CN109825565B - Cynoglossus semilaevis true and false male fish screening method based on fluorescent molecular marker system - Google Patents
Cynoglossus semilaevis true and false male fish screening method based on fluorescent molecular marker system Download PDFInfo
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Abstract
The invention relates to a fluorescence molecule marker-based cynoglossus semilaevis true and false male fish screening method. The method is developed based on Single Nucleotide Polymorphism (SNP) sites on a DNA sequence between Z/W sex chromosomes of cynoglossus semilaevis. Detection of molecular markers is based on a five-primer amplification-hindered mutation system. A SNP polymorphic site (named SNP _ chr _8936186_ C _ G) on a DNA sequence between Z/W sex chromosomes is searched and confirmed by utilizing bioinformatics analysis of the DNA sequence of the Z/W sex chromosomes of the cynoglossus semilaevis, and 1 group of fluorescent molecular marker primers based on PARMS are designed and verified. The high-throughput qPCR typing verification of 60 cynoglossus semilaevis samples with known sexes shows that the marker can successfully identify the true male fish and the false male fish in the samples. Compared with the existing method and the marker, the marker is quicker to operate, lower in cost and higher in detection rate, and can realize high-throughput detection.
Description
Technical Field
The invention belongs to the technical field of genetic sex identification and sex control of marine fishes in the aquatic biotechnology, and relates to a codominant high-throughput fluorescent molecular marker which is developed based on SNP loci between sex chromosomes Z/W of cynoglossus semilaevis and can be used for discriminating the sex of the cynoglossus semilaevis.
Background
Cynoglossus semilaevis (Cynogossus semilaevis) belongs to the order of Flounderiformes, the family of Cynoglossus semilaevis and the genus of Cynoglossus semilaevis, is a specific warm-warm offshore large-scale bottom-layer fish in the sea area of China, and is mainly distributed in coastal areas of the yellow sea and the Bohai sea. Because the natural resources of the cynoglossus semilaevis are less, the cynoglossus semilaevis is fresh and delicious in taste, smooth in mouthfeel and rich in nutrition, the cynoglossus semilaevis has a wide culture prospect and is an important and rare marine culture fish. The cynoglossus semilaevis is used as a flatfish, the growth rate of female individuals is more than 3 times that of male individuals (Chensong forest, etc., 2013), and the male fish has the characteristics of slow growth and small individual body size, so that the culture yield of the cynoglossus semilaevis is reduced, and the culture cost is increased. The culture of the cynoglossus semilaevis into the male fish can even reach 80 to 90 percent. Studies have found that some of the 80% to 90% proportion of male fish are false male. Studies have shown that the sex-determining mechanism of cynoglossus semilaevis is sex chromosomes Z and W, wherein the homochromosome ZZ is a genetic male individual and the heterochromosome ZW type is a genetic female individual (Zhuang et al, 2006; Zhouliqing, etc., 2005). The male fish of the cynoglossus semilaevis has ZZ type chromosome, and the female fish has ZW type chromosome. 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 imbalance of the proportion of physiological female fish in the cultured groups of the cynoglossus semilaevis (namely more and more male fish and less female fish) is caused, and the culture yield of the cynoglossus semilaevis is seriously influenced. Therefore, the development of the efficient molecular marker and the detection method for rapidly identifying the sex of the cynoglossus semilaevis, particularly for identifying true and false male fish, has important scientific significance and application value for genetic sex identification and production culture.
In the aspects of cynoglossus semilaevis gender specific molecular marker screening and genetic gender identification, a cynoglossus semilaevis female specific AFLP marker (Chen et al, 2007) is separated by a predecessor through an AFLP (amplified fragment length polymorphism) marker technology, and as the AFLP marker is dominant inheritance, ZW female and WW superfemale individuals cannot be distinguished in application, 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. Meanwhile, in the detection of the technology, 4% agarose gel is needed to help distinguish bands with small length difference (such as less than 50bp), and the high-concentration agarose is not easy to prepare, and has the problems of long electrophoresis time, high cost and the like, thereby further limiting the popularization of the method.
Therefore, the invention is particularly necessary to solve the problems by inventing a molecular marker capable of discriminating the genders of the cynoglossus semilaevis true and false male fish based on the codominance of the SNP.
