CN110616256A

CN110616256A - Multi-site cynoglossus semilaevis true and false male fish screening system based on SNaPshot technology and application

Info

Publication number: CN110616256A
Application number: CN201910762159.XA
Authority: CN
Inventors: 张博; 贾磊; 赵娜; 刘克奉; 何晓旭; 王群山; 赵东康; 鲍宝龙
Original assignee: TIANJIN BOHAI AQUATIC PRODUCT RESEARCH INSTITUTE
Current assignee: TIANJIN BOHAI AQUATIC PRODUCT RESEARCH INSTITUTE
Priority date: 2019-08-19
Filing date: 2019-08-19
Publication date: 2019-12-27
Anticipated expiration: 2039-08-19
Also published as: CN110616256B

Abstract

The invention relates to a multi-site cynoglossus semilaevis true and false male fish screening system based on an SNaPshot technology, which comprises 8 SNPs with a marking indication function, wherein the 8 SNPs are positioned on the same chromosome NC-024328.1, and the position information is as follows: SNP 1-13754040, SNP3-14518947, SNP4-14546665, SNP5-15320226, SNP6-14238006, SNP7-14546702, SNP9-14802286, and SNP 10-14802427. The system can only be operated on various genetic analysis instruments, can realize automatic typing and identify the male fish and the pseudo-male fish. Compared with the existing method for identifying the genetic sex of the cynoglossus semilaevis, the method is quicker to operate and higher in detection flux, and more importantly, the method has extremely high accuracy.

Description

Multi-site cynoglossus semilaevis true and false male fish screening system based on SNaPshot technology and application

Technical Field

The invention belongs to the technical field of genetic sex identification of marine fishes in aquatic biotechnology, and particularly relates to a system for discriminating true male fishes and false male fishes of cynoglossus semilaevis at multiple sites based on a SNaPshot technology and application thereof.

Background

The cynoglossus semilaevis is one of flatfish, the growth rate of female individuals is 2-4 times that of male individuals, the male fishes grow slowly, the individual bodies are small, the breeding yield of the cynoglossus semilaevis is reduced, and the breeding cost is increased. Research shows that the sex determining mechanism of the cynoglossus semilaevis is sex chromosomes Z and W, a normal male cynoglossus semilaevis has ZZ type chromosomes, and a normal female cynoglossus semilaevis has ZW type chromosomes. The pseudomale fish inherits female chromosomes, and the genotypes of the pseudomale fish are ZW types. But also shows male characteristics from both its physical characteristics and reproductive organs, and can produce fertile sperm and bred offspring like male fish. And if the pseudo-male fish is used as the male parent, the offspring can inherit the characteristics of the male parent to become the pseudo-male fish. Thus, through generation-by-generation accumulation, the proportion of physiological female cynoglossus semilaevis cultured population is unbalanced. The method has important application value for production and culture by screening and removing false male fish of cynoglossus semilaevis.

A plurality of cynoglossus semilaevis gender specific molecular markers have been developed. The cynoglossus semilaevis female specific AFLP marker (Chen et al, 2007) is separated by the predecessor through an AFLP (amplified fragment length polymorphism) marker technology, because the AFLP marker is dominant heredity, ZW female and WW superfemale individuals cannot be distinguished in application, and false negative results are difficult to avoid, so that false judgment is generated on ZZ male fish and ZW female fish. Through screening the cynoglossus semilaevis codominant sex specific microsatellite scaffold68-2 marker (Liuyang et al, 2014), a primer can be designed aiming at the codominant sex specific marker locus to identify the genetic sex of the cynoglossus semilaevis, but in practical application, the length difference of two PCR amplification product fragments from a Z chromosome and a W chromosome is small due to the fact that the INDEL in the microsatellite is short (less than 50bp), and the difficulty is caused when the length of the PCR amplification product fragments is judged through gel electrophoresis. In addition, the multiple marks are unit point marks, namely, a co-reaction system based on multiple SNPs is invented aiming at the mark of a certain specific site, and the sex judgment is carried out by using multiple sites together, so that the sex discrimination accuracy of the true male fish and the false male fish of the cynoglossus semilaevis can be greatly improved, and the gold standard for detecting the genetic sex of the fish is formed.

