Use of homozygous double v5 for breeding at snp locus associated with respiratory rate in pufferfish

Abstract

The application discloses application of homozygous double V type of respiratory frequency associated SNP site of Pseudorasbora parva in breeding, and belongs to the technical field of aquatic breeding. The double type is randomly formed by haploid I type, haploid II type and haploid III type. The bases at 433 and 727 of the hspa5 gene of the haploid I type are G and G respectively, the bases at 433 and 727 of the hspa5 gene of the haploid II type are G and A respectively, and the bases at 433 and 727 of the hspa5 gene of the haploid III type are A and A respectively. The homozygous double V type randomly formed by the haploid III type has a significant advantage in respiratory frequency and hspa5 gene expression compared to other double types. The homozygous double V type can be used for molecular assisted breeding of a new strain of Pseudorasbora parva resistant to high temperature, and the breeding process of the Pseudorasbora parva strain resistant to high temperature is accelerated.

Description

TECHNICAL FIELD

[0001] The application relates to application of homozygous double V type of respiratory frequency associated SNP site of Hippocampus guttulatus in breeding, and belongs to the technical field of aquatic breeding. BACKGROUND

[0002] The optimum breeding temperature of Hippocampus guttulatus is 18-20 DEG C, and high-temperature weather in summer seriously affects the survival rate of Hippocampus guttulatus. At present, there is still a lack of high-temperature-resistant strain of artificially bred Hippocampus guttulatus in China. Respiratory frequency is an important index for evaluating the high-temperature tolerance of Hippocampus guttulatus. The research team has bred the F2 generation of the high-temperature-resistant strain of Hippocampus guttulatus, and located the respiratory frequency associated SNP, so as to accelerate the breeding process of the high-temperature-resistant fine strain of Hippocampus guttulatus.

[0003] At present, there is no report on application of the respiratory frequency associated SNP site of Hippocampus guttulatus in breeding, and the application of the body weight / body length / body thickness / snout width / eye diameter associated SNP site of Hippocampus guttulatus in breeding is similar. However, the key genes of the body weight / body length / body thickness associated SNP can only be associated with the growth performance of Hippocampus guttulatus, and the key genes of the snout width / eye diameter associated SNP can only be associated with the feeding capacity of Hippocampus guttulatus, which cannot be associated with the respiratory frequency of Hippocampus guttulatus, and cannot be applied to screening of the high-temperature-resistant strain of Hippocampus guttulatus. SUMMARY

[0004] In order to solve the problems in the prior art, the application provides application of the double type of respiratory frequency associated SNP site of Hippocampus guttulatus in breeding, so as to promote the molecular assisted breeding of the new high-temperature-resistant strain of Hippocampus guttulatus and accelerate the breeding process of the high-temperature-resistant fine strain of Hippocampus guttulatus.

[0005] In order to achieve the above object, the application adopts the following technical scheme:

[0006] The application relates to application of homozygous double V type of respiratory frequency associated SNP site of Hippocampus guttulatus in breeding, and belongs to the technical field of aquatic breeding. 433 G 433 G 727 G 727 G 433 G 433 G 727 A 727 G 433G 433 A 727 A 727 , heterozygous double IV type G 433 A 433 A 727 A 727 and homozygous double V type A 433 A 433 A 727 A 727 The homozygous double V type has a significant advantage in respiratory rate and relative expression of the hspa5 gene compared to the other four double types, and the homozygous double V type can be used for molecular assisted breeding of the new heat-resistant strain of the Hippocampus kuda.

