Application of diplotype of high-association SNP (Single Nucleotide Polymorphism) site of rudon septentrionalis in breeding

Abstract

The invention discloses application of a diplotype of a high-association SNP (Single Nucleotide Polymorphism) site of a rudon septentrionalis body 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 269 and 469 positions of an MYF6 gene with relatively high genetic diversity, totally detecting three haplotypes in the W group and the M group, and randomly forming five diplotypes by the three haplotypes, compared with other four diplotypes, the homozygous diplotype V (G269G269C469C46969) has remarkable advantages in the aspects of average body height and MYF6 gene expression, can be used as a molecular marker related to the body height, 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 highly associated SNP sites in the body of greenfin pufferfish in breeding, and belongs to the field of aquatic breeding technology. Background Technology

[0002] Body height is an important indicator for evaluating the growth performance of greenfin filefish. Currently, because the parent stock of greenfin filefish mainly comes from wild capture, 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 height-associated SNP loci, aiming to increase the farmed yield of greenfin filefish.

[0004] Currently, there are no applications of highly associated SNP loci in greenfin filefish for breeding. The most similar one is a molecular marker of SNPs related to growth traits in greenfin filefish and its application (CN117925860A). However, this patent only identified single-base mutations associated with growth in greenfin filefish and did not establish a linkage disequilibrium relationship between body height 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 highly associated SNP sites in the body of 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 highly associated SNP locus in the body of the 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 269 and 469 of the MYF6 gene are A and A, respectively; in haploid type II, the bases at positions 269 and 469 are A and C, respectively; and in haploid type III, the bases at positions 269 and 469 are G and C, respectively. The nucleotide sequence of the MYF6 gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2. The three haploid types randomly form a homozygous diploid type I, A. 269 A 269 A 469 A 469 Heterozygous double type II A 269 A 269 A469 C 469 Homozygous double type III A 269 A 269 C 469 C 469 Heterozygous double type IV A 269 G 269 C 469 C 469 and homozygous double V-type G 269 G 269 C 469 C 469 Of the five diploid types, homozygous diploid V showed significant advantages over the other four diploid types in terms of average body height and MYF6 gene expression.

[0007] The advantage of this invention lies in the following: Association analysis of two SNP sites (located at positions 269 and 469 of the MYF6 gene, respectively) of the greenfin pufferfish's myogenic factor 6 (MYF6) gene with the body height of the greenfin pufferfish revealed a significant correlation between these two SNP sites and the body height of the greenfin pufferfish. Specifically, haplotype III (G... 269 C 469 Individuals were relatively tall. Further comparative analysis of body height 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 height and MYF6 gene expression between the fast-growing and normal populations of greenfin filefish. Specifically, the relative body height and MYF6 gene expression levels of the homozygous diploid type V (DV) greenfin filefish were significantly higher than the other four diploid types. Diploid type V (DV) can serve as a molecular marker related to body height 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 height 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 MYF6 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 height of the M group at 3 months, 4 months, and 5 months of age was significantly higher than that of the W group. Figure 1 ).

[0012] 2. Screening of SNP sites in the MYF6 gene Analysis of early sequencing data from the M and W populations of greenfin pufferfish identified two linkage-disequilibrium SNP sites at positions 269 and 469 of the MYF6 gene with high genetic diversity (PIC>0.5) (Table 1).

[0013] Table 1 SNP locus information

[0014] To further amplify the SNP sites at positions 269 and 469 of the MYF6 gene via PCR, primer sequences covering all coding regions of the MYF6 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 height 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 MYF6 gene in the greenfin pufferfish The nucleotide sequence of the MYF6 gene in the W population of the greenfin pufferfish is shown in SEQ ID NO: 1, and the nucleotide sequence of the MYF6 gene in the M population is shown in SEQ ID NO: 2. Correspondingly, the amino acid sequence encoded by the MYF6 gene in the W population of the greenfin pufferfish is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the MYF6 gene in the M population is shown in SEQ ID NO: 4.

[0022] Comparison of the MYF6 gene and the amino acids encoded by the MYF6 gene in the W and M populations revealed that the MYF6 gene coding region in the M population contained two variant sites, both missense mutations. 269 G 269 The codon change type at the site is AAT-AGT, encoding asparagine (N) and serine (S) located in exon 2, respectively. 469 C 469 The codon change type at the site is ATA-CTA, which encodes isoleucine (I) and leucine (L) located in the second exon, respectively.

[0023] Three haplotypes were detected in the W and M populations of greenfin pufferfish: haplotype I (HI, A) 269 A 469 ), haplotype II (HII, A) 269 C 469 ) and haplotype III (HIII, G) 269 C 469 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, A... 269 A 269 A 469 A 469 ), heterozygous double type II (DII, A) 269 A 269 A 469 C 469 ) and homozygous double type III (DIII, A 269 A 269 C 469 C 469 The frequencies of occurrence were 91.0%, 6.0%, and 3.0%, respectively. The specific information for the three double types is shown in Table 4.

