CN102226220A - Molecule marking method for identifying tilapia nilotica, oreochromis aureus and hybridized fish thereof - Google Patents
Molecule marking method for identifying tilapia nilotica, oreochromis aureus and hybridized fish thereof Download PDFInfo
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- CN102226220A CN102226220A CN201110163113XA CN201110163113A CN102226220A CN 102226220 A CN102226220 A CN 102226220A CN 201110163113X A CN201110163113X A CN 201110163113XA CN 201110163113 A CN201110163113 A CN 201110163113A CN 102226220 A CN102226220 A CN 102226220A
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- tilapia
- oreochromis aureus
- bolti
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Abstract
The invention relates to a molecule marking method for identifying tilapia nilotica, oreochromis aureus and hybridized fish thereof. The method is characterized by comprising the following steps: selecting three pairs of specific microsatellite marked primer combinations; sampling blood or fins from tilapia to be detected; extracting deoxyribonucleic acid (DNA) of a gene group; performing multiple polymerase chain reaction (PCR) amplifications on the DNA of the gene group of the tilapia to be detected; performing electrophoretic separation on the PCR product with a 8.0 percent non-denatured polyacrylamide gel; dying by argentation; and photographing to record the electrophoretic results. According to the results of multiple PCR electrophoretic detection, the tilapia nilotica, the oreochromis aureus and the hybridized fish thereof can be identified. By the method, the tilapia nilotica, the oreochromis aureus and the hybridized fish thereof can be quickly and effectively identified.
Description
Technical field
The invention belongs to the molecular marking technique field, relate to a kind of molecule marking method of differentiating bolti, Oreochromis aureus and hybridization fish thereof, is to the not discriminating of tilapia of the same race.
Background technology
Tilapia (Tilapia) originates in Africa, is worldwide breed variety, and this fish is introduced China the fifties in last century, has obtained very fast development, and existing China has been maximum in the world tilapia producing country and export State.At present, China's tilapia kind of mainly culturing have bolti (
Oreochromis niloticus), Oreochromis aureus (
Oreochromis aureus) and Buddhist nun difficult to understand hybridize tilapia, wherein Sarotherodon sp is 1 generation of hybridization of bolti (♀) and Oreochromis aureus (♂), have advantages such as male ratio height, growth is fast, disease resistance is strong, but its male ratio and breed performance and parent's germplasm purity is closely related.Owing to be easy to hybridization between the tilapia kind, and hybrid can educate, and the cross-fertilize seed form is very similar to the parent again, and traditional form discriminating conduct is difficult to accurately distinguish purebred and cross-fertilize seed again.Therefore, be easy to cause the tilapia germplasm to mix, cause good economic characters to be degenerated, this also is the outstanding problem that restricts China's tilapia breeding protection at present and culture development.
Bolti, Oreochromis aureus and their hybridization fish discriminating aspects thereof, scholars once utilized isoenzyme technique, RAPD technology, micro-satellite labeling technique etc. to set up biochemistry and the molecular genetic marker of distinguishing them.Yet isozyme is a gene expression product, but detection site is limited, and kind nearer to sibship or that hereditary basis is complicated is not easy to distinguish; Though the RAPD method is to differentiate different varieties from gene level, the RAPD method is very sensitive to the variation of reaction conditions, and the variant a little banding pattern that will cause increasing of many reaction factors changes, and stability and repeatability are relatively poor, thereby have limited its application.Microsatellite marker claims simple sequence repeating label (SSR) to compare with technology such as isozyme, RAPD, RFLP again, have polymorphism frequency height, detect easily, advantages such as good reproducibility, codominant Mendelian inheritance, be widely used in researchs such as kind and paternity test, carry out the research that multiplex PCR differentiates different tilapia kinds quickly and accurately and yet there are no report but use microsatellite marker.Adopt special micro-satellite labeling technique to carry out the hereditary property that multiplex PCR is analyzed bolti, Oreochromis aureus and their hybridization fish, can set up fast, accurately differentiate the method for different tilapia kinds.
Summary of the invention
Goal of the invention
The purpose of this invention is to provide a kind of molecule marking method of differentiating bolti, Oreochromis aureus and hybridization fish thereof, help fast, accurately differentiate tilapia not of the same race, protect kind tilapia, great using value arranged in seed selection and the breeding production.
