Specific DNA (deoxyribonucleic acid) fragment and primer for identifying sex of modestus septentrionalis, application of specific DNA fragment and primer and method for identifying sex of modestus septentrionalis

Abstract

The invention provides a specific DNA (deoxyribonucleic acid) fragment and primer for identifying the sex of septumnus virens, application of the specific DNA fragment and primer and a method for identifying the sex of septumnus virens, and relates to the technical field of fish sex identification. According to the invention, a section of male specific fragment of the rumedodon septentrionalis is screened in a whole genome range by comparing female and male rumedodon septentrionalis genomes. The sequence information of the fragment in male rumex septentrionalis is as shown in SEQ ID NO: 1, and the sex-specific DNA fragment of rumex septentrionalis is only specifically amplified in male individuals. In combination with a dissection method, experiments prove that the specific DNA fragment as a molecular marker can be used for sex identification of the rumex septentrionalis, so that molecular biological detection of the sex of the rumex septentrionalis is realized.

Description

Technical Field

[0001] The invention relates to the technical field of fish sex identification, in particular to a specific DNA fragment for identifying the sex of a greenfin pufferfish, a primer and an application thereof, and a method for identifying the sex of a greenfin pufferfish. Background Art

[0002] Greenfin pufferfish (Thamnaconus septentrionalis) is one of the important economic fish species in my country's coastal areas. It has excellent breeding characteristics and is easy to domesticate. It is particularly suitable for cage breeding due to its grazing and omnivorous characteristics. During the breeding process of greenfin pufferfish, the male-female ratio in the spawning pond of greenfin pufferfish seriously affects the ovulation rate and fertilization rate of fish. Therefore, it is necessary to artificially select male and female parents and adjust the male-female ratio in the spawning pond to the optimal ratio to maximize production efficiency. However, it is difficult to accurately distinguish the male and female of greenfin pufferfish by observation before the gonads mature.

[0003] Existing methods for sex identification of green-finned pufferfish can be divided into three categories: dissection, observation, and squeezing. The dissection method is to dissect the green-finned pufferfish and remove the gonadal tissue for observation and identification of male and female. This method is accurate, but the dissected green-finned pufferfish cannot survive, and this method is not suitable for sex identification of parents. The observation method is to identify the sex of the green-finned pufferfish by the skin color and the degree of abdominal bulge, but the skin color is affected by factors such as the environment. Once the fish body is stressed, it is almost impossible to distinguish between male and female by body color, and the degree of abdominal bulge is also affected by the amount of food intake. Therefore, the observation method cannot accurately identify the sex of the green-finned pufferfish. The squeezing method is to squeeze the abdomen when the gonads of the green-finned pufferfish are mature, and the sex is identified according to the type of gametes discharged from the genital pore. The accuracy of this method is affected by the degree of maturity of the gonads of the parent fish, and improper squeezing can also cause damage to the parents, or even death.

[0004] Therefore, it is necessary and urgent to research and develop a method for rapidly identifying the genetic sex of greenfin pufferfish, which can accurately identify the genetic sex of greenfin pufferfish and ensure the survival rate of the greenfin pufferfish to be tested.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The first purpose of the present invention is to provide a specific DNA fragment for identifying the sex of greenfin pufferfish, so as to solve the problem of molecular biological detection of the sex of greenfin pufferfish.

[0007] The second object of the present invention is to provide a primer for identifying the specific DNA fragment of the greenfin pufferfish.

[0008] The third object of the present invention is to provide a method for identifying the sex of greenfin pufferfish.

[0009] The fourth object of the present invention is to provide a reagent for identifying the sex of greenfin pufferfish.

[0010] The fifth object of the present invention is to provide a kit for identifying the sex of greenfin pufferfish.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0012] The invention provides a specific DNA fragment for identifying the sex of greenfin pufferfish, wherein the specific DNA fragment is a nucleotide sequence shown in SEQ ID NO:1.

[0013] Furthermore, the nucleotide sequence of SEQ ID NO: 1 is a DNA fragment unique to male greenfin pufferfish.

[0014] The invention provides primers for detecting the specific DNA fragment for identifying the sex of the greenfin pufferfish.

[0015] Further, the primers include an upstream primer and a downstream primer;

[0016] The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 2;

[0017] The nucleotide sequence of the downstream primer is shown in SEQ ID NO:3.

[0018] The invention provides an application of the specific DNA fragment for identifying the sex of greenfin pufferfish or the detection primer in identifying the sex of greenfin pufferfish.

