CN102912017A - Method for quickly and accurately identifying northern snakehead, Taiwan snakehead and hybrid snakehead - Google Patents
Method for quickly and accurately identifying northern snakehead, Taiwan snakehead and hybrid snakehead Download PDFInfo
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- CN102912017A CN102912017A CN2012103799040A CN201210379904A CN102912017A CN 102912017 A CN102912017 A CN 102912017A CN 2012103799040 A CN2012103799040 A CN 2012103799040A CN 201210379904 A CN201210379904 A CN 201210379904A CN 102912017 A CN102912017 A CN 102912017A
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Abstract
The invention relates to a method for quickly and accurately identifying northern snakehead, Taiwan snakehead and hybrid snakehead. The method comprises the steps as follows: 1) synthesizing primers; 2) extracting DNAs (Deoxyribose Nucleic Acids) from the northern snakehead, the Taiwan snakehead and the hybrid snakehead [the northern snakehead (Venus symbol) * the Taiwan snakehead (Mars symbol)]; 3) carrying out SRAP-PCR (Sequence-Related Amplified Polymorphism-Polymerase Chain Reaction) reaction by adopting a renaturation temperature change method; 4) carrying out electrophoresis detection; and 5) combining the primers capable of being used for identifying the northern snakehead, the Taiwan snakehead and the hybrid snakehead [the northern snakehead (Venus symbol) * the Taiwan snakehead (Mars symbol)], processing stripes by using a Gelpro32 software, marking the sizes of the stripes, and identifying in other sampled groups. According to the method, the northern snakehead, the Taiwan snakehead and the hybrid snakehead [the northern snakehead (Venus symbol) * the Taiwan snakehead (Mars symbol)] can be easily identified by utilizing an SRAP technology by cutting away a small amount of fin rays or other fish tissue parts only without killing fishes.
Description
Technical field
The present invention relates to the molecular biological technology of a kind of usefulness (SRAP) and identify the method for hybridization fingerling, in particular, the present invention relates to a kind of method of quick and precisely identifying snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)].
Background technology
Snakehead (Channa argus) and snakehead (Channa maculate) belong to Perciformes (Perciformes) murrel section (Channidae) Ophiocephalus.The first filial generation that the hybridized snakehead fish that obtains by snakehead and snakehead hybridization is, have the advantages such as growth is fast, output is high, disease is few, safe mass is high, the process of propagating artificially can adopt artificial diet to feed, greatly improve in the cultivation of former snakehead because adopting the chilled problems such as causing feed waste and pollution of waterhead of throwing something and feeding, become most important breed variety in the murrel section fish at Delta of Pearl River.Wherein, take snakehead as the hybridized snakehead fish of male parent as maternal and snakehead, because the advantage such as the hatching rate is high, surviving rate is high, proportion shared in Chinese murrel section fish cultivate is increasing.
SRAP (SRAP) is a kind of New molecular marker system that development in recent years is got up, the design of primers principle of its uniqueness, upstream primer can specific binding at exon or the promoter region of gene, downstream primer can be specifically and the intron pairing of gene, increase for genomic ORF, produce the polymorphism amplified production owing to individual intron, promotor and transcribed spacer are uneven in length.The SRAP technology has the characteristics such as easy, high codominance, repeatability is high, cost is low.This technology has been widely used in the aspects such as plant genetic map construction, crop genetic diversity analysis, the assignment of genes gene mapping and comparative genomics aspect plant.Aspect aquatic animal, SRAP reports to some extent at aspects such as Macrobrachium nipponensis, swimming crab and grass carps.
