Molecular markers, kits and methods for distinguishing Heilongjiang triangular bream, Qiantangjiang triangular bream and their hybrids

Abstract

Molecular markers, kits and methods for distinguishing Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids, relate to molecular markers, kits and methods for distinguishing fish of the genus Bream from different regions. The molecular marker primer pairs of the present invention are HLJFLf4 and HLJFLr4, and HLJFLf5 and HLJFLr5. Distinguishing methods: 1. DNA extraction; 2. Primer amplification; 3. Enzyme cutting; 4. Electrophoresis detection. The identification kit and method provided by the present invention can be used to directly distinguish and identify Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids, and the parental sources of the hybrids can be further distinguished. The method has high accuracy, simple operation process, short time, and an accuracy rate of 100%.

Description

Technical Field

[0001] The invention relates to a molecular marker, a kit and a differentiation method which can be used to differentiate bream fishes from different regions. Background Art

[0002] Fish of the genus Megalobrama are important medium-to-large freshwater economic fish in my country, among which the most famous is the amblycephalic bream (Wuchang fish), followed by the triangular bream, thick-jawed bream and Guangdong bream. In addition to the amblycephalic bream, the triangular bream has the most promising commercial breeding prospects. Studies have found that the triangular bream is similar to the amblycephalic bream in appearance, growth ability and meat quality, and is significantly better than the amblycephalic bream in resisting hemorrhagic disease caused by Aeromonas hydrophila (a disease that poses a huge threat to the amblycephalic bream industry). However, the variety breeding, breeding output and market recognition of the triangular bream are far inferior to those of the amblycephalic bream. Therefore, it is urgent to strengthen research on the germplasm characteristics of the triangular bream and the creation of new germplasm.

[0003] There are three main wild populations of triangular bream in my country, namely, triangular bream (MTQ, Qiantang River triangular bream) distributed in the Qiantang River Basin of Zhejiang Province, which is protected in the "Hangzhou National Qiantang River Triangular Bream Original Farm", triangular bream (MTJ) distributed in the Jinsha River Reservoir of Hong'an County, Hubei Province, which is protected in the "Hubei Hong'an Provincial Triangular Bream Original Farm", and triangular bream (MTH, Heilongjiang Triangular Bream) distributed in the Heilongjiang River Basin, also known as Faroese, is one of the famous "three flowers and five Luo" in the local area. Studies have found that MTH has a different genetic structure from MTQ and MTJ, and has undergone significant genetic differentiation, but MTQ and MTJ have the same genetic structure, and MTQ germplasm purity is higher. Therefore, MTH and MTQ are two ideal hybrid parents, suitable for the creation of new germplasm of excellent triangular bream. However, MTH and MTQ belong to two different geographical populations of the same species, and they are very similar in appearance. There is a lack of intuitive and obvious differences to distinguish the two populations, which is easy to cause confusion, and it is not convenient to compare the production performance of different hybrid combinations after hybridization, which brings difficulties to the creation and research of new germplasm. Therefore, there is an urgent need for a method that can distinguish the parents and hybrids when the Heilongjiang triangular bream and the Qiantangjiang triangular bream are hybridized, and then the hybrids can be tested at different stages under mixed culture conditions to see whether they have better production performance than the parents, so as to select new germplasm of superior triangular bream. Summary of the invention

[0004] In order to distinguish the parents and the hybrids when Heilongjiang triangular bream and Qiantangjiang triangular bream are hybridized, the present invention provides molecular marker primers, a kit and a distinguishing method for distinguishing Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids.

[0005] The present invention discloses a first molecular marker for distinguishing Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids. The primer pair of the molecular marker is primer HLJFLf4 and primer HLJFLr4. The nucleotide sequence of primer HLJFLf4 is shown in SEQ ID NO: 1; the nucleotide sequence of primer HLJFLr4 is shown in SEQ ID NO: 2.

