Application of Haplotype of SNP Loci Associated with the Body Weight of Pseudosciaena crocea in Breeding

Abstract

The invention discloses the application of the haplotype of SNPs associated with the body weight of large yellow croaker in breeding, belonging to the technical field of aquaculture breeding. The haplotype is randomly formed by haplotype I, haplotype II, and haplotype III. Among them, the bases at positions 226 and 727 of the exon of the prkaa1 gene of haplotype I are C and A respectively, the bases at positions 226 and 727 of the exon of the prkaa1 gene of haplotype II are C and T respectively, and the bases at positions 226 and 727 of the exon of the prkaa1 gene of haplotype III are A and T respectively. The homozygous haplotype V randomly formed by haplotype III has significant advantages in terms of average body weight and prkaa1 gene expression compared with other haplotypes. Haplotype V can be used for molecular-assisted breeding of fast-growing new strains of large yellow croaker, accelerating the breeding process of fast-growing fine varieties of large yellow croaker.

Description

Technical Field

[0001] The present invention relates to the application of the haplotype of SNPs associated with the body weight of large yellow croaker in breeding, and belongs to the technical field of aquaculture breeding. Background Art

[0002] Body weight is closely related to the yield of large yellow croaker, and the increase in body weight can bring significant economic benefits.

[0003] At present, there is no report on the application of SNPs associated with the body weight of large yellow croaker in breeding. The most similar one is the application of SNPs related to the sex of large yellow croaker. Chinese invention patent with the application number 201810586380.X discloses an SNP marker related to the genetic sex of large yellow croaker, its primer and application. However, it only locates the sex-related single base, and there is no genetic linkage disequilibrium genotype related to the body weight of large yellow croaker, so it cannot be applied to the screening of body weight dominant traits. Summary of the Invention

[0004] To solve the deficiencies of the prior art, the present invention provides the application of the haplotype of SNPs associated with the body weight of large yellow croaker in breeding, overcomes the limitations of various factors, strictly regulates the growth process of large yellow croaker, and can be used for molecular assisted breeding of fast-growing new strains of large yellow croaker.

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

[0006] The application of the haplotype of SNPs associated with the body weight of large yellow croaker in breeding, wherein the haplotype is randomly formed by haplotype I, haplotype II, and haplotype III. Among them, the bases at positions 226 and 727 of the exon of the prkaa1 gene in haplotype I are C and A respectively, the bases at positions 226 and 727 of the exon of the prkaa1 gene in haplotype II are C and T respectively, and the bases at positions 226 and 727 of the exon of the prkaa1 gene in haplotype III are A and T respectively. The nucleotide sequences of the prkaa1 gene are as shown in SEQ ID NO: 1 and SEQ ID NO: 2. The three haplotypes are randomly formed into five haplotypes, namely homozygous haplotype I C 226 C 226 A 727 A 727 , heterozygous haplotype II C 226 C 226 A 727 T 727 , homozygous haplotype III C 226 C 226 T 727 T 727 , heterozygous haplotype IV C 226 A 226 T 727 T 727 and homozygous haplotype V A226 A 226 T 727 T 727 Among them, the homozygous double V type has significant advantages over the other four double types in terms of average body weight and relative expression of the prkaa1 gene. The homozygous double V type can be used for molecular assisted breeding of fast-growing new strains of large yellow croaker.

[0007] The beneficial effects of the present invention are as follows: By performing association analysis on two SNP loci of the prkaa1 gene of large yellow croaker (located at exon 226 and 727 of the prkaa1 gene respectively) and the body weight of large yellow croaker, it is found that these two SNP loci are significantly correlated with body weight. Among them, the haplotype III type (HIII, A 226 T 727 ) individuals have significantly increased body weight. Through further comparison and analysis of the body weights of five double types (DI (C 226 C 226 A 727 A 727 ), DII (C 226 C 226 A 727 T 727 ), DIII (C 226 C 226 T 727 T 727 ), DIV (C 226 A 226 T 727 T 727 ), DV (A 226 A 226 T 727 T 727 )) individuals, it is found that there are significant differences in the body weight of large yellow croaker and the expression of the prkaa1 gene among different double types. Among them, the relative expression level of the prkaa1 gene and the body weight of large yellow croaker of DV (A 226 A 226 T 727 T 727 ) are significantly higher than those of the other four double types. The double V type (DV) can be used for molecular assisted breeding of fast-growing new strains of large yellow croaker, accelerating the breeding process of fast-growing fine varieties of large yellow croaker. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 is a comparison chart of the body weights of the W population and F population of large yellow croaker from 1 to 6 months old, * indicates P < 0.05;

[0009] Figure 2 is a comparison chart of the relative expression levels of the prkaa1 gene of five double-type large yellow croakers at 6 months old. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will be specifically introduced below in conjunction with the accompanying 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 were the common population of large yellow croaker (wild type, hereinafter referred to as the W population) and the fast-growing population (fast-growing type, hereinafter referred to as the F population), both of which were 5 months old, with good growth and development, and were kept in the Seahorse Research Center of Ludong University.

