Application of diplotype of SNP loci associated with body length in hippocampus distensae in breeding

Abstract

The invention discloses the application of the diplotype of the SNP site associated with the body length of the distended hippocampus in breeding, and belongs to the technical field of aquatic breeding. The diplotype is randomly formed by haplotype I, haplotype II, and haplotype III, wherein the bases at exons 534 and 601 of the acaca gene of haplotype I are T and C respectively, the bases at exons 534 and 601 of the acaca gene of haplotype II are T and A respectively, the bases at exons 534 and 601 of the acaca gene of haplotype III are A and A respectively, and the homozygous double V type randomly formed by haplotype III has significant advantages in average body length and acaca gene expression compared with other diplotypes, and the homozygous double V type can be used for molecular assisted breeding of new fast-growing strains of distended hippocampus, and accelerate the breeding process of fast-growing improved hippocampus.

Description

Technical Field

[0001] The invention relates to application of a diplotype of a hippocampus distended body length-related SNP site in breeding, and belongs to the technical field of aquatic breeding. Background Art

[0002] Body length is an important indicator for evaluating the growth performance of seahorses. At present, due to the disordered self-pollination of farmed seahorses and the lack of control over germplasm resources, the body length of farmed seahorses has been significantly reduced, greatly affecting the production of seahorses.

[0003] At present, there is still a lack of high-quality artificially cultivated seahorse strains. The research team has cultivated a fast-growing seahorse strain F 3 In the next generation, we will screen out the group with significantly increased body length and locate the SNP associated with body length, in order to increase the production of seahorse farming.

[0004] At present, there is no application of the body length-associated SNP loci of Distended hippocampus in breeding. The most similar is the microsatellite marker associated with the growth of the linear hippocampus. However, microsatellite markers can only roughly identify the linear hippocampus by the size of the electrophoresis fragment, and cannot accurately locate the body length-related SNPs, nor can they determine whether the linear hippocampus with growth advantages is homozygous and heritable. Summary of the invention

[0005] To address the deficiencies of the prior art, the present invention provides an application of the diplotype of SNP sites associated with body length of Hippocampus inflatus in breeding, which overcomes the limitations of multiple factors, strictly regulates the growth process of hippocampus, and can promote molecular-assisted breeding of new fast-growing strains of Hippocampus inflatus.

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

[0007] Application of diplotype of SNP site associated with hippocampal distension length in breeding, wherein the diplotype is randomly formed by haplotype I, haplotype II and haplotype III, wherein the bases at exon 534 and 601 of acaca gene of haplotype I are T and C respectively, the bases at exon 534 and 601 of acaca gene of haplotype II are T and A respectively, the bases at exon 534 and 601 of acaca gene of haplotype III are A and A respectively, the nucleotide sequence of acaca gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the three haplotypes randomly form a homozygous diplotype I T 534 T 534 C 601 C 601 , heterozygous double type II T 534 T 534 C 601 A 601 , homozygous double type III T534 T 534 A 601 A 601 , heterozygous double IV type T 534 A 534 A 601 A 601 and homozygous double V-type A 534 A 534 A 601 A 601 Among the 5 diplotypes, the homozygous diplotype V has significant advantages over the other 4 diplotypes in terms of average body length and relative expression of acaca gene. The homozygous diplotype V can be used for molecular assisted breeding of new fast-growing strains of Hippocampus inflatus.

[0008] The invention is beneficial in that: the association analysis of two SNP sites (located at exon 534 and 601 of the acaca gene) of the acetyl-CoA carboxylase gene (acaca gene) and the body length of the hippocampus distended was performed, and it was found that the two SNP sites were significantly correlated with the body length, among which haplotype III (A 534 A 601 ) individuals significantly increased in length. Further comparative analysis of the body length of five diplotypes, 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), revealed that the body length of hippocampus inflatus and the expression of acaca gene were significantly increased in the fast-growing strain F. 3 There were significant differences between the first and second generations and the normal population. Among them, the body length and relative expression of acaca gene of the homozygous double V type (DV) seahorse were significantly higher than those of the other four diplotypes. Double V type (DV) can be used as a molecular marker related to body length and applied to molecular assisted breeding of new fast-growing strains of seahorse, so as to improve the survival rate of fast-growing seahorse varieties in the seedling stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 It is a common population of hippocampus inflatus and a fast-growing strain F 3 Comparison chart of body length between 1-7 months of age;

[0010] Figure 2 It is a common population of hippocampus inflatus and a fast-growing strain F 3 Comparison chart of 6-month-old individuals, where A is the fast-growing strain F 3 generation, B is the common group;

[0011] Figure 3 This is a comparison chart of the relative expression levels of the acaca gene in five diplotypes of Hippocampus inflatus at 7 months of age. DETAILED DESCRIPTION

[0012] The present invention is described in detail below in conjunction with the accompanying drawings and examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples can be purchased from conventional biochemical reagent companies unless otherwise specified.

