CN114686595A - SNP molecular marker for sex identification of salangid and application thereof - Google Patents
SNP molecular marker for sex identification of salangid and application thereof Download PDFInfo
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Abstract
The invention discloses a SNP molecular marker for sex identification of salangid and application thereof, wherein the base sequence is as follows: SEQ ID NO.1, 373bp in length; the SNP molecular marker is the 271 th position in SEQ ID NO. 1. The method comprises the steps of obtaining Single Nucleotide Polymorphism (SNP) sites which are different between female and male silver dragon individuals and can represent sex of the female and male silver dragon through genome high-throughput sequencing comparison, obtaining an amplification sequence containing the SNP sites by combining primer amplification, realizing PCR amplification and sequencing of a sample to be detected through the primers on the basis, obtaining SNP site information of the sample, and identifying the sex of the sample to be detected. The SNP locus can realize accurate and rapid sex identification on the premise of not dissecting the silver dragon fish, and when the SNP locus is applied to the silver dragon fish culture, the mature artificial silver dragon fish culture technology is convenient to realize so as to realize mass production.
Description
Technical Field
The invention relates to the technical field of fish sex identification, in particular to a SNP molecular marker for sex identification of salangid and application thereof.
Background
The silver dragon fish is also called double-beard bone-tongue fish, is an ancient 'activated stone' fish, and has very important economic value as a famous and precious ornamental fish. At present, the vast majority of the domestic salangid depends on import, and the native mass culture of the salangid is difficult to realize; because the prior breeding technology of the salangid is limited, the artificial mass culture is difficult to realize. The main reason for limiting the breeding technique is the difficulty in sex differentiation of the male and female silver dragon fish.
The salangid has no specific amphoteric characteristic, cannot be distinguished in external morphology, and is not obvious in male and female fish even in the reproductive season. And the sex of the salangid cannot be judged by means of B-ultrasonic, CT and the like because the salangid has hard and thick scales. At present, the gonad can be identified only by dissecting and observing, and a simple and accurate method which can be identified without dissecting cannot be realized. The artificial propagation of the fish is greatly disturbed because no means for accurately judging the sex is available; because sex is difficult to distinguish, most farms can only adopt a semi-natural method for breeding, but cannot adopt a high-efficiency controllable artificial breeding mode, such as temporary breeding of male and female fish in different ponds, injection of an aphrodisiac and an oxytocin, and the like, so that the breeding efficiency and success rate are greatly limited. In order to break the situation that the silver dragon fish in China all depends on import, the artificial propagation of the silver dragon fish becomes a problem which needs to be solved urgently.
Therefore, there is a need for an effective sex identification method or marker for salangid to distinguish the sex of salangid, thereby overcoming the above problems.
Disclosure of Invention
The SNP locus can realize accurate and rapid sex identification on the premise of not dissecting the salangid, so that the normal growth and reproduction of the salangid are not influenced, when the SNP locus is applied to the breeding of the salangid, the sex identification is conveniently provided, the maturity and development of a breeding technology are promoted, and the reproduction rate and the success rate are improved.
The invention provides a SNP molecular marker for sex identification of salangid, which has the following base sequences: SEQ ID NO.1, 373bp in length; the SNP molecular marker is the 271 th position in SEQ ID NO. 1.
Further, when the 271 th base is A/G hybrid, the sample is male, and when the A/A pure, the sample is female. SEQ ID NO.1 is only a representative sequence showing one of the above-mentioned variations, and the corresponding other sequences comprising the above-mentioned variations should also be protected.
The invention also provides a primer pair for identifying or assisting in identifying the sex of the silver arowana, 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 salangid, 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 the genome DNA of the salangid to be detected;
(2) carrying out PCR amplification reaction by using the genomic DNA of the arowana to be detected as a template and utilizing an upstream primer F and a downstream primer R;
(3) and after the reaction is finished, analyzing, determining the genotype of the sample, and identifying the sex.
The method can realize the identification of the salangid on the premise of no dissection, and can realize the sequencing in batches by means of a plurality of sequencing methods or sequencing platforms, thereby being more beneficial to the application to the culture and research of the salangid on the basis, further quickly distinguishing the sex of the salangid and improving the identification and culture efficiency. The identification method is not only quick and simple, but also has accurate identification result shown 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 2. mu.l Taq MasterMix (Dye), 20. mu.l PrimerF 1.6. mu.l PrimerR 1.6. mu.l ddH2O15.8. mu.l and 1. mu.l genomic DNA.
