Multi-site large yellow croaker wild domestication population screening system based on agarose gel electrophoresis technology

Abstract

The invention belongs to the technical field of fish genetic identification, discloses a multi-site large yellow croaker wild domestication population identification system based on an agarose gel electrophoresis technology, and particularly discloses a group of InDel molecular markers for large yellow croaker wild and domestication population identification, and the InDel molecular markers comprise InDel11-5053756, InDel20-14004876, InDel3-28345321 and InDel21-17327392. The InDel molecular marker detection primer is used for amplification to obtain a PCR product, agarose gel can be used for detecting strip polymorphism, extra restriction enzyme digestion or complicated polyacrylamide gel electrophoresis operation is not needed, and identification of large yellow croaker wild and domesticated populations can be rapidly and accurately achieved.

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 identification system based on agarose gel electrophoresis technology. Background Art

[0002] Large yellow croaker (Larimichthys crocea) belongs to the order Perciformes, family Sciaenidae, genus Larimichthys, and is one of the most important marine economic fish species in China. In my country, large yellow croaker is distributed from the southern Yellow Sea in the north, through the East China Sea and the Taiwan Strait, to the east of the Leizhou Peninsula in the South China Sea in the south. In recent years, due to the expansion of large yellow croaker farming and the increase in the exchange of fry between different geographical regions, the distribution of farmed populations has far exceeded that of wild populations. Releasing activities have improved the resource base of large yellow croaker, but have also caused problems of mixed populations and low genetic diversity.

[0003] Resequencing refers to a bioinformatics method that obtains the genome sequence information of a living organism and compares it with an existing genome to find the differences between the sequence information. A large number of single nucleotide polymorphisms (SNPs), short insertions or deletions (Short InDels), structural variations (SVs) and copy number variations (CNVs) can be obtained through genome resequencing. With the rise of the next-generation sequencing platform, its high-throughput and low-cost advantages have led more and more species to join the ranks of high-throughput sequencing. By detecting variations in species individuals or groups and building a genetic variation database for the species, it is of great significance to promote the preservation of germplasm resources and the management of gene banks. According to the different needs for obtaining the scope of genomic genetic information, it can be divided into whole genome resequencing (WGS) and reduced-representation genome sequencing (RRGS). Whole genome resequencing refers to the use of high-throughput sequencing technology to completely sequence the genome of an organism and compare it with a reference genome. This technology can be used to analyze genetic differences between individuals, find disease-related genes or gene mutation sites, and reveal genome structure. With the continuous development of high-throughput sequencing technology, WGS technology has also been widely used. 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. Whole genome resequencing has gone through three stages with the development of sequencing technology, 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 fish, including carp (Cyprinus carpio), Atlantic salmon (Salmo salar), and goldfish (Carassius auratus). Currently, researchers can use whole genome resequencing to analyze the population structure and population history of fish, and discover genetic differences between fish populations.

[0004] Population genetics aims to study the genetic structure of a population and its changing patterns, mainly focusing on the distribution of genes in a population, the maintenance and changing patterns of gene frequencies and genotype frequencies. In population genetics, due to the traces left by natural or artificial selection on the genome, certain genes or genotypes increase in frequency in a population because they provide survival or reproductive advantages. These genes or genotypes are called selection signals. Identifying selection signals helps to understand the adaptation mechanism and evolutionary process of organisms and is conducive to the selection of improved varieties. Based on this concept, the detection of genomic selection signals is an important research field that can be used to determine which genes and genomic regions are affected by natural or artificial selection. The detection of genomic selection signals is based on the fixed index of genetic differentiation between populations (Fst) and nucleotide polymorphism (π) detection. Fst is an index that measures the degree of genetic differentiation between populations (ranging from 0 to 1). It is used to compare the diversity between subpopulations. The larger the Fst value, the higher the degree of differentiation between populations and the more distant the relationship. Nucleotide polymorphism π is a parameter that measures the polymorphism of a specific population. It is the mean of the nucleotide differences at each nucleotide site of 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 expression of nucleotide polymorphism π in population genetics, reflecting the level of genetic diversity of the population. By comparing the θπ values ​​of different populations, we can gain a preliminary understanding of the differences in genetic diversity between these populations.

