Pseudosciaena crocea wild and domesticated population typing identification method construction based on 10 general SNPs sites

Abstract

The invention belongs to the technical field of fish genetic identification, discloses construction of a large yellow croaker wild and domesticated population typing identification method based on 10 universal SNPs sites, and particularly discloses a group of SNP molecular markers for screening large yellow croaker wild and domesticated samples. The invention provides a group of SNP molecular markers for discriminating wild and domesticated samples of pseudosciaena crocea, and by detecting the SNP molecular markers, the identification of the wild and domesticated samples of the pseudosciaena crocea can be realized by only needing a multiple PCR reaction system and SNaPshot typing sequencing. The method can realize automatic typing and identification of large yellow croaker wild samples and large yellow croaker domestication samples only by operating on various genetic analysis instruments. Compared with the current method for identifying the pseudosciaena crocea population, the method provided by the invention has the advantages of quicker operation and higher detection flux, and more importantly, the method has extremely high accuracy and cumulative exclusion rate.

Description

Technical Field

[0001] The invention belongs to the technical field of fish genetic identification, and specifically relates to the construction of a typing identification method for wild and domesticated large yellow croaker populations based on 10 universal SNPs sites. Background Art

[0002] Large yellow croaker (Larimichthys crocea) belongs to the order Perciformes, family Sciaenidae, genus Larimichthys, commonly known as yellow croaker, also known as yellow croaker, big fresh, big yellow flower, etc. Large yellow croaker is a warm-temperate migratory near-shore fish living in the northwest Pacific Ocean. In my country, large yellow croaker is currently divided into three geographical populations: Daiqu, Min-Yuedong and Naozhou. Large yellow croaker is an important marine economic aquaculture fish in my country. It is famous for its tender and delicious meat, rich nutrition and beautiful body shape. It is deeply favored by consumers as a banquet dish. Large yellow croaker is rich in protein, mineral elements, amino acids and unsaturated fatty acids, and is an ideal protein source for humans. Wild large yellow croaker has higher content of beneficial mineral elements such as selenium (Se) and zinc (Zn), umami amino acids and polyunsaturated fatty acids (EPA, DHA, etc.) than farmed large yellow croaker, while farmed large yellow croaker is easier to increase protein and fatty acid content. Due to different market demands, the identification of wild / domesticated large yellow croaker population samples is of great significance.

[0003] Resequencing refers to a bioinformatics method that obtains genome sequence information of living organisms, compares it with existing genomes, and finds differences between sequence information to explore the genetic, evolutionary and biological characteristics of living organisms. Through genome resequencing, a large number of single nucleotide polymorphisms (SNPs), short fragment insertions or deletions (Short InDels), structural variations (SVs) and copy number variations (CNVs) can be obtained. These data information helps to reveal the characteristics of population differentiation and domestication selection areas, narrow the scope of candidate gene positioning, and lay the foundation for molecular breeding research. With the rise of the next-generation sequencing platform, its high-throughput and low-cost advantages have enabled more and more species to join the ranks of high-throughput sequencing. By detecting variations in species individuals or populations and building a genetic variation database for the species, it is of great significance to promote the preservation of germplasm resources and the management of gene banks. Whole genome resequencing (WGS) refers to the use of high-throughput sequencing technology to completely sequence the genome of an organism and compare it with a 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. With the continuous development of high-throughput sequencing technology, WGS technology has also been widely used. In recent years, whole genome resequencing has gradually been applied to population genetics studies of a variety of vertebrates, such as Korean cattle (Bos taurus var. coreana), domestic pigs (Sus scrofa var. domesticus), and jungle fowl (Gallus gallus). In fish, the application of whole genome resequencing is also becoming more and more common, including carp (Cyprinus carpio), Atlantic salmon (Salmo salar), and goldfish (Carassius auratus). Researchers used whole genome resequencing to analyze the population structure and population history of fish, and clarified the molecular mechanism of fish genetic traits by screening candidate genes and population genomic data sets related to genomic regions.

[0004] How to accurately and quickly identify wild and domesticated large yellow croaker populations is the core of large yellow croaker germplasm resource identification. In 2015, researchers used a strategy combining bacterial artificial chromosomes and whole-genome shotgun methods to sequence the whole genome of large yellow croaker and obtained a detailed map of the large yellow croaker genome. In 2019, researchers assembled a large yellow croaker reference genome using third-generation sequencing technology and high-throughput chromosome conformation capture technology. The highly accurate, chromosome-level large yellow croaker reference genome provides an important genomic resource to support the identification and evaluation of large yellow croaker germplasm resources. At present, the development of genetic-specific molecular markers has been applied in large yellow croaker. In 2022, researchers developed sex-specific molecular markers for the Daiqu population of large yellow croaker through the locus between dmrt1 and cfap157, providing a powerful tool for promoting sex-controlled breeding of the Daiqu population of large yellow croaker.

