Application of Haplotype of SNP Loci Associated with Eye Diameter of Hippocampus abdominalis in Breeding

Abstract

The present invention discloses the application of the haplotype of the eye diameter-related SNP locus of Hippocampus abdominalis in breeding, belonging to the technical field of aquaculture breeding. The haplotype is randomly formed by haplotype I, haplotype II, and haplotype III. Among them, the bases at exon 443 and 584 of the PIKFYVE gene of haplotype I are T and T respectively, the bases at exon 443 and 584 of the PIKFYVE gene of haplotype II are T and C respectively, and the bases at exon 443 and 584 of the PIKFYVE gene of haplotype III are C and C respectively. The homozygous haplotype V randomly formed by haplotype III has significant advantages in terms of average eye diameter and PIKFYVE gene expression compared with other haplotypes. The homozygous haplotype V can be used for molecular-assisted breeding of fast-growing new strains of Hippocampus abdominalis to accelerate the breeding process of fast-growing fine varieties of hippocampus.

Description

Technical Field

[0001] The present invention relates to the application of the haplotype of the eye diameter-related SNP locus of the distended hippocampus in breeding, belonging to the technical field of aquaculture breeding. Background Art

[0002] The sense of smell of the hippocampus is severely degraded, and its predation mainly relies on vision. The eye diameter is one of the important indicators of the visual ability of the hippocampus and is significantly correlated with the feeding ability of the hippocampus. Hippocampus seedlings need to feed on live Artemia nauplii, and the hatching time needs to be strictly controlled. For hippocampi with a smaller eye diameter, their visual ability is poor, and the inability to effectively prey on live Artemia nauplii will lead to death, seriously affecting the survival rate of seedlings and the aquaculture yield of hippocampi.

[0003] At present, there is still a lack of excellent cultured hippocampus strains artificially cultivated in China. The research team has cultivated the F3 generation of the fast-growing hippocampus strain, screened out a group with a significantly increased eye diameter, and located the eye diameter-related SNP, in order to improve the survival rate of hippocampus seedlings and the aquaculture yield.

[0004] At present, there is no report on the application of the eye diameter-related SNP locus of the distended hippocampus (Distended hippocampus) in breeding. The most similar one is the application of the snout width-related SNP locus of the distended hippocampus in breeding. However, the key gene (CSF2RB gene) of the snout width-related SNP can only be associated with the snout width of the hippocampus, and cannot be associated with the eye diameter trait of the hippocampus, and cannot be applied to the screening of the eye diameter size of the hippocampus. Even if hippocampi with a significantly increased snout width are screened out through the snout width-related SNP locus, only the limitation of the opening bait diameter is overcome. Since there are hippocampi with a smaller eye diameter among the hippocampi with a significantly increased snout width, the visual ability of these hippocampi with a smaller eye diameter is poor, and they will still die due to the inability to effectively prey on live Artemia nauplii, and ultimately will still seriously affect the survival rate of seedlings and the aquaculture yield of hippocampi. Summary of the Invention

[0005] To solve the deficiencies of the prior art, the present invention provides the application of the haplotype of the eye diameter-related SNP locus of the distended hippocampus in breeding, overcomes the limitations of the olfactory decline and poor vision of the hippocampus, improves the ability of seedlings to effectively prey on live Artemia nauplii, and can promote the development of industrialized breeding of the distended hippocampus.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions:

[0007] Application of homozygous diploid V type of SNP locus associated with eye diameter of Hippocampus abdominalis in breeding, wherein the diplotype is randomly formed by haplotype I, haplotype II, and haplotype III. Among them, the bases at positions 443 and 584 of the exon of the PIKFYVE gene in haplotype I are T and T respectively, the bases at positions 443 and 584 of the exon of the PIKFYVE gene in haplotype II are T and C respectively, and the bases at positions 443 and 584 of the exon of the PIKFYVE gene in haplotype III are C and C respectively. The nucleotide sequence of the PIKFYVE gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2. The 3 haplotypes are randomly formed into homozygous diploid I type T 443 T 443 T 584 T 584 , heterozygous diploid II type T 443 T 443 T 584 C 584 , homozygous diploid III type T 443 T 443 C 584 C 584 , heterozygous diploid IV type T 443 C 443 C 584 C 584 and homozygous diploid V type C 443 C 443 C 584 C 584 5 diplotypes. The homozygous diploid V type has significant advantages over the other 4 diplotypes in terms of average eye diameter and relative expression of the PIKFYVE gene. The homozygous diploid V type can be used for molecular assisted breeding of fast-growing new strains of Hippocampus abdominalis.