[0006] The second molecular marker for distinguishing Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids of the present invention comprises a primer pair of HLJFLf5 and HLJFLr5; wherein the nucleotide sequence of primer HLJFLf5 is shown in SEQ ID NO: 3; and the nucleotide sequence of primer HLJFLr5 is shown in SEQ ID NO: 4.

[0007] A kit for distinguishing Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids, comprising a molecular marker primer pair HLJFLf4 and HLJFLr4.

[0008] Furthermore, the kit also includes deionized water, 10× Taq Buffer, MgCl2, dNTPs, Taq DNA polymerase, 10× LabFD TM Buffert and ApaLI endonuclease.

[0009] Another kit for distinguishing Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids comprises a molecular marker primer pair HLJFLf5 and HLJFLr5.

[0010] Furthermore, the kit also includes deionized water, 10× Taq Buffer, MgCl2, dNTPs, Taq DNA polymerase, 10× LabFD TM Buffert and SmaI endonuclease.

[0011] The first method to distinguish Heilongjiang triangular bream from Qiantangjiang triangular bream and their hybrids:

[0012] 1. Extract genomic DNA from the fin rays of the fish sample to be tested;

[0013] 2. Using the extracted genomic DNA as a template, PCR amplification was performed using primers HLJFLf4 and HLJFLr4 to obtain PCR products;

[0014] 3. Use restriction endonuclease ApaLI to digest the PCR product in step 2;

[0015] 4. Perform electrophoresis on the products after enzyme digestion in step 3. The individuals with double bands of 173bp and 160bp in the electrophoresis results are Heilongjiang triangular bream, and the individuals with only 333bp band are Qiantangjiang triangular bream. The individuals with 333bp, 173bp and 160bp bands at the same time are hybrids of Heilongjiang triangular bream and Qiantangjiang triangular bream.

[0016] The second method to distinguish Heilongjiang triangle bream from Qiantangjiang triangle bream and their hybrids:

[0017] 1. Extract genomic DNA from the fin rays of the fish sample to be tested;

[0018] 2. Using the extracted genomic DNA as a template, PCR amplification was performed using primers HLJFLf5 and HLJFLr5 to obtain PCR products;

[0019] 3. Use restriction endonuclease SmaI to digest the PCR product in step 2;

[0020] 4. Perform electrophoresis on the products after enzyme digestion in step 3. The individual with only 456bp band is Heilongjiang triangular bream, the individual with double bands of 257bp and 199bp is Qiantangjiang triangular bream; the individual with all 456bp, 257bp and 199bp bands is a hybrid of Heilongjiang triangular bream and Qiantangjiang triangular bream.

[0021] Methods for distinguishing the hybrid parents of Heilongjiang triangular bream and Qiantangjiang triangular bream:

[0022] Step 1: using the genomic DNA of the hybrid individuals of Heilongjiang triangular bream and Qiantangjiang triangular bream as a template, and using primers HLJFLf1 (as shown in SEQ ID NO: 5) and primer HLJFLr1 (as shown in SEQ ID NO: 6) as primers to perform PCR amplification; PCR amplification obtains a 380 bp band;

[0023] Step 2: digest the PCR product of step 1 with restriction endonuclease Taq I;

[0024] Step 3: For the enzyme digestion product of step 2, gel electrophoresis, the corresponding individuals with double bands of 180 bp and 200 bp are hybrids with Heilongjiang triangular bream as the female parent; the corresponding individuals with only 380 bp band are hybrids with Qiantangjiang triangular bream as the female parent.

[0025] The two pairs of molecular marker primers of the invention can quickly, efficiently and stably distinguish Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids.

[0026] The two methods of the present invention can quickly, efficiently and stably distinguish Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids, and can also distinguish the maternal sources of the hybrids.

[0027] By adopting the identification kit and method provided by the present invention, the Heilongjiang triangular bream and the Qiantangjiang triangular bream and their hybrids can be directly distinguished and identified, and the parental sources of the hybrids can be further distinguished. The method has high accuracy, simple operation process, short time, and the accuracy rate reaches 100%.

