A DNA fragment for genetic sex identification of thick jawed bream and its application

Abstract

The present invention provides a DNA fragment for identifying the genetic sex of thick-jawed bream and its application. The present invention utilizes the target DNA fragment of male thick-jawed bream, which can not only quickly and accurately identify the genetic sex of thick-jawed bream, but also is not limited by the age of thick-jawed bream, and only needs to extract a trace amount of DNA of the thick-jawed bream to be detected, without causing significant impact and damage to the thick-jawed bream. The present invention provides assistance for the sex control breeding of thick-jawed bream, and is helpful for the population protection or promotion of breeding of thick-jawed bream.

Description

Technical Field

[0001] The invention relates to the field of molecular biology, and in particular to a DNA fragment for genetic sex identification of thick jaw bream and application thereof. Background Art

[0002] Megalobrama pellegrini belongs to the genus Megalobrama of the subfamily Cultrinae of the family Cyprinidae. Megalobrama pellegrini is relatively rare in the main stream and large tributaries of the upper reaches of the Yangtze River. There are still a certain number of them in some small slow-flowing tributaries, but their distribution range and population size have shown a clear downward trend. At present, research on the monoculture of adult Megalobrama pellegrini, as well as its biological characteristics and artificial propagation has made good progress, proving that Megalobrama pellegrini is an excellent aquaculture fish suitable for promotion of aquaculture in the Yangtze River Basin and has great development potential. However, Megalobrama pellegrini can spawn naturally in rivers, but not under artificial breeding conditions. Therefore, artificial propagation of Megalobrama pellegrini generally requires artificial induced spawning and artificial insemination to obtain a large number of fertilized eggs.

[0003] However, the thick-jawed bream has no secondary sexual characteristics, and it is difficult to identify the sex of the thick-jawed bream from its appearance. Too many female thick-jawed breams occupy a large number of breeding facilities, and the difficulty in determining the sex of the thick-jawed bream has caused great obstacles for thick-jawed bream farmers in the process of artificial breeding of thick-jawed bream. Therefore, developing a method to identify the sex of thick-jawed bream has great application value. Summary of the invention

[0004] The invention provides an application of a male thick-jawed bream target DNA fragment in identifying the genetic sex of the thick-jawed bream. The genetic sex of the thick-jawed bream can be identified quickly and accurately using the male thick-jawed bream target DNA fragment without being limited by the age of the thick-jawed bream.

[0005] The invention provides a primer pair for amplifying a target DNA fragment of a male thick-jawed bream. The accuracy of amplifying the target DNA fragment of the male thick-jawed bream by using the primer pair is high.

[0006] The invention provides a kit for identifying the genetic sex of thick-jawed bream, which can quickly and accurately identify the genetic sex of thick-jawed bream.

[0007] The present invention provides an application of the primer pair or the kit in identifying the genetic sex of thick jawed bream.

[0008] The present invention also provides a method for identifying the genetic sex of thick-jawed bream. By using the above-mentioned male thick-jawed bream target DNA fragment, the genetic sex of thick-jawed bream can be quickly and accurately identified, and only a trace amount of sample DNA is required, which will not cause significant impact and damage to the thick-jawed bream. At the same time, this method is not limited by the age of the thick-jawed bream.

[0009] The present invention provides an application of a male thick-jawed bream target DNA fragment in identifying the genetic sex of the thick-jawed bream, wherein the nucleotide sequence of the male thick-jawed bream target DNA fragment is shown in SEQ ID NO:1.

[0010] The invention provides a primer pair for amplifying a target DNA fragment of a male thick jaw bream, comprising a first primer and a second primer, wherein the nucleotide sequence of the first primer is shown in SEQ ID NO:2, and the nucleotide sequence of the second primer is shown in SEQ ID NO:3.

[0011] The invention provides a kit for genetic sex identification of thick jawed bream, comprising the above primer pair.

[0012] The kit as described above, wherein the kit further comprises at least one of water, PCR buffer, dNTPs, DNA polymerase, and loading buffer.

[0013] The present invention provides an application of the primer pair or the kit in identifying the genetic sex of thick jawed bream.