[0008] The beneficial effect of the present invention is that: the 2 SNP loci (located at positions 443 and 584 of the exon of the PIKFYVE gene respectively) of the lipid phosphoinositide catalytic enzyme encoding gene (PIKFYVE gene) of Hippocampus abdominalis are associated with the eye diameter of Hippocampus abdominalis. It is found that these 2 SNP loci are significantly correlated with the eye diameter. Among them, haplotype III (C 443 C 584The individual eye diameter is significantly increased. By further comparing and analyzing the eye diameters and the expression levels of the PIKFYVE gene in five types of diploid individuals, namely 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), it is found that there are significant differences in the eye diameters and the expression levels of the PIKFYVE gene between the fast-growing strain F3 generation and the common population of the puffy seahorse. Among them, the average eye diameter and the relative expression level of the PIKFYVE gene in the homozygous diploid type V (DV) puffy seahorse are significantly higher than those of the other four types of diploids. The homozygous diploid type V (DV) can be used as a molecular marker related to the eye diameter and applied to the molecular-assisted breeding of the fast-growing new strain of the puffy seahorse to improve the survival rate of the fast-growing fine varieties of seahorses at the seedling stage. Description of the Drawings

[0009] Figure 1 It is a comparison chart of the eye diameters of the common population of the puffy seahorse and the fast-growing strain F3 generation from 1 to 7 months old;

[0010] Figure 2 It is a comparison chart of the relative expression levels of the PIKFYVE gene in five types of diploid puffy seahorses at 3 months old. Detailed Implementation Modes

[0011] The present invention will be specifically introduced below in conjunction with the drawings and embodiments. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments can be purchased from conventional biochemical reagent companies unless otherwise specified.

[0012] I. Materials and Methods

[0013] 1. Experimental fish

[0014] The experimental fish are the common population of the puffy seahorse (wild type, hereinafter referred to as the W population) and the fast-growing strain F3 generation (mutant type, hereinafter referred to as the M population), all of which are 1 month old and have good growth and development. They are stored in the Seahorse Research Center of Ludong University.

[0015] The eye diameters of the M population used in the experiment from 3 to 7 months old are significantly higher than those of the W population ( Figure 1 ).

[0016] Two hundred tails are taken from each of the W population and the M population of the puffy seahorse and cultured in 4 glass tanks of 20 L, with 100 tails in each tank, and connected to the central circulation system, which has mechanical and biological filtration, ultraviolet sterilization, and a protein skimmer. The salinity is 31 - 32‰, the temperature is 18 - 19 °C, the pH is 8.2 - 8.3, and plastic plants are used as attachment substrates. Live brine shrimp are fed three times a day at 8:00, 12:00, and 16:00. Two hours after each feeding, the residual feed and feces in the glass tank are siphoned out.

[0017] 2. Screening of SNP loci of the gene encoding phosphatidylinositol lipid kinase (hereinafter referred to as the PIKFYVE gene)

[0018] By analyzing the sequencing data of the W population and M population of Hippocampus abdominalis, two linkage disequilibrium SNP loci at exon 443 and 584 of the PIKFYVE gene with high genetic diversity (PIC>0.5) were identified (Table 1).

[0019] Table 1 SNP locus information

[0020]

[0021] To further amplify the SNP loci at 443 and 584 of the PIKFYVE gene by PCR reaction, primer sequences covering all coding regions of the PIKFYVE gene were designed (Table 2).

[0022] Table 2 Primer information

[0023]

[0024] For a total of 400 Hippocampus abdominalis in the W population and M population, 1 / 3 of the dorsal fin was cut, DNA was extracted by the alkaline lysis method, partial gene fragments of the W population and M population were amplified by PCR, and the qualified PCR products were sent to Sangon Biotech (Shanghai, China) for sequencing.

[0025] 3. Growth comparison

[0026] Hippocampus abdominalis with the same SNP type were placed in the same glass tank for breeding, and the breeding conditions were the same as before. After 30 days (2 months old) and 60 days (3 months old) of breeding, the eye diameter and body weight were measured respectively, and the average eye diameter at 2 months old, the average eye diameter at 3 months old, and the average weight gain in 30 days from 2 months old to 3 months old were calculated.

[0027] 4. Real-time fluorescence quantitative PCR

[0028] For each SNP haplotype of Hippocampus abdominalis, 5 individuals were randomly selected, total RNA of the tail was extracted using RNAiso Plus, genomic DNA was removed from the RNA using the Prime Script RT kit, and cDNA was synthesized. Real-time quantitative PCR was performed on a CFX96 Touch™ real-time PCR detection system, and qRT-PCR detection was performed using SYBR Green Premix Ex Taq. The internal reference gene 18S was used as a control. The primers used are shown in Table 2. All experiments were repeated more than three times.