[0013] 1. Materials and Methods

[0014] 1. Experimental fish

[0015] The fish used in the experiment were the common population of Hippocampus inflatus (wild type, hereinafter referred to as W population) and the fast-growing strain F 3 The first generation (mutant type, hereinafter referred to as M group) were all 6 months old and grew well. They were kept in the Hippocampus Research Center of Ludong University.

[0016] The body length of the M group at 3-7 months of age was significantly higher than that of the W group ( Figure 1 ), and the average body length at 7 months of age increased by 13.85% ( Figure 2 ).

[0017] 200 Hippocampus W and M were taken and cultured in 4 20L glass tanks, 100 in each tank, and connected to a central circulation system with mechanical and biological filtration, UV sterilization and protein skimmer. Salinity 31-32‰, temperature 18-19℃, pH 8.2-8.3, plastic plants as substrate. Frozen mysids were fed three times a day at 8:00, 12:00 and 16:00. After 2 hours of each feeding, the residual feed and feces in the glass tank were siphoned out.

[0018] 2. Screening of SNP sites of acetyl-CoA carboxylase gene (hereinafter referred to as acaca gene)

[0019] By analyzing the resequencing data of Hippocampus W and M populations, two linkage disequilibrium SNP sites at exons 534 and 601 of the acaca gene with high genetic diversity (PIC>0.5) were identified (Table 1).

[0020] Table 1 SNP site information

[0021]

[0022] To further amplify the 534 and 601 SNP sites of the acaca gene by PCR reaction, primer sequences covering all coding regions of the acaca gene were designed ( Table 2 ).

[0023] Table 2 Primer information

[0024]

[0025] One third of the dorsal fin was cut off from 400 hippocampi from the W and M populations, and DNA was extracted by alkaline lysis. Partial gene fragments of the W and M populations were amplified by PCR, and the qualified PCR products were sent to Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing.

[0026] 3. Growth comparison

[0027] The same SNP type was placed in the same glass tank and reared under the same conditions as before. After 30 days of rearing (7 months old), the body length and average growth were measured.

[0028] 4. Real-time fluorescence quantitative PCR

[0029] Five tails of each SNP ploidy type were randomly selected from Hippocampus inflatus. Total RNA from the tails was extracted using RNAiso Plus. Genomic DNA was removed from RNA using the PrimeScript RT kit and cDNA was synthesized. Real-time quantitative PCR was performed in the 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.

[0030] 5. Statistical analysis

[0031] Data are presented 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.

[0032] 2. Results

[0033] 1. Analysis of SNP sites of the acaca gene in hippocampus

[0034] After testing, the nucleotide sequence of the acaca gene of the W population of Hippocampus inflatus is shown in SEQ ID NO: 1, and the nucleotide sequence of the acaca gene of the M population is shown in SEQ ID NO: 2. Correspondingly, the amino acid sequence encoded by the acaca gene of the W population of Hippocampus inflatus is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the acaca gene of the M population is shown in SEQ ID NO: 4.

[0035] Comparison of the acaca gene and the amino acids encoded by the acaca gene in the W and M populations revealed that there were two mutation sites in the acaca gene coding region of the M population, both of which were missense mutations. 534 A 534 The codon change type of the site is TTC-ATC, encoding phenylalanine and isoleucine in the second exon, respectively. 601 A601 The codon change type of the site is CTG-ATG, encoding alanine and aspartic acid located in the second exon, respectively.

[0036] Three haplotypes were detected in the W and M populations of Hippocampus inflatus: haplotype I (HI, T 534 C 601 ), haplotype II (HII, T 534 A 601 ) and haplotype III (HIII, A 534 A 601 ), the information of the three haplotypes is shown in Table 3.

[0037] Table 3 Information of three haplotypes

[0038]

[0039] 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.

[0040] The two haplotypes (HI, HII) of the W population randomly formed two diplotypes: homozygous diplotype I (DI, T 534 T 534 C 601 C 601 ) and heterozygous diploid type II (DII, T 534 T 534 C 601 A 601 ), the frequencies of occurrence were 85% and 15% respectively. The information of the two diplotypes is shown in Table 4.

[0041] The two haplotypes (HII and HIII) of the M population randomly formed three diplotypes: homozygous diplotype III (DIII, T 534 T 534 A 601 A 601 ), heterozygous double IV type (DIV, T 534 A 534 A 601 A 601 ) and homozygous double V type (DV, A 534 A 534 A 601 A 601 ), the frequencies of occurrence were 4%, 5%, and 91% respectively. The information of the three diplotypes is shown in Table 4.

[0042] Table 4 Information of five diplotypes

[0043]

[0044] 2. Analysis of body length of five diplotypes of Hippocampus inflatus

[0045] The statistical results of the average body length and average growth of the five diplotypes of Hippocampus inflatus at 6 and 7 months of age are shown in Table 5.

[0046] Table 5 Statistical results of average body length and average growth of five diplotypes at 6 and 7 months of age

[0047]

[0048] Note: Numbers with different lowercase letters in the same column indicate significant differences (p<0.05).

