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A microsatellite multiple fluorescent PCR method for E. coioides paternity testing

An oblique grouper, parent-child identification technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of larvae survival impact, larvae cannot be marked, etc., to improve accuracy, efficiency, and interpretation. accurate effect

Active Publication Date: 2017-07-14
SUN YAT SEN UNIV
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Problems solved by technology

Physical marking can be used to identify individuals, but this method has great limitations for juveniles, either because the larvae are too small to be marked, or because the marking has a greater impact on the survival of the larvae

Method used

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  • A microsatellite multiple fluorescent PCR method for E. coioides paternity testing
  • A microsatellite multiple fluorescent PCR method for E. coioides paternity testing
  • A microsatellite multiple fluorescent PCR method for E. coioides paternity testing

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Embodiment Construction

[0043] The specific embodiment of the present invention will be further described below in conjunction with examples.

[0044] The method for the microsatellite fluorescent multiplex PCR of the clotted grouper paternity test provided by the present embodiment comprises the following steps:

[0045] (1) Extraction of DNA from the grouper

[0046] Cut off the individual fin rays of the male parent, female parent and 30 offspring of the oblique grouper family and immediately store them in 95% ethanol. The male parent is marked as C, the female parent is marked as X, and the 30 offspring are marked as 1-30 , Genomic DNA was extracted using the phenol-chloroform method:

[0047] Use tweezers to remove the caudal fin preserved in alcohol, cut about 20 mg, gently press with filter paper to absorb excess alcohol, transfer to a 1.5 mL centrifuge tube pre-added with 580 μL tissue lysate and cut the tissue into pieces. Then add 20 μL of 20 mg / mL proteinase K, shake gently to mix, and pla...

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Abstract

A microsatellite multiple fluorescent PCR method for E. coioides paternity testing is disclosed. The method includes (1) a step of extracting individual DNAs of E. coioides; (2) a step of screening E. coioides polymorphic microsatellite primers; (3) a step of optimizing E. coioides eightfold PCR conditions and performing amplification; and (4) a step of performing paternity testing. The method combines microsatellite marking and a multiple fluorescent PCR technique, eight polymorphic microsatellite loci are screened and an eightfold fluorescent PCR system is built. The eightfold fluorescent PCR system is verified by utilizing the E. coioides family. The method is simple and rapid to operate and low in cost, can be popularized and applied in E. coioides germplasm identification, family management and improved-variety selection, and provides a novel technical means for assessment of enhancement and releasing effects.

Description

technical field [0001] The invention belongs to the technical field of paternity identification of grouper, in particular to a microsatellite fluorescent multiplex PCR method for parentage identification of grouper. Background technique [0002] The oblique-banded grouper (E.coioides) belongs to the Serranidae family (Serranidae), grouper subfamily (Epinephelinae), grouper genus (Epinephelus), and its distribution range is very wide, from Durban, South Africa, east to the Palau Islands and Fiji, north to the Ryukyu Islands of Japan, and south to the Arafura Sea and Australia (Heemstra & Randall 1993). Oblique-banded grouper is one of the few species of grouper that is widely cultivated artificially. Because of its delicious meat and high protein content, it has become an important commercial fish in the Asia-Pacific region. However, due to the destruction of the ecological environment and overfishing, the grouper was listed as a near-threatened species by the IUCN Red List ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6888C12Q2600/156C12Q2600/16
Inventor 张勇陈枭夏军红王翔赵密刘云李水生石和荣张海发林浩然
Owner SUN YAT SEN UNIV
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