[0029] 5. Statistical analysis

[0030] Data were expressed as mean ± sampling error and analyzed by one-way ANOVA using SPSS Statistics 17.0 software. Significance was set at p < 0.05 or p < 0.01.

[0031] II. Results

[0032] 1. Analysis of SNP Loci of PIKFYVE Gene in Hippocampus abdominalis

[0033] After detection, the nucleotide sequence of the PIKFYVE gene in the W population of Hippocampus abdominalis is shown in SEQ ID NO: 1, and the nucleotide sequence of the PIKFYVE gene in the M population is shown in SEQ ID NO: 2. Correspondingly, the amino acid sequence encoded by the PIKFYVE gene in the W population of Hippocampus abdominalis is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the PIKFYVE gene in the M population is shown in SEQ ID NO: 4.

[0034] Comparing the PIKFYVE gene and the amino acids encoded by the PIKFYVE gene between the W population and the M population, it was found that there were 2 mutation sites in the coding region of the PIKFYVE gene in the M population, both of which were missense mutations. The codon change type at the T 443 C 443 site was CTA - CCA, encoding leucine (L) and proline (P) located in the second exon respectively. The codon change type at the T 584 C 584 site was GTG - GCG, encoding valine (V) and alanine (A) located in the third exon respectively.

[0035] A total of 3 haplotypes were detected in the W population and M population of Hippocampus abdominalis, namely: Haplotype I (HI, T 443 T 584 ), Haplotype II (HII, T 443 C 584 ), and Haplotype III (HIII, C 443 C 584 ). The information of the 3 haplotypes is shown in Table 3.

[0036] Table 3 Information of 3 Haplotypes

[0037]

[0038] As can be seen from Table 3, the W population was mainly Haplotype I, with a small amount of Haplotype II and no Haplotype III. The M population was mainly Haplotype III, with a small amount of Haplotype II and no Haplotype I.

[0039] Two haplotypes (HI, HII) in the W population randomly formed 2 diploid types: homozygous diploid type I (DI, T 443 T443 T 584 T 584 ), and heterozygous diploid type II (DII, T 443 T 443 T 584 C 584 ), with frequencies of 63%, 37%, and 2%, respectively. The specific information of the two diploid types is shown in Table 4.

[0040] Two haplotypes (HII, HIII) in the M population randomly form three diploid types: homozygous diploid type III (DIII, T 443 T 443 C 584 C 584 ), heterozygous diploid type IV (DIV, T 443 C 443 C 584 C 584 ), and homozygous diploid type V (DV, C 443 C 443 C 584 C 584 ), with frequencies of 2%, 37%, and 61%, respectively. The specific information of the three diploid types is shown in Table 4.

[0041] Table 4 Information on 5 diploid types

[0042]

[0043] 2. Ocular diameter analysis of 5 diploid types of Hippocampus abdominalis

[0044] The statistical results of the average ocular diameters of 5 diploid types of Hippocampus abdominalis at 2 months and 3 months of age and the average weight gain in 30 days from 2 months to 3 months are shown in Table 5.

[0045] Table 5 Statistical results of the average ocular diameters and average weight gains of 5 diploid types at 2 months and 3 months of age

[0046]

[0047] Note: Different superscript lowercase letters of the numbers in the same column indicate significant differences (p < 0.05).

[0048] As can be seen from Table 5:

[0049] (1) At 2 months of age, the average ocular diameters of the three diploid types (DIII, DIV, DV) in the M population are slightly higher than those of the two diploid types (DI, DII) in the W population, but there are no significant differences in the average ocular diameters of the five diploid types ( p > 0.05);

[0050] (2)At 3 months old, the average eye diameter of the three haplotypes (DIII, DⅣ, DⅤ) in the M population was higher than that of the two haplotypes (DI, DII) in the W population. However, there was no significant difference in the average eye diameter among the four haplotypes DI, DII, DIII, and DⅣ ( p >0.05). Only the average eye diameter of the DⅤ haplotype was significantly higher than that of the other four haplotypes ( p <0.05);

[0051] (3)Within 30 days from 2 months old to 3 months old, there was no significant difference in the average weight gain among the four haplotypes DI, DII, DIII, and DⅣ ( p >0.05). Only the average weight gain of the DⅤ haplotype was significantly higher than that of the other four haplotypes ( p <0.05).

