Specific molecular markers, primers and kits for identifying southern catfish, catfish and their hybrids

Abstract

The present invention provides a specific molecular marker, primer and kit for rapid identification of southern catfish, catfish and their hybrids. The primer sequences are shown in SEQ ID: No.1 and SEQ ID: No.2. The specific molecular marker is located in the gene gsdf The molecular marker for the southern catfish is 595 bp long, with the nucleotide sequence shown in SEQ ID No. 3. The molecular marker for the catfish is 522 bp long, with the nucleotide sequence shown in SEQ ID No. 4. The identification process of the present invention requires only the clipping of a small number of fin rays, extraction of genomic DNA, PCR amplification using the primers, and subsequent agarose gel electrophoresis. It also has high accuracy and is not restricted by fish growth, allowing sampling and identification at various stages of breeding. It can be widely used in scientific research and promotion institutions, seedling breeding farms, and aquaculture businesses. It can be used to identify seedlings of the three catfish species (southern catfish, catfish, and their hybrids) and to establish germplasm standards for the southern catfish, catfish, and their hybrids.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and in particular relates to a molecular marker, primer, method and kit for accurately identifying southern catfish, catfish and their hybrids. Background Art

[0002] The southern catfish (Silurus meridionalis) and the silver catfish (Silurus asotus) both belong to the order Siluriformes, family Siluridae, and genus Silurus. Their appearance is strikingly similar, making them difficult to distinguish with the naked eye. The southern catfish, also known as the large-mouthed catfish, is primarily found in the Yangtze, Pearl, and Minjiang rivers. It boasts excellent aquaculture traits, including large size, rapid growth, strong adaptability, and delicious meat, making it an important large and medium-sized freshwater fish for economic aquaculture in my country. The silver catfish, also known as the native catfish or small-mouthed catfish, is smaller and grows more slowly than the southern catfish.

[0003] Compared with catfish, southern catfish has the advantages of no intramuscular spines, more delicate and tender meat, and high breeding profits, and is favored by fishery merchants and consumers. However, in view of the vast consumer market and significant economic benefits of southern catfish, some unscrupulous vendors in the market often use catfish or hybrid catfish of the two (southern catfish♀×catfish♂) to impersonate southern catfish for sale in order to obtain high profits. For aquaculture fishery merchants, it is difficult to accurately identify the fry when purchasing them, which can easily lead to unnecessary economic losses. Furthermore, hybrid breeding is common among Siluriformes fish. In order to evaluate the germplasm resources of Siluriformes fish, it is very necessary to carry out Siluriformes fish population identification.

[0004] DNA molecular markers, primers designed based on DNA sequence differences between species, can accurately identify species and are widely used in species identification, sex determination, and paternity testing. Due to the similar appearance of the southern catfish, catfish, and their hybrids, previous identification using morphological methods has been difficult and prone to errors. Therefore, the design of DNA molecular markers that can stably and efficiently identify the southern catfish, catfish, and their hybrids is of great scientific and practical significance. Summary of the Invention

[0005] The first object of the present invention is to provide a specific molecular marker for identifying southern catfish, catfish and their hybrids.

[0006] The second object of the present invention is to provide a primer for identifying southern catfish, catfish and their hybrids based on the specific molecular marker.

[0007] The third object of the present invention is to provide a detection kit for identifying southern catfish, catfish and their hybrids.

[0008] The fourth object of the present invention is to provide a method for identifying southern catfish, catfish and their hybrids.

[0009] The object of the present invention is achieved according to the following technical solutions:

[0010] A specific molecular marker for identifying southern catfish, Silurus meridionalis, and their hybrids. The molecular marker is located in the region from the second exon to the third exon of the gene gsdf. The molecular marker for the southern catfish is 595 bp long, with a nucleotide sequence as shown in SEQ ID No. 3, and is named SM. The molecular marker for the Silurus meridionalis is 522 bp long, with a nucleotide sequence as shown in SEQ ID No. 4, and is named SA.