Disclosure of Invention
1. Problems to be solved
The invention aims to provide a fluorescent molecular marker capable of discriminating the sex of a cynoglossus semilaevis, and solves the problems in the background technology by utilizing the sex-specific SNP locus typing of the cynoglossus semilaevis.
2. Technical scheme
In order to achieve the above object, the present invention provides the following technical solutions.
A sex discrimination technology for the genuine and counterfeit male cynoglossus semilaevis based on a fluorescent molecular marker system comprises the following steps:
s1, downloading public ZW chromosome genome sequences which have been disclosed without intellectual property rights;
s2, carrying out comparison analysis on the searched ZW chromosome homologous allele sequences, and screening out candidate SNP loci capable of distinguishing ZW sex chromosomes;
s3, designing a group of PARMS fluorescent labeled primers for cynoglossus semilaevis gender identification based on the screened SNP site SNP _ chr _8936186_ C _ G;
s4, extracting genome DNA from the cynoglossus semilaevis fin tissue by using a phenol chloroform extraction method;
s5, carrying out typing experiment by using real-time quantitative PCR (qPCR) to verify whether the fluorescence labeling of PARMS can identify male and female cynoglossus semilaevis.
Preferably, in the S2, a sex-specific SNP site SNP _ chr _8936186_ C _ G is selected from the cynoglossus semilaevis ZW sex chromosome DNA sequence: GCTGAAGGTGCAGTTGTGA [ C/G ], wherein C is a female fish-specific variation. The C/G genotype is female fish or pseudo-male fish, and the G/G genotype is true-male fish.
Preferably, the PARMS fluorescence labeled primers screened from S3 for cynoglossus semilaevis gender identification are:
8936186_PR_C:GAAGGTGACCAAGTTCATGCTGCTGAAGGTGCAGTTGTGAc；
8936186_PR_G:GAAGGTCGGAGTCAACGGATTGCTGAAGGTGCAGTTGTGAg；
8936186_PF:GGCAACAAGGACAGAATCTACATC。
3. advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
based on the discovered SNP loci between the cynoglossus semilaevis ZW sex chromosomes, the sex identification of male and female cynoglossus semilaevis can be realized only by a qPCR typing experiment. The method can realize automatic typing and identify males and females only by 5 mul of reaction system. Compared with the existing method for identifying the genetic sex of the cynoglossus semilaevis, the method has the advantages of rapider operation, lower cost and higher detection flux on the premise of ensuring the accuracy, and hundreds of samples can be detected by one-time reaction.
Drawings
FIG. 1 is a diagram of SNP-chr-8936186-C-G site PARMS fluorescent labeling qPCR typing results of sample No. 1-30 Cynoglossus semilaevis, black spot is homozygote ZZ male individual × 30 (3 replicates per sample); gray spots are heterozygous ZW females X30 (3 replicates per sample)
FIG. 2 is a diagram of SNP-chr-8936186-C-G site PARMS fluorescent labeling qPCR typing results of sample No. 31-60 Cynoglossus semilaevis, black spot is homozygote ZZ male individual × 30 (3 replicates per sample); gray spots are heterozygous ZW females X30 (3 replicates per sample)
Detailed Description
In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The genome sequence used by the invention is derived from the sequencing data of the whole genome of the cynoglossus semilaevis completed by the public published non-property protection of aquatic genome of the yellow sea aquatic research institute and the public domain of the cell engineering research laboratory.
