SNP marker associated with salt tolerance trait of largemouth bass and application thereof

Abstract

This invention belongs to the field of molecular biology and discloses a SNP marker associated with salt tolerance in largemouth bass and its application. This invention obtains an SNP molecular marker related to the strength of salt tolerance in largemouth bass by performing correlation analysis on the salt tolerance of extreme and sensitive individuals. The SNP molecular marker, as shown in SEQ ID NO:1, is located at position 501 and has three genotypes: TT, TG, and GG. The GG genotype largemouth bass exhibits a significantly longer survival time under salinity stress than the TG and TT genotypes. By detecting the genotype of this SNP molecular marker, largemouth bass parents with strong salt tolerance can be accurately selected, which can be effectively used for marker-assisted breeding of largemouth bass.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to an SNP marker related to salt tolerance traits in largemouth bass and its application. Background Technology

[0002] my country possesses abundant saline-alkali water and soil resources, and these resources are increasing year by year. According to incomplete statistics, there are approximately 690 million mu (16.7 million hectares) of saline-alkali water areas and 1.49 billion mu (8.9 million hectares) of saline-alkali land nationwide, distributed across 19 provinces, municipalities, and autonomous regions. However, due to the difficulty of remediation and the complexity of their types, most saline-alkali water areas remain idle, possessing enormous potential for development and utilization. (Largemouth bass...) Micropterus salmoides Largemouth bass is a popular freshwater economic fish that can survive in saline environments of 1‰ to 15‰. It exhibits excellent salt tolerance and has great potential for aquaculture and promotion in saline-alkali and tidal flat areas, making it an important candidate species for brackish water aquaculture. However, there is a lack of suitable high-quality species for saline-alkali water aquaculture. Promoting largemouth bass to saline-alkali water aquaculture will not only expand aquaculture space and increase the effective supply of aquatic products, but will also help restore saline-alkali farmland and improve the quality and area of ​​arable land. Currently, largemouth bass has been successfully cultured in some saline-alkali waters in Ningxia, Tianjin, Inner Mongolia, and Gansu, but its salt tolerance genetic basis remains unclear.

[0003] Genome-wide association studies (GWAS) are powerful tools for identifying genomic regions associated with complex traits, such as salt tolerance. By linking phenotypic variations to specific genetic loci, they provide insights into the genetic structure of these traits. Commonly used molecular markers include single nucleotide polymorphisms (SNPs), simple sequence repeats (SSRs), and insertions / deletions (Indels). Among these, SNP markers have become the preferred tool for elucidating the genetic mechanisms of complex traits due to their advantages such as high distribution density, high detection throughput, high degree of automation in genotyping, and low cost. Furthermore, SNP-based genotyping techniques can rapidly identify salt-tolerant candidate individuals. Combined with family breeding or genomic selection (GS) strategies, salt-tolerant dominant alleles can be selectively aggregated to breed new salt-tolerant largemouth bass varieties. Therefore, it is urgent to screen and identify new SNP loci associated with salt tolerance in largemouth bass. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. The present invention provides an SNP marker related to salt tolerance in largemouth bass, which can be used to breed largemouth bass with strong salt tolerance, providing a reliable basis for the breeding and improvement of salt-tolerant new varieties.

[0005] The first aspect of this invention aims to provide SNP molecular markers related to the salt tolerance of largemouth bass.

[0006] The second aspect of the present invention aims to provide the application of substances that detect the SNP molecular markers of the first aspect of the present invention in screening salt-tolerant largemouth bass or in preparing products for screening salt-tolerant largemouth bass.

[0007] A third 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.

[0008] A fourth aspect of the present invention is to provide a reagent kit.

[0009] The fifth aspect of this invention aims to provide the application of the SNP molecular marker of the first aspect of this invention, the primer set of the third aspect of this invention, and / or the kit of the fourth aspect of this invention.

[0010] The sixth aspect of this invention aims to provide a method for identifying and screening salt-tolerant largemouth bass.

[0011] The seventh aspect of this invention aims to provide the application of the method of the fifth aspect of this invention in the breeding of salt-tolerant largemouth bass.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] In a first aspect, the invention provides a molecular marker associated with salt tolerance of largemouth bass, the sequence of which is shown in SEQ ID NO:1, the SNP site being located at position 501 from the 5' end of the sequence shown in SEQ ID NO:1, and its polymorphism being T / G.

