Multi-site large yellow croaker wild domestication group screening method based on SNaPshot technology

Abstract

The invention belongs to the technical field of fish genetic identification, discloses a multi-site screening method for wild domestication groups of large yellow croakers based on an SNaPshot technology, and particularly discloses a group of SNP (Single Nucleotide Polymorphism) molecular markers for screening the wild domestication groups of the large yellow croakers, and the SNP molecular markers comprise at least three of SNP1-30187747, SNP1-30206146, SNP1-30225064, SNP13-42432874, SNP13-43081647, SNP13-43107208, SNP13-43228958 and SNP15-22344715. By detecting the SNP molecular marker, the identification of the wild domesticated population of the pseudosciaena crocea can be realized by only needing a multiple PCR reaction system and SNaPshot typing sequencing.

Description

Technical Field

[0001] The invention belongs to the technical field of fish genetic identification, and in particular relates to a multi-site large yellow croaker wild domesticated population screening 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, 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, and is distributed in many places. Large yellow croaker is an important marine economic farmed 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 yellow croaker has higher content of beneficial mineral elements such as selenium (Se), zinc (Zn), umami amino acids and polyunsaturated fatty acids (EPA, DHA, etc.) than farmed yellow croaker, while farmed yellow croaker is easier to increase the protein and fatty acid content.

[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 constructing 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. According to the different needs for obtaining the scope of genomic genetic information, it can be divided into whole genome resequencing (Whole Genome Sequencing, WGS) and reduced-representation genome sequencing (Reduced-Representation Genome Sequencing, RRGS). Based on the variation detection of simplified genome, the partial sequence of the whole genome is obtained, which is usually used to detect single nucleotide polymorphisms. The accuracy of detecting other types of variations is relatively low. Whole genome resequencing refers to the use of high-throughput sequencing technology to completely sequence the 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, reveal genome structure, etc. With the continuous development of high-throughput sequencing technology, WGS technology has also been widely used. The advancement of sequencing technology has strongly promoted the development of genomics, allowing complex genome sequences to be presented through accurate sequencing and precise assembly. With the development of sequencing technology, whole genome resequencing has also gone through three stages, namely Sanger sequencing technology, NGS technology, and PacBio and Nanopore technology. In recent years, whole genome resequencing has gradually been applied to a variety of vertebrates, such as Korean cattle (Bos taurus var.coreana), domestic pigs (Sus scrofa var.domesticus), and wild chickens (Gallusgallus) in population genetic studies. In fish, the application of whole genome resequencing is becoming more and more common, including common carp (Cyprinus carpio), Atlantic salmon, and goldfish (Carassius auratus). Researchers use whole genome resequencing to analyze the structure and historical evolution of fish populations, and to clarify the molecular mechanisms of fish genetic traits by screening candidate genes and population genomic data sets related to genomic regions.

[0004] Genome-wide selection signals refer to statistically significant genetic variations detected in the genome of a species. These variations may be the result of natural or artificial selection, reflecting the advantages of different genotypes in adapting to the environment. Detection of genomic selection signals is an important research field that can be used to determine on which genes and genomic regions natural or artificial selection acts. Currently, the commonly used methods mainly include methods based on allele frequency spectrum, methods based on linkage disequilibrium, and methods based on population differentiation. The method based on allele frequency spectrum is a statistical method for detecting positive selection signals. It detects selection signals by comparing the differences between the allele frequency spectra of two or more populations. This method is mainly used to detect strong selection signals. Commonly used allele frequency spectra include single nucleotide polymorphism frequency spectra and haplotype frequency spectra. The method based on linkage disequilibrium uses the linkage disequilibrium relationship between different sites on the genome to detect selection signals. This method can detect selection signals in low-frequency alleles. It usually uses a method based on haplotype frequency to determine the linkage disequilibrium relationship on the genome and detects selection signals by comparing the differences in linkage disequilibrium structure between different populations. The population differentiation-based method is a statistical method that uses the degree of differentiation of the genome in different populations to detect selection signals. This method can detect weaker selection signals and can be used to identify artificial selection. This method is often used to detect the effects of natural and artificial selection on the genome of species.

[0005] 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, Ao et al. used a strategy combining bacterial artificial chromosomes and whole-genome shotgun sequencing to sequence the whole genome of large yellow croaker and obtained a detailed map of the large yellow croaker genome. In 2019, Chen et al. assembled the large yellow croaker reference genome using third-generation sequencing technology (PacBio single-molecule 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, Yu et al. developed sex-specific molecular markers for the Daiqu population of large yellow croaker through the genome between dmrt1 and cfap157, providing a favorable tool for promoting sex-controlled breeding of the Daiqu population of large yellow croaker.

