A multi-locus discrimination system for wild and domesticated populations of pseudosciaena crocea based on agarose gel electrophoresis

Abstract

The application belongs to the technical field of fish genetic identification, and discloses a multi-site large yellow croaker wild domestication population discrimination system based on agarose gel electrophoresis technology, and specifically discloses a group of InDel molecular markers for identifying large yellow croaker wild and domestication populations, wherein the InDel molecular markers include InDel11-5053756, InDel20-14004876, InDel3-28345321 and InDel21-17327392. The PCR product amplified by the InDel molecular marker detection primer can be detected for band polymorphism by using agarose gel, without additional restriction endonuclease digestion or complicated polyacrylamide gel electrophoresis operation, so that the identification of the large yellow croaker wild and domestication populations can be quickly and accurately realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic identification of fish, and particularly relates to a multi-site Larimichthys crocea wild domestication population discrimination system based on agarose gel electrophoresis technology. BACKGROUND

[0002] Larimichthys crocea belongs to Perciformes, Sciaenidae and Larimichthys, and is one of the most important marine economic fish in China. In China, Larimichthys crocea is distributed in the north of the Yellow Sea, the East China Sea, the Taiwan Strait and the south of the Leizhou Peninsula in the South China Sea. In recent years, due to the expansion of Larimichthys crocea aquaculture and the increase of fry exchange between different geographical regions, the distribution of the aquaculture population has far exceeded that of the wild population. The release activities have improved the resource base of Larimichthys crocea, but also caused the problem of population mixing and low genetic diversity.

[0003] Re-sequencing is a bioinformatics method that obtains the genomic sequence information of an organism, compares it with the existing genome, and finds the differences between the sequence information. Through genome re-sequencing, a large number of single nucleotide polymorphisms (SNP), short fragment insertion or deletion (Short InDel), structural variation (SV) and copy number variation (CNV) sites can be obtained. With the rise of new generation sequencing platforms, their high throughput and low cost advantages have made more and more species join the ranks of high-throughput sequencing. By detecting variations in individuals or populations of a species, a genetic variation database for the species can be constructed, which is of great significance for promoting the conservation of germplasm resources and the management of gene banks. According to different needs for obtaining genomic genetic information, it can be divided into whole genome sequencing (WGS) and reduced-representation genome sequencing (RRGS). Whole genome sequencing refers to using high-throughput sequencing technology to sequence the complete genome of an organism and comparing it with the reference genome. This technology can be used to analyze genetic differences between individuals, find disease-related genes or gene mutation sites, and reveal genome structure, etc. With the continuous development of high-throughput sequencing technology, WGS technology has been widely applied. The progress of sequencing technology has strongly promoted the development of genomics, making it possible to accurately sequence and precisely assemble complex genomic sequences. Whole genome re-sequencing has experienced three stages with the development of sequencing technology, namely Sanger sequencing technology, NGS technology and PacBio and Nanopore technology. In recent years, whole genome re-sequencing has been gradually applied to a variety of fish, including Cyprinus carpio, Salmo salar and Carassius auratus. At present, researchers can use whole genome re-sequencing to analyze fish population structure and population historical evolution, and find genetic differences between fish populations.

[0004] Population genetics aims to study the genetic structure of populations and its variation rules. It mainly studies the distribution of genes, the maintenance and variation rules of gene frequency and genotype frequency in populations. In population genetics, due to the traces left on the genome by natural selection or artificial selection, some genes or genotypes increase in frequency in the population because they provide survival or reproductive advantages. These genes or genotypes are called selection signals. Identifying selection signals helps to understand biological adaptation mechanisms, evolutionary history and is beneficial to good breed selection. The concept of detecting genomic selection signals has emerged as an important research field, which can be used to determine which genes and genomic regions are affected by natural or artificial selection. Genomic selection signal detection is based on the detection of genetic differentiation fixation index (Fst) and nucleotide polymorphism (π) between populations. Fst is an index that measures the degree of genetic differentiation between populations (with a value range from 0 to 1). For comparison of subpopulation diversity, the larger the Fst value, the higher the degree of differentiation between populations, and the more distant the genetic relationship. Nucleotide polymorphism π is a parameter that measures the level of polymorphism in a particular population. It is the average of nucleotide differences at each nucleotide site between two randomly selected DNA sequences in the same population. The larger the π value, the higher the polymorphism of the corresponding subpopulation. θπ is a specific estimate or manifestation of nucleotide polymorphism π in population genetics, reflecting the level of genetic diversity in the population. By comparing the θπ values of different populations, we can preliminarily understand the differences in genetic diversity among these populations.