[0019] The present invention provides a method for identifying the sex of greenfin pufferfish, the method comprising:

[0020] The genomic DNA of the greenfin pufferfish to be tested was used as a template, PCR amplification was performed using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, and the sex of the greenfin pufferfish was determined based on the amplified product.

[0021] Furthermore, the genomic DNA of the greenfin pufferfish to be tested is extracted from the fin ray tissue of the greenfin pufferfish;

[0022] Preferably, 50 mg of fin ray tissue of the greenfin pufferfish is used to extract the genomic DNA of the greenfin pufferfish to be tested.

[0023] Furthermore, the method for identifying the sex of the greenfin pufferfish comprises:

[0024] (A) using the above primers to perform PCR amplification on the genomic DNA of the greenfin pufferfish to be tested, and detecting the length of the amplified fragment;

[0025] (B) Identifying the sex of the greenfin pufferfish to be tested based on the length of the amplified fragment.

[0026] When the amplified fragment is a 1086 bp band, the greenfin pufferfish to be tested has the nucleotide sequence shown in SEQ ID NO.1 and is identified as a male greenfin pufferfish;

[0027] When no amplified band appears, the greenfin pufferfish to be tested is identified as a female greenfin pufferfish.

[0028] The invention provides a reagent for identifying the sex of greenfin pufferfish, and the reagent comprises the above primers.

[0029] The present invention provides a kit for identifying the sex of greenfin pufferfish, which comprises the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3 or reagents containing the primers.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention screens a male-specific fragment of greenfin pufferfish in the whole genome by comparing the genomes of male and female greenfin pufferfish. The sequence information of the fragment in male greenfin pufferfish is shown in SEQ ID NO: 1. The greenfin pufferfish sex-specific DNA fragment is specifically amplified only in male individuals. The present application combines the dissection method and experimentally verifies that the specific DNA fragment can be used as a molecular marker for sex identification of greenfin pufferfish, thereby realizing the molecular biological detection of the sex of greenfin pufferfish.

[0032] The primers for identifying the sex of greenfin pufferfish provided by the present invention can be used to specifically amplify the specific DNA fragment (SEQ ID NO: 1) for identifying the sex of greenfin pufferfish, and then obtain the identification result of the sex of greenfin pufferfish by comparing the electrophoresis patterns.

[0033] The specific DNA fragment or the detection primer for identifying the sex of greenfin pufferfish provided by the present invention can be widely used in identifying the sex of greenfin pufferfish. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1This is an agarose gel electrophoresis diagram for sex identification of greenfin pufferfish provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0036] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] According to one aspect of the present invention, a specific DNA fragment for identifying the sex of greenfin pufferfish is provided, wherein the specific DNA fragment is the nucleotide sequence shown in SEQ ID NO:1.

[0038] The present invention screens a male-specific fragment of greenfin pufferfish in the whole genome by comparing the genomes of male and female greenfin pufferfish. The sequence information of the fragment in male greenfin pufferfish is shown in SEQ ID NO: 1. The greenfin pufferfish sex-specific DNA fragment is specifically amplified only in male individuals. The present application combines the dissection method and experimentally verifies that the specific DNA fragment can be used as a molecular marker for sex identification of greenfin pufferfish, and its accuracy can reach 100%, thereby realizing the molecular biological detection of the sex of greenfin pufferfish.

[0039] In a preferred embodiment of the present invention, the nucleotide sequence of SEQ ID NO: 1 is a DNA fragment specific to male greenfin pufferfish, which exists in the male genetic characteristic region of greenfin pufferfish but is missing in the genome of female greenfin pufferfish.

[0040] According to one aspect of the present invention, a primer is provided for detecting the specific DNA fragment for identifying the sex of greenfin pufferfish.

[0041] The primers for identifying the sex of greenfin pufferfish provided by the present invention can be used to specifically amplify the specific DNA fragment (SEQ ID NO: 1) for identifying the sex of greenfin pufferfish, and then obtain the identification result of the sex of greenfin pufferfish by comparing the electrophoresis patterns.

[0042] In a preferred embodiment of the present invention, the electrophoresis is agarose gel electrophoresis.

[0043] In a preferred embodiment of the present invention, the primers include an upstream primer and a downstream primer;

[0044] The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 2;

[0045] The nucleotide sequence of the downstream primer is shown in SEQ ID NO:3.

[0046] According to one aspect of the present invention, the specific DNA fragment for identifying the sex of greenfin pufferfish or the detection primer is used in identifying the sex of greenfin pufferfish.

