A method for identifying multiple sites of wild pescadorus reevesii based on SNaPshot technology

Abstract

The application belongs to the technical field of fish genetic identification, and discloses a multi-site identification method for wild large yellow croaker of Lutjanus Russellii based on SNaPshot technology, and specifically discloses a group of SNP molecular markers for identifying wild large yellow croaker of Lutjanus Russellii, wherein by detecting the SNP molecular markers, the identification of wild large yellow croaker of Lutjanus Russellii in wild large yellow croaker samples can be realized only by using a simple multiplex PCR reaction system and SNaPshot typing sequencing. The wild large yellow croaker samples of Lutjanus Russellii can be automatically typed and identified only by operating on various genetic analysis instruments. Compared with the method for identifying wild large yellow croaker of Lutjanus Russellii, the method is more accurate, faster and higher in detection flux, and has higher accuracy and cumulative exclusion rate.

Description

Technical Field

[0001] This invention belongs to the field of fish genetic identification technology, specifically relating to a multi-site identification method for wild large yellow croaker of the Naozhou group based on SNaPshot technology. Background Technology

[0002] Large yellow croaker (Larimichthys crocea) belongs to the order Perciformes, family Sciaenidae, and genus Larimichthys. It is commonly known as yellow croaker, and also called yellow fish, large fresh fish, and large yellow flower. It is a warm-temperate migratory coastal fish living in the northwestern Pacific Ocean. In my country, large yellow croaker is currently mainly divided into three geographical groups: the Daiqu group, the Min-Yuedong group, and the Naozhou group. They are distributed from the southern Yellow Sea in the north, through the East China Sea and the Taiwan Strait, to the area east of the Leizhou Peninsula in the South China Sea. The Min-Yuedong group large yellow croaker is mainly distributed in the waters near Fujian Province. Regarding the Min-Yuedong group large yellow croaker, my country took the lead in organizing scientific and technological personnel to conduct research on "artificial breeding and aquaculture techniques of large yellow croaker" in 1985, and mastered the artificial breeding techniques for broodstock in 1987. The large yellow croaker from Naozhou, which grows in the waters near Zhanjiang City, Guangdong Province, has high environmental requirements and is difficult to domesticate. Recently, a team in Zhanjiang, Guangdong Province, has also made a breakthrough in the artificial breeding of the large yellow croaker from Naozhou.

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

[0004] Maximum likelihood estimation (MLE), a classic statistical method, constructs a probabilistic model based on observational data and infers parameters by maximizing the likelihood function. Due to its theoretical rigor and flexibility, it is widely used in population genetics research. In fish population genotyping, this method is highly compatible with the characteristics of SNP data detected by resequencing: each SNP locus in a diploid individual can be considered as two independent samples, and the number of minor alleles naturally follows a binomial distribution, conforming to the random assignment assumption under Hardy-Weinberg equilibrium. Research practice shows that maximum likelihood estimation has been applied in salmon population tracing and Atlantic cod subpopulation analysis. By jointly calculating the probability contribution of multiple SNP loci, it can quantify the matching degree between the test sample and the reference population, combining computational efficiency and interpretability. Compared to Bayesian methods or machine learning models, MLE is more robust in small sample scenarios and avoids the risks of complex prior settings or overfitting. Based on the above biological rationale and technological maturity, using maximum likelihood estimation for population genotyping of large yellow croaker is feasible and can provide a reliable basis for accurately identifying the genetic background of fish.

[0005] Accurately and rapidly distinguishing the Naozhou group of large yellow croaker from other large yellow croaker populations is the core of Naozhou group large yellow croaker germplasm resource identification. In 2015, researchers used a strategy combining bacterial artificial chromosomes and whole-genome shotgun sequencing to sequence the entire large yellow croaker genome, obtaining a fine map of the large yellow croaker genome. In 2019, researchers assembled a 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 important genomic resources to support the identification and evaluation of large yellow croaker germplasm resources. Currently, the development of genetically specific molecular markers has been applied in large yellow croaker. In 2022, researchers developed sex-specific molecular markers for the large yellow croaker Daiqu population through the locus between dmrt1 and cfap157, providing a useful tool for promoting sex-controlled breeding of the large yellow croaker Daiqu population.

