A method for identifying a multiple SNP site yellowlip fish releasing population based on SNaPshot technology

Abstract

This invention belongs to the field of fish genetic identification technology and discloses a method for identifying released populations of yellow croaker (Croton tigrinum) based on SNaPshot technology using multiple SNP loci. This invention provides the application of a set of SNP molecular marker combinations in the identification of released yellow croaker populations. By detecting these SNP molecular markers, sample identification between released and wild populations of yellow croaker can be achieved using only a simple multiplex PCR reaction system and SNaPshot genotyping sequencing. It can be performed on various genetic analysis instruments, achieving automatic genotyping and identification of released yellow croaker population samples. Compared to current methods for identifying yellow croaker population samples, the identification method based on the above-mentioned SNP molecular marker construction has the advantages of accurate genotyping, rapid operation, high detection throughput, and extremely high accuracy and cumulative exclusion rate.

Description

Technical Field

[0001] This invention belongs to the field of fish genetic identification technology, specifically relating to a method for identifying multiple SNP loci in released populations of yellow croaker based on SNaPshot technology. Background Technology

[0002] Yellow croaker ( Bahaba taipingensis ) is a rare fish species inhabiting the East China Sea and South China Sea, belonging to the Sciaenidae family ( Sciaenidae The swim bladder of the Chinese yellow croaker is considered a highly valuable tonic and also has medicinal value in traditional Chinese medicine. In 2001, it was reported that in the high-end retail market, the swim bladder of the Chinese yellow croaker was worth more than seven times its weight in gold. This fish is recognized for its immense economic value. However, due to overfishing, human settlement, and industrial expansion, the population of Chinese yellow croakers has declined dramatically, plummeting by more than 90% since the 1930s. Therefore, in 2006, the International Union for Conservation of Nature (IUCN) listed the Chinese yellow croaker as a critically endangered (CR) species. The germplasm resources of the Chinese yellow croaker urgently need protection. In recent years, artificial breeding of the Chinese yellow croaker has been successful, reaching a certain scale, providing a foundation for the recovery of its germplasm resources.

[0003] Resequencing is a bioinformatics method that involves obtaining the genome sequence information of an organism, comparing it with existing genomes, and identifying differences in sequence information to explore the organism's genetics, evolution, and biological characteristics. In recent years, whole-genome resequencing has been increasingly applied to various vertebrates, such as the Korean cattle (Korean cattle). Bos taurus var. coreana ), domestic pigs ( Sus scrofa var. domesticus ), Red Junglefowl ( Gallus gallus Population genetic studies of vertebrates such as carp and others. In fish, whole-genome resequencing is also becoming increasingly common, including in carp (…). Cyprinus carpio Atlantic salmon Salmo salar ),goldfish( Carassius auratus Researchers used whole-genome resequencing to analyze fish population structure and historical evolution, and elucidated fish-specific genetic traits by screening candidate genes and population genome datasets related to genomic regions.

[0004] Releasing artificially bred yellow croaker populations into the wild can effectively increase their population size in the wild, which is a key approach to the restoration of yellow croaker germplasm resources. In this context, accurately identifying the released yellow croaker populations from the wild populations can help measure the survival status of artificially bred yellow croakers in the wild and effectively assess their ecological adaptability, which is of significant ecological importance.

[0005] Current methods for monitoring and evaluating the effectiveness of restocking and stock enhancement of the yellow croaker mainly rely on fisheries surveys, observation of recaptured samples, and implantation of electronic tags. However, yellow croaker resources in the wild are scarce and their distribution is elusive, resulting in a low recapture rate. Traditional tagging methods suffer from drawbacks such as large operational workload, easy detachment or failure, stress or damage to individuals, and difficulty in covering large-scale seedlings. Furthermore, they cannot reliably distinguish between artificially bred and released individuals and wild individuals in natural marine environments. As artificially bred offspring of the yellow croaker are obtained and gradually used for restocking and stock enhancement, the lack of accurate, rapid, low-damage, and scalable genetic identification methods will hinder the scientific assessment of the survival, dispersal, and replenishment of wild resources by released individuals. This will also impede subsequent population monitoring, parentage management, and optimization of conservation strategies. Therefore, there is an urgent need to establish a method for identifying released yellow croaker populations to achieve stable identification of the origin of released individuals. Summary of the Invention

[0006] This invention aims to at least solve one of the technical problems existing in the prior art. This invention constructs a multi-SNP locus identification method for released populations of *Cyprinus maculatus* based on SNaPshot technology, achieving stable identification of the origin of released individuals.