[0007] The present application has the advantages that: two SNP sites (located at 433 and 727 of the hspa5 gene, respectively) of the hspa5 gene (coding gene of heat shock protein 5) of the Hippocampus kuda are analyzed in association with the respiratory rate of the Hippocampus kuda, and it is found that the two SNP sites are significantly related to the respiratory rate, wherein the respiratory rate of the haplotype III (A 433 A 727 ) individual is significantly reduced. Through further comparative analysis of the respiratory rate and the expression of the hspa5 gene of the homozygous double I type (DI), the heterozygous double II type (DII), the homozygous double III type (DIII), the heterozygous double IV type (DIV), and the homozygous double V type (DV) five double types, it is found that there is a significant difference in the respiratory rate and the expression of the hspa5 gene of the Hippocampus kuda between the heat-resistant strain F2 generation and the ordinary population, wherein the average respiratory rate of the homozygous double V type (DV) of the Hippocampus kuda is significantly lower than that of the other four double types, the relative expression amount of the hspa5 gene is significantly higher than that of the other four double types, and the homozygous double V type (DV) can be used as a molecular marker related to the respiratory rate and applied to the molecular assisted breeding of the new heat-resistant strain of the Hippocampus kuda to accelerate the breeding process of the heat-resistant fine strain of the Hippocampus kuda. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 is a comparison chart of the respiratory rates of the 5-month-old Hippocampus kuda of the ordinary population and the heat-resistant strain F2 generation;

[0009] Figure 2 is a comparison chart of the relative expression amounts of the hspa5 gene of the five double types of the 5-month-old Hippocampus kuda. DETAILED DESCRIPTION

[0010] The present application will be specifically described below in combination with the drawings and examples. In the following examples, the experimental methods are conventional methods unless otherwise specified. The test materials used in the following examples can be purchased from conventional biochemical reagent companies unless otherwise specified.

[0011] I. Materials and methods

[0012] 1. Experimental fish

[0013] The experimental fish were the common population of the inflated red-belly seahorse (Wild Type, hereinafter referred to as W population) and the high-temperature-resistant strain F2 generation (Mutant Type, hereinafter referred to as M population), both 5 months old and well-grown. They were kept in the Seahorse Research Center of Ludong University.

[0014] The average respiratory frequency of the M population selected by the research team was significantly lower than that of the W population at 12:00-18:00 high-temperature period per day (P<0.05) (Fig. 1). Figure 1 ).

[0015] 200 tails of the W population and 200 tails of the M population of the inflated red-belly seahorse were each raised in two 40L glass tanks, 200 tails per tank, and connected to a central circulation system with mechanical and biological filtration, ultraviolet sterilization, and protein separator. The salinity was 31-32‰, the temperature was 18-19℃, the pH was 8.2-8.3, and plastic plants were used as attachment bases. Frozen brine shrimp was fed three times a day at 8:00, 12:00, and 16:00. After each feeding for 2 hours, the residual feed and feces in the glass tank were siphoned out.

[0016] 2. Screening of SNP sites of heat shock protein 5 encoding gene (hereinafter referred to as hspa5 gene)

[0017] Through analysis of the sequencing data of the W population and the M population of the inflated red-belly seahorse, two linkage disequilibrium SNP sites at 443 and 727 of the hspa5 gene with high genetic diversity (PIC>0.5) were identified (Table 1).

[0018] Table 1 SNP site information

[0019]

[0020] To further amplify the SNP sites at 433 and 727 of the hspa5 gene by PCR reaction, primer sequences covering all coding regions of the hspa5 gene were designed (Table 2).

[0021] Table 2 Primer information

[0022]

[0023] The 1 / 3 dorsal fin of 400 tails of the W population and the M population of the inflated red-belly seahorse was cut, and DNA was extracted by alkaline lysis. Partial gene fragments of the W population and the M population were amplified by PCR, and qualified PCR products were sent to Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing.

[0024] 3. High-temperature test

[0025] The 5-month-old seahorses with the same SNP type were put into the same glass tank for breeding, and the breeding temperature was increased to 22°C, and other breeding conditions were the same as before. The breathing rate was counted at 12:00 every day for 7 days.

[0026] 4. Real-time fluorescent quantitative PCR

[0027] After the high-temperature test, 5 seahorses of each SNP genotype were randomly selected, total RNA was extracted from the tail 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 in a CFX96 Touch™ real-time PCR detection system, and SYBR Green Premix Ex Taq was used for qRT-PCR detection. The internal reference gene 18S was used as a control. The primers used are shown in Table 2. All experiments were repeated more than 3 times.

[0028] 5. Statistical analysis

[0029] The data are expressed as mean ± standard error, and single factor analysis of variance was used with SPSS Statistics 17.0 software. Significance was p<0.05 or p<0.01.

