CN107217099A - A kind of SNP marker identified available for snakehead genetic sex and supermale fish and its application - Google Patents
A kind of SNP marker identified available for snakehead genetic sex and supermale fish and its application Download PDFInfo
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Abstract
The invention discloses a kind of SNP marker identified available for snakehead genetic sex and supermale fish, the SNP marker is SEQ ID NO：1~SEQ ID NO：The 82nd of nucleotide sequence shown in 3, its base is T/T, C/T or C/C.The SNP marker can identify snakehead genetic sex and supermale fish, also disclose the primer pair for the SNP marker identified available for snakehead genetic sex and supermale fish, and the application of identification snakehead genetic sex and the method and SNP marker of supermale fish in snakehead genetic sex and the identification of supermale fish, the primer pair can expand the genomic DNA of testing sample, genetic sex can be identified, 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 field, and in particular to one kind can be used for snakehead genetic sex and super
The SNP marker of milter identification and its application.
Background technology
Murrel is the important fresh water Quality and economy fish of China, with spur is few, meat is fresh and tender, it is nutritious the characteristics of, have
Stasis of blood life is new, and the effects such as nourishing is taken good care of is loved by consumers, it is easy to process, is one of important aquatic products of China, national annual production
It is the eighth-largest freshwater aquiculture kind of 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 overall speed of growth
Up to 30%, benefit highly significant, while promoting murrel aquaculture industry transition and upgrade, upgrading synergy.So market active demand all-male
Seed.And genetic sex identification is to carry out the key technology that all-male fish is cultivated, greatly accelerate cultivation process.
Current China south of the River and South China are mainly cultivated as hybridized snakehead fish, the Yellow River and to the north of main cultivation snakehead.With snakehead
It is that the black hybridized snakehead fish fast growth of maternal spot, seeding technique are simple for male parent, Ban Channa, seed occupation rate of market is big, is current
Main breed variety.Cultivate the black hybridized snakehead fish market prospects of supermale spot boundless.Spot crow hybridized snakehead fish is the institute using snakehead as male parent
With screen snakehead can be directly used for pseudo- raun and supermale fish identification molecular labeling be key problem in technology, by accelerate all-male spot crow it is miscellaneous
Murrel is handed over to cultivate speed, with huge industrial value and scientific value.
The content of the invention
First technical problem to be solved by this invention is to provide a kind of available 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 is to provide a kind of available for snakehead genetic sex and supermale fish mirror
The primer pair of fixed SNP marker, the primer pair can expand the genomic DNA of testing sample, it is possible to identify genetic sex, and puppet is female
Fish identifies with supermale fish in common milter offspring family.
3rd 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 is to provide above-mentioned available 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
The SNP marker identified in snakehead genetic sex and supermale fish, the SNP marker is SEQ ID NO：1~SEQ ID NO：Shown in 3
The 82nd of nucleotide sequence, its base is T/T, C/T or C/C.
When base is T/T homozygotes, 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 during C/T heterozygosis, during C/C homozygosis for male and
For supermale fish, wherein pseudo- raun is reversed for estrogenic, 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 homozygotes, sample is female, when sample is C/T heterozygosis
When sample for male；In being raised up seed with pseudo- raun (estrogenic is reversed, and genotype is C/T heterozygosis) and common milter (C/T),
Genotype be when being T/T homozygosis female, during C/T heterozygosis for male, be during C/C homozygosis male and be supermale fish.
Above-mentioned second technical problem to be solved of the present invention is achieved by the following technical solution：One kind can
The primer pair for the SNP marker identified for snakehead genetic sex and supermale fish, it includes sense primer to F and anti-sense primer to R,
Nucleotide sequence such as SEQ ID NO of the sense primer to F：Shown in 4, primer pair R nucleotide sequence such as SEQ downstream
ID NO：Shown in 5.
Sense primer is to F (Primer F)：5'AGTGTGTGAGTTTGAGGATGTC 3'；
Anti-sense primer is to R (Primer R)：5'CAAGTATATGAGCTGAGACCAG 3'.
Above-mentioned 3rd technical problem to be solved of the present invention is achieved by the following technical solution：One kind mirror
The method for determining snakehead genetic sex and supermale fish, comprises 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 anti-sense primer are carried out to R using above-mentioned sense primer
Pcr amplification reaction and melting curve are collected；
(3) analyzed after the completion of reacting, determine sample genotype, identify sex.