[0014] The present invention also provides a method for identifying the genetic sex of Megalobrama thunbergii, comprising the following steps:

[0015] Step 1, extracting DNA from a sample of Megalobrama to be tested;

[0016] Step 2, designing a first primer and a second primer for amplifying the male thick-jawed bream target DNA fragment according to the male thick-jawed bream target DNA fragment;

[0017] Step 3, using the first primer and the second primer to perform PCR amplification on the sample DNA;

[0018] Step 4: Determine the genetic sex of the thick-jawed bream to be tested according to the result of PCR amplification.

[0019] In the method as described above, the nucleotide sequence of the first primer is as shown in SEQ ID NO:2, and the nucleotide sequence of the second primer is as shown in SEQ ID NO:3.

[0020] The method as described above, wherein the genetic sex of the thick-jawed bream to be tested is determined based on the result of PCR amplification, comprising: performing agarose gel electrophoresis detection on the product after PCR amplification, and when a band of 377 bp appears in the detection result, or the sequence of the detection result is the nucleotide sequence shown in SEQ ID NO:1, the genetic sex of the thick-jawed bream is identified as male; when no band appears in the detection result, or the sequence of the detection result is not the nucleotide sequence shown in SEQ ID NO:1, the genetic sex of the thick-jawed bream is identified as female.

[0021] In the method as described above, the sample DNA of the thick-jawed bream to be tested is derived from at least one of the fin rays and mucus of the thick-jawed bream.

[0022] The method as described above, wherein in PCR amplification, the PCR reaction system is 25 μL in total, including 2 μL of DNA template, 1 μL of first primer, 1 μL of second primer, 12.5 μL of 2×Taq MasterMix and 8.5 μL of ddH2O; and / or,

[0023] In PCR amplification, the PCR amplification program includes pre-denaturation at 94°C for 3 min; denaturation at 94°C for 10 s, annealing at 56°C for 10 s, and extension at 72°C for 29 s, for a total of 35 cycles; and finally extension at 72°C for 5 min.

[0024] The present invention provides an application of a male thick-jawed bream target DNA fragment in identifying the genetic sex of the thick-jawed bream. Using the male thick-jawed bream target DNA fragment, not only can the genetic sex of the thick-jawed bream be identified quickly and accurately, but also the sex identification can be performed throughout the life cycle of the thick-jawed bream without being restricted by the age of the thick-jawed bream. The present invention provides assistance for sex-controlled breeding of thick-jawed bream, and is helpful for population protection or promotion of breeding of thick-jawed bream. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the electrophoresis test result of genetic sex identification of thick jaw bream in Example 1. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below. The specific implementation methods listed below are only for describing the principles and features of the present invention. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0027] A first aspect of the present invention provides an application of a male thick-jawed bream target DNA fragment in identifying the genetic sex of the thick-jawed bream, wherein the nucleotide sequence of the male thick-jawed bream target DNA fragment is shown in SEQ ID NO:1.

[0028] The present invention provides a male thick-jawed bream target DNA fragment (nucleotide sequence as shown in SEQ ID NO: 1) which is unique only in male thick-jawed bream by studying thick-jawed bream genomic DNA, with a length of 377 bp. The fragment is unique only in male thick-jawed bream and does not exist in female thick-jawed bream, and can be used as a target amplification sequence to identify the genetic sex of thick-jawed bream, thereby improving the accuracy of identification. Therefore, the genetic sex of thick-jawed bream can be quickly and accurately identified by using the male thick-jawed bream target DNA fragment, that is, the thick-jawed bream containing the male thick-jawed bream target DNA fragment is male, and the thick-jawed bream not containing the male thick-jawed bream target DNA fragment is female.

[0029] The second aspect of the present invention provides a primer pair for amplifying a target DNA fragment of a male thick-jawed bream, comprising a first primer and a second primer, the nucleotide sequence of the first primer is shown in SEQ ID NO:2, and the nucleotide sequence of the second primer is shown in SEQ ID NO:3.

[0030] Among them, the first primer is an upstream primer, and the second primer is a downstream primer.

