CN107217099B - A kind of SNP marker and its application can be used for snakehead genetic sex and the identification of supermale fish - Google Patents
A kind of SNP marker and its application can be used for snakehead genetic sex and the identification of supermale fish Download PDFInfo
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Abstract
The invention discloses a kind of SNP marker that can be used for snakehead genetic sex and the identification of supermale fish, the SNP marker is SEQ ID NO：1~SEQ ID NO：The 82nd of nucleotide sequence shown in 3, base T/T, C/T or C/C.The SNP marker can identify snakehead genetic sex and supermale fish, also disclose the primer pair that can be used for the SNP marker of snakehead genetic sex and the identification of supermale fish, and application of the method and SNP marker of identification snakehead genetic sex and supermale fish in snakehead genetic sex and the identification of supermale fish, the primer pair can expand the genomic DNA of sample to be tested, it can identify genetic sex, and identify by supermale fish in pseudo- raun and common milter offspring family, this method is simple and quick and accuracy rate is high.
Description
Technical field
The invention belongs to fish sex molecular identification technical fields, and in particular to one kind can be used for snakehead genetic sex and surpass
The SNP marker and its application of milter identification.
Background technique
Murrel is the important fresh water Quality and economy fish in China, has the characteristics that spur is few, meat is fresh and tender, full of nutrition, has
The stasis of blood is raw new, and nourishing is raised and other effects, is loved by consumers, easy to process, is one of important aquatic products in China, national annual output
It is the eighth-largest freshwater aquiculture kind in China more than 510,000 tons, the annual billions of tails of seed demand, market is huge.Murrel is to exist to show
The fish of sexual dimorphism are write, the male speed of growth is twice of female, and the cultivation of all-male murrel will improve the whole speed of growth
Up to 30%, benefit highly significant, while promoting murrel aquaculture industry transition and upgrade, upgrading synergy.So market urgent need all-male
Seed.And genetic sex identification is the key technology carrying out all-male fish and cultivating, and greatly accelerates cultivation process.
China Jiangnan and South China mainly cultivate as hybridized snakehead fish at present, the Yellow River and to the north of mainly cultivate snakehead.With snakehead
It is that the maternal spot crow hybridized snakehead fish speed of growth is fast, seeding technique is simple for male parent, Ban Channa, seed occupation rate of market is big, is current
Main breed variety.It is boundless to cultivate supermale spot crow hybridized snakehead fish market prospects.Spot crow hybridized snakehead fish is the institute using snakehead as male parent
It is key problem in technology to screen snakehead to can be directly used for the molecular labeling of pseudo- raun and the identification of supermale fish, by accelerating, all-male spot crow is miscellaneous
It hands over murrel to cultivate speed, there is huge industrial value and scientific value.
Summary of the invention
First technical problem to be solved by this invention, which is to provide one kind, can be used for snakehead genetic sex and supermale fish mirror
Fixed SNP marker, the SNP marker can identify snakehead genetic sex and supermale fish.
Second technical problem to be solved by this invention, which is to provide one kind, can be used for snakehead genetic sex and supermale fish mirror
The primer pair of fixed SNP marker, the primer pair can expand the genomic DNA of sample to be tested, it is possible to identify genetic sex, and will be pseudo- female
Supermale fish, which identifies, in fish and common milter offspring family comes.
Third technical problem to be solved by this invention is to provide a kind of side for identifying snakehead genetic sex and supermale fish
Method, this method is simple and quick and accuracy rate is high.
4th technical problem to be solved by this invention be to provide it is above-mentioned can be used for snakehead genetic sex and supermale fish mirror
Application of the fixed SNP marker in snakehead genetic sex and the identification of supermale fish.
Above-mentioned first technical problem to be solved by this invention is achieved by the following technical solution：One kind is available
In the SNP marker that snakehead genetic sex and supermale fish are identified, the SNP marker is SEQ ID NO：1~SEQ ID NO：Shown in 3
The 82nd of nucleotide sequence, base T/T, C/T or C/C.