The conventional aspect of identifying the hybridization fish of using is mainly identified from the morphology aspect.Yet Morphological Identification sometimes might not be accurate, has very strong subjective factor, and Morphological Identification can not be identified snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)] in the seed phase.For example, snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)] on morphology, snakehead easily is different from other two kinds of fishes, and snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)] be easy to obscure at morphology, both head decorative patterns all present " one eight eight ".The present invention only need cut off a small amount of fin ray or other Fish tissue part on the basis of not putting to death fingerling, utilize the SRAP technology just to be easy to distinguish the method for snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)]
Summary of the invention
The purpose of this invention is to provide a kind of method of quick and precisely identifying snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)], existing identify snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)] from morphology to overcome, exist subjectivity strong and can not effectively identify the defective of three kinds of fry kinds, authentication method of the present invention is more accurate, fast.
For realizing purpose of the present invention, technical scheme of the present invention is:
A kind of method of quick and precisely identifying snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)], the method comprising the steps of:
1) primer synthesis step: according to calendar year 2001 be the New molecular marker technology-SRAP of a kind of PCR-based of proposing of Li and doctor Quiros by California, USA university vegetable crop, synthetic primer;
2) extraction step of snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)] DNA: the pectoral fin of three kinds of fishes of clip, extract the DNA of tissue with phynol method, to 20ng/ μ l, Fang Ru – preserves below 20 ℃ the DNA concentration dilution;
3) SRAP-PCR reactions steps, wherein the renaturation temperature-switching method is adopted in the SRAP-PCR reaction; Specifically refer to: amplification program: 94 ℃ of denaturation 3min; 94 ℃ of denaturation 45s, 35 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 5 circulations; 94 ℃ of denaturation 45s, 50 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 35 circulations; 72 ℃ are extended 10min; 4 ℃ of preservations;
4) electrophoresis detection step: detect amplified production with 1.5% agarose, screening can be for the identification of the combination of primers of snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)], and verifies in the colony of other sampling;
5) identify snakehead, snakehead and hybridized snakehead fish with Gelpro32 software processes band, and according to the specific band that amplifies.The sample that can amplify 694,268 liang of bands is snakehead, and the sample that only amplifies 500 1 bands is snakehead, and can amplify simultaneously being of above three bands hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)].
In a preferred embodiment of the present invention, described step 1) the primer is that 5 Me make up with 6 Em in, totally 30 pairs of primers.
In a preferred embodiment of the present invention, described step 1) working concentration of primer is in: 20 μ M.
In a preferred embodiment of the present invention, described step 2) extraction of DNA comprises step in snakehead, snakehead and the hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)]:
1) sampling a small amount of pectoral fin originally shreds with eye scissors, puts into the 1.5ml centrifuge tube;
2) add lysate, lysate comprises Tris-HCl 25 μ l, SDS 50 μ l, EDTA 100 μ l, sterilized water 320 μ l, Proteinase K 6-10 μ l; Put into 55 ℃ of water-bath cracking 2-4 hour, become clarification and get final product;
3) add the saturated phenol that equates with the lysate volume, shake up 10min, during 12000rpm, 4 ℃ of centrifugal 20min;
4) extract supernatant liquor, replenish sterilized water to volume in the supernatant liquor that extracts to equate with the lysate volume, follow the saturated phenol that adding equates with the lysate volume, shake up 10min, during 12000rpm, 4 ℃ of centrifugal 20min;
5) extract supernatant liquor, replenishing sterilized water to volume in the supernatant liquor that extracts equates with the lysate volume, then add phenol, chloroform and the iso pentane alcohol mixture that equates with the lysate volume, wherein the ratio of phenol, chloroform and primary isoamyl alcohol is 25:24:1, shake up 10min, during 12000rpm, 4 ℃ of centrifugal 20min;
6) extract supernatant liquor, replenish sterilized water to volume in the supernatant liquor that extracts to equate with the lysate volume, follow chloroform that adding equates with the lysate volume and the mixture of primary isoamyl alcohol, the ratio 24:1 of chloroform and primary isoamyl alcohol wherein, shake up 10min, during 12000rpm, 4 ℃ of centrifugal 20min;
7) extract supernatant liquor, add the freezing raw spirit of double volume, have flocks to generate,, put in-20 ℃ and deposit 30min during without flocks as temporary, during 7000rpm, 4 ℃ of centrifugal 8min;
8) slowly outwell alcohol, be sure not to pour out DNA, wash once with 70% freezing alcohol, during 7000rpm, 4 ℃ of centrifugal 8min;
9) slowly outwell 70% freezing alcohol, after seasoning 1-2 hour, with the dissolving of 100 μ l sterilized waters ,-20 ℃ for subsequent use.