Through searching, the following patent publications related to the patent application of the invention are found:

a cynoglossus semilaevis true and false male fish screening method (CN109825565A) based on a fluorescent molecular marker system, and relates to a cynoglossus semilaevis true and false male fish screening method based on a fluorescent molecular marker system. The design and detection of molecular markers is based on a five primer amplification-hindered mutation system (i.e., PARMS). The method successfully searches and confirms SNP polymorphic sites on the DNA sequence between Z/W sex chromosomes by using bioinformatics analysis and molecular biology methods of the DNA sequence of the Z/W sex chromosomes of the cynoglossus semilaevis, and designs and verifies 1 group of fluorescent molecular markers based on PARMS. The high-throughput qPCR typing experiment verification of 60 cynoglossus semilaevis samples with known sexes shows that the PARMS molecular marker can successfully identify true male fishes and false male fishes in all samples. Compared with the existing identification method and the marker, the marker and the corresponding detection method have the advantages of rapider operation, lower cost and higher detection rate on the premise of ensuring the accuracy rate, and can realize high-throughput detection.

By contrast, the present patent application is substantially different from the above patent publications.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a multi-site cynoglossus semilaevis true and false male fish screening system based on the SNaPshot technology and application thereof. Compared with the existing method for identifying the genetic sex of the cynoglossus semilaevis, the method is quicker to operate and higher in detection flux, and more importantly, the method has extremely high accuracy.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a multi-site cynoglossus semilaevis true and false male fish screening system based on a SNaPshot technology comprises 8 SNPs with a marker indication function, wherein the 8 SNPs are positioned on the same chromosome NC-024328.1, and the position information is as follows: SNP 1-13754040, SNP3-14518947, SNP4-14546665, SNP5-15320226, SNP6-14238006, SNP7-14546702, SNP9-14802286, and SNP 10-14802427.

Furthermore, the 8 SNPs were obtained by simplified genome sequencing gbs (genomic by sequencing) sequencing and whole genome association analysis screening.

The names, sequences and lengths of the primers for the 8 SNPs are:

the application of the multi-site cynoglossus semilaevis true and false male fish screening system based on the SNaPshot technology in the aspect of screening the cynoglossus semilaevis true and false male fish.

The preparation method of the multi-site cynoglossus semilaevis true and false male fish screening system based on the SNaPshot technology comprises the following steps:

s1, after simplified genome sequencing is carried out on 45 male and female cynoglossus semilaevis, differential SNPs sites of the male and female cynoglossus semilaevis are screened by utilizing whole genome correlation analysis;

s2, in the found difference significant top300, defining two horizontal significance lines for the p value given by the whole genome association analysis, wherein the p value is transformed by-log 10, one is the p value when FDR is 0.05, and the other is the p value after Bonferroni correction is 0.05; selecting sites above a significance horizontal line for experimental verification; the Bonferroni corrected significant difference level is 6.987842, which is calculated as: log10(0.05/number of SNPs); choose 10^-770 different sites screened by the power are taken as candidate SNPs sites;

s3, selecting missense _ variable, 5_ prime _ UTR _ variable, downstream _ gene _ variable and updstream _ gene _ variable from 70 bit points, wherein the total 10 SNPs are used as final selection SNPs for mark verification and Snapshot system development;

based on the fact that the SNPs sites screened are located on the same chromosome NC _024328.1, SNP 1-13754040, SNP2-13471049, SNP3-14518947, SNP4-14546665, SNP5-15320226, SNP6-14238006, SNP7-14546702, SNP8-15087253, SNP9-14802286 and SNP10-14802427, a group of Snapshot marker primers for sex identification of cynoglossus semilaevis are designed;

s4, performing a typing experiment by using a multiple PCR reaction system, and verifying whether the 10-labeled reaction system can identify the genetic sex of the cynoglossus semilaevis;

s5, comparing the accuracy and the detection rate of the SNaPshot sequencing result with the detection result of the known marker, and proving that the reaction system can identify the genetic sex of the cynoglossus semilaevis.

