Application of diplotype of eye diameter associated SNP (Single Nucleotide Polymorphism) site of belly swelling hippocampus in breeding

Abstract

The invention discloses application of a diplotype of eye diameter associated SNP (Single Nucleotide Polymorphism) sites of belly swelling hippocampus in breeding, and belongs to the technical field of aquatic breeding. The haplotype is randomly formed by a haplotype I, a haplotype II and a haplotype III, basic groups at exons 443 and 584 of a PIKFYVE gene of the haplotype I are T and T respectively, basic groups at exons 443 and 584 of a PIKFYVE gene of the haplotype II are T and C respectively, basic groups at exons 443 and 584 of a PIKFYVE gene of the haplotype III are C and C respectively, and basic groups at exons 443 and 584 of a PIKFYVE gene of the haplotype III are T and C respectively. Compared with other diplotypes, the homozygous diplotype V randomly formed by the haplotype III has remarkable advantages in the aspects of average eye diameter and PIKFYVE gene expression, and the homozygous diplotype V can be used for auxiliary breeding of fast-growing new strain molecules of the belly-expanding hippocampus, so that the breeding process of fast-growing improved varieties of the hippocampus is accelerated.

Description

Technical Field

[0001] The present invention relates to the application of the haplotype of the SNP locus associated with the eye diameter 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 hippocampus with a smaller eye diameter, its 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 the hippocampus.

[0003] Currently, there is a lack of excellent cultured hippocampus strains artificially cultivated in China. The research team has cultivated the fast-growing strain F 3 generation of the hippocampus, and screened out a group with a significantly increased eye diameter, and located the SNP associated with the eye diameter, in order to improve the survival rate of hippocampus seedlings and the aquaculture yield.

[0004] Currently, there is no report on the application of the SNP locus associated with the eye diameter of the distended hippocampus (Distended hippocampus) in breeding. The most similar is the application of the SNP locus associated with the snout width of the distended hippocampus in breeding. However, the key gene (CSF2RB gene) of the SNP associated with the snout width 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 the hippocampus with a significantly increased snout width is screened out through the SNP locus associated with the snout width, it only overcomes the limitation of the mouth diameter of the initial bait. Since there are hippocampus with a smaller eye diameter among the hippocampus with a significantly increased snout width, the visual ability of these hippocampus 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 the hippocampus. Summary of the Invention

[0005] To solve the deficiencies of the prior art, the present invention provides the application of the haplotype of the SNP locus associated with the eye diameter 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: Application of homozygous diploid V type of SNP loci 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 of haplotype I are T and T respectively, the bases at positions 443 and 584 of the exon of the PIKFYVE gene of haplotype II are T and C respectively, and the bases at positions 443 and 584 of the exon of the PIKFYVE gene of 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 in average eye diameter and relative expression of the PIKFYVE gene compared with the other 4 diplotypes. The homozygous diploid V type can be used for molecular assisted breeding of fast-growing new strains of Hippocampus abdominalis.

[0007] The beneficial effect of the present invention is that: the association analysis between 2 SNP loci (located at positions 443 and 584 of the exon of the PIKFYVE gene) of the lipid phosphoinositide catalytic enzyme encoding gene (PIKFYVE gene) of Hippocampus abdominalis and the eye diameter of Hippocampus abdominalis is carried out, and it is found that these 2 SNP loci are significantly correlated with the eye diameter. Among them, the eye diameter of individuals of haplotype III (C 443 C 584 ) is significantly increased. Through further comparative analysis of the eye diameter and PIKFYVE gene expression of individuals of 5 diplotypes, namely homozygous diploid I type (DI), heterozygous diploid II type (DII), homozygous diploid III type (DIII), heterozygous diploid IV type (DIV) and homozygous diploid V type (DV), it is found that the eye diameter and the expression of the PIKFYVE gene of Hippocampus abdominalis in fast-growing strains F 3There are significant differences between generations and the general population. Among them, the average eye diameter and the relative expression level of the PIKFYVE gene in homozygous diploid V-type (DV) pregnant seahorses are significantly higher than those of the other four diploid types. Homozygous diploid V-type (DV) can be used as a molecular marker related to eye diameter and applied to molecular-assisted breeding of fast-growing new strains of pregnant seahorses to improve the survival rate of fast-growing high-quality seahorses at the seedling stage. Description of the Drawings

[0008] Figure 1 is a comparison chart of the eye diameters of the general population and the fast-growing strain F of pregnant seahorses 3 from 1 to 7 months old; Figure 2 is a comparison chart of the relative expression levels of the PIKFYVE gene in 3-month-old pregnant seahorses of five diploid types. Detailed Implementation Manner

[0009] 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.