[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 2019, researchers used the third-generation PacBio single-molecule sequencing technology and high-throughput chromosome conformation capture technology to assemble a large yellow croaker reference genome. The highly accurate large yellow croaker reference genome provides an important genomic resource to support the identification and evaluation of large yellow croaker germplasm resources. At present, the development of genetic-specific molecular markers has been applied in large yellow croaker. In 2022, researchers developed sex-specific molecular markers for the Daiqu population of large yellow croaker through the 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 genome resequencing and comparison of genetic marker data of yellow croaker populations distributed in the eastern and southern coastal waters of China, it was found that climate-driven habitat changes may have occurred between the Naozhou yellow croaker and the Min-Yuedong yellow croaker populations. However, there is currently a lack of accurate and rapid identification methods for wild / domesticated yellow croaker populations. The development of accurate and rapid identification methods for wild / domesticated yellow croaker using genetic-specific markers of yellow croaker can effectively promote the distribution research of yellow croaker populations in my country. 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 screened candidate genes associated with traits through Fst scanning combined with InDel sites. Based on the InDel data set obtained by whole genome resequencing of yellow croaker populations, agarose gel electrophoresis technology can be used to more intuitively show the genetic differences between different yellow croaker populations with the naked eye, and use this as a genetic marker to construct a population identification system. Summary of the invention

[0007] The purpose of the first aspect of the present invention is to provide a set of InDel 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 InDel 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 InDel 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 purpose of the fifth aspect of the present invention is to provide the application of the InDel 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 assisted breeding or identification of germplasm resources of large yellow croaker.

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

[0013] The purpose of the seventh 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 sixth aspect of the present invention in the identification and evaluation of large yellow croaker germplasm resources.

[0014] In order to achieve the above object, the technical solution adopted by the present invention is:

[0015] The first aspect of the present invention provides a group of InDel molecular markers for identifying wild and domesticated populations of large yellow croaker, wherein the InDel molecular markers include InDel11-5053756, InDel20-14004876, InDel3-28345321 and InDel21-17327392;

[0016] Among them, the InDel11-5053756 is located at position 5053756 of chromosome 11 NC_040021.1 of large yellow croaker, and there is an insertion / deletion of base G;

[0017] The InDel20-14004876 is located at position 14004876 of chromosome NC_040030.1 of large yellow croaker 20, and there is an insertion / deletion of base T;

[0018] The InDel3-28345321 is located at position 28345321 of chromosome NC_040013.1 of large yellow croaker 3, and there is an insertion / deletion of base T;

[0019] The InDel21-17327392 is located at position 17327392 of chromosome 21 NC_040031.1 of large yellow croaker, and there is an insertion / deletion of the base CAAA.

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

[0021] S1. After sequencing the whole genome of 395 wild and domesticated samples of large yellow croaker, the InDel sites with differences between wild and domesticated large yellow croaker were screened using population genetics selection signal analysis.

[0022] 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 between 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 4 candidate genes with non-synonymous mutations caused by InDel in exons were screened, and InDel verification was performed based on agarose gel electrophoresis technology.

[0023] The four InDels based on the above screening were located on four different chromosomes of large yellow croaker (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. A set of agarose gel electrophoresis primers that can be used for the identification of wild domesticated large yellow croaker samples was designed.

[0024] S3. Perform amplification experiments using the PCR reaction system to verify whether the four marker reaction systems can identify wild and domesticated populations of large yellow croaker;

[0025] S4. The agarose gel electrophoresis results were compared with the results of known marker detection for accuracy and detection rate, proving that the reaction system can identify wild and domesticated populations of large yellow croaker.

[0026] The second aspect of the present invention provides a primer set for amplifying a large yellow croaker genomic DNA fragment including the InDel molecular marker of the first aspect of the present invention.

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

[0028] 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.

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

[0030] The fourth aspect of the present invention provides the use of the InDel 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 identifying wild and / or domesticated populations of large yellow croaker.

