SNP molecular marker associated with body length of turbot (scophthalmus maximus) fed with fermented soybean meal and its application

Abstract

The present application relates to the SNP molecular marker of the body length correlation under the fermented soybean meal feeding of Scophthalmus maximus and application thereof, and belongs to the technical field of molecular marker, the body length trait of the present application is analyzed by using whole genome correlation analysis on Scophthalmus maximus, the SNP molecular marker of the significant correlation under the fermented soybean meal feeding of Scophthalmus maximus and body length trait is obtained, including at least one of 6 SNP sites, can be applied to the molecular assisted breeding of Scophthalmus maximus, the screening can be carried out at the genome level through the breeding method disclosed in the present application, the breeding cycle can be greatly shortened, the dependence on phenotypic traits is reduced, the selection breeding efficiency is improved, and good application prospect is obtained.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology, specifically, it relates to a SNP molecular marker associated with body length in turbot fed fermented soybean meal and its application. Background Technology

[0002] Turbot (Scophthalmus maximus) is an important economic fish species in cold-water marine aquaculture in northern my country, possessing high commercial value due to its rapid growth and delicious flesh. In traditional aquaculture, turbot growth is highly dependent on diets with high fishmeal content. However, with the global scarcity and rising prices of fishmeal resources, developing efficient and low-cost plant protein sources (such as soybean meal) to replace fishmeal has become an inevitable trend in the aquaculture feed industry. Studies have shown that anti-nutritional factors in ordinary soybean meal can induce physiological problems in turbot, such as intestinal inflammation and reduced digestibility. Although fermented soybean meal significantly reduces the content of anti-nutritional factors and improves palatability through microbial fermentation, in actual production, it has been found that turbot individuals with different genetic backgrounds have significant differences in their ability to utilize fermented soybean meal diets. This interaction between genotype and nutrient metabolism leads to significant differentiation in body length growth within the population under low fishmeal (high fermented soybean meal) feeding conditions.

[0003] Traditional breeding methods rely primarily on phenotypic selection, which is highly susceptible to environmental influences and has a long cycle, making it difficult to accurately screen for superior varieties with efficient conversion capabilities to new feeds. Although there have been reports on SNPs (single nucleotide polymorphisms) associated with growth traits in turbot in recent years, the contribution of genetic variation explained by a single SNP locus is usually limited. Haplotypes, composed of a group of physically linked SNP loci, provide richer genetic information and exhibit higher polymorphism and genetic stability compared to a single SNP. In the context of fermented soybean meal feeding, identifying haplotypes significantly associated with body length traits and applying them to early marker-assisted breeding (MAS) has significant economic and scientific value for developing new turbot varieties that are tolerant to plant protein fishmeal feed and possess excellent growth traits. Summary of the Invention

[0004] This invention addresses the aforementioned technical problems by providing a set of SNP molecular markers in the context of fermented soybean meal feeding, which can be used for molecular-assisted breeding of new strains of turbot with body length traits.

[0005] One of the objectives of this invention is to provide a SNP molecular marker associated with body length in turbot fed fermented soybean meal, wherein the molecular marker is at least one of the following: the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, and the molecular marker has an SNP site associated with body length in turbot at position 101, the allele of which is G or A, and the molecular marker is denoted as chr7:4184141;

[0006] The nucleotide sequence of the molecular marker is shown in SEQ ID NO.5. At position 101 of the molecular marker, there is an SNP site associated with the body length of turbot. The allele of this site is A or T. The molecular marker is denoted as chr7:1293734.

[0007] The nucleotide sequence of the molecular marker is shown in SEQ ID NO.9. At position 101 of the molecular marker, there is an SNP site associated with the body length of turbot. The allele of this site is T or A. The molecular marker is denoted as chr12:871680.

[0008] The nucleotide sequence of the molecular marker is shown in SEQ ID NO.13. At position 101 of the molecular marker, there is an SNP site associated with the body length of turbot, and the allele of this site is C or T. The molecular marker is denoted as chr12:24383879.

[0009] The nucleotide sequence of the molecular marker is shown in SEQ ID NO.17. At position 101 of the molecular marker, there is an SNP site associated with the body length of turbot, and the allele of this site is T or C; the molecular marker is denoted as chr15:1901316.