[0010] I. Materials and Methods 1. Experimental fish The experimental fish are the general population of pregnant seahorses (wild type, hereinafter referred to as the W population) and the fast-growing strain F 3 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.

[0011] 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 ).

[0012] Two hundred tails each of the W population and the M population of pregnant seahorses are respectively cultured in 4 20L glass tanks, with 100 tails in each tank, and connected to a central circulation system, which has mechanical and biological filtration, ultraviolet sterilization, and a protein separator. 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 artemia 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.

[0013] 2. Screening of SNP sites of the gene encoding phosphatidylinositol lipid kinase (hereinafter referred to as the PIKFYVE gene) By analyzing the sequencing data of the W population and the M population of pregnant seahorses, two linkage disequilibrium SNP sites at exon 443 and 584 of the PIKFYVE gene with high genetic diversity (PIC > 0.5) were identified (Table 1).

[0014] Table 1 SNP Locus Information

[0015] To further amplify the SNP loci at positions 443 and 584 of the PIKFYVE gene by PCR reaction, primer sequences covering all coding regions of the PIKFYVE gene were designed (Table 2).

[0016] Table 2 Primer Information

[0017] For a total of 400 bellied seahorses in the W population and the M population, 1 / 3 of the dorsal fin was cut, and DNA was extracted by the alkaline lysis method. Partial gene fragments of the W population and the M population were amplified by PCR, and the qualified PCR products were sent to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing.

[0018] 3. Growth Comparison Seahorses of 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 the 30 days from 2 months old to 3 months old were calculated.

[0019] 4. Real-time Fluorescent Quantitative PCR For each SNP haplotype of bellied seahorses, 5 tails were randomly selected. Total RNA from the tail was extracted 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, and qRT-PCR detection was carried out 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.

[0020] 5. Statistical Analysis The data were expressed in the form of mean ± sampling error and analyzed by one-way ANOVA using SPSS Statistics 17.0 software. The significance was at p < 0.05 or p < 0.01.

[0021] II. Results 1. Analysis of SNP Loci of the PIKFYVE Gene in Bellied Seahorses 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.

[0022] Comparing the PIKFYVE gene and the amino acid 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, T 443 C 443 The codon change type at the site was CTA-CCA, encoding leucine (L) and proline (P) located in the second exon, respectively, T 584 C 584 The codon change type at the site was GTG-GCG, encoding valine (V) and alanine (A) located in the third exon, respectively.

[0023] 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 specifically.

[0024] Table 3 Information of 3 haplotypes

[0025] As can be seen from Table 3, the W population is mainly Haplotype I, with a small amount of Haplotype II and no Haplotype III. The M population is mainly Haplotype III, with a small amount of Haplotype II and no Haplotype I.

[0026] Two haplotypes (HI, HII) in the W population randomly formed 2 diploid types: homozygous diploid I (DI, T 443 T 443 T 584 T 584 ), and heterozygous diploid II (DII, T 443 T 443 T 584 C 584 ). The frequencies of occurrence were 63% and 37% respectively. The information of the 2 diploid types is shown in Table 4 specifically.

[0027] Three diploid types are randomly formed from two haplotypes (HII, HIII) in population M: 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 occurrence frequencies of 2%, 37%, and 61% respectively. The specific information of the three diploid types is shown in Table 4.

[0028] Table 4 Information of 5 diploid types

[0029] 2. Ocular diameter analysis of Hippocampus abdominalis with 5 diploid types The statistical results of the average ocular diameters of Hippocampus abdominalis with 5 diploid types 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.