[0006] In 2023, Chen et al. compared the SNP marker data of yellow croaker populations distributed in the eastern and southern coastal waters of China based on genome resequencing and found that climate-driven habitat changes may have occurred between the Naozhou yellow croaker and the Min-Yuedong yellow croaker populations. In 2024, Yuan et al. used whole genome resequencing data of a large number of samples, including domesticated and wild populations, to conduct genetic structure analysis, indicating that there is 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 populations of yellow croaker. The use of genetic-specific markers of yellow croaker to develop accurate and rapid identification methods for wild / domesticated yellow croaker can effectively promote the distribution research of yellow croaker populations in 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 and domesticated populations of large yellow croaker.

[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, a gene chip or a 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 and / or domesticated populations of large yellow croaker.

[0011] The fifth aspect of the present invention aims to provide a method for identifying wild and / or domesticated populations of large yellow croaker

[0012] The purpose of the sixth aspect of the present invention is to provide the application of the method for identifying wild and / or domesticated populations of large yellow croaker according to 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 and domesticated populations of large yellow croaker is provided, wherein the SNP molecular markers include at least three of SNP1-30187747, SNP1-30206146, SNP1-30225064, SNP13-42432874, SNP13-43081647, SNP13-43107208, SNP13-43228958 and SNP15-22344715,

[0015] Among them, the SNP1-30187747, SNP1-30206146 and SNP1-30225064 are located at positions 30187747, 30206146 and 30225064 of chromosome 1 NC_040011.1 of large yellow croaker, respectively, and the polymorphisms are: A / G, A / G and T / C;

[0016] The SNP13-42432874, SNP13-43081647, SNP13-43107208 and SNP13-43228958 are located at positions 42432874, 43081647, 43107208 and 43228958 of chromosome 13 NC_040023.1 of large yellow croaker, and the polymorphisms are: G / A, C / T, G / A and G / A;

[0017] The SNP15-22344715 is located at position 22344715 of chromosome 15 NC_040023.1 of large yellow croaker, and the polymorphism is G / A.

[0018] In some embodiments of the present invention, the SNP molecular markers consist of SNP1-30187747, SNP1-30206146, SNP1-30225064, SNP13-42432874, SNP13-43081647, SNP13-43107208, SNP13-43228958 and SNP15-22344715.

[0019] In some embodiments of the present invention, the SNP molecular markers consist of SNP1-30187747, SNP1-30225064, SNP13-42432874, SNP13-43081647, SNP13-43107208, SNP13-43228958 and SNP15-22344715.

[0020] In some embodiments of the present invention, the SNP molecular markers consist of SNP1-30187747, SNP1-30225064, SNP13-42432874, SNP13-43081647, SNP13-43107208 and SNP13-43228958.

[0021] In some embodiments of the present invention, the SNP molecular markers consist of SNP1-30187747, SNP1-30225064, SNP13-42432874 and SNP13-43228958.

[0022] In some embodiments of the present invention, the SNP molecular markers consist of SNP1-30187747, SNP1-30225064 and SNP13-42432874.

[0023] The above 8 SNP molecular markers were obtained by the following steps:

[0024] S1. After sequencing the whole genome of 395 wild and domesticated large yellow croaker populations, the selection signal analysis of population genetics was used to screen for SNPs sites with differences in wild and domesticated large yellow croaker.

[0025] S2. Strong selection signals based on Fst & π were detected in the genome to explore the genetic differences caused by different living environments between wild and domesticated large yellow croaker. The top 2% of Fst and the top 5% of π ratios were used as selection signal regions for wild and domesticated large yellow croaker. Based on the selection signal analysis, candidate genes with extremely significant differences in allele frequencies between wild and farmed large yellow croaker populations were screened in the selection signal region (P<0.001), and a total of 139 genes were identified. On this basis, 8 candidate genes with non-synonymous mutations caused by SNPs in exons were screened for the next step of SNP verification.

