Multi-site sal ammoniac wild pseudosciaena crocea identification method based on SNaPshot technology

Abstract

The invention belongs to the technical field of fish genetic identification, discloses a multi-site sal-family wild large yellow croaker identification method based on an SNaPshot technology, and particularly discloses a group of SNP (Single Nucleotide Polymorphism) molecular markers for screening a sal-family wild large yellow croaker group. The identification of the sal-family wild pseudosciaena crocea in the pseudosciaena crocea wild population sample can be realized only by a simple multiple PCR reaction system and SNaPshot typing sequencing. And automatic typing and identification of the wild large yellow croaker sample of the large sal ammoniac family can be realized only by operating on various genetic analysis instruments. Compared with the existing method for identifying the wild large yellow croaker in the sal family, the method has the advantages that the typing is more accurate, the operation is quicker, the detection flux is higher, and meanwhile, the accuracy rate and the cumulative exclusion rate are higher.

Description

Technical Field

[0001] The invention belongs to the technical field of fish genetic identification, and in particular relates to a multi-locus Naozhou wild large yellow croaker identification method based on SNaPshot technology. Background Art

[0002] Large yellow croaker (Larimichthys crocea) belongs to the order Perciformes, family Sciaenidae, genus Larimichthys. Commonly known as yellow croaker, it is also known as yellow croaker, large fresh, or large yellow flower. Large yellow croaker is a warm-temperate migratory near-shore fish found in the northwest Pacific Ocean. In my country, large yellow croaker is primarily divided into three geographical populations: the Daiqu, Min-Yuedong, and Naozhou populations. These populations are found from the southern Yellow Sea in the north, through the East China Sea and the Taiwan Strait, to the east of the Leizhou Peninsula in the South China Sea. Large yellow croaker of the Min-Yuedong population is primarily found in the waters off the eastern Fujian and Yuedong coast (near Fujian Province). In 1985, my country pioneered research on artificial propagation and aquaculture techniques for large yellow croaker, focusing on the Min-Yuedong population. By 1987, China had mastered the artificial breeding techniques for aquaculture broodstock. The large yellow croaker of the Naozhou ethnic group, which grows in the waters of western Guangdong (near Zhanjiang City, Guangdong Province), has high environmental requirements and is difficult to domesticate. Recently, a relevant team in Zhanjiang, Guangdong Province has also made breakthroughs in the artificial breeding of the large yellow croaker of the Naozhou ethnic group.

[0003] Resequencing is a bioinformatics method that uses genome sequence information from living organisms to identify differences in sequence information and explore the genetic, evolutionary, and biological characteristics of living organisms by obtaining the genome sequence information of living organisms and comparing it with existing genomes. In recent years, whole-genome resequencing has been gradually applied to population genetic studies of various vertebrates, such as Korean cattle (Bos taurus var. coreana), domestic pigs (Susscrofa var. domesticus), and jungle fowl (Gallus gallus). The application of whole-genome resequencing is also becoming increasingly common in fish, including common carp (Cyprinus carpio), Atlantic salmon, and goldfish (Carassius auratus). Researchers have used whole-genome resequencing to analyze the population structure and historical evolution of fish populations, and have elucidated the molecular mechanisms of fish genetic traits by screening candidate genes and related genomic regions in population genomic datasets.

[0004] Maximum likelihood estimation (MLE), a classic statistical method, constructs probabilistic models based on observed data and infers parameters by maximizing the likelihood function. Due to its theoretical rigor and flexibility, it is widely used in population genetics research. In fish population typing, this method is highly compatible with the characteristics of SNP data from resequencing: each SNP locus in a diploid individual can be considered as two independent samples, and the number of minor alleles naturally follows a binomial distribution, which conforms to the random assortment assumption under Hardy-Weinberg equilibrium. Research has demonstrated that the maximum likelihood method has been applied to salmon population tracing and Atlantic cod subpopulation analysis. By jointly calculating the probabilistic contributions of multiple SNP loci, it can quantify the degree of match between the test sample and the reference population, combining computational efficiency with interpretable results. Compared to Bayesian methods or machine learning models, MLE is more robust in small sample settings and avoids complex priors or the risk of overfitting. Based on these biological plausibility and technical maturity, the maximum likelihood method is feasible for population typing of large yellow croaker and can provide a reliable basis for accurately identifying the genetic background of fish.

