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SNP (Single Nucleotide Polymorphism) molecular marker for improving disease resistance of procambarus clarkii and application

A technology of Crayfish and disease resistance, applied to SNP molecular markers and application fields for improving the disease resistance of Crayfish, can solve the problem of lack of transmission routes, difficult to define disease resistance traits, and disease resistance breeding of Crayfish. Slow progress and other problems, to achieve the effect of stable effect and excellent genotype disease resistance

Active Publication Date: 2021-10-15
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disease problem has hindered the development of Procambarus clarkii aquaculture
Although there has been some history of pathogen research in Procambarus clarkii, information about the pathogens, pathogenic conditions, and transmission routes of most diseases is still quite lacking.
In addition, the traditional forward selection method has many problems in the disease resistance breeding of Procambarus clarkii, for example, it is difficult to define the disease resistance traits, it is difficult to preserve the germplasm resources, and the disease resistance cannot be inherited stably, etc.
Therefore, the breeding of Procambarus clarkii for disease resistance is slow

Method used

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  • SNP (Single Nucleotide Polymorphism) molecular marker for improving disease resistance of procambarus clarkii and application
  • SNP (Single Nucleotide Polymorphism) molecular marker for improving disease resistance of procambarus clarkii and application
  • SNP (Single Nucleotide Polymorphism) molecular marker for improving disease resistance of procambarus clarkii and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] SNP site mining and disease resistance analysis of Crustin gene (GQ301202.1). A total of 176 Procambarus clarkii were selected for SNP site mining. A total of 90 Procambarus clarkii were selected for disease resistance analysis.

[0057] (1) Extract the hemolymph RNA of 176 individuals respectively, the specific method is as follows:

[0058] Use a 1ml syringe to extract 390-400μl (about 400μl) blood sample from the shrimp body, and mix it with ACD anticoagulant (formulation: 0.48g citric acid, 1.32g sodium citrate, 1.47g glucose, dissolved in 100ml double-distilled water) to 400μl, placed in EP tube, and placed on ice. Centrifuge at 800×g, 4°C for 20 minutes to separate blood cells; discard the supernatant, keep the white precipitate, add 200 μl of pre-cooled Trizol reagent, and use a grinder to grind until the white precipitate disappears, making the Trizol reagent solution pink, each sample Add 800μl Trizol reagent again; let it stand at room temperature for 5min,...

Embodiment 2

[0077] SNP site mining (screening) and disease resistance analysis for ALF gene (KU680792.1). A total of 176 Procambarus clarkii were selected for SNP site mining. A total of 90 Procambarus clarkii were selected for disease resistance analysis.

[0078] (1) Extract the hemolymph RNA of 176 Procambarus clarkii individuals respectively, and the specific steps are as follows:

[0079]390-400 μl (approximately 400 μl) blood sample was extracted from the shrimp of Procambarus clarkii using a 1ml syringe, and the volume ratio was 1:1 with ACD anticoagulant (0.48g citric acid, 1.32g sodium citrate, 1.47g glucose, dissolved in 100ml double distilled water) to 400μl, put in EP tube, and put on ice. Centrifuge at 800×g, 4°C for 20 minutes to separate blood cells; discard the supernatant, keep the white precipitate, add 200 μl of pre-cooled Trizol reagent, and use a grinder to grind until the white precipitate disappears, making the Trizol reagent pink. Add 800μl Trizol reagent; let s...

Embodiment 3

[0096] The Crustin gene and ALF gene were combined for SNP site mining and disease resistance analysis. A total of 176 Procambarus clarkii were selected for SNP site mining. A total of 90 Procambarus clarkii were selected for disease resistance analysis.

[0097] The Crustin gene and ALF gene were combined to further analyze the difference in disease resistance of different genotype combinations.

[0098] Specific steps are as follows:

[0099] (1) Extract the hemolymph RNA of 176 Procambarus clarkii individuals respectively, the specific method is as follows:

[0100] Use a 1ml syringe to extract 390-400μl (about 400μl) blood samples from Procambarus clarkii, and mix them with ACD anticoagulant (0.48g citric acid, 1.32g sodium citrate, 1.47g glucose, dissolved in 100ml double-distilled water) to 400μl, placed in EP tube, and placed on ice. Centrifuge at 800×g, 4°C for 20 minutes to separate blood cells; discard the supernatant, keep the white precipitate, add 200 μl of pr...

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Abstract

The invention belongs to the technical field of aquaculture molecular marker screening, and particularly relates to an SNP molecular marker for improving the disease resistance of procambarus clarkii and application. The method is characterized in that randomly collected procambarus clarkii RNA is respectively extracted, and cDNA is obtained through reverse transcription. A primer combination is designed according to procambarus clarkia disease-resistant gene Crutin gene and ALF gene sequences, a procambarus clarkia cDNA sample is subjected to PCR amplification through the primer combination, a target fragment is obtained, agarose gel electrophoresis is used for detection, and it is confirmed that the target fragment is amplified. A cDNA sequence of the procambarus clarkii is obtained by a first-generation sequencing means, sequence comparison is carried out, SNP sites in Crustin and AFL gene coding regions in a procambarus clarkii population are identified, and the two genes and a combination thereof are typed; and the differences of the disease resistance of the genotypes are compared, and screening is carried out to obtain the genotype with the strongest disease resistance and the combined marker primer thereof. The invention can be used for assisted selection.

Description

technical field [0001] The invention belongs to the technical field of aquaculture molecular marker screening, in particular to a SNP molecular marker for improving the disease resistance of Procambarus clarkii and its application. The present invention provides new resources for the screening of Procambarus clarkii disease-resistant molecular markers, and the application of the present invention can improve the auxiliary selection of disease-resistant Procambarus clarkii seedlings, and has certain advantages for the selection and breeding of Procambarus clarkii disease-resistant varieties or strains. Significance. Background technique [0002] Procambarus clarkii (Procambarus clarkii), also known as red swamp crayfish, is commonly known as "crayfish". Belonging to Arthropoda, Crustacea, Soft Armour, Decapod, Crawfishidae, Procambarus genus. The origin is located in northern Mexico and the southern United States, and was first introduced in the Honshu area of ​​Japan in 19...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6888C12Q1/6858C12Q2600/124C12Q2600/156C12Q2600/172C12Q2531/113C12Q2521/107Y02A50/30
Inventor 白旭峰任鑫彭国辉谭云飞彭波
Owner HUAZHONG AGRI UNIV
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