SNP (Single Nucleotide Polymorphism) molecular marker related to sexual gland weight character of fugu rubripes and application of SNP molecular marker

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker related to sexual gland weight traits of fugu rubripes and application of the SNP molecular marker, and relates to the technical field of biology. According to the invention, association analysis is carried out in combination with the sexual gland heavy character of the fugu rubripes, and the SNP molecular marker significantly related to the sexual gland heavy character of the fugu rubripes is successfully excavated. The SNP molecular marker is used for breeding of improved gonad breeds of fugu rubripes, and compared with traditional phenotype-based selective breeding and cross breeding means, the SNP molecular marker has higher accuracy and detection efficiency and has a wide application prospect.

Description

[0001] This application claims priority to the earlier application filed on October 15, 2025, with application number 202511473752.4 and entitled "SNP molecular markers related to gonadal weight traits of redfin pufferfish and their applications", the entire contents of which are set forth in this application. Technical Field

[0002] This invention relates to the field of biotechnology, and in particular to a SNP molecular marker associated with the gonadal weight trait of the red-finned pufferfish and its application. Background Technology

[0003] The redfin pufferfish is the most prized marine fish in the order Tetraodontiformes. Studies have shown that its muscles, mouth, and testes are non-toxic, and its testes are a highly valued food item. To increase the economic value of the redfin pufferfish, it is crucial to select for superior breeds with favorable gonadal traits. However, current breeding methods for redfin pufferfish primarily rely on selective breeding and hybridization, which have low breeding efficiency.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The primary objective of this invention is to provide an SNP molecular marker associated with the gonadal weight trait of the redfin pufferfish, in order to solve the aforementioned problems.

[0006] The second objective of this invention is to provide the application of the above-mentioned SNP molecular markers related to the gonadal weight trait of the red-finned pufferfish in the detection of the gonadal weight trait of the red-finned pufferfish.

[0007] A third objective of this invention is to provide the application of reagents for detecting the above-mentioned SNP molecular markers associated with the gonadal weight trait of red-finned pufferfish in the preparation of gonadal weight trait detection products for red-finned pufferfish.

[0008] A fourth objective of this invention is to provide a reagent for detecting gonadal weight characteristics in the red-finned pufferfish.

[0009] The fifth objective of this invention is to provide a kit for detecting gonadal weight traits in red-finned pufferfish.

[0010] The sixth objective of this invention is to provide the application of the above-mentioned SNP molecular markers related to the gonadal weight trait of redfin pufferfish in the breeding of redfin pufferfish.

[0011] The seventh objective of this invention is to provide the application of reagents for detecting the SNP molecular markers associated with the gonadal weight trait of the redfin pufferfish in the preparation of redfin pufferfish breeding products.

[0012] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a SNP molecular marker associated with the gonadal weight trait of the red-finned pufferfish, wherein the SNP molecular marker is: The genotype mutation at Chr 4:13241593 is AG; The genotype mutation at Chr 4:13819857 is CT; The genotype mutation at Chr 4:13274607 is CT.

[0013] Secondly, the present invention provides the application of the above-mentioned SNP molecular markers related to the gonadal weight trait of the red-finned pufferfish in the detection of the gonadal weight trait of the red-finned pufferfish.

[0014] Thirdly, the present invention provides the application of reagents for detecting the above-mentioned SNP molecular markers associated with the gonadal weight trait of red-finned pufferfish in the preparation of red-finned pufferfish gonadal weight trait detection products.

[0015] As a further technical solution, the product includes a reagent kit.

[0016] Fourthly, the present invention provides a reagent for detecting gonadal weight traits in redfin pufferfish, comprising primers and / or probes for detecting the SNP molecular marker.

[0017] Fifthly, the present invention provides a kit for detecting gonadal weight traits in redfin pufferfish, comprising the aforementioned reagent.

[0018] As a further technical solution, nucleic acid extraction reagents are also included.

[0019] Sixthly, the present invention provides the application of the above-mentioned SNP molecular markers related to the gonadal weight trait of redfin pufferfish in the breeding of redfin pufferfish.

[0020] In a seventh aspect, the present invention provides the application of reagents for detecting the above-mentioned SNP molecular markers related to the gonadal weight trait of redfin pufferfish in the preparation of redfin pufferfish breeding products.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention combines association analysis with the gonadal weight trait of the red-finned pufferfish to successfully identify SNP molecular markers significantly associated with this trait. Using these SNP markers for breeding superior red-finned pufferfish gonadal traits demonstrates higher accuracy and detection efficiency compared to traditional phenotype-based selection and hybridization breeding methods, and has broad application prospects. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 Histogram of gonadal density distribution for 260 red-finned pufferfish; Figure 2 The results of the correlation analysis of 260 redfin pufferfish. Detailed Implementation

[0024] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0025] In a first aspect, the present invention provides a SNP molecular marker associated with the gonadal weight trait of the red-finned pufferfish, wherein the SNP molecular marker is: The genotype mutation at Chr 4:13241593 is AG; The genotype mutation at Chr 4:13819857 is CT; The genotype mutation at Chr 4:13274607 is CT.