[0014] In some embodiments of the present invention, when the genotype of the SNP locus is GG, it is a largemouth bass with strong salt tolerance (i.e., salt-tolerant largemouth bass), and when the genotype of the SNP locus is TT, it is a largemouth bass with weak salt tolerance (i.e., salt-sensitive largemouth bass).

[0015] A second aspect of the present invention provides the use of substances that detect the SNP molecular markers of the first aspect of the present invention in screening for salt-tolerant largemouth bass or in preparing products for screening for salt-tolerant largemouth bass.

[0016] In some embodiments of the present invention, the substance comprises a substance for use in one or more detection techniques or methods selected from the group consisting of: Northern blotting, PCR, gene chip method, and nucleic acid sequencing.

[0017] In some embodiments of the present invention, PCR is used to detect the SNP molecular markers of the first aspect of the present invention.

[0018] In some embodiments of the present invention, the substance includes primers for PCR. Examples include an upstream primer with the nucleotide sequence 5'-GGGATCTACAACAGGAAAGG-3' (SEQ ID NO:2), a downstream primer with the nucleotide sequence 5'-TGAGTGTGCTAGAGACACTG-3' (SEQ ID NO:3), and a single-base extension primer with the nucleotide sequence 5'-ACAAACTAATGACATTCCCA-3' (SEQ ID NO:4).

[0019] In some embodiments of the present invention, the product includes at least one of reagents, reagent kits, test strips, chips, and systems.

[0020] A third aspect of the invention provides a primer set for amplifying the SNP molecular markers of the first aspect of the invention.

[0021] In some embodiments of the present invention, the primer set includes an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is 5'-GGGATCTACAACAGGAAAGG-3', and the nucleotide sequence of the downstream primer is 5'-TGAGTGTGCTAGAGACACTG-3'.

[0022] In some embodiments of the present invention, the primer set further includes a single-base extension primer with a nucleotide sequence of 5'-ACAAACTAATGACATTCCCA-3'.

[0023] A fourth aspect of the present invention provides a kit comprising the primer set of the third aspect of the present invention.

[0024] In some embodiments of the present invention, the kit further includes a buffer used in PCR.

[0025] In some embodiments of the present invention, the buffer used in the PCR is any reagent required for PCR amplification, such as DNA polymerase, dNTP, Taq enzyme, MgCl2, etc.

[0026] In some embodiments of the present invention, the kit further includes SAP, ExoI, and reagents for single-base extension reactions (such as SNaPshot Mix).

[0027] The fifth aspect of the present invention provides the use of the SNP molecular marker of the first aspect of the present invention, the primer set of the third aspect of the present invention, and / or the kit of the fourth aspect of the present invention in any one of (1) to (5):

[0028] (1) Assisted selection or breeding of salt-tolerant largemouth bass;

[0029] (2) To prepare products for auxiliary breeding or breeding of salt-tolerant largemouth bass;

[0030] (3) Identify the salt tolerance of largemouth bass;

[0031] (4) Prepare products for identifying the salt tolerance of largemouth bass;

[0032] (5) Management and development of germplasm resources of largemouth bass.

[0033] In some embodiments of the present invention, the selection is to screen for largemouth bass with excellent salt tolerance.

[0034] A sixth aspect of the present invention provides a method for identifying and screening salt-tolerant largemouth bass, wherein the salt tolerance of the largemouth bass is determined by detecting the genotype of the SNP molecular marker of the first aspect of the present invention in the genome of the largemouth bass to be tested, based on the genotype.

[0035] In some embodiments of the present invention, the method includes the following steps: using the DNA of the largemouth bass to be tested as a template, performing PCR amplification using the primer set in the third aspect of the present invention or the kit in the third aspect of the present invention to obtain PCR amplification products; performing sequencing analysis on the PCR amplification products to determine the genes of the SNP molecular markers of the first aspect of the present invention in the genome of the largemouth bass to be tested; or

[0036] The method includes the following steps: using the DNA of the largemouth bass to be tested as a template, performing PCR amplification using the upstream / downstream primers in the second aspect of the present invention to obtain PCR amplification products;

[0037] The PCR amplification product was treated with alkaline phosphatase to obtain product A;

[0038] Single-base extension primers were used to perform a single-base extension reaction on product A. After the reaction was completed, sequencing analysis was performed to identify the SNP molecular marker gene of the first aspect of the present invention in the genome of the largemouth bass to be tested.