[0014] The weights of the 5-month-old and 6-month-old F populations used in the experiment were significantly higher than those of the W population ( Figure 1 ).

[0015] Two hundred tails (400 tails in total) were taken from the 5-month-old W population and F population of large yellow croaker, and they were mixed and cultured in two rectangular cement ponds, with 100 tails of the W population and 100 tails of the F population in each pond (200 tails in total), and their fins were clipped for marking. The salinity was 31-32‰, the temperature was 18-19°C, and the pH was 8.2-8.3. Feeding was carried out at 8:00 and 16:00 every day, and the feeding amount each time was the same.

[0016] 2. Screening of SNP sites of the prkaa1 gene

[0017] By analyzing the sequencing data of the W population and F population of large yellow croaker, two linkage disequilibrium SNP sites at exon 226 and 727 of the protein kinase AMP-activated catalytic subunit α1 (prkaa1) 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 226 and 727 of the prkaa1 gene by PCR reaction, primer sequences covering all coding regions of the prkaa1 gene were designed (Table 2).

[0021] Table 2 Primer information

[0022]

[0023] For a total of 400 large yellow croaker in the W population and F population, 1 / 3 of the left ventral fin was clipped, and DNA was extracted by the alkaline lysis method. The partial gene fragments of the W population and F population were amplified by PCR, and the qualified PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China) for sequencing.

[0024] 3. Growth comparison

[0025] After 30 days of cultivation, the body weights and average weight gains of the W population and the F population were measured.

[0026] 4. Real-time fluorescence quantitative PCR

[0027] Five individuals of each of the five diploid types of large yellow croaker were randomly selected. Total RNA was extracted from the tails using RNAiso Plus. Genomic DNA was removed from the RNA using the PrimeScript RT kit, and cDNA was synthesized. Real-time quantitative PCR was performed on a CFX96 Touch™ real-time PCR detection system using SYBR Green Premix Ex Taq 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 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. SNP locus analysis of the prkaa1 gene in large yellow croaker

[0032] After detection, the nucleotide sequence of the prkaa1 gene in the W population of large yellow croaker is shown in SEQ ID NO: 1, and the nucleotide sequence of the prkaa1 gene in the F population is shown in SEQ ID NO: 2. Correspondingly, the amino acid sequence encoded by the prkaa1 gene in the W population of large yellow croaker is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the prkaa1 gene in the F population is shown in SEQ ID NO: 4.

[0033] Comparing the prkaa1 gene and the amino acid encoded by the prkaa1 gene between the W population and the F population, there were 2 variant sites in the coding region of the prkaa1 gene in the F population. Both of the two SNP sites were missense mutations. The codon change type at the C 226 A 226 site was CTT-ATT, encoding leucine (L) and isoleucine (I) located in the first exon respectively. The codon change type at the A 727 T 727 site was ACA-TCA, encoding threonine (T) and serine (S) located in the third exon respectively.

[0034] Three haplotypes were detected in the W population and the F population of large yellow croaker, namely: Haplotype I (HI, C 226A 727 ), Haplotype II (HII, C 226 T 727 ), and Haplotype III (HIII, A 226 T 727 ). The information of the three haplotypes is shown in Table 3 specifically.

[0035] Table 3 Information of Three Haplotypes

[0036]

[0037] As can be seen from Table 3, the W population is mainly Haplotype I, with a small amount of Haplotype II and no Haplotype III. The F population is mainly Haplotype III, with a small amount of Haplotype II and no Haplotype I.

[0038] Two haplotypes (HI, HII) in the W population randomly form three diplotypes: homozygous diplotype I (DI, C 226 C 226 A 727 A 727 ), heterozygous diplotype II (DII, C 226 C 226 A 727 T 727 ), and homozygous diplotype III (DIII, C 226 C 226 T 727 T 727 ). The frequencies of occurrence are 47%, 46%, and 7% respectively. The information of the three diplotypes is shown in Table 4 specifically.

[0039] Two haplotypes (HII, HIII) in the F population randomly form three diplotypes: homozygous diplotype III (DIII, C 226 C 226 T 727 T 727 ), heterozygous diplotype IV (DIV, C 226 A 226 T 727 T 727 ), and homozygous diplotype V (DV, A 226 A 226 T 727 T 727 ). The frequencies of occurrence are 2%, 53%, and 45% respectively. The information of the three diplotypes is shown in Table 4 specifically.

[0040] Table 4 Information of Five Diplotypes

[0041]

[0042] 2. Weight Analysis of Five Diplotype Large Yellow Croakers

[0043] The statistical results of the average body weight and average weight gain of five double - type large yellow croakers at 5 - month and 6 - month old are shown in Table 5 specifically.

