CN110616256B - Multi-site cynoglossus semilaevis true and false male fish screening system based on SNaPshot technology and application - Google Patents
Multi-site cynoglossus semilaevis true and false male fish screening system based on SNaPshot technology and application Download PDFInfo
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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, SNP10-02427. 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 has the advantages of quicker operation and higher detection flux, and more importantly, the method has extremely high accuracy.
Description
Technical Field
The invention belongs to the technical field of genetic sex identification of marine fishes in an aquatic biotechnology, and particularly relates to a multi-site cynoglossus semilaevis true and false male fish screening system 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 false male fish is used as a male parent, the offspring will inherit the characteristics of the male parent as the false 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 Amplified Fragment Length Polymorphism (AFLP) marker technology, and because the AFLP marker is dominant heredity, ZW female and WW superfemale individuals cannot be distinguished in application, false negative results are difficult to avoid, and thus false judgment is generated on ZZ male fish and ZW female fish. Through screening the cynoglossus semilaevis co-dominant sex specific microsatellite scaffold68-2 marker (Liuyang et al, 2014), a primer can be designed aiming at the co-dominant sex specific marker locus to identify the genetic sex of the cynoglossus semilaevis, but in practical application, the INDEL in the microsatellite is short (less than 50 bp), so that the length difference of two PCR amplification product fragments from a Z chromosome and a W chromosome is small, and the difficulty is caused when the length of the cynoglossus semilaevis is judged by 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 based on a fluorescent molecular marker system (CN 109825565A), a cynoglossus semilaevis true and false male fish screening method based on the 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 problem is as follows:
a multi-site cynoglossus semilaevis true and false male fish screening system based on the 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 SNP10-14802427.
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 discrimination 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 significance horizontal lines for a p value given by whole genome association analysis, wherein the p value is subjected to-log 10 conversion, one is the p value when FDR =0.05, and the other is the p =0.05 corrected by Bonferroni; selecting sites above a significance horizontal line for experimental verification; the corrected significant difference level of Bonferroni was 6.987842, which was calculated as: log10 (0.05/number of SNPs); choose 10 -7 70 different sites screened by the power are taken as candidate SNPs sites;
s3, selecting 10 SNPs of missense _ variable, 5/prime/UTR/variable, downstream _ gene _ variable and upstream _ gene _ variable from 70 bit points as final selection SNPs for mark verification and Snapshot system development;
based on the fact that the SNPs sites screened in the method are located on the same chromosome NC-024328.1, SNPs 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 sequencing result of the SNaPshot with the detection result of a 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 cynoglossus semilaevis gunther by using the multi-site cynoglossus semilaevis gunther true and false male fish discrimination system based on the SNaPshot technology comprises the following steps:
s1: extracting genome DNA from the fin tissue of the cynoglossus semilaevis by using a phenol-chloroform extraction method;
s2: constructing a reaction multiple PCR reaction system:
reaction procedures are as follows:
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: 10s at 96 ℃, 5s at 50 ℃, 30s at 60 ℃ and x 30cycles;
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. Mu.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 indicator SNPs into the same multiplex PCR reaction, and specifically amplifies and typing detects the indication accuracy of 10 sites in two groups of samples. 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 empty, 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 genetic sex identification of 45 Cynoglossus semilaevis Gunther by using known marker (patent application No. 201910063591. X) indel marker in the present invention; wherein, agarose electrophoresis shows that the fish with two bands at the positions of 620bp and 1.4kb is a false male fish, and the fish with one band at the position of 1.4kb is a male fish;
FIG. 3 is the result of agarose gel electrophoresis of physiological male fish of 40-fish cynoglossus semilaevis identified by known markers;
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 a 10 sample schematic of the original sequencing peak map of the first 45 cynoglossus semilaevis capture samples by the SNaPshot method;
FIG. 6 is a graph of the original sequencing peaks of the snaPshot of the second batch of 40 physiological male fish samples cultured by cynoglossus semilaevis, FIG. 10 samples.
Detailed Description
The present invention is described in detail below with reference to the following examples, which are intended to be illustrative and not limiting, 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.
A multi-locus cynoglossus semilaevis true-false male fish screening system based on a SNaPshot technology comprises 8 SNPs with a mark 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, SNP10-02427.
