CN114686596B - SNP molecular marker for sex identification of silver dragon fish and application thereof - Google Patents
SNP molecular marker for sex identification of silver dragon fish and application thereof Download PDFInfo
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- CN114686596B CN114686596B CN202011564604.0A CN202011564604A CN114686596B CN 114686596 B CN114686596 B CN 114686596B CN 202011564604 A CN202011564604 A CN 202011564604A CN 114686596 B CN114686596 B CN 114686596B
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention discloses an SNP molecular marker for sex identification of silver dragon fish and application thereof, wherein the base sequence is as follows: SEQ ID NO.1; the SNP molecular marker is the 243 rd position in SEQ ID NO. 1. The genome high-throughput sequencing comparison is adopted to obtain single nucleotide polymorphism Sites (SNP) which are different between the male and female individuals of the Agkistrodon halys and can characterize the sexes of the females and the males, and the amplification sequence containing the SNP sites is obtained by combining with the primer amplification, so that the PCR amplification and sequencing of the sample to be detected are realized by the primer, and the SNP site information of the sample is obtained, thereby identifying the sex of the sample to be detected. The SNP locus can realize accurate and rapid sex identification on the premise of not splitting the silver dragon fish, and is convenient to realize mature artificial silver dragon fish breeding technology when the SNP locus is applied to silver dragon fish breeding so as to realize mass production.
Description
Technical Field
The invention relates to the technical field of fish sex identification, in particular to an SNP molecular marker for identifying the sex of Agkistrodon halys and application thereof.
Background
The silver dragon fish is also called as double whisker bone tongue fish, is an old 'activated stone' fish, and has very important economic value as a rare ornamental fish. At present, most of domestic silver dragons are imported, and the silver dragons are difficult to realize native mass cultivation; because the current breeding technology of the silver dragon fish is limited, the artificial mass cultivation is difficult to realize. The main reason for limiting the reproduction technique is represented by the difficulty in distinguishing the male sex of silver Long Yuci.
The silver dragon fish has no amphoteric specificity, cannot be distinguished in external morphology, and has no obvious characteristics even in the breeding season. And because the silver dragon fish has hard and thick scales, the scales cannot be penetrated by means of B ultrasonic, CT and the like to judge the sex. At present, identification can only be carried out by dissecting and observing gonads, and a simple and accurate method for identifying without dissecting cannot be realized. The artificial propagation of the fish is greatly plagued only by the fact that a means for accurately judging the sex is not available; because the sexes are difficult to distinguish, most farms can only reproduce by adopting a semi-natural method, but cannot adopt a high-efficiency controllable artificial reproduction mode, such as temporary culture of male and female fishes in separate ponds, injection of oestrus accelerator, oestrus accelerator and the like, so that the reproduction efficiency and success rate are greatly limited. In order to break the situation that the silver dragon fish in China is totally dependent on import, artificial propagation of the silver dragon fish becomes a problem to be solved urgently.
Therefore, there is a need for an effective sex identification method or marker for silver Long Yuci to overcome the above problems.
Disclosure of Invention
The SNP locus can realize accurate and rapid sex identification without decomposing the silver dragon fish, so that the normal growth and reproduction of the silver dragon fish are not affected, and when the SNP locus is applied to the cultivation of the silver dragon fish, the sex identification is conveniently provided to promote the maturation and development of cultivation technology, and the reproduction rate and success rate are improved.
The invention provides an SNP molecular marker for sex identification of Agkistrodon halys, which has the following base sequence: SEQ ID NO.1, 340bp in length; the SNP molecular marker is the 243 rd position in SEQ ID NO. 1.
When the 243 th base is C/C homozygous, the sample is male, and when the 243 nd base is C/T heterozygous, the sample is female. SEQ ID NO.1 is only a representative sequence exhibiting one of the above-mentioned changes, and the corresponding other sequences comprising the above-mentioned changes should also be protected.
The invention also provides a primer pair for identifying or assisting in identifying the sex of the Agkistrodon halys, which comprises an upstream primer F and a downstream primer R, wherein the nucleotide sequence of the upstream primer F is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer R is shown as SEQ ID NO. 3.
The invention also provides a kit for identifying or assisting in identifying the sex of the Agkistrodon, which contains the primer pair, dNTP and DNA polymerase.
The invention also provides a sex identification method of the silver dragon fish, which comprises the following steps:
(1) Extracting genome DNA of the silver dragon fish to be detected;
(2) Taking genomic DNA of the silver dragon fish to be detected as a template, and carrying out PCR amplification reaction by using the upstream primer F and the downstream primer R;
(3) After the reaction is completed, analysis is carried out, the genotype of the sample is determined, and the sex is identified.
