CN112646870B - Lepidoptera sativa genetic sex identification primer and identification method - Google Patents
Lepidoptera sativa genetic sex identification primer and identification method Download PDFInfo
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- CN112646870B CN112646870B CN202110072267.1A CN202110072267A CN112646870B CN 112646870 B CN112646870 B CN 112646870B CN 202110072267 A CN202110072267 A CN 202110072267A CN 112646870 B CN112646870 B CN 112646870B
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Abstract
A primer and a method for identifying the genetic sex of brachymystax lenok relate to a primer and a method for identifying the genetic sex of fish. The invention provides a method for rapidly and accurately identifying the genetic sex of brachymystax lenok by using a molecular marker technology. The upstream primer sequence of the primer pair for identifying the genetic sex of the brachymystax lenok is 5'-GTGCCTGTCTGAGGTGTGAG-3'; the primer sequence downstream of the primer pair was 5'-AGCTGTGTCAGGTTGTCGTG-3'. The identification method comprises the following steps: 1. extracting DNA of an individual to be identified; 2. PCR amplification is carried out by using the primer; 3. gel electrophoresis, only 1 band of 235bp is female fish, and 207 band is male fish. The method can rapidly and accurately identify the genetic sex of the brachymystax lenok, save manpower and material resources, achieve proper female-male ratio in the parent cultivation and cultivation process, and save cultivation cost.
Description
Technical Field
The invention relates to a primer and a method for identifying the genetic sex of fish.
Background
Capelin [ Brachymystax lenok (Pallas) ] belonging to Salmoniformes (Salmoniformes), salmonidae (Salmonidae), brachypodisax is a rare, cold water fish in China, and is listed in red books of endangered animals in China in 1998, and endangered grades are endangered. The first maturation of the brachymystax lenok needs 3-4 years, the sex of the brachymystax lenok cannot be judged through the appearance form before sexual maturation, after sexual maturation, adult fish are dark in body color in the breeding season, fin bars at the front part of dorsal fin are blackened, and dark red spots appear on the body side. In different ages and different habitats, the body color of the fish is greatly changed, and the body color of the old fish is deeper than that of the young fish, so that the female and male can be hardly identified through marital color.
Disclosure of Invention
The invention provides a method for rapidly and accurately identifying the genetic sex of brachymystax lenok by using a molecular marker technology.
The upstream primer sequence of the primer pair for identifying the genetic sex of the brachymystax lenok is 5'-GTGCCTGTCTGAGGTGTGAG-3'; the primer sequence downstream of the primer pair was 5'-AGCTGTGTCAGGTTGTCGTG-3'.
The method for identifying the genetic sex of the brachymystax lenok comprises the following steps:
1. extracting DNA of an individual to be identified;
2. performing PCR amplification by using a primer, wherein the upstream primer sequence of the primer pair is 5'-GTGCCTGTCTGAGGTGTGAG-3'; the primer sequence downstream of the primer pair is 5'-AGCTGTGTCAGGTTGTCGTG-3';
3. gel electrophoresis, only 1 band of 235bp is female fish, and 207 band is male fish.
The method can rapidly and accurately identify the genetic sex of the brachymystax lenok, save manpower and material resources, achieve proper female-male ratio in the parent cultivation and cultivation process, and save cultivation cost.
Drawings
FIG. 1 is a gel electrophoresis chart of female and male fish of capelin identified by the method of the present invention, wherein lanes 1-3 are female fish of capelin, lanes 4-6 are male fish of capelin, and lane M is Marker.
Detailed Description
The technical scheme of the invention is not limited to the specific embodiments listed below, and also includes any combination of the specific embodiments.
The first embodiment is as follows: the primer sequence upstream of the primer pair for genetic sex determination of brachymystax lenok in the embodiment is 5'-GTGCCTGTCTGAGGTGTGAG-3'; the primer sequence downstream of the primer pair was 5'-AGCTGTGTCAGGTTGTCGTG-3'.
The second embodiment is as follows: the method for identifying genetic sex of brachymystax lenok according to the present embodiment:
1. extracting DNA of an individual to be identified;
2. performing PCR amplification by using a primer, wherein the upstream primer sequence of the primer pair is 5'-GTGCCTGTCTGAGGTGTGAG-3'; the primer sequence downstream of the primer pair is 5'-AGCTGTGTCAGGTTGTCGTG-3';
3. gel electrophoresis showed that only 1 band of 235bp was female fish and 207bp band was male fish (shown in FIG. 1).
In this embodiment, the male fish of brachymystax lenok amplified a band of about 207bp and a band of about 235 bp.
The method is convenient to operate, the sizes of the amplification products of the female and male fishes are only compared through agarose gel electrophoresis, and sequencing is not needed. The required equipment is simple, and the method has important application value in production.
The invention explores the genetic sex of the brachymystax lenok from the molecular level, is not limited by age, can be used for selecting early sex of the brachymystax lenok, and solves the problem that young fish cannot morphologically identify female and male.