[0005] How to accurately and quickly distinguish the large yellow croaker of the Naozhou ethnic group from other large yellow croaker populations is the core of the identification of the germplasm resources of the Naozhou ethnic group. In 2015, researchers used a strategy combining bacterial artificial chromosomes and whole-genome shotgun methods to sequence the whole genome of the large yellow croaker, and obtained a detailed map of the large yellow croaker genome. In 2019, researchers used third-generation sequencing technology (PacBio single-molecule sequencing technology) and high-throughput chromosome conformation capture technology to assemble the large yellow croaker reference genome. The highly accurate, chromosome-level large yellow croaker reference genome provides an important genomic resource to support the identification and evaluation of the germplasm resources of the large yellow croaker. At present, the development of genetic-specific molecular markers has been applied in large yellow croaker. In 2022, some researchers developed sex-specific molecular markers for the Daiqu population of large yellow croaker through the locus between dmrt1 and cfap157, providing a useful tool for promoting sex-controlled breeding of the Daiqu population of large yellow croaker.

[0006] In 2023, researchers used genome resequencing to compare SNP marker data from large yellow croaker populations distributed in the coastal waters of eastern and southern China and found that climate-driven habitat changes may have occurred between the Naozhou ethnic group and the Fujian-Yuedong ethnic group. In 2024, researchers used whole-genome resequencing data from a large number of samples, including domesticated and wild populations, to conduct genetic structure analysis, indicating that there is no obvious geographical structure in the wild populations along China's coast. This study's data overturned the long-standing view that they were divided into three genetic management units. Therefore, the Naozhou ethnic group large yellow croaker in western Guangdong, whose geographical distribution is controversial, the Fujian-Yuedong ethnic group large yellow croaker in Fujian waters, and the Huidong large yellow croaker population in the Pearl River Estuary in the intermediate geographical area were compared and analyzed to screen for population-specific molecular markers. In the South China Sea, it is difficult to distinguish wild large yellow croaker from the Naozhou ethnic group, wild large yellow croaker from the Fujian-Yuedong ethnic group, and wild large yellow croaker from Huidong based on morphology. Therefore, for the wild yellow croaker of the Naozhou ethnic group in western Guangdong, this group is currently easily confused with other South China Sea yellow croaker groups, and there is still a lack of accurate and rapid identification methods for this group. Developing an accurate and rapid identification method for the Naozhou ethnic group yellow croaker using genetically specific markers of the Naozhou ethnic group yellow croaker can effectively promote the distribution research of wild yellow croaker groups along the coast of my country. Summary of the Invention

[0007] The purpose of the first aspect of the present invention is to provide a set of SNP molecular markers for identifying wild large yellow croaker populations of the Naozhou tribe.

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

[0009] The third aspect of the present invention aims to provide a detection reagent, gene chip or kit.

[0010] The purpose of the fourth aspect of the present invention is to provide the use of the SNP molecular marker of the first aspect of the present invention, the primer set of the second aspect of the present invention or the detection reagent, gene chip or kit of the third aspect of the present invention in the identification of wild large yellow croaker populations of the Naozhou tribe.

[0011] The fifth aspect of the present invention is to provide a method for identifying wild large yellow croaker populations of the Naozhou tribe.

[0012] The purpose of the sixth aspect of the present invention is to provide the application of the method for identifying wild large yellow croaker populations of the Naozhou ethnic group of the fifth aspect of the present invention in the identification and evaluation of large yellow croaker germplasm resources.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] In a first aspect of the present invention, a group of SNP molecular markers for identifying wild large yellow croaker populations of the Naozhou ethnic group is provided, wherein the SNP molecular markers include SNP3-47505387, SNP7-9407565, SNP3-31085986, SNP3-42982617, SNP20-95328, SNP20-73728, SNP14-13509333, SNP17-8648833, SNP2-45873542, SNP10-28085288, SNP3-51328350, SNP20-6750079, SNP11-10317498 and SNP17-4124025;

[0015] Among them, the SNP3-47505387, SNP3-31085986, SNP3-42982617 and SNP3-51328350 are located at positions 47505387, 31085986, 42982617 and 51328350 of chromosome 3 NC_040013.1 of large yellow croaker, and the polymorphisms are: C / T, C / T, G / A and G / A;

[0016] The SNP7-9407565 is located at position 9407565 of chromosome 7 NC_040017.1 of large yellow croaker, and the polymorphism is: T / C;

[0017] The SNP20-95328, SNP20-73728 and SNP20-6750079 are located at positions 95328, 73728 and 6750079 of chromosome 20 NC_040030.1 of large yellow croaker, respectively, and the polymorphisms are G / C, A / G and T / A, respectively;

[0018] The SNP14-13509333 is located at position 13509333 of chromosome 14 NC_040024.1 of large yellow croaker, and the polymorphisms are: T / G;

[0019] The SNP17-8648833 and SNP17-4124025 are located at positions 8648833 and 4124025 of chromosome 17 NC_040027.1 of large yellow croaker, respectively, and the polymorphisms are T / G and A / G, respectively;

[0020] The SNP2-45873542 is located at position 45873542 of chromosome 2 NC_040012.1 of large yellow croaker, and the polymorphism is: T / C;

[0021] The SNP10-28085288 is located at position 28085288 of chromosome 10 NC_040020.1 of large yellow croaker, and the polymorphism is: A / G;

[0022] The SNP11-10317498 is located at position 10317498 of chromosome 11 NC_040021.1 of large yellow croaker, and the polymorphism is: T / C.