[0039] In some embodiments of the present invention, when the genotype of the SNP molecular marker is GG, it is a largemouth bass with strong salt tolerance (i.e., salt-tolerant largemouth bass), and when the genotype of the SNP molecular marker is TT, it is a largemouth bass with weak salt tolerance (i.e., salt-sensitive largemouth bass).

[0040] In some embodiments of the present invention, the DNA of the largemouth bass can be extracted using conventional methods in this technical field, including the phenol-chloroform method and various DNA extraction kits.

[0041] In some embodiments of the present invention, the PCR amplification reaction program is as follows: pre-denaturation at 90–94°C for 4–6 min; denaturation at 90–94°C for 20–35 s, annealing at 56–60°C for 30–35 s, extension at 70–72°C for 20–30 s, 35–37 cycles; and extension at 70–72°C for 3–5 min.

[0042] In some embodiments of the present invention, the alkaline phosphatase treatment includes mixing the PCR amplification product with SAP Mix and reacting it in a PCR instrument. The reaction program is 35-37°C for 35-40 min; 80-85°C for 12-16 min; 4°C for ∞.

[0043] In some embodiments of the present invention, the single-base extension reaction includes the following steps: mixing product A, single-base extension primers and SNaPshot Mix, and reacting in a PCR instrument. The reaction program is as follows: pre-denaturation at 94-96°C for 1-2 min; denaturation at 94-96°C for 2-5 s; annealing at 50-52°C for 3-5 s; extension at 60-62°C for 2-5 s; for 30-32 cycles.

[0044] In some embodiments of the present invention, the method for sequencing the PCR amplification products is not particularly limited, as long as the sequence of the PCR amplification product, i.e., the fragment containing the SNP marker, can be effectively obtained. The PCR amplification products can be detected using at least one method selected from first-generation gene sequencing, second-generation high-throughput gene sequencing, or third-generation high-throughput gene sequencing, or any other feasible method for determining the genotype. Thus, the genotype results can be obtained rapidly, efficiently, and accurately.

[0045] A seventh aspect of the invention provides the application of the method of the fifth aspect of the invention in the breeding of salt-tolerant largemouth bass.

[0046] The beneficial effects of this invention are:

[0047] This invention uses correlation analysis to identify the salt tolerance of largemouth bass in extreme and sensitive individuals, thereby obtaining a SNP molecular marker associated with the salt tolerance of largemouth bass. The SNP molecular marker, as shown in SEQ ID NO:1, is located at position 501 and contains three genotypes: TT, TG, and GG. The GG genotype largemouth bass exhibits significantly longer survival time under salinity stress than the TG and TT genotypes. By detecting the genotype of this SNP molecular marker, highly salt-tolerant largemouth bass parents can be accurately selected, which can be effectively used for marker-assisted breeding of largemouth bass. Furthermore, it allows for genotyping of largemouth bass parents according to actual breeding needs, selecting highly salt-tolerant genotype parents for reproduction to obtain largemouth bass offspring (fry) with high salt tolerance. This not only saves breeding time, reduces costs, and increases accuracy, but also accelerates the largemouth bass breeding process and increases the economic benefits of aquaculture in saline-alkali environments.

[0048] The present invention further provides primer sets, kits and detection methods for detecting SNP molecular markers, which can be used for rapid identification and screening of salt-tolerant largemouth bass.

[0049] Compared with existing technologies that use high-salt conditions to select largemouth bass with excellent salt tolerance, this invention can directly select largemouth bass parents with excellent salt tolerance by detecting SNP molecular markers. This will not cause lifelong irreversible damage to the largemouth bass or reduce their reproductive performance. It provides an effective screening tool for saltwater aquaculture of largemouth bass and greatly reduces the breeding cost of new salt-tolerant largemouth bass varieties. Attached Figure Description

[0050] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0051] Figure 1 The statistical results show the mean survival time of sensitive and salinity-tolerant families under salinity stress (14 ppt).

[0052] Figure 2 The results show the survival time comparison of the GG, TG, and TT genotypes of largemouth bass used for GWAS sequencing under salinity stress.