Further, the PCR amplification procedure in the PCR amplification reaction in step (2) is as follows: 94 ℃ for 2 min; then 35 cycles were performed including 94 ℃ 30s, 54 ℃ 30s, 72 ℃ 20 s; then the process is finished by keeping the temperature at 72 ℃ for 5 min.
Further, in step (3), Sanger sequencing is combined for analysis, the genotype of the sample is determined, and the sex is identified.
The invention also provides the SNP molecular marker for sex identification of the salangid, the primer pair, the kit and/or the method, and application of the SNP molecular marker, the primer pair, the kit and/or the method in sex identification and/or breeding of the salangid.
Compared with the prior art, the invention has the beneficial effects that: the method comprises the steps of obtaining Single Nucleotide Polypeptide (SNP) sites which are different between female and male silver dragon individuals and can represent sex of the female and male silver dragon through genome high-throughput sequencing comparison, obtaining an amplification sequence containing the SNP sites through primer amplification, realizing PCR amplification of a sample to be detected through the primers on the basis, obtaining SNP site information of the sample, and identifying the sex of the sample to be detected according to the genotype of the SNP sites. And the sex identification can be completed through PCR amplification and Sanger sequencing, and compared with other molecular identification methods with complicated operation and long time consumption, the method is more suitable for quickly identifying a large batch of samples. More importantly, the SNP locus according to the invention can simply, accurately and rapidly identify the sex of the silver dragon fish, does not need to dissect the silver dragon fish, basically does not affect the health condition of a detected sample, is convenient to realize the application in the silver dragon fish breeding technology, realizes the sex pairing of the silver dragon fish, greatly improves the breeding efficiency, the breeding success rate and the number of offspring of the silver dragon fish, solves the great industrial problem of the breeding of the silver dragon fish, and has important economic value and social value. And since the salangid is an old fish, the research on the sex differentiation and sex determination mechanism of the old fish has important scientific research significance for explaining the sex evolution and sex determination mechanism of the whole fish, the SNP molecular marker, the corresponding primer, the kit and the identification method provided by the invention can provide a basis for the research on the fish evolution and sex determination mechanism and the like in addition to the beneficial effects, and promote the development of the research direction.
Drawings
FIG. 1 is a representation of the electrophoresis results of the PCR products of a sample to be tested;
FIG. 2 shows a representative Sanger sequencing graph, a sequencing peak of a SNP marker site (position 271), an upper graph shows male (genotype AG), and a lower graph shows female (genotype AA).
Detailed Description
The drawings are only for purposes of illustration and are not to be construed as limiting the invention.
Example 1
In the embodiment, a high-throughput sequencing strategy is adopted, 5 female dragonfish samples and 5 male dragonfish samples are selected for DNA extraction, and a double-end genome DNA library with an insert fragment of 500bp is constructed according to the requirements of an Illumina library construction process. 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 150 bp.
And detecting the Single Nucleotide Polymorphism (SNP) sites of the sequenced female samples and the Single Nucleotide Polymorphism (SNP) sites of the male samples by adopting a high-throughput sequencing sequence comparison strategy, and screening the SNP sites which are completely the same between the female samples and the male samples and are different between the female samples and the male samples as sex identification molecular markers.
And (3) screening specific DNA molecular markers of the female sample and the male sample according to a high-throughput sequencing method, and finding that the gene sequences of the female sample and the male sample contain SNP molecular markers for distinguishing sexes. On the basis, the sequence which contains the SNP locus and can be used for PCR and Sanger sequencing to identify the sex of the silver dragon fish is obtained by amplifying according to the high-throughput sequencing data and the primer, specifically, the primer comprises an upstream primer F with the sequence shown as SEQ ID NO.2 and a downstream primer R with the sequence shown as SEQ ID NO.3, and the obtained sequence which contains the SNP locus is shown as SEQ ID NO. 1. And the SNP molecular marker is position 271 in SEQ ID NO. 1.