The method for identifying the true and false male fish of the multi-site cynoglossus semilaevis by using the discrimination system of the true and false male fish of the multi-site cynoglossus semilaevis based on the SNaPshot technology comprises the following steps:

s1: extracting genome DNA from the cynoglossus semilaevis fin tissue by a phenol-chloroform extraction method;

s2: constructing a reaction multiple PCR reaction system:

reaction procedure:

the pre-amplification products of each sample were mixed in equal proportions and 4. mu.l was used for digestion, and the digestion system was as follows after 10-fold dilution with ExoI enzyme:

pre-amplification product purification procedure: 1.0h at 37 ℃ and 20min at 75 ℃;

an extension system:

and (3) extension conditions: 96 ℃ for 10s, 50 ℃ for 5s, 60 ℃ for 30s, x 30 cycles;

s3: 3730xl on-machine detection

1) Adding 9 mul of mixed solution of molecular weight internal standard and formamide into each hole of a 96-hole plate, wherein the volume ratio of the mixed solution of the internal standard to the mixed solution of the formamide is 0.5: 8.5, 1.0 μ l of PCR product;

2) denaturation at 95 deg.C for 3min, and testing on computer.

The invention has the advantages and positive effects that:

1. the invention is based on a common detection system of 10 SNP loci, wherein the effective loci are 8, and the identification of the genetic sex of the cynoglossus semilaevis can be realized only by one multiplex PCR reaction system and the sequencing of the SNaPshot type. The system can only be operated on various genetic analysis instruments, and can realize automatic typing and identify male fishes and pseudo-male fishes. Compared with the existing method for identifying the genetic sex of the cynoglossus semilaevis, the method is quicker to operate and higher in detection flux, and more importantly, the method has extremely high accuracy and accumulative elimination rate.

2. The system is a multi-site cynoglossus semilaevis true and false male fish screening system based on the SNaPshot technology. The system is developed based on a plurality of Single Nucleotide Polymorphism (SNP) loci screened by whole genome association analysis (GWAS) of cynoglossus semilaevis female and male simplified genome sequencing. The design and detection of the system are based on the typing technology of the fluorescent labeling single base extension principle, namely SNaPshot. The system fuses 10 candidate indication SNPs into the same multiplex PCR reaction, and the indication accuracy of 10 sites in two groups of samples is detected through specific amplification and typing. Through the SNaPshot typing verification of 85 cynoglossus semilaevis samples with known sexes, the 8-locus SNPs marker system is proved to be capable of successfully identifying true and false male fishes in all samples. Compared with the existing identification method and the existing marker, the marker system and the detection method greatly improve the identification accuracy of the genuine and fake male cynoglossus semilaevis, can perform rapid and accurate gene typing on various genetic analyzers, realize the automation of SNP analysis, and can conveniently and efficiently realize high-throughput detection.

Drawings

FIG. 1 is a diagram of 10-locus SNP sequencing and typing results of 45 cynoglossus semilaevis according to the present invention; wherein, the homozygote allele 1 is a single base, and the allele 2 is the same as the single base and is marked as null, namely N-; heterozygote allele 1 and allele 2 are marked by actual bases and are marked as NX, and X is a mutation site;

FIG. 2 is a diagram of the identification of the genetic sex of 45 cynoglossus semilaevis by using known marker (application patent No.: 201910063591.X) indel marker in the present invention; wherein, agarose electrophoresis shows that pseudomale fish with two bands at the positions of 620bp and 1.4kb is present, and male fish with one band only at the position of 1.4kb is present;