[0053] Figure 3 The results of salt tolerance assessment of largemouth bass using the SNP markers provided by this invention. Detailed Implementation

[0054] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0055] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0056] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0057] Example 1: Screening of SNP markers associated with salt tolerance traits in largemouth bass

[0058] 1. Source of experimental fish and methods of salinity stress

[0059] The experimental fish used in this embodiment were derived from a family constructed by our team in May 2024 using the "Youlu No. 3" largemouth bass, a Taiwanese strain, and a salt-tolerant strain as parents. Twenty families of similar size (approximately 20g) were selected for an acute salinity stress experiment. Eighty fish from each family were randomly selected and housed individually in a 600L tank. Each tank was equipped with an air stone, and feeding was withheld the day before and during the experiment.

[0060] The salinity stress level (14 ppt) used in the formal experiment was determined based on the results of the preliminary experiment. The experimental brine was prepared using sun-dried salt and aerated for 24 hours before use. When increasing the salinity, it was initially increased from 4 ppt to 14 ppt gradually at a rate of 2 ppt / hour. During the experiment, samples were continuously taken from bass that began to die (before death) in each family. Tail fin samples were collected and preserved in anhydrous ethanol for DNA extraction. Mortality was recorded every 2 hours during the experiment, with the cessation of gill cover activity and lack of response to external stimuli as the criterion for death. Survival time was used as the standard for assessing salt tolerance. GWAS sequencing was performed on 248 individuals with extreme traits (including 124 salinity-tolerant and 124 salinity-sensitive individuals).

[0061] 2. Genome resequencing of salinity-sensitive and salinity-tolerant largemouth bass.

[0062] Whole-genome resequencing was performed on 248 largemouth bass. Genomic DNA was extracted using the phenol-chloroform method, and DNA integrity and purity were assessed by gel electrophoresis and Qubit assay. Quality-tested DNA samples were randomly fragmented into 350 bp fragments using a Covaris shredder, and libraries were constructed using the VAHTS Universal DNA Library Prep Kit (Novizan NDM607-01). After library quality control, sequencing was performed using a DNBSEQ-T7 PE150 sequencer. Raw data was processed through FastP / FastQC to obtain clean data. The clean data was aligned to the reference genome using bwa software, and PCR duplicates were removed using gatk. Based on the alignment results, SNP detection and filtering were performed using GATK software, and the detected SNP sites were annotated using ANNOVAR software. Using GAPIT3 software, based on genotype and phenotypic data, and with the first three principal components generated by Plink and the kinship matrix generated by TASSEL5 as covariates, association analysis was performed using multiple analytical models, including GLM, MLM, CMLM, ECMLM, Fast-LMM, Fast-LMM-Select, FarmCPU, and BLINK.

[0063] 3. Results

[0064] 3.1 Phenotypic Statistics During Salinity Stress

[0065] During the experiment, the number of deaths in all families at different time points generally followed a normal distribution. Families were differentiated into sensitive and tolerant groups based on survival time, and extreme phenotypes were selected for sequencing, including 124 sensitive individuals and 124 tolerant individuals. The average survival time of the sensitive family population was only 6.52 hours, while the average survival time of the salinity-tolerant family population was 47.69 hours. Figure 1 Statistical analysis showed a highly significant difference between the two phenotypes. P <0.001).

[0066] After filtering and quality control of 248 samples, a total of 2099.57G of clean base was obtained, with an average of 8.64G per sample. The proportion of high-quality bases (Q>30) was as high as 95.96%, and the average GC content was 40.78%. The average sequencing depth was 8.59X, and the average alignment rate was 97.62%. These data indicate that the library construction and sequencing were successful, laying the foundation for subsequent analysis.

[0067] To reveal the relationship between candidate SNPs and salt stress traits, comparisons of different analytical models suggest that the FarmCPU model yields more reliable results. The Bonferroni method was used to adjust... p The suggestive (chromosomal range) and significance (genome-wide) thresholds for the value were 3.86e-07 and 1.93e-08, respectively. This analysis identified 10 SNP loci associated with salinity tolerance in largemouth bass. Among them, chr4:9378904 had a significance of 0.002 and was significantly associated with the salt tolerance trait. This SNP molecular marker is located at position 9378904 on chromosome 4 of largemouth bass, at position 501 from the 5' end of the sequence shown in SEQ ID NO:1 (intronic region of the ip6k1 gene), and its polymorphism is T / G. Analysis showed that the average survival time of individuals with the GG genotype (39.41 h) was significantly higher than that of individuals with the TT (8.83) and TG genotypes (8.23 h). Moreover, 108 salt-tolerant individuals exhibited the GG genotype, accounting for 87.1% of salt-tolerant individuals, suggesting that this genotype is a highly accurate indicator of salt tolerance. Figure 2 ).