[0006] In 2023, researchers, based on genome resequencing and comparison of SNP marker data from large yellow croaker populations distributed in the coastal waters of eastern and southern China, discovered a possible climate-driven habitat change between the *Large Yellow Croaker* 'Naozhou' and *Large Yellow Croaker* 'Min-Yue East' populations. In 2024, researchers conducted genetic structure analysis using whole-genome resequencing data from a large sample including domesticated and wild populations, indicating that wild populations along the Chinese coast lack a clear geographical structure. This study overturned the long-held view of dividing them into three genetic management units. Therefore, comparative analysis was conducted on the geographically disputed *Large Yellow Croaker* 'Naozhou' population in western Guangdong, *Large Yellow Croaker* 'Min-Yue East' population in Fujian waters, and *Large Yellow Croaker* 'Huidong' population in the Pearl River Estuary in the intermediate geographical area, and population-specific molecular markers were screened. In the South China Sea, it is morphologically difficult to distinguish between wild *Large Yellow Croaker* 'Naozhou', wild *Large Yellow Croaker* 'Min-Yue East', and wild *Large Yellow Croaker* populations in Huidong. Therefore, the wild large yellow croaker of the Naozhou group in western Guangdong is currently easily confused with other large yellow croaker groups in the South China Sea, and there is a lack of accurate and rapid identification methods for this group. Developing an accurate and rapid identification method for the Naozhou group using genetically specific markers can effectively promote the study of the distribution of wild large yellow croaker groups along my country's coast. Summary of the Invention

[0007] The first objective of this invention is to provide a set of SNP molecular markers for the identification of wild large yellow croaker populations in the Naozhou group.

[0008] A second aspect of the present invention is to provide a primer set for amplifying the SNP molecular markers of the first aspect of the present invention.

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

[0010] The fourth aspect of this invention aims to provide the application of the SNP molecular markers of the first aspect of this invention, the primer sets of the second aspect of this invention, or the detection reagents, gene chips, or kits of the third aspect of this invention in the identification of wild large yellow croaker populations in Naozhou group.

[0011] The fifth objective of this invention is to provide a method for identifying populations of wild large yellow croaker from the Naozhou group.

[0012] The sixth aspect of this invention aims to provide the application of the method for identifying wild large yellow croaker populations from Naozhou in the identification and evaluation of large yellow croaker germplasm resources, as described in the fifth aspect of this invention.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

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

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

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

[0017] The SNPs 20-95328, 20-73728, and 20-6750079 are located at positions 95328, 73728, and 6750079 on chromosome 20 of large yellow croaker, respectively, with polymorphisms of G / C, A / G, and T / A, respectively.

[0018] The SNP14-13509333 is located at position 13509333 on chromosome 14 of large yellow croaker (NC_040024.1), and its polymorphism is T / G.

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

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

[0021] The SNP10-28085288 is located at position 28085288 on chromosome 10 of large yellow croaker (NC_040020.1), and its polymorphism is A / G.

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

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

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

[0025] S2. Based on the SNPs dataset, allele frequencies were calculated and chi-square tests were performed to screen candidate genes (P<0.001) that showed extremely significant differences in allele frequencies between the Naozhou wild large yellow croaker population and other wild large yellow croaker populations (Fujian and Guangdong eastern wild large yellow croaker, Huidong wild large yellow croaker). Fourteen candidate loci were obtained and were further validated by SNPs.