[0005] Based on genome resequencing and comparison of SNP marker data of yellow croaker populations distributed in the coastal waters of eastern and southern China, it was found that climate-driven habitat changes may have occurred between the Naozhou large yellow croaker and the Minyuedong large yellow croaker populations. 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 geographical structure of wild populations along the coast of China. The data of this study overturned the long-standing view that they were divided into three genetic management units. However, there is currently a lack of accurate and rapid identification methods for wild / domesticated large yellow croaker populations. The use of large yellow croaker genetic-specific markers to develop accurate and rapid identification methods for wild / domesticated large yellow croaker can effectively promote the distribution research of large yellow croaker populations in my country. Summary of the invention

[0006] The purpose of the first aspect of the present invention is 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 invention aims to provide a primer set for amplifying the SNP molecular marker of the first aspect of the present invention.

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

[0009] 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 and / or domesticated samples of large yellow croaker.

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

[0011] The purpose of the sixth aspect of the present invention is to provide the application of the method for identifying wild and / or domesticated large yellow croaker samples of the fifth aspect of the present invention in the identification and evaluation of large yellow croaker germplasm resources.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] In a first aspect of the present invention, a group of SNP molecular markers for identifying wild and domesticated samples of large yellow croaker is provided, 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] Among them, the SNP3-21436510 and SNP3-22072096 are located at positions 21436510 and 22072096 of chromosome 3 NC_040013.1 of large yellow croaker, respectively, and the polymorphisms are: C / T and T / A;

[0015] The SNP8-27317696 is located at position 27317696 of chromosome 8 NC_040018.1 of large yellow croaker, and the polymorphisms are: T / C;

[0016] The SNP11-5552678, SNP11-22820216, SNP11-3717127 and SNP11-5018155 are located at positions 5552678, 22820216, 3717127 and 5018155 of chromosome 11 of large yellow croaker NC_040021.1, respectively, and the polymorphisms are: C / T, A / G, A / T and T / C;

[0017] The SNP13-47551289 and SNP13-45288504 are located at positions 47551289 and 45288504 of chromosome 13 NC_040023.1 of large yellow croaker, respectively, and the polymorphisms are: C / T and C / G;

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

[0019] In some embodiments of the present invention, the SNP molecular markers are composed 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 present invention, the SNP molecular markers include 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 present invention, 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 present invention, 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 present invention, the SNP molecular markers consist of SNP3-21436510, SNP3-22072096, SNP8-27317696, SNP11-5552678 and SNP21-17196882.

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

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

[0026] S1. After sequencing the whole genome of 395 wild and domesticated samples of large yellow croaker, the selection signal analysis of population genetics was used to screen for SNPs sites with differences in wild and domesticated large yellow croaker;

[0027] S2. Allele frequency calculation and chi-square test were performed based on the SNPs data set to screen candidate genes with extremely significant differences in allele frequencies between wild and farmed large yellow croaker populations (P<0.001). Ten candidate loci were screened and the next step of SNP verification was carried out.

[0028] The SNPs sites based on the above screening are located on 5 different chromosomes of large yellow croaker (chromosome 3 (NC_040013.1), chromosome 8 (NC_040018.1), chromosome 11 (NC_040021.1), chromosome 13 (NC_040023.1), chromosome 21 (NC_040031.1)), 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, SNP21-17196882. A set of SNaPshot marker primers were designed for identification of wild domesticated samples of large yellow croaker.

[0029] S3. Use single / multiplex PCR reaction system to perform typing experiments to verify whether the 10 SNPs reaction system can identify wild domesticated samples of large yellow croaker;

[0030] S4. Compare the accuracy and detection rate of SNaPshot sequencing results with those of known marker detection to prove whether the above 10 SNPs combination can identify wild domesticated samples of large yellow croaker.

[0031] 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: 20.

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

[0033] The third aspect of the present invention provides a detection reagent, a gene chip or a kit comprising the primer set of the second aspect of the present invention.

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

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

[0036] In some embodiments of the present invention, the kit further comprises a single base extension primer.

[0037] In some embodiments of the present invention, the single-base extension primer is shown as SEQ ID NO: 20 to SEQ ID NO: 30.

[0038] 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 and / or domesticated large yellow croaker samples.