[0023] In some embodiments of the present invention, the above 14 SNP molecular markers are obtained by the following steps:

[0024] S1. After whole-genome sequencing of 395 large yellow croaker samples, population genetics selection signal analysis was used to screen for differential SNPs between the wild large yellow croaker population of the Naozhou tribe and other wild large yellow croaker populations (wild large yellow croaker of the Minyuedong tribe and wild large yellow croaker of Huidong);

[0025] S2. Allele frequency calculation and chi-square test were performed based on the SNPs dataset to screen candidate genes with extremely significant differences in allele frequency between the wild large yellow croaker population of the Ganzhou tribe and other wild large yellow croaker populations (wild large yellow croaker of the Fujian and Guangdong tribes, wild large yellow croaker of Huidong) (P<0.001). 14 candidate sites were screened and the next step of SNP verification was carried out.

[0026] The SNPs based on the above screening are located on 9 different chromosomes of large yellow croaker (chromosome 2 (NC_040012.1), chromosome 3 (NC_040013.1), chromosome 7 (NC_040017.1), chromosome 10 (NC_040020.1), chromosome 11 (NC_040021.1), chromosome 14 (NC_040024.1), chromosome 17 (NC_040027.1) and chromosome 20 (NC_040030.1)). The location information is as follows: A set of SNaPshot marker primers for identifying wild large yellow croaker populations of the Naozhou tribe were designed.

[0027] S3. Use singleplex / multiplex PCR reaction systems to conduct typing experiments to verify whether the 14 SNPs reaction system can identify wild large yellow croaker samples of the Naozhou tribe, the Fujian and Yuedong tribes, and the Huidong wild large yellow croaker populations;

[0028] S4. Compare the accuracy and detection rate of SNaPshot sequencing results with those of known marker detection to prove whether the above 14 SNPs combination can identify wild large yellow croaker samples of the Naozhou tribe.

[0029] The second aspect of the present invention provides a primer set for amplifying the SNP molecular marker of the first aspect of the present invention, wherein the nucleotide sequence of the primer set is shown as SEQ ID NO: 1 to SEQ ID NO: 28.

[0030] In some embodiments of the present invention, each two nucleic acid sequences in SEQ ID NO: 1 to SEQ ID NO: 28 constitute a pair of primers.

[0031] The third aspect of the present invention provides a detection reagent, a gene chip or a kit comprising the primer set according to the second aspect of the present invention.

[0032] In some embodiments of the present invention, the kit further comprises one or more of dNTPs, DNA polymerase, PCR reaction buffer, and Taq.

[0033] In some embodiments of the present invention, the kit further comprises SAP and ExoI.

[0034] In some embodiments of the present invention, the kit further comprises a single-base extension primer.

[0035] In some embodiments of the present invention, the single-base extension primer is shown as SEQ ID NO: 29 to SEQ ID NO: 42.

[0036] The fourth aspect of the present invention provides the use of the SNP molecular marker of the first aspect of the present invention, the primer set of the second aspect of the present invention or the detection reagent, gene chip or kit of the third aspect of the present invention in the identification of wild large yellow croaker populations of the Naozhou tribe.

[0037] The fifth aspect of the present invention provides a method for identifying wild large yellow croaker populations of the Naozhou ethnic group, comprising the step of using the primer set of the second aspect of the present invention or the detection reagent, gene chip or kit of the third aspect of the present invention to detect the SNP molecular marker of the first aspect of the present invention on the large yellow croaker sample to be tested.

[0038] This identification method is a multi-locus method for identifying wild large yellow croaker from the Naozhou ethnic group based on the SNaPshot technology. The method was developed based on multiple single nucleotide polymorphisms (SNPs) identified through whole-genome resequencing of large yellow croaker and selection signal analysis in population genetics. The design and detection of the method were based on SNaPshot, a typing technology based on the principle of fluorescent labeling single base extension. The system integrates 14 candidate indicator SNPs into a single multiplex PCR reaction, specifically amplifies and types the 14 loci to determine their accuracy in the test samples. SNaPshot typing validation of 108 large yellow croaker samples with known genotypes demonstrated that the 14-locus SNP marker system can successfully identify wild large yellow croaker from the Naozhou ethnic group in the vast majority of samples. Compared to current identification methods and markers, this marker system and detection method greatly improves the accuracy of identifying wild large yellow croaker from the Naozhou ethnic group. It can be used for rapid and accurate genotyping on a variety of genetic analyzers, automates SNP analysis, and facilitates efficient and convenient high-throughput testing.