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:
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 significance horizontal lines for a p value given by whole genome association analysis, wherein the p value is subjected to-log 10 conversion, one is the p value when FDR =0.05, and the other is the p =0.05 corrected by Bonferroni; selecting sites above a significance horizontal line for experimental verification; the Bonferroni corrected significant difference level was 6.987842, which was calculated as: log10 (0.05/number of SNPs); choose 10 -7 70 different sites screened by the power are taken as candidate SNPs sites;
s3, selecting 10 SNPs of missense _ variable, 5/prime/UTR/variable, downstream _ gene _ variable and upstream _ gene _ variable from 70 bit points as final selection SNPs for mark verification and Snapshot system development;
based on the SNPs locus screened on the same chromosome NC-024328.1, SNPs 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 labeled 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 sequencing result of the SNaPshot with the detection result of a 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 procedures are as follows:
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: 10s at 96 ℃, 5s at 50 ℃, 30s at 60 ℃ and x 30cycles;
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. Mu.l of PCR product;
2) Denaturation at 95 deg.C for 3min, and testing on machine.
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, after simplified genome sequencing (Genotype by sequencing) is carried out on 45 male and female cynoglossus semilaevis, GWAS (glow wire analysis) is utilized to analyze and screen different SNPs (single nucleotide polymorphism) sites of the male and female cynoglossus semilaevis;
s2, in the found difference significant top300, two horizontal significance lines (subjected to-log 10 conversion) are defined for the p value given by GWAS, wherein one is the p value when FDR =0.05, and the other is p =0.05 corrected by Bonferroni. The level of significant difference (-log 10 (0.05/number of SNPs)) after Bonferroni correction was 6.987842. Choose 10 -7 A total of 70 differential sites selected in the power were used as candidate SNPs (Table 1).
TABLE 1 selection of 10 -7 70 different sites selected in the power are taken as candidate SNPs sites
S3, selecting 10 SNPs (Table 2) from 70 sites, wherein the SNPs are missense _ variant, 5_ prime _ UTR _ variant, downstream _ gene _ variant and updstream _ gene _ variant.
TABLE 2 Final selection of SNPs
Based on the SNPs locus on the same chromosome (NC-024328.1), SNPs 1-13754040, SNP2-13471049, SNP3-14518947, SNP4-14546665, SNP5-15320226, SNP6-14238006, SNP7-14546702, SNP8-15087253, SNP9-14802286, SNP10-14802427, a set of Snapshoot marker primers for sex identification of Cynoglossus semilaevis Gunther was designed (Table 3);
and 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 the cynoglossus semilaevis selected in S3 is:
primers for sex identification of cynoglossus semilaevis selected in S3 of Table 3
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 procedures are as follows:
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 ℃ 10s,50 ℃ 5s,60 ℃ 30s, x 30cycles.
S3:3730xl on-machine detection
1) Adding a molecular weight internal standard and a formamide mixed solution into each hole of a 96-hole 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 solution is 0.5:8.5 9 μ l, 1.0 μ l 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 (SNP 2-13471049, SNP8-15087253) did not show differences in sex between the two species of fish. 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 (see figure 2), which shows that 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 a second round of screening. The statistics of the genetic sex for the SNaPshot assay of 40 samples (see fig. 4) show that each SNP in the 8-locus SNaPshot system effectively shows the difference between male and pseudomale. 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 genetic sex information of 40 male Cynoglossus semilaevis cultured in the second batch
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 appended claims, and therefore, the scope of the invention is not limited to the disclosure of the embodiments and drawings.