[0068]

[0069] Note: "[ ]" indicates the location of the SNP molecular marker.

[0070] Example 2: Validation of the association between SNP markers and salt tolerance traits in largemouth bass

[0071] 1. Source of experimental fish and extraction of genomic DNA

[0072] Five hundred largemouth bass purchased from Liangshi Aquatic Seed Industry Co., Ltd. in Sanshui District, Foshan City, Guangdong Province were randomly selected. After being injected with PIT electronic tags, their tail fins were harvested. Genomic DNA was extracted from the fin rays using a marine animal tissue genomic DNA extraction kit (Tiangen). The quality of the genomic DNA was assessed by 1.0% agarose gel electrophoresis, and the concentration was determined using a UV spectrophotometer (Eppendorf, AG2231). The DNA was stored at -20℃ for later use.

[0073] 2. Genotyping Primer Design

[0074] Based on the genomic sequence of approximately 500 bp before and after the chr4:9378904 site, as shown in SEQ ID NO:1, PCR reaction primers and single-base extension primers were designed and synthesized.

[0075] The nucleotide sequences of the PCR reaction primers Primer F / R are 5'-GGGATCTACAACAGGAAAGG-3' (SEQ ID NO:2); 5'-TGAGTGTGCTAGAGACACTG-3' (SEQ ID NO:3);

[0076] The nucleotide sequence of the base extension primer is 5'-ACAAACTAATGACATTCCCA-3' (SEQ ID NO:4).

[0077] 3. Fertility test

[0078] (1) PCR amplification reaction

[0079] Prepare the PCR Master mix by mixing 5 μL of 2×Taq PCR Master Mix (catalog number 4992920), 0.5 μL of Primer F / R (0.1 nmol / mL), and 3 μL of ddH2O. After mixing, centrifuge at low speed with vortexing. Then, add 9 μL of PCR Master mix to each well, followed by 1 μL of template DNA (20 ng / μL). Mix well and perform PCR amplification. The amplification conditions are: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 20 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 3 min.

[0080] (2) Treatment of product alkaline phosphatase

[0081] After the PCR reaction, the PCR product was treated with shrimp alkaline phosphatase (SAP) (catalog number 783901000UN) to remove free dNTPs from the system. The SAP Mix reaction solution was prepared as follows: SAP 0.5 μL, SAP buffer 0.5 μL, ExoI (Thermo, catalog number EN0582, concentration 20 U / μL) 0.2 μL, and H2O to 0.8 μL. 2 μL of SAP mix was added to a 384-well PCR plate, followed by 4 μL of PCR product, for a total reaction volume of 6 μL. The reaction program was: 37℃ for 40 min; 85℃ for 15 min; 4℃ infinity.

[0082] (3) Single base extension reaction (SNaPshot reaction)

[0083] After alkaline phosphatase treatment, a single-base extension reaction was performed in a total volume of 5 μL. The system consisted of: 3 μL of alkaline phosphatase-treated PCR product; 0.5 μL of Snapshot Mix reagent; 1 μL of extension primer (SEQ ID NO:4); and H2O added to a final volume of 5 μL. The reaction conditions were: 95℃ pre-denaturation for 1 min; 96℃ denaturation for 5 s, 52℃ annealing for 5 s, and 60℃ extension for 5 s, for 30 cycles.

[0084] (4) Sequencing

[0085] 2 μL of the SNaPshot reaction product was added to 8 μL of deionized formamide containing 0.8% LIZ120, denatured at 95 °C for 5 min, then rapidly cooled to -20 °C, and then sequenced at 3730 xL.

[0086] 4. Comparison of salt tolerance under acute salinity stress

[0087] After a week of temporary rearing, acute salinity stress was applied. One hundred largemouth bass with genotypes GG, TG, and TT at position 501 of SEQ ID NO:1 were selected based on genotyping results. The salinity was adjusted to 14 ppt at a rate of 2 ppt / h. After reaching 14 ppt, the fish were observed every four hours, dead fish were promptly removed, survival time was recorded, and tail fins were collected, soaked in anhydrous ethanol, and stored at 4°C for later use.

