Application of homozygous double V-type of respiratory rate associated SNP (Single Nucleotide Polymorphism) site of belly-expanding hippocampus in breeding

Abstract

The invention discloses an application of a homozygous double V type of a respiratory rate associated SNP (Single Nucleotide Polymorphism) site of a seahorse belly distension in breeding, and belongs to the technical field of aquatic breeding. Wherein the haplotype is randomly formed by a haplotype I, a haplotype II and a haplotype III, basic groups at the positions 433 and 727 of the hspa5 gene of the haplotype I are G and G respectively, basic groups at the positions 433 and 727 of the hspa5 gene of the haplotype II are G and A respectively, and basic groups at the positions 433 and 727 of the hspa5 gene of the haplotype III are A and A respectively. Compared with other haplotypes, the homozygous double V type randomly formed by the haplotype III has remarkable advantages in the aspects of respiratory rate and hspa5 gene expression, and the homozygous double V type can be used for auxiliary breeding of high-temperature-resistant new strain molecules of the belly-expanding hippocampus, so that the breeding process of high-temperature-resistant improved varieties of the hippocampus 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: 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 433 G 433A 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 Pelvicachromis kribensis.

[0006] The present application has the advantages that: the 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 Pelvicachromis kribensis are analyzed in association with the respiratory rate of the Pelvicachromis kribensis, and it is found that the two SNP sites are significantly related to the respiratory rate, wherein the respiratory rate of the individual of the haplotype III (A 433 A 727 ) is significantly reduced. Through further comparative analysis of the respiratory rate and the expression of the hspa5 gene of the individuals 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 Pelvicachromis kribensis between the heat-resistant strain F2 generation and the ordinary population, wherein the average respiratory rate of the homozygous double V type (DV) Pelvicachromis kribensis 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 Pelvicachromis kribensis, so as to accelerate the breeding process of the heat-resistant fine strain of the Pelvicachromis kribensis. BRIEF DESCRIPTION OF DRAWINGS

[0007] Figure 1 is a comparison chart of the respiratory rates of the ordinary population and the heat-resistant strain F2 generation of the Pelvicachromis kribensis at the age of 5 months; Figure 2 is a comparison chart of the relative expression amounts of the hspa5 gene of the five double types of the Pelvicachromis kribensis at the age of 5 months. DETAILED DESCRIPTION

[0008] The present application will be specifically introduced below in combination with the drawings and examples. In the following examples, the experimental methods are all conventional methods unless otherwise specified. The test materials used in the following examples can be purchased from conventional biochemical reagent companies unless otherwise specified.

[0009] I. Materials and methods 1. Experimental fish The experimental fish were the common group of S. notata (wild type, hereinafter referred to as W group) and the high-temperature-resistant strain F2 generation (mutant type, hereinafter referred to as M group), both 5 months old and in good growth and development. They were stored in the S. notata Research Center of Ludong University.

[0010] The average respiratory rate of the M group selected and bred by the research team was significantly lower than that of the W group during the high-temperature period from 12:00 to 18:00 every day. Figure 1 ).

[0011] Two hundred S. notata of the W group and the M group were respectively raised in two 40 L glass tanks, 200 in each 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.

[0012] 2. Screening of SNP sites of heat shock protein 5 encoding gene (hereinafter referred to as hspa5 gene) Through the analysis of the sequencing data of the W group and the M group of S. notata, two linkage disequilibrium SNP sites at 443 and 727 of the hspa5 gene with high genetic diversity (PIC>0.5) were identified (Table 1).

[0013] Table 1 SNP site information

[0014] 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).

[0015] Table 2 Primer information

[0016] The 1 / 3 dorsal fins of 400 S. notata of the W group and the M group were cut, and DNA was extracted by alkaline lysis. Partial gene fragments of the W group and the M group were amplified by PCR, and qualified PCR products were sent to Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing.

[0017] 3. High-temperature test 5-month-old S. notata of the same SNP type were placed in the same glass tank for breeding, and the breeding temperature was increased to 22℃, and other breeding conditions were the same as before. After 7 days of breeding, the respiratory rate was counted at 12:00 every day.

[0018] 4. Real-time fluorescent quantitative PCR After the high temperature test, 5 tails of each SNP haplotype of the A. bicolor were randomly selected, total RNA was extracted from the tails 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 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 3 times.

[0019] 5、Statistical analysis The data are expressed as mean ± standard error, and single factor variance analysis was performed using SPSS Statistics 17.0 software. Significance was p<0.05 or p<0.01.

[0020] II、Results 1、Analysis of SNP sites of hspa5 gene of A. bicolor After detection, the nucleotide sequence of the hspa5 gene of the W population of A. bicolor was as shown in SEQ ID NO: 1, and the nucleotide sequence of the hspa5 gene of the M population of A. bicolor was as shown in SEQ ID NO: 2. Correspondingly, the amino acid sequence encoded by the hspa5 gene of the W population of A. bicolor was as shown in SEQ ID NO: 3, and the amino acid sequence encoded by the hspa5 gene of the M population of A. bicolor was as shown in SEQ ID NO: 4.

[0021] 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.

[0022] A total of 3 haplotypes were detected in the W population and the M population of A. bicolor, which were haplotype I (HI, G 433 G 727 ), haplotype II (HII, G 433 A 727 ), and haplotype III (HIII, A 433 A 727 ). The information of the 3 haplotypes is shown in Table 3.

[0023] Table 3 3 haplotype information

[0024] From Table 3, W population is mainly haplotype I, with a small amount of haplotype II, and no haplotype III. M population is mainly haplotype III, with a small amount of haplotype II, and no haplotype I.