Specifically, it is represented in the sequence as follows:
AGTACCGTCCTGGACATCAgctgagataaatggacagttttcccagaagaagatcccgatctaacctaaaacaaaccc atttgaacactgaaaaattcacctatattttgaaagttacacaactcatcactttaaagcaataggcgtggacaatatacctgtgggcggagcttcg gggctctatcacagccatcaaaagtgaatgtatcaatcgagtgcagtacagcataaaatgggattcaaacctgtgtccttcaaacacaaagtgcc [ A/G ] ggtctaaccacgaatgccacctactgacgggctctgcgttatagatctgtccaccattcccgtgccagtgtggaaagggaacaCTA GACACGATTCACCCAG. For convenience, capitalized letters are used to highlight specific sites, and in the above sequences, capitalized letters are used as primer sequences or SNP sites.
The primer information is shown in the following table:
and in the above-mentioned sequences containing SNP sites, the correspondence between SNP sites and males and females is shown in the following table:
| serial number | Type of mark | Position of | Male genotype | |
| 1 | |
271 | AG | AA |
Therefore, the SNP molecular marker can be used for distinguishing the sex of the salangid.
Example 2
In order to verify the accuracy of the sex identification of the salangid by the SNP sites, the verification of the SNP molecular markers in example 1 is performed in this example, and whether the molecular markers detected by the salangid are accurate is verified by using PCR and Sanger sequencing methods. Specifically, genomic DNA was extracted from a non-high-throughput sequencing sample, the specificity of primers was verified by PCR and Sanger sequencing, and the PCR and Sanger sequencing were compared with the physiological sex of the silver dragon fish sample to determine whether the SNP molecular markers and primers in example 1 can be used for sex identification of silver dragon fish.
1. Sample preparation
60 samples of the salangid, containing 30 female samples and 30 male samples, were selected for validation and the following table is sample information:
experimental validation sample
2. Genomic DNA extraction
The method adopts a general column type genome DNA extraction kit for extraction, and the DNA extraction kit is purchased from Beijing kang, a century science and technology limited company, and has a product number of: CW2298M, and the extraction process was performed according to the kit instructions.
3. PCR amplification
1) Reagent consumable
DNA polymerase: 2 Taq MasterMix (Dye) (available from Beijing kang, a century science and technology Co., Ltd., cat # CW 0682L); extracting the genomic DNA of the sample to be detected in the step 2; primer: comprises PrimerF and PrimerR, the nucleotide sequences of PrimerF and PrimerR are respectively shown in SEQ ID NO.2 and SEQ ID NO.3, and the PrimerF and PrimerR are synthesized by Huada gene; ddH2O。
2) PCR reaction System (shown in the following Table)
3) PCR reaction conditions (as shown in the following Table)
4. Agarose gel picture
And detecting whether the PCR amplification is successful or not, and performing agarose gel electrophoresis. Specifically, the conditions include: the glue concentration is 1%, the voltage is 180V, and the time is 20 min; marker: m is DM2000, available from Beijing kang, century science and technology, cat #: CW 0632M; m1 is Super DNA Marker, purchased from Beijing kang, century science and technology Co., Ltd, cat #: CW 2583M. The electrophoresis results of the PCR products are shown in FIG. 1.
5. Sanger sequencing assay results
After PCR amplification, Sanger sequencing analysis was performed. As a result, sex information based on the SNP site reaction was consistent with sex information confirmed by physiological dissection as shown in the following Table. The sex identification of the salangid male and female can be accurately realized through the primer F, R and the sequence containing the SNP locus, and compared with the traditional male and female identification, the sex identification method does not need biological dissection.
Comparison of sample sequencing genotype and sex determination result with dissecting genotype and sex determination result
Example 3
The embodiment provides a sex identification method for a salangid, which comprises the following steps:
(1) extracting the genome DNA of the salangid to be detected;
(2) carrying out PCR amplification reaction by using genome DNA of the silver dragon fish to be detected as a template and using an upstream primer F and a downstream primer R with sequences respectively shown as SEQ ID NO.2 and SEQ ID NO. 3;
(3) after the reaction is finished, Sanger sequencing is combined for analysis, the genotype of the sample is determined, and the sex is identified according to the 271 th position in the sequence SEQ ID NO. 1.
Specifically, the reaction system and the amplification procedure used in the PCR amplification reaction in step (2) were the same as those in example 2.