[0026] The SNPs identified through the above screening are located on nine different chromosomes of the large yellow croaker (chromosomes 2 (NC_040012.1), 3 (NC_040013.1), 7 (NC_040017.1), 10 (NC_040020.1), 11 (NC_040021.1), 14 (NC_040024.1), 17 (NC_040027.1), and 20 (NC_040030.1)). Their locations are as follows: SNP3-47505387, SNP7-9407565, SNP3-31085986, SNP3-42982617, SNP20-95328, SNP20-73728, SNP14-13509333, SNP17-8648833, SNP2-45873542, SNP10-28085288, SNP3-51328350, SNP20-6750079, SNP11-10317498, and SNP17-4124025. A set of SNaPshot marker primers was designed for the identification of wild large yellow croaker populations in the Naozhou group.

[0027] S3. Use single / multiplex PCR reaction system to perform typing experiments to verify whether the 14 SNPs reaction system can identify wild large yellow croaker samples from Naozhou wild large yellow croaker, wild large yellow croaker from Fujian and Guangdong, and wild large yellow croaker from Huidong.

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

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

[0030] In some embodiments of the present invention, SEQ ID NO:1 to SEQ ID NO:28 are arranged in sequence to form a primer pair of two nucleic acid sequences.

[0031] A third aspect of the present invention provides detection reagents, gene chips, or kits 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 includes SAP and ExoI.

[0034] In some embodiments of the present invention, the kit further includes single-base extension primers.

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

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

[0037] A fifth aspect of the present invention provides a method for identifying a population of wild large yellow croaker from Naozhou, 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 in the large yellow croaker sample to be tested.

[0038] This identification method is a multi-site population identification method for wild large yellow croaker (Cetacea naan) based on SNaPshot technology. This method was developed based on multiple single nucleotide polymorphism (SNP) sites screened through whole-genome resequencing and population genetic selection signal analysis of large yellow croaker. The method design and detection are based on SNaPshot, a genotyping technique using fluorescently labeled single-base extension. The system integrates 14 candidate indicator SNPs into a single multiplex PCR reaction, specifically amplifying and genotyping the 14 sites to detect their accuracy in the tested samples. SNaPshot genotyping validation on 108 known-genotyped large yellow croaker samples demonstrated that the 14-site SNP marker system can successfully identify the vast majority of wild large yellow croaker (Cetacea naan) in the samples. Compared to current identification methods and markers, this marker system and detection method significantly improve the accuracy of identifying wild large yellow croaker (Cetacea naan), enabling rapid and accurate genotyping on various genetic analyzers, automating SNP analysis, and facilitating convenient and efficient high-throughput detection.

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

[0040] (1) Using the DNA of the large yellow croaker sample to be tested as a template, PCR amplification is performed using the primer set of 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 PCR amplification products.

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

[0042] (3) Analyze the frequency of the genotypes of the SNP molecular markers, score them, construct ROC curves, and determine that the large yellow croaker sample to be tested is a wild large yellow croaker of the Naozhou tribe.

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

[0044] If the frequency of the CC genotype of SNP3-47505387 in the SNP molecular marker is greater than that of the TT genotype, then 1 point is awarded; otherwise, 0 points are awarded.

[0045] If the frequency of the CC genotype of SNP7-9407565 in the SNP molecular marker is greater than that of the TT genotype, then 1 point is awarded; otherwise, 0 points are awarded.

[0046] If the frequency of the CC genotype of SNP3-31085986 in the SNP molecular marker is greater than that of the TT genotype, then 1 point is awarded; otherwise, 0 points are awarded.

[0047] If the frequency of the AA genotype of SNP3-42982617 in the SNP molecular marker is greater than that of the GG genotype, then 1 point is awarded; otherwise, 0 points are awarded.

[0048] If the frequency of the CC genotype of SNP20-95328 in the SNP molecular marker is greater than that of the GG genotype, then 1 point is awarded; otherwise, 0 points are awarded.

[0049] If the frequency of the GG genotype of SNP20-73728 in the SNP molecular marker is greater than that of the AA genotype, then 1 point is awarded; otherwise, 0 points are awarded.

