Application of Homozygous Diploid V-Type of SNP Loci Associated with Rainbow Trout Body Weight in Breeding

Abstract

The present invention discloses the application of the homozygous diploid V type of rainbow trout body weight-associated SNP loci in breeding, belonging to the technical field of aquaculture breeding. Among them, the diploid type is randomly formed by haploid type I, haploid type II, and haploid type III. Among them, the bases at positions 434 and 520 of the exon of the IGFBP2 gene of haploid type I are T and C respectively, the bases at positions 434 and 520 of the exon of the IGFBP2 gene of haploid type II are G and C respectively, and the bases at positions 434 and 520 of the exon of the IGFBP2 gene of haploid type III are G and A respectively. The homozygous diploid V type randomly formed by haploid type III has significant advantages in terms of average body weight and IGFBP2 gene expression compared with other diploid types. The homozygous diploid V type can be used for molecular-assisted breeding of fast-growing new strains of rainbow trout, accelerating the breeding process of fast-growing fine varieties of rainbow trout.

Description

Technical Field

[0001] The present invention relates to the application of the homozygous diploid V type of rainbow trout body weight-associated SNP loci in breeding, and belongs to the technical field of aquaculture breeding. Background Art

[0002] Body weight is an important indicator for evaluating the growth performance of rainbow trout. At present, rainbow trout farming mainly relies on imports, lacking control over germplasm resources, resulting in uneven growth performance of rainbow trout farming varieties, which greatly restricts the development of rainbow trout farming. Currently, there is still a lack of artificial breeding technology for rainbow trout in China.

[0003] At present, there is no report on the application of rainbow trout body weight-associated SNP loci in breeding. The most similar one is a rainbow trout high-temperature-resistant SNP molecular marker and its application. However, it only discloses the positions of high-temperature-resistant related SNPs and does not analyze the trait correlation, unable to accurately locate the body weight-related SNPs. At the same time, it is impossible to determine whether the rainbow trout with growth advantages is homozygous and inheritable. Summary of the Invention

[0004] To solve the deficiencies of the prior art, the present invention provides the application of the diplotype of rainbow trout body weight-associated SNP loci in breeding, in order to overcome the limitations of various factors, strictly regulate the growth process of rainbow trout, and promote the increase in rainbow trout production.

[0005] To achieve the above objectives, the present invention adopts the following technical solutions:

[0006] The application of the homozygous diploid V type of rainbow trout body weight-associated SNP loci in breeding, wherein the diplotype is randomly formed by haploid type I, haploid type II, and haploid type III. Among them, the bases at positions 434 and 520 of the exon of the IGFBP2 gene of haploid type I are T and C respectively, the bases at positions 434 and 520 of the exon of the IGFBP2 gene of haploid type II are G and C respectively, and the bases at positions 434 and 520 of the exon of the IGFBP2 gene of haploid type III are G and A respectively. The nucleotide sequence of the IGFBP2 gene is as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The three haplotypes randomly form homozygous diploid type I T 434 T 434 C 520 C 520 、heterozygous diploid type II T 434 G 434 C 520 C 520 、homozygous diploid type III G 434 G 434 C 520 C 520 、heterozygous diploid type IV G 434 G 434 C 520 A520 and homozygous double V-type G 434 G 434 A 520 A 520 There are 5 double types. The homozygous double V-type has significant advantages over the other 4 double types in terms of average body weight and relative expression of the IGFBP2 gene. The homozygous double V-type can be used for molecular-assisted breeding of fast-growing new strains of rainbow trout.

[0007] The beneficial effects of the present invention are as follows: Two SNP loci of the rainbow trout insulin-like growth factor binding protein 2 (IGFBP2) gene (located at exon 434 and 520 of the IGFBP2 gene respectively) were associated with the body weight of rainbow trout. It was found that these two SNP loci were significantly correlated with the body weight of rainbow trout. Among them, the body weight of individuals of haplotype III (G 434 A 520 ) was significantly increased. By further comparing and analyzing the body weight and IGFBP2 gene expression of individuals of 5 double types, namely 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), it was found that there were significant differences in the body weight of rainbow trout and the expression of the IGFBP2 gene between the fast-growing population and the common population. Among them, the average body weight and relative expression of the IGFBP2 gene of homozygous double V-type (DV) rainbow trout were significantly higher than those of the other 4 double types. The homozygous double V-type (DV) can be used as a molecular marker related to body weight and applied to molecular-assisted breeding of fast-growing rainbow trout populations to accelerate the breeding process of fast-growing fine varieties of rainbow trout. Description of the Drawings

[0008] Figure 1 It is a comparison chart of the body weights of rainbow trout W population and M population from 1 to 5 months old;

[0009] Figure 2 It is a comparison chart of the relative expression levels of the IGFBP2 gene of 5 double types of 5-month-old rainbow trout. Detailed Embodiments

[0010] 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.

