetic data analysis, improve Accuracy, the effect of improving work efficiency

Active Publication Date: 2015-05-13
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

[0004] Although the construction of a microsatellite multiplex PCR system for Abalone rugosa has been reported (Nie Hongtao et al., 2013), each combination of this system has only 3 microsatellite markers, which are separated by 6% denaturing polyacrylamide gel electrophoresis and visualized by silver staining. color, therefore, less efficient and subject to subjective influence

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  • Method for carrying out parentage identification and paternity identification by utilizing multiple haliotis discus hannai Ino fluorescence-labeled microsatellite
  • Method for carrying out parentage identification and paternity identification by utilizing multiple haliotis discus hannai Ino fluorescence-labeled microsatellite
  • Method for carrying out parentage identification and paternity identification by utilizing multiple haliotis discus hannai Ino fluorescence-labeled microsatellite

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Embodiment

[0078] A mating combination of 10♀×10♂P. Two different sets of multiplex PCR reaction systems were used for amplification.

[0079] Specifically, the reaction conditions are: Taq DNA Master Mix (ABI) 7.5ul, Primer Mix 1ul (see Table 1 for the concentration of each primer), GC Enchaner 1.5ul, template DNA (40-80ng / ul) 1ul, double distilled water to make up to 15ul. PCR reaction conditions were denaturation at 94°C for 4 min, followed by 13 cycles (denaturation at 94°C for 30 s; annealing temperature decreased from 59°C to 53°C, annealing temperature decreased by 0.5°C per cycle, annealing time was 45 s; extension at 72°C for 45 s) , followed by 22 cycles (denaturation at 94°C for 30s; annealing at 53°C for 45s; extension at 72°C for 45s), and finally, extension at 72°C for 10 min, and storage at 4°C.

[0080] The two groups of PCR samples obtained above were subjected to microsatellite typing using an ABI 3730xl automatic DNA sequencer, and ROX fluorescent molecules were use...

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Abstract

The invention belongs to the field of marine biotechnology, and particularly relates to a method for carrying out parentage identification and paternity identification by utilizing a multiple haliotis discus hannai Ino fluorescence-labeled microsatellite. Multiple PCR reaction is adopted by the fluorophore-labeled haliotis discus hannai Ino microsatellite, so that the microsatellite can be applied to distinguishment of different parentages of haliotis discus hannai Ino or haliotis discus hannai Ino paternity identification. Two combinations disclosed by the invention both integrate original nine microsatellite PCR amplification and detection to one time, the detection cost is lowed, and the work efficiency is improved. An ABI 3730xl full-automatic DNA sequenator is used for performing microsatellite classification, classification error caused by denaturing polyacrylamide gel electrophoresis separation and sensitive silver staining is avoided, and the accuracy of paternity identification is improved. The data analysis in studying of haliotis discus hannai Ino genetics is more standardized and normalized by the method established in the invention, and data communication and sharing among different researchers are facilitated.

Description

technical field [0001] The invention belongs to the technical field of marine organisms, and specifically relates to a method for family identification and paternity identification by using multiple wrinkled disc abalone fluorescently marked microsatellites. Background technique [0002] Haliotis discus hannai Ino is a kind of mollusk with important economic value. Because of its delicious meat and rich nutrition, it has already become one of the important mariculture objects in my country. With the continuous improvement of people's living standards, the demand for wrinkled pan abalone is increasing day by day, so various genetic breeding techniques have also been developed in this species. Establishing a family with excellent traits and relatively stable is one of the main tasks at present. However, due to the long growth cycle of Abalone wrinkle and the frequent operation of the breeding process, the breeding and cultivation process is easily polluted by exogenous familie...

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q2600/156C12Q2600/16
Inventor 张振李加琦孙辉姜冰冰
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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