[0011] The invention relates to a primer for identifying southern catfish, catfish and their hybrids. The upstream primer for PCR amplification of the above-mentioned specific molecular marker is F, and its nucleotide sequence is shown in SEQ ID No.1; the downstream primer is R, and its nucleotide sequence is shown in SEQ ID No.2.

[0012] A detection kit for identifying southern catfish, catfish and their hybrids, the kit contains the above-mentioned specific primers, the upstream primer is F, its nucleotide sequence is shown in SEQ ID No.1, and the downstream primer is R, its nucleotide sequence is shown in SEQ ID No.2.

[0013] A method for identifying southern catfish, catfish and their hybrids comprises the following steps:

[0014] A. PCR Amplification: Fin rays of the southern catfish, Silurus meridionalis, and their hybrids were collected to extract genomic DNA. Using the resulting genomic DNA as a template, PCR amplification was performed using the upstream primer (5'-GAGCCTGATGTGTCCGTTCCT-3') and the downstream primer (5'-AACTTGCAGCATTGGAGGTGTG-3') shown. The resulting PCR amplification product was compared with the gsdf-specific molecular markers described above for the southern catfish and Silurus meridionalis.

[0015] B. Result Interpretation: Only a 595 bp DNA fragment was amplified from the genomic DNA of the test individual, and the nucleotide sequence was shown as SEQ ID No. 3, indicating that the species was Silurus meridionalis; only a 522 bp DNA fragment was amplified from the genomic DNA of the test individual, and the nucleotide sequence was shown as SEQ ID No. 4, indicating that the species was Silurus meridionalis; two DNA fragments, the 595 bp and 522 bp DNA fragments, were amplified from the genomic DNA of the test individual, indicating that the species was a hybrid of Silurus meridionalis and Silurus meridionalis.

[0016] The application of the above-mentioned specific molecular markers or primers in identifying southern catfish, catfish and their hybrids.

[0017] The application of the above-mentioned specific molecular markers or primers in the preparation of a kit for identifying southern catfish, catfish and their hybrids.

[0018] The present invention has the following beneficial effects:

[0019] The present invention obtains specific sequences in the genomes of southern catfish and catfish through a specific design method and a large number of experimental verifications, and screens out a pair of specific primers that can accurately identify southern catfish, catfish and their hybrids. The identification process only requires cutting a small amount of fin rays, extracting genomic DNA, and using the primers for PCR amplification and then performing agarose gel electrophoresis; at the same time, the accuracy rate is high. This method has the advantages of simple operation, low cost, high efficiency and stability, accuracy and reliability, and is not restricted by the growth of the fish body, and can be sampled and identified at each breeding stage. This kit can serve scientific research and promotion units that study southern catfish and catfish, seedling production enterprises (breeding farms) and breeding merchants. On the one hand, it can be used to identify seedlings of three catfish (southern catfish, catfish and its hybrids). On the other hand, it can be used for the formulation of germplasm standards for southern catfish, catfish and its hybrids. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram of the gsdf gene structure on the autosomes of the southern catfish and catfish described in the present invention. The boxes represent exons 1-5, and the introns are represented by black lines. The numbers below the boxes and above the lines represent the lengths of the exons and introns, respectively. The position, sequence and amplification length of the two positions F and R primers are marked in the figure.

[0021] Figure 2 This is a sequence alignment diagram of Silurus meridionalis and Silurus siliqua amplified by the primers of the present invention. The primer positions are marked with arrows, the black background is the consistent sequence of Silurus meridionalis and Silurus siliqua, "-" indicates a missing sequence, SM indicates Silurus meridionalis, and SA indicates Silurus siliqua.

