Application of diplotype of body length associated SNP (Single Nucleotide Polymorphism) site of modestus septentrionalis in breeding

Abstract

The invention discloses an application of a diplotype of a body length associated SNP (Single Nucleotide Polymorphism) site of modestus septentrionalis in breeding, and belongs to the technical field of aquatic breeding. The method comprises the following steps: analyzing early-stage sequencing data of a W group and an M group of navodon septentrionalis to identify two linkage imbalance SNP sites at 268 and 532 positions of an IGFBP2 gene with higher genetic diversity, totally detecting three haplotypes in the W group and the M group, randomly forming five diplotypes by the three haplotypes, compared with other four diplotypes, the homozygous diplotype V (A268A268C532C532) has remarkable advantages in the aspects of average body length and IGFBP2 gene expression, can be used as a molecular marker related to the body length, and is applied to molecule-assisted breeding of a new fast-growing strain of the navodon septentrionalis so as to improve the yield of a fast-growing improved variety of the navodon septentrionalis.

Description

Technical Field

[0001] This invention relates to the application of diploids of body length-associated SNP sites in the breeding of greenfin pufferfish, and belongs to the field of aquatic breeding technology. Background Technology

[0002] Body length is an important indicator for evaluating the growth performance of greenfin filefish. Currently, because the parent fish of greenfin filefish mainly come from wild catches, there is a lack of control over germplasm resources. Disorderly self-pollination has led to a significant decline in the growth performance of farmed greenfin filefish, which greatly restricts the development of industrialized greenfin filefish farming.

[0003] Currently, my country lacks high-quality, artificially bred greenfin filefish (Pteranodon spp.) strains. Our research team screened out a fast-growing population of greenfin filefish from existing farmed populations and located body length-related SNP loci, aiming to increase the farmed yield of greenfin filefish.

[0004] Currently, there are no applications of body length-related SNP loci in greenfin filefish for breeding. The most similar one is a molecular SNP marker associated with growth traits in greenfin filefish and its application (CN117925860A). However, this patent only identified a single-base mutation associated with greenfin filefish growth and did not establish a linkage disequilibrium relationship between body length and genotype, which has significant limitations in practical production. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of diploids of body length-related SNP sites in greenfin pufferfish in breeding, in order to overcome the limitations of various factors, strictly regulate the growth process of greenfin pufferfish, and promote molecular-assisted breeding of new fast-growing strains of greenfin pufferfish.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The application of homozygous diploid type V, a body length-associated SNP locus in greenfin pufferfish, in molecular-assisted breeding of a new fast-growing strain of greenfin pufferfish. The diploid type is randomly formed from haploid type I, haploid type II, and haploid type III. In haploid type I, the bases at positions 268 and 532 of the IGFBP2 gene are C and T, respectively; in haploid type II, the bases at positions 268 and 532 are C and C, respectively; and in haploid type III, the bases at positions 268 and 532 are A and C, respectively. The nucleotide sequence of the IGFBP2 gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2. The three haploid types randomly form a homozygous diploid type I C. 268 C 268 T 532 T 532 Heterozygous double type II C 268 C268 T 532 C 532 Homozygous double type III C 268 C 268 C 532 C 532 Heterozygous double type IV C 268 A 268 C 532 C 8532 and homozygous double V-type A 268 A 268 C 532 C 532 Of the five diploid types, homozygous diploid V showed significant advantages over the other four diploid types in terms of average body length and IGFBP2 gene expression.