FIG. 3 is a diagram showing the result of agarose gel electrophoresis of a physiological male fish of a 40-tailed cynoglossus semilaevis identified by a known marker according to the present invention;

FIG. 4 is a statistical chart of the locus information identified by the SNaPshot system developed by the present invention for a physiological male fish of a 40-tailed cynoglossus semilaevis;

FIG. 5 is an exemplary 10 samples of the original sequencing peak map of the SNaPshot of the first 45 cynoglossus semilaevis sea-capture samples;

FIG. 6 is an example of the original sequencing peak value of the SNaPshot of the second batch of 40 physiological male fish samples cultured by cynoglossus semilaevis Gunther, 10 samples.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

Preferably, the 8 SNPs are obtained by GBS sequencing and whole genome association analysis screening.

Preferably, the names, sequences and lengths of the primers of the 8 SNPs are as follows:

s2: constructing a reaction multiple PCR reaction system:

reaction procedure:

an extension system:

s3: 3730xl on-machine detection

2) denaturation at 95 deg.C for 3min, and testing on computer.

Specifically, a multi-site cynoglossus semilaevis true and false male fish screening system based on the SNaPshot technology, which comprises the following preparation steps:

s1, carrying out simplified genome sequencing (Genotype by sequencing) on 45 male and female cynoglossus semilaevis, and analyzing and screening different SNPs sites of the male and female cynoglossus semilaevis by utilizing GWAS (glow wire analysis);

s2, in finding the difference significant top300, two horizontal significance lines are defined for the p value given by GWAS (conversion of-log 10 has been performed), one is the p value at FDR 0.05, and the other is p 0.05 corrected by Bonferroni. The level of significant difference (-log10(0.05/number of SNPs)) after Bonferroni correction was 6.987842. Choose 10^-7A total of 70 differential sites selected in the power were used as candidate SNPs (Table 1).

TABLE 1 selection of 10^-770 different sites selected in the power are taken as candidate SNPs sites

S3, selecting missense _ variable, 5_ private _ UTR _ variable, downstream _ gene _ variable and updstream _ gene _ variable from 70 sites to be used as final selection SNPs for mark verification and Snapshot system development (Table 2).

TABLE 2 Final selection of SNPs

Based on the fact that the SNPs sites screened are located on the same chromosome (NC _024328.1), SNP 1-13754040, SNP2-13471049, SNP3-14518947, SNP4-14546665, SNP5-15320226, SNP6-14238006, SNP7-14546702, SNP8-15087253, SNP9-14802286 and SNP10-14802427, a group of Snapshot marker primers for sex identification of cynoglossus semilaevis are designed (Table 3);

s4, performing a typing experiment by using the multiple PCR reaction system, and verifying whether the 10-labeled reaction system can identify the genetic sex of the cynoglossus semilaevis.

S5, comparing the accuracy and the detection rate of the SNaPshot sequencing result with the detection result of a known marker (application patent number: 201910063591. X).

Preferably, the primer for identifying the sex of cynoglossus semilaevis selected in S3 is:

primer for identifying cynoglossus semilaevis gender screened in Table 3S3

The candidate SNP locus used by the invention is derived from whole genome association analysis (GWAS) screening after sequencing of the degenerate genome of female and male cynoglossus semilaevis.

A multi-site cynoglossus semilaevis true and false male fish screening system based on a SNaPshot technology comprises the following steps:

s1: extracting genome DNA from 40 cynoglossus semilaevis fin tissues of known genetic sex screened by other markers by using a phenol-chloroform extraction method;

the identification of known markers of the genetic sex of 40 cynoglossus semilaevis is shown in figure 3;

s2: constructing a reaction multiple PCR reaction system:

reaction procedure:

pre-amplification product purification procedure: 1.0h at 37 ℃ and 20min at 75 ℃.

An extension system:

and (3) extension conditions: 96 ℃ for 10s, 50 ℃ for 5s, 60 ℃ for 30s, x 30 cycles.