When base is T/T homozygote, sample is female, and when sample is C/T heterozygosis, sample is male；With pseudo- raun and
During common milter raises up seed, genotype is female when being T/T homozygosis, is male when C/T heterozygosis, when C/C homozygosis for male and
For supermale fish, wherein pseudo- raun is estrogenic reverse, genotype is C/T heterozygosis, and common milter genotype is C/T heterozygosis.
It is specific as follows：
Raun：
CAGTGTGTGAGTTTGAGGATGTCAACAAATGCATAAAGTT/
CGCATAAAAACTGCTGAGCTGGGCTCTTCGATGCAATCAGGT/TGCTGGTCTCAGCTCATATACTTG
Milter：
CAGTGTGTGAGTTTGAGGATGTCAACAAATGCATAAAGTT/
CGCATAAAAACTGCTGAGCTGGGCTCTTCGATGCAATCAGGC/TGCTGGTCTCAGCTCATATACTTG
Supermale fish：
CAGTGTGTGAGTTTGAGGATGTCAACAAATGCATAAAGTT/
CGCATAAAAACTGCTGAGCTGGGCTCTTCGATGCAATCAGGC/CGCTGGTCTCAGCTCATATACTTG
Overstriking italicized bases are SNP site, and when base is T/T homozygote, sample is female, when sample is C/T heterozygosis
When sample be male；With pseudo- raun (estrogenic reverses, and genotype is C/T heterozygosis) and during common milter (C/T) raises up seed,
Genotype is female when being T/T homozygosis, is male when C/T heterozygosis, and when C/C homozygosis is male and is supermale fish.
Above-mentioned second technical problem to be solved of the invention is achieved by the following technical solution：One kind can
Primer pair for the SNP marker that snakehead genetic sex and supermale fish are identified comprising upstream primer to F and downstream primer to R,
Nucleotide sequence such as SEQ ID NO of the upstream primer to F：Shown in 4, the nucleotide sequence of primer pair R such as SEQ downstream
ID NO：Shown in 5.
Upstream primer is to F (Primer F)：5'AGTGTGTGAGTTTGAGGATGTC 3'；
Downstream primer is to R (Primer R)：5'CAAGTATATGAGCTGAGACCAG 3'.
Above-mentioned third technical problem to be solved of the invention is achieved by the following technical solution：A kind of mirror
The method for determining snakehead genetic sex and supermale fish, includes the following steps：
(1) genomic DNA of snakehead to be measured is extracted；
(2) using the genomic DNA of snakehead to be measured as template, F and downstream primer carry out R using above-mentioned upstream primer
Pcr amplification reaction and melting curve are collected；
(3) it is analyzed after the reaction was completed, determines sample genotype, identify gender.
In above-mentioned identification snakehead genetic sex and the method for supermale fish：
The reaction system used when pcr amplification reaction in step (2) for 20 μ L, including：ABI Meltdoctor mix 10
0.6 μ L, primer R of μ L, primer F 0.6 μ L, 1 μ L of DNA profiling, 7.8 μ L of sterile water.
PCR amplification program is when pcr amplification reaction in step (2)：50 DEG C of 2min, 95 DEG C of 10min；Then 40 are carried out
Circulation, including 95 DEG C of 15s, 58 DEG C of 1min；Then 95 are warming up to 0.3 DEG C/s speed after 95 DEG C of unwindings 15s, 60 DEG C of 1min
DEG C, it is collected simultaneously fluorescence signal, last 60 DEG C of holdings 15s terminates.
It selects 75 DEG C~77.5 DEG C sections to carry out melting curve phenotypic analysis using HRM software in step (3), determines sample
Genotype identifies gender.
Above-mentioned 4th technical problem to be solved of the invention is achieved by the following technical solution：Above-mentioned
Can be used for snakehead genetic sex and supermale fish identification SNP marker snakehead genetic sex and supermale fish identification in application.