In a preferred embodiment of the present invention, described step 2) in, the volumetric molar concentration of the Tris-HCl aqueous solution is 1M, the pH value is 8.0.
In a preferred embodiment of the present invention, described step 2) in, the concentration of SDS is 10%.
In a preferred embodiment of the present invention, described step 2) in, the volumetric molar concentration of the EDTA aqueous solution is 0.5M.
The present invention compares with traditional Morphological Identification, a kind of method of quick and precisely identifying snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)] is provided, existing identify snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)] from morphology to overcome, exist subjectivity strong and can not effectively identify the defective of three kinds of fry kinds, authentication method of the present invention is more accurate, and is objective.
Description of drawings
The specific band figure of Fig. 1 for amplifying with Gelpro32 software processes band.Wherein A is the combination of primer Me3-Em5, and B is the combination of primer Me4-Em5; The 1-5 hybridized snakehead fish, 6-10 snakehead, 11-15 snakehead, M-D2000.
Embodiment
Below in conjunction with specific embodiment, further specify the present invention.
Embodiment
1. primer is synthetic: according to calendar year 2001 be the New molecular marker technology-SRAP of a kind of PCR-based of proposing of Li and doctor Quiros by California, USA university vegetable crop, the synthetic primer sequence numbering is subordinate list 1;
Subordinate list 1
2. the extraction step of snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)] DNA:
1) [(a small amount of pectoral fin of ♀) ╳ snakehead (♂) shreds with eye scissors and to put into the 1.5ml centrifuge tube snakehead to get snakehead, snakehead and hybridized snakehead fish;
2) add lysate, lysate comprises Tris-HCl 25 μ l, SDS 50 μ l, and EDTA 100 μ l, sterilized water 320 μ l, Proteinase K 5 μ l put into 55 ℃ of water-bath cracking 2-4 hour, become clarification and get final product;
3) add and the isopyknic saturated phenol of lysate (500 μ l), shake up 10min, during 12000rpm, 4 ℃ of centrifugal 20min;
4) extract supernatant liquor, supernatant liquor less than 500 μ l replenish with sterilized water, add the saturated phenol of equivalent (500 μ l), shake up 10min, during 12000rpm, and 4 ℃ of centrifugal 20min;
5) extract supernatant liquor, supernatant liquor less than 500 μ l replenish with sterilized water, add the mixture of equivalent (500 μ l) phenol, chloroform and primary isoamyl alcohol, wherein the ratio of phenol, chloroform and primary isoamyl alcohol is 25:24:1, shakes up 10min, during 12000rpm, 4 ℃ of centrifugal 20min;
6) extract supernatant liquor, supernatant liquor less than 500 μ l replenish with sterilized water, add the mixture of equivalent (500 μ l) chloroform and primary isoamyl alcohol, and wherein the ratio of chloroform and primary isoamyl alcohol is 24:1, shakes up 10min, during 12000rpm, and 4 ℃ of centrifugal 20min;
7) extract supernatant liquor, add the freezing raw spirit of double volume, have flocks generate (without flocks, put-20 ℃ as temporary, 30min), during 7000rpm, 4 ℃ of centrifugal 8min;
8) slowly outwell alcohol, be sure not to pour out DNA, wash once with 70% freezing alcohol, during 7000rpm, 4 ℃ of centrifugal 8min; Slowly outwell 70% freezing alcohol, seasoning 1-2 hour, with the dissolving of 100 μ l sterilized waters ,-20 ℃ for subsequent use.