And a third specific embodiment: the present embodiment differs from the second embodiment in that: step two, PCR amplification reaction system was 20. Mu.l, including 2 XPCR mix 10. Mu.l, concentration 30 ng/. Mu.l of individual DNA to be identified 2. Mu.l, concentration 10. Mu.M of each of the upstream and downstream primers was 1. Mu.l, and the remainder was made up with ultrapure water. Other steps and parameters are the same as those of the second embodiment.
The specific embodiment IV is as follows: the difference between this embodiment and the second or third embodiment is that: the second PCR procedure was 95℃pre-denatured for 3min,95℃denatured for 30sec, 60℃annealed for 30sec, 72℃extended for 30sec,33 cycles, 72℃extended for 5min. Other steps and parameters are the same as those of the second or third embodiment.
Under the condition of the embodiment, the target sequence is thoroughly denatured, the activity of Taq enzyme is not affected, and the primer extension is complete, so that the effective amplification amount can be achieved.
Example 1
Population acquisition of known gender: identifying female and male fish when spawning at a cold water fish experimental station of Bohai sea of China aquatic science institute, collecting 50 fish samples of female and male fish, cutting fin sample, attaching to filter paper, and drying in shade.
A method for identifying genetic sex of brachymystax lenok:
1. extracting DNA of an individual to be identified: extracting DNA by using an animal tissue genome DNA kit, measuring the concentration of the DNA by using Qubit3, and diluting to 30 ng/. Mu.l for later use;
2. performing PCR amplification by using a primer, wherein the upstream primer sequence of the primer pair is 5'-GTGCCTGTCTGAGGTGTGAG-3'; the primer sequence downstream of the primer pair is 5'-AGCTGTGTCAGGTTGTCGTG-3'; the PCR amplification reaction system is 20 μl, comprising 2×PCR mix 10 μl, concentration of 30 ng/. Mu.l of individual DNA to be identified 2 μl, concentration of 1 μl of each of the upstream and downstream primers of 10 μM, and the remainder is made up with ultrapure water; the PCR reaction procedure was 95℃pre-denatured for 3min,95℃denatured for 30sec, 60℃annealed for 30sec, 72℃extended for 30sec,33 cycles, 72℃extended for 5min;
3. gel electrophoresis: the PCR product was obtained and 6. Mu.l was analyzed by 2.0% agarose gel electrophoresis.
Identification result:
all 50 female fishes are amplified into 1 band with the size of 235bp; the 207bp bands are amplified by 50 males, and the 235bp bands can be amplified by partial males; the authentication result is consistent with the sampling.
Claims (5)
1. A genetic sex identification primer for brachymystax lenok, characterized in that the upstream primer sequence of the identification primer pair is 5'-GTGCCTGTCTGAGGTGTGAG-3'; the primer sequence downstream of the primer pair was 5'-AGCTGTGTCAGGTTGTCGTG-3'.
2. A method for identifying genetic sex of brachymystax lenok using the primer according to claim 1, characterized in that the method is carried out according to the following steps:
1. extracting DNA of an individual to be identified;
2. performing PCR amplification using the primer of claim 1;
3. gel electrophoresis, only 1 band of 235bp is female fish, and 207 band is male fish.
3. The method for identifying genetic sex of brachymystax lenok according to claim 2, characterized in that the PCR amplification reaction system of step two is 20. Mu.l, comprising 2. Mu.l of 2 XPCR mix 10. Mu.l, 2. Mu.l of DNA of the individual to be identified at a concentration of 30 ng/. Mu.l, 1. Mu.l of each of the upstream and downstream primers at a concentration of 10. Mu.M, the remainder being made up with ultrapure water.
4. A method for identifying the genetic sex of brachymystax lenok according to claim 2 or 3, characterized in that the step two PCR reaction procedure is 95 ℃ pre-denaturation for 3min,95 ℃ denaturation for 30sec, 60 ℃ annealing for 30sec, 72 ℃ extension for 30sec,33 cycles, 72 ℃ extension for 5min.
5. The method for identifying genetic sex of brachymystax lenok according to claim 2, wherein the male fish of brachymystax lenok is amplified out of a band of 207bp and a band of 235bp is partially amplified.
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| CN2020112767310 | 2020-11-16 | ||
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Citations (1)
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| CN110042168A (en) * | 2019-05-28 | 2019-07-23 | 中国水产科学研究院黑龙江水产研究所 | For distinguishing primer pair, kit and the method for fine-scaled graphite and Hucho taimen |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042168A (en) * | 2019-05-28 | 2019-07-23 | 中国水产科学研究院黑龙江水产研究所 | For distinguishing primer pair, kit and the method for fine-scaled graphite and Hucho taimen |
Non-Patent Citations (1)
| Title |
|---|
| 野生细鳞鱼的生物学特性及繁殖力;白庆利等;《水产学杂志》;第69-73页 * |
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