[0031] The present invention designs a primer pair for amplifying a target DNA fragment of male thick-jawed bream based on a target DNA fragment of male thick-jawed bream, taking into account the primer length, the GC base content in the primer, and the primer annealing temperature. Therefore, the primer pair of the present invention can specifically amplify a target DNA fragment of male thick-jawed bream in male thick-jawed bream, but will not amplify a target DNA fragment of male thick-jawed bream in female thick-jawed bream, and has a high accuracy rate.

[0032] The third aspect of the present invention provides a kit for genetic sex identification of thick jaw bream, comprising the above-mentioned primer pair.

[0033] Since the kit includes the primer pair provided in the second aspect of the present invention, the kit also has the ability to quickly and accurately identify the genetic sex of thick-jawed bream.

[0034] In a specific embodiment, the kit further comprises at least one of water, PCR buffer, dNTPs, DNA polymerase, and loading buffer.

[0035] A fourth aspect of the present invention provides a use of the above primer pair or the above kit in identifying the genetic sex of thick-jawed bream.

[0036] The above primer pair or the above kit can be used to quickly and accurately identify the genetic sex of Megalobrama thick jaw. Figure 1 The electrophoresis test result of genetic sex identification of thick jaw bream provided by one embodiment of the present invention is as follows: Figure 1As shown, when a band of 377 bp appears in the test result, the genetic sex of the thick-jawed bream is male; when no band appears in the test result, the genetic sex of the thick-jawed bream is female.

[0037] A fifth aspect of the present invention provides a method for identifying the genetic sex of Megalobrama sutchuenensis, comprising the following steps:

[0038] Step 1, extracting DNA from a sample of Megalobrama to be tested;

[0039] Step 2, designing a first primer and a second primer for amplifying the male thick-jawed bream target DNA fragment according to the male thick-jawed bream target DNA fragment;

[0040] Step 3, using the first primer and the second primer to perform PCR amplification on the sample DNA;

[0041] Step 4: Determine the genetic sex of the thick-jawed bream to be tested according to the result of PCR amplification.

[0042] According to the male thick-jawed bream target DNA fragment provided by the first aspect of the present invention, a first primer and a second primer for amplifying the male thick-jawed bream target DNA fragment can be designed, and then the first primer and the second primer are used to perform PCR amplification on the sample DNA of the sturgeon to be detected, and the genetic sex of the thick-jawed bream to be detected can be determined according to the result of PCR amplification, that is, the sex of the thick-jawed bream to be detected is determined to be male or female. The identification method provided by the present invention only needs to extract a trace amount of DNA from the thick-jawed bream to be detected, which will not cause significant impact and damage to the thick-jawed bream, and will not be limited by the age of the thick-jawed bream. The genetic sex identification can be performed throughout the life cycle of the thick-jawed bream.

[0043] The nucleotide sequence of the first primer is shown as SEQ ID NO:2, and the nucleotide sequence of the second primer is shown as SEQ ID NO:3.

[0044] According to the first primer and the second primer, the genetic sex of the thick-jawed bream can be identified quickly and accurately.

[0045] In addition, according to the results of PCR amplification, the genetic sex of the thick-jawed bream to be tested is determined, which also includes: performing agarose gel electrophoresis on the product after PCR amplification, when the test result shows a band of 377bp, or the sequence of the test result is the nucleotide sequence shown in SEQ ID NO:1, the genetic sex of the thick-jawed bream is identified as male; when the test result does not show a band, or the sequence of the test result is not the nucleotide sequence shown in SEQ ID NO:1, the genetic sex of the thick-jawed bream is identified as female.

[0046] Since the length of the target DNA fragment of male thick-jawed bream is 377 bp, and the nucleotide sequence is shown in SEQ ID NO: 1, the genetic sex of the thick-jawed bream to be tested can be identified based on the presence or absence of bands detected by agarose gel electrophoresis and the size of the bands or the sequence of the test results, that is, the sex of the thick-jawed bream to be tested can be identified as female or male. Only when a band appears in the test result, and the band size is 377 bp, or the sequence of the test result is the nucleotide sequence shown in SEQ ID NO: 1, can the genetic sex of the thick-jawed bream be determined to be male; only when no band appears in the test result, or the sequence of the test result is not the nucleotide sequence shown in SEQ ID NO: 1, can the genetic sex of the thick-jawed bream be determined to be female.