[0050] If the frequency of the TT genotype of SNP14-13509333 in the SNP molecular marker is greater than that of the GG genotype, then 1 point is awarded; otherwise, 0 points are awarded.

[0051] If the frequency of the TT genotype of SNP17-8648833 in the SNP molecular marker is greater than that of the GG genotype, then 1 point is awarded; otherwise, 0 points are awarded.

[0052] If the frequency of the TT genotype of SNP2-45873542 in the SNP molecular marker is greater than that of the CC genotype, then 1 point is awarded; otherwise, 0 points are awarded.

[0053] If the frequency of the GG genotype of SNP10-28085288 in the SNP molecular marker is greater than that of the AA genotype, then 1 point is awarded; otherwise, 0 points are awarded.

[0054] If the frequency of the GG genotype of SNP3-51328350 in the SNP molecular marker is greater than that of the AA genotype, then 1 point is awarded; otherwise, 0 points are awarded.

[0055] If the frequency of the AA genotype of SNP20-6750079 in the SNP molecular marker is greater than that of the TT genotype, then 1 point is awarded; otherwise, 0 points are awarded.

[0056] If the frequency of the CC genotype of SNP11-10317498 in the SNP molecular marker is greater than that of the TT genotype, then 1 point is awarded; otherwise, 0 points are awarded.

[0057] If the frequency of the GG genotype of SNP17-4124025 in the SNP molecular marker is greater than that of the AA genotype, then 1 point is awarded; otherwise, 0 points are awarded.

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

[0059] In some embodiments of the present invention, the cut-off value of the ROC curve (i.e., the maximum value of the ROC curve sensitivity (sensitivity%) + specificity (specificity%)) is set as the discrimination threshold. When the total score of the large yellow croaker sample to be tested is higher than the threshold, it is a wild large yellow croaker population from Naozhou, otherwise it is another wild large yellow croaker population (such as wild large yellow croaker from Fujian and Guangdong, and wild large yellow croaker from Huidong).

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

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

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

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

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

[0065] In some embodiments of the present invention, the identification method further includes using the maximum likelihood estimation method to perform genotyping verification of population samples, including calculating the overall log-likelihood value of all samples and all loci using the maximum likelihood method, and classifying the sample with the highest overall log-likelihood value based on the number of minor alleles as the corresponding reference population. Specific rules are as follows:

[0066]

[0067] Among them, A, B, and C are reference populations for wild large yellow croaker from Naozhou, wild large yellow croaker from Fujian and Guangdong, and wild large yellow croaker from Huidong, respectively.

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

[0069] The beneficial effects of this invention are:

[0070] This invention provides a set of SNP molecular markers for identifying wild large yellow croaker populations of the Naozhou group. By detecting these SNP molecular markers, the identification of wild large yellow croaker from the Naozhou group in wild population samples can be achieved using only a simple multiplex PCR reaction system and SNaPshot genotyping sequencing. The method can be automated by operating on various genetic analysis instruments, enabling automatic genotyping and identification of wild large yellow croaker samples from the Naozhou group. Compared to current methods for identifying wild large yellow croaker from the Naozhou group, this method offers more accurate genotyping, faster operation, higher throughput, and higher accuracy and cumulative exclusion rate. Attached Figure Description

[0071] Figure 1 Partial amplification electrophoresis image of SNaPshot primers designed for SNPs.

[0072] Figure 2 This is a peak diagram of SNaPshot detection for SNPs.

[0073] Figure 3 The ROC curves for the multi-site discrimination method of wild large yellow croaker from Naozhou based on SNPs and other wild large yellow croaker populations (wild large yellow croaker from Fujian and Guangdong, and wild large yellow croaker from Huidong) are shown. Among them, A is the ROC curve of the discrimination method of wild large yellow croaker from Naozhou based on 14 SNPs and wild large yellow croaker from Fujian and Guangdong; B is the ROC curve of the discrimination method of wild large yellow croaker from Naozhou based on 14 SNPs and wild large yellow croaker from Huidong. Detailed Implementation

[0074] The present invention will be further described in detail below through specific embodiments.