[0039] In some embodiments of the present invention, the identification method comprises the following steps:

[0040] (1) using the DNA of the large yellow croaker sample to be tested as a template, performing PCR amplification using the primer set of the second aspect of the present invention or the detection reagent, gene chip or kit of the third aspect of the present invention to obtain a PCR amplification product;

[0041] (2) performing SNaPshot sequencing analysis on the PCR amplification product to obtain the genotype of the SNP molecular marker of the first aspect of the present invention in the genome of the large yellow croaker to be tested;

[0042] (3) Analyze the frequency of the genotype of the SNP molecular marker, score it, and construct a ROC curve to determine whether the large yellow croaker sample to be tested is a wild large yellow croaker of the Naozhou tribe.

[0043] In some embodiments of the present invention, the scoring rules in step (3) are as follows:

[0044] When the frequency of CC genotype of SNP3-47505387 in the SNP molecular marker is greater than that of TT genotype, 1 point is recorded, otherwise 0 point is recorded;

[0045] When the frequency of CC genotype of SNP7-9407565 in the SNP molecular marker is greater than that of TT genotype, a score of 1 is recorded, otherwise a score of 0 is recorded;

[0046] When the frequency of CC genotype of SNP3-31085986 in the SNP molecular marker is greater than that of TT genotype, a score of 1 is given, otherwise a score of 0 is given;

[0047] When the frequency of the AA genotype of SNP3-42982617 in the SNP molecular marker is greater than that of the GG genotype, a score of 1 is recorded, otherwise a score of 0 is recorded;

[0048] When the frequency of CC genotype of SNP20-95328 in the SNP molecular marker is greater than that of GG genotype, 1 point is recorded, otherwise 0 point is recorded;

[0049] When the frequency of the GG genotype of SNP20-73728 in the SNP molecular marker is greater than that of the AA genotype, a score of 1 is given, otherwise a score of 0 is given;

[0050] When the frequency of the TT genotype of SNP14-13509333 in the SNP molecular marker is greater than that of the GG genotype, a score of 1 is recorded, otherwise a score of 0 is recorded;

[0051] When the frequency of the TT genotype of SNP17-8648833 in the SNP molecular marker is greater than that of the GG genotype, a score of 1 is recorded, otherwise a score of 0 is recorded;

[0052] When the frequency of the TT genotype of SNP2-45873542 in the SNP molecular marker is greater than that of the CC genotype, a score of 1 is recorded, otherwise a score of 0 is recorded;

[0053] When the frequency of the GG genotype of SNP10-28085288 in the SNP molecular marker is greater than that of the AA genotype, a score of 1 is recorded, otherwise a score of 0 is recorded;

[0054] When the frequency of the GG genotype of SNP3-51328350 in the SNP molecular marker is greater than that of the AA genotype, a score of 1 is given, otherwise a score of 0 is given;

[0055] When the frequency of the AA genotype of SNP20-6750079 in the SNP molecular marker is greater than that of the TT genotype, a score of 1 is recorded, otherwise a score of 0 is recorded;

[0056] When the frequency of CC genotype of SNP11-10317498 in the SNP molecular marker is greater than that of TT genotype, a score of 1 is given, otherwise a score of 0 is given;

[0057] When the frequency of the GG genotype of SNP17-4124025 in the SNP molecular marker is greater than that of the AA genotype, a score of 1 is recorded, otherwise a score of 0 is recorded.

[0058] Generally, on this basis, if a screening method is constructed based on x loci (x≤8), the total score of the screening method is 2x (number of alleles*2).

[0059] In some embodiments of the present invention, 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 discrimination threshold. When the total score of the large yellow croaker sample to be tested is higher than the threshold, it is a wild large yellow croaker population of the Naozhou tribe, otherwise it is other wild large yellow croaker populations (such as wild large yellow croaker of the Fujian and Guangdong tribes, and wild large yellow croaker of Huidong).

[0060] In some embodiments of the present invention, the PCR amplification in step (1) is multiplex PCR amplification.

[0061] In some embodiments of the present invention, the reaction procedure of the PCR amplification is pre-denaturation at 92-96°C for 3-6 min; denaturation at 92-96°C for 28-35 s, annealing at 58-62°C for 45-55 s, extension at 70-72°C for 45-55 s, 32-37 cycles; extension at 70-72°C for 8-12 min.

[0062] In some embodiments of the present invention, the PCR amplification product is subjected to digestion, single-base extension, purification, and other treatments before sequencing.

[0063] In some embodiments of the present invention, the digestion reaction system includes 8-12 μL PCR amplification product, 0.2-0.4 U SAP and 0.05-0.2 U ExoI; the digestion reaction conditions are 35-38° C., 55-65 min; 70-77° C., 12-16 min.

[0064] In some embodiments of the present invention, the reaction conditions for the single base extension are 94-97° C. for 8-13 s, 46-52° C. for 3-7 s, and 58-62° C. for 28-32 s, for 25-30 cycles.