[0007] The advantage of this invention lies in the following: Association analysis of two SNP sites (located at positions 268 and 532 of the insulin-like growth factor binding protein 2 gene, respectively) with the body length of the greenfin pufferfish revealed that these two SNP sites were significantly correlated with the body length of the greenfin pufferfish. Specifically, haplotype III (A...) 268 C 532 Individuals are relatively long. Further comparative analysis of body length among five diploid types—homozygous diploid type I (DI), heterozygous diploid type II (DII), homozygous diploid type III (DIII), heterozygous diploid type IV (DIV), and homozygous diploid type V (DV)—revealed significant differences in body length and IGFBP2 gene expression between fast-growing and normal populations of greenfin filefish. Specifically, homozygous diploid type V (DV) greenfin filefish exhibited significantly higher body length and relative IGFBP2 gene expression levels than the other four diploid types. Diploid type V (DV) can serve as a molecular marker related to body length and can be applied to molecular-assisted breeding of fast-growing greenfin filefish strains to improve the yield of high-quality fast-growing greenfin filefish. Attached Figure Description

[0008] Figure 1 This is a comparison of the body length of greenfin pufferfish from a normal population and a fast-growing population aged 1-5 months. * indicates P<0.05. Figure 2 This is a comparison of the relative expression levels of the IGFBP2 gene among five diploid types of greenfin pufferfish at 5 months of age. Detailed Implementation

[0009] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments can be purchased from conventional biochemical reagent companies.

[0010] I. Materials and Methods 1. Experimental materials The experimental materials were a common population of greenfin triggerfish (wild group, hereinafter referred to as W group) and the fast-growing F2 generation (mutant group, hereinafter referred to as M group), both 4 months old and in good condition. They are preserved at the Fisheries Research Center of Ludong University.

[0011] The body length of the M group at 4 months and 5 months of age used in the experiment was significantly higher than that of the W group. Figure 1 ).

[0012] 2. Screening of IGFBP2 gene SNP sites Analysis of early sequencing data from the M and W populations of greenfin pufferfish identified two linkage-disequilibrium SNP sites at positions 268 and 532 of the IGFBP2 gene, which exhibited high genetic diversity (PIC>0.5) (Table 1).

[0013] Table 1 SNP locus information

[0014] To further amplify the SNP sites at positions 268 and 532 of the IGFBP2 gene via PCR, primer sequences covering all coding regions of the IGFBP2 gene were designed (Table 2).

[0015] Table 2 Primer Information

[0016] Two hundred greenfin triggerfish (M group) and two 200 greenfin triggerfish (W group) were collected from each of the four-month-old groups, totaling 400 fish. These were reared in two separate 20L glass tanks. Specifically, the M group (200 fish) was housed in one tank, and the W group (200 fish) in the other. Both tanks were connected to a central circulation system equipped with mechanical and biological filtration, UV sterilization, and a protein skimmer. The salinity was 31–32‰, the temperature 18–19°C, and the pH 8.2–8.3. Plastic plants were used as a substrate. The fish were fed a formulated diet three times daily (08:00, 12:00, and 16:00). Two hours after each feeding, any remaining feed and feces were siphoned out of the tanks.

[0017] One-third of the dorsal fin was cut from 400 greenfin pufferfish from populations M and W. DNA was extracted using the alkaline cleavage method. Partial gene fragments from populations M and W were amplified by PCR. The qualified PCR products were sent to Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing.

[0018] 3. Growth Comparison Diploid strains of the same SNP were cultured in the same glass tank under the same conditions. After 30 days (5 months of age), body length and average growth were measured.

[0019] 4. Real-time quantitative PCR Five fish of each SNP diploid type were randomly selected from the greenfin pufferfish. Total RNA was extracted from the tails using RNAiso Plus, and genomic DNA was removed from the RNA using the Prime Script RT kit to synthesize cDNA. Real-time quantitative PCR was performed on a CFX96 Touch™ real-time PCR detection system using SYBR Green Premix Ex Taq for qRT-PCR detection. The 18S internal control gene was used as a reference. Primers used are listed in Table 2. All experiments were performed in at least three replicates.

[0020] 5. Statistical Analysis Data are expressed as mean ± sampling error and analyzed using one-way ANOVA with SPSS Statistics 17.0 software. Significance is defined as p < 0.05 or p < 0.01.