[0075] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0076] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

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

[0078] Example 1

[0079] A set of SNPs (Small Indices) based on SNaPshot technology were used to distinguish a multi-locus wild large yellow croaker population from Naozhou group from other wild large yellow croaker populations (Fujian-Guangdong wild large yellow croaker, Huidong wild large yellow croaker). These SNPs are located on nine different chromosomes of the large yellow croaker (chromosomes 2 (NC_040012.1), 3 (NC_040013.1), 7 (NC_040017.1), 10 (NC_040020.1), 11 (NC_040021.1), 14 (NC_040024.1), 17 (NC_040027.1) and... On chromosome 20 (NC_040030.1), the following positions are listed: SNP3-47505387, SNP7-9407565, SNP3-31085986, SNP3-42982617, SNP20-95328, SNP20-73728, SNP14-13509333, SNP17-8648833, SNP2-45873542, SNP10-28085288, SNP3-51328350, SNP20-6750079, SNP11-10317498, and SNP17-4124025.

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

[0081] A total of 576.43G of raw whole-genome resequencing data was generated from 395 large yellow croaker individuals, with an average of 13405.42M of raw data per sample. Based on this, a total of 574.60G of filtered data was generated, with an average of 13362.97M per sample. 99.48% of reads were located to the large yellow croaker reference genome. Allele frequencies were calculated and chi-square tests were performed on the SNP dataset (P<0.05). Fourteen SNPs with the highest allele frequencies between the Naozhou wild large yellow croaker population and other wild large yellow croaker populations (Fujian-Guangdong wild large yellow croaker, Huidong wild large yellow croaker) that passed the chi-square test were selected as candidate specific genetic difference loci. SNP amplification and validation based on SNaPshot technology were then performed.

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

[0083] Table 1. Specific genotypes of the 14 SNPs

[0084]

[0085]

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

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

[0088]

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

[0090] Table 3. Extended primer sequence information for the 14 SNPs

[0091]

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

[0093] Example 2

[0094] A method for differentiating a multi-site population of wild large yellow croaker (Cyprinus naanensis) from other wild large yellow croaker populations (wild large yellow croaker from Fujian and Guangdong, and wild large yellow croaker from Huidong) based on SNaPshot technology includes the following steps:

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

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

[0097] Table 4 Multiplex PCR reaction system

[0098]

[0099]

[0100] Table 5 Multiplex PCR reaction procedure

[0101]

[0102] The amplified products were digested. The digestion system is shown in Table 6. The digestion conditions were 37℃ for 60 min and 75℃ for 15 min.

[0103] Table 6 Digestive System

[0104]

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

[0106] Table 7 Extended Reaction System

[0107]

[0108] The extension product was purified by adding 0.5 μL of CIP to 6 μL of the extension product; 37℃ for 1.0 h, then 75℃ for 15 min.

[0109] S3: 3730XL sequencer detection

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

[0111] 2) After 3 minutes at 95℃, place in an ice bath and then detect using a 3730XL sequencer;

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

[0113] S4: Based on the SNaPshot validation results, the differences in allele frequencies at the above 14 loci among the populations of wild large yellow croaker from Naozhou, wild large yellow croaker from Fujian and Guangdong, and wild large yellow croaker from Huidong were analyzed (P<0.05).

[0114] S5: Construct a population identification method for wild large yellow croaker from Naozhou, wild large yellow croaker from Fujian and Guangdong, and wild large yellow croaker from Huidong based on the above 14 SNPs.

