Development of a genotyping method for wild and domesticated populations of large yellow croaker based on 10 common snps

Abstract

The present application belongs to the technical field of fish genetic identification, discloses a large yellow croaker wild and domestication population typing identification method based on 10 universal SNPs sites, and specifically discloses a set of SNP molecular markers for identifying large yellow croaker wild and domestication samples. The present application provides a set of SNP molecular markers for identifying large yellow croaker wild and domestication samples. By detecting the SNP molecular markers, the identification of large yellow croaker wild and domestication samples can be realized only by using a multiplex PCR reaction system and SNaPshot typing sequencing. The operation on various genetic analysis instruments can realize automatic typing and identification of large yellow croaker wild samples and large yellow croaker domestication samples. Compared with the current method for identifying large yellow croaker populations, the method is faster, has higher detection flux, and more importantly, has very high accuracy and cumulative exclusion rate.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic identification of fish, and particularly relates to a method for constructing a Larimichthys crocea wild and domesticated population typing identification method based on 10 universal SNPs sites. BACKGROUND

[0002] Larimichthys crocea belongs to Perciformes, Sciaenidae, and Larimichthys, and is commonly known as yellow croaker, yellow fish, and large yellow croaker. Larimichthys crocea is a warm-temperate migratory coastal fish living in the northwest Pacific Ocean. In China, Larimichthys crocea is mainly divided into three geographical populations, namely, Daiqu population, Min-Yue East population, and Naozhou population. Larimichthys crocea is an important marine economic fish for aquaculture in China. It is famous for its tender and delicious meat, rich nutrition, and beautiful body shape, and is favored by consumers as a feast dish. Larimichthys crocea contains rich protein, mineral elements, amino acids, and unsaturated fatty acids, and is an ideal protein source for humans. Wild Larimichthys crocea has higher contents of beneficial mineral elements such as selenium (Se) and zinc (Zn), delicious amino acids, and polyunsaturated fatty acids (EPA, DHA, etc.), while farmed Larimichthys crocea is easier to improve the contents of protein and fatty acids. Due to different market demands, it is of great significance to distinguish wild / farmed Larimichthys crocea population samples.

[0003] Re-sequencing is a bioinformatics method that explores the genetic, evolutionary and biological characteristics of living organisms by obtaining their genomic sequence information, comparing it with existing genomes, and finding differences between the sequence information. Through genome re-sequencing, a large number of single nucleotide polymorphisms (SNP), short insertions or deletions (Short InDel), structural variations (SV) and copy number variations (CNV) sites can be obtained. These data help to reveal the characteristics of population differentiation and domestication selection regions, and narrow down the range of candidate genes, laying the foundation for molecular breeding research. With the rise of new generation sequencing platforms, their high throughput and low cost have made more and more species join the ranks of high-throughput sequencing. By detecting variations in individuals or populations of a species, a genetic variation database for the species can be constructed, which is of great significance for promoting the preservation of germplasm resources and the management of gene banks. Whole genome sequencing (WGS) refers to the use of high-throughput sequencing technology to sequence the entire genome of an organism and compare it with the reference genome. This technology can be used to analyze genetic differences between individuals, find disease-related genes or gene mutation sites, and reveal genome structure, etc. With the continuous development of high-throughput sequencing technology, WGS technology has been widely applied. In recent years, whole genome re-sequencing has been gradually applied to the population genetic research of various vertebrates, such as Korean cattle (Bos taurus var. coreana), domestic pigs (Sus scrofa var. domesticus), and original chickens (Gallus gallus). In fish, the application of whole genome re-sequencing is also becoming more and more common, including common carp (Cyprinus carpio), Atlantic salmon (Salmo salar), and goldfish (Carassius auratus). Researchers have used whole genome re-sequencing to analyze fish population structure and population history, and by screening candidate genes and population genomic datasets related to genomic regions, they have elucidated the molecular mechanisms of fish genetic traits.