[0039] The fifth aspect of the present invention provides a method for identifying wild and / or domesticated large yellow croaker samples, 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.

[0040] This identification method is a multi-locus identification method for wild and domesticated large yellow croaker samples based on SNaPshot technology. This method was developed based on multiple single nucleotide polymorphism (SNP) sites screened by whole genome resequencing of large yellow croaker and selection signal analysis of population genetics. The design and detection of the method is based on the typing technology SNaPshot, which is based on the principle of fluorescent labeling single base extension. The system integrates 10 candidate indicator SNPs into the same multiplex PCR reaction, specifically amplifies and types the indication accuracy of the 10 sites in the two groups of samples. Through the SNaPshot typing verification of 240 large yellow croaker samples with known typing, it is proved that the 10-site SNPs marker system can successfully identify the wild / domesticated classification of large yellow croaker in most samples. Compared with current identification methods and markers, this marker system and detection method greatly improves the accuracy of identifying wild and domesticated populations of large yellow croaker. It can perform rapid and accurate genotyping on a variety of genetic analyzers, realizes the automation of SNP analysis, and can conveniently and efficiently realize high-throughput detection.

[0041] In some embodiments of the present invention, the identification method comprises the following steps:

[0042] (1) using the DNA of the large yellow croaker sample to be tested as a template, and 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;

[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 invention in the genome of the large yellow croaker to be tested;

[0044] (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 wild large yellow croaker or domesticated large yellow croaker.

[0045] In some embodiments of the present invention, the scoring rules 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 the CC genotype, a score of 1 is recorded, otherwise a score of 0 is recorded;

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

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

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

[0050] When the frequency of the GG genotype of SNP11-22820216 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;

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

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

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

[0054] When the frequency of the GG genotype of SNP13-45288504 in the SNP molecular marker is greater than the CC genotype, a score of 1 is recorded, otherwise a score of 0 is recorded;

[0055] When the frequency of the GG genotype of SNP21-17196882 in the SNP molecular marker is greater than that of the AA genotype, 1 point is recorded, otherwise 0 point is recorded.

[0056] 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).

[0057] 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 sample, otherwise it is a domesticated large yellow croaker sample.

[0058] In some embodiments of the present invention, the PCR amplification in step (1) is singleplex / multiplex PCR amplification.

[0059] 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 28-35 s, extension at 70-72°C for 20-30 s, 32-37 cycles; extension at 70-72°C for 8-12 min.

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

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

[0062] 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, 58-62° C. for 28-32 s, and 25-30 cycles.

[0063] A sixth aspect of the present invention provides the application of the method for identifying wild and / or domesticated large yellow croaker samples of the fifth aspect of the present invention in the identification and evaluation of large yellow croaker germplasm resources.

[0064] The beneficial effects of the present invention are:

[0065] The present invention provides a set of SNP molecular markers for identifying wild and domesticated samples of large yellow croaker. By detecting the SNP molecular markers, only one multiplex PCR reaction system and SNaPshot typing and sequencing are required to identify wild and domesticated samples of large yellow croaker. Only a variety of genetic analysis instruments are required to automatically type and identify wild and domesticated samples of large yellow croaker. Compared with the current method for identifying large yellow croaker populations, this method is faster to operate and has a higher detection throughput. More importantly, this method has an extremely high accuracy rate and cumulative exclusion rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 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 large yellow croaker, X142, X143, and E73 are known wild samples of large yellow croaker, N is a negative control with no sample, and M is a maker.

[0067] Figure 2 This is the peak graph detected by SNaPshot.

[0068] Figure 3 Figure 3 is the ROC curve corresponding to the multi-locus large yellow croaker wild / domesticated sample discrimination method based on SNPs; where A is the ROC curve of the discrimination method based on H1, H2, H3, H4, H5, H6, H7, H8, H9 and H10, B is the ROC curve of the discrimination method based on H1, H2, H3, H4, H5, H7, H8, H9 and H10, C is the ROC curve of the discrimination method based on H1, H2, H3, H4, H5, H8, H9 and H10, D is the ROC curve of the discrimination method based on H1, H2, H3, H4, H5, H9 and H10, E is the ROC curve of the discrimination method based on H1, H2, H3, H4 and H10, and F is the ROC curve of the discrimination method based on H1, H2 and H10. DETAILED DESCRIPTION

[0069] The present invention is further described in detail below through specific examples.

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

[0071] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0072] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0073] Example 1

[0074] A set of SNPs for identification of wild domesticated samples of large yellow croaker based on SNaPshot technology were constructed. The SNPs were located on five different chromosomes of large yellow croaker (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). )), 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.