[0065] In some embodiments of the present invention, the screening method further includes using the maximum likelihood estimation method to perform typing verification on the population samples, including using the maximum likelihood method to calculate the overall log-likelihood value of all sites in all samples, and classifying the sample with the largest overall log-likelihood value calculated based on the number of minor alleles in the sample to be tested as the corresponding reference population. The specific rules are as follows:

[0066]

[0067] Among them, A, B, and C are the reference groups of wild yellow croaker of the Ganzhou ethnic group, wild yellow croaker of the Fujian and Guangdong ethnic group, and wild yellow croaker of Huidong, respectively.

[0068] The sixth aspect of the present invention provides the application of the method for identifying wild large yellow croaker populations of the Naozhou ethnic group according to the fifth aspect of the present invention in the identification and evaluation of large yellow croaker germplasm resources.

[0069] The beneficial effects of the present invention are:

[0070] The present invention provides a set of SNP molecular markers for identifying wild large yellow croaker populations of the Naozhou ethnic group. By detecting these SNP molecular markers, a simple multiplex PCR reaction system and SNaPshot typing and sequencing are required to identify wild large yellow croaker samples from the Naozhou ethnic group. This method can be operated on a variety of genetic analysis instruments to automatically type and identify large samples of wild large yellow croaker from the Naozhou ethnic group. Compared to current methods for identifying wild large yellow croaker from the Naozhou ethnic group, this method provides more accurate typing, faster operation, higher detection throughput, and higher accuracy and cumulative exclusion rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 This is a partial electrophoresis diagram of the amplification using SNaPshot primer mix designed for SNPs.

[0072] Figure 2 This is the peak graph of SNaPshot detection for SNPs.

[0073] Figure 3 Figure 3 is the ROC curve corresponding to the multi-locus SNPs-based discrimination method for the wild large yellow croaker population of the Naozhou ethnic group and other wild large yellow croaker populations (wild large yellow croaker of the Fujian-Yuedong ethnic group and wild large yellow croaker of Huidong); among them, A is the ROC curve of the discrimination method for the wild large yellow croaker of the Naozhou ethnic group and the wild large yellow croaker of the Fujian-Yuedong ethnic group based on 14 SNPs sites; B: The ROC curve of the discrimination method for the wild large yellow croaker of the Naozhou ethnic group and the wild large yellow croaker of Huidong based on 14 SNPs sites. DETAILED DESCRIPTION

[0074] The present invention is further described in detail below through specific examples.

[0075] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0076] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0077] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0078] Example 1

[0079] A set of SNPs based on SNaPshot technology were identified in the wild large yellow croaker population of the Naozhou tribe and other wild large yellow croaker populations (wild large yellow croaker of the Minyuedong tribe and wild large yellow croaker of Huidong). The SNPs were located on 9 different chromosomes of large yellow croaker (chromosome 2 (NC_040012.1), chromosome 3 (NC_040013.1), chromosome 7 (NC_040017.1), chromosome 10 (NC_040020.1), chromosome 11 (NC_040021.1), chromosome 14 (NC_040024.1), chromosome 17 (NC_040027.1) and chromosome 2 (NC_040028.1). On chromosome 20 (NC_040030.1), the position information is as follows: SNP3-47505387, SNP7-9407565, SNP3-31085986, SNP3-42982617, SNP20-95328, SNP20-73728, SNP14-13509333, SNP17-8648833, SNP2-45873542, SNP10-28085288, SNP3-51328350, SNP20-6750079, SNP11-10317498 and SNP17-4124025.

[0080] The above 14 SNPs were obtained through whole genome reassessment, variant detection, and allele frequency (Chi-square test (P < 0.05) screening. The specific methods are as follows:

[0081] A total of 576.43 gigabytes of whole-genome resequencing raw data was generated from 395 large yellow croaker individuals, with an average of 13,405.42 megabytes of raw data per sample. This data was then used to generate a total of 574.60 gigabytes of filtered data, with an average of 13,362.97 megabytes per sample. 99.48% of reads were mapped to the large yellow croaker reference genome. Allele frequencies were calculated and chi-squared tests were performed based on the SNP dataset (P < 0.05). Fourteen SNPs with the highest allele frequency differences between the wild large yellow croaker population of the Naozhou tribe and other wild large yellow croaker populations (including wild large yellow croaker from the Fujian and Guangdong tribes and wild large yellow croaker from Huidong) were identified as candidate specific genetically distinct loci. These SNPs were then validated using the SNaPshot technology.

[0082] The specific genotypes of the 14 SNPs are shown in Table 1 .

[0083] Table 1 Specific genotypes of 14 SNPs

[0084]

[0085]

[0086] The corresponding amplification primer sequences were designed for the above 14 SNPs, and the primer sequences and amplification lengths are shown in Table 2. The names, sequences, extension lengths, and extension bases of the corresponding extension primers designed for the 14 SNPs are shown in Table 3.