[0005] How to accurately and quickly identify wild and domesticated populations of large yellow croaker is the core of large yellow croaker germplasm resource identification. In 2019, researchers used the third-generation PacBio single-molecule sequencing technology and high-throughput chromosome conformation capture technology to assemble the large yellow croaker reference genome. The highly accurate large yellow croaker reference genome provides an important genomic resource for supporting the identification and evaluation of large yellow croaker germplasm resources. Currently, 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 by genome between dmrt1 and cfap157, providing a powerful tool for promoting sex-controlled breeding of the Daiqu population of large yellow croaker.

[0006] In 2023, based on the genetic marker data of the genome resequencing alignment of the large yellow croaker populations distributed in the coastal areas of eastern and southern China, it was found that climate-driven habitat changes may have occurred between the Niaozhou large yellow croaker population and the Min-Yue-Dong large yellow croaker population. However, there is currently a lack of accurate and rapid identification methods for wild / domesticated large yellow croaker populations, and the development of a method for accurately and rapidly identifying wild / domesticated large yellow croaker using large yellow croaker genetic specific markers can effectively promote the distribution research of large yellow croaker populations in China. InDel is the insertion or deletion of a fragment sequence at a certain position in the genome, and is one of the molecular genetic markers. Researchers screen trait-associated candidate genes by Fst scanning combined with InDel site selection. Based on the InDel dataset obtained by whole genome resequencing of large yellow croaker populations, combined with agarose gel electrophoresis technology, the genetic differences between different large yellow croaker populations can be more directly observed by the naked eye, and used as genetic markers to construct a population identification system. SUMMARY

[0007] The first aspect of the present application aims to provide a set of InDel molecular markers for identifying wild and domesticated large yellow croaker populations.

[0008] The second aspect of the present application aims to provide a primer set for amplifying the InDel molecular markers of the first aspect of the present application.

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

[0010] The fourth aspect of the present application aims to provide the use of the InDel molecular markers of the first aspect of the present application, the primer set of the second aspect of the present application or the detection reagent, the gene chip or the kit of the third aspect of the present application in the identification of wild and / or domesticated large yellow croaker populations.

[0011] The fifth aspect of the present application aims to provide the use of the InDel molecular markers of the first aspect of the present application, the primer set of the second aspect of the present application or the detection reagent, the gene chip or the kit of the third aspect of the present application in the assisted breeding of large yellow croaker or the identification of germplasm resources.

[0012] The sixth aspect of the present application aims to provide a method for identifying wild and / or domesticated large yellow croaker populations.

[0013] The seventh aspect of the present application aims to provide the use of the method for identifying wild and / or domesticated large yellow croaker populations of the sixth aspect of the present application in the identification and evaluation of large yellow croaker germplasm resources.

[0014] To achieve the above-mentioned objectives, the technical solutions adopted by the present application are as follows:

[0015] In a first aspect, the application provides a set of InDel molecular markers for identifying wild and domesticated populations of Pseudosciaena crocea, comprising InDel11-5053756, InDel20-14004876, InDel3-28345321 and InDel21-17327392.

[0016] The InDel11-5053756 is located at position 5053756 of chromosome 11 of Pseudosciaena crocea NC_040021.1, and an insertion / deletion of base G exists.

[0017] The InDel20-14004876 is located at position 14004876 of chromosome 20 of Pseudosciaena crocea NC_040030.1, and an insertion / deletion of base T exists.

[0018] The InDel3-28345321 is located at position 28345321 of chromosome 3 of Pseudosciaena crocea NC_040013.1, and an insertion / deletion of base T exists.

[0019] The InDel21-17327392 is located at position 17327392 of chromosome 21 of Pseudosciaena crocea NC_040031.1, and an insertion / deletion of base CAA exists.

[0020] In some embodiments of the application, the four InDel molecular markers described above are obtained by the following steps:

[0021] S1, after whole genome sequencing of 395 wild and domesticated samples of Pseudosciaena crocea, screening for InDel sites different between wild and domesticated Pseudosciaena crocea using population genetics selection signal analysis;

[0022] S2, detecting strong selection signals based on Fst and π in the genome to mine genetic differences caused by different living environments of wild and domesticated Pseudosciaena crocea. The top 2% of Fst and the top 5% of π ratio are selected as the selection signal region of wild and domesticated Pseudosciaena crocea. On the basis of selection signal analysis, candidate genes with extremely significant differences in allele frequency between wild and domesticated Pseudosciaena crocea are screened (P<0.001) in the selection signal region. Four candidate genes with InDel causing non-synonymous mutations in exons are screened, and InDel verification is performed based on agarose gel electrophoresis technology.