[0026] This invention combines the gonadal weight trait of the red-finned pufferfish with correlation analysis and successfully identified SNP molecular markers that are significantly associated with the gonadal weight trait of the red-finned pufferfish.

[0027] Secondly, the present invention provides the application of the above-mentioned SNP molecular markers related to the gonadal weight trait of the red-finned pufferfish in the detection of the gonadal weight trait of the red-finned pufferfish.

[0028] The SNP molecular marker provided by the present invention can be used for the detection of gonadal weight traits in redfin pufferfish.

[0029] Thirdly, the present invention provides the application of reagents for detecting the above-mentioned SNP molecular markers associated with the gonadal weight trait of red-finned pufferfish in the preparation of red-finned pufferfish gonadal weight trait detection products.

[0030] The SNP molecular marker of the present invention is significantly correlated with the gonadal weight trait of the red-finned pufferfish. Therefore, the gonadal weight trait of the red-finned pufferfish can be detected using the reagent that detects the SNP molecular marker.

[0031] In some alternative implementations, the product includes a reagent kit.

[0032] Fourthly, the present invention provides a reagent for detecting gonadal weight traits in redfin pufferfish, comprising primers and / or probes for detecting the SNP molecular marker.

[0033] This reagent can detect the gonadal weight trait of the redfin pufferfish by detecting SNP molecular markers.

[0034] Fifthly, the present invention provides a kit for detecting gonadal weight traits in redfin pufferfish, comprising the aforementioned reagent.

[0035] This kit enables the detection of gonadal weight traits in the red-finned pufferfish by detecting SNP molecular markers.

[0036] In some optional implementations, a nucleic acid extraction reagent is also included. The DNA of the sample to be tested is obtained using the nucleic acid extraction reagent.

[0037] Sixthly, the present invention provides the application of the above-mentioned SNP molecular markers related to the gonadal weight trait of redfin pufferfish in the breeding of redfin pufferfish.

[0038] The SNP molecular marker provided by this invention is significantly correlated with the gonadal weight trait of the red-finned pufferfish. Using this SNP molecular marker for the breeding of superior red-finned pufferfish gonadal varieties demonstrates higher accuracy and detection efficiency compared to traditional phenotype-based selection and hybridization breeding methods, and has broad application prospects.

[0039] In a seventh aspect, the present invention provides the application of reagents for detecting the above-mentioned SNP molecular markers related to the gonadal weight trait of redfin pufferfish in the preparation of redfin pufferfish breeding products.

[0040] The SNP molecular marker provided by this invention is significantly correlated with the gonadal weight trait of the redfin pufferfish. Therefore, the reagent for detecting this SNP molecular marker can be used to breed superior varieties of redfin pufferfish.

[0041] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0042] Example 1 1. Materials and Methods 1.1 Experimental Sample Collection Twenty-sixty male red-finned pufferfish were randomly selected from Tangshan Yuanhetai Aquatic Products Company. Growth traits (gona weight) were measured. Fin tissue was collected from each individual and preserved in 95% ethanol for subsequent analysis at 4°C.

[0043] 1.2 DNA extraction, library construction and sequencing Genomic DNA was extracted from 260 *Pueraria lobata* samples using the TIANDamp Marine Animal DNA Extraction Kit from Tiangen Biotech (Beijing) Co., Ltd., following the manufacturer's instructions. DNA quality and concentration were analyzed using NanoDrop 2000 and 1.5% agarose gel electrophoresis, respectively. A DNA library with an insert size of approximately 300 bp was constructed using the VAHTS UniversalPlus DNA Library Prep Kit from Nanjing Novizan Biotechnology Co., Ltd. The constructed DNA library was subjected to 150 bp paired-end sequencing on the Illumina Novaseq 6000 platform at Novogene (Beijing).

[0044] 1.3 Sequencing quality control and single nucleotide polymorphism (SNP) identification The raw data were quality controlled using Fastp software with default parameters. BWA was used to align the filtered data to the reference genome of *Putrachea rubra* (GCF_901000725.2, https: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCF / 901 / 000 / 725 / GCF_901000725.2_fTakRub1.2 / ). SAMtools was used to convert the alignment results to BAM format and sort them. GATK software was used to remove duplicate reads and perform variant detection. The HaplotypeCaller model in GATK was used to identify variants, and genotyping was performed using the GenotypeGVCFs parameter. SNPs were screened according to the hard filtering criteria of GATK software. Further screening was performed using PLINK software, retaining dimorphic SNPs with a minor allele frequency ≥0.05 and a genotype deletion rate ≤0.95.

[0045] 1.4 Genome-wide association analysis (GWAS) A genome-wide association study of gonadal weight was conducted using a mixture linear model (MLM) from GEMMA (Zhou and Stephens, 2014). The model was defined as follows: Y = Xα + Zβ + Wl + e.