[0028] Two populations lacking intuitive and obvious differences are prone to confusion. The present invention can distinguish between parents and hybrids when the two are hybridized, compare the production performance of different hybrid combinations, and further detect whether the hybrids have better production performance than the parents at different stages under mixed culture conditions, so as to select excellent new germplasm of triangular bream. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the electrophoresis diagram of the enzyme digestion product of step 4 in Example 1;

[0030] Figure 2 This is the electrophoresis diagram of the enzyme digestion product of step 4 in Example 2;

[0031] Figure 3 This is the electrophoresis diagram of the enzyme digestion product in step 3 of Example 3. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not 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.

[0033] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0034] Specific implementation method 1: This implementation method distinguishes the molecular markers of Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids. The primer pair of the molecular marker is primer HLJFLf4 and primer HLJFLr4; wherein the nucleotide sequence of primer HLJFLf4 is shown in SEQ ID NO: 1; the nucleotide sequence of primer HLJFLr4 is shown in SEQ ID NO: 2.

[0035] Specific implementation method 2: This implementation method is a molecular marker for distinguishing Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids. The primer pair of the molecular marker is primer HLJFLf5 and primer HLJFLr5; wherein the nucleotide sequence of primer HLJFLf5 is shown in SEQ ID NO: 3; the nucleotide sequence of primer HLJFLr5 is shown in SEQ ID NO: 4.

[0036] Specific implementation method three: This implementation method is a kit for distinguishing Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids, and the kit includes a molecular marker primer pair HLJFLf4 and HLJFLr4.

[0037] Specific embodiment 4: The difference between this embodiment and specific embodiment 3 is that the kit also includes deionized water, 10×Taq Buffer, MgCl2, dNTPs, Taq DNA polymerase, 10×LabFD TM Buffert and ApaLI endonuclease. The other steps and parameters of this embodiment are the same as those of the third embodiment.

[0038] Specific implementation method 5: This implementation method provides a kit for distinguishing Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids, and the kit includes a molecular marker primer pair HLJFLf5 and HLJFLr5.

[0039] Specific embodiment 6: The difference between this embodiment and specific embodiment 5 is that the kit also includes deionized water, 10×Taq Buffer, MgCl2, dNTPs, Taq DNA polymerase, 10×LabFD TM Buffert and SmaI endonuclease. The other steps and parameters of this embodiment are the same as those of the fifth embodiment.

[0040] Specific implementation method seven: This implementation method distinguishes Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids:

[0041] 1. Extract genomic DNA from the fin rays of the fish sample to be tested;

[0042] 2. Using the extracted genomic DNA as a template, PCR amplification was performed using primers HLJFLf4 and HLJFLr4 to obtain PCR products; wherein the PCR amplification system is as follows:

[0043]

[0044]

[0045] ;PCR amplification conditions: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 30 s, a total of 30 cycles, then 72℃ extension for 5 min, and 4℃ insulation;

[0046] 3. Use restriction endonuclease ApaLI to digest the PCR product in step 2; wherein the digestion reaction system is as follows:

[0047]

[0048] ; Enzyme digestion conditions: 37℃ water bath digestion for 30min;

[0049] 4. Perform electrophoresis on the products after enzyme digestion in step 3. The individuals with double bands of 173bp and 160bp in the electrophoresis results are Heilongjiang triangular bream, and the individuals with only 333bp band are Qiantangjiang triangular bream. The individuals with 333bp, 173bp and 160bp bands at the same time are hybrids of Heilongjiang triangular bream and Qiantangjiang triangular bream.

[0050] Specific implementation method eight: This implementation method distinguishes Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids:

[0051] 1. Extract genomic DNA from the fin rays of the fish sample to be tested;

[0052] 2. Using the extracted genomic DNA as a template, PCR amplification was performed using primers HLJFLf5 and HLJFLr5 to obtain PCR products; wherein the PCR amplification system is as follows:

[0053]

[0054] ;PCR amplification conditions: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 30 s, a total of 30 cycles, then 72℃ extension for 5 min, and 4℃ insulation;

[0055] 3. Use restriction endonuclease SmaI to digest the PCR product in step 2; wherein the digestion reaction system is as follows:

[0056]

[0057] ; Enzyme digestion conditions: 25℃ water bath digestion for 30min;

[0058] 4. Perform electrophoresis on the products after enzyme digestion in step 3. The individual with only 456bp band is Heilongjiang triangular bream, the individual with double bands of 257bp and 199bp is Qiantangjiang triangular bream; the individual with all 456bp, 257bp and 199bp bands is a hybrid of Heilongjiang triangular bream and Qiantangjiang triangular bream.