[0047] The specific DNA fragment or the detection primer for identifying the sex of greenfin pufferfish provided by the present invention can be widely used in identifying the sex of greenfin pufferfish.

[0048] According to one aspect of the present invention, a method for identifying the sex of greenfin pufferfish comprises:

[0049] The genomic DNA of the greenfin pufferfish to be tested was used as a template, PCR amplification was performed using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, and the sex of the greenfin pufferfish was determined based on the amplified product.

[0050] Specifically, the method for identifying the sex of greenfin pufferfish comprises:

[0051] (A) using the primers shown in SEQ ID NO:2 and SEQ ID NO:3 to perform PCR amplification on the genomic DNA of the greenfin pufferfish to be tested, and detecting the length of the amplified fragment;

[0052] (B) Identifying the sex of the greenfin pufferfish to be tested based on the length of the amplified fragment.

[0053] When the amplified fragment is a 1086 bp band, the greenfin pufferfish to be tested has the nucleotide sequence shown in SEQ ID NO.1 and is identified as a male greenfin pufferfish;

[0054] When no amplified band appears, the greenfin pufferfish to be tested is identified as a female greenfin pufferfish.

[0055] In a preferred embodiment of the present invention, the genomic DNA of the greenfin pufferfish to be tested is extracted from the fin ray tissue of the greenfin pufferfish;

[0056] Preferably, 50 mg of fin ray tissue of the greenfin pufferfish is used to extract the genomic DNA of the greenfin pufferfish to be tested.

[0057] As a preferred embodiment, the present application uses the fin ray tissue of the greenfin pufferfish to extract the genomic DNA of the greenfin pufferfish to be tested. Since the fin ray tissue of the greenfin pufferfish lacks organs such as muscles and blood vessels, cutting 50 mg of fin ray does almost no damage to the fish body, ensuring the survival rate of the tested fish.

[0058] According to one aspect of the present invention, a reagent for identifying the sex of greenfin pufferfish comprises the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0059] According to one aspect of the present invention, a kit for identifying the sex of greenfin pufferfish comprises the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3 or reagents containing the same.

[0060] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0061] Example 1 Sex-specific DNA markers of greenfin pufferfish

[0062] The applicant compared the genomes of male and female greenfin pufferfish and screened a male-specific fragment of greenfin pufferfish in the whole genome. The sequence information of the fragment in male greenfin pufferfish is shown in SEQ ID NO:1.

[0063]

[0064] Based on the above-mentioned specific DNA fragment for identifying the sex of greenfin pufferfish (SEQ ID NO: 1), the applicant has designed and developed primers for identifying the specific DNA fragment for identifying the sex of greenfin pufferfish. The primers include an upstream primer and a downstream primer;

[0065] The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 2;

[0066] The specific sequence of SEQ ID NO.2 is as follows: 5'—CCGTTTGTCTTATCAGGC—3';

[0067] The nucleotide sequence of the downstream primer is shown in SEQ ID NO:3.

[0068] The specific sequence of SEQ ID NO.3 is as follows: 5'—CAGCCATGACCAGTGTTC—3'.

[0069] Example 2 Sex Identification of Greenfin Pufferfish

[0070] The genomic DNA of the greenfin pufferfish whose sex was determined by dissection was amplified by PCR using the primers of the present invention, and the accuracy and specificity of the method of the present invention were verified by comparing the agarose gel electrophoresis patterns. The specific steps of the method are as follows:

[0071] 1. Take 10 male and 10 female greenfin pufferfish adult fish identified by dissection, cut off 10 male and 10 female greenfin pufferfish partial fin rays, put them into anhydrous ethanol, and store them at -20℃ for genomic DNA extraction;

[0072] 2. The genomic DNA was extracted by phenol-chloroform method, and the obtained genomic DNA was uniformly diluted to 50 ng / μl as a template;

[0073] 3. PCR amplification using sex-specific primers:

[0074] (1) PCR amplification system: The total reaction volume is 25 μL, including 2.5 μL 10× PCR buffer (Mg-free) 2+ ), 1.5 μL MgCl 2 (25mmol / L), 2μL dNTPs (2.5mmol / L each), 0.75μL primer GS-F (10μmol / L), 0.75μL primer GS-R (10μmol / L), 0.1μL Taq DNA polymerase (5U / μL), 1μL template DNA (50ng / μL), and the remainder is sterile double distilled water.