[0075] The above 10 SNPs were obtained through whole genome resequencing and population genetics analysis screening. The specific methods are as follows:

[0076] A total of 576.43G whole genome resequencing raw data were generated for 395 large yellow croaker individuals, with an average of 13405.42M raw data generated per sample. On this basis, a total of 574.60G filtered data were generated, with an average of 13362.97M generated per sample. 99.48% of reads were mapped to the large yellow croaker reference genome, and a total of 78370338 raw SNPs were obtained. After filtering and screening, a total of 2041211 high-quality SNPs were obtained. Allele frequency calculation and chi-square test (P<0.05) were performed based on the SNPs dataset. The 10 SNPs with the highest allele frequency difference between wild large yellow croaker populations and domesticated large yellow croaker populations and passing the chi-square test were selected as candidate specific genetic difference sites and SNPs expansion verification based on SNaPshot technology was performed.

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

[0078] Table 1 Specific genotypes of the 10 SNPs

[0079]

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

[0081] Table 2 Amplification primer sequences corresponding to the 10 SNPs

[0082]

[0083]

[0084] Note: R is the degenerate base of GA, and M is the degenerate base of AC.

[0085] Table 3 Extension primer sequences corresponding to the 10 SNPs

[0086]

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

[0088] Example 2

[0089] A multi-site identification method for wild domesticated large yellow croaker samples based on SNaPshot technology comprises the following steps:

[0090] S1: Extract genomic DNA from the fin tissue of the tested large yellow croaker population using the phenol-chloroform extraction method;

[0091] 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.

[0092] Table 4 Multiplex PCR reaction system

[0093]

[0094]

[0095] Table 5 Multiplex PCR reaction program

[0096]

[0097] The amplified product was digested. The digestion system was as shown in Table 6. The digestion conditions were 37°C for 60 min and 75°C for 15 min.

[0098] Table 6 Digestion system

[0099]

[0100] The digested product was extended. The extension reaction system was shown in Table 7. The extension conditions were 96°C for 10 s, 50°C for 5 s, 60°C for 30 s, and 27 cycles.

[0101] Table 7 Extension reaction system

[0102]

[0103] The extension product was purified: 0.5 μL CIP was added to 6 μL of the extension reaction product; incubated at 37°C for 1.0 h and at 75°C for 15 min.

[0104] S3: 3730XL sequencer detection

[0105] 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;

[0106] 2) Incubate at 95°C for 3 minutes, then place on ice, and perform sequencing using a 3730XL sequencer;

[0107] 3) Data analysis: Import the raw data files obtained from the test into the analysis software for analysis;

[0108] S4: Combined with the SNaPshot validation results, the differences in allele frequencies of the above 10 SNPs between wild and domesticated populations of large yellow croaker were analyzed (P<0.05);

[0109] S5: Construct a method for distinguishing wild and domesticated large yellow croaker samples based on the above 10 SNPs

[0110] Combined with the genotypes in Table 1, the alleles with a higher frequency in the wild population of large yellow croaker than in the domesticated population were scored as 1, and the alleles with a lower frequency in the wild population of large yellow croaker than in the domesticated population were scored as 0. A method for distinguishing large yellow croaker wild / domesticated samples based on the above 10 SNPs was constructed. Generally, if a method is constructed based on x sites (x≤8), the total score of the discrimination method is 2x (number of alleles*2).

[0111] All samples to be tested were scored, and based on the total score of the 10 SNPs site scoring results, the total score data sets of wild populations and domesticated populations were constructed respectively. The data sets were input into GraphPad Prism 8 to construct the receiver operating characteristic curve (ROC curve, the method was feasible when the ROC curve area (AUC) was greater than 0.9). 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%)), and the samples with scores higher than the threshold were wild samples of large yellow croaker, and the samples with scores lower than the threshold were domesticated samples of large yellow croaker.

[0112] Example 3

[0113] The multi-site large yellow croaker wild and domesticated sample identification method based on SNaPshot technology in Example 2 was used to identify known wild large yellow croaker and domesticated large yellow croaker populations, with 120 wild large yellow croakers and 120 domesticated large yellow croakers.

[0114] 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 the SNaPshot detection peaks are shown in Figure 2 The specific genotype analysis results are shown in Table 8. The screening method based on the above 10 SNPs is shown in the attached Figure 3 As shown, by combining 10 SNPs ( Figure 3 In Figure A), the constructed ROC curve area (AUC) was 0.9999, the sensitivity of the optimal threshold was 100.00% (i.e., the probability of a wild large yellow croaker being identified by this evaluation method was 100.00%), and the specificity was 99.17% (i.e., the probability of a domesticated large yellow croaker being excluded by this evaluation method was 99.17%), where an AUC area greater than 0.9 indicated that the method was feasible.