[0023] The four InDels screened above are located on four different chromosomes (chromosome 3 (NC_040013.1), chromosome 11 (NC_040021.1), chromosome 20 (NC_040030.1) and chromosome 21 (NC_040031.1)) of P. olivaceus, and the position information is as follows: InDel3-28345321, InDel11-5053756, InDel20-14004876 and InDel21-17327392. A set of agarose gel electrophoresis primers for identifying wild and domesticated samples of P. olivaceus are designed.

[0024] S3, using a PCR reaction system to perform amplification experiment, verifying whether the four marker reaction systems can identify wild and domesticated populations of P. olivaceus;

[0025] S4, comparing the results of agarose gel electrophoresis with the results of known marker detection in terms of accuracy and detection rate, proving that the reaction system can identify wild and domesticated populations of P. olivaceus.

[0026] In a second aspect of the present application, a primer set is provided, which is used for amplifying a P. olivaceus genomic DNA fragment containing the InDel molecular marker of the first aspect of the present application.

[0027] In some embodiments of the present application, the nucleotide sequence of the primer set is shown in SEQ ID NO: 1-14.

[0028] In a third aspect of the present application, a detection reagent, a gene chip or a kit is provided, comprising the primer set of the second aspect of the present application.

[0029] In some embodiments of the present application, the kit further comprises one or more of dNTPs, polymerase (DNA polymerase and exonuclease), PCR reaction buffer, Taq, PCR stabilizer and enhancer.

[0030] In a fourth aspect of the present application, the InDel molecular marker of the first aspect of the present application, the primer set of the second aspect of the present application or the detection reagent, the gene chip or the kit of the third aspect of the present application is applied in the identification of wild and / or domesticated populations of P. olivaceus.

[0031] In a fifth aspect of the present application, the InDel molecular marker of the first aspect of the present application, the primer set of the second aspect of the present application or the detection reagent, the gene chip or the kit of the third aspect of the present application is applied in the assisted breeding of P. olivaceus or the identification of germplasm resources.

[0032] In a sixth aspect of the present application, a method for identifying a wild and / or domesticated population of Larimichthys crocea is provided, comprising the step of detecting the InDel molecular marker of the first aspect of the present application in a sample of Larimichthys crocea to be tested using the primer set of the second aspect of the present application or the detection reagent, gene chip or kit of the third aspect of the present application.

[0033] In some embodiments of the present application, the identification method comprises the following steps:

[0034] (1) using the DNA of the sample of Larimichthys crocea to be tested as a template, performing PCR amplification using the primer set of the second aspect of the present application or the detection reagent, gene chip or kit of the third aspect of the present application to obtain a PCR amplification product;

[0035] (2) performing gel electrophoresis on the PCR amplification product to obtain the genotype of the InDel molecular marker of the first aspect of the present application in the genome of the sample of Larimichthys crocea to be tested according to the amplified fragments;

[0036] (3) analyzing the frequency of the genotype of the InDel molecular marker, performing scoring, and constructing a ROC curve to determine whether the sample of Larimichthys crocea to be tested is a wild Larimichthys crocea or a domesticated Larimichthys crocea.

[0037] In some embodiments of the present application, the genotype determination in step (2) is as follows: the larger band in the same locus is the In fragment, the smaller band is the Del fragment, only the genotype with a large band is counted as In / In, only the genotype with a small band is counted as Del / Del, and the genotype with one large band and one small band is counted as In / Del.

[0038] In some embodiments of the present application, the scoring rule in step (3) is as follows:

[0039] When the frequency of the D / D genotype of InDel11-5053756, InDel20-14004876, InDel3-28345321 and InDel21-17327392 is greater than the frequency of the I / I genotype, 1 point is scored, otherwise 0 point is scored.

[0040] In some embodiments of the present application, the cut-off value of the ROC curve (i.e. the maximum value of the sensitivity (sensitivity %) + specificity (specificity %) of the ROC curve) is set as the discrimination threshold value, and when the total score of the sample of Larimichthys crocea to be tested is higher than the threshold value, it is a wild population of Larimichthys crocea, otherwise it is a domesticated population of Larimichthys crocea.