[0047] In a specific embodiment, Figure 1 The electrophoresis test result of genetic sex identification of thick jaw bream provided by one embodiment of the present invention is as follows: Figure 1 As shown, when a band of 377 bp appears in the test result, the genetic sex of the thick-jawed bream is male; when no band appears in the test result, the genetic sex of the thick-jawed bream is female.

[0048] When the identification is performed according to the above steps, the sample DNA of the thick jaw bream to be detected is derived from at least one of the fin rays and mucus of the thick jaw bream. The genomic DNA of the sample of the thick jaw bream to be detected can be extracted by conventional technical means in the art, such as the CTAB method.

[0049] Further, in PCR amplification, the PCR reaction system is 25 μL in total, including 2 μL of DNA template, 1 μL of the first primer, 1 μL of the second primer, 12.5 μL of 2×Taq MasterMix and 8.5 μL of ddH2O; and / or,

[0050] In PCR amplification, the PCR amplification program includes pre-denaturation at 94°C for 3 min; denaturation at 94°C for 10 s, annealing at 56°C for 10 s, and extension at 72°C for 29 s, for a total of 35 cycles; and finally extension at 72°C for 5 min.

[0051] The use of the above PCR reaction system helps to improve the stability and accuracy of the PCR reaction, thereby ensuring the accuracy of identifying the genetic sex of the thick-jawed bream; the use of the above PCR amplification program helps to improve the specificity of the PCR reaction, further improving the accuracy of identifying the genetic sex of the thick-jawed bream.

[0052] Finally, the PCR amplification product is detected according to the conventional agarose gel electrophoresis detection method in the art, and the genetic sex of the thick-jawed bream to be detected can be identified based on the presence or absence of bands displayed by agarose gel electrophoresis and the size of the bands or the sequence of the detection results.

[0053] The technical solution of the present application will be further explained below in conjunction with specific embodiments.

[0054] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers. The reagents used are commercially available or publicly available unless otherwise specified.

[0055] The primer sequences involved in the following examples are shown in Table 1:

[0056] Table 1

[0057]

[0058]

[0059] Example 1: Identification of genetic sex of Megalobrama thunbergii

[0060] During the breeding season of the thick-jawed bream, the thick-jawed bream that ejaculates sperm is marked as male fish, and the thick-jawed bream that ovulates eggs is marked as female fish, and the fin rays of 12 male thick-jawed breams and 12 female thick-jawed breams are taken, respectively. DNA of the above 24 thick-jawed breams is extracted from the fin rays of the thick-jawed bream using a DNA extraction kit.

[0061] The upstream primer TTF83F and the downstream primer TTF83R were designed according to the target DNA fragment of male thick-jawed bream. The nucleotide sequence of the target DNA fragment of male thick-jawed bream is shown in SEQ ID NO:1, and the fragment size is 377 bp; the nucleotide sequence of the upstream primer TTF83F is shown in SEQ ID NO:2, and the primer length is 18 bp; the nucleotide sequence of the downstream primer TTF83R is shown in SEQ ID NO:3, and the primer length is 19 bp; the annealing temperature of the upstream primer TTF83F and the downstream primer TTF83R is 56°C.

[0062] The DNA of the thick jawed bream was amplified by PCR using the upstream primer TTF83F and the downstream primer TTF83R to obtain the PCR amplified product. The PCR reaction system was 25 μL, including 2 μL of DNA template, 1 μL of upstream primer TTF83F, 1 μL of downstream primer TTF83R, 12.5 μL of 2×Taq MasterMix and 8.5 μL of ddH2O. The PCR amplification program included 94℃ pre-denaturation for 3 min; 94℃ denaturation for 10 s, 56℃ annealing for 10 s, 72℃ extension for 29 s, a total of 35 cycles; and finally 72℃ extension for 5 min, and stored at 4℃.

[0063] Add the product after PCR amplification to 2% agarose gel, set the voltage to 120V, the time to 30 minutes, perform electrophoresis, and use a gel illuminator to observe the agarose gel electrophoresis results. When the test result shows a band of 377bp, the genetic sex of the thick-jawed bream is identified as male; when the test result shows no band, the genetic sex of the thick-jawed bream is identified as female.