[0031] The fifth aspect of the present invention provides the use of the InDel 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 assisted breeding or identification of germplasm resources of large yellow croaker.

[0032] The sixth 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 InDel molecular marker of the first aspect of the present invention on a large yellow croaker sample to be tested.

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

[0034] (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;

[0035] (2) performing gel electrophoresis on the PCR amplification product, and obtaining the genotype of the InDel molecular marker of the first aspect of the present invention in the genome of the large yellow croaker to be tested according to the amplified fragment;

[0036] (3) Analyze the frequency of the genotype of the InDel 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.

[0037] In some embodiments of the present invention, the genotype in step (2) is determined as follows: within the same site, the larger band is the In fragment, the smaller band is the Del fragment, the genotype with only the large band is counted as In / In, the genotype with only the small band is counted as Del / Del, and the presence of one large band and one small band is counted as In / Del.

[0038] In some embodiments of the present invention, the scoring rules in step (3) are 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 given, otherwise 0 point is given.

[0040] 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.

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

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

[0043] The identification method of the present invention is based on 4 effective InDel sites, and only 4 PCR reaction systems and instant agarose gel electrophoresis are needed to realize rapid and simple identification of wild and domesticated samples of large yellow croaker. The method can be operated with simple reagents and analytical instruments, and can realize rapid and accurate identification of wild and domesticated samples of large yellow croaker. Compared with the current method for identifying large yellow croaker populations, the method is faster to operate, has lower detection requirements, and has a higher accuracy rate and extremely fast detection efficiency.

[0044] The seventh 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 sixth aspect of the present invention in the identification and evaluation of large yellow croaker germplasm resources.

[0045] The beneficial effects of the present invention are:

[0046] The present invention provides a set of InDel molecular markers for wild and domesticated populations of large yellow croaker. The PCR products obtained by amplification using the InDel molecular marker detection primers of the present invention can be detected by using agarose gel to detect band polymorphism, without the need for additional restriction endonuclease digestion or complicated polyacrylamide gel electrophoresis operations, and can quickly and accurately achieve identification of wild and domesticated populations of large yellow croaker. The InDel molecular marker is a co-dominant marker, and has the advantages of stable amplification, high accuracy, convenient and rapid detection, etc.

[0047] The identification method provided by the present invention is a multi-site large yellow croaker wild domesticated 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 4 candidate indicator InDels into 4 PCR reactions, specifically amplifies and types the indication accuracy of the 4 sites in two groups of samples. Through agarose gel electrophoresis verification of 240 large yellow croaker samples with known typing, it is proved that the 4-site InDel markers can successfully identify the wild / domesticated classification of large yellow croaker in most samples. Compared with the current identification methods and markers, this method greatly improves the efficiency of distinguishing wild and domesticated populations of large yellow croaker, can use simple instruments and equipment for rapid and accurate genotyping, and can conveniently and efficiently realize the typing identification of large yellow croaker populations. The method has the advantages of simple operation, low cost and short time consumption, high efficiency and applicability, providing an efficient identification method for wild and domesticated populations of large yellow croaker, and providing favorable genetic resources for large yellow croaker breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The results of signal analysis are selected for wild and domesticated large yellow croaker populations, where A is the Fst value distribution of wild and domesticated large yellow croaker populations 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 calculated with a step size of 10 kb in a 50 kb sliding window.

[0049] Figure 2 Amplification results of agarose gel electrophoresis primers D1 and I1 system designed for InDel (targeting E1 and E2 sites).

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

[0051] The present invention is further described in detail below through specific examples.

[0052] 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.

[0053] 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.

