Application of large yellow croaker body weight associated SNP site diplotype in breeding

Abstract

The invention discloses an application of a large yellow croaker body weight associated SNP locus diplotype in breeding, and belongs to the technical field of aquatic product breeding. The haplotype is randomly formed by a haplotype I, a haplotype II and a haplotype III, basic groups at exons 226 and 727 of the prkaa1 gene of the haplotype I are C and A respectively, basic groups at exons 226 and 727 of the prkaa1 gene of the haplotype II are C and T respectively, basic groups at exons 226 and 727 of the prkaa1 gene of the haplotype III are A, T, C and T respectively, and basic groups at exons 226 and 727 of the prkaa1 gene of the haplotype III are C and T respectively. Compared with other plotypes, the homozygous double V type randomly formed by the haplotype III has remarkable advantages in average weight and prkaa1 gene expression, and the double V type can be used for large yellow croaker fast-growing new line molecule assisted breeding, so that the breeding process of large yellow croaker fast-growing improved varieties is accelerated.

Description

Technical Field

[0001] The invention relates to application of a diplotype of a SNP site associated with large yellow croaker weight in breeding, and belongs to the technical field of aquatic breeding. Background Art

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

[0003] At present, there is no report on the application of SNP loci associated with the weight of large yellow croaker in breeding. The most similar is the application of SNPs associated with the sex of large yellow croaker. The Chinese invention patent with application number 201810586380.X discloses a SNP marker associated with the genetic sex of large yellow croaker, its primers and application. However, it only locates a single base related to sex, and there is no genetic linkage disequilibrium genotype associated with the weight of large yellow croaker. Therefore, it cannot be used for the screening of weight advantage traits. Summary of the invention

[0004] To address the deficiencies of the prior art, the present invention provides an application of a diplotype of a SNP site associated with large yellow croaker weight in breeding, overcomes the limitations of multiple 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: Application of diplotypes of SNP sites associated with large yellow croaker weight in breeding, wherein the diplotype is randomly formed by haplotype I, haplotype II, and haplotype III, wherein the bases at exons 226 and 727 of the prkaa1 gene of haplotype I are C and A, respectively, the bases at exons 226 and 727 of the prkaa1 gene of haplotype II are C and T, respectively, the bases at exons 226 and 727 of the prkaa1 gene of haplotype III are A and T, and the nucleotide sequence of the prkaa1 gene is shown in SEQ ID NO: 1 and SEQ ID NO: 2. The three haplotypes randomly form five diplotypes, namely, homozygous diplotype I, C, and T. 226 C 226 A 727 A 727 , heterozygous double type II C 226 C 226 A 727 T 727 , homozygous double type III C 226 C 226 T 727 T 727 , heterozygous double IV type C 226 A 226 T 727 T 727 and homozygous double V-type A 226A 226 T 727 T 727 Among them, the homozygous diploid V type has significant advantages over the other four diplotypes in average body weight and relative expression of the prkaa1 gene. The homozygous diploid V type can be used for molecular assisted breeding of new fast-growing strains of large yellow croaker.

[0006] The invention is beneficial in that: the association analysis of two SNP sites of the prkaa1 gene of large yellow croaker (located at exons 226 and 727 of the prkaa1 gene, respectively) and the weight of large yellow croaker is performed, and it is found that the two SNP sites are significantly correlated with the weight, among which haplotype III (HIII, A 226 T 727 ) individuals had significantly higher body weight. 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 The body weight of yellow croaker (DV) and the expression of prkaa1 gene were significantly different in different diplotypes. 226 A 226 T 727 T 727 ) The relative expression level of prkaa1 gene and body weight of large yellow croaker were significantly higher than those of the other four diplotypes. The double V type (DV) can be used for molecular assisted breeding of new fast-growing strains of large yellow croaker and accelerate the breeding process of fast-growing improved varieties of large yellow croaker. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 This is a comparison of the body weight of large yellow croaker W group and F group from 1 to 6 months old, * indicates P < 0.05; Figure 2 This is a comparison chart of the relative expression levels of the prkaa1 gene in five diplotypes of large yellow croaker at 6 months of age. DETAILED DESCRIPTION

[0008] 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.