[0115] Based on the genotypes in Table 1, alleles in the Naozhou wild large yellow croaker population with a higher frequency than other wild large yellow croaker populations (i.e., the Fujian-Guangdong wild large yellow croaker and the Huidong wild large yellow croaker population) are scored as 1 point, while alleles in the Naozhou wild large yellow croaker population with a lower frequency than other wild populations are scored as 0 points. A method for identifying the Naozhou wild large yellow croaker, Fujian-Guangdong wild large yellow croaker, and Huidong wild large yellow croaker populations based on the aforementioned 14 SNP loci is constructed. Generally, if a method is constructed based on x loci (x≤10), the total score of the identification method is 2x (number of alleles * 2).

[0116] All samples were scored, and total score datasets for wild and domesticated populations were constructed based on the total scores of the 14 SNPs. These datasets were then input into GraphPad Prism 8 to construct receiver operating characteristic (ROC) curves (where an area under the ROC curve (AUC) greater than 0.9 indicates a feasible method). A cut-off value was set as the discrimination threshold (the cut-off value is the maximum of the ROC curve's sensitivity% + specificity%). Samples with scores greater than or equal to the threshold of 15 were classified as wild large yellow croaker from the Naozhou group, while samples with scores less than the threshold of 15 were classified as wild large yellow croaker from the Fujian-Guangdong and Huidong groups.

[0117] S6: Based on the analysis of allele frequency differences in the tested samples, and since the allele frequencies conform to a binomial distribution Bin(2, p), the maximum likelihood estimation method is used for genotyping of the population samples: the overall log-likelihood value of all loci in all samples is calculated using the maximum likelihood method, and the sample with the highest overall log-likelihood value calculated based on the number of secondary alleles is classified into the corresponding reference population. Specific identification rules are as follows:

[0118]

[0119] Among them, A, B, and C are reference populations for wild large yellow croaker from Naozhou, wild large yellow croaker from Fujian and Guangdong, and wild large yellow croaker from Huidong, respectively.

[0120] Example 3

[0121] The multi-site identification method based on SNaPshot technology used in Example 2 to identify wild large yellow croaker populations from Naozhou and other wild large yellow croaker populations (wild large yellow croaker from Fujian and Guangdong, and wild large yellow croaker from Huidong) was used to identify known wild large yellow croaker populations. Among them, there were 50 wild large yellow croaker from Naozhou, 30 wild large yellow croaker from Fujian and Guangdong, and 28 wild large yellow croaker from Huidong.

[0122] Electrophoresis images of PCR reaction products from some large yellow croaker samples are shown below. Figure 1 As shown in the figure. SNaPshot analysis of the above-mentioned large yellow croaker yielded the following partial SNaPshot peak diagram: Figure 2 The specific gene frequency detection results are shown in Table 8. The individual sample screening method constructed based on the above 14 SNPs is as follows: Figure 3As shown, the area under the ROC curve (AUC) for wild large yellow croaker from Naozhou and wild large yellow croaker from Fujian and Guangdong, constructed using combinations of 14 SNPs, was 0.9110. The sensitivity of the optimal threshold was 96.7% (i.e., the probability of identifying a wild large yellow croaker from Naozhou using this evaluation method was 96.7%), and the specificity was 78.0% (i.e., the probability of excluding a wild large yellow croaker from Fujian and Guangdong using this evaluation method was 78.0%). Figure 3 The method is considered feasible if the area under the ROC curve (AUC) is greater than 0.9. Simultaneously, by combining 14 SNPs, the area under the ROC curve (AUC) for wild large yellow croaker from Naozhou and wild large yellow croaker from Huidong was constructed, with an AUC of 0.9096. The sensitivity of the optimal threshold is 89.3% (i.e., the probability of identifying a wild large yellow croaker from Naozhou using this evaluation method is 89.3%), and the specificity is 78.0% (i.e., the probability of excluding a wild large yellow croaker from Huidong using this evaluation method is 78.0%). Figure 3 (B) , where an AUC area greater than 0.9 indicates a feasible method. In summary, in this system, the individual sample identification method constructed based on 14 SNPs has a high discrimination rate and can be applied to the identification of wild large yellow croaker samples from the Naozhou group (Naozhou large yellow croaker, Fujian-Guangdong wild large yellow croaker, and Huidong wild large yellow croaker). Further, based on the analysis of allele frequency differences in the tested samples, the maximum likelihood estimation method was used to select another 30 samples from the Naozhou wild large yellow croaker group for group affiliation determination. The overall log-likelihood values ​​calculated based on the three reference groups are shown in Table 9. It can be seen that the overall log-likelihood value of the Naozhou wild large yellow croaker group is the largest. The tested sample was identified as belonging to the Naozhou wild large yellow croaker group, consistent with the reference classification.