[0011] I. Materials and Methods

[0012] 1. Experimental fish

[0013] The experimental fish used were rainbow trout common population (wild type, hereinafter referred to as W population) and fast-growing population (mutant type, hereinafter referred to as M population), both of which were 4 months old and had good growth and development.

[0014] The body weight of the M population at 5 months old used in the experiment was significantly higher than that of the W population ( Figure 1 ).

[0015] Two hundred rainbow trouts each from the W population and the M population of rainbow trout were respectively cultured in 4 glass aquariums of 20 L, with 100 fish in each aquarium, and connected to a central circulation system, which has mechanical and biological filtration, ultraviolet sterilization and a protein skimmer. The salinity was 31–32‰, the temperature was 18-19°C, the pH was 8.2-8.3, and plastic plants were used as attachment substrates. The formulated feed was 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 aquariums were siphoned out.

[0016] 2. Screening of SNP loci of the IGFBP2 gene

[0017] By analyzing the sequencing data of the W population and the M population of rainbow trout, two linkage disequilibrium SNP loci at exon 434 and 520 of the IGFBP2 gene with relatively high genetic diversity (PIC>0.5) were identified (Table 1).

[0018] Table 1 SNP locus information

[0019]

[0020] To further amplify the SNP loci at 434 and 520 of the IGFBP2 gene by PCR reaction, primer sequences covering all coding regions of the IGFBP2 gene were designed (Table 2).

[0021] Table 2 Primer information

[0022]

[0023] For a total of 400 rainbow trouts from the W population and the M population (200 fish in each population), 1 / 3 of the dorsal fin was cut, and after extracting DNA by the alkaline lysis method, partial gene fragments of the W population and the M population were amplified by PCR. Subsequently, the qualified PCR products were sent to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing.

[0024] 3. Growth comparison

[0025] Rainbow trouts of the same SNP type were placed in the same glass aquarium for cultivation, and the feeding conditions were the same as before. After 30 days of cultivation (5 months old), the average body weight was measured.

[0026] 4. Real-time fluorescence quantitative PCR

[0027] For each SNP haplotype of rainbow trout at 5 months old, 5 tails were randomly selected, and total RNA was extracted from the tails using RNAiso Plus. 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 was detected 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.

[0028] 5. Statistical analysis

[0029] 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.

[0030] II. Results

[0031] 1. Analysis of SNP sites in the rainbow trout IGFBP2 gene

[0032] After detection, the nucleotide sequence of the IGFBP2 gene in the W population of rainbow trout is shown in SEQ ID NO: 1, and the nucleotide sequence of the IGFBP2 gene in the M population is shown in SEQ ID NO: 2. Correspondingly, the amino acid sequence encoded by the IGFBP2 gene in the W population of rainbow trout is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the IGFBP2 gene in the M population is shown in SEQ ID NO: 4.

[0033] Comparing the IGFBP2 gene and the amino acids encoded by the IGFBP2 gene in the W population and the M population, there are 2 variant sites in the coding region of the IGFBP2 gene in the M population, both of which are missense mutations. The codon change type at the T 434 G 434 site is ATC-AGC, encoding isoleucine (I) and serine (S) located in the 3rd exon respectively. The codon change type at the C 520 A 520 site is CGC-AGC, encoding arginine (R) and serine (S) located in the 3rd exon respectively.

[0034] A total of 3 haplotypes were detected in the W population and M population of rainbow trout, namely: haplotype I (HI, T 434 C 520 ), haplotype II (HII, G 434 C 520 ), and haplotype III (HIII, G 434 A 520 ). The information of the 3 haplotypes is shown in Table 3 specifically.

[0035] Table 3 Information of 3 haplotypes

[0036]

[0037] As can be seen from Table 3, only haplotype I (HI, T 434 C 520 ), and haplotype II (HII, G 434 C 520 ) exist in the W population, with frequencies of 80% and 20% respectively. Only haplotype II (HII, G 434 C 520 ) and haplotype III (HIII, G 434 A 520 ) exist in the M population, with frequencies of 21% and 79% respectively.

[0038] Three diploid types are randomly formed in the W population: homozygous diploid I (DI, T 434 T 434 C 520 C 520 ), heterozygous diploid II (DII, T 434 G 434 C 520 C 520 ), and homozygous diploid III (DIII, G 434 G 434 C 520 C 520 ), with frequencies of 67%, 26%, and 7% respectively. Three diploid types are randomly formed in the M population: homozygous diploid III (DIII, G 434 G 434 C 520 C 520 ), heterozygous diploid IV (DIV, G 434 G 434 C 520 A 520 ), and homozygous diploid V (DV, G 434 G 434 A 520 A 520 ), with frequencies of 3%, 36%, and 61% respectively. The specific information of the 5 diploid types is shown in Table 4.