[0041] In some embodiments of the present application, the PCR amplification in step (1) is multiplex PCR amplification.

[0042] In some embodiments of the present application, the reaction procedure of the PCR amplification is 92-96 DEG C pre-denaturation for 8-12 min; 92-96 DEG C denaturation for 28-35 s, 58-60 DEG C annealing for 30-40 s, 70-72 DEG C extension for 40-50 s, 32-37 cycles; 70-72 DEG C extension for 3-6 min.

[0043] The present application is based on the identification method of four effective InDel sites, and only four PCR reaction systems and instant agarose gel electrophoresis can realize the rapid and simple identification of wild and domesticated samples of Pseudosciaena crocea. The method can be operated by using simple reagents and analysis instruments, and can realize the rapid and accurate identification of wild samples and domesticated samples of Pseudosciaena crocea. Compared with the current method for identifying Pseudosciaena crocea population, the method is faster and requires lower detection, and has higher accuracy and extremely fast detection efficiency.

[0044] In a seventh aspect, the present application provides application of the identification method of wild and / or domesticated population of Pseudosciaena crocea in the sixth aspect of the present application in identification and evaluation of Pseudosciaena crocea germplasm resources.

[0045] The present application has the following beneficial effects:

[0046] The present application provides a set of InDel molecular markers for wild and domesticated populations of Pseudosciaena crocea. The PCR products amplified by the detection primer of the InDel molecular marker are detected by agarose gel, without additional restriction enzyme digestion or complicated polyacrylamide gel electrophoresis operation, so that the identification of wild and domesticated populations of Pseudosciaena crocea can be realized quickly and accurately. The InDel molecular marker is a co-dominant marker, and has the advantages of stable amplification, high accuracy, convenient detection, rapidness and the like.

[0047] The identification method provided by the application is a multi-site large yellow croaker wild domestication sample identification system based on agarose gel electrophoresis technology, which is developed based on multiple DNA fragment insertion and deletion (InDel) sites screened by whole genome resequencing and population genetics selection signal analysis of large yellow croaker. The design and detection of the method are based on PCR amplification reaction and agarose gel electrophoresis technology. The system fuses four candidate indicator InDels into four PCR reactions, and specifically amplifies and types four sites to detect the indication accuracy of the four sites in two groups of samples. Through agarose gel electrophoresis verification of 240 known typed large yellow croaker samples, it is proved that the four site InDel markers can successfully identify the wild / domestic classification of large yellow croaker in most samples. Compared with the current identification method and markers, the method greatly improves the identification efficiency of wild and domestic populations of large yellow croaker, can use simple instrument equipment for rapid and accurate genotyping, and can conveniently and efficiently realize the genotyping identification of large yellow croaker population. It has the advantages of simple operation, low cost, short time-consuming and high efficiency and applicability, and provides an efficient identification method for wild and domestic populations of large yellow croaker, and provides beneficial gene resources for large yellow croaker breeding. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 Figure 1 is the selection signal analysis results of wild large yellow croaker population and domesticated large yellow croaker population, wherein A is the Fst value distribution of wild large yellow croaker population and domesticated large yellow croaker population calculated at 10kb step in 50kb sliding window, and B is the π ratio distribution of wild large yellow croaker population and domesticated large yellow croaker population calculated at 10kb step in 50kb sliding window.

[0049] Figure 2 Figure 4 is the agarose gel electrophoresis primer D1 and I1 system amplification results (for E1 and E2 sites) designed for InDel.

[0050] Figure 3 Figure 5 is the ROC curve corresponding to the multi-site rapid identification method of wild and domestic populations of large yellow croaker in Example 2. DETAILED DESCRIPTION

[0051] The content of the application will be further described in detail through specific examples.

[0052] It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application.

[0053] In order to make the purpose, technical scheme and advantages of the embodiments of the application more clear, the technical scheme in the embodiments of the application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are used. If the used reagents or instruments are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0054] The features and performances of the present application are further described in detail below in combination with examples.

[0055] Example 1

[0056] A group of InDel sites based on agarose gel electrophoresis technology for multi-site discrimination of wild and domesticated populations of Pseudosciaena crocea, the InDel sites are located on 4 different chromosomes of Pseudosciaena crocea (chromosome 3 (NC_040013.1), chromosome 11 (NC_040021.1), chromosome 20 (NC_040030.1) and chromosome 21 (NC_040031.1)), and the position information is as follows: InDel3-28345321, InDel11-5053756, InDel20-14004876 and InDel21-17327392.