3.SRAP-PCR the renaturation temperature-switching method is adopted in reaction: amplification program is 94 ℃ of denaturation 3min; 94 ℃ of denaturation 45s, 35 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 5 circulations; 94 ℃ of denaturation 45s, 50 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 35 circulations; 72 ℃ are extended 10min; 4 ℃ of preservations.
4. electrophoresis detection: detect amplified production with 1.5% agarose, screening can be for the identification of the combination of primers of snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)], and verifies in the colony of other sampling;
5. with Gelpro32 software processes band, the example that is combined as such as primer Me3-Em5 among the figure A amplifies obvious three bands among the 1-5, and size is respectively 594,500, and 268; Amplify an obvious band among the 6-10, size is 500; Amplifying obvious two bands among the 11-15 is 694,268;
6. identify snakehead, snakehead and hybridized snakehead fish according to the specific band that amplifies.The sample that can amplify 694,268 liang of bands is snakehead, and the sample that only amplifies 500 1 bands is snakehead, and can amplify simultaneously being of above three bands hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)];
7. same method, shown in the B figure, the hybridized snakehead fish stripe size is 375,271; Snakehead tape size is 271; The snakehead stripe size is 375; Find in the diagram, snakehead also has the specific band of 611 sizes except having 375 band;
8. use Me3-Em5, the combination primer of Me4-Em5 is identified other sampling colonies, finds to obtain same conclusion, and this illustrates that these two pairs of primers have versatility, can be good at identifying snakehead, snakehead and hybridized snakehead fish.
Claims (7)
1. a method of quick and precisely identifying snakehead, snakehead and hybridized snakehead fish is characterized in that, comprises step:
1) primer synthesis step: according to calendar year 2001 be the New molecular marker technology-SRAP of a kind of PCR-based of proposing of Li and doctor Quiros by California, USA university vegetable crop, synthetic primer;
2) extraction step of snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)] DNA: the pectoral fin of three kinds of fishes of clip, extract the DNA of tissue with phynol method, to 20ng/ μ l, Fang Ru – preserves below 20 ℃ the DNA concentration dilution;
3) SRAP-PCR reactions steps, wherein the renaturation temperature-switching method is adopted in the SRAP-PCR reaction; Specifically refer to: amplification program: 94 ℃ of denaturation 3min; 94 ℃ of denaturation 45s, 35 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 5 circulations; 94 ℃ of denaturation 45s, 50 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 35 circulations; 72 ℃ are extended 10min; 4 ℃ of preservations;
4) electrophoresis detection step: detect amplified production with 1.5% agarose, screening can be for the identification of the combination of primers of snakehead, snakehead and hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)], and verifies in the colony of other sampling;
5) identify snakehead, snakehead and hybridized snakehead fish with Gelpro32 software processes band, and according to the specific band that amplifies.The sample that can amplify 694,268 liang of bands is snakehead, and the sample that only amplifies 500 1 bands is snakehead, and can amplify simultaneously being of above three bands hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)].
2. the method for claim 1 is characterized in that, described step 1) in the primer to be 5 Me make up with 6 Em, totally 30 pairs of primers.
3. the method for claim 1 is characterized in that, described step 1) in the working concentration of primer be: 20 μ M.