In above-mentioned identification snakehead genetic sex and the method for supermale fish：
The reaction system used in step (2) during pcr amplification reaction for 20 μ L, including：ABI Meltdoctor mix 10
0.6 μ L, primer R of μ L, primer F 0.6 μ L, the μ L of DNA profiling 1, the μ L of sterilized water 7.8.
PCR amplification programs are during 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 15s that unwind, 60 DEG C of 1min
DEG C, while collecting fluorescence signal, last 60 DEG C keep 15s to terminate.
75 DEG C~77.5 DEG C interval progress melting curve phenotypic analysis are selected using HRM softwares in step (3), sample is determined
Genotype, identifies sex.
Above-mentioned 4th technical problem to be solved of the present invention is achieved by the following technical solution：Above-mentioned
Available for the application of snakehead genetic sex and the SNP marker of supermale fish identification in snakehead genetic sex and the identification of supermale fish.
The present invention has advantages below：
(1) primer provided using the present invention expands the genomic DNA of testing sample, then by based on quantitative fluorescent PCR
The high-resolution melting curve analysis of instrument, it is possible to identify the uniformity of genetic sex, genotype and phenotype is 98% is accurate in family
Rate 100%, and supermale fish in pseudo- raun and common milter offspring family can be identified；
(2) the whole detection process of authentication method of the present invention can be completed in one day, simple and quick and accuracy rate is high；
(3) of the invention SNP site and detection method be it is currently the only can be to snakehead while carrying out common sex identification
The molecule labelling method identified with supermale fish.
Brief description of the drawings
Fig. 1 is using HRM softwares to carry out Genotyping to different sexes snakehead in embodiment 3.
Embodiment
Embodiment 1
What the present embodiment was provided can be used for the SNP marker that snakehead genetic sex and supermale fish are identified, the SNP marker is SEQ
ID NO：1~SEQ ID NO：The of nucleotide sequence shown in 3 (respectively correspond to SNP marker 1, SNP marker 2, SNP markers 3)
82, its base is 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 homozygotes, sample is female, when sample is C/T heterozygosis
When sample for male；In being raised up seed with pseudo- raun (estrogenic is reversed, and genotype is C/T heterozygosis) and common milter (C/T),
Genotype be when being T/T homozygosis female, during C/T heterozygosis for male, be during C/C homozygosis male and be supermale fish.
Embodiment 2
What the present embodiment was provided can be used for the primer pair of snakehead genetic sex and the SNP marker of supermale fish identification, and it includes
Sense primer is to F and anti-sense primer to R, wherein nucleotide sequence such as SEQ ID NO of the sense primer to F：Shown in 4, downstream is drawn
Nucleotide sequence such as SEQ ID NO of the thing to R：Shown in 5.
Specially：
Sense primer is to F (Primer F)：5'AGTGTGTGAGTTTGAGGATGTC 3'；
Anti-sense primer is to R (Primer R)：5'CAAGTATATGAGCTGAGACCAG 3'.
Embodiment 3
Identification snakehead genetic sex and the method for supermale fish that the present embodiment is provided, comprise the following steps：
(1) sample DNA is extracted
Extracting the genomic DNA of sample can be extracted using ordinary skill in the art means, including phenol-chloroform method and each
DNA extraction kit is planted, 20ng μ L are diluted to sterilized water after extracting STb gene.
(2) sequence for including SNP site is expanded using following primer：
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 homozygotes, sample is female, when sample is C/T heterozygosis
When sample for male；In being raised up seed with pseudo- raun (estrogenic is reversed, and genotype is C/T heterozygosis) and common milter (C/T),
Genotype be when being T/T homozygosis female, during C/T heterozygosis for male, be during C/C homozygosis male and be supermale fish.
(3) PCR amplifications 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 subjected to amplification and melting curve collection on stepone plus real-time PCRs, react
After the completion of analyzed using HRM softwares, sample genotype is determined, so as to identify sex.
Wherein PCR reaction systems are 20 μ L, including：
The μ L of 10 μ L, primer F of ABI Meltdoctor mix, 0.6 μ L, primer R 0.6, DNA profiling 1 μ L, it is sterile
The μ L of water 7.8.Reaction is carried out in special 96 orifice plate of quantitative PCR, high printing opacity shrouding mould covering sealing.
Amplification and testing conditions are：
Prepared by above-mentioned reaction system after reagent, reaction plate is placed in ABI stepone plus real-time PCRs
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 15s that unwind, 60 DEG C of 1min, while collecting fluorescence signal, last 60 DEG C
15s is kept to terminate.