[0039] Table 4 Information of 5 diploid types

[0040]

[0041] 2. Weight analysis of 5 diploid types of rainbow trout

[0042] The statistical results of the average weight of 5 diploid types of rainbow trout at 5 months of age are shown in Table 5.

[0043] Table 5 Statistical results of the average body weight of 5 five - month - old double types

[0044]

[0045] Note: Different lowercase letters superscripted on the numbers within the same column indicate significant differences (p < 0.05).

[0046] As can be seen from Table 5: At 5 months of age, the average body weight of homozygous double - type V (DV) rainbow trout was significantly higher than that of the other 4 double - types (p < 0.05).

[0047] 3. Analysis of the relative expression levels of the IGFBP2 gene in 5 double - types of rainbow trout

[0048] The relative expression levels of the IGFBP2 gene in 5 double - types of 5 - month - old rainbow trout, namely homozygous double - type I (DI), heterozygous double - type II (DII), homozygous double - type III (DIII), heterozygous double - type IV (DIV), and homozygous double - type V (DV), were 1.37 ± 0.11, 1.38 ± 0.10, 1.38 ± 0.11, 1.48 ± 0.21, and 2.31 ± 0.25 respectively. The comparison of the relative expression levels of the IGFBP2 gene is as Figure 2 shown.

[0049] As Figure 2 can be seen: The relative expression level of the IGFBP2 gene in the DV type was significantly higher than that of the other 4 double - types (p < 0.05), and there were no significant differences in the relative expression levels of the IGFBP2 gene among the DI type, DII type, DIII type, and DIV type (p > 0.05).

[0050] III. Conclusion

[0051] 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 linked - disequilibrium SNP loci were identified at positions 434 and 520 of the exon of the IGFBP2 gene with relatively high genetic diversity (PIC > 0.5). There are 3 haplotypes (haplotype I, haplotype II, and haplotype III) among them. These 3 haplotypes randomly form 5 double - types (homozygous double - type I, heterozygous double - type II, homozygous double - type III, heterozygous double - type IV, and homozygous double - type V). These two SNP loci in the exon of the IGFBP2 gene result in missense mutations of codons, and the change in the amino - acid sequence may lead to changes in protein function.

[0052] Body weight is an important indicator for evaluating the growth performance of fish. To verify whether the mutations of two SNP sites in the exon of the IGFBP2 gene are associated with the body weight of rainbow trout, the present invention identified the W population and M population of rainbow trout. After a 1-month growth comparison experiment, the growth performance of different types of SNPs was statistically analyzed. The results showed that: the body weight of the homozygous diploid type V (DV, G 434 G 434 A 520 A 520 ) was significantly higher than that of the other four diploid types. Therefore, it was concluded that the simultaneous mutations at positions 434 and 520 (haploid type III SNP G 434 A 520 ) could significantly increase the body weight of rainbow trout. Existing studies have shown that the IGFBP2 gene is the coding gene of insulin-like growth factor-binding protein 2 and plays an important role in growth and development. There are 2 variant sites in the coding region of the rainbow trout IGFBP2 gene. The codon change at the T 434 G 434 site changed isoleucine (I) to serine (S), and the codon change at the C 520 A 520 site changed arginine (R) to serine (S), thus significantly increasing the expression level of the IGFBP2 gene and promoting weight gain. The homozygous diploid type V of rainbow trout (DV, G 434 G 434 A 520 A 520 ) showed a significant weight advantage and could be used for molecular-assisted breeding of fast-growing new strains of rainbow trout to increase production.

[0053] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention and are not limitations on 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 list 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 diploid V type of SNP loci associated with rainbow trout body weight in breeding, characterized in that, Among them, The double type is randomly formed by haploid type I, haploid type II, and haploid type III. Among them, the bases at exon 434 and 520 of the IGFBP2 gene in haploid type I are T and C respectively, the bases at exon 434 and 520 of the IGFBP2 gene in haploid type II are G and C respectively, and the bases at exon 434 and 520 of the IGFBP2 gene in haploid type III are G and A respectively. The nucleotide sequence of the IGFBP2 gene is shown in SEQ ID NO: 1 or SEQ ID NO:

2. The 3 haplotypes randomly form homozygous double type I T 434 T 434 C 520 C 520 , heterozygous double type II T 434 G 434 C 520 C 520 , homozygous double type III G 434 G 434 C 520 C 520 , heterozygous double type IV G 434 G 434 C 520 A 520 and homozygous double type V G 434 G 434 A 520 A 520 There are 5 double types. Homozygous double type V has significant advantages in average body weight and relative expression of the IGFBP2 gene compared with the other 4 double types. Homozygous double type V can be used for molecular assisted breeding of fast-growing new strains of rainbow trout.

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