[0087] Table 2 Amplification primer sequences corresponding to the 14 SNPs

[0088]

[0089] Note: In the above table, Y is the degenerate base CT.

[0090] Table 3 Extension primer sequence information corresponding to 14 SNPs

[0091]

[0092] Note: In the above table, K is the degenerate base GT; Y is the degenerate base CT; R is the degenerate base GA; M is the degenerate base CA.

[0093] Example 2

[0094] A multi-site identification method for wild large yellow croaker populations of the Naozhou tribe and other wild large yellow croaker populations (wild large yellow croaker of the Min-Yue-Dong tribe and wild large yellow croaker of Huidong tribe) based on SNaPshot technology comprises the following steps:

[0095] S1: Genomic DNA was extracted from the fin tissue of the tested large yellow croaker population using the phenol-chloroform extraction method;

[0096] S2: Construct a multiplex PCR reaction system for typing experiments. The multiplex PCR reaction system is shown in Table 4, and the reaction procedure is shown in Table 5.

[0097] Table 4 Multiplex PCR reaction system

[0098]

[0099]

[0100] Table 5 Multiplex PCR reaction program

[0101]

[0102] The amplified product was digested using the digestion system shown in Table 6. The digestion conditions were 37°C for 60 min and 75°C for 15 min.

[0103] Table 6 Digestion system

[0104]

[0105] The digested product was extended. The extension reaction system is shown in Table 7. The extension conditions were 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s, for 27 cycles.

[0106] Table 7 Extension reaction system

[0107]

[0108] The extension product was purified: 0.5 μL CIP was added to 6 μL of the extension reaction product; incubate at 37°C for 1.0 h, then at 75°C for 15 min.

[0109] S3: 3730XL sequencer detection

[0110] 1) Add 9 μL of molecular weight internal standard and formamide mixture and 1 μL of product to each well of a 96-well plate;

[0111] 2) Incubate at 95°C for 3 minutes, then place on ice and analyze using a 3730XL sequencer.

[0112] 3) Data analysis: Import the raw data files obtained from the test into the analysis software for analysis;

[0113] S4: Combined with the SNaPshot validation results, the differences in the allele frequencies of the above 14 loci among the wild large yellow croaker of the Naozhou tribe, the wild large yellow croaker of the Minyuedong tribe, and the wild large yellow croaker of Huidong were analyzed (P<0.05);

[0114] S5: Construct a method for identifying wild large yellow croaker of the Naozhou ethnic group, wild large yellow croaker of the Minyuedong ethnic group, and wild large yellow croaker of Huidong based on the above 14 SNPs

[0115] Based on the genotypes in Table 1, alleles with a higher frequency in the Naozhou wild large yellow croaker population than in other wild large yellow croaker populations (i.e., the Fujian-Yuedong wild large yellow croaker population and the Huidong wild large yellow croaker population) were assigned a score of 1, while alleles with a lower frequency in the Naozhou wild large yellow croaker population than in other wild large yellow croaker populations were assigned a score of 0. A discrimination method for the Naozhou wild large yellow croaker population, the Fujian-Yuedong wild large yellow croaker population, and the Huidong wild large yellow croaker population was constructed based on the above 14 SNPs. Generally, if a method is constructed based on x loci (x ≤ 10), the total score of the discrimination method is 2x(number of alleles * 2).

[0116] All samples were scored, and based on the total score of the 14 SNPs loci, total score datasets were constructed for wild and domesticated populations. The datasets were input into GraphPad Prism 8 to construct receiver operating characteristic curves (ROC curves; an ROC curve area (AUC) greater than 0.9 indicated that the method was feasible). The cut-off value was set as the discrimination threshold (the cut-off value was the maximum value of the ROC curve sensitivity (sensitivity%) + specificity (specificity%)). Samples with a score greater than or equal to the threshold of 15 points were classified as wild large yellow croaker from the Naozhou ethnic group, while samples with a score less than the threshold of 15 points were classified as wild large yellow croaker from the Minyuedong ethnic group and the Huidong wild large yellow croaker population.

[0117] S6: Based on the analysis of allele frequency differences in the test samples, since the allele frequencies conform to the binomial distribution Bin(2, p), the maximum likelihood estimation method is used to type the population samples: the overall log-likelihood value of all loci in all samples is calculated according to the maximum likelihood method. The sample with the largest overall log-likelihood value calculated based on the number of minor alleles in the test sample is classified as the corresponding reference population. The specific identification rules are as follows:

[0118]

[0119] Among them, A, B, and C are the reference groups of wild yellow croaker of the Ganzhou ethnic group, wild yellow croaker of the Fujian and Guangdong ethnic group, and wild yellow croaker of Huidong, respectively.