S3: 3730xl on-machine detection

1) Adding a molecular weight internal standard and formamide mixed liquor into each well of a 96-well plate (the molecular internal standard is a quantitative marker commonly used in the industry and has a trade name of Rox500, and the volume ratio of the internal standard to the formamide mixed liquor is 0.5: 8.5) 9. mu.l, 1.0. mu.l of PCR product;

2) denaturation at 95 deg.C for 3min, and testing on computer.

The figure of the identification result of the 8 effective sites Snapshot marker system of 40 cynoglossus semilaevis is shown in figure 4.

Results and discussion:

in the first round of the detection result of the SNaPshot, 10 pairs of primers are subjected to multiple polymerase chain reaction in the same system, and 10 mononucleotide polymorphic sites are detected in 45 cynoglossus semilaevis samples by using SNaPshot mini-sequencing. However, only 8 single nucleotide polymorphisms had different genotypes between males and pseudomales (see FIG. 1), and the two single nucleotide polymorphisms (SNP2-13471049, SNP8-15087253) could not show the sex of the two fishes differently. The sex information of the samples in the SNaPshot assay results are shown in table 4 and fig. 5. The result of the SNaPshot is completely consistent with the detection result of the developed INDEL marker (shown in figure 2), and the detection result of the 8-site single nucleotide polymorphism of the system can well reflect the genetic sex of the cynoglossus semilaevis.

Meanwhile, in order to further verify the universal applicability of the system, 40 additional cynoglossus semilaevis physiological male fish samples are taken from an aquaculture farm and subjected to second-round screening. SnaPshot test genetic sex statistics for 40 samples (see FIG. 4) show that each SNP in the 8-site SnaPshot system is effective in showing differences between males and pseudomales. In combination with the agarose gel electrophoresis validation of known Indel markers (see fig. 3), 13 false males and 27 true males were all detected in agreement with the snap shot system (see table 5 and fig. 6). The eight SNPs sites are indicated to have universality on the indication of the genetic sex of the cynoglossus semilaevis.

TABLE 4 genetic sex information of the first 45 cynoglossus semilaevis individuals captured by sea

TABLE 5 second batch of 40 male cynoglossus semilaevis cultured individual genetic sex information

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.

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Claims

1. A multi-site cynoglossus semilaevis true and false male fish screening system based on the SNaPshot technology is characterized in that: the system comprises 8 SNPs with marker indication effects, the 8 SNPs are positioned on the same chromosome NC-024328.1, and the position information is as follows: SNP 1-13754040, SNP3-14518947, SNP4-14546665, SNP5-15320226, SNP6-14238006, SNP7-14546702, SNP9-14802286, and SNP 10-14802427.

2. The SNaPshot technology-based true and false male cynoglossus semilaevis screening system according to claim 1, which is characterized in that: the 8 SNPs are obtained by GBS sequencing and whole genome association analysis screening.

3. The SNaPshot technology-based true and false male cynoglossus semilaevis screening system according to claim 1 or 2, characterized in that: the names and sequences of the primers of the 8 SNPs are as follows:

4. the application of the SNaPshot technology-based multi-site Cynoglossus semilaevis male fish screening system in screening of Cynoglossus semilaevis male fish according to any one of claims 1 to 3.

5. The method for preparing a discrimination system for true and false male cynoglossus semilaevis based on the SNapshot technology as claimed in any one of claims 1 to 3, which is characterized in that: the method comprises the following steps:

6. The method for identifying the true and false male fish of the multi-site cynoglossus semilaevis by using the screening system for the true and false male fish of the multi-site cynoglossus semilaevis based on the SNapshot technology as claimed in any one of claims 1 to 3, which is characterized in that: the method comprises the following steps:

s2: constructing a reaction multiple PCR reaction system:

reaction procedure:

an extension system:

s3: 3730xl on-machine detection

2) denaturation at 95 deg.C for 3min, and testing on computer.