Application of the homozygous double V type of SNP loci associated with hippocampal thickness in breeding

Abstract

The invention discloses the application of homozygous double V type of SNP site associated with body thickness of hippocampus inflatus 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 227 and 300 of CSF2RB gene of haplotype I are T and C respectively, the bases at exons 227 and 300 of CSF2RB gene of haplotype II are T and A respectively, the bases at exons 227 and 300 of CSF2RB gene of haplotype III are G and A respectively, the homozygous double V type randomly formed by haplotype III has significant advantages in average body thickness and CSF2RB 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 hippocampus inflatus, and accelerate the breeding process of fast-growing improved hippocampus.

Description

Technical Field

[0001] The invention relates to application of homozygous double V-type of hippocampal corpus bulgais thickness-related SNP sites in breeding, belonging to the technical field of aquatic breeding. Background Art

[0002] The size of the body thickness is closely related to the production of seahorses. At present, due to the disordered self-pollination of farmed seahorses and the lack of control over germplasm resources, the seahorse germplasm resources have declined, and the body thickness of farmed seahorses has been significantly reduced, which has greatly affected the weight and production of seahorses.

[0003] At present, there is still a lack of artificially bred high-quality seahorse breeding strains. The research team has bred the F3 generation of seahorse fast-growing strains, screened out the group with significantly improved body thickness, and located the SNP associated with body thickness, in order to increase the production of seahorse breeding.

[0004] At present, there is no report on the application of SNP loci associated with body thickness of Distended hippocampus in breeding. Similar to this is the application of SNP loci associated with body length of Distended hippocampus in breeding. However, the key gene of body length-associated SNP can only be associated with hippocampal body length, not hippocampal body thickness, and cannot be used for body thickness screening. Summary of the invention

[0005] To address the deficiencies of the prior art, the present invention provides an application of a diplotype of a SNP locus associated with body thickness in hippocampus inflatus in breeding, in order to select hippocampus inflatus with larger body thickness, increase the yield of hippocampus inflatus, and promote the development of factory-scale breeding of hippocampus inflatus.

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

[0007] Application of homozygous diploid V type of SNP loci associated with hippocampal distention thickness in breeding, wherein the diploid type is randomly formed by haplotype I, haplotype II, and haplotype III, wherein the bases at exon 227 and 300 of the CSF2RB gene of haplotype I are T and C, respectively, the bases at exon 227 and 300 of the CSF2RB gene of haplotype II are T and A, respectively, the bases at exon 227 and 300 of the CSF2RB gene of haplotype III are G and A, respectively, the nucleotide sequence of the CSF2RB gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the three haplotypes randomly form a homozygous diploid I type T 227 T 227 C 300 C 300 , heterozygous double type II T 227 T 227 C 300 A 300 , homozygous double type III T227 T 227 A 300 A 300 , heterozygous double IV type T 227 G 227 A 300 A 300 and homozygous double V-type G 227 G 227 A 300 A 300 Among the 5 diplotypes, the homozygous diplotype V has significant advantages over the other 4 diplotypes in average body thickness and relative expression of the CSF2RB 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 two SNP sites (located at exons 227 and 300 of the CSF2RB gene (the gene encoding colony stimulating factor 2 receptor subunit β) of hippocampus distended are analyzed for association with the body thickness of hippocampus distended, and it is found that the two SNP sites are significantly correlated with body thickness, among which haplotype III (G 227 A 300 ) individuals were significantly thicker. Further comparative analysis of the body thickness and CSF2RB gene expression of five diplotypes, including homozygous diploid I (DI), heterozygous diploid II (DII), homozygous diploid III (DIII), heterozygous diploid IV (DIV), and homozygous diploid V (DV), revealed that there were significant differences in the body thickness and CSF2RB gene expression of Hippocampus inflatus between the F3 generation of the fast-growing strain and the common population. Among them, the average body thickness and relative expression of CSF2RB gene of the homozygous diploid V (DV) Hippocampus inflatus were significantly higher than those of the other four diplotypes. Homozygous diploid V (DV) can be used as a molecular marker related to body thickness and applied to molecular assisted breeding of new fast-growing strains of Hippocampus inflatus to increase the yield of seahorses. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a comparison of the body thickness of the common population and the fast-growing strain F3 generation of hippocampus inflatus from 1 to 7 months old;

[0010] Figure 2 This is a comparison chart of the relative expression levels of the CSF2RB gene in five diplotypes of 4-month-old hippocampus. DETAILED DESCRIPTION

[0011] 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.