Technical scheme
A kind of molecule marking method of differentiating bolti, Oreochromis aureus and hybridization fish thereof is characterized in that, adopts following extraction step:
(1) select for use 3 pairs of special micro-satellite primers combinations to carry out multi-PRC reaction, wherein the special micro-satellite primers of this 3 couple is as follows:
CCCTCTGTTTCCATCTCA；GATACCTGTCCATACCTCCTC；
GCTCGCTCCAGAAAAATCAC；GTCAAAAAGGCATGGCAAAG；
CTGCACTTTTACTGAGGG；TGGGAGATTAACAGAATAACA
(2), adopt the DNA extraction method to extract genomic dna from tilapia sampling blood to be measured or fin ray;
(3) genomic dna with step (2) is a template, the amplification of the selected 3 pairs of micro-satellite primers combination carrying out of step (1) multiplex PCR;
(4) the PCR product of step (3) is carried out electrophoretic separation with 8.0% non-denaturing polyacrylamide gel, after the argentation dyeing, the Taking Pictures recording electrophoresis result.
(5) according to the electrophoresis detection result of multiplex PCR, differentiate bolti, Oreochromis aureus and their hybridization fish.
In the step (3), when carrying out pcr amplification, PCR reaction cumulative volume is 15 μ L, wherein contains 10 * reaction buffer, 1.5 μ L, Mg
2+2 mmol/L, dNTP 200 μ mol/L, 3 couples of each 0.2 μ mol/L of upstream and downstream primer, Taq enzyme 0.4U, DNA 50 ng～100 ng supply volume with the sterilization bi-distilled water.
In the step (3), the PCR reaction conditions is: 94 ℃ of 3 min; 94 ℃ of 20 s, 52 ℃ of annealing 20s, 72 ℃ of 20s, 25～30 circulations; 72 ℃ are extended 5 min.
The present invention's step specific as follows realizes:
(1) select for use 3 pairs of special microsatellite marker combination of primers to carry out multi-PRC reaction.
(2), adopt the DNA extraction method to extract genomic dna from tilapia sampling blood to be measured or fin ray.
(3) genomic dna with step (2) is a template, the amplification of the selected 3 pairs of micro-satellite primers combination carrying out of step (1) multiplex PCR.
(4) the PCR product of step (3) is carried out electrophoretic separation with 8.0% non-denaturing polyacrylamide gel, after the argentation dyeing, the Taking Pictures recording electrophoresis result.
(5) according to the electrophoresis detection result of multiplex PCR, just can differentiate bolti, Oreochromis aureus and their hybridization fish: in primer GM017 amplified production district, 1 or 2 of bolti band, clip size is about 144～148bp; 1 of Oreochromis aureus band, clip size is about 160bp; The hybridization fish band all is 2, and wherein 1 bar segment size is about 144～148bp, and 1 bar segment size is about 160bp in addition.In primer UNH948 amplified production district, 1 or 2 of bolti band, clip size is about 170～178bp; Oreochromis aureus band 1 or 2, clip size is about 196～202bp; The hybridization fish band all is 2, and wherein 1 bar segment size is about 170～178bp, and 1 bar segment size is about 196～202bp in addition.In primer GM440 amplified production district, 1 or 2 of bolti band, clip size is about 262～266bp; 1 of Oreochromis aureus band, clip size is about 290bp; The hybridization fish band all is 2, and wherein 1 bar segment size is about 262～266bp, and 1 bar segment size is about 290bp in addition.
Selected 3 pairs of micro-satellite primers pcr amplification reaction in bolti, Oreochromis aureus and hybridization fish thereof is stable among the present invention, clear, the not of the same race tilapia amplified fragments difference of amplified fragments is obvious, and these 3 pairs of micro-satellite primers annealing temperatures are close, every pair of primer amplification fragment differs more than 20bp, and separately pcr amplification product mutually is independent of each other in same system multi-PRC reaction.Wherein the special micro-satellite primers of this 3 couple is as follows for the special micro-satellite primers of this 3 couple:
CCCTCTGTTTCCATCTCA；GATACCTGTCCATACCTCCTC；
GCTCGCTCCAGAAAAATCAC；GTCAAAAAGGCATGGCAAAG；
CTGCACTTTTACTGAGGG；TGGGAGATTAACAGAATAACA
PCR reaction cumulative volume is 15 μ L among the present invention, wherein contains 10 * reaction buffer, 1.5 μ L, Mg
2+2 mmol/L, dNTP 200 μ mol/L, 3 couples of each 0.2 μ mol/L of upstream and downstream primer,
TaqEnzyme 0.4U, DNA 50 ng～100 ng supply volume with the sterilization bi-distilled water.