[0057] The above 4 InDels are obtained by Whole Genome Resequencing sequencing and population genetics selection signal analysis screening, and the specific method is as follows:

[0058] After whole genome sequencing of 395 wild and domesticated samples of Pseudosciaena crocea, the InDel sites of wild and domesticated Pseudosciaena crocea are screened by population genetics selection signal analysis, specifically, a total of 576.43G of whole genome resequencing raw data is generated for 395 Pseudosciaena crocea individuals, and an average of 13405.42M of raw data is generated for each sample, on the basis of which a total of 574.60G of filtered data is generated, and an average of 13362.97M is generated for each sample. 99.48% of reads are located to the Pseudosciaena crocea reference genome, and a total of 78370338 original InDels are obtained. After filtering and screening, a total of 2041211 high-quality InDels are obtained. Based on the InDel dataset, strong selection signals based on fixation index (Fst) and genetic polymorphism ratio (π) are detected as significant genetic differences between wild Pseudosciaena crocea population and domesticated Pseudosciaena crocea population. The specific screening conditions are that the top 2% of Fst values and the top 5% of π ratios. Further, on the basis of selection signal analysis, 4 InDels with extremely significant differences in allele frequency between wild Pseudosciaena crocea population and domesticated Pseudosciaena crocea population (P<0.001) are screened in the selection signal region as candidate InDels for verification.

[0059] Selection signal analysis of wild Pseudosciaena crocea population and domesticated Pseudosciaena crocea population Figure 1Figure 2A) Fst value distribution of wild group (Wild) and domesticated group (Breed) of P. major in 50 kb sliding window with 10 kb step size; Figure 2B) π ratio distribution of wild group (Wild) and domesticated group (Breed) of P. major in 50 kb sliding window with 10 kb step size Figure 1

[0060] The specific genotypes of the four InDels are shown in Table 1.

[0061] Table 1 Specific genotypes of the four InDels

[0062]

[0063] For InDels, In refers to large fragment insertion, and Del refers to large fragment deletion. The large fragment mutation information of each site is as follows:

[0064] E1: AG→A, i.e. deletion or insertion of base G at position 5053756 of chromosome 11 (NC_040021.1);

[0065] E2: AT→A, i.e. deletion or insertion of base T at position 14004876 of chromosome 20 (NC_040030.1);

[0066] E3: GT→G, i.e. deletion or insertion of base T at position 28345321 of chromosome 3 (NC_040013.1);

[0067] E4: TCAAA→T, i.e. deletion or insertion of bases CAAA at position 17327392 of chromosome 21 (NC_040031.1).

[0068] Corresponding amplification primer sequences were designed for the above four InDels, and the primer sequences are shown in Table 2.

[0069] Table 2 Amplification primer sequences

[0070]

[0071]

[0072] Note: The primer sequences of E1-E4 four sites have deletion mutation sequences, so the sequence matching the deletion mutation sequence of the primer region needs to be added to the reaction system. For example, taking the primer of E1 as an example, E1-iR4 and E1-dR4 primer region deletion mutation sequence match, and E1-dR4 needs to be added to the amplification system. In the table, Y is a degenerate base of C / T, K is a degenerate base of G / T, R is a degenerate base of G / A, and M is a degenerate base of A / C. ​

[0073] Example 2

[0074] A rapid identification method of multi-site wild and domesticated populations of Pseudosciaena crocea based on agarose gel electrophoresis technology, comprising the following steps:

[0075] S1: Extract genomic DNA from the fin tissue of Pseudosciaena crocea by phenol-chloroform extraction method;

[0076] S2: Construct the PCR reaction system, the PCR reaction system is shown in Table 4, and the reaction program is shown in Table 5;

[0077] Site grouping: The four sites are grouped into the following four groups (Table 3):

[0078] D1 and D2 system: Amplification for corresponding site deletion Del typing;

[0079] I1 and I2 system: Amplification for corresponding site insertion Insert typing.

[0080] Table 3 Site grouping

[0081]

[0082]

[0083] Table 4 PCR reaction system

[0084]

[0085] Table 5 PCR reaction program

[0086]

[0087] S3: Agarose gel electrophoresis instrument detection

[0088] Prepare agarose gel with a concentration of 2.5%;

[0089] Add 3 μL product or DNA marker in the gel hole; 100V electrophoresis for 35 minutes;

[0090] Gel imaging instrument takes electrophoretogram;

[0091] Data analysis: Arrange the electrophoretogram information into excel table, "I" represents insertion typing, and "D" represents deletion typing.

