Specific DNA fragment combination for distinguishing female acipenser baeri from female huso dauricus and application thereof

Abstract

The application belongs to the field of fish species and gender identification in the field of aquaculture, and discloses a specific DNA fragment combination for distinguishing female Russian sturgeon and female Kaluga sturgeon and application thereof. The application is a gender-specific DNA fragment for distinguishing female Russian sturgeon and female Kaluga sturgeon, which is successfully screened through conventional PCR amplification, sequencing and sequence comparison analysis, and a primer is designed to accurately and quickly identify the gender of Russian sturgeon and Kaluga sturgeon, distinguish female Russian sturgeon and female Kaluga sturgeon, and identify the gender and maternal origin of the hybrid offspring, and the primer sequence, PCR reaction system and conditions are disclosed. The identification method is simple, fast, accurate and has little harm to the fish body, and solves the problems of gender identification of Russian sturgeon and Kaluga sturgeon, maternal identification of hybrid sturgeon and caviar identification, which will help the development of sturgeon caviar industry.

Description

Technical Field

[0001] This invention belongs to the field of fish species and sex identification in aquaculture, specifically involving the combination and application of specific DNA fragments for distinguishing female Russian sturgeon and female Kaluga sturgeon. Background Technology

[0002] Sturgeon (collectively known as sturgeon), belonging to the subclass Actinopterygii and the infraclass Cartilaginous fish, are among the oldest fish species. Sturgeon are large fish with significant economic value, primarily in their caviar. Sturgeon caviar is made by curing sturgeon eggs and is rich in essential amino acids, highly unsaturated fatty acids (EPA, DHA), inorganic salts, vitamins A, B, and D, as well as trace elements such as calcium, copper, magnesium, iron, and selenium. Most caviar currently on the market comes from species such as the Russian sturgeon, short-snout sturgeon, European sturgeon, and Kaluga sturgeon. To protect wild sturgeon populations and enrich sturgeon species diversity, researchers are actively engaged in sturgeon hybridization breeding. Although there are differences in chromosome ploidy among some different sturgeon species, they still exhibit a high degree of similarity at the genomic sequence level, providing a basis for intra- and inter-generic hybridization.

[0003] Russian sturgeon and Kaluga sturgeon are not only two core sources of sturgeon caviar, but also recognized as among the most famous and precious species. Currently, researchers are conducting hybridization of the two species to obtain varieties with superior traits. However, effectively distinguishing between purebreds, hybrids, and caviar origins at the molecular level remains challenging. Publicly available information on sturgeon sex identification involving both Russian sturgeon and Kaluga sturgeon, such as application / patent numbers 201910066586.4 and 202410028698.1, does not mention the ability to distinguish female Russian sturgeon and female Kaluga sturgeon using sex-specific marker DNA fragments. While SSR technology is commonly used for sturgeon germplasm identification, there are no reports of specific DNA sequences used to differentiate between these two species.

[0004] To address the issues of germplasm and sex identification, this invention obtains a set of DNA sequences and corresponding detection primers through conventional PCR amplification and sequence alignment. These sequences can not only effectively distinguish the maternal origins of female Russian sturgeon, female Kaluga sturgeon, and their hybrid offspring, but can also be used for sex identification. Summary of the Invention

[0005] The first objective of this invention is to provide a specific combination of DNA fragments for identifying the maternal origin of female Russian sturgeon, female Kaluga sturgeon, or hybrid sturgeon, said combination of fragments comprising the polynucleotides shown in SEQ ID NO.1 and SEQ ID NO.2.

[0006] Another object of the present invention is to provide a reagent for detecting the above-mentioned DNA fragment combinations.

[0007] The final objective of this invention is to provide the application of the aforementioned DNA fragment combinations and reagents. The DNA fragments or reagents can be used to distinguish the maternal origins of female Russian sturgeon, female Kaluga sturgeon, and hybrid sturgeon, solving the problems of germplasm and sex identification of the maternal origins of female Russian sturgeon, female Kaluga sturgeon, and hybrid sturgeon, and promoting the development of sturgeon farming and the caviar industry.

[0008] To achieve the above objectives, the present invention adopts the following technical measures:

[0009] A specific DNA fragment combination for identifying the maternal origin of female Russian sturgeon, female Kaluga sturgeon, or hybrid sturgeon, the fragment combination comprising the polynucleotides shown in SEQ ID NO.1 and SEQ ID NO.2, wherein the hybrid sturgeon is Russian sturgeon × Kaluga sturgeon.

[0010] The scope of protection of this invention also includes:

[0011] Reagents for detecting the above DNA fragment combinations;

[0012] The reagents described above, preferably PCR primers, can all be used to detect the above-mentioned DNA fragment combinations, and all can realize the present invention.

[0013] The primers described above are preferably F: 5`-CCGAACCATGTGACTTGCGTA-3' and R: 5`-TGTCATTCCGCACAAATCCTT-3'. This invention provides one primer pair for the detection of the sequences shown in SEQ ID NO.1 and SEQ ID NO.2. Other primer pairs with added or removed bases can also be used to detect these sequences.