[0052] 3. Analysis of the relative expression levels of the PIKFYVE gene in five haplotypes of Hippocampus abdominalis

[0053] The relative expression levels of the PIKFYVE gene in three-month-old Hippocampus abdominalis of five haplotypes, namely homozygous haplotype I (DI), heterozygous haplotype II (DII), homozygous haplotype III (DIII), heterozygous haplotype IV (DIV), and homozygous haplotype V (DⅤ), were 1.24±0.13, 1.28±0.19, 1.28±0.16, 1.29±0.18, and 1.76±0.17, respectively. The comparison of the relative expression levels of the PIKFYVE gene is shown in Figure 2 .

[0054] As Figure 2 can be seen, there was no significant difference in the relative expression levels of the PIKFYVE gene among the four haplotypes DI, DII, DIII, and DⅣ (p>0.05). The relative expression level of the PIKFYVE gene of the DⅤ type in the M population was significantly higher than that of the other four haplotypes (p<0.05).

[0055] III. Conclusion

[0056] Single nucleotide polymorphisms (SNPs) are DNA genetic polymorphisms caused by single nucleotide changes in the genome. They are widely distributed in the genome, have stable heredity, and are very important molecular markers. In this invention, two linkage disequilibrium SNP loci were identified at positions 443 and 584 of the exon of the PIKFYVE gene with relatively high genetic diversity (PIC>0.5), and there are three haplotypes among them (haplotype I, haplotype II, and haplotype III). These three haplotypes randomly form five diploid types (homozygous diploid type I, heterozygous diploid type II, homozygous diploid type III, heterozygous diploid type IV, and homozygous diploid type V). These two SNP loci in the exon of the PIKFYVE gene lead to missense mutations in the codons, and the change in the amino acid sequence may lead to a change in the protein function.

[0057] Eye diameter is an important indicator for evaluating the feeding performance of fish, which determines the food intake and weight gain rate of seahorses. In order to verify whether the mutations of the two SNP loci in the exon of the PIKFYVE gene are associated with the eye diameter of the pot-bellied seahorse, this invention identified the common population and the F3 generation of the fast-growing strain. After a 30-day growth comparison experiment, the eye diameters and weight gains of different types of SNPs were statistically analyzed. The results showed that: compared with the common population, the eye diameter of the homozygous diploid type V (DⅤ, C 443 C 584 ), randomly formed by haplotype III (HIII C 443 C 443 C 584 C 584 ), had a significant advantage. Therefore, it was concluded that the simultaneous mutations at positions 443 and 584 (haplotype III SNP C 443 C 584 ), could significantly increase the eye diameter of the pot-bellied seahorse. Existing studies have shown that the PIKFYVE gene is the coding gene of a lipid phosphoinositide catalase and plays an important role in growth and development. In the F3 generation of the fast-growing strain, the leucine in the second exon of the haplotype III SNP (C 443 C 584 ), was changed to proline, and the valine in the third exon was changed to alanine, thus significantly increasing the expression level of the PIKFYVE gene and promoting the increase in eye diameter. The pot-bellied seahorse homozygous diploid type V (DⅤ, C 443 C 443 C 584 C 584 ), showed a significant advantage in eye diameter, and could be used for molecular-assisted breeding of new fast-growing strains of pot-bellied seahorses to improve the efficiency of preying on live bait and its own yield.

[0058] It should be noted that the above embodiments are merely examples given to clearly illustrate the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation manners here. Any obvious changes or modifications derived from the technical solution of the present invention still fall within the protection scope of the present invention.

Claims

1. Application of homozygous diploid V type of SNP locus related to eye diameter of Hippocampus abdominalis in breeding, characterized in that, Among them, The diploid type is randomly formed by haploid type I, haploid type II, and haploid type III. Among them, the bases at positions 443 and 584 of the exon of the PIKFYVE gene in haploid type I are T and T respectively, the bases at positions 443 and 584 of the exon of the PIKFYVE gene in haploid type II are T and C respectively, and the bases at positions 443 and 584 of the exon of the PIKFYVE gene in haploid type III are C and C respectively. The nucleotide sequence of the PIKFYVE gene is shown as SEQ ID NO: 1 or SEQ ID NO:

2. The three haplotypes randomly form homozygous diploid type I T 443 T 443 T 584 T 584 , heterozygous diploid type II T 443 T 443 T 584 C 584 , homozygous diploid type III T 443 T 443 C 584 C 584 , heterozygous diploid type IV T 443 C 443 C 584 C 584 and homozygous diploid type V C 443 C 443 C 584 C 584 There are 5 diploid types. Homozygous diploid type V has significant advantages over the other 4 diploid types in terms of average eye diameter and relative expression of the PIKFYVE gene. Homozygous diploid type V can be used for molecular-assisted breeding of fast-growing new strains of the belly-swollen seahorse.

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