[0059] Specific implementation method 9: the method for distinguishing the hybrid parents of Heilongjiang triangular bream and Qiantangjiang triangular bream in this implementation method:

[0060] Step 1: Using the genomic DNA of the hybrid individuals of Heilongjiang triangular bream and Qiantangjiang triangular bream as a template, PCR amplification was performed using primers HLJFLf1 (as shown in SEQ ID NO: 5) and primer HLJFLr1 (as shown in SEQ ID NO: 6) as primers; wherein the PCR amplification system is as follows:

[0061]

[0062] ; PCR amplification conditions: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 30s, a total of 30 cycles, then 72℃ extension for 5min, 4℃ insulation; PCR amplification obtained a 380bp band;

[0063] Step 2: digest the PCR product of step 1 with restriction endonuclease Taq I; wherein the digestion reaction system is as follows:

[0064]

[0065] ; Enzyme digestion conditions: 65℃ water bath digestion for 30min;

[0066] Step 3: For the enzyme digestion product of step 2, gel electrophoresis, the corresponding individuals with double bands of 180 bp and 200 bp are hybrids with Heilongjiang triangular bream as the female parent; the corresponding individuals with only 380 bp band are hybrids with Qiantangjiang triangular bream as the female parent.

[0067] Example 1

[0068] Methods for distinguishing Heilongjiang triangular bream from Qiantangjiang triangular bream and their hybrids:

[0069] 1. Extract genomic DNA from the fin rays of the fish sample to be tested (about 60 mg of fin ray tissue per fish);

[0070] 2. Using the extracted genomic DNA as a template, PCR amplification was performed using primers HLJFLf4 and HLJFLr4 to obtain PCR products; wherein the PCR amplification system is as follows:

[0071]

[0072] ;PCR amplification conditions: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 30 s, a total of 30 cycles, then 72℃ extension for 5 min, and 4℃ insulation;

[0073] 3. Use restriction endonuclease ApaLI to digest the PCR product in step 2; wherein the digestion reaction system is as follows:

[0074]

[0075]

[0076] ; Enzyme digestion conditions: 37℃ water bath digestion for 30min;

[0077] 4. The products after enzyme digestion in step 3 were subjected to 1.5% agarose gel electrophoresis. The individuals with double bands of 173bp and 160bp in the electrophoresis results were Heilongjiang triangular bream, and the individuals with only 333bp band were Qiantangjiang triangular bream. The individuals with 333bp, 173bp and 160bp bands at the same time were hybrids of Heilongjiang triangular bream and Qiantangjiang triangular bream.

[0078] In this embodiment, the PCR products of step 2 are all 333 bp; among them, the DNA fragment of the PCR product of Heilongjiang triangular bream is sequenced as shown in SEQ ID NO: 7; the DNA fragment of the PCR product of Qiantangjiang triangular bream is sequenced as shown in SEQ ID NO: 8.

