Specific DNA fragments and primers for identifying the sex of greenfin pufferfish, their applications, and method for identifying the sex of greenfin pufferfish

Abstract

The present invention provides a specific DNA fragment, primers, and applications thereof for identifying the sex of greenfin pufferfish, as well as a method for identifying the sex of greenfin pufferfish, relating to the technical field of fish sex identification. The present invention compares the genomes of male and female greenfin pufferfish and screens a male-specific fragment of greenfin pufferfish across the entire genome. The sequence information of this fragment in male greenfin pufferfish is shown in SEQ ID NO:1, and 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 this specific DNA fragment can be used as a molecular marker for sex identification of greenfin pufferfish, thereby achieving molecular biological detection of the sex of greenfin pufferfish.

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 greenfin pufferfish, a primer and application thereof, and a method for identifying the sex of greenfin pufferfish. Background Art

[0002] Greenfin pufferfish ( Thamnaconus septentrionalis Greenfin pufferfish (Pulveridae) are one of the most important economic fish species along my country's coast. They have excellent aquaculture traits and are easily domesticated. Their grazing and omnivorous nature makes them particularly well-suited for cage culture. However, during the breeding process, the sex ratio in the spawning ponds severely affects the ovulation and fertilization rates of the fish. Therefore, manual screening of male and female parents is necessary to adjust the sex ratio in the spawning ponds to the optimal level and maximize production efficiency. However, before the gonads of greenfin pufferfish mature, accurate sex determination by observation is difficult.

[0003] Existing methods for sexing greenfin pufferfish can be categorized into three types: dissection, observation, and squeezing. The dissection method involves dissecting the pufferfish, removing gonadal tissue, and observing to identify the sex. While accurate, the dissected pufferfish will not survive, making this method unsuitable for sexing parental pufferfish. The observation method uses skin color and abdominal swelling to determine sex. However, skin color is affected by environmental factors, and once the fish is stressed, it becomes nearly impossible to distinguish between males and females based on body color. The abdominal swelling is also affected by food intake. Therefore, observation is not an accurate method for sexing greenfin pufferfish. The squeezing method, on the other hand, involves squeezing the abdomen of the pufferfish when its gonads are mature, and determining sex based on the type of gametes released from the genital pore. The accuracy of this method is affected by the maturity of the pufferfish's gonads, and improper squeezing can also damage or even kill the pufferfish.

[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] A 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 adopted:

[0012] The present 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] Furthermore, 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 present 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, comprising:

[0020] The genomic DNA of the greenfin pufferfish to be tested was used as a template, and PCR amplification was performed using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3. 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) PCR amplification of the genomic DNA of the target greenfin pufferfish was performed using the above primers, and the length of the amplified fragment was detected;

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

[0026] When the amplified fragment is a 1083 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 present invention provides a reagent for identifying the sex of greenfin pufferfish, which 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] By comparing the genomes of male and female greenfin pufferfish, the present invention screened for a male-specific fragment within the entire genome. The sequence information of this fragment in male greenfin pufferfish is shown in SEQ ID NO:1. The greenfin pufferfish sex-specific DNA fragment was specifically amplified only in male individuals. This application, combined with dissection methods, experimentally verified that this specific DNA fragment can be used as a molecular marker for sex identification in greenfin pufferfish, thereby achieving molecular biological sex detection of greenfin pufferfish.

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

[0033] The specific DNA fragment for identifying the sex of greenfin pufferfish or the detection primer 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 embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any 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 following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts 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] By comparing the genomes of male and female greenfin pufferfish, the present invention identified a male-specific fragment within the entire genome. The sequence information of this fragment in male greenfin pufferfish is shown in SEQ ID NO:1. The greenfin pufferfish sex-specific DNA fragment was specifically amplified only in male individuals. This application, combined with dissection methods, experimentally verified that this specific DNA fragment can be used as a molecular marker for sex identification of greenfin pufferfish with 100% accuracy, thereby achieving molecular biological sex detection of greenfin pufferfish.