[0120] Example 3

[0121] The multi-site identification method of Example 2 based on SNaPshot technology of the wild large yellow croaker population of the Naozhou ethnic group and other wild large yellow croaker populations (wild large yellow croaker of the Fujian-Guangdong ethnic group and wild large yellow croaker of Huidong) was used to identify the known wild large yellow croaker populations, among which 50 wild large yellow croaker of the Naozhou ethnic group, 30 wild large yellow croaker of the Fujian-Guangdong ethnic group and 28 wild large yellow croaker of Huidong were identified.

[0122] The electrophoresis diagram of PCR reaction products of some large yellow croaker samples is as follows: Figure 1 By performing SNaPshot analysis on the above large yellow croaker, some of its SNaPshot detection peaks are shown as follows. Figure 2 The specific gene frequency detection results are shown in Table 8. The individual sample identification method based on the above 14 SNPs is as follows Figure 3As shown, among them, through the combination of 14 SNPs, the ROC curve area (AUC) of wild large yellow croaker of the Naozhou tribe and wild large yellow croaker of the Min-Yuedong tribe was constructed to be 0.9110, the sensitivity of the optimal threshold was 96.7% (that is, the probability of a wild large yellow croaker of the Naozhou tribe being identified by this evaluation method was 96.7%), and the specificity was 78.0% (that is, the probability of a wild large yellow croaker of the Min-Yuedong tribe being excluded by this evaluation method was 78.0%) ( Figure 3 A), where the AUC area is greater than 0.9, indicating that the method is feasible; at the same time, through the combination of 14 SNPs, the ROC curve area (AUC) of wild large yellow croaker of the Naozhou tribe and wild large yellow croaker of Huidong was constructed to be 0.9096, the sensitivity of the optimal threshold was 89.3% (that is, the probability of a wild large yellow croaker of the Naozhou tribe being identified by this evaluation method is 89.3%), and the specificity was 78.0% (that is, the probability of a wild large yellow croaker of Huidong being excluded by this evaluation method is 78.0%) ( Figure 3 B), where the AUC area is greater than 0.9, indicating that the method is feasible. In summary, in this system, the individual sample identification method constructed based on 14 SNPs has a high discrimination rate and can be applied to the identification of wild large yellow croaker samples from the Naozhou ethnic group (Nanzhou ethnic group large yellow croaker, Fujian and Guangdong ethnic group wild large yellow croaker, and Huidong wild large yellow croaker). Further, based on the analysis of the allele frequency differences in the test samples, 30 samples from the Naozhou ethnic group wild large yellow croaker population were taken using the maximum likelihood estimation method to determine the group to which they belonged. The overall log-likelihood values calculated based on the three reference groups are shown in Table 9. It can be seen that the overall log-likelihood value of the Naozhou ethnic group wild large yellow croaker population is the largest. The group to which the sample to be tested belongs was identified as the Naozhou ethnic group wild large yellow croaker population, which is consistent with the reference typing.

[0123] In summary, among the identification methods, the population identification method constructed based on 14 SNPs is reliable and can be used to identify wild large yellow croaker populations of the Naozhou ethnic group (wild large yellow croaker of the Naozhou ethnic group in western Guangdong, wild large yellow croaker of the Naozhou ethnic group in eastern Fujian and Guangdong, and wild large yellow croaker of Huidong).

[0124] Table 8 Specific gene frequency detection results

[0125]

[0126]

[0127] Table 9 Overall log-likelihood values calculated based on three wild reference populations

[0128]

[0129] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A group of SNP molecular markers for screening wild large yellow croaker populations of the Naozhou tribe, the SNP molecular markers including SNP3-47505387, SNP7-9407565, SNP3-31085986, SNP3-42982617, SNP20-95328, SNP20-73728, SNP14-13509333, SNP17-8648833, SNP2-45873542, SNP10-28085288, SNP3-51328350, SNP20-6750079, SNP11-10317498 and SNP17-4124025; in, The SNP3-47505387, SNP3-31085986, SNP3-42982617 and SNP3-51328350 are located at positions 47505387, 31085986, 42982617 and 51328350 of chromosome 3 NC_040013.1 of large yellow croaker, respectively, and the polymorphisms are: C / T, C / T, G / A and G / A; The SNP7-9407565 is located at position 9407565 of chromosome 7 NC_040017.1 of large yellow croaker, and the polymorphism is: T / C; The SNP20-95328, SNP20-73728 and SNP20-6750079 are located at positions 95328, 73728 and 6750079 of chromosome 20 NC_040030.1 of large yellow croaker, respectively, and the polymorphisms are G / C, A / G and T / A, respectively; The SNP14-13509333 is located at position 13509333 of chromosome 14 NC_040024.1 of large yellow croaker, and the polymorphisms are: T / G; The SNP17-8648833 and SNP17-4124025 are located at positions 8648833 and 4124025 of chromosome 17 NC_040027.1 of large yellow croaker, respectively, and the polymorphisms are T / G and A / G, respectively; The SNP2-45873542 is located at position 45873542 of chromosome 2 NC_040012.1 of large yellow croaker, and the polymorphism is: T / C; The SNP10-28085288 is located at position 28085288 of chromosome 10 NC_040020.1 of large yellow croaker, and the polymorphism is: A / G; The SNP11-10317498 is located at position 10317498 of chromosome 11 NC_040021.1 of large yellow croaker, and the polymorphism is: T / C.