[0004] How to accurately and quickly identify wild and domesticated populations of large yellow croaker is the core of large yellow croaker germplasm identification. In 2015, researchers used a combination of bacterial artificial chromosome and whole genome shotgun sequencing strategy to sequence the whole genome of large yellow croaker and obtained a fine map of the genome of large yellow croaker. In 2019, researchers used third-generation sequencing technology and high-throughput chromosome conformation capture technology to assemble a reference genome of large yellow croaker. The highly accurate and chromosome-level reference genome of large yellow croaker provides important genomic resources for supporting the identification and evaluation of large yellow croaker germplasm. Currently, the development of genetic specific molecular markers has been applied in large yellow croaker. In 2022, researchers developed a gender-specific molecular marker for the Daiqu population of large yellow croaker by locating between dmrt1 and cfap157, which provides a powerful tool for promoting gender control breeding of the Daiqu population of large yellow croaker.

[0005] Based on the SNP marker data of large yellow croaker populations distributed in the coastal areas of eastern and southern China, it was found that there may be climate-driven habitat changes between the Naozhou population of large yellow croaker and the Minyue population of large yellow croaker. Genetic structure analysis using whole-genome resequencing data of a large number of samples including domesticated and wild populations showed that there was no obvious geographic structure in the wild populations along the coast of China. The data of this study refuted the long-held view of dividing them into three genetic management units. However, there is currently a lack of accurate and rapid identification method for wild / domesticated populations of large yellow croaker, and the development of genetic specific markers for large yellow croaker can effectively promote the distribution research of large yellow croaker populations in China. SUMMARY

[0006] The first aspect of the present application aims to provide a set of SNP molecular markers for identifying wild and domesticated samples of large yellow croaker.

[0007] The second aspect of the present application aims to provide a primer set for amplifying the SNP molecular markers of the first aspect of the present application.

[0008] The third aspect of the present application aims to provide a detection reagent, a gene chip or a kit.

[0009] The fourth aspect of the present application aims to provide the application of the SNP molecular markers of the first aspect of the present application, the primer set of the second aspect of the present application, or the detection reagent, the gene chip or the kit of the third aspect of the present application in the identification of wild and / or domesticated samples of large yellow croaker.

[0010] The fifth aspect of the present application aims to provide a method for identifying wild and / or domesticated samples of large yellow croaker

[0011] The sixth aspect of the present application aims to provide an application of the wild and / or domesticated sample discrimination method of the fifth aspect of the present application in the identification and evaluation of the genetic resources of Pseudosciaena crocea.

[0012] In order to achieve the above-mentioned purpose, the technical solution adopted by the present application is:

[0013] The first aspect of the present application provides a set of SNP molecular markers for discriminating wild and domesticated samples of Pseudosciaena crocea, wherein the SNP molecular markers include at least three of SNP3-21436510, SNP3-22072096, SNP8-27317696, SNP11-5552678, SNP11-22820216, SNP11-3717127, SNP11-5018155, SNP13-47551289, SNP13-45288504 and SNP21-17196882,

[0014] The SNP3-21436510 and SNP3-22072096 are sequentially located at the 21436510th and 22072096th positions of the chromosome 3 NC_040013.1 of Pseudosciaena crocea, and the polymorphisms are C / T and T / A, respectively.

[0015] The SNP8-27317696 is located at the 27317696th position of the chromosome 8 NC_040018.1 of Pseudosciaena crocea, and the polymorphism is T / C.

[0016] The SNP11-5552678, SNP11-22820216, SNP11-3717127 and SNP11-5018155 are sequentially located at the 5552678th, 22820216th, 3717127th and 5018155th positions of the chromosome 11 NC_040021.1 of Pseudosciaena crocea, and the polymorphisms are C / T, A / G, A / T and T / C, respectively.

[0017] The SNP13-47551289 and SNP13-45288504 are sequentially located at the 47551289th and 45288504th positions of the chromosome 13 NC_040023.1 of Pseudosciaena crocea, and the polymorphisms are C / T and C / G, respectively.

[0018] The SNP21-17196882 is located at the 17196882th position of the chromosome 21 NC_040031.1 of Pseudosciaena crocea, and the polymorphism is G / A.

[0019] In some embodiments of the application, the SNP molecular markers consist of SNP3-21436510, SNP3-22072096, SNP8-27317696, SNP11-5552678, SNP11-22820216, SNP11-3717127, SNP11-5018155, SNP13-47551289, SNP13-45288504, and SNP21-17196882.