[0079] Eight triangular breams with clear population relationships were selected and numbered and tested using the method of this embodiment (wherein individuals numbered 1, 3, 7, and 8 were Heilongjiang triangular breams, individuals numbered 2 and 6 were hybrids of Heilongjiang triangular breams and Qiantangjiang triangular breams, and individuals numbered 4 and 5 were Qiantangjiang triangular breams); the products after enzyme digestion in step 3 were loaded into the corresponding swimming lanes according to the individual numbers, and the electrophoresis results of the enzyme digestion products were as follows: Figure 1 shown. Figure 1 The electrophoresis results show that the 333bp bands of the individuals numbered 1, 3, 7, and 8 are cut into double bands of 173bp and 160bp (the double bands are not separated in the figure, and do not affect the discrimination), indicating that these four individuals are Heilongjiang triangular bream; the individuals numbered 2 and 6 have three bands of 333bp, 173bp, and 160bp (173bp and 160bp are not separated in the figure, and do not affect the discrimination), indicating that these two individuals are hybrids of Heilongjiang triangular bream and Qiantangjiang triangular bream; the individuals numbered 4 and 5 only have a band of 333bp, indicating that these two individuals are Qiantangjiang triangular bream. The identification results of this embodiment are consistent with the actual situation, proving that the accuracy of the method of the present invention in identifying Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids is 100%.

[0080] Example 2

[0081] Methods for distinguishing Heilongjiang triangular bream from Qiantangjiang triangular bream and their hybrids:

[0082] 1. Extract genomic DNA from the fin rays of the fish sample to be tested (about 60 mg of fin ray tissue per fish);

[0083] 2. Using the extracted genomic DNA as a template, PCR amplification was performed using primers HLJFLf5 and HLJFLr5 to obtain PCR products; wherein the PCR amplification system is as follows:

[0084]

[0085]

[0086] ;PCR amplification conditions: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 30 s, a total of 30 cycles, then 72℃ extension for 5 min, and 4℃ insulation;

[0087] 3. Use restriction endonuclease SmaI to digest the PCR product in step 2; wherein the digestion reaction system is as follows:

[0088]

[0089] ; Enzyme digestion conditions: 25℃ water bath digestion for 30min;

[0090] 4. The products after enzyme digestion in step 3 were detected by 1.5% agarose gel electrophoresis. The corresponding individuals with only 456bp band were Heilongjiang triangular bream, and the corresponding individuals with double bands of 257bp and 199bp were Qiantangjiang triangular bream; the individuals with all the 456bp, 257bp and 199bp bands were hybrids of Heilongjiang triangular bream and Qiantangjiang triangular bream.

[0091] In this embodiment, the PCR products in step 2 are all 456 bp; among them, the DNA fragment of the PCR product of Heilongjiang triangular bream is sequenced as shown in SEQ ID NO: 9; the DNA fragment of the PCR product of Qiantangjiang triangular bream is sequenced as shown in SEQ ID NO: 10.

[0092] Eight triangular breams with clear population relationships were selected and numbered and tested using the method of this embodiment (wherein individuals numbered 1, 7, and 8 were Heilongjiang triangular breams, individuals numbered 2, 3, and 6 were hybrids of Heilongjiang triangular breams and Qiantangjiang triangular breams, and individuals numbered 4 and 5 were Qiantangjiang triangular breams); the products after enzyme digestion in step 3 were loaded into the corresponding swimming lanes according to the individual numbers, and the electrophoresis results of the enzyme digestion products were as follows: Figure 2 shown. Figure 2The electrophoresis results showed that the individuals numbered 1, 7, and 8 only had a band of 456 bp, indicating that these three individuals were Heilongjiang triangular bream; the individuals numbered 2, 3, and 6 had three bands of 456 bp, 257 bp, and 199 bp, indicating that these three individuals were hybrids of Heilongjiang triangular bream and Qiantangjiang triangular bream; the individuals numbered 4 and 5 had two bands of 257 bp and 199 bp, indicating that these two individuals were Qiantangjiang triangular bream. The identification results of this embodiment are consistent with the actual situation, proving that the accuracy of the method of the present invention in identifying Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids is 100%.