(4) the use of instrument is that ABI stepone plus enter performing PCR amplification and signal collection, reaction utilizes instrument after terminating
Software stepone 2.3 carries out initial analysis, preserves file；ABI high-resolution melting curve analysis softwares HRM is recycled to open
The file of above-mentioned preservation, selects 75 DEG C~77.5 DEG C interval progress melting curve partings.
As a result：After being analyzed using HRM, the genotype of sample can differentiate, as shown in fig. 1, and 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) homozygosis
Type, is accredited as supermale fish.
Application Example one
Using above-mentioned SNP marker primer and detection method to the tail parent population of snakehead 100 (bred, physiology sex determine, its
The middle tail of raun 50, the tail of milter 50) carry out sex checking.
(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, absolute ethyl alcohol preservation, normal temperature are put into
Under take back laboratory；The fin ray sample that absolute ethyl alcohol is preserved washes away alcohol with aseptic double-distilled water, utilizes omega animal tissues
DNA extraction kit extracts genomic DNA；The DNA of extraction detects DNA integrality, NanoQ through 1% agarose gel electrophoresisTM
Microspectrophotometer (Bo Ao) detectable concentration, and it is diluted to deionized water final concentration 20ng/ μ L.
(2) HRM parting reaction mixtures are prepared
10 μ L, primer F of ABI Meltdoctor mix, 0.6 μ L, primer R 0.6 μ L, the μ L of DNA profiling 1 is sterile
The μ L of water 7.8.Reaction is carried out in special 96 orifice plate of ABI stepone plus quantitative PCRs, high printing opacity shrouding mould covering sealing.
(3) machine testing on
Using ABI stepone plus real-time PCRs, software is stepone 2.3, and parameter by specification is set,
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
DEG C unwind 15s, and 95 DEG C are warming up to 0.3 DEG C/s speed after 60 DEG C of 1min, while fluorescence signal is collected, last 60 DEG C of holdings 15s
Terminate.
(4) result
After program operation is finished, using the software initial analyses of stepone 2.3, file is preserved.Again by the text of preservation
Part is opened with HRM3.0.1 softwares, and selection temperature range is analyzed, the tail of TT genotype 50 in 50 tails female parent population individual；50 tails
T/C genotype is 48 tails, the tail of TT types 2 in physiological male individual.
Application Example two
Using the primer and detection method of above-mentioned SNP marker, to the same tail juvenile fish of family snakehead 200, (family female parent swashs to be female
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 extractions, physiology sex identification, PCR amplification and HRM classifying methods are identical with Application Example one.
(2) result passes through the tail of T/C genotype 103, the tail of T/T genotype 51 in HRM software partings, 200 tails individual；C/C bases
Because type is 46 tails, meet the 2 of common milter in offspring, supermale fish and raun:1:1 expection, illustrates that the mark of the present 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 example.
The those of ordinary skill of the technical field takes scope according to above present disclosure and each parameter, and this can be achieved
The purpose of invention.
The ＞ China's Pearl River Fishery Research Institute of Aquatic Science Research Institute of ＜ 110
A kind of SNP markers identified available for snakehead genetic sex and supermale fish of the ＞ of ＜ 120 and its application
〈210〉1
〈211〉106
〈212〉DNA
The ＞ SNP markers 1 of ＜ 223
〈400〉1
CAGTGTGTGA GTTTGAGGAT GTCAACAAAT GCATAAAGTT CGCATAAAAA CTGCTGAGCT 60
GGGCTCTTCG ATGCAATCAG GTTGCTGGTC TCAGCTCATA TACTTG 106
〈210〉2
〈211〉106
〈212〉DNA
The ＞ SNP markers 2 of ＜ 223
〈400〉2
CAGTGTGTGA GTTTGAGGAT GTCAACAAAT GCATAAAGTT CGCATAAAAA CTGCTGAGCT 60
GGGCTCTTCG ATGCAATCAG GCTGCTGGTC TCAGCTCATA TACTTG 106
〈210〉3
〈211〉106
〈212〉DNA
The ＞ SNP markers 3 of ＜ 223
〈400〉3
CAGTGTGTGA GTTTGAGGAT GTCAACAAAT GCATAAAGTT CGCATAAAAA CTGCTGAGCT 60
GGGCTCTTCG ATGCAATCAG GCCGCTGGTC TCAGCTCATA TACTTG 106
〈210〉4
〈211〉22
〈212〉DNA
The ＞ sense primers of ＜ 223 are to F
〈400〉4
AGTGTGTGAG TTTGAGGATG TC 22
〈210〉5
〈211〉22
〈212〉DNA
The ＞ anti-sense primers of ＜ 223 are to R
〈400〉5
CAAGTATATG AGCTGAGACC AG 22
Claims (8)
1. a kind of SNP marker identified available for snakehead genetic sex and supermale fish, it is characterized in that：The SNP marker is SEQ
ID NO：1~SEQ ID NO：The 82nd of nucleotide sequence shown in 3, its base is T/T, C/T or C/C.