[0022] Figure 3 The results are as follows: 8 southern catfish, 8 catfish and 8 hybrid catfish were species identified using the primers of the present invention; M represents the DNA molecular weight standard DL2000. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0024] Example 1

[0025] A method for obtaining molecular markers and primers for identifying southern catfish, catfish, and their hybrids comprises the following steps:

[0026] The southern catfish and the Silurus are two closely related species belonging to the order Siluriformes, family Siluridae, and genus Silurus. The laboratory previously conducted whole-genome sequencing on the southern catfish and the Silurus. Whole-genome comparison analysis found that the DNA sequences of the southern catfish and the Silurus were highly homologous, especially in the gene region, with a homology of more than 90%. The gsdf gene sequences of the southern catfish and the Silurus were extracted and compared and analyzed. It was found that the gsdf gene structures of the two were the same, both with 5 exons, and the exon sequence homology was as high as 96.7%, but there were significant differences in intron length, such as Figure 1 Taking advantage of the difference in the length of the second intron of the gsdf gene between the southern catfish and the Silurus catfish (398 bp in the southern catfish and 328 bp in the Silurus catfish), we designed primers F (5'-GAGCCTGATGTGTCCGTTCCT-3', as shown in SEQ ID NO. 1) and R (5'-AACTTGCAGCATTGGAGGTGTG-3', as shown in SEQ ID NO. 2) at the consensus sequences in the second and third exons, respectively. PCR amplification and sequencing analysis were performed on the genomes of the southern catfish, the Silurus catfish, and their hybrids. The results showed that the length of the sequence amplified by this primer combination was clearly species-specific. The product amplified by the primers was cut out of the agarose gel containing the amplified product with a blade, the target fragment was recovered, cloned into the Peasy-T3 vector, and then transformed into Escherichia coli competent cells. The positive clones were screened by the blue-white screening method, and the white spot single colonies were picked. The positive clones were identified by PCR and sequenced using an ABI3730 sequencer. It was finally determined that the primer combination could amplify a specific band of 595 bp in length in southern catfish individuals, named SM, and its nucleotide sequence is shown in SEQID NO: 3. A specific band of 522 bp in length was amplified in catfish individuals, named SA, and its nucleotide sequence is shown in SEQID NO: 4. Two bands of 595 bp and 522 bp were amplified simultaneously in hybrid catfish individuals. The sequence alignment analysis of SM and SA is shown in the figure. Figure 2 .

[0027] Example 2

[0028] A method for identifying southern catfish, catfish and their hybrids comprises the following steps:

[0029] A. Genomic DNA extraction: 10 mg of fin ray of southern catfish, catfish and their hybrids were cut and placed in lysis buffer [10 mM Tris-HCl (pH 8.0) + 100 mM EDTA (pH 8.0) + 100 mM NaCl + 5 mg / ml The cell suspension was homogenized in 600 μl of SDS-containing solution, proteinase K at a final concentration of 20 mg / ml and ribonuclease A (RNase A) at a final concentration of 100 μg / ml were added, and digested in a 55°C water bath until clear. 600 μl of a phenol-chloroform-isoamyl alcohol (volume ratio of 25:24:1) mixture was added and mixed thoroughly. The mixture was centrifuged at 12,000 rpm for 10 minutes. The supernatant was aspirated and DNA was extracted by adding a phenol-chloroform-isoamyl alcohol (volume ratio of 25:24:1) mixture again. The DNA was centrifuged for 10 minutes, the supernatant was aspirated, and an equal volume of isopropanol was added to precipitate the DNA. The DNA was centrifuged for 10 minutes, and the supernatant was discarded. The DNA precipitate was washed with 70% ethanol by volume, dried naturally, and then dissolved in TE buffer to prepare a solution with a concentration of 20 ng / μl (OD260 / OD280 = 1.76-1.80). The solution was stored at -20°C for later use.

[0030] B. PCR Amplification: Using the obtained genomic DNA as a template, PCR amplification was performed using the upstream primer F shown in SEQ ID NO. 1 and the downstream primer R shown in SEQ ID NO. 2. PCR amplification parameters were: initial denaturation at 94°C for 3 minutes, followed by 32 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 40 seconds, with a final extension at 72°C for 10 minutes. The resulting PCR products were subjected to 2% agarose gel electrophoresis.