4. the method for claim 1 is characterized in that, described step 2) in, the extraction of DNA comprises step in snakehead, snakehead and the hybridized snakehead fish [snakehead (♀) ╳ snakehead (♂)]:
1) sampling a small amount of pectoral fin originally shreds with eye scissors, puts into the 1.5ml centrifuge tube;
2) add lysate, lysate comprises Tris-HCl 25 μ l, SDS 50 μ l, EDTA 100 μ l, sterilized water 320 μ l, Proteinase K 6-10 μ l; Put into 55 ℃ of water-bath cracking 2-4 hour, become clarification and get final product;
3) add the saturated phenol that equates with the lysate volume, shake up 10min, during 12000rpm, 4 ℃ of centrifugal 20min;
4) extract supernatant liquor, replenish sterilized water to volume in the supernatant liquor that extracts to equate with the lysate volume, follow the saturated phenol that adding equates with the lysate volume, shake up 10min, during 12000rpm, 4 ℃ of centrifugal 20min;
5) extract supernatant liquor, replenishing sterilized water to volume in the supernatant liquor that extracts equates with the lysate volume, then add phenol, chloroform and the iso pentane alcohol mixture that equates with the lysate volume, wherein the ratio of phenol, chloroform and primary isoamyl alcohol is 25:24:1, shake up 10min, during 12000rpm, 4 ℃ of centrifugal 20min;
6) extract supernatant liquor, replenish sterilized water to volume in the supernatant liquor that extracts to equate with the lysate volume, follow chloroform that adding equates with the lysate volume and the mixture of primary isoamyl alcohol, wherein the ratio of chloroform and primary isoamyl alcohol is 24:1, shake up 10min, during 12000rpm, 4 ℃ of centrifugal 20min;
7) extract supernatant liquor, add the freezing raw spirit of double volume, have flocks to generate,, put in-20 ℃ and deposit 30min during without flocks as temporary, during 7000rpm, 4 ℃ of centrifugal 8min;
8) slowly outwell alcohol, be sure not to pour out DNA, wash once with 70% freezing alcohol, during 7000rpm, 4 ℃ of centrifugal 8min;
9) slowly outwell 70% freezing alcohol, after seasoning 1-2 hour, with the dissolving of 100 μ l sterilized waters ,-20 ℃ for subsequent use.
5. method as claimed in claim 4 is characterized in that, in a preferred embodiment of the present invention, and described step 2) in, the volumetric molar concentration of the Tris-HCl aqueous solution is 1M, the pH value is 8.0.
6. method as claimed in claim 4 is characterized in that, described step 2) in, the concentration of SDS is 10%.
7. method as claimed in claim 4 is characterized in that, described step 2) in, the volumetric molar concentration of the EDTA aqueous solution is 0.5M.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103290131A (en) * | 2013-06-18 | 2013-09-11 | 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 |
| CN106119378A (en) * | 2016-07-06 | 2016-11-16 | 中国水产科学研究院珠江水产研究所 | SNP site and detection method thereof for snakehead sex identification |
| CN106480226A (en) * | 2016-12-29 | 2017-03-08 | 武汉达邦生物科技有限公司 | Ophicephalus arguss male molecular labeling primer and application |
| CN107217099A (en) * | 2017-06-28 | 2017-09-29 | 中国水产科学研究院珠江水产研究所 | A kind of SNP marker identified available for snakehead genetic sex and supermale fish and its application |
| CN107641656A (en) * | 2017-10-25 | 2018-01-30 | 安徽科技学院 | A kind of snakehead single nucleotide polymorphism and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134604A (en) * | 2010-12-30 | 2011-07-27 | 厦门大学 | Marker and method for identifying single nucleotide polymorphism of bostrichthys sinensis group |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134604A (en) * | 2010-12-30 | 2011-07-27 | 厦门大学 | Marker and method for identifying single nucleotide polymorphism of bostrichthys sinensis group |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103290131A (en) * | 2013-06-18 | 2013-09-11 | 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 |
| CN106119378A (en) * | 2016-07-06 | 2016-11-16 | 中国水产科学研究院珠江水产研究所 | SNP site and detection method thereof for snakehead sex identification |
| CN106119378B (en) * | 2016-07-06 | 2019-01-01 | 中国水产科学研究院珠江水产研究所 | SNP site and its detection method for snakehead sex identification |
| CN106480226A (en) * | 2016-12-29 | 2017-03-08 | 武汉达邦生物科技有限公司 | Ophicephalus arguss male molecular labeling primer and application |
| CN107217099A (en) * | 2017-06-28 | 2017-09-29 | 中国水产科学研究院珠江水产研究所 | A kind of SNP marker identified available for snakehead genetic sex and supermale fish and its application |
| 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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