[0054] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0055] Example 1

[0056] A set of InDel sites identified from wild domesticated populations of large yellow croaker by multi-locus agarose gel electrophoresis technology, the InDel sites are located on four different chromosomes of large yellow croaker (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 four InDels were obtained through whole genome resequencing and population genetics selection signal analysis screening. The specific method is as follows:

[0058] After whole genome sequencing of 395 wild and domesticated samples of yellow croaker, population genetics selection signal analysis was used to screen the InDel sites of wild and domesticated differences in yellow croaker. Specifically, a total of 576.43G whole genome resequencing raw data was generated for 395 yellow croaker individuals, and an average of 13405.42M raw data was generated for each sample. On this basis, a total of 574.60G filtered data was generated, and an average of 13362.97M was generated for each sample. 99.48% of the reads were mapped to the yellow croaker reference genome, and a total of 78370338 raw InDels were obtained. After filtering and screening, a total of 2041211 high-quality InDels were obtained. Based on the InDel dataset, strong selection signals based on the fixation index (Fst) and the genetic polymorphism ratio (π) were detected as significant genetic differences between wild and domesticated yellow croaker populations. The specific screening conditions were the top 2% of the Fst value and the top 5% of the π ratio. Furthermore, based on the selection signal analysis, four InDels 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 and verified as candidate InDels.

[0059] Analysis of selection signals between wild and domesticated large yellow croaker populations ( Figure 1A) Distribution of Fst values ​​of wild yellow croaker population (Wild) and domesticated yellow croaker population (Breed) calculated in a 50kb sliding window with a step size of 10kb; distribution of π ratio values ​​of wild yellow croaker population (Wild) and domesticated yellow croaker population (Breed) calculated in a 50kb sliding window with a step size of 10kb ( Figure 1 (middle B).

[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 InDel, In is a large fragment insertion, Del is a large fragment deletion, and the large fragment mutation information of each site is:

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

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

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

[0067] E4: TCAAA→T, i.e., there is a deletion or insertion of the base 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: There are deletion mutation sequences in the primers of the four sites E1 to E4, so a sequence matching the deletion mutation sequence in the primer region needs to be added to the reaction system. Taking the primer of E1 as an example, the deletion mutation sequence in the primer region of E1-iR4 matches that of E1-dR4, and needs to be added to the amplification system together with E1-dR4. In the table, Y is the degenerate base of C / T, K is the degenerate base of G / T, R is the degenerate base of G / A, and M is the degenerate base of A / C.

[0073] Example 2

[0074] A rapid identification method for wild and domesticated populations of large yellow croaker based on agarose gel electrophoresis technology at multiple sites comprises the following steps:

[0075] S1: Genomic DNA was extracted from fin tissue of large yellow croaker using phenol-chloroform extraction method;

[0076] S2: construct a PCR reaction system, the PCR reaction system is shown in Table 4, and the reaction procedure is shown in Table 5;

[0077] Site grouping: The 4 sites were grouped in batches: divided into the following 4 groups (Table 3):

[0078] D1 and D2 systems: amplification of Del typing for deletion of corresponding loci;

[0079] I1 and I2 systems: amplification of Insert typing at the corresponding sites.

[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 detection

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

[0089] Add 3 μL of product or DNA marker to the gel wells; electrophoresis at 100 V for 35 minutes;

[0090] The electrophoresis pattern was captured by gel imaging instrument;

[0091] Data analysis: The electrophoresis pattern information was organized into an excel table, where “I” represents insertion typing and “D” represents deletion typing.

[0092] S4: Combined with the agarose gel electrophoresis verification results, the difference in allele frequencies of the above four loci between the wild population and the domesticated population of large yellow croaker was analyzed (P<0.05). The alleles with a higher frequency in the wild population of large yellow croaker than in the domesticated population were scored as 1 point, and the alleles with a lower frequency in the wild population of large yellow croaker than in the domesticated population were scored as 0 points. A method for discriminating wild / domesticated samples of large yellow croaker based on the above four loci was constructed. Generally, on this basis, if a method is constructed based on x loci (x≤4), the total score of the discrimination method is 2x (number of alleles*2), and all validation samples are scored, and a receiver operating characteristic curve (ROC curve) is constructed based on the scoring results. The method is feasible when the ROC curve area (AUC) is greater than 0.9, and the cut-off value is set as the discrimination threshold (the cut-off value is the maximum value of the ROC curve sensitivity (sensitivity%) + specificity (specificity%)). Samples with scores higher than the threshold are wild samples of large yellow croaker, and samples with scores lower than the threshold are domesticated samples of large yellow croaker.