[0009] 1. Materials and Methods 1. Experimental fish The fish used in the experiment were a common group of large yellow croaker (wild type, hereinafter referred to as W group) and a fast-growing group (fast-growing type, hereinafter referred to as F group), both of which were 5 months old, with good growth and development, and were kept in the Hippocampus Research Center of Ludong University.

[0010] The body weight of the F group at 5 and 6 months of age was significantly higher than that of the W group ( Figure 1 ).

[0011] 200 5-month-old large yellow croakers from the W group and F group (400 in total) were taken and mixed in two rectangular cement pools, with 100 from each group (200 in total) in each pool, and their fins were clipped and marked. Salinity was 31-32‰, temperature was 18-19℃, and pH was 8.2-8.3. Feeding was carried out at 8:00 and 16:00 every day, and the feeding amount was the same each time.

[0012] 2. Screening of SNP sites in the prkaa1 gene By analyzing the sequencing data of the W and F populations of large yellow croaker, two linkage disequilibrium SNP sites at exons 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).

[0013] Table 1 SNP site information

[0014] To further amplify the 226 and 727 SNP sites of the prkaa1 gene by PCR reaction, primer sequences covering all coding regions of the prkaa1 gene were designed ( Table 2 ).

[0015] Table 2 Primer information

[0016] A total of 400 large yellow croakers from the W and F populations were cut off one-third of their left ventral fins, and DNA was extracted by the alkaline splitting method. Partial gene fragments of the W and F populations were amplified by PCR, and the qualified PCR products were sent to Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China) for sequencing.

[0017] 3. Growth comparison After 30 days of culture, the body weight and average weight gain of the W and F groups were measured.

[0018] 4. Real-time fluorescence quantitative PCR Five tails of each of the five diplotypes of large yellow croaker were randomly selected, and 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 SYBR Green Premix Ex Taq was used 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.

[0019] 5. Statistical analysis 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.

[0020] 2. Results 1. Analysis of SNP sites of prkaa1 gene in large yellow croaker After testing, the nucleotide sequence of the prkaa1 gene of the W population of large yellow croaker is shown in SEQ ID NO: 1, and the nucleotide sequence of the prkaa1 gene of the F population is shown in SEQ ID NO: 2. Correspondingly, the amino acid sequence encoded by the prkaa1 gene of the W population of large yellow croaker is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the prkaa1 gene of the F population is shown in SEQ ID NO: 4.

[0021] Comparison of the prkaa1 gene and the amino acids encoded by the prkaa1 gene between the W and F populations revealed that there were two variant sites in the coding region of the prkaa1 gene in the F population, both of which were missense mutations. 226 A 226 The codon change type of the site is CTT-ATT, encoding leucine (L) and isoleucine (I) located in the first exon, A 727 T 727 The codon change type at the site is ACA-TCA, encoding threonine (T) and serine (S) located in the third exon, respectively.

[0022] Three haplotypes were detected in the W and F populations of large yellow croaker: haplotype I (HI, C 226 A 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.

[0023] Table 3 Information of three haplotypes

[0024] As shown in Table 3, the W population is mainly haplotype I, with a small amount of haplotype II and no haplotype III, and the F population is mainly haplotype III, with a small amount of haplotype II and no haplotype I.

[0025] The two haplotypes (HI, HII) of the W population randomly formed three diplotypes: homozygous diplotype I (DI, C 226 C 226 A 727 A 727 ), heterozygous diploid type II (DII, C 226 C 226 A 727 T 727 ) and homozygous double type III (DIII, C 226 C 226 T 727 T 727 ), the frequencies of occurrence were 47%, 46% and 7% respectively. The information of the three diplotypes is shown in Table 4.