The present invention has the following advantages that：
(1) using the genomic DNA of primer amplification sample to be tested provided by the invention, then by being based on quantitative fluorescent PCR
The high-resolution melting curve analysis of instrument, it is possible to identify the consistency of genetic sex, genotype and phenotype is 98%, accurate in family
Rate 100%, and supermale fish in pseudo- raun and common milter offspring family can be identified；
(2) the entire detection process of identification method of the present invention can be completed in one day, and simple and quick and accuracy rate is high；
(3) SNP site of the invention and detection method are currently the only to snakehead while can to carry out common sex identification
With the molecule labelling method of supermale fish identification.
Detailed description of the invention
Fig. 1 is to carry out Genotyping to different sexes snakehead using HRM software in embodiment 3.
Specific embodiment
Embodiment 1
The SNP marker provided in this embodiment that can be used for snakehead genetic sex and the identification of supermale fish, the SNP marker are SEQ
ID NO：1~SEQ ID NO：Of nucleotide sequence shown in 3 (respectively corresponding SNP marker 1, SNP marker 2, SNP marker 3)
82, base T/T, C/T or C/C.
It is specific as follows：
Raun：
CAGTGTGTGAGTTTGAGGATGTCAACAAATGCATAAAGTT/
CGCATAAAAACTGCTGAGCTGGGCTCTTCGATGCAATCAGGT/TGCTGGTCTCAGCTCATATACTTG
Milter：
CAGTGTGTGAGTTTGAGGATGTCAACAAATGCATAAAGTT/
CGCATAAAAACTGCTGAGCTGGGCTCTTCGATGCAATCAGGC/TGCTGGTCTCAGCTCATATACTTG
Supermale fish：
CAGTGTGTGAGTTTGAGGATGTCAACAAATGCATAAAGTT/
CGCATAAAAACTGCTGAGCTGGGCTCTTCGATGCAATCAGGC/CGCTGGTCTCAGCTCATATACTTG。
Overstriking italicized bases are SNP site, and when base is T/T homozygote, sample is female, when sample is C/T heterozygosis
When sample be male；With pseudo- raun (estrogenic reverses, and genotype is C/T heterozygosis) and during common milter (C/T) raises up seed,
Genotype is female when being T/T homozygosis, is male when C/T heterozygosis, and when C/C homozygosis is male and is supermale fish.
Embodiment 2
The primer pair of the SNP marker provided in this embodiment that can be used for snakehead genetic sex and the identification of supermale fish comprising
Upstream primer to F and downstream primer to R, wherein nucleotide sequence such as SEQ ID NO of the upstream primer to F：Shown in 4, downstream is drawn
Nucleotide sequence such as SEQ ID NO of the object to R：Shown in 5.
Specially：
Upstream primer is to F (Primer F)：5'AGTGTGTGAGTTTGAGGATGTC 3'；
Downstream primer is to R (Primer R)：5'CAAGTATATGAGCTGAGACCAG 3'.
Embodiment 3
The method of identification snakehead genetic sex and supermale fish provided in this embodiment, includes the following steps：
(1) sample DNA extracts
The genomic DNA for extracting sample can be extracted using ordinary skill in the art means, including phenol-chloroform method and each
Kind of DNA extraction kit is extracted and is diluted to 20ng μ L with sterile water after total DNA.
(2) sequence using following primer amplification comprising SNP site：
Primer is (underscore part in sequence)：
Primer F：5'AGTGTGTGAGTTTGAGGATGTC 3'
Primer R：5'CAAGTATATGAGCTGAGACCAG 3'
Extension increasing sequence is：
Raun：
CAGTGTGTGAGTTTGAGGATGTCAACAAATGCATAAAGTT/
CGCATAAAAACTGCTGAGCTGGGCTCTTCGATGCAATCAGGT/TGCTGGTCTCAGCTCATATACTTG
Milter：
CAGTGTGTGAGTTTGAGGATGTCAACAAATGCATAAAGTT/
CGCATAAAAACTGCTGAGCTGGGCTCTTCGATGCAATCAGGC/TGCTGGTCTCAGCTCATATACTTG
Supermale fish：
CAGTGTGTGAGTTTGAGGATGTCAACAAATGCATAAAGTT/
CGCATAAAAACTGCTGAGCTGGGCTCTTCGATGCAATCAGGC/CGCTGGTCTCAGCTCATATACTTG。
Overstriking italicized bases are SNP site, and when base is T/T homozygote, sample is female, when sample is C/T heterozygosis
When sample be male；With pseudo- raun (estrogenic reverses, and genotype is C/T heterozygosis) and during common milter (C/T) raises up seed,
Genotype is female when being T/T homozygosis, is male when C/T heterozygosis, and when C/C homozygosis is male and is supermale fish.
