CN108588231A - Mandarin sturgeon Relationship iden- tification kit and its application - Google Patents
Mandarin sturgeon Relationship iden- tification kit and its application Download PDFInfo
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Abstract
The invention discloses mandarin sturgeon Relationship iden- tification kit and its application processes, belong to animal molecular genetics field.This kit provides the double PCR reaction system of 5 groups of mandarin sturgeon microsatellite markers.Inbreeding between mandarin sturgeon can be avoided using this kit with the affiliation between Rapid identification individual.The amplification of this kit has the polymorphism and stability of height, and the financial resources that are consumed of this kit and manpower all reduce half than traditional PCR reactions, have huge practical value.
Description
Technical field
The present invention relates to a kind of mandarin sturgeon Relationship iden- tification kit and its applications, belong to animal molecular genetics neck
Domain.
Background technology
Mandarin sturgeon is that distribution latitude is minimum in the kind Acipenseridae fish of the whole world more than 20, and the maximum fish of build is that China is distinctive
Species.The seventies in last century, the reproductive population in the Changjiang river can reach more than 10,000 tails, due to environmental pollution, overfishing, inhabit
The factors mandarin sturgeons such as ground forfeiture are endangered.The individual but also right of endangered species is not only protected to the protection of endangered species
The gene of endangered species, which also has, to be understood and is protected, and then formulates good breeding policy, prevents inbreeding.But until
Today is extremely limited to the hereditary Journal of Sex Research of mandarin sturgeon, and urgent need studies mandarin sturgeon from molecular orientation.
Microsatellite marker is optimal a kind of mode in current research animals on the brink of extinction conservative genetics, and microsatellite has more
State property height, spreads all over the advantages such as whole gene group at codominant inheritance, is widely used in animal heredity Journal of Sex Research.Microsatellite marker is more
Weight PCR is the PCR reactions for adding two pairs or more primers in same PCR reaction systems, while amplifying multiple nucleic acid fragments.It is more
Weight PCR has many advantages, such as high efficiency, systematicness, economical and convenient.Microsatellite marker multiplex PCR in relation to mandarin sturgeon is domestic and international
It has not been reported.The present invention provides the affiliations between a kind of identification individual using mandarin sturgeon microsatellite double PCR system
Kit realizes the measurement of the affiliation between mandarin sturgeon individual and then determines the affiliation between individual, for China
The genetic management of sturgeon, artificial propagation breeding is formulated, Analysis of Genetic Background provides solid technical support.
Invention content
Identifying that the relationship between individual is closed using mandarin sturgeon microsatellite double PCR system the object of the present invention is to provide a kind of
The kit of system and the identification method that the affiliation between the individual of mandarin sturgeon is realized using this kit, and then determine
Affiliation between individual avoids inbreeding between mandarin sturgeon individual.
Technical solution
5 groups of China in the kit of the affiliation between individual are measured using mandarin sturgeon microsatellite double PCR system
Micro-satellite primers in the double PCR reaction system of sturgeon microsatellite marker are as follows：
The mandarin sturgeon Relationship iden- tification kit and its application, the kit further includes PCR reagent, described
Reagent be：10 × PCR Buffer 2-3ul, 2.5mmol/L dNTP 0.1-1ul, MgCl21-2ul, every group of reaction system
The upstream and downstream primer each 0.5-2ul, Taq enzyme 0.1-0.5ul, DNA profiling 1-5ul, ultra-pure water 10-15ul of two pairs of primers.
Further preferably the kit includes PCR reagent, and the reagent is：10×PCR Buffer
2.5ul, 2.5mmol/L dNTP 0.5ul, MgCl21.5ul, every group of reaction system two 1ul each to the upstream and downstream primer of primer,
Taq enzyme 0.3ul, DNA profiling 3ul, ultra-pure water 13.2ul.
In the kits for measuring the affiliation between individual using mandarin sturgeon microsatellite double PCR system
The method of affiliation between magnificent sturgeon individual, includes the following steps：Take mandarin sturgeon tissue sample；Extract mandarin sturgeon sample DNA；
Using the micro-satellite primers in the double PCR reaction system of 5 groups of mandarin sturgeon microsatellite markers shown in claim 1 to mandarin sturgeon
Sample carries out PCR amplification；Pcr amplification product is separated by electrophoresis on 10% polyacrylamide gel；According to separating resulting
Count the genotype of pcr amplification product；According to the genotypic results of each individual with NTsys softwares draw it is each individual between
Clustering figure, determine individual between affiliation.The PCR response procedures are：94 DEG C of pre-degeneration 1-5min；94℃
It is denaturalized 25-35s, 56 DEG C of renaturation 25-35s of annealing temperature, 72 DEG C of extension 25-35s, 25-35 recycles；72 DEG C of extension 8-15min；
4 DEG C of preservations.The PCR response procedures are：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 56 DEG C of renaturation 30s of annealing temperature,
72 DEG C of extension 30s, 30 cycles；72 DEG C of extension 10min；4 DEG C of preservations.
Compared with prior art, the present invention has the following advantages that：
The heredity that mandarin sturgeon Relationship iden- tification kit provided by the invention and its application may be implemented to mandarin sturgeon is more
Sample detection, individual identification, can also identify the distance of the affiliation between individual, close relative is numerous between avoiding mandarin sturgeon individual
It grows.And the financial resources and manpower that kit provided by the invention is consumed all reduce half than traditional PCR, have huge
Promotional value.