[0026] The SNPs sites based on the above screening are located on three different chromosomes of large yellow croaker (chromosome 1 (NC_040011.1), chromosome 13 (NC_040023.1), chromosome 15 (NC_040025.1)), and the position information is as follows: SNP1-30187747, SNP1-30206146, SNP1-30225064, SNP13-42432874, SNP13-43081647, SNP13-43107208, SNP13-43228958, SNP15-22344715. A set of SNaPshot marker primers that can be used for the identification of wild domesticated populations of large yellow croaker was designed;

[0027] S3. Use single / multiplex PCR reaction system to perform typing experiments to verify whether the 8 SNPs reaction system can identify wild domesticated populations of large yellow croaker;

[0028] S4. Compare the accuracy and detection rate of SNaPshot sequencing results with those of known marker detection to prove whether the above 8 SNPs combination can identify wild domesticated populations of large yellow croaker.

[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:16.

[0030] In some embodiments of the present invention, every two nucleic acid sequences in SEQ ID NO: 1 to SEQ ID NO: 16 constitute a pair of primer sets in order.

[0031] 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.

[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: 17 to SEQ ID NO: 24.

[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 and / or domesticated populations of large yellow croaker.

[0037] The fifth aspect of the present invention provides a method for identifying wild and / or domesticated populations of large yellow croaker, 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 population to be tested.

[0038] The identification method was developed based on multiple single nucleotide polymorphism (SNP) sites screened by whole genome resequencing and population genetics selection signal analysis of large yellow croaker. 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 8 candidate indicator SNPs into the same multiplex PCR reaction, specifically amplifies and types the 8 sites to detect the indication accuracy in two groups. Through the SNaPshot typing verification of 240 large yellow croaker groups with known typing, it is proved that the 8-site SNP marker system can successfully identify the wild / domesticated classification of large yellow croaker in most groups. Compared with the current identification methods and markers, this marker system and detection method greatly improves the accuracy of identification of wild and domesticated large yellow croaker populations, can be quickly and accurately genotyped on a variety of genetic analyzers, realizes the automation of SNP analysis, and can conveniently and efficiently realize high-throughput detection.

[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, 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;

[0041] (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;

[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 population to be tested is wild large yellow croaker or domesticated large yellow croaker.

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

[0044] When the frequency of the AA genotype of SNP1-30187747 in the SNP molecular marker is greater than the GG genotype, a score of 1 is recorded, otherwise a score of 0 is recorded;

[0045] When the frequency of the GG genotype of SNP1-30206146 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;

[0046] When the frequency of the TT genotype of SNP1-30225064 in the SNP molecular marker is greater than the CC genotype, 1 point is recorded, otherwise 0 point is recorded;

[0047] When the frequency of the GG genotype of SNP13-42432874 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;

[0048] When the frequency of the TT genotype of SNP13-43081647 in the SNP molecular marker is greater than the CC genotype, a score of 1 is recorded, otherwise a score of 0 is recorded;

[0049] When the frequency of the GG genotype of SNP13-43107208 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;

[0050] When the frequency of the GG genotype of SNP13-43228958 in the SNP molecular marker is greater than the AA genotype, a score of 1 is recorded, otherwise a score of 0 is recorded;

[0051] When the frequency of the GG genotype of SNP15-22344715 in the SNP molecular marker is greater than that of the AA genotype, 1 point is recorded, otherwise 0 point is recorded.

[0052] 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).

[0053] 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 population to be tested is higher than the threshold, it is a wild large yellow croaker population, otherwise it is a domesticated large yellow croaker population.

[0054] In some embodiments of the present invention, the PCR amplification in step (1) is singleplex / multiplex PCR amplification.

[0055] In some embodiments of the present invention, the reaction procedure for 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.

[0056] In some embodiments of the present invention, the PCR amplification product is subjected to digestion, single base extension, purification, and other treatments before sequencing.

[0057] 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.

[0058] 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.

[0059] A sixth aspect of the present invention provides the application of the method for identifying wild and / or domesticated populations of large yellow croaker according to the fifth aspect of the present invention in the identification and evaluation of large yellow croaker germplasm resources.

[0060] The beneficial effects of the present invention are:

[0061] The present invention provides a group of SNP molecular markers for identifying wild and domesticated populations of large yellow croaker. By detecting the SNP molecular markers, identification of wild and domesticated populations of large yellow croaker can be achieved with only one multiplex PCR reaction system and SNaPshot typing and sequencing. Only a variety of genetic analysis instruments are needed to automatically type and identify wild and domesticated populations 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

[0062] Figure 1 Selection signal analysis for wild and domesticated large yellow croaker populations; A is the Fst value distribution of wild and domesticated large yellow croaker populations (Wild) calculated with a step size of 10 kb in a 50 kb sliding window, and B is the π ratio distribution of wild and domesticated large yellow croaker populations (Breed) calculated with a step size of 10 kb in a 50 kb sliding window.