The method can realize identification of the silver dragon fish without decomposition, and can realize batch sequencing by means of the multiple sequencing methods or the sequencing platforms, and on the basis, the method is more beneficial to being applied to cultivation and research of the silver dragon fish, so that the sex of the silver dragon fish is rapidly distinguished, and the identification and cultivation efficiency is improved. The identification method is quick and simple, and has the advantages that the identification result is accurate in more than one embodiment of the invention.
Further, the reaction system used in the PCR amplification reaction in step (2) was 40. Mu.l, including 20. Mu.l of 2X Taq Master mix (Dye), 1.6. Mu.l of Primer F, 1.6. Mu.l of Primer R, 15.8. Mu.l of ddH2O, and 1. Mu.l of genomic DNA.
Further, the PCR amplification procedure in the PCR amplification reaction in the step (2) is as follows: 94 ℃ for 2min; then 35 cycles were performed, including 94℃for 30s,56℃for 30s, and 72℃for 20s; then the temperature is maintained at 72 ℃ for 5 min.
Further, in step (3), the analysis is performed in combination with Sanger sequencing to determine the genotype of the sample and identify the sex.
The invention also provides the SNP molecular marker for sex identification of the silver dragon fish, the primer pair, the kit and/or the application of the method in sex identification of the silver dragon fish and/or cultivation of the silver dragon fish.
Compared with the prior art, the invention has the beneficial effects that: the genome high-throughput sequencing comparison is adopted to obtain single nucleotide polymorphism Sites (SNP) which are different between the male and female individuals of the Agkistrodon halys and can characterize the sexes of the females and the males, and the amplification sequence containing the SNP sites is obtained by combining with the primer amplification, so that the PCR amplification of the sample to be detected is realized by the primer on the basis, the SNP site information of the sample is obtained, and the sex of the sample to be detected is identified according to the genotype of the SNP site. And the sex identification can be finished through PCR amplification and Sanger sequencing, and compared with other molecular identification methods with complicated operation and long time consumption, the sex identification method is more suitable for rapidly identifying a large number of samples. More importantly, the SNP locus provided by the invention can simply, accurately and rapidly identify the sex of the Agkistrodon, does not need to dissect the Agkistrodon, basically does not influence the health condition of a tested sample, is convenient to realize application on the Agkistrodon breeding technology, realizes sex pairing of the Agkistrodon, greatly improves the breeding efficiency, the breeding success rate and the number of offspring of the Agkistrodon, solves the important industrial problem of breeding the Agkistrodon, and has important economic and social values. Moreover, as the silver dragon fish is an ancient fish, the research on sex differentiation and sex determination mechanisms of the silver dragon fish has important scientific significance for explaining the sex evolution and sex determination mechanisms of the whole fish, so that the SNP molecular marker, the corresponding primer, the kit and the identification method provided by the invention not only have the beneficial effects, but also can provide a foundation for the research on the fish evolution and sex determination mechanisms and the like, and promote the development of the research direction.
Drawings
FIG. 1 is a representative diagram of the electrophoresis result of a PCR product of a sample to be tested;
FIG. 2 is a representation of Sanger sequencing, showing the sequencing peaks of SNP marker loci (position 243), the upper panel being male (genotype CC) and the lower panel being female (genotype CT).
Detailed Description
The drawings are for illustrative purposes only and are not to be construed as limiting the invention.
Example 1
In the embodiment, a high-throughput sequencing strategy is adopted, 5 female dragon fish samples and 5 male fish samples are selected for DNA extraction, and a double-end genome DNA library with the insert size of 500bp is constructed according to the requirement of an Illumina library construction flow. And then, carrying out high-throughput sequencing on the genome by adopting an Illumina NovaSeq sequencing platform, wherein the sequencing quantity of each sample is 30Gb, and the sequencing strategy is Pair-End 150bp.
And detecting single nucleotide polymorphism Sites (SNP) of the sequenced female samples and single nucleotide polymorphism Sites (SNP) of the male samples by adopting a high-throughput sequencing sequence comparison strategy, and screening SNP sites which are identical between female samples, identical between male samples and different between male and female samples as sex identification molecular markers.