[0020] In some embodiments of the application, the SNP molecular markers consist of SNP3-21436510, SNP3-22072096, SNP8-27317696, SNP11-5552678, SNP11-22820216, SNP11-5018155, SNP13-47551289, SNP13-45288504, and SNP21-17196882.

[0021] In some embodiments of the application, the SNP molecular markers consist of SNP3-21436510, SNP3-22072096, SNP8-27317696, SNP11-5552678, SNP11-22820216, SNP13-47551289, SNP13-45288504, and SNP21-17196882.

[0022] In some embodiments of the application, the SNP molecular markers consist of SNP3-21436510, SNP3-22072096, SNP8-27317696, SNP11-5552678, SNP11-22820216, SNP13-45288504, and SNP21-17196882.

[0023] In some embodiments of the application, the SNP molecular markers consist of SNP3-21436510, SNP3-22072096, SNP8-27317696, SNP11-5552678, and SNP21-17196882.

[0024] In some embodiments of the application, the SNP molecular markers consist of SNP3-21436510, SNP3-22072096, and SNP21-17196882.

[0025] In some embodiments of the application, the above 10 SNP molecular markers are obtained by the following steps:

[0026] S1, after whole genome sequencing of 395 wild and domesticated samples of Pseudosciaena crocea, the difference SNPs sites between wild and domesticated Pseudosciaena crocea are screened by population genetics selection signal analysis;

[0027] S2, on the basis of SNPs data set, allele frequency calculation and chi-square test are carried out, and the candidate genes with extremely significant difference in allele frequency between wild Pseudosciaena crocea population and cultured Pseudosciaena crocea population are screened (P<0.001), 10 candidate sites are screened, and the next step of SNP verification is carried out.

[0028] The screened SNPs sites are located on 5 different chromosomes (chromosome 3 (NC_040013.1), chromosome 8 (NC_040018.1), chromosome 11 (NC_040021.1), chromosome 13 (NC_040023.1) and chromosome 21 (NC_040031.1)) of Pseudosciaena crocea, and the position information is as follows: SNP3-21436510, SNP3-22072096, SNP8-27317696, SNP11-5552678, SNP11-22820216, SNP11-3717127, SNP11-5018155, SNP13-47551289, SNP13-45288504 and SNP21-17196882. A set of SNaPshot marker primers for identifying wild and domesticated samples of Pseudosciaena crocea are designed.

[0029] S3, a single / multiple PCR reaction system is used for typing experiment to verify whether the 10 SNPs reaction systems can identify wild and domesticated samples of Pseudosciaena crocea;

[0030] S4, the SNaPshot sequencing results are compared with the results of known marker detection in terms of accuracy and detection rate, and whether the above 10 SNPs combinations can identify wild and domesticated samples of Pseudosciaena crocea is proved.

[0031] In a second aspect of the present application, a primer set for amplifying the SNP molecular marker of the first aspect of the present application is provided, and the nucleotide sequence of the primer set is shown in SEQ ID NO:1-SEQ ID NO:20.

[0032] In some embodiments of the present application, the SEQ ID NO:1-SEQ ID NO:20 form a pair of primer sets in order every two nucleic acid sequences.

[0033] In a third aspect of the present application, a detection reagent, a gene chip or a kit comprising the primer set of the second aspect of the present application is provided.

[0034] In some embodiments of the present application, the kit further comprises one or more of dNTPs, DNA polymerase, PCR reaction buffer, Taq.

[0035] In some embodiments of the present application, the kit further comprises SAP and ExoI.

[0036] In some embodiments of the present application, the kit further comprises single base extension primers.

[0037] In some embodiments of the present application, the single base extension primers are as shown in SEQ ID NO: 20-30.

[0038] In a fourth aspect of the present application, there is provided the use of the SNP molecular marker of the first aspect of the present application, the primer set of the second aspect of the present application, or the detection reagent, gene chip or kit of the third aspect of the present application in the discrimination of wild and / or domesticated samples of P. major.