[0027] In population M, two haplotypes (HII, HIII) are randomly generated, and two diploids are formed: heterozygous diploid type IV (DIV, A). 269 G 269 C 469 C 469 ) and homozygous double V type (DV, G) 269 G 269 C 469 C 469 The frequencies of occurrence were 67.0% and 33.0% respectively. The specific information for the two double types is shown in Table 4.

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

[0029] 2. Body height analysis of five diploid forms of the greenfin pufferfish The statistical results of the average body height 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 height and average growth rate of five diploid strains at 4 and 5 months of age.

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

[0032] As shown in Table 5: (1) At 4 and 5 months of age, the body height of DIV in the M group was not significantly different from that of the three diploids (DI, DII, DIII) in the W group (p>0.05), while the body height of DV in the M group 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, the average growth of DIV in the M group was not significantly different from that of the three diploids (DI, DII, DIII) in the W group (p>0.05), while the average growth of DV in the M group was significantly higher than that of the other four diploids (p<0.05).

[0034] 3. Relative expression levels of the MYF6 gene in five diploid types of greenfin pufferfish The relative expression levels of the MYF6 gene in 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)) of the greenfin pufferfish at 5 months of age were 0.66±0.08, 0.66±0.07, 0.67±0.08, 0.69±0.10, and 1.22±0.16, respectively. The comparison of the relative expression levels of the MYF6 gene is shown below. Figure 2 As shown.

[0035] Depend on Figure 2 It can be seen that the relative expression level of MYF6 gene of type DV in population M is significantly higher than that of the other four diploids (p<0.05); the relative expression level of MYF6 gene of type DIV in population M is not significantly different from that of the three diploids (type DI, type DII, and type DIII) in population W (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 269 and 469 of the MYF6 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 MYF6 gene lead to codon missense mutations, and the changes in amino acid sequence may result in alterations in protein function.

[0037] Body height is an important indicator for evaluating the growth performance of fish. To verify whether mutations at two SNP sites in the MYF6 gene are associated with body height in the greenfin pufferfish, this invention identified populations W and M. A 30-day growth comparison experiment was conducted, and the average body height and average growth of different SNP diploids were statistically analyzed. The results showed that compared to population W, the M population had a higher average body height (HIII, G...). 269 C 469 Randomly generated homozygous double V type (DV, G) 269 G 269 C 469 C 469 The height of the genotype 269 SNP G is significantly superior. Therefore, it can be concluded that simultaneous mutations at positions 269 and 469 (haplotype III SNP G) indicate a significant advantage. 269 C 469 This can significantly increase the body height of the greenfin pufferfish. Previous studies have shown that MYF6 is a myogenic factor that plays an important role in growth and development. The haplotype III SNP (G) in the M population... 269 C 469 The expression level of the MYF6 gene was significantly increased by changing asparagine to serine and isoleucine to leucine in the second exon.

[0038] These results provide an explanation: MYF6 significantly promotes muscle fiber proliferation by maintaining the functional stability of mature muscle fibers, ultimately accelerating the increase in body height in the greenfin pufferfish. Haplotype III SNP (G 269 C 469 The mutation increases the expression of the MYF6 gene, enhances muscle fiber proliferation, and thus increases the doubling of type DV G in the M population. 269 G 269 C 469 C 469 Body height of greenfin pufferfish.

[0039] Therefore, the greenfin triggerfish is a homozygous double type V (DV, G) 269 G 269 C 469 C 469 It exhibits a significant advantage in body height 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 highly associated SNP sites in the body of greenfin triggerfish in molecular-assisted breeding of fast-growing new strains of greenfin triggerfish, characterized by, in, The diploid type is randomly formed from haplotypes I, II, and III. In haplotype I, the bases at positions 269 and 469 of the MYF6 gene are A and A, respectively; in haplotype II, the bases at positions 269 and 469 are A and C, respectively; and in haplotype III, the bases at positions 269 and 469 are G and C, respectively. The nucleotide sequence of the MYF6 gene is shown in SEQ ID NO: 1 or SEQ ID NO:

2. The three haplotypes randomly form a homozygous diploid type I, A. 269 A 269 A 469 A 469 Heterozygous double type II A 269 A 269 A 469 C 469 Homozygous double type III A 269 A 269 C 469 C 469 Heterozygous double type IV A 269 G 269 C 469 C 469 and homozygous double V-type G 269 G 269 C 469 C 469 Of the five diploid types, homozygous diploid V showed significant advantages over the other four diploid types in terms of average body height and MYF6 gene expression.

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