[0021] II. Results 1. SNP locus analysis of the IGFBP2 gene in greenfin pufferfish The nucleotide sequence of the IGFBP2 gene in the W population of the greenfin pufferfish is shown in SEQ ID NO: 1, and the nucleotide sequence of the IGFBP2 gene in the M population is shown in SEQ ID NO: 2. Correspondingly, the amino acid sequence encoded by the IGFBP2 gene in the W population of the greenfin pufferfish is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the IGFBP2 gene in the M population is shown in SEQ ID NO: 4.

[0022] Comparison of the IGFBP2 gene and the amino acids encoded by the IGFBP2 gene in the W and M populations revealed that the IGFBP2 gene coding region in the M population contained two variant sites, both of which were missense mutations. 268 A 268 The codon change type at the site is CCG-ACG, encoding proline (P) and threonine (T) located in exon 2, respectively. 532 C 532 The codon change type at the site is TGC-CGC, which encodes cysteine ​​(C) and arginine (R) located in the third exon, respectively.

[0023] Three haplotypes were detected in the W and M populations of greenfin pufferfish: haplotype I (HI, C...). 268 T 532 ), haplotype II (HII, C) 268 C532 ) and haplotype III (HIII, A) 268 C 532 The information for the three haplotypes is shown in Table 3.

[0024] Table 3. Information on the three haplotypes

[0025] As shown in Table 3, the W population is mainly haplotype I, with a small amount of haplotype II and no haplotype III, while the M population is mainly haplotype III, with a small amount of haplotype II and no haplotype I.

[0026] The W population randomly generates three diploid types from two haplotypes (HI, HII): homozygous diploid type I (DI, C). 268 C 268 T 532 T 532 ), heterozygous double type II (DII, C) 268 C 268 T 532 C 532 ) and homozygous double type III (DIII, C 268 C 268 C 532 C 532 The frequencies of occurrence were 73.0%, 26.0%, and 1.0%, respectively. The specific information for the three double types is shown in Table 4.

[0027] The M population randomly generates three diploid types from two haplotypes (HII, HIII): homozygous diploid type III (DIII, C... 268 C 268 C 532 C 532 ), heterozygous double type IV (DIV, C) 268 A 268 C 532 C 532 ) and homozygous double V type (DV, A 268 A 268 C 532 C 532 The frequencies of occurrence were 6.0%, 85.0%, and 9.0%, respectively. The specific information for the three double types is shown in Table 4.

[0028] Table 4 Information on Five Types of Double Forms

[0029] 2. Body length analysis of five diploid forms of the greenfin pufferfish The statistical results of the average body length and average growth of the five diploid types of greenfin pufferfish at 4 and 5 months of age are shown in Table 5.

[0030] Table 5. Statistical results of average body length and average growth of five diploid types at 4 and 5 months of age.

[0031] Note: Different lowercase letters in the superscript of numbers within the same column indicate significant differences (p<0.05).

[0032] As shown in Table 5: (1) At 4 and 5 months of age, there was no significant difference in the average body length of the four diploids DI, DII, DIII and DIV (p>0.05), while the average body length of DV was significantly higher than that of the other four diploids (p<0.05).

[0033] (2) During the 30 days from 4 months to 5 months of age, there was no significant difference in the average growth of the four diploid types DI, DII, DIII and DIV (p>0.05), while the average growth of DV was significantly higher than that of the other four diploid types (p<0.05).

[0034] 3. Relative expression levels of IGFBP2 gene in five diploids of greenfin pufferfish The relative expression levels of the IGFBP2 gene at 5 months of age for five diploid types in greenfin pufferfish—homozygous diploid type I (DI), heterozygous diploid type II (DII), homozygous diploid type III (DIII), heterozygous diploid type IV (DIV), and homozygous diploid type V (DⅤ)—were 2.31±0.26, 2.31±0.25, 2.32±0.16, 2.34±0.25, and 3.31±0.32, respectively. The comparison of relative IGFBP2 gene expression levels is shown below. Figure 2 As shown.