[0007] The first objective of this invention is to provide the application of SNP molecular marker combinations in the identification of released populations of yellow croaker.

[0008] The second aspect of the present invention aims to provide a primer set for amplifying the SNP molecular marker combinations of the first aspect of the present invention.

[0009] A third aspect of the present invention is to provide a detection kit.

[0010] The fourth aspect of this invention aims to provide the application of the primer set of the second aspect of this invention or the detection kit of the third aspect of this invention in the identification of released populations of yellow croaker.

[0011] The fifth objective of this invention is to provide a method for identifying released populations of yellow croaker.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the application of SNP molecular marker combinations in the identification of released populations of yellow croaker, wherein the SNP molecular marker combinations include SNP2-18194705, SNP11-4501424, SNP11-4641490, SNP11-5557114, SNP11-4082555, SNP11-6432431, SNP14-12088975, SNP14-12088995 and SNP14-11987418; The SNP2-18194705 is located at position 18194705 on chromosome 2 of the yellow croaker, and its polymorphism is G / A; the nucleotide sequence of chromosome 2 of the yellow croaker can be found in GenBank:CM143070.1; The SNPs 11-4501424, 4641490, 5557114, 4082555, and 6432431 are located at positions 4501424, 4641490, 5557114, 4082555, and 6432431, respectively, on chromosome 11 of the yellow croaker. Their polymorphisms are T / C, T / C, T / C, G / C, and T / C, respectively. The nucleotide sequence of chromosome 11 of the yellow croaker is available in GenBank:CM143079.1. The SNPs 14-12088975, 12088995, and 11987418 are located at positions 12088975, 12088995, and 11987418 of chromosome 14 of the yellow croaker, respectively, with polymorphisms of C / T, A / T, and C / A, respectively. The nucleotide sequence of chromosome 14 of the yellow croaker can be found in GenBank:CM143082.1.

[0013] In some embodiments of the present invention, the above-mentioned nine SNP molecular markers are obtained by the following steps: S1. After whole-genome resequencing of 107 samples from the released and wild populations of yellow croaker, allele frequency calculation and Fisher's exact test (FDR<0.05) were used to screen for differential SNPs between the released and wild populations of yellow croaker. S2. Nine SNPs with the highest allele frequency differences between the released and wild populations of yellow croaker were selected as candidate specific genetic difference loci and expanded verification based on SNaPshot technology. Based on the above screening, the SNP loci are located on three different chromosomes of the yellow croaker (chromosome 2 (chr 2), chromosome 11 (chr 11), and chromosome 14 (chr 14)), with the following location information: SNP14-11987418, SNP2-18194705, SNP11-4501424, SNP11-4641490, SNP11-5557114, SNP11-4082555, SNP11-6432431, SNP14-12088975, and SNP14-12088995. A set of SNaPshot marker primers for identifying released yellow croaker populations was designed. S3. Use multiplex PCR reaction system to perform typing experiments to verify whether the 9 SNPs reaction system can identify yellow croaker released samples in a yellow croaker population composed of released (recaptured) yellow croaker and wild yellow croaker. S4. Based on the SNaPshot sequencing results, scores are generated for each sample, and a receiver operating characteristic (ROC) curve is constructed based on the sum of the sample scores. By comparing the accuracy and specificity with the known subtypes of the samples, the threshold for identifying the yellow croaker released population samples is determined, and the reliability of the reaction system for identifying yellow croaker released population samples is verified by whether the area under the ROC curve (AUC) is greater than 0.9.

[0014] In a second aspect, the present invention provides a primer set for amplifying the SNP molecular marker combination of the first aspect of the present invention, the nucleotide sequences of the primer set being shown in SEQ ID NO:1-SEQ ID NO:16.

[0015] In some embodiments of the present invention, the SEQ ID NO:1-SEQ ID NO:16 are arranged in sequence to form a primer pair of every two nucleic acid sequences.