[0046] In this model, Y is the vector of phenotypic observations. α and β represent fixed effects related to population structure and genotype, respectively. l represents a polygenic effect, assumed to follow a distribution of l ~ N(0, G), where G is the standardized kinship matrix representing the variance of the genetic effect. X, Z, and W are the design matrices for α, β, and l, respectively, and e represents the residuals. The Bonferroni correction method was used to set the significance threshold for p-values ​​to p < 0.05 / n, and the suggestive association threshold to p < 1 / n, where n is the number of SNPs used in GWAS. Manhattan plots and QQ plots were plotted using the CMplot2 package in R 4.3.0 for GWAS visualization. Significant SNPs exceeding the thresholds were considered candidate SNPs associated with the trait, and these candidate SNPs were annotated using SNPEff.

[0047] 1.5 Candidate SNP Genotyping Validation Genotyping was validated using PCR amplification. An additional 56 individuals were selected in the validation population for locus genotyping confirmation. Specific primers for candidate loci are listed in Table 1. PCR reactions were performed on an A300-PCR instrument using TransGene (Beijing) PCR Mix, with the following program: 95 ℃ pre-denaturation for 3 min; 35 cycles (94 ℃ 20 s, 58 ℃ 20 s, 72 ℃ 40 s); final extension at 72 ℃ for 5 min. Amplified products were sequenced using an ABI 3730xL DNA analyzer to determine genotypes. The obtained genotyping data were analyzed using IBM SPSS Statistics 26 for one-way ANOVA.

[0048] Table 1. Primers for candidate marker genotype verification

[0049] 2 Results 2.1 Statistical analysis of growth traits Phenotypic information on gonadal weight collected from 260 male red-finned pufferfish individuals is summarized in Table 2. The mean gonadal weight was 963.93 ± 94.91 g, with a coefficient of variation of 9.85%. Furthermore, histograms showed that these three traits were approximately normally distributed, with a Shapiro-Wilk test > 0.05. Figure 1 ).

[0050] Table 2. Statistical data on gonadal weight phenotype of 260 redfin pufferfish.

[0051] 2.2 SNP detection and genome-wide association analysis A total of 5,264,855 original SNPs were identified from all samples, with an average sequencing depth of 11.18 × 10⁻⁶. After quality control and biallelic screening, 1,697,490 SNPs were retained for further analysis. Gonadal weight GWAS analysis was performed using a mixed linear model in GEMMA. For gonadal weight GWAS analysis of the trait, SNPs were ranked according to the p-value of the trait-related SNPs in the GWAS analysis (…). Figure 2 SNP loci significantly associated with gonadal weight traits were screened. Among these loci, loci 13241593, 13819857, and 13274607 on chromosome 4 of the redfin pufferfish were precisely located.

[0052] 2.5 Validation of Significant SNP Genotyping To verify the relationship between SNP loci Chr 4:13241593, Chr 4:13819857, and Chr 4:13274607 and gonadal weight trait, 56 individuals were screened for SNP genotyping verification targeting gonadal weight. The results are shown in Table 3.

[0053] Table 3. Comparative analysis of gonadal heavy SNP loci among different genotypes in the validation population.

[0054] As shown in Table 3, the gonadal weight of the AG genotype at the Chr 4:4:13241593 locus was significantly higher than that of the AA and AG genotypes, indicating that the AG genotype is the dominant genotype; the gonadal weight of the CT genotype at the Chr 4:13819857 locus was significantly higher than that of the CC and TT genotypes, indicating that the CT genotype is the dominant genotype; and the gonadal weight of the CT genotype at the Chr 4:13274607 locus was significantly higher than that of the CC and TT genotypes, indicating that the CT genotype is the dominant genotype.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A SNP molecular marker associated with gonadal traits of Takifugu rubripes, characterized in that, The SNP molecular marker is: The genotype mutation of site Chr 4:13241593 is AG; The genotype mutation of site Chr 4:13819857 is CT; The genotype mutation of site Chr 4:13274607 is CT.

2. Application of the SNP molecular marker related to the gonad weight trait of P. rockfelleri in gonad weight trait detection of P. rockfelleri.

3. Application of reagents for detecting the SNP molecular marker related to the gonad weight trait of P. rockfelleri in preparation of gonad weight trait detection products of P. rockfelleri.

4. Use according to claim 3, characterized in that, The products include kits.

5. A reagent for detecting a gonadal heavy trait of Takifugu rubripes, characterized by, The reagents include primers and / or probes for detecting the SNP molecular marker.

6. A kit for detecting gonadal weight traits in Takifugu rubripes, comprising, The reagents include the reagents of claim 5.

7. The kit of claim 6, wherein The reagents further include nucleic acid extraction reagents.

8. Application of the SNP molecular marker related to the gonad weight trait of P. rockfelleri in breeding of P. rockfelleri.

9. Application of reagents for detecting the SNP molecular marker related to the gonad weight trait of P. rockfelleri in preparation of breeding products of P. rockfelleri.

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