(3) PCR amplification and high-resolution melting curve analysis
Sample DNA templates, Primer F, Primer R, meltdoctor master mix are prepared according to the instructions provided
Reaction mixture, then reaction plate is carried out to amplification and melting curve collection, reaction on stepone plus real-time PCR
It is analyzed after the completion using HRM software, determines sample genotype, to identify gender.
Wherein PCR reaction system is 20 μ L, including：
10 μ L, primer F of ABI Meltdoctor mix, 0.6 μ L, primer R, 0.6 μ L, DNA profiling 1 μ L, it is sterile
7.8 μ L of water.Reaction carries out in dedicated 96 orifice plate of quantitative PCR, the covering sealing of high light transmission sealing plate mould.
Amplification and testing conditions are：
After preparing reagent by above-mentioned reaction system, reaction plate is placed in ABI stepone plus real-time PCR
In, following procedure is set：50 DEG C of 2min, 95 DEG C of 10min；Then 40 circulations, including 95 DEG C of 15s, 58 DEG C of 1min are carried out；
Then 95 DEG C are warming up to 0.3 DEG C/s speed after 95 DEG C of unwindings 15s, 60 DEG C of 1min, are collected simultaneously fluorescence signal, last 60 DEG C
15s is kept to terminate.
(4) the use of instrument is that ABI stepone plus carries out PCR amplification and signal collection, utilizes instrument after reaction
Software stepone 2.3 carries out preliminary analysis, saves file；ABI high-resolution melting curve analysis software HRM is recycled to open
The file of above-mentioned preservation selects 75 DEG C~77.5 DEG C sections to carry out melting curve parting.
As a result：After being analyzed using HRM, the genotype of sample can identify, and as shown in fig. 1, wherein variant 1 is
(T/C) heterozygous is accredited as milter；Variant 2 is that (TT) is homozygous, is accredited as raun, and variant 3 is (CC) homozygous
Type is accredited as supermale fish.
Application Example one
Using above-mentioned SNP marker primer and detection method to 100 tail parent population of snakehead (bred, physiology gender determine,
Middle 50 tail of raun, 50 tail of milter) carry out gender verifying.
(1) test fish clip fin ray is extracted into genomic DNA
Using scissors clip snakehead part tail fin about 0.5 × 0.5cm of alcohol disinfecting, it is put into dehydrated alcohol preservation, room temperature
Under take back laboratory；The fin ray sample that dehydrated alcohol saves is washed away into alcohol with aseptic double-distilled water, utilizes omega animal tissue
DNA extraction kit extracts genomic DNA；Integrality of the DNA of extraction through 1% agarose gel electrophoresis detection DNA, NanoQTM
Microspectrophotometer (Bo Ao) detectable concentration, and final concentration 20ng/ μ L is diluted to deionized water.
(2) HRM parting reaction mixture is prepared
10 μ L, primer F of ABI Meltdoctor mix, 0.6 μ L, primer R 0.6 μ L, 1 μ L of DNA profiling is sterile
7.8 μ L of water.Reaction carries out in dedicated 96 orifice plate of ABI stepone plus quantitative PCR, the covering sealing of high light transmission sealing plate mould.
