CN108277287B - Molecular marker, kit and distinguishing method for distinguishing black-dragon-river triangular bream from megalobrama fish - Google Patents
Molecular marker, kit and distinguishing method for distinguishing black-dragon-river triangular bream from megalobrama fish Download PDFInfo
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Abstract
A molecular marker, a kit and a distinguishing method for distinguishing triangular bream in Heilongjiang and Megalobrama fish relate to a molecular marker for distinguishing Megalobrama fish and application thereof. The primer pair of the molecular marker is HLJFLf1 and HLJFLr 1. The kit comprises a molecular marker primer pair HLJFLf1 and HLJFLr 1. The distinguishing method comprises the following steps: firstly, extracting DNA of a fin ray; secondly, performing PCR amplification by using HLJFLf1 and HLJFLr1 as primers; thirdly, carrying out enzyme digestion on the PCR product by using restriction enzyme TaqI; and fourthly, performing electrophoresis detection, wherein the corresponding individual with double bands of 200bp and 180bp is the triangular bream in Heilongjiang according to the electrophoresis result. The method can quickly, efficiently and stably distinguish the representative geographical population of the black-dragon-river triangular bream and the south-bream fishes under the condition of no hybridization.
Description
Technical Field
The invention relates to a molecular marker for distinguishing megalobrama fishes and application thereof.
Background
The existing megalobrama fishes in China comprise 4 types of megalobrama amblycephala at the upstream of the Yangtze river, megalobrama amblycephala (Megalobrama amblycephala) in large lakes at the middle and lower reaches of the Yangtze river, Guangdong amblycephala in various water systems of the Zhujiang river and the Hainan and megalobrama amblycephala in various water systems which are widely distributed. Currently, there exist 3 representative geographical populations of triangular breams, which are qiantangjiang triangular breams, changjiang triangular breams, and black longjiang triangular breams, respectively. The black-dragon-river triangular bream is a French bream in a famous fish 'three flowers and five rollers' specially owned by the black-dragon-river basin, and is also the only naturally distributed bream fish in the north of the yellow river. As the local medium-large economic famous fish, the fish has strong stress resistance, high fat and delicious taste and great development potential; on the other hand, long-term geographical isolation and unique habitat may make it harbor key genes or markers required for future megalobrama fish breeding. Unfortunately, long term harsher capture and pollution has obliterated populations inhabiting the Songhua, Nen, mirror lake and Xingkai lakes for many years, and a few individuals are now visible each year in the far river segment, which is the black dragon river of the middle and Russian kings. Compared with the general 3-year accessibility and maturity of the megalobrama amblycephala in south China, the megalobrama amblycephala in black Longjiang can propagate initially in 5 years, and the megalobrama amblycephala can be completely killed by high-intensity fishing and slow population supplement. In conclusion, germplasm resource protection of the triangular breams in Heilongjiang is still slow.
The megalobrama amblycephala species have very similar forms, especially in the early growth stage before sexual maturity. From the existing morphological research, the visual and obvious difference is not available to distinguish the black-dragon-river triangular bream from the megalobrama fish. When the endangered triangular bream resources of Heilongjiang are artificially propagated and rescued, a rapid and accurate identification method needs to be established for ensuring the germplasm purity of the harvested parent fish, which has important significance for protecting and developing the precious local germplasm resources.
Disclosure of Invention
The invention aims to provide a molecular marker, a kit and a distinguishing method for distinguishing triangular bream in Heilongjiang from Megalobrama fish.
The invention discloses a primer pair for distinguishing molecular markers of black-dragon-river triangular breams and southern-bream fishes, which is HLJFLf1 and HLJFLr1, and has the specific sequences as follows:
HLJFLf1：5′-GCATCTGGCTTCAATCTCA-3′
HLJFLr1：5′-GATTTGCTGAGCGTAGGG-3′。
the kit for distinguishing the black-dragon-river triangular bream from the megalobrama fish comprises a molecular marker primer pair HLJFLf1 and HLJFLr 1.
Further, the kit also comprisesIncluding 10 XTaq Buffer, MgCl2dNTPs, Taq DNA polymerase, RE Buffer solution 10 xbuffer, bovine serum albumin BSA and TaqI endonuclease.
The invention also provides application of the molecular marker in distinguishing and identifying the triangular breams in Heilongjiang and the megalobrama fishes. The specific method comprises the following steps:
firstly, extracting genome DNA of fin rays of the bream fishes to be detected for later use;
secondly, performing PCR amplification by taking the extracted genomic DNA as a template and HLJFLf1 and HLJFLr1 as primers to obtain a PCR product;
thirdly, carrying out enzyme digestion on the PCR product by using restriction enzyme TaqI;
and fourthly, carrying out electrophoresis detection on the enzyme digestion product by using 1.5% agarose gel, wherein the corresponding individual with double bands of 200bp and 180bp is the black-dragon-river triangular bream, and the individual non-black-dragon-river triangular bream with double bands of 200bp and 180bp is not shown in the electrophoresis result, thus finishing the distinguishing identification.