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

[0124] Table 8. Specific gene frequency detection results

[0125]

[0126]

[0127] Table 9 shows the overall log-likelihood values ​​calculated based on three wild reference populations.

[0128]

[0129] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of SNP molecular marker combinations in the differentiation of wild large yellow croaker populations from Naozhou group and other wild large yellow croaker populations, wherein the other wild large yellow croaker populations are wild large yellow croaker from Fujian and Guangdong and wild large yellow croaker from Huidong, and the SNP molecular marker combinations include SNP3-47505387, SNP7-9407565, SNP3-31085986, SNP3-42982617, SNP20-95328, SNP20-73728, SNP14-13509333, SNP17-8648833, SNP2-45873542, SNP10-28085288, SNP3-51328350, SNP20-6750079, SNP11-10317498 and SNP17-4124025; in, The SNPs 3-47505387, 31085986, 42982617, and 51328350 are located at positions 47505387, 31085986, 42982617, and 51328350 on chromosome 3 of large yellow croaker, respectively, and their polymorphisms are C / T, C / T, G / A, and G / A, respectively. The SNP7-9407565 is located at position 9407565 on chromosome 7 of large yellow croaker NC_040017.1, and its polymorphism is T / C; The SNPs 20-95328, 20-73728, and 20-6750079 are located at positions 95328, 73728, and 6750079 on chromosome 20 of large yellow croaker, respectively, with polymorphisms of G / C, A / G, and T / A, respectively. The SNP14-13509333 is located at position 13509333 on chromosome 14 of large yellow croaker (NC_040024.1), and its polymorphism is T / G. The SNP17-8648833 and SNP17-4124025 are located at positions 8648833 and 4124025 respectively on chromosome 17 of large yellow croaker NC_040027.1, with polymorphisms of T / G and A / G respectively; The SNP2-45873542 is located at position 45873542 on chromosome 2 of large yellow croaker NC_040012.1, and its polymorphism is T / C; The SNP10-28085288 is located at position 28085288 on chromosome 10 of large yellow croaker (NC_040020.1), and its polymorphism is A / G. The SNP11-10317498 is located at position 10317498 on chromosome 11 of large yellow croaker NC_040021.1, and its polymorphism is T / C.

2. A detection kit comprising a primer set for amplifying the SNP molecular marker combination of claim 1, the nucleotide sequence of the primer set being shown in SEQ ID NO:1 to SEQ ID NO:28, and further comprising a single-base extension primer, the nucleotide sequence of the single-base extension primer being shown in SEQ ID NO:29 to SEQ ID NO:

42.

3. The application of the detection kit according to claim 2 in the differentiation of wild large yellow croaker populations from Naozhou group and other wild large yellow croaker populations, wherein the other wild large yellow croaker populations are wild large yellow croakers from Fujian and Guangdong and wild large yellow croakers from Huidong.

4. A method for distinguishing a population of wild large yellow croaker from Naozhou from other wild large yellow croaker populations, wherein the other wild large yellow croaker populations are wild large yellow croaker from Fujian and Guangdong and wild large yellow croaker from Huidong, wherein the method includes the step of using the detection kit of claim 2 to detect the SNP molecular marker combination of claim 1 in the large yellow croaker sample to be tested.

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