[0075] The sex-specific primers are as follows:

[0076] GS-F: 5'-GCCCGTTTGTCTTATCAGGC-3' (SEQ ID NO: 2);

[0077] GS-R: 5'-GCAGCCATGACCAGTGTTC-3' (SEQ ID NO: 3);

[0078] (2) PCR amplification procedure: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 25 cycles; extension at 72°C for 10 min; storage at 4°C.

[0079] 4. Agarose gel electrophoresis detection and result analysis:

[0080] The PCR products amplified with sex-specific primers (GS-F / R) can be detected by electrophoresis using 1.5% agarose gel, constant voltage 120V, and electrophoresis time of about 40 minutes. Figure 1 .

[0081] Figure 1 This is an agarose gel electrophoresis diagram for sex identification of the greenfin pufferfish provided in this example.

[0082] in, Figure 1 1 to 10 represent the 10 male greenfin pufferfish to be tested in step 1 above; Figure 1 10 to 20 represent the 10 female greenfin pufferfish to be tested in step 1 above; Figure 1 M in the figure indicates DNA molecular weight standard DL2000.

[0083] Depend on Figure 1 It can be seen that among all the greenfin pufferfish tested, only the male individuals had specific amplification, with a band size of 1086 bp, while all the female individuals tested had no amplification. The results are consistent with the sex confirmed by anatomical histological observation. Therefore, it is shown that the primers of the present invention can accurately identify the sex of the greenfin pufferfish.

[0084] In summary, the method for identifying the sex of the greenfin pufferfish provided by the present invention is to cut 50 mg of fin rays of the greenfin pufferfish to extract genomic DNA, and to perform PCR amplification on the sex-specific DNA molecular markers of the greenfin pufferfish, and to identify the sex according to the agarose gel image of the amplification result. This method has high accuracy and causes almost no damage to the fish body, and can be used as an accurate and minimally invasive method for identifying the sex of the greenfin pufferfish.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A specific DNA fragment for identifying the sex of greenfin pufferfish, characterized in that: The specific DNA fragment is the nucleotide sequence shown in SEQ ID NO:

1.

2. The specific DNA fragment for identifying the sex of greenfin pufferfish according to claim 1, characterized in that: The nucleotide sequence of SEQ ID NO: 1 is a DNA fragment specific to male greenfin pufferfish.

3. Primers for detecting the specific DNA fragment for identifying the sex of greenfin pufferfish as described in claim 1.

4. The primer according to claim 3, characterized in that The primers include an upstream primer and a downstream primer; The nucleotide sequence of the upstream primer is shown in SEQ ID NO: 2; The nucleotide sequence of the downstream primer is shown in SEQ ID NO:

3.

5. Use of the specific DNA fragment for identifying the sex of greenfin pufferfish according to claim 1 or 2 or the primer according to claim 3 or 4 in identifying the sex of greenfin pufferfish.

6. A method for identifying the sex of greenfin pufferfish, characterized in that: The method comprises: The genomic DNA of the greenfin pufferfish to be tested is used as a template, PCR amplification is performed using the primers described in claim 3 or 4, and the sex of the greenfin pufferfish is determined based on the amplified product.

7. The method for identifying the sex of greenfin pufferfish according to claim 6, characterized in that: The genomic DNA of the greenfin pufferfish to be tested is extracted from the fin ray tissue of the greenfin pufferfish; Preferably, 50 mg of fin ray tissue of the greenfin pufferfish is used to extract the genomic DNA of the greenfin pufferfish to be tested.

8. The method for identifying the sex of greenfin pufferfish according to claim 6, characterized in that: The method for identifying the sex of greenfin pufferfish comprises: (A) using the primers described in claim 3 or 4 to perform PCR amplification on the genomic DNA of the greenfin pufferfish to be tested, and detecting the length of the amplified fragment; (B) identifying the sex of the greenfin pufferfish to be tested based on the length of the amplified fragment; When the amplified fragment is a 1086 bp band, the greenfin pufferfish to be tested has the nucleotide sequence shown in SEQ ID NO.1 and is identified as a male greenfin pufferfish; When no amplified band appears, the greenfin pufferfish to be tested is identified as a female greenfin pufferfish.

9. A reagent for identifying the sex of greenfin pufferfish, characterized in that: The reagent comprises the primer according to claim 3 or 4.

10. A kit for identifying the sex of greenfin pufferfish, characterized in that: The kit comprises the primer according to claim 3 or 4 or the reagent according to claim 9.

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