And (3) carrying out specific DNA molecular marker screening on the female sample and the male sample according to high-throughput sequencing, and finding that the gene sequence contains SNP molecular markers for distinguishing gender. On the basis, the primer is combined with high-throughput sequencing data for amplification to obtain a sequence which contains SNP loci and can be used for PCR and Sanger sequencing to identify the male sex of silver Long Yuci, specifically, the primer comprises an upstream primer F with a sequence shown as SEQ ID NO.2 and a downstream primer R with a sequence shown as SEQ ID NO.3, and the obtained sequence which contains SNP loci is shown as SEQ ID NO. 1. And the SNP molecular marker is the 243 rd position in SEQ ID NO. 1.
Specifically, the expression is as follows:
GTTCCCATCTAGGTTCCACTctttccttagccttgatccccatacttccaggataggccctgctacccggactggcataagtggttgaggatcgtgactgaacgagtgagtacatgtcacttcataactgcagtgtcattgtgcttaaggatataggagtaaaggataagtgtaggctgagctacagcatgataattttttttttttaaaaactgtctagaattaaagaattctgagctcat [ C/T ] ggattgattctacactgctgtgggcatcgagtcagtcctttgttgaggcttgtgcggtgcaggcctcttcaggtGTGGCTATGATGCGTGATGGGAT. For convenience of presentation, special sites are highlighted in uppercase letters, which are primer sequences or SNP sites in the above sequences.
Primer information is shown in the following table:
and in the sequence containing the SNP locus, the correspondence between the SNP locus and the male and female is shown in the following table:
| sequence number | Marking type | Position of | Male genotype | |
| 1 | |
243 | CC | CT |
Therefore, the SNP molecular marker can be used for distinguishing the sex of the silver dragon fish.
Example 2
In order to verify the accuracy of SNP locus identification of the sex of the Agkistrodon, the verification of the SNP molecular markers in the embodiment 1 is carried out, and whether the molecular markers detected by the Agkistrodon are accurate or not is verified by PCR and Sanger sequencing methods. Specifically, genomic DNA was extracted from non-high throughput sequencing samples, the specificity of the primers was verified by PCR and Sanger sequencing, and the SNP molecular markers and primers of example 1 were confirmed to be able to be used for sex identification of the silver dragon fish by cross-comparison with the physiological sex of the silver dragon fish samples.
1. Sample preparation
60 samples of silver dragon fish were selected and verified to contain 30 female samples and 30 male samples, and the following table is sample information:
experiment verifies sample
2. Genomic DNA extraction
The extraction is carried out by adopting a general column type genome DNA extraction kit, wherein the DNA extraction kit is purchased from Beijing kang as century science and technology Co., ltd., product number: CW2298M, the extraction procedure was performed with reference to the kit instructions.
3. PCR amplification
1) Reagent consumable
DNA polymerase: taq Master mix (Dye) (available from Beijing kang as century technology Co., ltd., product number: CW 0682L); the genome DNA of the sample to be detected extracted in the step 2; primer: the Primer F and PrimerR nucleotide sequences are respectively shown as SEQ ID NO.2 and SEQ ID NO.3 and are synthesized by the Huada genes; ddH 2 O。
2) PCR reaction system (shown in the following table)
3) PCR reaction conditions (shown in the following Table)
4. Agarose gel diagram
And detecting whether the PCR amplification is successful or not, and performing agarose gel electrophoresis. Specifically, the conditions include: glue concentration 1%, voltage 180V, time 20min; marker: m is DM2000, purchased from Beijing kang as century science and technology Co., ltd., product number: CW0632M. The result of electrophoresis of the PCR products is shown in FIG. 1.
5. Sanger sequencing identification results
After PCR amplification, sanger sequencing analysis was performed. The results are shown in the following table, and the sex information confirmed by physiological anatomy is consistent with sex information of SNP locus reaction. The sex identification of the fingerlings can be accurately realized through the primer F, R and the sequence containing the SNP locus, and compared with the traditional sex identification, the sex identification does not need biological cleavage.
Sample sequencing genotyping sex determination and dissecting genotyping sex determination results contrast
Example 3
The embodiment provides a sex identification method for silver dragon fish, which comprises the following steps:
(1) Extracting genome DNA of the silver dragon fish to be detected;
(2) Taking genomic DNA of the silver dragon fish to be detected as a template, and carrying out PCR amplification reaction by using an upstream primer F and a downstream primer R with sequences shown as SEQ ID NO.2 and SEQ ID NO.3 respectively;
(3) After the reaction is completed, the sample genotype is determined by combining Sanger sequencing, and the sex is identified according to the 243 rd position in the sequence SEQ ID NO. 1.