[0088] 5. Experimental Results

[0089] Under 14 ppt salinity stress, the average survival time of the largemouth bass with the GG genotype at position 501 of SEQ ID NO:1 was 32.17 h, significantly longer than that of the TT genotype (12.2 h) and TG genotype (22.21 h). This result is consistent with the results obtained from sequencing individuals, indicating that the largemouth bass with the GG genotype at position 501 of SEQ ID NO:1 exhibits significantly better salt tolerance than the TT and TG genotypes, and can be used as a molecular marker for screening salt-tolerant largemouth bass. Figure 3 Furthermore, among individuals with the GG genotype, only one individual survived for 6 hours, while the survival times of the other tested individuals were all higher than 14 hours, with the longest survival time being 42 hours. However, among individuals with the TT genotype, only one individual survived for as long as 22 hours, while the survival times of the remaining individuals did not exceed 12 hours.

[0090] 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. A SNP molecular marker associated with salt tolerance in largemouth bass, characterized in that, The sequence of the SNP molecular marker is shown in SEQ ID NO:

1.

2. The SNP molecular marker according to claim 1, characterized in that, When the genotype of the SNP locus is GG, it is a largemouth bass with strong salt tolerance; when the genotype of the SNP locus is TT, it is a largemouth bass with weak salt tolerance.

3. The application of the SNP molecular marker described in claim 1 or 2 in screening salt-tolerant largemouth bass or preparing products for screening salt-tolerant largemouth bass, wherein when the genotype of the SNP molecular marker is GG, it is a largemouth bass with strong salt tolerance, and when the genotype of the SNP molecular marker is TT, it is a largemouth bass with weak salt tolerance.

4. The application according to claim 3, characterized in that, The substance comprises substances selected from one or more detection techniques or methods chosen from the group consisting of: Northern blotting, PCR, gene chip method, and nucleic acid sequencing.

5. The application according to claim 4, characterized in that, The product includes at least one of reagents, reagent kits, test strips, chips, and systems.

6. A primer set for detecting the SNP molecular marker as described in claim 1 or 2; The primer set includes an upstream primer, a downstream primer, and a single-base extension primer, wherein, The upstream primer has the nucleotide sequence 5'-GGGATCTACAACAGGAAAGG-3', the downstream primer has the nucleotide sequence 5'-TGAGTGTGCTAGAGACACTG-3', and the single-base extension primer has the nucleotide sequence 5'-ACAAACTAATGACATTCCCA-3'.

7. A kit comprising the primer set of claim 6.

8. The use of the primer set of claim 6 and / or the kit of claim 7 in any one of (1) to (5): (1) Assisted selection or breeding of salt-tolerant largemouth bass; (2) To prepare products for auxiliary breeding or breeding of salt-tolerant largemouth bass; (3) Identify the salt tolerance of largemouth bass; (4) Prepare products for identifying the salt tolerance of largemouth bass; (5) Management and development of salt-tolerant largemouth bass germplasm resources; When the genotype of the SNP molecular marker described in claim 1 or 2 in the genome of the largemouth bass is GG, it is a largemouth bass with strong salt tolerance; when the genotype of the SNP molecular marker described in claim 1 or 2 in the genome of the largemouth bass is TT, it is a largemouth bass with weak salt tolerance.

9. A method for identifying and screening salt-tolerant largemouth bass, comprising detecting the genotype of the SNP molecular marker as described in claim 1 or 2 in the genome of the largemouth bass to be tested, and determining the salt tolerance of the largemouth bass to be tested based on the genotype; when the genotype of the SNP molecular marker is GG, it is a largemouth bass with strong salt tolerance, and when the genotype of the SNP molecular marker is TT, it is a largemouth bass with weak salt tolerance.

10. The method according to claim 9, characterized in that, The method includes the following steps: using the DNA of the largemouth bass to be tested as a template, performing PCR amplification using the upstream and downstream primers described in claim 6 or the kit described in claim 7 to obtain PCR amplification products; performing sequencing analysis on the PCR amplification products to determine the genotype of the SNP molecular markers described in claim 1 or 2 in the genome of the largemouth bass to be tested; or; The method includes the following steps: using the DNA of the largemouth bass to be tested as a template, performing PCR amplification using the upstream primer and downstream primer described in claim 6 to obtain the PCR amplification product; The PCR amplification product was treated with alkaline phosphatase to obtain product A; The single-base extension primers described in claim 6 were used to perform a single-base extension reaction on product A. After the reaction was completed, sequencing analysis was performed to determine the genotype of the SNP molecular marker described in claim 1 or 2 in the genome of the largemouth bass to be tested.

11. The application of the method of claim 9 or 10 in the breeding of salt-tolerant largemouth bass.

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