[0012] 1. Materials and Methods

[0013] 1. Experimental fish

[0014] The fish used in the experiment were the common population of H. inflata (wild type, hereinafter referred to as W population) and the fast-growing strain F3 generation (mutant type, hereinafter referred to as M population), both of which were 1 month old and had good growth and development. They were kept in the Hippocampus Research Center of Ludong University.

[0015] The body thickness of the M group at 4-7 months of age was significantly higher than that of the W group ( Figure 1 ).

[0016] 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. Live Artemia were fed three times a day at 8:00, 12:00 and 16:00. After 2h of each feeding, the residual feed and feces in the glass tank were siphoned out.

[0017] 2. Screening of SNP sites in the gene encoding colony stimulating factor 2 receptor subunit β (CSF2RB) (hereinafter referred to as CSF2RB gene)

[0018] By analyzing the sequencing data of Hippocampus W and M populations, two linkage disequilibrium SNP sites at exons 227 and 300 of the CSF2RB gene with high genetic diversity (PIC>0.5) were identified (Table 1).

[0019] Table 1 SNP site information

[0020]

[0021] To further amplify the 227 and 300 SNP sites of the CSF2RB gene by PCR reaction, primer sequences covering all coding regions of the CSF2RB gene were designed ( Table 2 ).

[0022] Table 2 Primer information

[0023]

[0024] 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.

[0025] 3. Growth comparison

[0026] The same SNP type was placed in the same glass tank and reared under the same conditions. After 60 days (3 months of age) and 90 days (4 months of age), the average body thickness and average weight gain were measured.

[0027] 4. Real-time fluorescence quantitative PCR

[0028] Five tails of each SNP ploidy type were randomly selected from 4-month-old hippocampus. Total RNA from the tails was extracted using RNAiso Plus. Genomic DNA was removed from RNA using the Prime Script RT kit and cDNA was synthesized. Real-time quantitative PCR was performed in the CFX96 Touch™ real-time PCR detection system, and qRT-PCR 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] 2. Results

[0032] 1. Analysis of SNP sites of CSF2RB gene in hippocampus

[0033] After testing, the nucleotide sequence of the CSF2RB gene of the W population of Hippocampus inflatus is shown in SEQ ID NO: 1, and the nucleotide sequence of the CSF2RB gene of the M population is shown in SEQ ID NO: 2. Correspondingly, the amino acid sequence encoded by the CSF2RB gene of the W population of Hippocampus inflatus is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the CSF2RB gene of the M population is shown in SEQ ID NO: 4.

[0034] Comparison of the CSF2RB gene and the amino acids encoded by the CSF2RB gene in the W and M populations revealed that there were two mutation sites in the CSF2RB gene coding region of the M population, both of which were missense mutations. 227 G 227 The codon change type of the site is TTT-TGT, encoding phenylalanine (F) and cysteine ​​(C) located in the first exon, respectively. 300 A 300 The codon change type of the site is CCT-CAT, encoding proline (P) and histidine (H) located in the second exon, respectively.

[0035] Three haplotypes were detected in the W and M populations of Hippocampus inflatus: haplotype I (HI, T 227 C 300 ), haplotype II (HII, T 227 A 300 ) and haplotype III (HIII, G 227 A 300 ), the information of the three haplotypes is shown in Table 3.

[0036] Table 3 Information of three haplotypes

[0037]

[0038] 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.

[0039] The two haplotypes (HI, HII) of the W population randomly formed two diplotypes: homozygous diplotype I (DI, T 227 T 227 C 300 C 300 ) and heterozygous diploid type II (DII, T 227 T 227 C 300 A 300 ), the frequencies of occurrence were 58% and 42% respectively. The information of the two diplotypes is shown in Table 4.

[0040] The two haplotypes (HII and HIII) of the M population randomly formed three diplotypes: homozygous diplotype III (DIII, T 227 T 227 A 300 A 300 ), heterozygous double IV type (DIV, T 227 G 227 A 300 A 300 ) and homozygous double V type (DV, G 227 G 227 A 300 A 300 ), the frequencies of occurrence were 1%, 47%, and 52% respectively. The information of the three diplotypes is shown in Table 4.

[0041] Table 4 Information of five diplotypes

[0042]

[0043] 2. Analysis of body thickness of five diplotypes of Hippocampus distensus

[0044] The statistical results of the average body thickness and average weight gain of the five diplotypes of Hippocampus inflatus at 3 and 4 months of age are shown in Table 5.

[0045] Table 5 Statistical results of average body thickness and average weight gain of five diplotypes at 3 and 4 months of age

[0046]

[0047] Note: Numbers with different lowercase letters in the same column indicate significant differences (p<0.05).