Example 4
In this embodiment, the SNP molecular marker with the sequence shown in SEQ ID No.1, the upstream primer F and the downstream primer R with the sequences shown in SEQ ID No.2 and SEQ ID No.3, the kit containing the primer F, R, and/or the application of the identification method of embodiment 3 in sex identification and/or breeding of arowana argentea are provided.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.
SEQUENCE LISTING
<110> Zhujiang aquatic product institute of China aquatic product science institute
<120> SNP molecular marker for sex identification of salangid and application thereof
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 373
<212> DNA
<213> unknown
<400> 1
agtaccgtcc tggacatcag ctgagataaa tggacagttt tcccagaaga agatcccgat 60
ctaacctaaa acaaacccat ttgaacactg aaaaattcac ctatattttg aaagttacac 120
aactcatcac tttaaagcaa taggcgtgga caatatacct gtgggcggag cttcggggct 180
ctatcacagc catcaaaagt gaatgtatca atcgagtgca gtacagcata aaatgggatt 240
caaacctgtg tccttcaaac acaaagtgcc aggtctaacc acgaatgcca cctactgacg 300
ggctctgcgt tatagatctg tccaccattc ccgtgccagt gtggaaaggg aacactagac 360
acgattcacc cag 373
<210> 2
<211> 19
<212> DNA
<213> unknown
<400> 2
agtaccgtcc tggacatca 19
<210> 3
<211> 19
<212> DNA
<213> unknown
<400> 3
ctgggtgaat cgtgtctag 19
Claims (9)
1. An SNP molecular marker for sex identification of salangid is characterized in that the base sequence is as follows: SEQ ID NO.1, 373bp in length; the SNP molecular marker is the 271 th site in SEQ ID NO. 1.
2. The SNP molecular marker for sex identification of silver dragon fish according to claim 1, wherein when the 271 th base is A/G hybrid, the sample is male, and when the A/A pure, the sample is female.
3. A primer pair for identifying or assisting in identifying the sex of the silver dragon fish 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 sex determination or assisted sex determination of salangid, comprising the primer pair of claim 3, dNTPs and DNA polymerase.
5. The sex identification method of the salangid is characterized by comprising the following steps of:
(1) extracting the genome DNA of the salangid to be detected;
(2) carrying out PCR amplification reaction by using the genome DNA of the arowana to be detected as a template and the upstream primer F and the downstream primer R in claim 3;
(3) and after the reaction is finished, analyzing, determining the genotype of the sample, and identifying the sex.
6. The method for sex identification of silver dragon fish according to claim 5, wherein the reaction system used in the PCR amplification reaction in step (2) is 40. mu.l, including 2. mu.l Taq MasterMix (Dye) 20. mu.l PrimerF 1.6. mu.l PrimerR 1.6. mu.l ddH2O15.8. mu.l and genomic DNA 1. mu.l.
7. The method for sex identification of silver dragon fish according to claim 5, wherein the PCR amplification procedure in the PCR amplification reaction in step (2) is: 94 ℃ for 2 min; then 35 cycles were performed including 94 ℃ 30s, 54 ℃ 30s, 72 ℃ 20 s; then the process is finished by keeping the temperature at 72 ℃ for 5 min.
8. The sex determination method of silver dragon fish according to claim 5, characterized in that in the step (3), the sample genotype is determined and the sex is determined by analyzing in combination with Sanger sequencing.
9. The SNP molecular marker for sex identification of silver dragon fish according to any one of claims 1 to 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 to 8, and the application of the SNP molecular marker for sex identification of silver dragon fish and/or the application of the method in breeding silver dragon fish.
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Citations (1)
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| CN107217099A (en) * | 2017-06-28 | 2017-09-29 | 中国水产科学研究院珠江水产研究所 | A kind of SNP marker identified available for snakehead genetic sex and supermale fish and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107217099A (en) * | 2017-06-28 | 2017-09-29 | 中国水产科学研究院珠江水产研究所 | A kind of SNP marker identified available for snakehead genetic sex and supermale fish and its application |
Non-Patent Citations (1)
| Title |
|---|
| 林晓煜等: "大黄鱼性别特异SNP标记的开发与验证", 《水产学报》 * |
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