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<210> 12
<211> 20
<212> DNA
<213> SNP-9-R(Unknown)
<400> 12
<210> 13
<211> 21
<212> DNA
<213> SNP-10-F(Unknown)
<400> 13
<210> 14
<211> 22
<212> DNA
<213> SNP-10-R(Unknown)
<400> 14
tgactgtgag ttcacggtct cc 22
<210> 15
<211> 30
<212> DNA
<213> SNP-1-YS-R(Unknown)
<400> 15
ttttttttgg agctggactt cctcttctcc 30
<210> 16
<211> 36
<212> DNA
<213> SNP-3-YS-R(Unknown)
<400> 16
tttttttttt ttttttgctc ggaccagccc atctac 36
<210> 17
<211> 47
<212> DNA
<213> SNP-4-YS-R(Unknown)
<400> 17
tttttttttt tttttttttt tttttttagt ccaacagcac ggccgac 47
<210> 18
<211> 42
<212> DNA
<213> SNP-5-YS-F(Unknown)
<400> 18
tttttttttt tttttttttt tttccggcat ctcaccgtag ga 42
<210> 19
<211> 52
<212> DNA
<213> SNP-6-YS-F(Unknown)
<400> 19
tttttttttt tttttttttt tttttttttt tctccatcct cctcaatttc tg 52
<210> 20
<211> 69
<212> DNA
<213> SNP-7-YS-R(Unknown)
<400> 20
tttttttttt tttttttttt tttttttttt tttttttttt tttttttttg agctccagcc 60
ctctggcca 69
<210> 21
<211> 61
<212> DNA
<213> SNP-9-YS-F(Unknown)
<400> 21
tttttttttt tttttttttt tttttttttt tttttttttt tatgggaggc tgatggtgaa 60
t 61
<210> 22
<211> 72
<212> DNA
<213> SNP-10-YS-F(Unknown)
<400> 22
tttttttttt tttttttttt tttttttttt tttttttttt ttttttttta cacaagtgca 60
ttatgggtat ac 72
Claims (5)
1. A8 SNPs co-detection primer combination for discriminating true and false male cynoglossus semilaevis based on the SNaPshot technology is characterized in that: the 8 SNPs are located 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, SNP10-14802427.
2. The SNaPshot technology-based 8 SNPs co-detection primer combination for discriminating true male fish and false male fish of cynoglossus semilaevis at multiple sites according to claim 1, characterized in that: the 8 SNPs are obtained by GBS sequencing and whole genome association analysis screening.
3. The SNaPshot technology-based 8 SNPs co-detection primer combination for true and false male fish screening of cynoglossus semilaevis at multiple sites 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 8 SNPs co-detection primer combination for the discrimination of the true and false male cynoglossus semilaevis based on the SNaPshot technology in the discrimination of the true and false male cynoglossus semilaevis according to any one of claims 1 to 3.
5. The method for identifying the true and false male fish of the multi-site cynoglossus semilaevis by using the 8 SNPs co-detection primer combination for screening 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 to3, which is characterized in that: the method 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:
after the pre-amplification products of each sample are mixed in equal proportion, 4 mul of the pre-amplification products are used for digestion, and a digestion system is as follows after 10 times of ExoI enzyme dilution:
Pre-amplification product purification procedure: 1.0h at 37 ℃ and 20min at 75 ℃;
an extension system:
And (3) extension conditions: 10s at 96 ℃, 5s at 50 ℃, 30s at 60 ℃ and x 30cycles;
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 internal standard to the mixed solution of the formamide is 0.5:8.5, 1.0. Mu.l of PCR product;
2) Denaturation at 95 deg.C for 3min, and testing on computer.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101225437A (en) * | 2007-12-01 | 2008-07-23 | 中国水产科学研究院黄海水产研究所 | Tongue sole molecular marker auxiliary sex control method |
| CN109536624A (en) * | 2019-01-22 | 2019-03-29 | 天津渤海水产研究所 | For screening the fluorescent molecule tagging and testing method of Cynoglossus semilaevis true and false milter property |
| CN109825565A (en) * | 2019-01-22 | 2019-05-31 | 天津渤海水产研究所 | A kind of Cynoglossus semilaevis true and false milter discriminating method based on fluorescent molecule label system |
-
2019
- 2019-08-19 CN CN201910762159.XA patent/CN110616256B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101225437A (en) * | 2007-12-01 | 2008-07-23 | 中国水产科学研究院黄海水产研究所 | Tongue sole molecular marker auxiliary sex control method |
| CN109536624A (en) * | 2019-01-22 | 2019-03-29 | 天津渤海水产研究所 | For screening the fluorescent molecule tagging and testing method of Cynoglossus semilaevis true and false milter property |
| CN109825565A (en) * | 2019-01-22 | 2019-05-31 | 天津渤海水产研究所 | A kind of Cynoglossus semilaevis true and false milter discriminating method based on fluorescent molecule label system |
Non-Patent Citations (2)
| Title |
|---|
| New locus reveals the genetic architecture of sex reversal in the Chinese tongue sole (Cynoglossus semilaevis);CUI，Y.等;《Heredity》;20180809;319-326 * |
| Single nucleotide polymorphism markers with applications in aquaculture and assessment of its impact on natural populations;WENNE,R.;《Aquat. Living Resour.》;20181231;第2号文章 * |
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