Application of anti-WSSV candidate molecular marker of nLvALF1 gene of Litopenaeus vannamei to genetic breeding of Litopenaeus vannamei

A molecular marker and molecular marker-assisted technology, applied in the field of aquatic animal genetics and breeding, can solve problems such as slow progress, and achieve the effects of strong pertinence, short cycle and simple operation

Inactive Publication Date: 2016-10-12
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

Because WSSV is highly pathogenic and lethal, and virus carriers will vertically transmit the virus to their offspring, the traditional selection work guided by traditional quantitative genetics theory has progressed slowly

Method used

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  • Application of anti-WSSV candidate molecular marker of nLvALF1 gene of Litopenaeus vannamei to genetic breeding of Litopenaeus vannamei
  • Application of anti-WSSV candidate molecular marker of nLvALF1 gene of Litopenaeus vannamei to genetic breeding of Litopenaeus vannamei

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Experimental program
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Embodiment 1

[0017] Example 1: Obtaining of nLvALF1g-114-T>C marker

[0018] Feed the diseased shrimps infected with WSSV to death for challenge. After the challenge is successful, collect the first dead shrimp as a sensitive group, and collect the last dead shrimp as a resistant group. Use genomic data to obtain the full-length sequence of the nLvALF1 gene, and Design primers to amplify the genomic DNA sequence, obtain the SNP sites in the sequence, obtain the SNP sites in the nLvALF1 gene, and use sequencing technology to genotype these SNP sites in the resistant population and sensitive population, and compare the SNP The difference between the genotype and gene frequency distribution of the markers in the resistant group and the sensitive group was selected, and the loci with significant differences between the two groups were screened out (see references Jingwen Liu, Yang Yu, Fuhua Li*, Xiaojun Zhang, Jianhai Xiang, 2014.A new anti-lipopolysaccharide factor (ALF) gene with its SNP pol...

Embodiment 2

[0021] Application in disease resistance breeding

[0022] 1. Extraction of DNA from breeding materials

[0023] In the breeding population, one swimming leg of the candidate broodstock was clipped, and the genomic DNA of each broodstock was extracted and labeled using the Tiangen Plant Genomic DNA Extraction Kit (Tiangen, China). Use NanoDrop 1000 to detect the DNA concentration and purity of the extracted DNA, and the detected DNA concentration>50ng / μl and purity OD 260 / 280 The individual DNA of 1.7-1.9 is detected by 1% agarose gel electrophoresis, and the individual whose DNA fragment is complete as a result of electrophoresis is selected for use.

[0024] 2. Amplification of marker sequences

[0025] Individuals obtained in the above manner will be amplified using nLvA1F and nLvA1R primers. Specifically:

[0026] The amplification system is as follows:

[0027]

[0028] The PCR reaction program was: pre-denaturation at 94°C for 5 minutes, followed by 35 cycles of a...

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Abstract

The invention relates to application of an anti-WSSV candidate molecular marker of the nLvALF1 gene of Litopenaeus vannamei to genetic breeding of Litopenaeus vannamei, belonging to the field of genetic breeding of aquatic livestock. Through preliminary study, three SNP markers (P<0.05) significantly correlated with anti-WSSV are screened out from the nLvALF1 gene by using a correlation analysis method. In the invention, resistance lines and non-resistance lines of the three SNP markers in other genetic breeding materials are subjected to SNP genotyping so as to eventually screen out one SNP marker, located at a site nLvALF1g-114-T>C, significantly correlated with anti-WSSV; the genotype frequency of the CC type of the site in resistance lines is substantially higher than the genotype frequency of the CC type of the site in non-resistance lines; and a method for molecular marker-aided genetic breeding of anti-WSSV is established by using the marker. The invention provides a candidate technical means for molecular marker-aided breeding for disease resistance and lays foundation for research on breeding of an anti-WSSV variety of Litopenaeus vannamei.

Description

technical field [0001] The invention belongs to the field of aquatic animal genetics and breeding, and relates to the application of a WSSV-resistant candidate molecular marker of the nLvALF1 gene of Litopenaeus vannamei in the genetic breeding of prawns. Background technique [0002] my country is a big country in prawn farming. The output of prawn farming has reached more than 1.5 million tons, ranking first in the world. Among them, the prawn species with the largest farming scale and output is Litopenaeus vannamei, with an output of about 1.2 million tons. In recent years, with the expansion of aquaculture scale and the refinement of aquaculture models, the demand for high-yield and disease-resistant improved varieties is increasing. The healthy development of Penaeus prawn industry is of great significance. [0003] The genetic selection of Litopenaeus vannamei has been carried out for nearly 30 years. Under the guidance of the theory of quantitative genetics, the gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 李富花于洋刘敬文相建海张晓军
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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