阅读说明：本技术 一种细鳞鲑遗传性别鉴定引物以及鉴定方法 (Scleroderma lenok genetic sex identification primer and identification method ) 是由 佟广香 张庆渔 张永泉 匡友谊 尹家胜 于 2021-01-20 设计创作，主要内容包括：一种细鳞鲑遗传性别鉴定引物以及鉴定方法,它涉及一种鱼类的遗传性别鉴定引物以及鉴定方法。本发明提供了一种利用分子标记技术快速准确鉴定细鳞鲑的遗传性别的方法。本发明细鳞鲑遗传性别鉴定引物对上游引物序列为5’-GTGCCTGTCTGAGGTGTGAG-3’；引物对下游引物序列为5’-AGCTGTGTCAGGTTGTCGTG-3’。鉴定方法：一、待鉴定个体DNA提取；二、用引物进行PCR扩增；三、凝胶电泳,仅有1条235bp条带的为雌鱼,有207bp条带的为雄鱼。利用本发明方法可以快速、准确鉴定细鳞鲑的遗传性别,节省人力和物力,在亲本培育和养殖过程中做到合适的雌、雄比例,节约养殖成本。(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 sequence of the upstream primer of the brachymystax lenok genetic sex identification primer pair is 5'-GTGCCTGTCTGAGGTGTGAG-3'; the primer sequence downstream of the primer pair is 5'-AGCTGTGTCAGGTTGTCGTG-3'. The identification method comprises the following steps: firstly, extracting DNA of an individual to be identified; secondly, PCR amplification is carried out by using a primer; and thirdly, performing gel electrophoresis, wherein only 1 band with 235bp is female fish, and a band with 207bp is male fish. The method can quickly and accurately identify the genetic sex of the brachymystax lenok, saves manpower and material resources, achieves proper female-male ratio in the parent cultivation and breeding process, and saves the breeding cost.)

1. The brachymystax lenok genetic sex identifying primer is characterized in that the sequence of an upstream primer of the identifying primer pair is 5'-GTGCCTGTCTGAGGTGTGAG-3'; the primer sequence downstream of the primer pair is 5'-AGCTGTGTCAGGTTGTCGTG-3'.

2. A method for identifying the genetic sex of brachymystax lenok using the primer of claim 1, characterized in that the method is carried out by the steps of:

firstly, extracting DNA of an individual to be identified;

secondly, carrying out PCR amplification by using the primer of claim 1;

and thirdly, performing gel electrophoresis, wherein only 1 band with 235bp is female fish, and a band with 207bp is male fish.

3. The method for genetically sexing brachymystax lenok according to claim 2, wherein the second PCR amplification reaction system is 20 μ l, and comprises 2 XPCR mix 10 μ l, 2 μ l of DNA of the individual to be identified with a concentration of 30ng/μ l, 1 μ l of each of the upstream and downstream primers with a concentration of 10 μ M, and the balance of ultrapure water.

4. The method for genetic sex identification of brachymystax lenok according to claim 2 or 3, wherein the step two PCR reaction program is pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 30sec, extension at 72 ℃ for 30sec, 33 cycles, and extension at 72 ℃ for 5 min.

5. The method for genetically determining the genetic sex of brachymystax lenok according to claim 2, wherein the androctopus lenok partially amplifies a 235bp band in addition to a 207bp band.

Technical Field

The invention relates to a primer and a method for identifying genetic sex of fish.

Background

Brachymystax lenok (Pallas) belongs to the Salmoniformes and Salmonidae, and Brachymystax is a rare and rare cold water fish in China, and is listed in Chinese endangered animal red book in 1998 with endangered grade being endangered. The first sexual maturity of the brachymystax lenok needs 3-4 years, the sex of the brachymystax lenok cannot be judged through appearance morphology before sexual maturity, after sexual maturity, the body color of the adult fish is dark in the reproduction season, fin lines in the front of dorsal fins are blackened, and hidden red spots appear on the body side. In different age sizes and different habitat environments, the body color of the fish is greatly changed, the color of the old fish is darker than that of the young fish, and the male and the female can not be easily identified through marriage 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 sequence of the upstream primer of the brachymystax lenok genetic sex identification primer pair is 5'-GTGCCTGTCTGAGGTGTGAG-3'; the primer sequence downstream of the primer pair is 5'-AGCTGTGTCAGGTTGTCGTG-3'.

