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SNP (Single Nucleotide Polymorphism) molecular marker for sex identification of silver arowana and application thereof

A molecular marker, silver dragon fish technology, applied in the direction of recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., can solve problems such as limiting reproductive efficiency and success rate, inability to penetrate scales, and gender indistinguishability, etc. Achieve important economic value and social value, solve major industrial problems, and improve reproductive efficiency

Active Publication Date: 2022-07-01
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And because silver arowana has hard and thick scales, B-ultrasound, CT and other means cannot penetrate the scales to determine gender
At present, identification can only be made by anatomical observation of the gonads, and a simple, accurate and identification method that does not require anatomy cannot be realized
It is precisely because there is no means to accurately determine the sex that the artificial reproduction of this fish has caused great trouble; because the sex is difficult to distinguish, most farms can only use semi-natural methods for breeding, instead of more efficient and controllable methods. Artificial breeding methods, such as separate ponds for male and female fish, injection of aphrodisiacs and oxytocins, etc., greatly limit the reproductive efficiency and success rate

Method used

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  • SNP (Single Nucleotide Polymorphism) molecular marker for sex identification of silver arowana and application thereof
  • SNP (Single Nucleotide Polymorphism) molecular marker for sex identification of silver arowana and application thereof
  • SNP (Single Nucleotide Polymorphism) molecular marker for sex identification of silver arowana and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] In this example, a high-throughput sequencing strategy was used to select 5 female dragon fish samples and 5 male fish samples for DNA extraction. According to the requirements of the Illumina library construction process, a double-ended genomic DNA library with an insert size of 500 bp was constructed. Then high-throughput sequencing was performed on the genome using the Illumina NovaSeq sequencing platform, with a sequencing volume of 30 Gb per sample and a sequencing strategy of Pair-End 150 bp.

[0025] Using a high-throughput sequencing sequence alignment strategy, the single nucleotide polymorphism sites (SNPs) of the sequenced female samples and the single nucleotide polymorphism sites (SNPs) of the male samples were detected, and the female samples were screened. are identical between male and female samples, and the different SNP loci of male and female samples are used as molecular markers for sex identification.

[0026] The female samples and male samples we...

Embodiment 2

[0035] In order to verify the accuracy of the SNP site to identify the gender of Arowana, this example carried out the verification of the SNP molecular markers in Example 1, and verified whether the molecular markers detected by Arowana were accurate by PCR and Sanger sequencing. Specifically, genomic DNA was extracted from the non-high-throughput sequencing samples, the specificity of the primers was verified by PCR and Sanger sequencing, and the physiological sex of the silver arowana samples was compared with each other to confirm the SNP molecular markers in Example 1 , Whether the primers can be used for gender identification of silver arowana.

[0036] 1. Sample preparation

[0037] Select 60 silver arowana samples, including 30 female samples and 30 male samples, for verification. The following table shows the sample information:

[0038] Experimental verification samples

[0039]

[0040]

[0041]

[0042] 2. Genomic DNA extraction

[0043] Extraction was ca...

Embodiment 3

[0062] The present embodiment provides a method for gender identification of silver arowana, comprising the following steps:

[0063] (1) extracting the genomic DNA of the silver arowana to be tested;

[0064] (2) using the genomic DNA of the silver arowana to be tested as a template, using the upstream primer F and the downstream primer R whose sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively, to carry out PCR amplification reaction;

[0065] (3) After the reaction is completed, the Sanger sequencing is combined for analysis to determine the genotype of the sample, and the gender is identified according to the 243rd position in the sequence SEQ ID NO.1.

[0066] Specifically, the reaction system and amplification procedure used in the PCR amplification reaction in step (2) are the same as those in Example 2.

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Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker for sex identification of silver arowana and application of the SNP molecular marker. The base sequence of the SNP molecular marker is SEQ ID NO.1; the SNP molecular marker is the 243 site in SEQ ID NO. 1. Single nucleotide polypeptide sites (SNP) which have differences between male and female silver arowana individuals and can characterize sex are obtained through genome high-throughput sequencing comparison, primer amplification is combined to obtain an amplification sequence containing the SNP sites, on the basis, PCR amplification and sequencing of a to-be-detected sample are achieved through primers, sample SNP site information is obtained, and the SNP sites can be used for detecting the sex of the to-be-detected sample. Therefore, the gender of the to-be-detected sample is identified. By utilizing the SNP site, accurate and rapid sex identification can be realized on the premise that the silver arowana is not dissected, and when the SNP site is applied to silver arowana culture, a mature artificial silver arowana culture technology is convenient to realize, so that mass production is realized.

Description

technical field [0001] The invention relates to the technical field of fish sex identification, and more particularly, to a SNP molecular marker for sex identification of silver arowana and its application. Background technique [0002] Silver arowana, also known as double-bearded tongue fish, is an ancient "living fossil" fish, which has very important economic value as a precious ornamental fish. At present, the vast majority of silver arowanas in China rely on imports, and it is difficult for silver arowana to be cultivated locally; because the current breeding technology of silver arowana is limited, it is difficult to achieve large-scale artificial culture. The main reason for restricting reproductive technology is the difficulty in distinguishing male and female Arowana. [0003] Arowana has no sex-specific characteristics and cannot be distinguished in external morphology, even in the reproductive season, the characteristics of male and female fish are not obvious. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6879C12Q1/6869C12N15/11
CPCC12Q1/6888C12Q1/6879C12Q1/6869C12Q2600/156C12Q2531/113C12Q2535/101Y02A40/81
Inventor 牟希东刘奕刘超杨叶欣宋红梅汪学杰徐猛顾党恩房苗胡隐昌
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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