[0039] In a fifth aspect of the present application, there is provided a method for discriminating wild and / or domesticated samples of P. major, comprising the step of detecting the SNP molecular marker of the first aspect of the present application in a sample of P. major to be tested using the primer set of the second aspect of the present application or the detection reagent, gene chip or kit of the third aspect of the present application.

[0040] The discrimination method is a multi-site discrimination method for wild and domesticated samples of P. major based on SNaPshot technology. The method is developed based on a plurality of single nucleotide polymorphism (SNP) sites screened by whole genome resequencing and population genetics selection signal analysis. The design and detection of the method are based on the typing technology of SNaPshot which is a single base extension typing technology based on fluorescent labeling. The system combines 10 candidate indicator SNPs into the same multiplex PCR reaction, and specifically amplifies and types to detect the indication accuracy of the 10 sites in the two groups of samples. Through SNaPshot typing verification on 240 tails of known typing samples of P. major, it is proved that the 10 site SNP marker system can successfully identify most of the wild / domesticated classification of P. major in the samples. Compared with the current identification method and marker, the marker system and detection method greatly improve the recognition accuracy of wild and domesticated populations of P. major, can be quickly and accurately genotyped on various genetic analyzers, realize the automation of SNP analysis, and can realize high-throughput detection conveniently and efficiently.

[0041] In some embodiments of the present application, the discrimination method comprises the following steps:

[0042] (1) using the DNA of the sample of the P. major to be tested as a template, performing PCR amplification using the primer set of the second aspect of the present application or the detection reagent, gene chip or kit of the third aspect of the present application to obtain a PCR amplification product;

[0043] (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 application in the genome of the P. major to be tested;

[0044] (3) analyzing the frequency of the genotype of the SNP molecular marker, scoring, and constructing a ROC curve to determine whether the sample of the P. major to be tested is a wild P. major or a domesticated P. major.

[0045] In some embodiments of the present application, the rules of the scoring in step (3) are as follows:

[0046] When the frequency of the TT genotype of SNP3-21436510 in the SNP molecular marker is greater than that of the CC genotype, score 1, otherwise score 0;

[0047] When the frequency of the AA genotype of SNP3-22072096 in the SNP molecular marker is greater than that of the TT genotype, score 1, otherwise score 0;

[0048] When the frequency of the TT genotype of SNP8-27317696 in the SNP molecular marker is greater than that of the CC genotype, score 1, otherwise score 0;

[0049] When the frequency of the TT genotype of SNP11-5552678 in the SNP molecular marker is greater than that of the CC genotype, score 1, otherwise score 0;

[0050] When the frequency of the GG genotype of SNP11-22820216 in the SNP molecular marker is greater than that of the AA genotype, score 1, otherwise score 0;

[0051] When the frequency of the TT genotype of SNP11-3717127 in the SNP molecular marker is greater than that of the AA genotype, score 1, otherwise score 0;

[0052] When the frequency of the TT genotype of SNP11-5018155 in the SNP molecular marker is greater than that of the CC genotype, score 1, otherwise score 0;

[0053] When the frequency of the CC genotype of SNP13-47551289 in the SNP molecular marker is greater than that of the TT genotype, score 1, otherwise score 0;

[0054] When the frequency of GG genotype of SNP 13-45288504 in the SNP molecular marker is greater than that of CC genotype, score 1, otherwise score 0;

[0055] When the frequency of GG genotype of SNP 21-17196882 in the SNP molecular marker is greater than that of AA genotype, score 1, otherwise score 0.

[0056] Generally, if a discrimination method is constructed based on x loci (x≤8), the total score of the discrimination method is 2x (allele number*2).

[0057] In some embodiments of the present application, the cut-off value of the ROC curve (i.e. the maximum value of sensitivity (sensitivity %) + specificity (specificity %) of the ROC curve) is set as the discrimination threshold value, and when the total score of the sample to be tested is higher than the threshold value, the sample is a wild sample of P. major, otherwise it is a domesticated sample of P. major.

[0058] In some embodiments of the present application, the PCR amplification in step (1) is single / multiple PCR amplification.