[0093] Example 3

[0094] Methods for distinguishing the hybrid parents of Heilongjiang triangular bream and Qiantangjiang triangular bream:

[0095] Step 1: Using the genomic DNA of the hybrid individuals of Heilongjiang triangular bream and Qiantangjiang triangular bream as a template, PCR amplification was performed using primers HLJFLf1 (as shown in SEQ ID NO: 5) and primer HLJFLr1 (as shown in SEQ ID NO: 6) as primers; wherein the PCR amplification system is as follows:

[0096]

[0097] 100ng / μL genomic DNA 0.5μL

[0098] ; PCR amplification conditions: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 30s, a total of 30 cycles, then 72℃ extension for 5min, 4℃ insulation; PCR amplification obtained a 380bp band;

[0099] Step 2: digest the PCR product of step 1 with restriction endonuclease Taq I; wherein the digestion reaction system is as follows:

[0100]

[0101] ; Enzyme digestion conditions: 65℃ water bath digestion for 30min;

[0102] Step 3: Perform 1.5% agarose gel electrophoresis on the enzyme digestion products of step 2. The corresponding individuals with double bands of 180 bp and 200 bp are hybrids with Heilongjiang triangular bream as the female parent; the corresponding individuals with only a 380 bp band are hybrids with Qiantangjiang triangular bream as the female parent.

[0103] Eight hybrid breams with clear parental relationships were selected (one of the male and female parents was the Heilongjiang bream and the other was the Qiantangjiang bream), and the numbering was followed by detection using the method of this embodiment (wherein the individuals numbered 1, 5-7 were hybrids with the Qiantangjiang bream as the female parent and the Heilongjiang bream as the male parent; the individuals numbered 2-4, 8 were hybrids with the Heilongjiang bream as the female parent and the Qiantangjiang bream as the male parent); the products after enzyme digestion in step 2 were loaded into the corresponding swimming lanes according to the individual numbers, and the electrophoresis results of the enzyme digestion products were as follows: Figure 3 shown. Figure 3 The electrophoresis results showed that the individuals numbered 1, 5-7 had a single band of 380 bp, indicating that these four individuals were hybrids whose maternal parent was Qiantangjiang triangular bream and whose paternal parent was Heilongjiang triangular bream; the individuals numbered 2-4, 8 had double bands of 180 bp and 200 bp (the double bands were not separated in the figure and did not affect the identification), indicating that these four individuals were hybrids whose maternal parent was Heilongjiang triangular bream and whose paternal parent was Qiantangjiang triangular bream. The identification results of this embodiment are consistent with the actual situation, proving that the accuracy of the method of the present invention in identifying the parental origin of the hybrid of Heilongjiang triangular bream and Qiantangjiang triangular bream is 100%.

Claims

1. A molecular marker for distinguishing Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids, characterized in that: The primer pair of the molecular marker is primer HLJFLf5 and primer HLJFLr5; wherein the nucleotide sequence of primer HLJFLf5 is shown in SEQ ID NO:3; and the nucleotide sequence of primer HLJFLr5 is shown in SEQ ID NO:

4.

2. A kit for distinguishing Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids, characterized in that: The kit comprises the molecular marker primer pair HLJFLf5 and HLJFLr5 described in claim 1.

3. The kit according to claim 2, characterized in that The kit also includes deionized water, 10× Taq Buffer, MgCl2, dNTPs, Taq DNA polymerase, 10×LabFD TM Buffert and SmaI endonuclease.

4. A method for distinguishing Heilongjiang triangular bream and Qiantangjiang triangular bream and their hybrids, characterized in that: The method proceeds as follows:

1. Extract genomic DNA from the fin rays of the fish sample to be tested; 2. Using the extracted genomic DNA as a template, PCR amplification was performed using primers HLJFLf5 and HLJFLr5 to obtain PCR products; 3. Use restriction endonuclease SmaI to digest the PCR product in step 2; Fourth, the product after the enzyme digestion in step 3 is subjected to electrophoresis detection. The corresponding individual with only 456bp band in the electrophoresis result is Heilongjiang triangular bream, and the corresponding individual with double bands of 257bp and 199bp is Qiantangjiang triangular bream; the individual with 456bp, 257bp and 199bp bands at the same time is a hybrid of Heilongjiang triangular bream and Qiantangjiang triangular bream; The nucleotide sequence of primer HLJFLf5 is shown in SEQ ID NO:3; the nucleotide sequence of primer HLJFLr5 is shown in SEQ ID NO:4.

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