[0030] 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

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

[0032] As can be seen from Table 5: (1) At 2 months of age, the average ocular diameters of the three diploid types (DIII, DIV, DV) in population M are slightly higher than those of the two diploid types (DI, DII) in population W, but there are no significant differences in the average ocular diameters of the five diploid types ( p > 0.05); (2) At 3 months of age, the average ocular diameters of the three diploid types (DIII, DIV, DV) in population M are higher than those of the two diploid types (DI, DII) in population W, but there are no significant differences in the average ocular diameters of the four diploid types DI, DII, DIII, and DIV ( p > 0.05). Only the average ocular diameter of the DV diploid type is significantly higher than those of the other four diploid types ( p < 0.05); (3) Within 30 days from 2 months to 3 months of age, there are no significant differences in the average weight gains of the four diploid types DI, DII, DIII, and DIV ( p > 0.05). Only the average weight gain of the DV diploid type is significantly higher than those of the other four diploid types.p <0.05).

[0033] 3. Relative Expression Analysis of PIKFYVE Gene in Five Types of Doubled Pygmy Seahorses The relative expression levels of the PIKFYVE gene in three - month - old pure - bred doubled type I (DI), heterozygous doubled type II (DII), pure - bred doubled type III (DIII), heterozygous doubled type IV (DIV), and pure - bred doubled type V (DV) of five types of doubled pygmy seahorses 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 .

[0034] It can be seen from Figure 2 that there was no significant difference in the relative expression levels of the PIKFYVE gene among the four doubled types of DI, DII, DIII, and DIV (p > 0.05), and the relative expression level of the PIKFYVE gene in the DV type of the M population was significantly higher than that of the other four doubled types of the PIKFYVE gene (p < 0.05).

[0035] III. Conclusion 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). There are three haplotypes (haplotype I, haplotype II, and haplotype III) among them. These three haplotypes randomly form five doubled types (pure - bred doubled type I, heterozygous doubled type II, pure - bred doubled type III, heterozygous doubled type IV, and pure - bred doubled type V). These two SNP loci in the exon of the PIKFYVE gene lead to missense mutations of codons, and the change of the amino - acid sequence may lead to the change of protein function.

[0036] 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 pygmy seahorses, this invention identified the common population and the fast - growing strain F 3 generation. 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 homozygous doubled type V (DV, C 3 C 443 C 584 ) randomly formed by the haplotype III (HIII C 443 C 443 C 584 C584 ) has a significant advantage in eye diameter. Therefore, it is concluded that the simultaneous mutation at positions 443 and 584 (haplotype III SNP C 443 C 584 ) can significantly increase the eye diameter of Hippocampus abdominalis. Existing studies have shown that the PIKFYVE gene is the coding gene of a lipid phosphoinositide catalytic enzyme and plays an important role in growth and development. The haplotype III SNP (C 3 C 443 C 584 ) in the F generation of the fast-growing strain changes leucine in the second exon to proline and valine in the third exon to alanine, thus significantly increasing the expression level of the PIKFYVE gene and promoting the increase in eye diameter. The homozygous diploid type V (DV, C 443 C 443 C 584 C 584 ) of Hippocampus abdominalis shows a significant advantage in increased eye diameter and can be used for molecular-assisted breeding of new fast-growing strains of Hippocampus abdominalis to improve the efficiency of preying on live bait and its own yield.

[0037] 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 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 enumerate all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. Application of the homozygous double V type of SNP loci associated with eye diameter of hippocampus inflatus in breeding, characterized in that: in, The diplotype is randomly formed by haplotype I, haplotype II, and haplotype III, wherein 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, and the nucleotide sequence of the PIKFYVE gene is shown in SEQ ID NO: 1 or SEQ ID NO:

2. The three haplotypes randomly form a homozygous diplotype I T 443 T 443 T 584 T 584 , heterozygous double type II T 443 T 443 T 584 C 584 , homozygous double type III T 443 T 443 C 584 C 584 , heterozygous double IV type T 443 C 443 C 584 C 584 and homozygous double V-type C 443 C 443 C 584 C 584 Among the 5 diplotypes, the homozygous diplotype V has significant advantages over the other 4 diplotypes in terms of average eye diameter and relative expression of the PIKFYVE gene. The homozygous diplotype V can be used for molecular assisted breeding of new fast-growing strains of Hippocampus inflatus.

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