[0044] Table 5 Statistical results of the average body weight and average weight gain of five double - type large yellow croakers at 5 - month and 6 - month old

[0045]

[0046] Note: Different lowercase letters superscripted on the numbers within the same column indicate significant differences (p < 0.05).

[0047] As can be seen from Table 5: At 5 - month and 6 - month old, there were no significant differences in the average body weight and average weight gain among the first three double - types (DI, DII, DVIII) (p > 0.05). The average body weight and average weight gain of the latter two double - types (DIV, DV) were significantly higher (p < 0.05) than those of the first three double - types (DI, DII, DVIII). Among them, the average body weight and average weight gain of DV were the highest.

[0048] 3. Analysis of the relative expression levels of the prkaa1 gene in five double - type large yellow croakers

[0049] The relative expression levels of the prkaa1 gene in five double - type large yellow croakers (DI, DII, DIII, DIV, and DV) at 6 - month old were 0.62 ± 0.06, 0.63 ± 0.05, 0.66 ± 0.08, 0.68 ± 0.08, and 1.12 ± 0.07 respectively. The comparison of the relative expression levels of the prkaa1 gene is as Figure 2 shown.

[0050] From Figure 2 it can be seen that: The relative expression level of the prkaa1 gene of the DV type in the F population was significantly higher (p < 0.05) than that of the other four double - types. There were no significant differences in the relative expression levels of the prkaa1 gene among the DI, DII, DIII, and DIV types (p > 0.05).

[0051] III. Conclusion

[0052] 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 226 and 727 of the exon of the prkaa1 gene with relatively high genetic diversity (PIC > 0.5). There are three haplotypes (haplotype I, haplotype II, and haplotype III) among them. These three haplotypes randomly form five double - types (double - type I, double - type II, double - type III, double - type IV, and double - type V). The two SNP loci in the exon of the prkaa1 gene lead to missense mutations of codons, and the change of the amino - acid sequence may lead to the change of protein function.

[0053] Body weight is an important indicator for evaluating fish production and determines the weight gain rate of large yellow croaker. To verify whether the mutations of two SNP sites in the exon of the prkaa1 gene are associated with the body weight of large yellow croaker, the present invention identified different ploidies of the W population and F population of large yellow croaker. Through a 30-day growth comparison experiment, the average body weight and average weight gain of different ploidies were statistically analyzed. It was found that compared with the W population, the heterozygous diploid type IV (DIV, C 226 T 727 ), randomly formed by the haploid type III (HIII, A 226 A 226 T 727 T 727 ), and the homozygous diploid type V (DV, A 226 A 226 T 727 T 727 ), had a significant body weight advantage, and the body weight of the homozygous diploid type V (DV) was even greater. Therefore, it was concluded that the simultaneous mutations at positions 226 and 727 (haploid type III SNP A 226 T 727 ), could significantly increase the body weight of large yellow croaker. Existing studies have shown that the catalytic subunit α1 of protein kinase AMP-activated plays an important role in growth and development. In the homozygous diploid type V (DV, A 226 A 226 T 727 T 727 ), the leucine (L) in the first exon was changed to isoleucine (I), and the threonine (T) in the third exon was changed to serine (S), thus significantly increasing the relative expression level of the prkaa1 gene and promoting weight gain.

[0054] Therefore, large yellow croaker of the diploid type V (DV, A 226 A 226 T 727 T 727 ), showed a significant body weight advantage and could be used for molecular-assisted breeding of fast-growing new strains of large yellow croaker to increase production.

[0055] 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 haplotype of SNP locus associated with body weight of large yellow croaker in breeding, characterized in that, The diploid types are randomly formed by haploid type I, haploid type II, and haploid type III. Among them, the bases at positions 226 and 727 of the prkaa1 gene in haploid type I are C and A respectively, the bases at positions 226 and 727 of the prkaa1 gene in haploid type II are C and T respectively, and the bases at positions 226 and 727 of the prkaa1 gene in haploid type III are A and T respectively. The nucleotide sequences of the prkaa1 gene are shown in SEQ ID NO: 1 and SEQ ID NO:

2. The three haploid types randomly form five diploid types, namely homozygous diploid type I C 226 C 226 A 727 A 727 , heterozygous diploid type II C 226 C 226 A 727 T 727 , homozygous diploid type III C 226 C 226 T 727 T 727 , heterozygous diploid type IV C 226 A 226 T 727 T 727 and homozygous diploid type V A 226 A 226 T 727 T 727 . Among them, homozygous diploid type V has significant advantages in terms of average body weight and relative expression of the prkaa1 gene compared with the other four diploid types. Homozygous diploid type V can be used for molecular assisted breeding of fast-growing new strains of large yellow croaker.

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