[0031] C. Result evaluation: When only a 595 bp specific band is amplified from the genomic DNA of the tested southern catfish, catfish, and its hybrid, and the nucleotide sequence is shown as SEQ ID No. 3, the species is determined to be the southern catfish; when only a 522 bp specific band is amplified from the genomic DNA of the tested southern catfish, catfish, and its hybrid, and the nucleotide sequence is shown as SEQ ID No. 4, the species is determined to be the catfish; when two bands, namely the 595 bp and 522 bp specific bands, are simultaneously amplified from the genomic DNA of the tested southern catfish, catfish, and its hybrid, the species is determined to be the southern catfish and a catfish hybrid.

[0032] The results of population identification of 8 southern catfish, 8 southern catfish and 8 southern catfish-catfish hybrids using the above method are shown in Figure 3 Only the 595bp-long specific band was amplified in all southern catfish individuals, and only the 522bp-long Y chromosome-specific band was amplified in all catfish individuals. However, both the 595bp-long specific band and the 522bp-long specific band were amplified in hybrid individuals, which was consistent with the theoretical results.

[0033] Example 3

[0034] A kit for rapid identification of southern catfish, catfish and their hybrids:

[0035] The kit contains primers for identifying southern catfish, catfish and their hybrids; the primer sequences are as follows:

[0036] Upstream primer F: 5′-GAGCCTGATGTGTCCGTTCCT-3′ (SEQ ID NO: 1)

[0037] Downstream primer R: 5′-AACTTGCAGCATTGGAGGTGTG-3′ (SEQ ID NO: 2)

[0038] At the same time, the kit also contains reagents required for PCR amplification reaction: 2×PCR mix Buffer and ddH2O.

[0039] The method for using the kit comprises the following steps:

[0040] S1. Extract genomic DNA from the sample to be tested;

[0041] S2. PCR amplification of S1 genomic DNA using the above primers;

[0042] S3. Subject the PCR amplification product of S2 to agarose gel electrophoresis and analyze the electrophoresis results. If the amplified product is the above-mentioned single electrophoresis band of 595 bp in length, it is southern catfish; if the amplified product is the above-mentioned single electrophoresis band of 522 bp in length, it is catfish; if the amplified product is the above-mentioned two electrophoresis bands of 595 bp and 522 bp in length, it is a hybrid of southern catfish and catfish.

Claims

1. A specific molecular marker for identifying southern catfish, catfish and their hybrids, characterized by: The specific molecular marker is located in the gene gsdf The molecular marker of southern catfish is 595 bp in length, and its nucleotide sequence is shown in SEQ ID No. 3, which is named SM. The molecular marker of catfish is 522 bp in length, and its nucleotide sequence is shown in SEQ ID No. 4, which is named SA.

2. A method for identifying southern catfish, catfish and their hybrids, characterized in that: The following steps are involved: A. PCR Amplification: Fin rays of the southern catfish, Silurus meridionalis, and their hybrids to be tested are collected and genomic DNA is extracted. PCR amplification is performed using the obtained genomic DNA as a template using an upstream primer having a nucleotide sequence as shown in SEQ ID No. 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID No.

2. The obtained PCR amplification product is then compared with the specific molecular markers of the southern catfish and Silurus meridionalis as described in claim 1. B. Interpretation of the results: Only a 595 bp DNA fragment was amplified from the genomic DNA of the test individual, with the nucleotide sequence shown in SEQ ID No. 3, indicating that the specimen was Silurus meridionalis. Only a 522 bp DNA fragment was amplified from the genomic DNA of the test individual, with the nucleotide sequence shown in SEQ ID No. 4, indicating that the specimen was Silurus meridionalis. Two DNA fragments, the 595 bp and 522 bp DNA fragments, were amplified from the genomic DNA of the test individual, indicating that the specimen was a hybrid of Silurus meridionalis and Silurus meridionalis.

3. Use of the specific molecular marker as claimed in claim 1 in identifying southern catfish, catfish and their hybrids.

4. Use of the specific molecular marker according to claim 1 in preparing a kit for identifying southern catfish, catfish and their hybrids.

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