[0016] In some embodiments of the present invention, the primer set further includes single-base extension primers, the nucleotide sequences of which are shown in SEQ ID NO:17-SEQ ID NO:25.

[0017] A third aspect of the present invention provides a detection kit comprising the primer set of the second aspect of the present invention.

[0018] In some embodiments of the present invention, the kit further comprises dNTPs, DNA polymerase, PCR reaction buffer, SAP, CIP, and ExoI One or more of them.

[0019] A fourth aspect of the present invention provides the application of the primer set of the second aspect of the present invention or the detection kit of the third aspect of the present invention in the identification of released populations of yellow croaker.

[0020] A fifth aspect of the present invention provides a method for identifying a released population of yellow croaker, comprising the step of using the primer set of the second aspect of the present invention or the detection kit of the third aspect of the present invention to detect the SNP molecular marker combination described in the first aspect of the present invention in a yellow croaker sample to be tested.

[0021] This identification method is based on SNaPshot technology for identifying multiple SNP sites in released populations of the yellow croaker. This method is developed based on multiple single nucleotide polymorphisms (SNPs) identified through whole-genome resequencing and allele frequency screening of the yellow croaker. The method design and detection are based on SNaPshot, a genotyping technique using fluorescently labeled single-base extension. Nine candidate indicator SNPs are fused into a multiplex PCR reaction, specifically amplified, and genotyped to determine the accuracy of these nine sites in the tested samples. Validation using SNaPshot on 71 known-genotyped yellow croaker samples demonstrates that the nine-site SNP marker system can successfully identify the vast majority of released (recaptured) yellow croaker samples. This marker system and detection method significantly improve the accuracy of identifying released (recaptured) yellow croaker samples, enabling rapid and accurate genotyping on various genetic analyzers, automating SNP analysis, and facilitating convenient and efficient high-throughput detection.

[0022] In some embodiments of the present invention, the identification method includes the following steps: Using the DNA of the yellow croaker sample to be tested as a template, PCR amplification was performed using the primer set of the second aspect of the present invention or the detection kit of the third aspect of the present invention to obtain PCR amplification products. SNaPshot sequencing analysis of the PCR amplification products was performed to obtain the genotype of the SNP molecular marker combination described in the first aspect of the present invention in the genome of the yellow croaker to be tested. The frequencies of the genotypes of the SNP molecular marker combinations were analyzed, scored, and ROC curves were constructed to determine whether the yellow croaker samples to be tested belonged to the released yellow croaker population.

[0023] In some embodiments of the present invention, the scoring rules in step (3) are as follows: When the allele of SNP2-18194705 in the SNP molecular marker combination is G, 1 point is awarded; otherwise, 0 points are awarded. If the allele of SNP11-4501424 in the SNP molecular marker combination is T, then 1 point is awarded; otherwise, 0 points are awarded. If the allele of SNP11-4641490 in the SNP molecular marker combination is T, then 1 point is awarded; otherwise, 0 points are awarded. When the allele of SNP11-5557114 in the SNP molecular marker combination is T, 1 point is awarded; otherwise, 0 points are awarded. If the allele of SNP11-4082555 in the SNP molecular marker combination is C, then 1 point is awarded; otherwise, 0 points are awarded. If the allele of SNP11-6432431 in the SNP molecular marker combination is T, then 1 point is awarded; otherwise, 0 points are awarded. When the allele of SNP14-12088975 in the SNP molecular marker combination is C, 1 point is awarded; otherwise, 0 points are awarded. If the allele of SNP14-12088995 in the SNP molecular marker combination is A, then 1 point is awarded; otherwise, 0 points are awarded. If the allele of SNP14-11987418 in the SNP molecular marker combination is C, then 1 point is awarded; otherwise, 0 points are awarded.

[0024] Generally, if an identification method is constructed based on x loci, then the total number of identification methods is 2x (number of alleles). 2).

[0025] In some embodiments of the present invention, the total score of the sample corresponding to the cut-off value of the ROC curve (i.e., the maximum value of the ROC curve sensitivity (sensitivity%) + specificity (specificity%)) is set as the identification threshold. When the total score of the yellow croaker sample to be tested is higher than the threshold, it is a yellow croaker released fish population sample; otherwise, it is a yellow croaker wild population sample.