[0059] In some embodiments of the present application, the reaction procedure of the PCR amplification is 92-96℃ pre-denaturation for 3-6min; 92-96℃ denaturation for 28-35s, 58-62℃ annealing for 28-35s, 70-72℃ extension for 20-30s, 32-37 cycles; 70-72℃ extension for 8-12min.

[0060] In some embodiments of the present application, the PCR amplification product is subjected to digestion, single-base extension, purification and the like before sequencing.

[0061] In some embodiments of the present application, the reaction system of the digestion comprises 8-12μL PCR amplification product, 0.2-0.4U SAP and 0.05-0.2U ExoI; the reaction conditions of the digestion are 35-38℃, 55-65min; 70-77℃, 12-16min.

[0062] In some embodiments of the present application, the reaction conditions of the single-base extension are 94-97℃ for 8-13s, 46-52℃ for 3-7s, 58-62℃ for 28-32s, 25-30 cycles.

[0063] In the sixth aspect of the present application, the discrimination method of wild and / or domesticated sample of P. major in the fifth aspect of the present application is applied to the identification and evaluation of P. major germplasm resources.

[0064] The present application has the following beneficial effects:

[0065] The application provides a set of SNP molecular markers for identifying wild and domesticated samples of Pseudosciaena crocea. By detecting the SNP molecular markers, wild and domesticated samples of Pseudosciaena crocea can be identified only by using a multiplex PCR reaction system and SNaPshot typing sequencing. The wild and domesticated samples of Pseudosciaena crocea can be identified only by operating on various genetic analysis instruments, realizing automatic typing. Compared with the current method for identifying Pseudosciaena crocea populations, the method is faster, has higher detection throughput, and more importantly, has a high accuracy and cumulative exclusion rate. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1 For the results of single amplification (top) or mixed amplification (bottom) of SNaPshot primers designed for SNPs, in the figure, C3, B45, Y9 and Y2 are known domesticated samples of Pseudosciaena crocea, X142, X143 and E73 are known wild samples of Pseudosciaena crocea, N is a sample negative control, and M is a marker.

[0067] Figure 2 For the SNaPshot detection peak chart.

[0068] Figure 3 For the ROC curve of the multi-site wild / domesticated sample identification method of Pseudosciaena crocea based on SNPs; wherein, A is the ROC curve of the identification method based on H1, H2, H3, H4, H5, H6, H7, H8, H9 and H10, B is the ROC curve of the identification method based on H1, H2, H3, H4, H5, H7, H8, H9 and H10, C is the ROC curve of the identification method based on H1, H2, H3, H4, H5, H8, H9 and H10, D is the ROC curve of the identification method based on H1, H2, H3, H4, H5, H9 and H10, E is the ROC curve of the identification method based on H1, H2, H3, H4 and H10, and F is the ROC curve of the identification method based on H1, H2 and H10. DETAILED DESCRIPTION

[0069] The content of the application will be further described in detail through specific examples.

[0070] It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application.

[0071] In order to make the purpose, technical scheme and advantages of the embodiments of the application more clear, the technical scheme in the embodiments of the application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturer are used. If the used reagents or instruments are not specified by the manufacturer, they are all conventional products that can be purchased in the market.

[0072] The features and performances of the present application are further described in detail below in combination with examples.

[0073] Example 1

[0074] A set of SNaPshot technology-based multi-site SNPs for distinguishing wild and domesticated samples of Pseudosciaena crocea, the SNPs being located on five different chromosomes (chromosome 3 (NC_040013.1), chromosome 8 (NC_040018.1), chromosome 11 (NC_040021.1), chromosome 13 (NC_040023.1), and chromosome 21 (NC_040031.1)) of Pseudosciaena crocea, the position information being as follows: SNP3-21436510, SNP3-22072096, SNP8-27317696, SNP11-5552678, SNP11-22820216, SNP11-3717127, SNP11-5018155, SNP13-47551289, SNP13-45288504, and SNP21-17196882.