[0026] The two haplotypes (HII and HIII) of the F population randomly formed three diplotypes: homozygous diplotype III (DIII, C 226 C 226 T 727 T 727 ), heterozygous double IV type (DIV, C 226 A 226 T 727 T 727 ) and homozygous double V type (DV, A 226 A 226 T 727 T 727 ), with frequencies of 2%, 53% and 45% respectively. The information of the three diplotypes is shown in Table 4.

[0027] Table 4 Information of five diplotypes

[0028] 2. Weight analysis of five diplotypes of large yellow croaker The statistical results of the average body weight and average weight gain of the five diplotype large yellow croakers at 5 and 6 months of age are shown in Table 5.

[0029] Table 5 Statistical results of average body weight and average weight gain of five diplotype large yellow croakers at 5 and 6 months of age

[0030] Note: Numbers with different lowercase letters in the same column indicate significant differences (p<0.05).

[0031] It can be seen from Table 5 that at 5 and 6 months of age, there was no significant difference in the average body weight and average weight gain of the first three diplotypes (DI, DII, DVIII) (p>0.05), and the average body weight and average weight gain of the last two diplotypes (DIV, DV) were significantly (p<0.05) higher than those of the first three diplotypes (DI, DII, DVIII), among which DV had the highest average body weight and average weight gain.

[0032] 3. Analysis of relative expression of prkaa1 gene in five diplotypes of large yellow croaker The relative expression levels of prkaa1 gene in the five diplotypes of large yellow croaker DI, DII, DIII, DIV and DV at 6 months of age were 0.62±0.06, 0.63±0.05, 0.66±0.08, 0.68±0.08 and 1.12±0.07, respectively. Figure 2 shown.

[0033] Depend on Figure 2 It can be seen that the relative expression level of DⅤ type prkaa1 gene in the F 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 DI, DII, DIII and DⅣ types of prkaa1 genes (p>0.05).

[0034] Conclusion 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 226 and 727 of the prkaa1 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 (double type I, double type II, double type III, double type IV, and double type V). The two SNP sites in the exons of the prkaa1 gene lead to codon missense mutations, and changes in the amino acid sequence may lead to changes in protein function.

[0035] Body weight is an important indicator for evaluating fish production and determines the weight gain rate of large yellow croaker. In order to verify whether the mutation of two SNP sites in the exon of the prkaa1 gene is associated with the weight of large yellow croaker, the present invention identified different ploidy types of large yellow croaker W population and F population, and statistically analyzed the average body weight and average weight gain of different ploidy types after a 30-day growth comparison experiment. It was found that compared with the W population, haplotype III (HIII, A 226 T 727) Randomly formed heterozygous double type IV (DIV, C 226 A 226 T 727 T 727 ) and homozygous double V type (DV, A 226 A 226 T 727 T 727 ) has a significant advantage in weight, and the weight of homozygous double type V (DV) is greater. Therefore, it is concluded that the simultaneous mutations at 226 and 727 (single type III SNP A 226 T 727 ) can significantly increase the weight of large yellow croaker. Studies have shown that the catalytic subunit α1 of protein kinase AMP activation plays an important role in growth and development. 226 A 226 T 727 T 727 ) The leucine (L) in the first exon is changed to isoleucine (I), and the threonine (T) in the third exon is changed to serine (S), thereby significantly increasing the relative expression of the prkaa1 gene and promoting weight gain.

[0036] Therefore, the double V type (DV, A 226 A 226 T 727 T 727 ) Large yellow croaker showed a significantly improved weight advantage, which can be used for molecular assisted breeding of new fast-growing strains of large yellow croaker to increase yield.

[0037] 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 diplotype of SNP loci associated with large yellow croaker weight in breeding, characterized in that: The diplotype is randomly formed by haplotype I, haplotype II, and haplotype III, wherein the bases at exon 226 and 727 of the prkaa1 gene of haplotype I are C and A, respectively, the bases at exon 226 and 727 of the prkaa1 gene of haplotype II are C and T, respectively, and the bases at exon 226 and 727 of the prkaa1 gene of haplotype III are A and T, respectively. The nucleotide sequence of the prkaa1 gene is shown in SEQ ID NO: 1 and SEQ ID NO:

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

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