[0014] The application of reagents for detecting the above-mentioned DNA fragment combinations in the identification of maternal germplasm of female Russian sturgeon, female Kaluga sturgeon, or hybrid sturgeon, wherein the hybrid sturgeon is Russian sturgeon × Kaluga sturgeon.

[0015] A method for identifying the maternal germplasm source of female Russian sturgeon, female Kaluga sturgeon, or hybrid sturgeon, comprising the following steps: detecting the presence of the polynucleotide shown in SEQ ID NO.1 or SEQ ID NO.2 in the genome to be tested by genome sequencing or PCR; if the polynucleotide shown in SEQ ID NO.1 is present, the individual is a female Russian sturgeon; if the polynucleotide shown in SEQ ID NO.2 is present, the individual is a female Kaluga sturgeon; if the hybrid sturgeon (Russian sturgeon × Kaluga sturgeon) contains the polynucleotide shown in SEQ ID NO.1, its maternal parent is Russian sturgeon; if it contains the polynucleotide shown in SEQ ID NO.2, its maternal parent is Kaluga sturgeon.

[0016] Compared with the prior art, the present invention has the following advantages and effects:

[0017] The sex-specific DNA fragment combination provided by this invention achieves 100% accuracy in verifying the germplasm and sex of female Russian sturgeon, female Kaluga sturgeon, and hybrid sturgeon (Russian sturgeon × Kaluga sturgeon) maternal sources using conventional PCR amplification. Compared with previous methods using microsatellite (SSR) technology to identify sturgeon germplasm, this technology is accurate, simple, and rapid, while also having the function of sex detection, thus contributing to the development of the sturgeon farming industry. Attached Figure Description

[0018] Figure 1 This is a schematic diagram showing the sequence alignment of female sex-specific DNA fragments of Russian sturgeon and female sex-specific DNA fragments of Kaluga sturgeon.

[0019] Figure 2 A schematic diagram illustrating the results of identifying female sex-specific DNA fragments in Russian sturgeon;

[0020] In the figure, lanes 1-12 show no band amplification in male individuals, while lanes 13-24 show a specific 935 bp band amplification in female individuals. C represents the negative control, and M represents the DL2000 DNA marker.

[0021] Figure 3 A schematic diagram illustrating the results of identifying female sex-specific DNA fragments in the Kaluga sturgeon;

[0022] In the figure, lanes 1-12 show no band amplification in male individuals, while lanes 13-24 show a 726 bp specific band amplified in female individuals. C represents the negative control, and M represents the DL2000 DNA marker.

[0023] Figure 4 This is a schematic diagram showing the results of identifying female sex-specific DNA fragments in hybrid sturgeon (Russian sturgeon ♀ × Kaluga sturgeon ♂).

[0024] In the figure, lanes 1-12 show no band amplification in male individuals, while lanes 13-24 show a specific 935 bp band amplification in female individuals. C represents the negative control, and M represents the DL2000 DNA marker. Detailed Implementation

[0025] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0026] Example 1:

[0027] Obtaining female sex-specific DNA fragment sequences from Russian sturgeon and Kaluga sturgeon:

[0028] Twelve male and twelve female Russian sturgeon and 12 male Kaluga sturgeon were identified using paraffin sections of gonadal tissue. Their whole-genome DNA was extracted using a high-salt method. Universal primers were designed, PCR amplification was performed, and Sanger sequencing was used to obtain two common but distinct female-specific DNA fragments. Corresponding primers were designed for population validation of the effectiveness of these methods. Finally, female-specific DNA fragments were obtained for Russian sturgeon (SEQ ID NO.1) and Kaluga sturgeon (SEQ ID NO.2). No homologous sequences were found in the GenBank database, and the sequence differences between the two fragments enabled the differentiation of the two female sturgeon species. Figure 1 Therefore, the DNA fragments shown in SEQ ID NO.1 and SEQ ID NO.2 can be used as a molecular marker combination for the identification of the two female sturgeon species.

[0029] The primers designed for the above DNA fragment combinations are:

[0030] F: 5`-CCGAACCATGTGACTTGCGTA-3' and R: 5`-TGTCATTCCGCAAAATCCTT-3'.

[0031] Using the primers described above, a 935 bp band was specifically amplified in female Russian sturgeon (SEQ ID NO.1), while no band was amplified in male Russian sturgeon. A 726 bp band was specifically amplified in female Kaluga sturgeon (SEQ ID NO.2), while no band was amplified in male Kaluga sturgeon.

[0032] Example 2:

[0033] Application of primers for detecting DNA fragment combinations obtained in Example 1 in female Russian sturgeon and female Kaluga sturgeon:

[0034] 1) Fin tissue samples of 12 male and 12 female Russian sturgeon were preserved in anhydrous ethanol. Genomic DNA was extracted using the high-salt method, diluted to 50 ng / μL, and stored at -20℃ for later use.