[0075] The above 10 SNPs are obtained through Whole Genome Resequencing sequencing and population genetics analysis screening, and the specific method is as follows:

[0076] A total of 576.43G Whole Genome Resequencing raw data is generated for 395 Pseudosciaena crocea individuals, with an average of 13405.42M raw data generated per sample. On this basis, a total of 574.60G filtered data is generated, with an average of 13362.97M per sample. 99.48% of the reads are located to the Pseudosciaena crocea reference genome, and a total of 78370338 original SNPs are obtained. After filtering and screening, a total of 2041211 high-quality SNPs are obtained. Based on the SNPs data set, allele frequency calculation and chi-square test (P<0.05) are performed, and 10 SNPs with the highest difference in allele frequency between wild Pseudosciaena crocea population and domesticated Pseudosciaena crocea population and passing the chi-square test are selected as candidate specific genetic difference sites and subjected to SNaPshot technology-based SNP expansion verification.

[0077] The specific genotypes of the 10 SNPs are shown in Table 1.

[0078] Table 1 Specific genotypes of 10 SNPs

[0079]

[0080] Corresponding peripheral amplification primer sequences are designed for the above 10 SNPs, and the primer sequences are shown in Table 2.

[0081] Table 2 Sequences of the amplification primers corresponding to the 10 SNPs

[0082]

[0083]

[0084] Note: R is a degenerate base for GA, and M is a degenerate base for AC.

[0085] Table 3 Sequences of the extension primers corresponding to the 10 SNPs

[0086]

[0087] Note: In Table 3, R is a degenerate base for A / G, Y is a degenerate base for C / T, and S is a degenerate base for G / C.

[0088] Example 2

[0089] A multi-site wild domesticated sample discrimination method for Pseudosciaena crocea based on SNaPshot technology, comprising the following steps:

[0090] S1: Extracting genomic DNA from the fin tissue of the Pseudosciaena crocea to be tested by using phenol-chloroform extraction method;

[0091] S2: Constructing a multiplex PCR reaction system for typing experiment, and the multiplex PCR reaction system is shown in Table 4, and the reaction program is shown in Table 5.

[0092] Table 4 Multiplex PCR reaction system

[0093]

[0094]

[0095] Table 5 Multiplex PCR reaction program

[0096]

[0097] Digesting the amplification product, and the digestion system is shown in Table 6, and the digestion condition is 37℃ for 60 min; 75℃ for 15 min.

[0098] Table 6 Digestion system

[0099]

[0100] Extending the product after digestion, and the extension reaction system is shown in Table 7, and the extension condition is 96℃ for 10 s, 50℃ for 5 s, 60℃ for 30 s, 27 cycles.

[0101] Table 7 Extension reaction system

[0102]

[0103] Purification of the extension product: 6 μL of the extension reaction product was added with 0.5 μL CIP; 37°C for 1.0 h, 75°C for 15 min.

[0104] S3: 3730XL sequencer detection

[0105] 1) Add 9 μL of molecular weight marker and formamide mixture and 1 μL of product to each well of a 96-well plate;

[0106] 2) After 3 min at 95°C, ice bath, 3730XL sequencer detection;

[0107] 3) Data analysis: import the raw data file obtained by detection into the analysis software for analysis;

[0108] S4: Combine the SNaPshot verification results to analyze the significance (P < 0.05) of the difference in allele frequency at the above 10 SNPs between the wild and domesticated groups of P. major;

[0109] S5: Construct a P. major wild / domesticated sample discrimination method based on the above 10 SNPs

[0110] Based on the genotypes in Table 1, the alleles with a higher frequency in the wild group of P. major than in the domesticated group were scored as 1, and the alleles with a lower frequency in the wild group of P. major than in the domesticated group were scored as 0. A P. major wild / domesticated sample discrimination method based on the above 10 SNPs was constructed. Generally, if a method is constructed based on x sites (x ≤ 8) on this basis, the total score of the discrimination method is 2x (allele number * 2).

[0111] All samples to be tested were scored, and based on the total score of the 10 SNP site scores, the total score datasets of the wild and domesticated groups were constructed, and the datasets were input into GraphPad Prism 8 to construct a receiver operating characteristic curve (ROC curve, and an area under the ROC curve (AUC) greater than 0.9 indicates that the method is feasible). The cut-off value was set as the discrimination threshold (the cut-off value is the maximum value of the sensitivity (sensitivity %) and specificity (specificity %) of the ROC curve), and the sample score was higher than the threshold value, which was the wild sample of P. major, and the sample score was lower than the threshold value, which was the domesticated sample of P. major.