2. the SNP marker according to claim 1 identified available for snakehead genetic sex and supermale fish, it is characterized in that：When
When base is T/T homozygotes, sample is female, and when sample is C/T heterozygosis, sample is male；It is numerous with pseudo- raun and common milter
Grow in offspring, genotype be when being T/T homozygosis female, during C/T heterozygosis for male, be during C/C homozygosis male and be supermale fish, its
Middle pseudo- raun is reversed for estrogenic, and genotype is C/T heterozygosis, and common milter genotype is C/T heterozygosis.
3. a kind of primer pair for the SNP marker identified available for snakehead genetic sex and supermale fish, it is characterized in that：It includes upstream
Primer pair F and anti-sense primer are to R, nucleotide sequence such as SEQ ID NO of the sense primer to F：Shown in 4, primer downstream
To R nucleotide sequence such as SEQ ID NO：Shown in 5.
4. a kind of identify snakehead genetic sex and the method for supermale fish, it is characterized in that comprising 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, using sense primer described in claim 2 to F and anti-sense primer to R,
Carry out pcr amplification reaction and melting curve is collected；
(3) analyzed after the completion of reacting, determine sample genotype, identify sex.
5. identification snakehead genetic sex according to claim 4 and the method for supermale fish, it is characterized in that：PCR in step (2)
The reaction system used during amplified reaction for 20 μ L, including：The μ L of 10 μ L, primer F of ABI Meltdoctor mix 0.6,
The μ L of primer R 0.6, the μ L of DNA profiling 1, the μ L of sterilized water 7.8.
6. identification snakehead genetic sex according to claim 4 and the method for supermale fish, it is characterized in that：PCR in step (2)
PCR amplification programs are during amplified reaction：50 DEG C of 2min, 95 DEG C of 10min；Then 40 circulations, including 95 DEG C of 15s, 58 DEG C are carried out
1min；Then 95 DEG C are warming up to 0.3 DEG C/s speed after 95 DEG C of 15s that unwind, 60 DEG C of 1min, while collecting fluorescence signal, finally
60 DEG C keep 15s to terminate.
7. identification snakehead genetic sex according to claim 4 and the method for supermale fish, it is characterized in that：It is sharp in step (3)
Select 75 DEG C~77.5 DEG C intervals to carry out melting curve phenotypic analysis with HRM softwares, determine sample genotype, identify sex.
8. described in claim 1 can be used for the SNP marker that snakehead genetic sex and supermale fish are identified 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 |
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| CN112746111A (en) * | 2021-01-20 | 2021-05-04 | 广东梁氏水产种业有限公司 | Northern snakehead male molecular marker primer and application thereof |
| CN113930525A (en) * | 2021-11-19 | 2022-01-14 | 中国科学院水生生物研究所 | Specific sequence for snakehead sex identification and application |
| CN114686594A (en) * | 2020-12-25 | 2022-07-01 | 中国水产科学研究院珠江水产研究所 | SNP molecular marker suitable for sex identification of salangid and application thereof |
| CN114686595A (en) * | 2020-12-25 | 2022-07-01 | 中国水产科学研究院珠江水产研究所 | SNP molecular marker for sex identification of salangid and application thereof |
| CN116732157A (en) * | 2023-03-28 | 2023-09-12 | 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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| 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 |
| CN114686594A (en) * | 2020-12-25 | 2022-07-01 | 中国水产科学研究院珠江水产研究所 | SNP molecular marker suitable for sex identification of salangid and application thereof |
| CN114686595A (en) * | 2020-12-25 | 2022-07-01 | 中国水产科学研究院珠江水产研究所 | SNP molecular marker for sex identification of salangid and application thereof |
| 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 |
| CN112746111A (en) * | 2021-01-20 | 2021-05-04 | 广东梁氏水产种业有限公司 | Northern snakehead male molecular marker primer and application thereof |
| CN113930525A (en) * | 2021-11-19 | 2022-01-14 | 中国科学院水生生物研究所 | Specific sequence for snakehead sex identification and application |
| 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 |
| CN116732157A (en) * | 2023-03-28 | 2023-09-12 | 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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