The application of the sex-specific SNP site detection technology provided by the invention comprises the following specific steps:
s1: selecting ZW chromosome homologous allele sequences and searching candidate SNP loci;
s2: designing a Primer pair capable of amplifying an allele sequence containing a candidate SNP locus by combining with Primer3Plus online Primer design software;
s3: sending the PCR product amplified in the second step to a first generation sequencing, and determining a sex-specific SNP site SNP _ chr _8936186_ C _ G according to a first generation sequencing result;
s4: and designing a group of PARMS fluorescent labeled primers for cynoglossus semilaevis gender identification by combining Primer3Plus online Primer design software based on the SNP site SNP _ chr _8936186_ C _ G verified in the third step. The PARMS fluorescent labeling primers are as follows:
8936186_PR_C:GAAGGTGACCAAGTTCATGCTGCTGAAGGTGCAGTTGTGAc；
8936186_PR_G:GAAGGTCGGAGTCAACGGATTGCTGAAGGTGCAGTTGTGAg；
8936186_PF:GGCAACAAGGACAGAATCTACATC。
wherein 8936186_ PR _ C is connected with FAM fluorophore, and the sequence of the FAM fluorophore is as follows: GAAGGTGACCAAGTTCATGCT, respectively; 8936186_ PR _ G is connected with HEX fluorophore, and the HEX fluorophore sequence is GAAGGTCGGAGTCAACGGATT
S5: extracting genome DNA from 60 cynoglossus semilaevis fin tissues with known genetic sex by a phenol-chloroform extraction method;
s6: preparing a 10 XPARMS primer mother solution according to 200 reaction requirements:
s7: the typing was performed automatically using 5. mu.l of PARMS Touchdown PCR (Touchdown PCR) reaction system and ABI Q6 platform with own analytical software.
| Reagent/sample | Dosage of |
| 2×PARMS Master Mix | 2.5μl |
| 10 XPARMS primer mother liquor | 0.5μl |
| DNA template(50ng) | Adding water to 5 μ l |
| Total volume of single reaction | 5μl |
The environmental parameters required by the qPCR PARMS touchDown PCR reaction program are as follows: step 1: 3min at 94 ℃; step 2: 20s at 94 ℃; and 3, step 3: at 65 ℃ (-0.8 ℃ per cycle) for 1 min; and 4, step 4: returning to the step 2, and performing 10 cycles; and 5, step 5: 20s at 94 ℃; and 6, step 6: 1min at 57 ℃; and 7, step 7: go back to step 5, 30 cycles.
S8: and judging the sex of the cynoglossus semilaevis through a qPCR automatic typing result.
Table one: and (3) marking and identifying the result of PARMS at SNP _ chr _8936186_ C _ G site of 60 cynoglossus semilaevis.
The accuracy of the identification result of the technology is determined by taking the identification result of the morphological sex of the cynoglossus semilaevis as a comparison.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which shall fall within the scope of the claimed invention. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
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<120> fluorescence molecular marker system-based cynoglossus semilaevis true and false male fish screening method
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Claims (2)
1. The method for discriminating the true male fish from the false male fish of the cynoglossus semilaevis based on a fluorescent molecular marker system is characterized in that SNP marker sites SNP _ chr _8936186_ C _ G screened from a genomic DNA sequence of the cynoglossus semilaevis are as follows:
GCTGAAGGTGCAGTTGTGA [ C/G ], wherein C is female fish specific variation, C/G genotype is female fish or pseudo-male fish, and G/G genotype is true-male fish.
2. A marker primer for detecting sex-specific SNP sites of cynoglossus semilaevis according to claim 1, wherein the primer sequence is as follows:
8936186_PR_C：GAAGGTGACCAAGTTCATGCTGCTGAAGGTGCAGTTGTGAc；
8936186_PR_G：GAAGGTCGGAGTCAACGGATTGCTGAAGGTGCAGTTGTGAg；
8936186_PF：GGCAACAAGGACAGAATCTACATC；
wherein 8936186_ PR _ C is connected with FAM fluorophore, and the sequence of the FAM fluorophore is as follows: GAAGGTGACCAAGTTCATGCT, respectively; 8936186_ PR _ G is linked to a HEX fluorophore with a HEX fluorophore sequence of GAAGGTCGGAGTCAACGGATT.
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| CN110616256B (en) * | 2019-08-19 | 2022-11-22 | 天津渤海水产研究所 | Multi-site cynoglossus semilaevis true and false male fish screening system based on SNaPshot technology and application |
| CN111676297A (en) * | 2020-06-19 | 2020-09-18 | 安徽微分基因科技有限公司 | Molecular marker and detection method for rapidly discriminating true and false male fish of cynoglossus semilaevis based on RAA-LFD |
| CN113832219A (en) * | 2020-06-23 | 2021-12-24 | 安徽微分基因科技有限公司 | Nano-upgrade ultrahigh-flux SNP genotyping method based on PARMS technology |
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