[0030] II. Results

[0031] 1. Analysis of hspa5 gene SNP sites in seahorses

[0032] After detection, the nucleotide sequence of the hspa5 gene of the W population of seahorses is shown in SEQ ID NO: 1, and the nucleotide sequence of the hspa5 gene of the M population of seahorses is shown in SEQ ID NO: 2. Correspondingly, the amino acid sequence encoded by the hspa5 gene of the W population of seahorses is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the hspa5 gene of the M population of seahorses is shown in SEQ ID NO: 4.

[0033] By comparing the hspa5 gene and the amino acid encoded by the hspa5 gene of the W population and the M population, it was found that the M population hspa5 gene coding region had a total of 2 variation sites, both of which were missense mutations, G 433 A 433 The codon change type of the G site was GGG-AGG, which encoded glycine (G) and arginine (R) located in the 2nd exon, respectively. 727 A 727 The codon change type of the G site was GAA-AAA, which encoded glutamic acid (E) and lysine (K) located in the 3rd exon, respectively.

[0034] Three haplotypes were detected in W and M populations of A. inflatus, which were haplotype I (HI, G 433 G 727 ), haplotype II (HII, G 433 A 727 ) and haplotype III (HIII, A 433 A 727 ), respectively. The information of the three haplotypes was shown in Table 3.

[0035] Table 3 Information of three haplotypes

[0036]

[0037] From Table 3, it could be seen that W population was mainly haplotype I, with a small amount of haplotype II, and no haplotype III; M population was mainly haplotype III, with a small amount of haplotype II, and no haplotype I.

[0038] Two haplotypes (HI, HII) of W population randomly formed two diplotypes, which were homozygous diplotype I (DI, G 433 G 433 G 727 G 727 ) and heterozygous diplotype II (DII, G 433 G 433 G 727 A 727 ), with the frequencies of 71% and 29%, respectively. The information of the two diplotypes was shown in Table 4.

[0039] Two haplotypes (HII, HIII) of M population randomly formed three diplotypes, which were homozygous diplotype III (DIII, G 433 G 433 A 727 A 727 ), heterozygous diplotype IV (DIV, G 433 A 433 A 727 A 727 ) and homozygous diplotype V (DV, A 433 A 433 A 727 A 727 ), with the frequencies of 22%, 43% and 35%, respectively. The information of the three diplotypes was shown in Table 4.

[0040] Table 4 Information of five diplotypes

[0041]

[0042] 2. Analysis of respiratory frequency of five diplotypes of A. inflatus

[0043] The statistical results of the 7-day average respiratory rate of the five diploid types of the distended hippocampus after the high-temperature test are shown in Table 5.

[0044] Table 5. Statistical results of the 7-day average respiratory rate of five diploid strains of the distended hippocampus.

[0045]

[0046] Note: Different lowercase letters in the superscript of numbers in the same column indicate significant differences (p<0.05).

[0047] As shown in Table 5, under high temperature conditions (22℃), the respiratory rates of the three diploid types (DIII, DIV, DV) in population M were all lower than those of the two diploid types (DI, DII) in population W. Among them, the respiratory rates of homozygous diploid type III (DIII) and heterozygous diploid type IV (DIV) in population M were not significantly different from those of the two diploid types (DI, DII) in population W (p>0.05), while the respiratory rate of homozygous diploid type V (DV) in population M was significantly different from those of the two diploid types (DI, DII) in population W (p<0.05).

[0048] 3. Relative expression levels of the hspa5 gene in five diploid types of the bloated hippocampus

[0049] After the high-temperature test, the relative expression levels of the hspa5 gene in five diploid types of the distended hippocampus—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 measured to be 3.78±0.69, 3.80±0.61, 3.85±0.58, 4.12±0.73, and 5.82±0.96, respectively. The comparison of the relative expression levels of the hspa5 gene is shown below. Figure 2 As shown.

[0050] Depend on Figure 2 It can be seen that the relative expression level of hspa5 gene in type DV of population M is significantly higher than that in the other four diploids (p<0.05), while there is no significant difference in the relative expression level of hspa5 gene in types DI, DII, DIII and DIV (p>0.05).