[0026] In some embodiments of the present invention, the PCR amplification in step (1) is multiplex PCR amplification.

[0027] In some embodiments of the present invention, the PCR amplification reaction program is as follows: pre-denaturation at 92-96℃ for 3-6 min; denaturation at 92-96℃ for 28-35 s, annealing at 58-62℃ for 25-35 s, extension at 70-72℃ for 45-55 s, for 32-37 cycles; and extension at 70-72℃ for 8-12 min.

[0028] In some embodiments of the present invention, the PCR amplification products are subjected to digestion, single-base extension, purification, and other treatments before sequencing.

[0029] In some embodiments of the present invention, the digestion reaction system comprises 3-5 μL of PCR amplification product 1-2 USAP and 0.4-0.6 U ExoI The digestion reaction conditions are 35-38℃ for 55-65 min; 70-77℃ for 18-25 min.

[0030] In some embodiments of the present invention, the reaction conditions for the monobasic extension are 94-97°C for 8-13s, 46-52°C for 3-7s, 58-62°C for 28-32s, for 25-30 cycles.

[0031] The beneficial effects of this invention are: This invention provides the application of a set of SNP molecular marker combinations in the identification of released populations of the yellow croaker. By detecting these SNP molecular markers, the identification of samples between released and wild populations of yellow croaker can be achieved using only a simple multiplex PCR reaction system and SNaPshot genotyping sequencing. The method can be automated on various genetic analysis instruments, enabling automatic genotyping and identification of released yellow croaker samples. Compared to current methods for identifying yellow croaker population samples, the identification method based on the above-mentioned SNP molecular marker construction offers advantages such as accurate genotyping, rapid operation, high detection throughput, and extremely high accuracy and cumulative exclusion rate.

[0032] Given that the yellow croaker is a Class I protected animal in China, and that artificial breeding technology has only recently been established, the release of artificially bred yellow croakers helps promote the stability of their populations in natural marine areas. Simultaneously, there is an urgent need to establish a method for identifying released yellow croaker populations to scientifically assess the release effect and contribute to the resource assessment of yellow croakers in their natural marine environments. Therefore, the method provided in this invention is of great importance and uniqueness. Compared to current methods for identifying yellow croaker population samples, this method offers more accurate typing, faster operation, higher detection throughput, and higher accuracy and cumulative exclusion rate. Attached Figure Description

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a peak diagram of SNaPshot detection for SNPs.

[0034] Figure 2 The ROC curves for the multi-SNP locus identification method of yellow croaker released population samples based on SNaPshot technology are shown. Detailed Implementation

[0035] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0036] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0037] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0038] Example 1 A set of SNPs identified in a released population of *Croton tigrinum* using SNaPshot technology were analyzed. These SNPs were located on three different chromosomes of *Croton tigrinum*: chromosome 2 (chr 2, NCBI sequence number: CM143070.1), chromosome 11 (chr11, NCBI sequence number: CM143079.1), and chromosome 14 (chr 2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 ... 14, NCBI sequence number: CM143082.1) (Referring to the latest whole genome data of Yellow Croaker (NCBI accession number: GCA_054948935.1)), the position information is as follows: SNP14-11987418, SNP2-18194705, SNP11-4501424, SNP11-4641490, SNP11-5557114, SNP11-4082555, SNP11-6432431, SNP14-12088975, SNP14-12088995.

[0039] The above 9 SNPs were obtained through whole-genome resequencing, SNP detection, and allele frequency screening (Fisher exact test (FDR < 0.05)). The specific methods are as follows: A total of 576.43 G of raw whole-genome resequencing data was generated from 107 individuals of *Croton tigrinum* (including 92 representative individuals from released populations and 15 wild individuals), with an average of 13405.42 M of raw data per sample. Based on this, a total of 574.60 G of filtered data was generated, with an average of 13362.97 M per sample. 99.48% of reads were mapped to the *Croton tigrinum* reference genome. Allele frequencies were calculated and Fisher's exact test (FDR < 0.05) was performed on the SNP dataset. Nine SNPs with the highest allele frequency differences between the released (recaptured) and wild populations of *Croton tigrinum*, and which passed Fisher's exact test, were selected as candidate specific genetic difference loci. These SNPs were then further validated using SNaPshot technology.