[0035] 2) Twelve fin tissue samples from each of the known male and female Kaluga sturgeon were preserved in anhydrous ethanol. Genomic DNA was extracted using the high-salt method, diluted to 50 ng / μL, and stored at -20℃ for later use.

[0036] 3) PCR amplification:

[0037] The reaction system consisted of approximately 50 ng of sturgeon DNA obtained in step 1) or 2); 1.5 U of Taq polymerase; 2.5 μl of 10× amplification buffer; 200 μM concentrations of the four dNTPs; 0.2 μM final concentrations of the forward and reverse primers; and ddH2O added to 25 μl. The primers were F: 5'-CCGAACCATGTGACTTGCGTA-3' and R: 5'-TGTCATTCCGCACAAATCCTT-3'.

[0038] The PCR reaction conditions corresponding to the primers were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 7 min; and storage at 4℃.

[0039] 4) Results are as follows Figure 2 and Figure 3 As shown:

[0040] After PCR amplification, a 1.5% agarose gel was prepared for electrophoresis detection. Figure 2 The results showed that female Russian sturgeon amplified a specific 935bp band, while male Russian sturgeon individuals were unable to amplify the band. Figure 3 The results showed that female Kaluga sturgeon amplified a specific 726bp band, while male Kaluga sturgeon did not amplify the specific band. The interspecific and sex determination results using the specific DNA fragment combinations provided by this invention are completely consistent with the actual situation.

[0041] Example 3:

[0042] Application of primers for detecting DNA fragment combinations obtained in Example 1 in the identification of hybrid sturgeon (Russian sturgeon ♀ × Kaluga sturgeon ♂):

[0043] 1) Fin tissue samples of 12 male and 12 female hybrid sturgeon (Russian sturgeon ♀ × Kaluga sturgeon ♂) were preserved in anhydrous ethanol, and their genomic DNA was extracted using the high-salt method, diluted to 50 ng / μL and stored at -20℃ for later use.

[0044] 2) PCR amplification:

[0045] The reaction system consisted of approximately 50 ng of template DNA; 1.5 U of Taq polymerase; 2.5 μl of 10× amplification buffer; 200 μM concentrations of four dNTPs; 0.2 μM final concentrations of forward and reverse primers; and ddH2O added to a final volume of 25 μl. The primers were F: 5'-CCGAACCATGTGACTTGCGTA-3' and R: 5'-TGTCATTCCGCACAAATCCTT-3'.

[0046] The PCR reaction conditions corresponding to the primers were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 7 min; and storage at 4℃.

[0047] 3) Results are as follows Figure 4 As shown:

[0048] After PCR amplification, a 1.5% agarose gel was prepared for electrophoresis. A specific 935bp band was amplified in female hybrid sturgeon individuals whose maternal parent was Russian sturgeon. No specific band was amplified in male hybrid sturgeon individuals. Figure 4 This is completely consistent with the actual situation.

Claims

1. A specific combination of DNA fragments for identifying the maternal origin of female Russian sturgeon, female Kaluga sturgeon, or hybrid sturgeon, said combination of fragments comprising the polynucleotides shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. The application of the reagent for detecting the DNA fragment combination described in claim 1 in the identification of maternal germplasm of female Russian sturgeon, female Kaluga sturgeon, or hybrid sturgeon, wherein the hybrid sturgeon is obtained by crossing Russian sturgeon and Kaluga sturgeon, and the application process includes: The presence of the polynucleotide shown in SEQ ID NO.1 or SEQ ID NO.2 in the genome to be tested indicates the presence of a female Russian sturgeon individual if the polynucleotide shown in SEQ ID NO.1 is present, and a female Kaluga sturgeon individual if the polynucleotide shown in SEQ ID NO.2 is present. If the hybrid sturgeon contains the polynucleotide shown in SEQ ID NO.1, its maternal parent is a Russian sturgeon; if it contains the polynucleotide shown in SEQ ID NO.2, its maternal parent is a Kaluga sturgeon.

3. The application according to claim 2, wherein the reagent is a primer.

4. The application according to claim 3, wherein the primers are F: 5'-CCGAACCATGTGACTTGCGTA-3' and R: 5'-TGTCATTCCGCACAAATCCTT-3'.

5. A method for identifying the maternal germplasm of female Russian sturgeon, female Kaluga sturgeon, or hybrid sturgeon, comprising the following steps: The presence of the polynucleotide shown in SEQ ID NO.1 or SEQ ID NO.2 in the genome of the target organism is detected by genome sequencing or PCR. If the polynucleotide shown in SEQ ID NO.1 is present, the organism is a female Russian sturgeon; if the polynucleotide shown in SEQ ID NO.2 is present, the organism is a female Kaluga sturgeon. If the hybrid sturgeon contains the polynucleotide shown in SEQ ID NO.1, its maternal parent is a Russian sturgeon; if it contains the polynucleotide shown in SEQ ID NO.2, its maternal parent is a Kaluga sturgeon. The hybrid sturgeon mentioned above was obtained by crossing Russian sturgeon and Kaluga sturgeon.

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