[0051] III. Conclusion

[0052] SNPs are DNA genetic polymorphisms caused by single nucleotide changes in the genome, widely distributed in the genome, and have stable heredity, which are very important molecular markers. The present application identifies two linkage disequilibrium SNP sites at 433 and 727 of the hspa5 gene with high genetic diversity (PIC>0.5), and they exist in three haplotypes (haplotype I, haplotype II and haplotype III). The three haplotypes randomly form five double haplotypes (homozygous double haplotype I, heterozygous double haplotype II, homozygous double haplotype III, heterozygous double haplotype IV and homozygous double haplotype V). The two SNP sites of the hspa5 gene cause codon missense mutations, and the change of amino acid sequence may cause the change of protein function.

[0053] Respiratory frequency is an important indicator for evaluating the growth of fish at high temperature. In order to verify whether the mutation of the two SNP sites of the hspa5 gene is associated with the respiratory frequency of the Hippocampus kuda, the present application identifies the common population and the high-temperature-resistant strain F2 generation, and the respiratory frequency of different types of SNPs is counted after 7-day high-temperature test. The results show that: compared with the common population, the respiratory frequency of the homozygous double haplotype V (D V, A 433 A 727 ) formed randomly by the haplotype III (H III, A 433 A 433 A 727 A 727 ) of the high-temperature-resistant strain F2 generation has a significant advantage. Therefore, it is concluded that the simultaneous mutation at 433 and 727 (haplotype III SNP A 433 A 727 ) can significantly reduce the respiratory frequency of the Hippocampus kuda at high temperature. It has been shown that the hspa5 gene is a member of the heat shock protein 70 family, and is closely related to the high-temperature adaptation mechanism of fish. The glycine in the second exon of the haplotype III SNP (A 433 A 727 ) of the high-temperature-resistant strain F2 generation is changed to arginine, and the glutamic acid in the third exon is changed to lysine. The haplotype III SNP A 433 A 727 SNP mutation improves the expression of the hspa5 gene, and the hspa5 gene reduces oxidative damage by enhancing the activity of antioxidant enzymes, thereby maintaining the respiratory function of the homozygous double haplotype V (D V, A 433 A 433 A 727 A 727 ) of the Hippocampus kuda of the high-temperature-resistant strain F2 generation. Therefore, the homozygous double haplotype V (D V, G 227 G 227 A 300 A 300 A 433 A 433 A 727 A727 ) High temperature can show stable respiratory rate, can be used for abdominal hippocampus new strain molecular assisted breeding of high temperature resistance.

[0054] It should be noted that the above examples are merely examples for clearly illustrating the present application, and are not intended to limit the modes of implementation of the present application. On the basis of the above description, other different forms of changes or variations can also be made by those of ordinary skill in the art. Here, all the implementation modes cannot be exhausted. Any obvious changes or variations derived from the technical scheme of the present application are still within the scope of protection of the present application.

Claims

1. The application of homozygous double V-type of respiratory rate-associated SNP loci in bloated hippocampus in breeding, characterized by, in, The diploid type is randomly formed from haplotypes I, II, and III. In haplotype I, the bases at positions 433 and 727 of the hspa5 gene are G and G, respectively; in haplotype II, the bases at positions 433 and 727 are G and A, respectively; and in haplotype III, the bases at positions 433 and 727 are A and A, respectively. The nucleotide sequence of the hspa5 gene is shown in SEQ ID NO: 1 or SEQ ID NO:

2. The three haplotypes randomly form a homozygous diploid type I (G). 433 G 433 G 727 G 727 Hybrid double type II G 433 G 433 G 727 A 727 homozygous double type III G 433 G 433 A 727 A 727 Heterozygous double type IV G 433 A 433 A 727 A 727 and homozygous double V-type A 433 A 433 A 727 A 727 Five diploid types were identified. The homozygous diploid V type showed significant advantages over the other four diploid types in terms of respiratory rate and relative expression of the hspa5 gene. The homozygous diploid V type can be used for molecular-assisted breeding of new heat-resistant strains of bloated seahorses.

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