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

28.

3. The primer set according to claim 2, characterized in that Each two nucleic acid sequences in SEQ ID NO: 1 to SEQ ID NO: 28 constitute a pair of primers in sequence.

4. A detection reagent, gene chip or kit comprising the primer set according to claim 2 or 3.

5. Use of the SNP molecular marker according to claim 1, the primer set according to claim 2 or 3, or the detection reagent, gene chip or kit according to claim 4 in the identification of wild large yellow croaker populations of the Naozhou tribe.

6. A method for identifying a population of wild large yellow croaker of the Naozhou ethnic group, comprising the step of detecting the SNP molecular marker of claim 1 on a large yellow croaker sample using the primer set of claim 2 or 3 or the detection reagent, gene chip or kit of claim 4.

7. The identification method according to claim 6, characterized in that: The screening method comprises the following steps: (1) Using the DNA of the large yellow croaker sample to be tested as a template, PCR amplification is performed using the primer set of claim 2 or 3 or the detection reagent, gene chip or kit of claim 4 to obtain a PCR amplification product; (2) performing SNaPshot sequencing analysis on the PCR amplification product to obtain the genotype of the SNP molecular marker according to claim 1 in the genome of the large yellow croaker to be tested; (3) Analyze the frequency of the genotype of the SNP molecular marker, score it, and construct a ROC curve to determine whether the large yellow croaker sample to be tested is a wild large yellow croaker of the Naozhou tribe.

8. The identification method according to claim 7, characterized in that: The scoring rule in step (3) is as follows: when the frequency of CC genotype of SNP3-47505387 in the SNP molecular marker is greater than that of TT genotype, 1 point is scored, otherwise 0 point is scored; When the frequency of CC genotype of SNP7-9407565 in the SNP molecular marker is greater than that of TT genotype, a score of 1 is recorded, otherwise a score of 0 is recorded; When the frequency of CC genotype of SNP3-31085986 in the SNP molecular marker is greater than that of TT genotype, a score of 1 is given, otherwise a score of 0 is given; When the frequency of the AA genotype of SNP3-42982617 in the SNP molecular marker is greater than that of the GG genotype, a score of 1 is recorded, otherwise a score of 0 is recorded; When the frequency of CC genotype of SNP20-95328 in the SNP molecular marker is greater than that of GG genotype, 1 point is recorded, otherwise 0 point is recorded; When the frequency of the GG genotype of SNP20-73728 in the SNP molecular marker is greater than that of the AA genotype, a score of 1 is given, otherwise a score of 0 is given; When the frequency of the TT genotype of SNP14-13509333 in the SNP molecular marker is greater than that of the GG genotype, a score of 1 is recorded, otherwise a score of 0 is recorded; When the frequency of the TT genotype of SNP17-8648833 in the SNP molecular marker is greater than that of the GG genotype, a score of 1 is recorded, otherwise a score of 0 is recorded; When the frequency of the TT genotype of SNP2-45873542 in the SNP molecular marker is greater than that of the CC genotype, a score of 1 is recorded, otherwise a score of 0 is recorded; When the frequency of the GG genotype of SNP10-28085288 in the SNP molecular marker is greater than that of the AA genotype, a score of 1 is recorded, otherwise a score of 0 is recorded; When the frequency of the GG genotype of SNP3-51328350 in the SNP molecular marker is greater than that of the AA genotype, a score of 1 is given, otherwise a score of 0 is given; When the frequency of the AA genotype of SNP20-6750079 in the SNP molecular marker is greater than that of the TT genotype, a score of 1 is recorded, otherwise a score of 0 is recorded; When the frequency of CC genotype of SNP11-10317498 in the SNP molecular marker is greater than that of TT genotype, a score of 1 is given, otherwise a score of 0 is given; When the frequency of the GG genotype of SNP17-4124025 in the SNP molecular marker is greater than that of the AA genotype, a score of 1 is recorded, otherwise a score of 0 is recorded.

9. The identification method according to claim 8, characterized in that: The cut-off value of the ROC curve is set as the discrimination threshold. When the total score of the large yellow croaker sample to be tested is higher than the threshold, it belongs to the wild large yellow croaker population of the Naozhou tribe.

10. Use of the method for identifying wild large yellow croaker populations of the Naozhou ethnic group according to any one of claims 6 to 9 in the identification and evaluation of large yellow croaker germplasm resources.

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