[0115] In addition, when the number of loci was reduced to 9 (SNPs loci included the combination of H1, H2, H3, H4, H5, H7, H8, H9, and H10), 8 (SNPs loci included the combination of H1, H2, H3, H4, H5, H8, H9, and H10), 7 (SNPs loci included the combination of H1, H2, H3, H4, H5, H9, and H10), 5 (SNPs loci included the combination of H1, H2, H3, H4, and H10), or 3 (SNPs loci included the combination of H1, H2, and H10), the AUC areas were all greater than 0.9, with a sensitivity of more than 96% and a specificity of more than 95% ( Figure 3 B~F in the middle). It shows that the above methods can be applied to the identification of wild large yellow croaker and domesticated large yellow croaker.

[0116] Table 8 Genotype analysis results of wild and domesticated large yellow croaker populations

[0117]

[0118]

[0119] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A group of SNP molecular markers for identifying wild and domesticated samples of large yellow croaker, the SNP molecular markers comprising 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, in, The SNP3-21436510 and SNP3-22072096 are located at positions 21436510 and 22072096 of chromosome 3 NC_040013.1 of large yellow croaker, respectively, and the polymorphisms are: C / T and T / A; The SNP8-27317696 is located at position 27317696 of chromosome 8 NC_040018.1 of large yellow croaker, and the polymorphisms are: T / C; The SNP11-5552678, SNP11-22820216, SNP11-3717127 and SNP11-5018155 are located at positions 5552678, 22820216, 3717127 and 5018155 of chromosome 11 of large yellow croaker NC_040021.1, respectively, and the polymorphisms are: C / T, A / G, A / T and T / C; The SNP13-47551289 and SNP13-45288504 are located at positions 47551289 and 45288504 of chromosome 13 NC_040023.1 of large yellow croaker, respectively, and the polymorphisms are: C / T and C / G; The SNP21-17196882 is located at position 17196882 of chromosome 21 NC_040031.1 of large yellow croaker, and the polymorphism is G / A.

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:

20.

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: 20 constitute a pair of primer sets in order.

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 and / or domesticated large yellow croaker samples.

6. A method for identifying wild and / or domesticated large yellow croaker samples, comprising the step of using the primer set described in claim 2 or 3 or the detection reagent, gene chip or kit described in claim 4 to detect the SNP molecular marker described in claim 1 on the large yellow croaker sample to be tested.

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, and performing PCR amplification using the primer set described in claim 2 or 3 or the detection reagent, gene chip or kit described in 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 described in 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 wild large yellow croaker or domesticated large yellow croaker.

8. The identification method according to claim 7, characterized in that: The scoring rule in step (3) is as follows: when the frequency of the TT genotype of SNP3-21436510 in the SNP molecular marker is greater than the CC genotype, 1 point is scored, otherwise 0 point is scored; When the frequency of the AA genotype of SNP3-22072096 in the SNP molecular marker is greater than the TT genotype, a score of 1 is recorded, otherwise a score of 0 is recorded; When the frequency of the TT genotype of SNP8-27317696 in the SNP molecular marker is greater than the CC genotype, 1 point is recorded, otherwise 0 point is recorded; When the frequency of the TT genotype of SNP11-5552678 in the SNP molecular marker is greater than the CC genotype, a score of 1 is recorded, otherwise a score of 0 is recorded; When the frequency of the GG genotype of SNP11-22820216 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 TT genotype of SNP11-3717127 in the SNP molecular marker is greater than the AA genotype, 1 point is recorded, otherwise 0 point is recorded; When the frequency of the TT genotype of SNP11-5018155 in the SNP molecular marker is greater than the CC genotype, 1 point is recorded, otherwise 0 point is recorded; When the frequency of CC genotype of SNP13-47551289 in the SNP molecular marker is greater than that of TT genotype, 1 point is recorded, otherwise 0 point is recorded; When the frequency of the GG genotype of SNP13-45288504 in the SNP molecular marker is greater than the CC genotype, a score of 1 is recorded, otherwise a score of 0 is recorded; When the frequency of the GG genotype of SNP21-17196882 in the SNP molecular marker is greater than that of the AA genotype, 1 point is recorded, otherwise 0 point 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 is a wild large yellow croaker sample, otherwise it is a domesticated large yellow croaker sample.

10. Application of the screening method according to any one of claims 6 to 9 in the identification and evaluation of large yellow croaker germplasm resources.

Images