[0063] Figure 2 Results of single amplification (top) or mixed amplification (bottom) of SNaPshot primers designed for SNPs. In the figure, C3, B7, Y1, and Y2 are known domesticated populations of large yellow croaker, X142, X143, A91, and E93 are known wild populations of large yellow croaker, N is a negative control with no sample, and M is a maker.

[0064] Figure 3 This is the peak graph detected by SNaPshot.

[0065] Figure 4Figure 3 is the ROC curve corresponding to the multi-locus large yellow croaker wild / domesticated population discrimination method based on SNPs; where A is the ROC curve of the discrimination method based on H1, H2, H3, H4, H5, H6, H7 and H8, B is the ROC curve of the discrimination method based on H1, H2, H3, H4, H5, H6 and H7, C is the ROC curve of the discrimination method based on H1, H3, H4, H5, H6, H7 and H8, D is the ROC curve of the discrimination method based on H1, H3, H4, H5, H6 and H7, E is the ROC curve of the discrimination method based on H1, H3, H4 and H7, and F is the ROC curve of the discrimination method based on H1, H3 and H4. DETAILED DESCRIPTION

[0066] The present invention is further described in detail below through specific examples.

[0067] 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.

[0068] 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.

[0069] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0070] Example 1

[0071] A set of SNPs identified from wild domesticated populations of large yellow croaker based on multi-locus SNaPshot technology, the SNPs are located on three different chromosomes of large yellow croaker (chromosome 1 (NC_040011.1), chromosome 13 (NC_040023.1) and chromosome 15 (NC_040025.1)), and the position information is as follows: SNP1-30187747, SNP1-30206146, SNP1-30225064, SNP13-42432874, SNP13-43081647, SNP13-43107208, SNP13-43228958 and SNP15-22344715.

[0072] The above 8 SNPs were obtained through whole genome resequencing and population genetics selection signal analysis screening. The specific methods are as follows:

[0073] 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 for each sample. On this basis, a total of 574.60G filtered data were generated, with an average of 13362.97M generated for each sample. 99.48% of the 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. Based on the SNPs dataset, a strong selection signal based on the fixation index (Fst) and the genetic polymorphism ratio (π) was detected as a significant genetic difference between the wild large yellow croaker population and the domesticated large yellow croaker population ( Figure 1 ). The specific screening conditions were the top 2% of Fst values ​​and the top 5% of π ratios, and a total of 622 differentially expressed genes were screened. Furthermore, based on the selection signal analysis, 139 candidate genes with extremely significant differences in allele frequencies between wild and domesticated large yellow croaker populations (P<0.001) were screened in the selection signal region. Among them, 8 candidate genes with non-synonymous mutations caused by SNPs in exons were screened and verified as candidate SNPs.

[0074] The specific genotypes of the eight SNPs are shown in Table 1.

[0075] Table 18 SNPs specific genotypes

[0076]

[0077] Corresponding peripheral amplification primer sequences and extension primers were designed for the above 8 SNPs, and the primer sequences are shown in Tables 2 and 3.

[0078] Table 2 Amplification primer sequences corresponding to 8 SNPs

[0079]

[0080]

[0081] Table 3 Extension primer sequences corresponding to 8 SNPs

[0082]

[0083] Note: R is the degenerate base of GA.

[0084] Example 2

[0085] A multi-site large yellow croaker wild domesticated population screening method based on SNaPshot technology, comprising the following steps:

[0086] S1: Extract genomic DNA from the fin tissue of the tested large yellow croaker population using the phenol-chloroform extraction method;

[0087] 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.

[0088] Table 4 Multiplex PCR reaction system

[0089]

[0090] Table 5 Multiplex PCR reaction program

[0091]

[0092] 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.

[0093] Table 6 Digestion system

[0094]

[0095] 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.

[0096] Table 7 Extension reaction system

[0097]

[0098] 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.

[0099] S3: 3730XL sequencer detection

[0100] 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;

[0101] 2) Incubate at 95°C for 3 minutes, then place on ice and perform detection using a 3730 sequencer;

[0102] 3) Data analysis: Import the raw data files obtained from the detection into the analysis software for analysis.

[0103] S4: Combined with the SNaPshot verification results, the significance of the differences in the allele frequencies of the above 8 SNPs between the wild and domesticated populations of large yellow croaker was analyzed (P<0.05).

[0104] S5: Construct a method to distinguish wild and domesticated populations of large yellow croaker based on the above 8 SNPs

[0105] 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 populations based on the above 8 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).