Specifically, the reaction system and the amplification procedure used in the PCR amplification reaction in the step (2) were the same as those in example 2.
Example 4
In this example, there is provided the use of SNP molecular markers of which the sequences are shown as SEQ ID NO.1, upstream primer F and downstream primer R of which the sequences are shown as SEQ ID NO.2 and SEQ ID NO.3 respectively, a kit comprising primer F, R and/or the identification method of example 3 in the sex identification and/or cultivation of Agrocybe aegerita.
It should be understood that the foregoing examples of the present invention are merely illustrative of the present invention and are not intended to limit the present invention to the specific embodiments thereof. Any modification, equivalent replacement, improvement, etc. that comes within the spirit and principle of the claims of the present invention should be included in the protection scope of the claims of the present invention.
SEQUENCE LISTING
<110> Zhujiang aquatic institute of China aquatic science institute
<120> SNP molecular marker for sex identification of Agkistrodon halys and application thereof
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 340
<212> DNA
<213> unknown
<400> 1
gttcccatct aggttccact ctttccttag ccttgatccc catacttcca ggataggccc 60
tgctacccgg actggcataa gtggttgagg atcgtgactg aacgagtgag tacatgtcac 120
ttcataactg cagtgtcatt gtgcttaagg atataggagt aaaggataag tgtaggctga 180
gctacagcat gataattttt tttttttaaa aactgtctag aattaaagaa ttctgagctc 240
atcggattga ttctacactg ctgtgggcat cgagtcagtc ctttgttgag gcttgtgcgg 300
tgcaggcctc ttcaggtgtg gctatgatgc gtgatgggat 340
<210> 2
<211> 20
<212> DNA
<213> unknown
<400> 2
gttcccatct aggttccact 20
<210> 3
<211> 23
<212> DNA
<213> unknown
<400> 3
atcccatcac gcatcatagc cac 23
Claims (9)
1. The SNP molecular marker for sex identification of the Agkistrodon halys is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, wherein the 243 th position of the sequence is C or T.
2. The SNP molecular marker for sex determination of silver dragon fish according to claim 1, wherein when the 243 th base is C/C homozygous, the sample is male, and when it is C/T heterozygous, the sample is female.
3. The primer pair for identifying or assisting in identifying the sex of the Agkistrodon halys is characterized by comprising an upstream primer F and a downstream primer R, wherein the nucleotide sequence of the upstream primer F is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer R is shown as SEQ ID NO. 3.
4. A kit for identifying or assisting in identifying the sex of Agkistrodon halys, which is characterized by comprising the primer pair of claim 3, dNTPs and DNA polymerase.
5. The sex identification method for the silver dragon fish is characterized by comprising the following steps of:
(1) Extracting genome DNA of the silver dragon fish to be detected;
(2) Taking genomic DNA of the silver dragon fish to be detected as a template, and carrying out PCR amplification reaction by using the upstream primer F and the downstream primer R according to claim 3;
(3) After the reaction is completed, analyzing, determining the genotype of the sample, and identifying the sex; when the 243 th base in the nucleotide sequence of SEQ ID NO.1 is C/C homozygous, the sample is male, and when the sample is C/T heterozygous, the sample is female.
6. The method according to claim 5, wherein the reaction system used in the PCR amplification reaction in the step (2) is 40. Mu.l, which comprises 2*Taq MasterMix 20. Mu.l, primer F1.6. Mu.l, primer R1.6. Mu.l, ddH2O 15.8. Mu.l, and genomic DNA 1. Mu.l.
7. The method for sex determination of silver dragon fish according to claim 5, wherein the PCR amplification procedure in the PCR amplification reaction in the step (2) is: 94 ℃ for 2min; then 35 cycles were performed, including 94℃for 30s,56℃for 30s, and 72℃for 20s; then the temperature is maintained at 72 ℃ for 5 min.
8. The method of sex determination of croaker of claim 5, wherein in step (3) the analysis is performed in combination with Sanger sequencing to determine the genotype of the sample and determine the sex.
9. Use of the SNP molecular marker for sex determination of silver dragon fish according to any one of claims 1-2, the primer pair according to claim 3, the kit according to claim 4 and/or the method according to any one of claims 5-8 for sex determination of silver dragon fish.
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| CN114686597B (en) * | 2020-12-25 | 2023-04-28 | 中国水产科学研究院珠江水产研究所 | SNP molecular marker for sex identification of silver dragon fish and application thereof |
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