[0064] like Figure 1 As shown in the figure, M is a DNA molecular weight marker (Marker), specifically DL 2000Plus. Figure 1 It can be seen that all 12 male thick-jawed breams showed a band of 377 bp; and none of the 12 female thick-jawed breams showed any band. In addition, to ensure the accuracy of the experiment, a blank control was added in this example, and the 12 male thick-jawed breams were repeatedly tested. The results of this experiment were consistent with the results of genetic sex identification of thick-jawed breams obtained by breeding period marking, with an accuracy rate of 100%.

[0065] Therefore, the genetic sex of the thick-jawed bream identified by the present invention is consistent with the genetic sex of the thick-jawed bream obtained by the breeding period marker, indicating that the present invention can accurately identify the genetic sex of the thick-jawed bream. The present invention utilizes the target DNA fragment of the male thick-jawed bream, which can not only quickly and accurately identify the genetic sex of the thick-jawed bream, but also is not limited by the age of the thick-jawed bream, and sex identification can be performed throughout the life cycle of the thick-jawed bream; and only a trace amount of DNA of the thick-jawed bream to be tested needs to be extracted, which will not cause significant impact and damage to the thick-jawed bream. The present invention provides assistance for sex-controlled breeding of thick-jawed bream, and is helpful for population protection or promotion of breeding of thick-jawed bream.

[0066] 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 the technical solutions described in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An application of a male thick-jawed bream target DNA fragment in identifying the genetic sex of thick-jawed bream, characterized in that: The nucleotide sequence of the male thick-jawed bream target DNA fragment is shown in SEQ ID NO:

1.

2. A primer pair for amplifying a target DNA fragment of a male thick jawed bream, characterized in that: It comprises a first primer and a second primer, wherein the nucleotide sequence of the first primer is shown as SEQ ID NO:2, and the nucleotide sequence of the second primer is shown as SEQ ID NO:

3.

3. A kit for genetic sex identification of thick jawed bream, characterized in that: Comprising the primer pair described in claim 2.

4. The kit according to claim 3, characterized in that The kit further comprises at least one of water, PCR buffer, dNTPs, and DNA polymerase.

5. Use of the primer pair according to claim 2 or the kit according to any one of claims 3 to 4 in identifying the genetic sex of Megalobrama thick jaws.

6. A method for identifying the genetic sex of thick jawed bream, characterized in that: The steps include: Step 1, extracting DNA from a sample of Megalobrama to be tested; Step 2, designing a first primer and a second primer for amplifying the male thick-jawed bream target DNA fragment according to claim 1; Step 3, using the first primer and the second primer to perform PCR amplification on the sample DNA; Step 4: Determine the genetic sex of the thick-jawed bream to be tested according to the result of the PCR amplification.

7. The method according to claim 6, characterized in that The nucleotide sequence of the first primer is shown in SEQ ID NO:2, and the nucleotide sequence of the second primer is shown in SEQ ID NO:

3.

8. The method according to claim 7, characterized in that According to the result of the PCR amplification, the genetic sex of the thick-jawed bream to be detected is determined, including: performing agarose gel electrophoresis detection on the product after the PCR amplification, when the detection result shows a band of 377bp, or the sequence of the detection result is the nucleotide sequence shown in SEQ ID NO:1, the genetic sex of the thick-jawed bream is identified as male; when the detection result does not show a band, or the sequence of the detection result is not the nucleotide sequence shown in SEQ ID NO:1, the genetic sex of the thick-jawed bream is identified as female.

9. The method according to claim 6, characterized in that The sample DNA of the thick-jawed bream to be detected is derived from at least one of the fin rays and mucus of the thick-jawed bream.

10. The method according to any one of claims 6 to 9, characterized in that: In the PCR amplification, the PCR reaction system is 25 μL in total, including 2 μL of DNA template, 1 μL of the first primer, 1 μL of the second primer, 12.5 μL of 2×Taq MasterMix and 8.5 μL of ddH2O; and / or, In the PCR amplification, the PCR amplification program includes 94°C pre-denaturation for 3 minutes; 94°C denaturation for 10 seconds, 56°C annealing for 10 seconds, 72°C extension for 29 seconds, a total of 35 cycles; and finally 72°C extension for 5 minutes.

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