[0106] Scores were given to all the tested populations, and based on the total scores of the 8 SNPs site scoring results, total score data sets of wild populations and domesticated populations were constructed respectively. The data sets were input into GraphPad Prism 8 to construct receiver operating characteristic curves (ROC curves, where 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%)). The populations with scores higher than the threshold were wild populations of large yellow croaker, and the populations with scores lower than the threshold were domesticated populations of large yellow croaker.

[0107] Example 3

[0108] The multi-site large yellow croaker wild and domesticated population 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 respectively.

[0109] The electrophoresis diagram of PCR reaction products of some large yellow croaker samples is as follows: Figure 2 By performing SNaPshot analysis on the above large yellow croaker, some of the SNaPshot detection peaks are shown in Figure 3 The specific genotype analysis results are shown in Table 8. Based on the above 8 SNPs, the identification method was constructed as follows Figure 4 As shown, through the combination of 8 SNPs ( Figure 4 The constructed ROC curve area (AUC) was 0.9469, the sensitivity of the optimal threshold was 93.3% (i.e., the probability of a wild large yellow croaker being identified by this evaluation method was 93.3%), and the specificity was 89.2% (i.e., the probability of a domesticated large yellow croaker being excluded by this evaluation method was 89.2%). The method was feasible when the AUC area was greater than 0.9.

[0110] In addition, when the SNPs sites are reduced to 7 (such as SNPs sites including a combination of H1, H2, H3, H4, H5, H6 and H7, or a combination of H1, H3, H4, H5, H6, H7 and H8), 5 (such as SNPs sites including a combination of H1, H3, H4, H5, H6 and H7), 4 (such as SNPs sites including a combination of H1, H3, H4, and H7), 3 (such as SNPs sites including a combination of H1, H3 and H4), the steps of Example 2 are used to construct the identification methods. The verification shows that the AUC area of ​​each identification method is greater than 0.9, and has a sensitivity of more than 90% and a specificity of more than 70% ( Figure 4 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.

[0111] Table 8 Genotype analysis results of wild and domesticated large yellow croaker populations

[0112]

[0113] Example 4

[0114] In order to further verify the universal applicability of the multi-site large yellow croaker wild and domesticated population screening method based on SNaPshot technology in Example 2, 30 additional known large yellow croaker wild / domesticated populations were selected for a second round of screening. Scoring was performed based on the SNaPshot test results of the 30 populations, of which 12 large yellow croaker wild populations and 18 large yellow croaker domesticated populations were screened, which were consistent with the known typing of large yellow croaker. This shows that the 8 SNPs site mixed screening method has universal applicability in indicating large yellow croaker wild / domesticated populations.

[0115] 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 populations of large yellow croaker, the SNP molecular markers comprising at least three of SNP1-30187747, SNP1-30206146, SNP1-30225064, SNP13-42432874, SNP13-43081647, SNP13-43107208, SNP13-43228958 and SNP15-22344715, in, The SNP1-30187747, SNP1-30206146 and SNP1-30225064 are located at positions 30187747, 30206146 and 30225064 of chromosome 1 NC_040011.1 of large yellow croaker, respectively, and the polymorphisms are: A / G, A / G and T / C; The SNP13-42432874, SNP13-43081647, SNP13-43107208 and SNP13-43228958 are located at positions 42432874, 43081647, 43107208 and 43228958 of chromosome 13 NC_040023.1 of large yellow croaker, and the polymorphisms are: G / A, C / T, G / A and G / A; The SNP15-22344715 is located at position 22344715 of chromosome 15 NC_040023.1 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:

16.

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: 16 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 populations of large yellow croaker.

6. A method for identifying wild and / or domesticated populations of large yellow croaker, 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 population 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 population 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 AA genotype of SNP1-30187747 in the SNP molecular marker is greater than the GG genotype, 1 point is scored, otherwise 0 point is scored; When the frequency of the GG genotype of SNP1-30206146 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 SNP1-30225064 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 GG genotype of SNP13-42432874 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 SNP13-43081647 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 SNP13-43107208 in the SNP molecular marker is greater than the AA genotype, a score of 1 is recorded, otherwise a score of 0 is recorded; When the frequency of the GG genotype of SNP13-43228958 in the SNP molecular marker is greater than the AA genotype, a score of 1 is recorded, otherwise a score of 0 is recorded; When the frequency of the GG genotype of SNP15-22344715 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 population to be tested is higher than the threshold, it is a wild large yellow croaker population, otherwise it is a domesticated large yellow croaker population.

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.

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