[0025] W population randomly forms 2 dihaplotype types (HI, HII) into 2 dihaplotype types: homozygous dihaplotype I (DI, G 433 G 433 G 727 G 727 ) and heterozygous dihaplotype II (DII, G 433 G 433 G 727 A 727 ), with frequencies of 71% and 29% respectively. The information of 2 dihaplotype types is shown in Table 4.

[0026] M population randomly forms 2 haplotype types (HII, HIII) into 3 dihaplotype types: homozygous dihaplotype III (DIII, G 433 G 433 A 727 A 727 ), heterozygous dihaplotype IV (DIV, G 433 A 433 A 727 A 727 ) and homozygous dihaplotype V (DV, A 433 A 433 A 727 A 727 ), with frequencies of 22%, 43% and 35% respectively. The information of 3 dihaplotype types is shown in Table 4.

[0027] Table 4 5 dihaplotype information

[0028] 2. Analysis of respiratory frequency of 5 dihaplotype types of hippocampus The statistical results of 7-day average respiratory frequency of 5 dihaplotype types of hippocampus after high temperature test are shown in Table 5.

[0029] Table 5 Statistical results of 7-day average respiratory frequency of 5 dihaplotype types of hippocampus

[0030] Note: The same column indicates significant difference (p<0.05) in the same column.

[0031] From table 5 can be known: in high temperature environment (22 DEG C), the respiratory frequency of 3 double types (DIII, DIV, DV) of M population is lower than that of 2 double types (DI, DII) of W population, wherein, the respiratory frequency of homozygous double III type (DIII) and heterozygous double IV type (DIV) of M population has no significant difference (p>0.05) with that of 2 double types (DI, DII) of W population, and the respiratory frequency of homozygous double V type (DⅤ) of M population has significant difference (p<0.05) with that of 2 double types (DI, DII) of W population.

[0032] 3、hspa5 gene relative expression amount analysis of 5 double types of P. veleris After high temperature test, it is detected that the hspa5 gene relative expression amounts of homozygous double I type (DI), heterozygous double II type (DII), homozygous double III type (DIII), heterozygous double IV type (DIV) and homozygous double V type (DⅤ) of P. veleris are 3.78±0.69, 3.80±0.61, 3.85±0.58, 4.12±0.73 and 5.82±0.96 respectively. Figure 2

[0033] From Figure 2 It can be known that the hspa5 gene relative expression amount of DⅤ type of M population is significantly higher than that of other four double types (p<0.05), and the hspa5 gene relative expression amounts of DI type, DII type, DIII type and DⅣ type have no significant difference (p>0.05).

[0034] Three, conclusion SNPs are DNA genetic polymorphisms caused by single nucleotide changes in genome, are widely distributed in genome, have stable heredity and are very important molecular markers. Two linkage disequilibrium SNP sites are identified at 433 and 727 of hspa5 gene with high genetic diversity (PIC>0.5) in the application, and three haplotypes (haplotype I, haplotype II and haplotype III) exist. The three haplotypes randomly form five double types (homozygous double I type, heterozygous double II type, homozygous double III type, heterozygous double IV type and homozygous double V type). The two SNP sites of hspa5 gene cause codon missense mutation, and the change of amino acid sequence can cause the change of protein function.

[0035] ​Respiration rate is an important indicator for evaluating fish growth under high temperature, in order to verify whether the mutation of two SNP sites of the hspa5 gene is associated with the respiration rate of the Hippocampus kuda, the common population and the high-temperature-resistant line F2 generation are identified, and the respiration rates of different types of SNPs are counted after 7-day high-temperature test. 433 A 727 433 A 433 A 727 A 727 The respiration rate of the homozygous double type V (DV, A 433 A 727 ) randomly formed in the high-temperature-resistant line F2 generation has a significant advantage compared with the common population. Therefore, it is concluded that the simultaneous mutation at positions 433 and 727 (haplotype III SNP A 433 A 727 ) can significantly reduce the respiration rate of the Hippocampus kuda under high temperature. Previous studies have 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 line 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 433 A 727 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 type V (DV, A 227 G 227 A 300 A 300 A 433 A 433 A 727 A 727 ) of the Hippocampus kuda of the high-temperature-resistant line F2 generation. Therefore, the homozygous double type V (DV, G 227 G 227 A 300 A 300 A 433 A 433 A 727 A 727 ) of the Hippocampus kuda under high temperature shows stable respiration rate, and can be used for molecular-assisted breeding of new high-temperature-resistant lines of the Hippocampus kuda.

[0036] It should be noted that the above examples are merely examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or modifications can be made by those skilled in the art. Here, all the embodiments cannot be exhausted. Any obvious changes or modifications derived from the technical scheme of the present application are still within the scope of protection of the present application.

Claims

1. Use of homozygous double V5 for breeding at the respiratory frequency-associated SNP locus of the B. ventricosa, characterized in that, wherein The double type is randomly formed by haplotype I, haplotype II and haplotype III, wherein the bases at 433 and 727 of the hspa5 gene of the haplotype I are G and G respectively, the bases at 433 and 727 of the hspa5 gene of the haplotype II are G and A respectively, and the bases at 433 and 727 of the hspa5 gene of the haplotype III are A and A respectively, the nucleotide sequence of the hspa5 gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the three haplotypes are randomly formed into homozygous double type I G 433 G 433 G 727 G 727 , heterozygous 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 type V A 433 A 433 A 727 A 727 The homozygous double type V 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 type V can be used for molecular assisted breeding of a new strain of the Iniopteryx inflata.

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