[0048] From Table 5, we can see that:

[0049] (1) At 3 months of age, there was no significant difference in the average body thickness of the four diplotypes: homozygous diplotype I (DI), heterozygous diplotype II (DII), homozygous diplotype III (DIII) and heterozygous diplotype IV (DIV) (p>0.05), while the average body thickness of homozygous diplotype V (DV) was significantly higher than that of the other four diplotypes (p<0.05);

[0050] (2) At 4 months of age, there was no significant difference in the average body thickness of the four diplotypes: homozygous diplotype I (DI), heterozygous diplotype II (DII), homozygous diplotype III (DIII) and heterozygous diplotype IV (DIV) (p>0.05), while the average body thickness of homozygous diplotype V (DV) was significantly higher than that of the other four diplotypes (p<0.05).

[0051] (3) During the 30 days from 3 to 4 months of age, there was no significant difference in the average weight gain of the four diplotypes, namely, homozygous diploid type I (DI), heterozygous diploid type II (DII), homozygous diploid type III (DIII) and heterozygous diploid type IV (DIV) (p>0.05). The average food intake weight gain of homozygous diploid type V (DV) was significantly higher than that of the other four diplotypes (p<0.05).

[0052] 3. Analysis of relative expression of CSF2RB gene in five diplotypes of Hippocampus distensus

[0053] The relative expression levels of CSF2RB gene in 4-month-old hippocampus with 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 0.82±0.06, 0.82±0.05, 0.83±0.06, 0.89±0.08 and 1.12±0.07, respectively. Figure 2 shown.

[0054] Depend on Figure 2It can be seen that the relative expression level of CSF2RB gene of type DⅤ in the M population was significantly higher than that of the other four diplotypes (p<0.05), and there was no significant difference in the relative expression levels of CSF2RB gene of type DI, type DII, type DIII and type DⅣ (p>0.05).

[0055] Conclusion

[0056] 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 227 and 300 of the CSF2RB 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 CSF2RB gene lead to codon missense mutations, and changes in the amino acid sequence may lead to changes in protein function.

[0057] Body thickness is an important indicator for improving fish production and determines the weight gain rate of seahorses. In order to verify whether the two SNP site mutations in the exon of the CSF2RB gene are associated with the body thickness of the distended hippocampus, the present invention identified the common population and the F3 generation of the fast-growing strain, and statistically analyzed the body thickness and weight gain of different types of SNPs after a three-month growth comparison experiment. The results showed that compared with the common population, the haplotype III (HIII G 227 A 300 ) randomly formed homozygous double V type (DV, G 227 G 227 A 300 A 300 ) has a significant advantage in body thickness. Therefore, it is concluded that the simultaneous mutations at 227 and 300 (haplotype III SNPG 227 A 300 ) can significantly increase the body thickness of hippocampus bulging. Studies have shown that the CSF2RB gene is the gene encoding colony stimulating factor 2 receptor subunit β and plays an important role in body thickness development. 227 A 300 ) The phenylalanine (F) in the first exon is changed to cysteine ​​(C), and the proline (P) in the second exon is changed to histidine (H), thereby significantly increasing the expression of the CSF2RB gene and promoting the increase of body thickness. 227 G 227 A 300 A 300) showed a significantly improved body thickness advantage and can be used for molecular assisted breeding of new fast-growing strains of seahorses to increase yield.

[0058] 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 SNP loci associated with hippocampal distension thickness in breeding, characterized in that: in, The diplotype is randomly formed by haplotype I, haplotype II, and haplotype III, wherein the bases at exon 227 and 300 of the CSF2RB gene of haplotype I are T and C, respectively, the bases at exon 227 and 300 of the CSF2RB gene of haplotype II are T and A, respectively, and the bases at exon 227 and 300 of the CSF2RB gene of haplotype III are G and A, respectively. The nucleotide sequence of the CSF2RB gene is shown in SEQ ID NO: 1 or SEQ ID NO:

2. The three haplotypes randomly form a homozygous diplotype I T 227 T 227 C 300 C 300 , heterozygous double type II T 227 T 227 C 300 A 300 , homozygous double type III T 227 T 227 A 300 A 300 , heterozygous double IV type T 227 G 227 A 300 A 300 and homozygous double V-type G 227 G 227 A 300 A 300 Among the 5 diplotypes, the homozygous diplotype V has significant advantages over the other 4 diplotypes in average body thickness and relative expression of the CSF2RB gene. The homozygous diplotype V can be used for molecular assisted breeding of new fast-growing strains of Hippocampus inflatus.

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