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

[0041] The present invention provides primers for identifying a specific DNA fragment for sex of the greenfin pufferfish. The primers can be used to specifically amplify the specific DNA fragment for sex identification of the greenfin pufferfish (SEQ ID NO: 1), and then obtain the sex identification result of the 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 for identifying the sex of greenfin pufferfish or the detection primer 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, and PCR amplification was performed using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3. The sex of the greenfin pufferfish was determined based on the amplified product.

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

[0051] (A) PCR amplification of the genomic DNA of the target greenfin pufferfish was performed using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, and the length of the amplified fragment was detected;

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

[0053] When the amplified fragment is a 1083 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 fin ray tissue from the greenfin pufferfish to extract genomic DNA from the greenfin pufferfish to be tested. Because the greenfin pufferfish's fin ray tissue lacks organs such as muscle and blood vessels, cutting 50mg of fin ray is almost harmless to the fish, 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 is provided, comprising 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 with reference to embodiments.

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

[0062] By comparing the genomes of male and female greenfin pufferfish, the applicant identified a male-specific fragment within the entire genome. The sequence information of this fragment in male greenfin pufferfish is shown as 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 designed and developed primers for identifying the sex of greenfin pufferfish using the specific DNA fragment. 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 SEQ ID NO. 3 sequence is as follows: 5'—CAGCCATGACCAGTGTTC—3'.

[0069] Example 2 Sex Identification of Greenfin Pufferfish

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

[0071] 1. Obtain 10 male and 10 female adult greenfin pufferfish identified by autopsy. Cut off 10 male and 10 female fin rays, respectively, and place them in anhydrous ethanol and store at -20°C for genomic DNA extraction.

[0072] 2. Genomic DNA was extracted using the phenol-chloroform method and diluted to 50 ng / µl for use 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 MgCl2 (25 mmol / L), 2 μL dNTPs (2.5 mmol / L each), 0.75 μL primer GS-F (10 μmol / L), 0.75 μL primer GS-R (10 μmol / L), 0.1 μL Taq DNA polymerase (5 U / μL), 1 μL template DNA (50 ng / μL), and the balance is sterile double-distilled water.

[0075] 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; 25 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s; extension at 72°C for 10 min; and storage at 4°C.

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

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

[0081] Figure 1 The agarose gel electrophoresis diagram for sex identification of greenfin pufferfish provided in this embodiment. Figure 1 1 to 10 represent the 10 male green-finned pufferfish tested in step 1 above; Figure 1 10 to 20 represent the 10 female greenfin pufferfish tested in step 1 above; Figure 1 M in the middle represents DNA molecular weight standard DL2000.

[0082] Depend on Figure 1 As can be seen, among all greenfin pufferfish tested, only male individuals showed specific amplification, with a band size of 1083 bp, while no amplification was observed in any of the female individuals tested. This result is consistent with the sex confirmed by anatomical histological observations. Therefore, it is demonstrated that the primers of the present invention can accurately identify the sex of greenfin pufferfish.

[0083] In summary, the method for identifying the sex of greenfin pufferfish provided by the present invention involves cutting 50 mg of fin rays from the greenfin pufferfish to extract genomic DNA, performing PCR amplification on the sex-specific DNA molecular markers of the greenfin pufferfish, and identifying the sex based on the agarose gel image of the amplification results. 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 greenfin pufferfish.

[0084] 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with 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, wherein The nucleotide sequence of SEQ ID NO: 1 is a DNA fragment unique to male greenfin pufferfish.

3. Primers for detecting the specific DNA fragment for identifying the sex of greenfin pufferfish according to claim 1, wherein 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.

4. 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 in identifying the sex of greenfin pufferfish.

5. 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, and the sex of the greenfin pufferfish is determined based on the amplified product.

6. The method for identifying the sex of greenfin pufferfish according to claim 5, wherein: The genomic DNA of the greenfin pufferfish to be tested is extracted from the fin ray tissue of the greenfin pufferfish.

7. The method for identifying the sex of greenfin pufferfish according to claim 6, wherein: The extraction method is: 50 mg of fin ray tissue of the greenfin pufferfish was 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 5, wherein: The method for identifying the sex of greenfin pufferfish comprises: (A) using the primers described in claim 3 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 1083 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.

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

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