[0035] Depend on Figure 2 It can be seen that the relative expression level of type DV IGFBP2 gene is significantly higher than that of the other four diploids (p<0.05); there is no significant difference in the relative expression level of type DI, type DII, type DIII and type DIV IGFBP2 gene (p>0.05).

[0036] III. Conclusion SNPs (Symptoms of Non-Positive Nucleotides) are DNA genetic polymorphisms caused by single nucleotide changes in the genome. They are widely distributed throughout the genome, possess stable heritability, and are very important molecular markers. This invention identified two linkage disequilibrium SNP sites at positions 268 and 532 of the IGFBP2 gene, which exhibit high genetic diversity (PIC > 0.5). These sites have three haplotypes (haplotype I, haplotype II, and haplotype III). These three haplotypes randomly form five diplotypes (homozygous diplotype I, heterozygous diplotype II, homozygous diplotype III, heterozygous diplotype IV, and homozygous diplotype V). These two SNP sites in the IGFBP2 gene lead to codon missense mutations, and the changes in amino acid sequence may result in alterations in protein function.

[0037] Body length is an important indicator for evaluating the growth performance of fish. To verify whether mutations at two SNP sites in the IGFBP2 gene are associated with the body length of the greenfin pufferfish, this invention identified populations W and M. A 30-day growth comparison experiment was conducted, and the average body length and average growth of different SNP diploids were statistically analyzed. The results showed that compared to population W, population M had a higher average body length (HIII, A...). 268 C 532 Randomly generated homozygous double V type (DV, A) 268 A 268 C 532 C 532 The body length of the genotype 3 SNP A is significantly advantageous. Therefore, it is concluded that simultaneous mutations at positions 268 and 532 (haplotype III SNP A) indicate a significant advantage. 268 C 532 This can significantly increase the body length of the greenfin pufferfish. Previous studies have shown that IGFBP2 plays an important role in growth and development. The haplotype III SNP (A) in the M population... 268 C 532 The expression level of the IGFBP2 gene was significantly increased by changing proline to threonine in the second exon and cysteine ​​to arginine in the third exon.

[0038] These results provide an explanation: IGFBP2 releases free IGF-1 through a conformational change, allowing it to bind to the IGF-1 receptor (IGF-1R), activating the downstream PI3K-Akt and mTOR signaling pathways. These two pathways are core pathways for fish growth regulation, significantly promoting muscle cell proliferation and protein synthesis, ultimately accelerating the body length growth of the greenfin pufferfish. Haplotype III SNPs (A 268 C 532 The mutation increases the expression of the IGFBP2 gene, enhances the binding ability of IGF-1 to its receptor, and thus increases the double-digit DV type A in the M population. 268 A 268 C 532 C 532The body length of the greenfin pufferfish.

[0039] Therefore, the greenfin triggerfish is a homozygous double type V (DV, A) 268 A 268 C 532 C 532 It exhibits a significant advantage in body length and can be used for molecular-assisted breeding of new fast-growing strains of greenfin triggerfish to increase yield.

[0040] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. The application of homozygous double-V type of body length-related SNP loci in molecular-assisted breeding of fast-growing new strains of greenfin triggerfish, characterized in that, in, The diploid type is randomly formed from haplotypes I, II, and III. Specifically, haplotype I has C and T bases at positions 268 and 532, respectively; haplotype II has C and C bases at positions 268 and 532, respectively; and haplotype III has A and C bases at positions 268 and 532, respectively. The nucleotide sequence of the IGFBP2 gene is shown in SEQ ID NO: 1 or SEQ ID NO:

2. The three haplotypes randomly form a homozygous diploid type I C. 268 C 268 T 532 T 532 Heterozygous double type II C 268 C 268 T 532 C 532 Homozygous double type III C 268 C 268 C 532 C 532 Heterozygous double type IV C 268 A 268 C 532 C 8532 and homozygous double V-type A 268 A 268 C 532 C 532 Of the five diploid types, homozygous diploid V showed significant advantages over the other four diploid types in terms of average body length and IGFBP2 gene expression.

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