Amplification PCR reaction is all finished on the PCR instrument among the present invention.The PCR reaction conditions is: 94 ℃ of 3 min; 94 ℃ of 20 s, 52 ℃ of annealing 20s, 72 ℃ of 20s, 25～30 circulations; 72 ℃ are extended 5 min.
Beneficial effect
Compared with prior art, the application adopts special micro-satellite labeling technique to carry out the hereditary property that multiplex PCR is analyzed bolti, Oreochromis aureus and their hybridization fish, stable reaction, good reproducibility can distinguish purebred bolti, Oreochromis aureus and their filial generation exactly.The application once can detect a plurality of microsatellite locus simultaneously, has reduced detection time and has reduced the detection cost.
Description of drawings
Fig. 1 bolti, Oreochromis aureus and their hybridization fish multiplex PCR electrophoresis detection result, wherein M: molecular weight standard
1-13: bolti
14-20: Sarotherodon sp
21-28: Buddhist nun tilapia difficult to understand
29-40: Oreochromis aureus
Embodiment:
Embodiment 1:
A kind of molecule marking method of bolti, Oreochromis aureus and hybridization fish thereof of differentiating of the present invention adopts following technological step:
Wherein the special micro-satellite primers of this 3 couple is as follows:
CCCTCTGTTTCCATCTCA；GATACCTGTCCATACCTCCTC；
GCTCGCTCCAGAAAAATCAC；GTCAAAAAGGCATGGCAAAG；
CTGCACTTTTACTGAGGG；TGGGAGATTAACAGAATAACA。
Embodiment 2:
A kind of molecule marking method of bolti, Oreochromis aureus and hybridization fish thereof of differentiating of the present invention adopts following technological step:
Ao Ni hybridization tilapia (bolti ♀ * Oreochromis aureus ♂) 8 tails are adopted from fishing ground, Yixing, China Aquatic Science Research Institute fresh water fishery research centre.Every tail fish is got 0.1g tail fin fin ray, extracting genomic dna.The genomic dna of hybridizing tilapia with Buddhist nun difficult to understand is a template, uses primer GM017, and 3 pairs of primers of UNH948 and GM440 are to carrying out the multiplex PCR amplification, and PCR reaction cumulative volume is 15 μ L, wherein contains 10 * reaction buffer, 1.5 μ L, MgC1
22 mmol/L, dNTP 200 μ mol/L, 3 couples of each 0.2 μ mol/L of upstream and downstream primer,
TaqEnzyme 0.4U, DNA 50 ng supply volume with the sterilization bi-distilled water.The PCR reaction conditions is: 94 ℃ of 3 min; 94 ℃ of 30 s, 52 ℃ of annealing 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ are extended 8 min.After the PCR reaction finished, product carried out electrophoretic separation with 8.0% non-denaturing polyacrylamide gel, after the argentation dyeing, and the Taking Pictures recording electrophoresis result.Electrophoresis detection finds that in primer GM017 amplified production district, Buddhist nun's hybridization fish band difficult to understand all is 2, and wherein 1 bar segment size is about 144～148bp, and 1 bar segment size is about 160bp in addition.In primer UNH948 amplified production district, Buddhist nun's hybridization fish band difficult to understand all is 2, and wherein 1 bar segment size is about 170～178bp, and 1 bar segment size is about 196～202bp in addition.In primer GM440 amplified production district, Buddhist nun's hybridization fish band difficult to understand all is 2, and wherein 1 bar segment size is about 262～266bp, and 1 bar segment size is about 290bp in addition.