[0010] The nucleotide sequence of the molecular marker is shown in SEQ ID NO.21. At position 101 of the molecular marker, there is an SNP site associated with the body length of turbot, and the allele of this site is T or A; the molecular marker is denoted as chr15:1783670.

[0011] A second objective of this invention is to provide a set of SNP molecular marker combinations associated with body length in turbot fed fermented soybean meal, wherein the SNP molecular marker combination is one of the following: chr7:4184141 and chr12:871680; chr7:4184141, chr12:871680 and chr15:1901316; chr7:1293734, chr12:871680 and chr15:1783670; chr7:4184141, chr7:1293734, chr12:871680 and chr15:1901316; chr The definitions of the SNP molecular markers are as described above. (The list includes chr7:4184141, chr7:1293734, chr12:871680, chr12:24383879, chr15:1901316, and chr15:1783670.)

[0012] The third technical objective of this invention is to provide primers for amplifying the SNP molecular marker, wherein the primers are capable of amplifying the SNP molecular marker.

[0013] The fourth technical objective of this invention is to provide the application of the SNP molecular markers in the breeding of turbot body length traits; the application involves selecting individuals with dominant genotypes as parents for body length trait breeding. For example, individuals with the original genotype of molecular marker chr7:4184141 are selected as GG individuals, individuals with the mutant genotype of molecular marker chr7:1293734 are selected as TT individuals, individuals with the original genotype of molecular marker chr12:871680 are selected as TT individuals, individuals with the mutant genotype of molecular marker chr12:24383879 are selected as TT individuals, individuals with the original genotype of molecular marker chr15:1901316 are selected as TT individuals, and individuals with the mutant genotype of molecular marker chr15:1783670 are selected as AA individuals.

[0014] The fifth technical objective of this invention is to provide the application of the molecular marker combination or the primers of the molecular marker in the breeding of turbot body length trait; the application is to select individuals with the dominant genotype combination in the molecular marker combination as parents for breeding body length trait.

[0015] The sixth technical objective of this invention is to provide the application of the primers in the breeding of turbot body length traits, using the primers to amplify turbot individuals and screen individuals with the dominant genotype among the molecular markers as parents for breeding body length traits.

[0016] The beneficial effects of this invention compared with the prior art are as follows: This invention uses genome-wide association analysis to analyze the body length trait of turbot and obtains SNP molecular markers that are significantly associated with the body length trait, including at least one of the six SNP loci. This can be applied to molecular-assisted breeding of turbot. By screening at the genome level using the breeding method disclosed in this invention, the breeding cycle can be significantly shortened, the dependence on phenotypic traits can be reduced, and the efficiency of selection breeding can be improved, which has good application prospects. Attached Figure Description

[0017] Figure 1 Body length frequency distribution diagram;

[0018] Figure 2 The Manhattan plot uses chromosomes as the horizontal axis and the vertical axis to show the -log of each SNP. 10 (p) value. Detailed Implementation

[0019] To better understand the technical content of this invention, specific embodiments and accompanying drawings are described below. Unless otherwise specified, the experimental methods in the following examples are conventional methods. The instruments, reagents, and kits used in the experiments are all commercially available.

[0020] I. Materials and Methods

[0021] The turbot used in the experiment came from Yantai Development Zone Tianyuan Aquatic Products Co., Ltd. One thousand 4-month-old turbot were used, with an initial weight of 22±0.43 cm, a body length of 8.55±0.25 cm, and a condition factor of 3.22±0.18 g / cm². 3 Turbot were fed with 50% soybean meal instead of fishmeal. The feed composition is shown in Table 1. After 8 weeks, 351 fish were randomly selected from the fish population.

[0022] Table 1 Feed composition

[0023] .

[0024] II. Phenotypic Trait Analysis

[0025] The body length of the collected samples was measured, and the phenotypic data were statistically analyzed, including minimum, maximum, mean, standard deviation, and coefficient of variation. The results are shown in Table 2. The normality of the turbot body length measurement data was verified, and the results are as follows: Figure 1 Table 2;

[0026] Table 2 Body length data of turbot samples

[0027] .