[0112] Example 3

[0113] The SNaPshot technology-based multi-site discrimination method of Larimichthys crocea wild domestication samples of Example 2 was used to discriminate between known wild Larimichthys crocea and domesticated Larimichthys crocea populations, each of which had 120 samples.

[0114] The electropherogram of the PCR reaction product of some Larimichthys crocea samples is shown in Figure 2. Figure 1 The SNaPshot analysis of the above Larimichthys crocea samples produced some SNaPshot detection peak charts, as shown in Figure 3. Figure 2 The specific genotype analysis results are shown in Table 8. Based on the above 10 SNPs, a discrimination method was constructed, as shown in Figure 4. Figure 3 Figure 3 The area of the ROC curve (AUC) constructed by the combination of the 10 SNPs (A) was 0.9999, the sensitivity of the optimal threshold was 100.00% (i.e., the probability that a wild Larimichthys crocea sample was identified by this evaluation method was 100.00%), and the specificity was 99.17% (i.e., the probability that a domesticated Larimichthys crocea sample was excluded by this evaluation method was 99.17%), and the AUC area was greater than 0.9, indicating that the method was feasible.

[0115] In addition, when the number of sites was reduced to 9 (SNP sites including the combination of H1, H2, H3, H4, H5, H7, H8, H9, and H10), 8 (SNP sites including the combination of H1, H2, H3, H4, H5, H8, H9, and H10), 7 (SNP sites including the combination of H1, H2, H3, H4, H5, H9, and H10), 5 (SNP sites including the combination of H1, H2, H3, H4, and H10), or 3 (SNP sites including the combination of H1, H2, and H10), the AUC area was greater than 0.9, and the sensitivity was greater than 96% and the specificity was greater than 95% (B-F in Table 1). Figure 3

[0116] Table 8 Genotype analysis results of wild Larimichthys crocea and domesticated Larimichthys crocea populations

[0117]

[0118]

[0119] The above embodiments of the present application are described in detail in conjunction with the accompanying drawings, but the present application is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by a person of ordinary skill in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.​​

Claims

1. A combination of SNP molecular markers for identifying wild and domesticated samples of Pseudosciaena crocea, wherein the combination of SNP molecular markers is a combination of SNP3-21436510, SNP3-22072096, SNP8-27317696, SNP11-5552678, and SNP21-17196882; or the combination of SNP molecular markers is a combination of SNP3-21436510, SNP3-22072096, and SNP21-17196882. wherein The SNP3-21436510 and SNP3-22072096 are located at positions 21436510 and 22072096 of chromosome 3 of Pseudosciaena crocea NC_040013.1, respectively, and the polymorphisms are C / T and T / A, respectively. The SNP8-27317696 is located at position 27317696 of chromosome 8 of Pseudosciaena crocea NC_040018.1, and the polymorphism is T / C. The SNP11-5552678 is located at position 5552678 of chromosome 11 of Pseudosciaena crocea NC_040021.1, and the polymorphism is C / T. The SNP21-17196882 is located at position 17196882 of chromosome 21 of Pseudosciaena crocea NC_040031.1, and the polymorphism is G / A.

2. A set of primer sets, characterized in that, The nucleotide sequences of the primer sets are shown in SEQ ID NO: 1-SEQ ID NO:

20.

3. The primer set of claim 2, wherein, Each two nucleic acid sequences of SEQ ID NO: 1-SEQ ID NO: 20 form a pair of primer sets in order. 4.A detection reagent or kit comprising the primer set of claim 2 or 3. 5.Use of the primer set of claim 2 or 3 or the detection reagent or kit of claim 4 in identifying wild and / or domesticated samples of Pseudosciaena crocea. 6.A method for identifying wild and / or domesticated samples of Pseudosciaena crocea, comprising the step of detecting the SNP molecular markers of claim 1 in a sample of Pseudosciaena crocea to be tested using the primer set of claim 2 or 3 or the detection reagent or kit of claim 4.

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