Wherein the special micro-satellite primers of this 3 couple is as follows:
CCCTCTGTTTCCATCTCA；GATACCTGTCCATACCTCCTC；
GCTCGCTCCAGAAAAATCAC；GTCAAAAAGGCATGGCAAAG；
CTGCACTTTTACTGAGGG；TGGGAGATTAACAGAATAACA。
Embodiment 3:
A kind of molecule marking method of bolti, Oreochromis aureus and hybridization fish thereof of differentiating of the present invention adopts following technological step:
Oreochromis aureus 12 tails, Buddhist nun's hybridization difficult to understand tilapia (bolti ♂ * Oreochromis aureus ♀) 7 tails are adopted from fishing ground, Yixing, China Aquatic Science Research Institute fresh water fishery research centre.Every tail fish is got 30 μ L hemocyte extracting genomic dnas.With these genomic dnas is template, uses primer GM017, and 3 pairs of primers of UNH948 and GM440 are to carrying out the multiplex PCR amplification, and PCR reaction cumulative volume is 15 μ L, wherein contains 10 * reaction buffer, 1.5 μ L, MgC1
22 mmol/L, dNTP 200 μ mol/L, 3 couples of each 0.2 μ mol/L of upstream and downstream primer,
TaqEnzyme 0.4U, DNA 70 ng supply volume with the sterilization bi-distilled water.The PCR reaction conditions is: 94 ℃ of 3 min; 94 ℃ of 30 s, 52 ℃ of annealing 30s, 72 ℃ of 30s, 28 circulations; 72 ℃ are extended 8 min.After the PCR reaction finished, product carried out electrophoretic separation with 8.0% non-denaturing polyacrylamide gel, after the argentation dyeing, and the Taking Pictures recording electrophoresis result.The electrophoresis detection discovery, in primer GM017 amplified production district, 1 of Oreochromis aureus band, clip size is about 160bp; Buddhist nun's hybridization fish band difficult to understand all is 2, and wherein 1 bar segment size is about 144～148bp, and 1 bar segment size is at 160bp in addition.In primer UNH948 amplified production district, Oreochromis aureus band 1 or 2, clip size is about 196～202bp; Buddhist nun's hybridization fish band difficult to understand all is 2, and wherein 1 bar segment size is about 170～178bp, and 1 bar segment size is about 196～202bp in addition.In primer GM440 amplified production district, 1 of Oreochromis aureus band, clip size is about 290bp; Buddhist nun's hybridization fish band difficult to understand all is 2, and wherein 1 bar segment size is about 262～266bp, and 1 bar segment size is about 290bp in addition.
Wherein the special micro-satellite primers of this 3 couple is as follows:
CCCTCTGTTTCCATCTCA；GATACCTGTCCATACCTCCTC；
GCTCGCTCCAGAAAAATCAC；GTCAAAAAGGCATGGCAAAG；
CTGCACTTTTACTGAGGG；TGGGAGATTAACAGAATAACA。
SEQUENCE?LISTING
＜110〉China Aquatic Science Research Academy Fresh Water Fishery Research Center
＜120〉a kind of molecule marking method of differentiating bolti, Oreochromis aureus and hybridization fish thereof
<130>
<160> 6
<170> PatentIn?version?3.3
<210> 1
<211> 18
<212> DNA
＜213〉artificial sequence
<400> 1
ccctctgttt?ccatctca 18
<210> 2
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 2
gatacctgtc?catacctcct?c 21
<210> 3
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 3
gctcgctcca?gaaaaatcac 20
<210> 4
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 4
gtcaaaaagg?catggcaaag 20
<210> 5
<211> 18
<212> DNA
＜213〉artificial sequence
<400> 5
ctgcactttt?actgaggg 18
<210> 6
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 6
tgggagatta?acagaataac?a 21
Claims (3)
1. a molecule marking method of differentiating bolti, Oreochromis aureus and hybridization fish thereof is characterized in that, adopts following extraction step:
(1) select for use 3 pairs of special micro-satellite primers combinations to carry out multi-PRC reaction, wherein the special micro-satellite primers of this 3 couple is as follows:
CCCTCTGTTTCCATCTCA；GATACCTGTCCATACCTCCTC；
GCTCGCTCCAGAAAAATCAC；GTCAAAAAGGCATGGCAAAG；
CTGCACTTTTACTGAGGG；TGGGAGATTAACAGAATAACA
(2), adopt the DNA extraction method to extract genomic dna from tilapia sampling blood to be measured or fin ray;
(3) genomic dna with step (2) is a template, the amplification of the selected 3 pairs of micro-satellite primers combination carrying out of step (1) multiplex PCR;
(4) the PCR product of step (3) is carried out electrophoretic separation with 8.0% non-denaturing polyacrylamide gel, after the argentation dyeing, the Taking Pictures recording electrophoresis result;
(5) according to the electrophoresis detection result of multiplex PCR, differentiate bolti, Oreochromis aureus and their hybridization fish.