The specific sequences of the HLJFLf1 and the HLJFLr1 in the step two are as follows:
HLJFLf1：5′-GCATCTGGCTTCAATCTCA-3′
HLJFLr1：5′-GATTTGCTGAGCGTAGGG-3′。
the invention has the beneficial effects that:
the method can quickly, efficiently and stably distinguish the representative geographical population of the black-dragon-river triangular bream and the south-bream fishes (the Qiantangjiang triangular bream, the Changjiang triangular bream, the Liangzi lake megalobrama amblycephala, the Ihe megalobrama amblycephala, the Longxi river megalobrama amblycephala and the West river Guangdong megalobrama amblycephala) under the condition of no hybridization.
By adopting the molecular marker and the method provided by the invention, the fishes of the triangular breams in Heilongjiang and the Megalobrama in south China can be directly distinguished and identified. The method has the advantages of high accuracy, simple operation process, short time consumption and 100% accuracy.
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FIG. 1 is a graph showing the results of electrophoresis in example 1.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
The first embodiment is as follows: the primer pair of the molecular marker for distinguishing the black-dragon-river triangular bream from the megalobrama fish in the embodiment is HLJFLf1 and HLJFLr1, and the specific sequence is as follows:
HLJFLf1：5′-GCATCTGGCTTCAATCTCA-3′
HLJFLr1：5′-GATTTGCTGAGCGTAGGG-3′。
the second embodiment is as follows: the molecular marker of the embodiment is applied to distinguishing the triangular breams in the Heilongjiang river and the megalobrama fishes.
The third concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: the specific method for distinguishing the fishes of the genuses of the triangular breams of the black dragon river and the megalobrama by the molecular marker comprises the following steps:
firstly, extracting genome DNA of fin rays of the bream fishes to be detected for later use;
secondly, performing PCR amplification by taking the extracted genomic DNA as a template and HLJFLf1 and HLJFLr1 as primers to obtain a PCR product;
thirdly, carrying out enzyme digestion on the PCR product by using restriction enzyme TaqI;
carrying out electrophoresis detection on the enzyme digestion product by using 1.5% agarose gel, wherein the corresponding individual with double bands of 200bp and 180bp is the triangular bream in black dragon river as the electrophoresis result, and the individual non-triangular bream in black dragon river with double bands of 200bp and 180bp is not shown, namely, the distinguishing identification is completed;
wherein HLJFLf1 nucleotide sequence is: 5'-GCATCTGGCTTCAATCTCA-3'
The HLJFLr1 nucleotide sequence is as follows: 5'-GATTTGCTGAGCGTAGGG-3' are provided. The rest is the same as the first embodiment.
The fourth concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: the PCR amplification system in the second step is as follows:
PCR amplification conditions: denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles, extension at 72 ℃ for 5min, and heat preservation at 4 ℃. The rest is the same as the first embodiment.
The fifth concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: the enzyme digestion reaction system in the third step is as follows:
enzyme cutting conditions are as follows: enzyme digestion is carried out for 2h in water bath at 65 ℃. The rest is the same as the first embodiment.
The following examples are given to illustrate the present invention, and the following examples are carried out on the premise of the technical solution of the present invention, and give detailed embodiments and specific procedures, but the scope of the present invention is not limited to the following examples.
Example 1:
the experimental samples selected in this example were representative geopopulations of black-and south-bream fish (qianshijiang triangular bream, changjiang triangular bream, Liangzi lake megalobrama amblycephala, Ihe megalobrama amblycephala, Longxi river megalobrama amblycephala, and West river Guangdong megalobrama amblycephala). The accuracy of the molecular marker of the invention for distinguishing the fishes of the triangular breams of black dragon river and the fishes of the megalobrama of south breams is verified by using the following method.
Firstly, extracting genome DNA of fin rays of the bream fishes to be detected for later use; the specific method comprises the following steps:
(1) taking fin ray tissue (about 60mg per tail) of a sample to be identified, shearing, placing in a 1.5mL EP tube, adding 600 mu L of tissue extract and 10 mu L of proteinase K, and digesting overnight on a shaker at 56 ℃ and 150 rpm;
(2) adding 600 μ L Tris saturated phenol, inverting for 10 min, centrifuging at 12000rpm for 10 min, collecting the upper water phase, adding equal volume of phenol-chloroform-isoamyl alcohol (24:23:1), and mixing for 10 min;
(4) centrifuging at 12000rpm for 10 min, collecting upper water phase, adding equal volume of chloroform-isoamyl alcohol (23:1), and mixing for 10 min;
(5) centrifuging at 12000rpm for 10 min, collecting upper water phase, adding 2 times volume of anhydrous glacial ethanol (-20 deg.C), and horizontally shaking until white floccule appears;
(6) centrifuging at 10000rpm for 10 minutes until white DNA precipitate is visible at the bottom of the tube, picking the precipitate with toothpick into 1.5mL EP tube filled with 500 μ L of 70% glacial ethanol in volume concentration, centrifuging at 8000rpm for 5 minutes, pouring off the ethanol, and naturally drying;
(7) adding appropriate amount of TE buffer solution, placing in 55 deg.C water bath to dissolve DNA for 4 hr, and storing at-20 deg.C for use.