[0040] The specific information for the 9 SNPs is shown in Table 1.

[0041] Table 1. Detailed information on the 9 SNPs

[0042] For each of the nine SNPs mentioned above, corresponding amplification primer sequences were designed. The primer sequences and amplification lengths are shown in Table 2. For each of the nine SNPs, the names, sequences, extension lengths, and extension bases of the corresponding extension primers are shown in Table 3.

[0043] Table 2. Names, sequences, and amplification lengths of the amplification primers for the 9 SNPs.

[0044] Table 3. Names, sequences, and extension lengths of the extension primers for the 9 SNPs.

[0045] Example 2 A method for differentiating between released and wild populations of yellow croaker (Caucasus simonii) based on SNaPshot technology includes the following steps: S1: Genomic DNA was extracted from the fin tissue of the yellow croaker using the phenol-chloroform extraction method; S2: Construct a multiplex PCR reaction. The multiplex PCR reaction system is shown in Table 4, and the reaction procedure is shown in Table 5. Table 4 Multiplex PCR reaction system

[0046] Table 5 Reaction Procedure

[0047] Pre-amplification product quality control: Load 2µL of sample and perform 2% agarose gel electrophoresis; The amplified products were digested. The digestion system is shown in Table 6. The digestion conditions were 37℃ for 60 min and 75℃ for 20 min. Table 6 Digestive System

[0048] The digested product was extended, and the extension reaction system is shown in Table 7. The extension conditions were 96℃ for 10s, 50℃ for 5s, 60℃ for 30s, and 30 cycles. Table 7 Extended Reaction System

[0049] The extended product was purified. Purification of the extension product: 6 μL of the extension reaction product was added to 1.0 μL of CIP; incubated at 37℃ for 1 h, then at 75℃ for 15 min; S3: 3500 sequencer detection 1) Add 9 μL of a mixture of molecular weight internal standard and formamide and 1 μL of PCR product to each well of a 96-well plate; 2) After incubation at 95℃ for 3 minutes, place in an ice bath and then detect using a 3500 sequencer; 3) Data analysis: Import the raw data files obtained from the detection into the analysis software for analysis.

[0050] S4: Constructing a method for differentiating between released and wild populations of yellow croaker based on the above 9 SNPs. Based on the SNaPshot validation results, the significant differences in allele frequencies at the above 9 loci between released (recaptured) yellow croaker and wild yellow croaker were analyzed. P <0.05). For the identification of released (recaptured) yellow croaker samples: Referring to Table 1, among the 9 differential SNPs, the dominant allele in the released yellow croaker population (the allele with a higher frequency in the released yellow croaker population than in the wild yellow croaker population) is scored as 1 point, and the dominant allele in the wild yellow croaker population (the allele with a lower frequency in the released yellow croaker population than in the wild yellow croaker population) is scored as 0 points. A method for identifying yellow croaker released population samples based on the above 9 loci is constructed. Generally, if a method is constructed based on x loci, the total score of the identification method is 2 × (number of alleles). 2).

[0051] A total score was assigned to each tested sample, and a Receiver Occurrence Recognition (ROC) curve (GraphPad Prism 8) was constructed based on the scores of all tested samples. An ROC curve area (AUC) greater than 0.9 indicated a feasible method. The cut-off value obtained from the ROC curve was set as the discrimination threshold (the cut-off value is the maximum of the sum of the sensitivity% and specificity% of the ROC curve). For this identification method, a discrimination threshold of 7 was obtained. A single *Cyprinus fulvidraco* sample with a score greater than the threshold of 7 was classified as a *Cyprinus fulvidraco* released population sample, while a sample with a score less than or equal to the threshold of 7 was classified as a wild-type *Cyprinus fulvidraco* population sample.

[0052] Example 3 The method for identifying the released and wild populations of yellow croaker based on SNaPshot technology, as described in Example 2, was used to identify known yellow croaker populations. Among them, there were 57 released (recaptured) yellow croaker individuals and 14 wild yellow croaker individuals.