[0092] S4: Combined with the results of agarose gel electrophoresis, the significance of the difference in allele frequency between the wild and domesticated populations of P. major at the above four loci was analyzed (P < 0.05). The alleles with higher frequency in the wild population than in the domesticated population were scored as 1, and the alleles with lower frequency in the wild population than in the domesticated population were scored as 0. A method for identifying wild / domesticated samples of P. major based on the above four loci was constructed. Generally, if a method is constructed based on x loci (x < 4), the total score of the identification method is 2x (allele number * 2), the scores of all validation samples are calculated based on the score results, and a receiver operating characteristic (ROC) curve is constructed based on the score results. If the area under the ROC curve (AUC) is greater than 0.9, the method is feasible, the cut-off value is set as the threshold value (the cut-off value is the maximum value of the sensitivity (sensitivity %) and the specificity (specificity %) of the ROC curve), and the sample score higher than the threshold value is the wild sample of P. major, and the sample score lower than the threshold value is the domesticated sample of P. major.

[0093] Example 2

[0094] The known wild and domesticated P. major populations were identified using the rapid identification method of Example 2, and each of the wild and domesticated P. major populations had 120 samples.

[0095] The specific genotype analysis results of the P. major samples are shown in Table 6, and the electrophoretograms of the PCR reaction products of some P. major samples are shown in Figure 2 The detection results of the identification method constructed based on the four InDels are shown in Figure 3 The area under the ROC curve (AUC) constructed is 0.9771, the sensitivity of the optimal threshold value is 95.7% (i.e., the probability of identifying a wild P. major sample through the identification method is 95.7%), and the specificity is 91.6% (i.e., the probability of excluding a domesticated P. major sample through the identification method is 91.6%), and the AUC area greater than 0.9 is the feasible method. The threshold value of the total score of the identification method constructed based on the above four InDels is 3 points, and based on this, the score greater than 3 points is identified as a domesticated P. major sample, and the score less than or equal to 3 points is identified as a wild P. major sample. For actual detection, whether there are more than three In fragments in the agarose gel electrophoresis results can be observed by naked eye to analyze the P. major population (more than three In fragments are domesticated P. major samples, and less than or equal to three In fragments are wild P. major samples, the larger band in the same locus is the In fragment, the smaller band is the Del fragment, only the large band genotype is In / In, only the small band genotype is Del / Del, and one large band and one small band are In / Del).

[0096] In summary, the identification method based on the four InDels has high discrimination rate and fast discrimination efficiency, and can be applied to rapid discrimination of wild and domesticated Pseudosciaena crocea.

[0097] Table 6 Genotype analysis results

[0098]

[0099] The embodiments of the present application are described in detail above with reference to the drawings, but the present application is not limited to the above-described embodiments, and various changes can be made within the scope of knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. The application of a set of InDel molecular markers in the identification of wild and domesticated populations of large yellow croaker, characterized by: The InDel molecular markers consist of InDel11-5053756, InDel20-14004876, InDel3-28345321 and InDel21-17327392; Among them, InDel11-5053756 is located at position 5053756 of chromosome 11 NC_040021.1 of the large yellow croaker, and there is an insertion / deletion of the base G; The InDel20-14004876 is located at position 14004876 on chromosome 20 of the large yellow croaker, NC_040030.1, and there is an insertion / deletion of the base T. The InDel3-28345321 is located at position 28345321 on chromosome 3 of the large yellow croaker, NC_040013.1, and there is an insertion / deletion of the base T. The InDel21-17327392 is located at position 17327392 on chromosome 21 of the large yellow croaker, NC_040031.1, and contains an insertion / deletion of the base CAAA.

2. A primer set for amplifying a large yellow croaker genomic DNA fragment containing the InDel molecular marker described in claim 1, wherein the nucleotide sequence of the primer set is shown in SEQ ID NO:1 to SEQ ID NO:

14.

3. A detection reagent comprising the primer set as described in claim 2.

4. A kit comprising the primer set of claim 2.

5. The application of the primer set of claim 2, the detection reagent of claim 3, or the kit of claim 4 in the identification of wild and domesticated populations of large yellow croaker.

6. A method for identifying wild and domesticated populations of large yellow croaker, comprising the step of detecting the InDel molecular marker described in claim 1 in a large yellow croaker sample using the primer set described in claim 2, the detection reagent described in claim 3, or the kit described in claim 4.

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