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Molecular marker for hemorrhagic disease resistance trait of grass carp and application of molecular marker

An anti-bleeding disease and molecular marker technology, which is applied in the fields of application, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of time-consuming and labor-consuming, and achieve accurate detection, high throughput and fast speed Effect

Inactive Publication Date: 2015-12-09
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional breeding methods are time-consuming and labor-intensive, so molecular breeding methods based on molecular markers are gradually recognized by researchers.

Method used

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  • Molecular marker for hemorrhagic disease resistance trait of grass carp and application of molecular marker
  • Molecular marker for hemorrhagic disease resistance trait of grass carp and application of molecular marker
  • Molecular marker for hemorrhagic disease resistance trait of grass carp and application of molecular marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Grass carp transcriptome sequencing

[0044] (1) Infection test

[0045] The aquarium required for the test was soaked in 0.05% potassium permanganate solution for 24 hours, cleaned, filled with clean water, installed the oxygenation pump and heating rod, adjusted the temperature to 28°C-30°C, and turned on the oxygenation pump. Temporary breeding of experimental fish. Before the GCRV virus infection, the grass carp were randomly divided into five groups (60 fish / group) and temporarily raised in the prepared aquarium, and the palatable pellet feed was fed twice a day at a fixed point, time, and quantity, and the residual bait and metabolic waste were cleaned up in time . For about a week, prepare for the virus infection test after the daily physiological activities of the fish school are normal and the vitality is active. Grasscarp reovirus (GCRV-097) stock solution was donated by Institute of Hydrobiology, Chinese Academy of Sciences. The virus suspensio...

Embodiment 2

[0064] Example 2: RIG-I gene transcript verification and association analysis

[0065] (1) DNA extraction of grass carp spleen and head kidney tissue

[0066] Genomic DNA was extracted by the chloroform method. The specific operation steps are as follows:

[0067] 1) Take 0.05g of tissue (mung bean size) and put it in a 1.5mLEP tube, add 200μL of tissue extract, and grind it thoroughly. Add 400 μL of tissue extract and 60 μL of 1 mg / mL proteinase K, put in a 55°C water bath (for about 3 hours) or overnight at 37°C, and digest until the liquid is clear;

[0068] 2) Add 600 μL Tris-saturated phenol, mix thoroughly for 10 minutes, and centrifuge at 12,000 rpm for 10 minutes;

[0069] 3) Transfer the supernatant to a new EP tube, add 500 μL chloroform for extraction, mix thoroughly for 10 minutes, and centrifuge at 12,000 rpm for 10 minutes;

[0070] 4) Pipette the supernatant into a new EP tube, add 2 times the volume of ice ethanol (-20°C), precipitate DNA at -20°C for at le...

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Abstract

The invention belongs to the technical field of preparing a molecular marker of fish, and particularly relates to the molecular marker for a hemorrhagic disease resistance trait of grass carp and the application of the molecular marker. The molecular marker is obtained through grass carp RIG-I (Retinoic Acid-Induced Gene I) gene cloning and transcriptome sequencing; a nucleotide sequence of the molecular marker is as shown in a sequence table SEQ ID NO.1, and the insertion (deletion) (which is as shown in a sequence table SEQ ID NO.2) of amino acids located in 211 to 215 of RIG-I protein of the grass carp is caused by the insertion (the deletion) of a segment of 15bp base fragments of a basic group located in 665 of the SEQ ID NO.1. A GCRV (Grass Carp Reovirus) infection test shows that the death rate of gene inserting type grass carp is obviously lower than that of deletion type grass carp, thereby showing that the mutation of the segment of basic group of the RIG-I gene of the grass carp is related to the grass carp hemorrhagic disease resistance trait. According to the molecular marker disclosed by the invention, a new molecular marker is provided for marker assisted selection of the hemorrhagic disease resistance trait of the grass carp.

Description

technical field [0001] The invention belongs to the technical field of preparation of fish molecular markers, and in particular relates to a molecular marker and application of grass carp anti-hemorrhagic disease traits. The molecular marker is cloned from the RIG-I gene mutation, and the mutated RIG-I gene fragment can be used as a molecular marker and application of grass carp hemorrhagic disease resistance traits. Background technique [0002] Grass carp (Ctenopharyngodonidella, English name grasscarp) is a large-scale economic fish of Cyprinidae endemic to my country. In the process of raising grass carp fry to edible fish, the four major infectious diseases of hemorrhage, enteritis, gill rot and red skin are very easy to break out and prevail, which makes the survival rate of fish fry low, generally only about 30% (Su Jianguo, Yang Chunrong. 2000. Progress in Immunological Research of Major Infectious Diseases of Grass Carp. Advances in Veterinary Medicine, 21(4):183-1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/11C12Q1/68
Inventor 苏建国万全元饶友亮
Owner HUAZHONG AGRI UNIV
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