[0093] Example 2

[0094] The rapid identification method of Example 2 was used to screen known wild large yellow croaker and domesticated large yellow croaker populations, with 120 wild large yellow croakers and 120 domesticated large yellow croakers respectively.

[0095] The specific genotype analysis results of the large yellow croaker samples are shown in Table 6. The electrophoresis diagrams of the PCR reaction products of some large yellow croaker samples are shown in Figure 2 The results of the identification method based on the four InDels are shown in Figure 3 As shown, the constructed ROC curve area (AUC) is 0.9771, the sensitivity of the optimal threshold is 95.7% (that is, the probability of a wild large yellow croaker being identified by this identification method is 95.7%), and the specificity is 91.6% (that is, the probability of a domesticated large yellow croaker being excluded by this identification method is 91.6%), wherein an AUC area greater than 0.9 indicates that the method is feasible. The threshold for the total score of the identification method based on the above four InDels is 3 points. On this basis, samples with a score greater than 3 points are identified as domesticated large yellow croaker samples, and samples with a score less than or equal to 3 points are identified as wild large yellow croaker samples. According to the actual detection situation, the large yellow croaker population can be analyzed by observing with the naked eye whether there are more than three In fragments among the four InDels in the agarose gel electrophoresis result diagram (more than three In fragments are domesticated large yellow croaker samples, less than or equal to three In fragments are wild large yellow croaker samples, the larger band in the same site is the In fragment, the smaller band is the Del fragment, the genotype with only large bands is counted as In / In, the genotype with only small bands is counted as Del / Del, and the presence of one large band and one small band is counted as In / Del).

[0096] In summary, the identification method constructed based on four InDels has a high discrimination rate and extremely fast discrimination efficiency, and can be applied to the rapid discrimination of wild and domesticated large yellow croaker.

[0097] Table 6 Genotype analysis results

[0098]

[0099] 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 set of InDel molecular markers for identifying wild and domesticated populations of large yellow croaker, characterized in that: The InDel molecular markers include InDel11-5053756, InDel20-14004876, InDel3-28345321 and InDel21-17327392; wherein, InDel11-5053756 is located at position 5053756 of chromosome 11 NC_040021.1 of large yellow croaker, and there is an insertion / deletion of base G; The InDel20-14004876 is located at position 14004876 of chromosome NC_040030.1 of large yellow croaker 20, and there is an insertion / deletion of base T; The InDel3-28345321 is located at position 28345321 of chromosome NC_040013.1 of large yellow croaker 3, and there is an insertion / deletion of base T; The InDel21-17327392 is located at position 17327392 of chromosome 21 NC_040031.1 of large yellow croaker, and there is an insertion / deletion of the base CAAA.

2. A primer set, which is used to amplify a large yellow croaker genomic DNA fragment containing the InDel molecular marker described in claim 1.

3. The primer set according to claim 2, characterized in that The nucleotide sequences of the primer set are shown in SEQ ID NO:1 to SEQ ID NO:

14.

4. A detection reagent, gene chip or kit, comprising the primer set according to claim 2 or 3.

5. Use of the InDel 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. Use of the InDel 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 assisted breeding or identification of germplasm resources of large yellow croaker.

7. 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 InDel molecular marker described in claim 1 on a large yellow croaker sample to be tested.

8. The identification method according to claim 7, characterized in that: The identification 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 gel electrophoresis on the PCR amplification product, and obtaining the genotype of the InDel molecular marker according to claim 1 in the genome of the large yellow croaker to be tested according to the amplified fragment; (3) Analyze the frequency of the genotype of the InDel 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.

9. The identification method according to claim 8, characterized in that: The scoring rules in step (3) are as follows: When the frequency of the Del / Del genotype of InDel11-5053756, InDel20-14004876, InDel3-28345321 and InDel21-17327392 is greater than the frequency of the In / In genotype, 1 point is given, otherwise 0 point is given.

10. Use of the identification method according to any one of claims 7 to 9 in the identification and evaluation of large yellow croaker germplasm resources.

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