2. the molecule marking method of differentiating bolti, Oreochromis aureus and hybridization fish thereof according to claim 1 is characterized in that, in the step (3), when carrying out pcr amplification, PCR reaction cumulative volume is 15 μ L, wherein contains 10 * reaction buffer, 1.5 μ L, Mg
2+2 mmol/L, dNTP 200 μ mol/L, 3 couples of each 0.2 μ mol/L of upstream and downstream primer, Taq enzyme 0.4U, DNA 50 ng～100 ng supply volume with the sterilization bi-distilled water.
3. the molecule marking method of differentiating bolti, Oreochromis aureus and hybridization fish thereof according to claim 1 is characterized in that in the step (3), the PCR reaction conditions is: 94 ℃ of 3 min; 94 ℃ of 20 s, 52 ℃ of annealing 20s, 72 ℃ of 20s, 25～30 circulations; 72 ℃ are extended 5 min.
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| CN201110163113XA CN102226220A (en) | 2011-06-17 | 2011-06-17 | Molecule marking method for identifying tilapia nilotica, oreochromis aureus and hybridized fish thereof |
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| CN201110163113XA CN102226220A (en) | 2011-06-17 | 2011-06-17 | Molecule marking method for identifying tilapia nilotica, oreochromis aureus and hybridized fish thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365872A (en) * | 2017-09-15 | 2017-11-21 | 中国水产科学研究院黑龙江水产研究所 | The method for identifying the three pieces of fish difference migration colonies in the Suifenhe |
| CN110241230A (en) * | 2019-06-26 | 2019-09-17 | 中国水产科学研究院淡水渔业研究中心 | Identify method, kit and the application of Oreochromis aureus, bolti and its hybrid generation |
| CN110331217A (en) * | 2019-08-15 | 2019-10-15 | 中国水产科学研究院珠江水产研究所 | A kind of microsatellite marker paternity identification primer, method and application suitable for bolti, Oreochromis aureus and its cenospecies |
| CN111286547A (en) * | 2020-03-26 | 2020-06-16 | 茂名市伟业罗非鱼良种场 | Tilapia and microsatellite identification primer and method for genetic diversity of Tilapia |
| CN113637764A (en) * | 2021-05-14 | 2021-11-12 | 中山大学 | Detection primer for microsatellite related to body color of tilapia and application of detection primer |
-
2011
- 2011-06-17 CN CN201110163113XA patent/CN102226220A/en active Pending
Non-Patent Citations (3)
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| 宋红梅等: "三种罗非鱼的微卫星分子鉴定和遗传结构分析", 《农业生物技术学报》 * |
| 张庭等: "奥利亚罗非鱼微卫星位点的筛选", 《广东海洋大学学报》 * |
| 李建林等: "尼罗罗非鱼、奥利亚罗非鱼及其杂交后代微卫星标记鉴别和种质纯度分析", 《上海海洋大学学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365872A (en) * | 2017-09-15 | 2017-11-21 | 中国水产科学研究院黑龙江水产研究所 | The method for identifying the three pieces of fish difference migration colonies in the Suifenhe |
| CN110241230A (en) * | 2019-06-26 | 2019-09-17 | 中国水产科学研究院淡水渔业研究中心 | Identify method, kit and the application of Oreochromis aureus, bolti and its hybrid generation |
| CN110241230B (en) * | 2019-06-26 | 2022-10-14 | 中国水产科学研究院淡水渔业研究中心 | Method and kit for identifying Oreochromis mossambicus and Nile tilapia and hybrid filial generation thereof, and application of kit |
| CN110331217A (en) * | 2019-08-15 | 2019-10-15 | 中国水产科学研究院珠江水产研究所 | A kind of microsatellite marker paternity identification primer, method and application suitable for bolti, Oreochromis aureus and its cenospecies |
| CN111286547A (en) * | 2020-03-26 | 2020-06-16 | 茂名市伟业罗非鱼良种场 | Tilapia and microsatellite identification primer and method for genetic diversity of Tilapia |
| CN113637764A (en) * | 2021-05-14 | 2021-11-12 | 中山大学 | Detection primer for microsatellite related to body color of tilapia and application of detection primer |
| CN113637764B (en) * | 2021-05-14 | 2023-05-30 | 中山大学 | Detection primer of microsatellite with tilapia body color and application thereof |
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Application publication date: 20111026 |