The method for identifying the genetic sex of the brachymystax lenok comprises the following steps:

firstly, extracting DNA of an individual to be identified;

secondly, PCR amplification is carried out by using primers, and the sequence of the primer on the upstream of the primer pair is 5'-GTGCCTGTCTGAGGTGTGAG-3'; the sequence of the downstream primer of the primer pair is 5'-AGCTGTGTCAGGTTGTCGTG-3';

and thirdly, performing gel electrophoresis, wherein only 1 band with 235bp is female fish, and a band with 207bp is male fish.

The method can quickly and accurately identify the genetic sex of the brachymystax lenok, saves manpower and material resources, achieves proper female-male ratio in the parent cultivation and breeding process, and saves the breeding cost.

Drawings

FIG. 1 is a gel electrophoresis chart of female and male brachymystax lenok identified by the method of the present invention, wherein lanes 1-3 are female brachymystax lenok, lanes 4-6 are male brachymystax lenok, and lane M is Marker.

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 upstream primer sequence of the brachymystax lenok genetic sex identification primer pair is 5'-GTGCCTGTCTGAGGTGTGAG-3'; the primer sequence downstream of the primer pair is 5'-AGCTGTGTCAGGTTGTCGTG-3'.

The second embodiment is as follows: the method for identifying the genetic sex of the brachymystax lenok comprises the following steps:

firstly, extracting DNA of an individual to be identified;

secondly, PCR amplification is carried out by using primers, and the sequence of the primer on the upstream of the primer pair is 5'-GTGCCTGTCTGAGGTGTGAG-3'; the sequence of the downstream primer of the primer pair is 5'-AGCTGTGTCAGGTTGTCGTG-3';

and thirdly, performing gel electrophoresis, wherein only 1 235bp band is female fish, and a 207bp band is male fish (shown in figure 1).

In this embodiment, the brachymystax lenok milt partially amplified a 235bp band in addition to a 207bp band.

The method is convenient to operate, and the sizes of the female and male fish amplification products are compared by agarose gel electrophoresis for identification without sequencing. The required equipment is simple, and the method has important application value in the production aspect.

The invention explores the genetic sex of the brachymystax lenok from the molecular level, is not limited by the age, can be used for selecting the early sex of the brachymystax lenok, and solves the problem that the young fish can not identify the female and the male morphologically.

The third concrete implementation mode: the present embodiment is different from the second embodiment in that: the PCR amplification reaction system of the second step is 20 mul, comprising 2 XPCR mix 10 mul, 2 mul of individual DNA to be identified with the concentration of 30 ng/mul, 1 mul of each upstream primer and downstream primer with the concentration of 10 mul, and the rest is complemented with ultrapure water. Other steps and parameters are the same as those in the second embodiment.

The fourth concrete implementation mode: the present embodiment differs from the second or third embodiment in that: the PCR reaction program of the second step is pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 30sec, extension at 72 ℃ for 30sec, 33 cycles, and extension at 72 ℃ for 5 min. Other steps and parameters are the same as those in the second or third embodiment.

Under the condition of the embodiment, the target sequence is thoroughly denatured, the activity of Taq enzyme is not influenced, the primer is completely extended, and effective amplification amount can be achieved.

Example 1

Population acquisition of known gender: identifying male and female fish when the Bohai sea cold water fish experimental station of the institute of aquatic products science, Heilongjiang aquatic products, China lays eggs, collecting 50 tails of male and female fish samples respectively, shearing fin strip samples, and pasting the fin strip samples on filter paper for drying in the shade.

The method for identifying the genetic sex of the brachymystax lenok comprises the following steps:

firstly, extracting DNA of an individual to be identified: extracting DNA by using an animal tissue genome DNA kit, determining the DNA concentration by using the Qubit3, and diluting to 30 ng/mul for later use;

secondly, PCR amplification is carried out by using primers, and the sequence of the primer on the upstream of the primer pair is 5'-GTGCCTGTCTGAGGTGTGAG-3'; the sequence of the downstream primer of the primer pair is 5'-AGCTGTGTCAGGTTGTCGTG-3'; the PCR amplification reaction system is 20 mu l, and comprises 2 XPCR mix 10 mu l, 2 mu l of individual DNA to be identified with the concentration of 30 ng/mu l, 1 mu l of each upstream primer and downstream primer with the concentration of 10 mu M, and the balance of ultrapure water; the PCR reaction program comprises pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 30sec, and extension at 72 ℃ for 30sec, and has 33 cycles and extension at 72 ℃ for 5 min;

thirdly, gel electrophoresis: mu.l of the obtained PCR product was analyzed by 2.0% agarose gel electrophoresis.

And (3) identification result:

only 1 band is amplified from all 50 female fishes, and the size is 235 bp; 207bp bands are amplified in 50 male fishes, and 235bp bands can be amplified in part of the male fishes; the identification result is consistent with the sampling.