[0028] III. Genomic Data

[0029] Genomic DNA was extracted from the obtained turbot samples and sent to Meiji Biotechnology Co., Ltd. for whole-genome resequencing and variant detection. After the DNBSeq T7TM sequencing data was processed, quality control was performed on the processed data to filter out low-quality data and obtain high-quality data. The clean data was aligned to the reference genome sequence using BWA-MEME software to determine the sequence location. The BAM file was corrected using the Best Practices workflow of GATK software to obtain the vcf file of the population genotype.

[0030] A total of 2,277.59 G reads were obtained, with a sequencing Q30 of 94.91% and a GC content of 42.99%. Through variant detection analysis, a total of 3,952,962 SNPs and 1,026,431 InDels were obtained.

[0031] IV. Genome-wide association analysis

[0032] Based on the variation data (VCF format) of the experimental population, we standardized the genome-wide SNP data and performed GWAS analysis. First, we used bcftools to extract variant sites on autosomes and retain SNP types. Then, we used VCFtools for strict quality control, retaining only bis-allelic SNPs (min / max alleles = 2), with a deletion rate ≤5%, minor allele frequency (MAF) ≥0.05, variation quality value ≥30, and average sequencing depth ≥10. After quality control, the genotype data was imputed using Beagle (v27Feb25.75f) and converted to PLINK binary format for subsequent analysis.

[0033] Based on the quality-controlled VCF data, the first 10 principal components (PC1–PC10) were calculated using PLINK to correct for population structure. The first 8 principal components were then identified using a rockfall plot and included as covariates in the model. The genomic relationship matrix (GRM) was constructed using GEMMA (-gk 2), and genome-wide association analysis was subsequently performed within GEMMA based on a linear mixture model (LMM). Phenotypic values ​​were used as the response variable, and PC1–PC8 were used as covariates. Simultaneously, inter-individual phylogenetic relationships were corrected for, effectively reducing the potential interference of population structure and genetic correlations on the association results.

[0034] Linear Mixture Model (LMM) can be formally represented as:

[0035] ;

[0036] in, This is a vector of phenotypic values ​​for the individual's body length trait. Design a matrix for fixed effects. This is a fixed effects vector, including the population mean and covariates (such as population structure variables obtained from principal component analysis). Design a matrix for random effects; This represents the additive genetic effect in an individual. Random residual vector. Genome-wide significance threshold adjusted using Bonferroni: threshold is... The result is the Manhattan diagram. Figure 2 Based on the absolute value of the effect (Beta), the core SNP loci that contributed most to the increase in body length of turbot were screened. The screened SNPs were further screened, and the final 6 SNP loci are shown in Table 3. The genotypes and phenotypic traits corresponding to the screened SNPs are shown in Table 4, and the molecular marker locus information is shown in Table 5.

[0037] Table 3. Six SNP sites

[0038] ;

[0039] Table 4. Screening for SNPs and their corresponding genotypes and phenotypic traits

[0040] ;

[0041] Note: The enhancement or reduction of effect refers to the quality of the trait after mutation at this site compared with the original genotype, that is, the body length becomes larger or smaller after mutation at this site compared with the original genotype.

[0042] Table 5. Molecular marker site information

[0043] .

[0044] V. Validation in different groups

[0045] Different batches of juvenile turbot were selected for validation. The validation population consisted of 300 turbot randomly selected from those fed a 50% soybean meal diet for 12 weeks. Body length was first statistically analyzed, including minimum, maximum, mean, standard deviation, and coefficient of variation. The results are shown in Table 6. Screening analysis was performed on the six SNP loci mentioned above.

[0046] The GG gene at the chr7:4184141 locus represents a 9.91% increase over the GA gene, indicating that individuals with the GG genotype at this locus are turbot with a dominant gene.

[0047] The TT gene at the chr7:1293734 locus is 15.42% higher than that of AA and 11.24% higher than that of AT. Individuals with the TT genotype at this locus are those with the dominant gene.

[0048] The TT gene at the chr12:871680 locus is 11.23% higher than the TA gene, indicating that this locus represents the dominant gene in turbot individuals with the TT genotype.