Description of the drawings
Fig. 1 is the clustering figure between individual.
Specific implementation mode
Embodiment 1
48 tail mandarin sturgeons are taken from research institute of Acipenser Sinensis Institute of China Three Gorges Corporation, acquire the fin ray of mandarin sturgeon, are extracted
The genomic DNA of mandarin sturgeon, extracting mode take traditional phenol-chloroform to extract approach.
Each individual takes 0.1g tail fins to be put in the centrifuge tube of 1.5ml, shreds, and 450 μ LSTE extraction buffers (10 are added
Mmol/L Tris-HCl, pH8.0；1mmol/L EDTA, pH8.0), 35 μ L SDS (10%), 15 μ L Proteinase Ks (0.2%).
Centrifuge tube is put into and is placed in 55 DEG C of water-bath 1 hour of water-bath to clear.
700ul Tris saturated phenols are added in centrifuge tube, are placed on mixing 30 minutes on oscillator, 12000 turns at 4 DEG C/
Min centrifuges 10min, and supernatant is transferred to (the 1mL tube heads that attention is cut flat with tip in another clean eppendorf pipe
Aspirate supernatant, to prevent obscuring lower sediment).
The imitative alcohol mixture of isometric phenol is added in supernatant, and (phenol, chloroform, isoamyl alcohol ratio are 25：24：1), oscillation is mixed
After even 15min, 12000 turns/min centrifuges 10min at 4 DEG C, sucts clear liquid in another new Eppendorf pipes.
It is added isometric chloroform in supernatant, after oscillation mixing 15min, 12000 turns/min is from 10min at 4 DEG C, in absorption
Clear liquid.
The absolute ethyl alcohol 1mL that -20 DEG C of precoolings are added precipitates DNA, collects precipitation.
Precipitation is washed with 70% ethyl alcohol twice, and 200 μ L TE (10mmol/LTris-HCl, pH 8.0 are added after dry；
0.1mmol/L EDTA, pH 8.0), room temperature fully dissolves.
48 mandarin sturgeon individuals are carried out using mandarin sturgeon Relationship iden- tification kit provided by the invention and its application
PCR reacts, and PCR reaction systems are 25ul：10 × PCR Buffer 2.5ul, 2.5mmol/L dNTP 0.5ul, MgCl2
1.5ul, every group of reaction system two 1ul each to the upstream and downstream primer of primer, Taq enzyme 0.3ul, DNA profiling 3ul, ultra-pure water
13.2ul.PCR response procedures are：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, annealing temperature renaturation 30s, 72 DEG C of extension 30s,
30 cycles；72 DEG C of extension 10min；4 DEG C of preservations.PCR product carries out electrophoresis and silver staining with 10% polyacrylamide gel.
The size of the allele segment for each individual that polyacrylamide gel is shown, root are judged with software BIO-PROFIL
According to each individual the allele size on each microsatellite locus with NTsys softwares draw it is each individual between cluster
Analysis chart is illustrated in fig. 1 shown below.The affiliation between each individual is may determine that according to cluster analysis result, identifies whole
Mandarin sturgeon individual, to realize the identification of the affiliation on mandarin sturgeon molecular genetic level.It can be obtained from Fig. 1, the
1,3,14,43,32,34,35,37,27 and No. 41 individuals are a family, and affiliation is closer, should not be matched between them
Breeding.4th, 30,8 and No. 33 individual is a family, and affiliation is closer, should not match breeding between them.Remaining
Body is a family, and affiliation is closer, should not match breeding between them.
SEQUENCE LISTING
<110>Acipenser Sinensis Institute of China Three Gorges Corporation
<120>Mandarin sturgeon Relationship iden- tification kit and its application
<130> 20
<160> 20
<170> PatentIn version 3.5
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<211> 26
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gcaacttata cacaaataca gtgggt 26
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<212> DNA
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tattgcatga agctgtgcgc 20
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<212> DNA
<213>Artificial sequence
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aggggcgaat gatactgcac 20
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<212> DNA
<213>Artificial sequence
<400> 5
agctgcttct acaccgacac 20
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<212> DNA
<213>Artificial sequence
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gtgcccaaga tagcgcaaaa 20
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence
<400> 7
cgaatgaaat tgacgcaaac 20
<210> 8
<211> 20
<212> DNA
<213>Artificial sequence
<400> 8
ccatttattt tggccaccag 20
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence
<400> 9
gctttgcgca ctctttccat 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<400> 10
ggcactccac tccactgtac 20
<210> 11
<211> 22
<212> DNA
<213>Artificial sequence
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aaagggaact tcatcttttc ca 22
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<400> 12
gttttgccat gccaatcttt 20
<210> 13
<211> 25
<212> DNA
<213>Artificial sequence
<400> 13
tcagtgcatt aacttacatt ttgca 25
<210> 14
<211> 22
<212> DNA
<213>Artificial sequence
<400> 14
agagtcctct tcatgacaca ca 22
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<400> 15
tctggatagc tggccttctg 20
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence
<400> 16
aactgtgcaa aaggggaaga 20
<210> 17
<211> 18
<212> DNA
<213>Artificial sequence
<400> 17
aaagcgcgct gtttgtgt 18
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<211> 20
<212> DNA
<213>Artificial sequence
<400> 18
aaacaaatcc aggagcgaag 20
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<211> 20
<212> DNA
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<400> 19
agccccatct gcaatactgt 20
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<211> 21
<212> DNA
<213>Artificial sequence
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ctgatggcat atcacatgct t 21
Claims (8)
1. mandarin sturgeon Relationship iden- tification kit, which is characterized in that the kit includes that 10 pairs of micro-satellite primers are as follows：
2. mandarin sturgeon Relationship iden- tification kit, which is characterized in that including mandarin sturgeon microsatellite marker described in claim 1
5 groups of double PCR reaction systems micro-satellite primers.