(3) machine testing on
Using ABI stepone plus real-time PCR, software is stepone 2.3, and parameter by specification is arranged,
Operation program is 50 DEG C of 2min, 95 DEG C of 10min；Then 40 circulations, including 95 DEG C of 15s, 58 DEG C of 1min are carried out；Then 95
95 DEG C are warming up to 0.3 DEG C/s speed after DEG C unwinding 15s, 60 DEG C of 1min, is collected simultaneously fluorescence signal, last 60 DEG C of holdings 15s
Terminate.
(4) result
After running by program, using 2.3 software preliminary analysis of stepone, file is saved.Again by the text of preservation
Part is opened with HRM3.0.1 software, and temperature range is selected to be analyzed, 50 tail of TT genotype in 50 tail female parent population individuals；50 tails
T/C genotype is 48 tails, 2 tail of TT type in physiological male individual.
Application Example two
Using above-mentioned SNP marker primer and detection method to same 200 tail juvenile fish of family snakehead (family female parent be it is female swash
Disposition reverses fish, and is identified as T/C genotype；Male parent is common milter, is identified as T/C genotype), carry out genotype mirror
It is fixed.
(1) DNA extraction, physiology sex identification, PCR amplification and HRM classifying method are identical as Application Example one.
(2) result passes through HRM software parting, 103 tail of T/C genotype in 200 tails individual, 51 tail of T/T genotype；C/C base
Because type is 46 tails, meet the 2 of common milter in offspring, supermale fish and raun:1:1 expection illustrates that label of the invention can be same
When identification for three kinds of fishes.
Upper embodiment is used merely to explain the present invention, and protection scope of the present invention is not intended to be limited to above embodiments.
Based on the contents of the disclosure of the present invention and the scope of each parameter, this can be achieved in the those of ordinary skill of the technical field
The purpose of invention.
110 > China's Pearl River Fishery Research Institute of Aquatic Science Research Institute of <
A kind of SNP marker and its application that can be used for snakehead genetic sex and the identification of supermale fish of 120 > of <
〈210〉1
〈211〉106
〈212〉DNA
223 > SNP marker 1 of <
〈400〉1
CAGTGTGTGA GTTTGAGGAT GTCAACAAAT GCATAAAGTT CGCATAAAAA CTGCTGAGCT 60
GGGCTCTTCG ATGCAATCAG GTTGCTGGTC TCAGCTCATA TACTTG 106
〈210〉2
〈211〉106
〈212〉DNA
223 > SNP marker 2 of <
〈400〉2
CAGTGTGTGA GTTTGAGGAT GTCAACAAAT GCATAAAGTT CGCATAAAAA CTGCTGAGCT 60
GGGCTCTTCG ATGCAATCAG GCTGCTGGTC TCAGCTCATA TACTTG 106
〈210〉3
〈211〉106
〈212〉DNA
223 > SNP marker 3 of <
〈400〉3
CAGTGTGTGA GTTTGAGGAT GTCAACAAAT GCATAAAGTT CGCATAAAAA CTGCTGAGCT 60
GGGCTCTTCG ATGCAATCAG GCCGCTGGTC TCAGCTCATA TACTTG 106
〈210〉4
〈211〉22
〈212〉DNA
223 > upstream primer of < is to F
〈400〉4
AGTGTGTGAG TTTGAGGATG TC 22
〈210〉5
〈211〉22
〈212〉DNA
223 > downstream primer of < is to R
〈400〉5
CAAGTATATG AGCTGAGACC AG 22
Claims (8)
1. a kind of SNP marker that can be used for snakehead genetic sex and the identification of supermale fish, it is characterized in that：The SNP marker is SEQ
ID NO：1 ~ SEQ ID NO：Sequence shown in 3, the base that the sequence is the 82nd are T/T, C/T or C/C.
2. the SNP marker according to claim 1 that can be used for snakehead genetic sex and the identification of supermale fish, it is characterized in that：When
When base is T/T homozygote, sample is female, and when sample is C/T heterozygosis, sample is male；It is numerous with pseudo- raun and common milter
It grows in offspring, it is male when C/T heterozygosis that genotype, which is female when being T/T homozygosis, and when C/C homozygosis is male and is supermale fish,
Middle puppet raun is estrogenic reverse, and genotype is C/T heterozygosis, and common milter genotype is C/T heterozygosis.
