CN114686596A - 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 PDF

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CN114686596A
CN114686596A CN202011564604.0A CN202011564604A CN114686596A CN 114686596 A CN114686596 A CN 114686596A CN 202011564604 A CN202011564604 A CN 202011564604A CN 114686596 A CN114686596 A CN 114686596A
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sex
snp
salangid
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silver dragon
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CN114686596B (en
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牟希东
刘奕
刘超
杨叶欣
宋红梅
汪学杰
徐猛
顾党恩
房苗
胡隐昌
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Pearl River Fisheries Research Institute CAFS
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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; the SNP molecular marker is the 243 th site 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

SNP molecular marker for sex identification of salangid and application thereof
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 silver dragon fish has hard and thick scales, so the scales cannot be penetrated by means of B ultrasonic, CT and the like to judge the sex. 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 fishes in 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 determination 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 determination is conveniently provided, the maturity and development of the 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, 340bp in length; the SNP molecular marker is the 243 th site in SEQ ID NO. 1.
When the base 243 is C/C pure, the sample is male, and when the base is C/T hybrid, the sample is female. SEQ ID NO.1 is only a representative sequence showing 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 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 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 the genome DNA of the arowana to be detected as a template and the upstream primer F and the downstream primer R of claim 3;
(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 TaqMasterMix (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, 56 ℃ 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 fussy 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 SNP marker loci (position 243), an upper graph showing male (genotype CC) and a lower graph showing female (genotype CT).
Detailed Description
The drawings are only for purposes of illustration and are not to be construed as limiting the invention.
Example 1
In this example, a high-throughput sequencing strategy was employed to select 5 female and 5 male samples for DNA extraction, and a double-ended genomic DNA library with an insert size of 500bp was constructed according to the Illumina library construction process requirements. And then carrying out high-throughput sequencing on the genome by adopting an IlluminaNovaSeq 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 the 243 th site in SEQ ID NO. 1.
Specifically, it is represented in the sequence as follows:
GTTCCCATCTAGGTTCCACTctttccttagccttgatccccatacttccaggataggccctgctacccggactggcataagtggttgaggatcgtgactgaacgagtgagtacatgtcacttcataactgcagtgtcattgtgcttaaggatataggagtaaaggataagtgtaggctgagctacagcatgataattttttttttttaaaaactgtctagaattaaagaattctgagctcat [ C/T ] ggattgattctacactgctgtgggcatcgagtcagtcctttgttgaggcttgtgcggtgcaggcctcttcaggtGTGGCTATGATGCGTGATGGGAT. 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:
Figure BDA0002861492490000041
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 Female genotype
1 SNP markers 243 CC CT
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
Figure BDA0002861492490000051
Figure BDA0002861492490000061
Figure BDA0002861492490000071
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 TaqMasterMix (dye) (available from Beijing kang, 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, wherein the nucleotide sequences of PrimerF and PrimerR are respectively shown as SEQ ID NO.2 and SEQ ID NO.3 and are synthesized by Huada gene; ddH2O。
2) PCR reaction System (shown in the following Table)
Figure BDA0002861492490000072
3) PCR reaction conditions (as shown in the following Table)
Figure BDA0002861492490000073
Figure BDA0002861492490000081
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. 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 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 of the salangid does not need to be performed by biological dissection.
Comparison of sample sequencing genotype and sex determination result with dissecting genotype and sex determination result
Figure BDA0002861492490000082
Figure BDA0002861492490000091
Figure BDA0002861492490000101
Figure BDA0002861492490000111
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 243 st 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, an SNP molecular marker having a sequence shown in SEQ ID No.1, an upstream primer F and a downstream primer R having sequences shown in SEQ ID No.2 and SEQ ID No.3, a kit comprising primer F, R, and/or an application of the identification method of embodiment 3 in sex identification and/or breeding of arowana virgata 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 claims of the present invention should be included in the protection scope of the claims of the present invention.
Figure BDA0002861492490000121
Figure BDA0002861492490000131
SEQUENCE LISTING
<110> Zhujiang aquatic research institute of Chinese aquatic science research institute
<120> SNP molecular marker for sex identification of salangid 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. An SNP molecular marker for sex determination of salangid is characterized in that the base sequence is as follows: SEQ ID NO.1, 340bp in length; the SNP molecular marker is the 243 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 base 243 is C/C pure, the sample is male, and when the base is C/T hybrid, 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 identification or assisted sex identification 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 of 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, and includes 2 TaqMasterMix (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, 56 ℃ 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 (2)

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Publication number Priority date Publication date Assignee Title
WO2014129982A1 (en) * 2013-02-19 2014-08-28 Agricultural Research Development Agency (Public Organization) A method of determining the sex of indonesian red arowanas
WO2022134451A1 (en) * 2020-12-25 2022-06-30 中国水产科学研究院珠江水产研究所 Snp molecular marker for identification of sex of osteoglossum bicirrhosum, and use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014129982A1 (en) * 2013-02-19 2014-08-28 Agricultural Research Development Agency (Public Organization) A method of determining the sex of indonesian red arowanas
WO2022134451A1 (en) * 2020-12-25 2022-06-30 中国水产科学研究院珠江水产研究所 Snp molecular marker for identification of sex of osteoglossum bicirrhosum, and use thereof

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Title
G H YUE等: "A strain-specific and a sex-associated STS marker for Asian arowana (Scleropages formosus, Osteoglossidae)", 《AQUACULTURE RESEARCH》 *
NANTARIKA CHANSUE等: "Asian Arowana (Scleropages formosus) Sex Determination Using Different Methods", 《THE ANNUAL CONFERENCE ON FISHERIES》 *
XIDONG MU等: "Identification of candidate sex-specific genomic regions in male and female Asian arowana genomes", 《GIGASCIENCE》 *
田媛: "双须骨舌鱼雌雄鉴别与性腺发育研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 *
田媛等: "双须骨舌鱼形态特征与核型分析", 《上海海洋大学学报》 *

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