[0049] The TT gene at the chr12:24383879 locus was 19.83% higher than that of CC and 16.87% higher than that of CT, indicating that individuals with the TT genotype at this locus were dominant.

[0050] The TT gene at the chr15:1901316 locus was 14.35% higher than that of CC and 6.47% higher than that of TC. Individuals with the TT genotype at this locus are those with the dominant gene.

[0051] The AA gene at the chr15:1783670 locus was 11.43% higher than that of TT and 6.21% higher than that of TA, indicating that individuals with the AA genotype at this locus were dominant.

[0052] See Table 7 for specific locus and phenotypic data;

[0053] Table 6. Verification group phenotypes

[0054] ;

[0055] Table 7. Loci and Phenotypes

[0056] .

[0057] Different combinations of SNP loci have different effects on body length traits, as shown in Table 8.

[0058] Table 8. Phenotypic data of individuals with mutant combinations

[0059] ;

[0060] Note: The SNP loci for typical genotype combinations from left to right are chr7:4184141, chr7:1293734, chr12:871680, chr12:24383879, chr15:1901316, and chr15:1783670.

[0061] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection of the present invention.

Claims

1. A SNP molecular marker associated with the lower body length of turbot fed fermented soybean meal, characterized in that, The molecular marker is any one of the following: the nucleotide sequence of the molecular marker is as shown in SEQ ID NO.1, and there is an SNP site associated with the body length of turbot at position 101 of the molecular marker, the allele of the site is G or A, and the molecular marker is denoted as chr7:4184141. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

5. At position 101 of the molecular marker, there is an SNP site associated with the body length of turbot. The allele of this site is A or T. The molecular marker is denoted as chr7:1293734. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

9. At position 101 of the molecular marker, there is an SNP site associated with the body length of turbot. The allele of this site is T or A. The molecular marker is denoted as chr12:871680. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

13. At position 101 of the molecular marker, there is an SNP site associated with the body length of turbot. The allele of this site is C or T. The molecular marker is denoted as chr12:24383879. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

17. At position 101 of the molecular marker, there is an SNP site associated with the body length of turbot. The allele of this site is T or C. The molecular marker is denoted as chr15:1901316. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

21. At position 101 of the molecular marker, there is an SNP site associated with the body length of turbot. The allele of this site is T or A. The molecular marker is denoted as hr15:1783670.

2. The primers for amplifying the SNP molecular marker as described in claim 1, characterized in that, The primers are capable of amplifying the SNP molecular markers, and the nucleotide sequences of the primers are shown in SEQ ID NO.2-4, SEQ ID NO.6-8, SEQ ID NO.10-12, SEQ ID NO.14-16, SEQ ID NO.18-20 and SEQ ID NO.22-24.

3. The application of the SNP molecular marker described in claim 1 in the breeding of turbot body length trait, characterized in that, The application involves selecting individuals with a dominant genotype as breeding parents for body length traits. The individuals with the dominant genotype are turbot with the GG genotype at the chr7:4184141 locus. Turbot individuals with the TT genotype at the chr7:1293734 locus are the dominant genotype. Turbot individuals with the TT genotype at the chr12:871680 locus are the dominant genotype. Turbot individuals with the TT genotype at the chr12:24383879 locus are the dominant genotype. Turbot individuals with the TT genotype at the chr15:1901316 locus are the dominant genotype. The turbot with the AA genotype at the chr15:1783670 locus is the dominant genotype.

4. The application of the primers described in claim 2 in the breeding of turbot body length traits, characterized in that, The primers were used to amplify turbot individuals, and individuals with the dominant genotype among the SNP molecular markers described in claim 1 were selected as breeding parents for body length traits; the individuals with the dominant genotype were turbot individuals with the GG genotype at the chr7:4184141 locus. Turbot individuals with the TT genotype at the chr7:1293734 locus are the dominant genotype. Turbot individuals with the TT genotype at the chr12:871680 locus are the dominant genotype. Turbot individuals with the TT genotype at the chr12:24383879 locus are the dominant genotype. Turbot individuals with the TT genotype at the chr15:1901316 locus are the dominant genotype. The turbot with the AA genotype at the chr15:1783670 locus is the dominant genotype.

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