[0053] The above-mentioned yellow croaker was analyzed using SNaPshot, and some of the SNaPshot peaks are shown in the figure below. Figure 1 The specific gene frequency detection results are shown in Table 8. The ROC curves for identifying the released population samples of *Cyprinus macranthum* based on the above 9 SNPs are shown in Table 8. Figure 2As shown in the figure, the area under the ROC curve (AUC) was 0.9987, and the cut-off value (i.e., the maximum of sensitivity (Sensitivity%) + specificity (Specificity%)) was 198.25%. At this point, the sensitivity was 100% (meaning that the probability of identifying a released (recaptured) yellow croaker using this identification method was 100%), and the specificity was 98.25% (meaning that the probability of excluding a wild yellow croaker using this identification method was 98.25%). The corresponding sample score at this point was 7 points, which is the judgment threshold. AUC > 0.9 indicates that the identification method is reliable. Specific detection results are shown in Table 9.

[0054] Table 8 Specific Genotype Detection Results

[0055] Table 9 Specific Test Results

[0056] In summary, the multi-SNP locus yellow croaker released (recaptured) population samples based on SNaPshot technology have an extremely high identification rate and can be applied to the identification of yellow croaker released populations.

[0057] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. Application of SNP molecular marker combinations in the identification of released populations of yellow croaker, wherein the SNP molecular marker combinations include SNP2-18194705, SNP11-4501424, SNP11-4641490, SNP11-5557114, SNP11-4082555, SNP11-6432431, SNP14-12088975, SNP14-12088995 and SNP14-11987418; in, The SNP2-18194705 is located at position 18194705 on chromosome 2 of the yellow croaker, and its polymorphism is G / A. The SNPs 11-4501424, 4641490, 5557114, 4082555, and 6432431 are located at positions 4501424, 4641490, 5557114, 4082555, and 6432431 on chromosome 11 of the yellow croaker, respectively, and their polymorphisms are T / C, T / C, T / C, G / C, and T / C, respectively. The SNPs 14-12088975, 12088995, and 11987418 are located at positions 12088975, 12088995, and 11987418 on chromosome 14 of the yellow croaker, respectively, and their polymorphisms are C / T, A / T, and C / A, respectively.

2. A primer set for amplifying the SNP molecular marker combination described in claim 1, characterized in that, The nucleotide sequences of the primer set are shown in SEQ ID NO:1-SEQ ID NO:

18.

3. The primer set according to claim 2, characterized in that, The SEQ ID NO:1-SEQ ID NO:18 sequences form a primer pair for every two nucleic acid sequences in sequence.

4. The primer set according to claim 2, characterized in that, The primer set also includes single-base extension primers, the nucleotide sequences of which are shown in SEQ ID NO:19-SEQ ID NO:

27.

5. A detection kit comprising the primer set according to any one of claims 2-4.

6. The detection kit according to claim 5, characterized in that, The kit also contains dNTPs, DNA polymerase, PCR reaction buffer, SAP, and ExoI One or more of them.

7. The application of the primer set according to any one of claims 2-4 or the detection kit according to claim 5 or 6 in the identification of released populations of yellow croaker.

8. A method for identifying a released population of yellow croaker, comprising the step of using the primer set of any one of claims 2-4 or the detection kit of claim 5 or 6 to detect the SNP molecular marker combination of claim 1 in the yellow croaker sample to be tested.

9. The identification method according to claim 8, characterized in that, The identification method includes the following steps: Using the DNA of the yellow croaker sample to be tested as a template, PCR amplification was performed using the primer set described in any one of claims 2-4 or the detection kit described in claim 5 or 6 to obtain PCR amplification products; The PCR amplification products were subjected to SNaPshot sequencing analysis to obtain the genotype of the SNP molecular marker combination described in claim 1 in the genome of the yellow croaker to be tested. The frequencies of the genotypes of the SNP molecular marker combinations were analyzed, scored, and ROC curves were constructed to determine whether the yellow croaker samples to be tested belonged to the released yellow croaker population.

10. The identification method according to claim 9, characterized in that, The cut-off value of the ROC curve is set as the identification threshold. When the total score of the yellow croaker sample to be tested is higher than the threshold, it is considered a yellow croaker release group.

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