3. the mandarin sturgeon Relationship iden- tification kit described in claim 2, which is characterized in that the kit includes
PCR reagent, the reagent are：10 × PCR Buffer 2-3ul, 2.5mmol/L dNTP 0.1-1ul, MgCl21-2ul,
Every group of reaction system two 0.5-2ul each to the upstream and downstream primer of primer, Taq enzyme 0.1-0.5ul, DNA profiling 1-5ul, ultra-pure water
10-15ul。
4. the mandarin sturgeon Relationship iden- tification kit described in claim 3, which is characterized in that the kit includes
PCR reagent, the reagent are：10 × PCR Buffer 2.5ul, 2.5mmol/L dNTP 0.5ul, MgCl21.5ul, often
Group reaction system two 1ul each to the upstream and downstream primer of primer, Taq enzyme 0.3ul, DNA profiling 3ul, ultra-pure water 13.2ul.
5. taking claim 2-3 any one of them mandarin sturgeon Relationship iden- tifications kit in authentication between magnificent sturgeon individual
Affiliation on application.
6. the application described in claim 5, which is characterized in that include the following steps：Take mandarin sturgeon tissue sample；Extract mandarin sturgeon
Sample DNA；Utilize the double PCR reaction system for 5 groups of mandarin sturgeon microsatellite markers that this kit shown in claim 1 provides
In micro-satellite primers to mandarin sturgeon sample carry out PCR amplification；Pcr amplification product is carried out on polyacrylamide gel electrophoresis point
From；The genotype of pcr amplification product is counted according to separating resulting；According to the genotypic results NTsys softwares of each individual
The clustering figure between each individual is drawn, determines the affiliation between individual.
7. the application described in claim 6, which is characterized in that the PCR response procedures are：94 DEG C of pre-degeneration 1-5min；94
DEG C denaturation 25-35s, 56 DEG C of annealing temperature renaturation 25-35s, 72 DEG C of extension 25-35s, 25-35 recycles；72 DEG C of extension 8-
15min；4 DEG C of preservations.
8. the application described in claim 7, which is characterized in that the PCR response procedures are：94 DEG C of pre-degeneration 3min；94℃
It is denaturalized 30s, 56 DEG C of renaturation 30s of annealing temperature, 72 DEG C of extension 30s, 30 recycle；72 DEG C of extension 10min；4 DEG C of preservations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810312499.8A CN108588231B (en) | 2018-04-09 | 2018-04-09 | Chinese sturgeon genetic relationship identification kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810312499.8A CN108588231B (en) | 2018-04-09 | 2018-04-09 | Chinese sturgeon genetic relationship identification kit and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108588231A true CN108588231A (en) | 2018-09-28 |
| CN108588231B CN108588231B (en) | 2021-08-03 |
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ID=63621266
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810312499.8A Active CN108588231B (en) | 2018-04-09 | 2018-04-09 | Chinese sturgeon genetic relationship identification kit and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498613A (en) * | 2014-12-31 | 2015-04-08 | 中国水产科学研究院长江水产研究所 | SSR fluorescence labeling primer for paternity test of Chinese sturgeon and application |
| CN106811540A (en) * | 2017-03-22 | 2017-06-09 | 淮阴师范学院 | It is a kind of to identify female ussuriensis, male individual microsatellite marker and specific primer and application |
-
2018
- 2018-04-09 CN CN201810312499.8A patent/CN108588231B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498613A (en) * | 2014-12-31 | 2015-04-08 | 中国水产科学研究院长江水产研究所 | SSR fluorescence labeling primer for paternity test of Chinese sturgeon and application |
| CN106811540A (en) * | 2017-03-22 | 2017-06-09 | 淮阴师范学院 | It is a kind of to identify female ussuriensis, male individual microsatellite marker and specific primer and application |
Non-Patent Citations (2)
| Title |
|---|
| 孙丽婷等: "中华鲟资源及其遗传多样性研究进展", 《渔业信息与战略》 * |
| 辛苗苗: "基于SSR的中华鲟亲子鉴定和遗传特性研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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