[0049] From Table 5, we can see that:

[0050] (1) At 6 months of age, the average body lengths of the three diplotypes (DIII, DIV, and DV) in the M population were significantly different ( p <0.05) were higher than the two diplotypes (DI and DII) of the W population, among which the average body length of the DV type was significantly ( p <0.05) higher than the other four diplotypes;

[0051] (2) At 7 months of age, the average body lengths of the three diplotypes (DIII, DIV, and DV) of the M population were significantly different ( p <0.05) was higher than the DI type in the W population. The average body length of the DIII and DIV types in the M population was not significantly different from that of the DII type in the W population, but the average body length of the DV type in the M population was significantly ( p <0.05) higher than the other four diplotypes;

[0052] (3) Within 30 days from 6 to 7 months of age, the average growth of the three diplotypes (DIII, DIV, and DV) in the M population was significant ( p <0.05) were higher than the two diplotypes (DI and DII) in the W population, and the average increase of the DV type in the M population was significantly ( p <0.05) were higher than those of type DIII and type DIV in the M population.

[0053] 3. Analysis of relative expression of acaca gene in five diplotypes of Hippocampus inflatus

[0054] The relative expression levels of acaca gene in Hippocampus distensus at 7 months of age were 0.92±0.09, 0.92±0.11, 0.97±0.15, 0.98±0.11, and 1.36±0.14, respectively. Figure 3 shown.

[0055] Depend on Figure 3 It can be seen that the relative expression level of the DⅤ type acaca gene in the M population was significantly higher (p<0.05) than that of the other four diplotypes, and there was no significant difference in the relative expression levels of the DI type, DII type, DIII type and DⅣ type acaca gene (p>0.05).

[0056] Conclusion

[0057] SNPs are DNA genetic polymorphisms caused by changes in a single nucleotide in the genome. They are widely distributed in the genome, have stable heritability, and are very important molecular markers. The present invention identified two linkage disequilibrium SNP sites at exons 534 and 601 of the acaca gene with high genetic diversity (PIC>0.5), which 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). The two SNP sites in the exons of the acaca gene lead to codon missense mutations, and changes in the amino acid sequence may lead to changes in protein function.

[0058] Body length is an important indicator for evaluating fish growth performance. In order to verify whether the mutation of two SNP sites in the exon of the acaca gene is associated with the body length of Hippocampus inflatus, the present invention identified the common population and the fast-growing strain F 3 After 30 days of growth comparison experiment, the average body length and average growth of different types of SNP were calculated. The results showed that compared with the normal population, the fast-growing strain F 3 The haplotype III (HIII, A 534 A 601 ) randomly formed homozygous double V type (D V, A 534 A 534 A 601 A 601 ) has a significant advantage in body length. Therefore, it is concluded that the simultaneous mutation of positions 534 and 601 (haplotype III SNP A 534 A 601 ) can significantly increase the body length of hippocampus bulging. Studies have shown that the acaca gene is the coding gene for acetyl-CoA carboxylase and plays an important role in growth and development. 3 The haplotype III SNP (A 534 A 601 ) The phenylalanine in the second exon is changed to isoleucine, and the alanine in the second exon is changed to aspartic acid, thereby significantly increasing the expression of the acaca gene and promoting the increase in body length.

[0059] In the pyruvate metabolic pathway, acetyl-CoA carboxylase catalyzes pyruvate to form malonyl-CoA, which ultimately accelerates fatty acid biosynthesis and promotes growth. 534 A 601 The SNP mutation increases the expression of the acaca gene, speeding up the decomposition of pyruvate and the synthesis of malonyl-CoA, thereby increasing the number of fast-growing lines F 3 The homozygous double V type (DV, A 534 A 534 A 601 A 601 ) The body length of Hippocampus distended. Hippocampus distended homozygous double V type (D V, A 534 A 534 A 601 A 601 ) showed a significantly improved body length advantage and can be used for molecular assisted breeding of new fast-growing strains of Hippocampus inflatus.

[0060] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled 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 embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. Application of the homozygous double V type of the SNP locus associated with hippocampal distension length in breeding, characterized in that: in, The diplotype is randomly formed by haplotype I, haplotype II, and haplotype III, wherein the bases at exon 534 and 601 of the acaca gene of haplotype I are T and C, respectively, the bases at exon 534 and 601 of the acaca gene of haplotype II are T and A, respectively, and the bases at exon 534 and 601 of the acaca gene of haplotype III are A and A, respectively. The nucleotide sequence of the acaca gene is shown in SEQ ID NO: 1 or SEQ ID NO:

2. The three haplotypes randomly form a homozygous diplotype I T 534 T 534 C 601 C 601 , heterozygous double type II T 534 T 534 C 601 A 601 , homozygous double type III T 534 T 534 A 601 A 601 , heterozygous double IV type T 534 A 534 A 601 A 601 and homozygous double V-type A 534 A 534 A 601 A 601 Among the 5 diplotypes, the homozygous diplotype V has significant advantages over the other 4 diplotypes in terms of average body length and relative expression of acaca gene. The homozygous diplotype V can be used for molecular assisted breeding of new fast-growing strains of Hippocampus inflatus.

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