Secondly, performing PCR amplification by taking the extracted genomic DNA as a template and HLJFLf1 and HLJFLr1 as primers to obtain a PCR product;
HLJFLf1：5′-GCATCTGGCTTCAATCTCA-3′
HLJFLr1：5′-GATTTGCTGAGCGTAGGG-3′。
thirdly, carrying out enzyme digestion on the PCR product by using restriction enzyme TaqI;
and fourthly, carrying out electrophoresis detection on the enzyme digestion product by using 1.5 percent agarose gel, and selecting individuals with double bands of 200bp and 180bp respectively as electrophoresis results to finish distinguishing identification.
The PCR amplification system in the second step is as follows:
PCR amplification conditions: denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles, extension at 72 ℃ for 5min, and heat preservation at 4 ℃.
The enzyme digestion reaction system in the third step is as follows:
enzyme cutting conditions are as follows: enzyme digestion is carried out for 2h in water bath at 65 ℃.
The electrophoresis results are shown in FIG. 1. The number of the sequence 1-10 is 200bp and 180bp double bands, so that the sequence is a Heilongjiang triangular bream; the number of the megalobrama amblycephala is 11 to 20; the number 21-30 is that of the roof beam lake megalobrama amblycephala; the number 31-40 is that of the megalobrama amblycephala in the Ihe; the number of the triangular bream is 41 to 50; the number of the triangular bream is 51 to 60; the number is 61-70 West river Guangdong bream. As can be seen, only 200bp and 180bp double bands appear in the gel electrophoresis lane of the individual Megalobrama amblycephala in Heilongjiang, which proves that the accuracy of the embodiment reaches 100%.
Claims (5)
1. A kit for distinguishing triangular bream in Heilongjiang and Megalobrama fish is characterized by comprising a molecular marker primer pair HLJFLf1 and HLJFLr1, wherein the specific sequence is as follows:
HLJFLf1：5'- GCATCTGGCTTCAATCTCA- 3'
HLJFLr1：5'-GATTTGCTGAGCGTAGGG- 3'；
the kit also comprises 10 piecesTaq Buffer、MgCl2、dNTPs、TaqDNA polymerase, RE Buffer 10 x Buffer, bovine serum albumin BSA and TaqI endonuclease.
2. The use of the kit of claim 1 for distinguishing triangular breams in the Heilongjiang river from Megalobrama amblycephala in species.
3. The use of claim 2, wherein the kit of claim 1 is used for distinguishing the fishes of the genu of the triangular bream of black dragon river and the fishes of the genu of the megalobrama, and the specific method is as follows:
firstly, extracting genome DNA of fin rays of the bream fishes to be detected for later use;
secondly, performing PCR amplification by taking the extracted genomic DNA as a template and HLJFLf1 and HLJFLr1 as primers to obtain a PCR product;
thirdly, carrying out enzyme digestion on the PCR product by using restriction enzyme TaqI;
carrying out electrophoresis detection on the enzyme digestion product by using 1.5% agarose gel, wherein the corresponding individual with the electrophoresis result of 200bp and 180bp double bands is the black-dragon-river triangular bream, and otherwise, the corresponding individual is not the black-dragon-river triangular bream, namely, completing the distinguishing identification;
wherein HLJFLf1 nucleotide sequence is: 5'-GCATCTGGCTTCAATCTCA-3'
The HLJFLr1 nucleotide sequence is as follows: 5'-GATTTGCTGAGCGTAGGG-3' are provided.
4. The use according to claim 3, wherein the reaction system for PCR amplification in step two is as follows:
PCR amplification conditions: denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extending for 5min at 72 ℃, and preserving heat at 4 ℃.
5. The use according to claim 3, characterized in that the reaction system of step three enzymatic cleavage is as follows:
enzyme cutting conditions are as follows: enzyme digestion is carried out for 2h in water bath at 65 ℃.
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| CN111850138B (en) * | 2020-07-31 | 2021-11-19 | 中国水产科学研究院黑龙江水产研究所 | Molecular marker, kit and method for distinguishing triangular bream and megalobrama amblycephala in Heilongjiang and hybrid thereof |
| CN114717302B (en) * | 2022-04-08 | 2022-10-04 | 中国水产科学研究院黑龙江水产研究所 | Molecular marker, kit and method for distinguishing genders of triangular breams |
| CN118147318A (en) * | 2024-03-29 | 2024-06-07 | 中国水产科学研究院黑龙江水产研究所 | Molecular marker, kit and distinguishing method for distinguishing Heilongjiang triangular bream and Qianyangjiang triangular bream and hybrid thereof |
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Application publication date: 20180713 Assignee: SHENYANG HUATAI FISHERIES CO.,LTD. Assignor: Heilongjiang River Fisheries Research Institute of fisheries, Chinese Academy of Fishery Sciences Contract record no.: X2022230000109 Denomination of invention: A Molecular Marker, Kit and Differentiation Method for Distinguishing Triglobrama and Megalobrama Granted publication date: 20210622 License type: Common License Record date: 20221013 |