3. a kind of primer pair for the SNP marker that can be used for snakehead genetic sex and the identification of supermale fish, it is characterized in that：It includes upstream
Primers F and downstream primer R, the nucleotide sequence of the upstream primer F such as SEQ ID NO：Shown in 4, the core of primer R downstream
Nucleotide sequence such as SEQ ID NO：Shown in 5.
4. a kind of method for identifying snakehead genetic sex and supermale fish, it is characterized in that including the following steps：
（1）Extract the genomic DNA of snakehead to be measured；
（2）Using the genomic DNA of snakehead to be measured as template, using upstream primer F described in claim 3 and downstream primer R, into
Row PCR amplified reaction and melting curve are collected；
（3）It is analyzed after the reaction was completed, determines sample genotype, identify gender.
5. the method for identification snakehead genetic sex and supermale fish according to claim 4, it is characterized in that：Step（2）Middle PCR
The reaction system used when amplified reaction for 20 μ L, including：10 μ L, primer F of ABI Meltdoctor mix, 0.6 μ L,
0.6 μ L, DNA template of primer R 1 μ L, 7.8 μ L of sterile water.
6. the method for identification snakehead genetic sex and supermale fish according to claim 4, it is characterized in that：Step（2）In
PCR amplification program is when pcr amplification reaction：50 DEG C of 2min, 95 DEG C of 10min；Then 40 circulations, including 95 DEG C are carried out
15s, 58 DEG C of 1min；Then 95 DEG C are warming up to 0.3 DEG C/s speed after 95 DEG C of unwindings 15s, 60 DEG C of 1min, are collected simultaneously fluorescence
Signal, last 60 DEG C of holdings 15s terminate.
7. the method for identification snakehead genetic sex and supermale fish according to claim 4, it is characterized in that：Step（3）Middle benefit
It selects 75 DEG C ~ 77.5 DEG C sections to carry out melting curve phenotypic analysis with HRM software, determines sample genotype, identify gender.
8. the SNP marker described in claim 1 that can be used for snakehead genetic sex and the identification of supermale fish in snakehead genetic sex and
Application in the identification of supermale fish.
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| CN107641656A (en) * | 2017-10-25 | 2018-01-30 | 安徽科技学院 | A kind of snakehead single nucleotide polymorphism and application thereof |
| EP3620536A1 (en) | 2018-09-07 | 2020-03-11 | Consejo Superior De Investigaciones Científicas | Method for predicting sex in fish |
| CN114686595B (en) * | 2020-12-25 | 2023-04-18 | 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 |
| CN114686594B (en) * | 2020-12-25 | 2023-05-02 | 中国水产科学研究院珠江水产研究所 | SNP molecular marker suitable for sex identification of silver dragon fish and application thereof |
| CN112746111B (en) * | 2021-01-20 | 2022-11-29 | 广东梁氏水产种业有限公司 | Northern snakehead male molecular marker primer and application thereof |
| CN113930525B (en) * | 2021-11-19 | 2022-11-29 | 中国科学院水生生物研究所 | Specific sequence for snakehead sex identification and application |
| CN116732157B (en) * | 2023-03-28 | 2024-06-14 | 中国海洋大学 | Universal molecular marker for sex and variety identification of snakeheads, macula maculata and hybrid snakeheads |
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| CN103276068B (en) * | 2013-05-20 | 2014-07-02 | 中国水产科学研究院珠江水产研究所 | Marking primer and method for identifying channa argus and channa maculate as well as F1-generation channa argus*channa maculate and channa maculate*channa argus hybridized by channa argus and channa maculate |
| CN103290131B (en) * | 2013-06-18 | 2014-10-08 | 徐鹏 | Primer pair and kit for distinguishing Channa argus and Channa maculata, and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) detection method |
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