SNP molecular marker associated with condition factor of turbot (scophthalmus maximus) fed with fermented soybean meal and its application
By screening SNP molecular markers that are significantly associated with the fatness trait of turbot through genome-wide association analysis, the problem of traditional breeding methods being difficult to accurately screen individuals for efficient conversion of fermented soybean meal diets has been solved, enabling precise breeding of turbot fatness trait and shortening the breeding cycle.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- OCEAN UNIV OF CHINA
- Filing Date
- 2026-04-08
- Publication Date
- 2026-06-26
AI Technical Summary
Traditional breeding methods are insufficient to accurately screen turbot individuals with high conversion efficiency to fermented soybean meal diets. This results in significant differentiation in the growth of body condition within the population under low fishmeal and high fermented soybean meal feeding conditions, and the contribution of existing SNP loci to explain genetic variation is limited.
Genome-wide association analysis was used to screen out SNP molecular markers that were significantly associated with the condition of turbot, including 6 SNP loci and their combinations, for marker-assisted breeding. Individuals with the dominant genotype were selected as parents for breeding the condition trait.
By shortening the breeding cycle, reducing reliance on phenotypic traits, and improving selection breeding efficiency, precise screening of the fatness trait in turbot and the improvement of breeding efficiency have been achieved.
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Figure CN121992117B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular marker technology, specifically, it relates to a SNP molecular marker associated with condition factor in turbot fed fermented soybean meal and its application. Background Technology
[0002] turbot( Scophthalmus maximus Turbot 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 the growth of body condition 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 conditionability 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 fattening traits.
[0005] One of the objectives of this invention is to provide a SNP molecular marker associated with the condition of turbot fed fermented soybean meal. 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 there is an SNP site associated with the condition of turbot at position 101 of the molecular marker, the allele of which is A or G, and the molecular marker is denoted as chr11:6173666.
[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 fatness of turbot. The allele of this site is A or T. The molecular marker is denoted as chr11:6752389.
[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 fatness of turbot. The allele of this site is T or A. The molecular marker is denoted as chr11:6557587.
[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 fatness of turbot, and the allele of this site is T or C; the molecular marker is denoted as chr11:6163141.
[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 fatness of turbot, and the allele of this site is T or C; the molecular marker is denoted as chr11:6193220.
[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 fatness of turbot, and the allele of this site is T or C; the molecular marker is denoted as chr11:6730072.
[0011] A second objective of this invention is to provide a set of SNP molecular marker combinations associated with condition rating in turbot fed fermented soybean meal, wherein the SNP molecular marker combination is one of the following: chr11:6173666 and chr11:6752389; chr11:6173666 and chr11:6193220; chr11:6173666, chr11:6752389 and chr11:6557587; hr11:6173666, chr11:6752389, chr11:6557587 and chr11:6163141; The SNP molecular markers are defined as follows: chr11:6752389, chr11:6557587, chr11:6163141, and chr11:6193220; chr11:6173666, chr11:6752389, chr11:6557587, chr11:6163141, and chr11:6193220; chr11:6173666, chr11:6752389, chr11:6557587, chr11:6163141, chr11:6193220, and chr11:6730072.
[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 molecular marker.
[0013] The fourth technical objective of this invention is to provide the application of the SNP molecular marker in the breeding of turbot for the condition trait; the application involves selecting individuals with the dominant genotype in the molecular marker as parents for breeding the condition trait. Examples include chr11:6173666-AA, chr11:6752389-AA, chr11:6557587-TT, chr11:6163141-TT, chr11:6193220-TT, or chr11:6730072-TT. Individuals with the original genotype AA based on the molecular marker chr11:6173666, the original genotype AA based on the molecular marker chr11:6752389, the original genotype TT based on the molecular marker chr11:6557587, the original genotype TT based on the molecular marker chr11:6163141, the original genotype TT based on the molecular marker chr11:6193220, and the original genotype TT based on the molecular marker chr11:6730072 were selected.
[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 for the condition trait; the application is to select individuals with the genotype of the molecular marker combination as parents for breeding the condition trait.
[0015] The sixth technical objective of this invention is to provide the application of the primers in the breeding of turbot for the condition trait, using the primers to amplify turbot individuals and screening individuals with the original genotype in the molecular markers as parents for breeding the condition trait.
[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 condition of turbot and obtains SNP molecular markers that are significantly associated with condition, 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 This is a frequency distribution diagram of body fat percentage;
[0018] Figure 2 The Hatton plot uses chromosomes as the horizontal axis and the vertical axis to represent 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 group. The condition factor, also known as the body fat coefficient, is defined as the ratio of the fish's body weight to the cube of its body length. The calculation formula is: K = W / L 3 Where: K: fatness, W: total weight of the fish (in grams), L: body length of the fish (in centimeters).
[0022] Table 1 Feed composition
[0023] .
[0024] II. Phenotypic Trait Analysis
[0025] The body condition 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 condition measurement data was verified, and the results are as follows: Figure 1 Table 2;
[0026] Table 2. Fattening 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 body condition trait in an individual. 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 improving the condition 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. 6 SNP sites
[0038] ;
[0039] Table 4. Screening for SNPs and their corresponding genotypes and phenotypic traits
[0040] ;
[0041] Note: The reduction in efficacy refers to the trait resulting from the mutation at this locus compared to the original genotype; that is, a decrease in body condition after the mutation at this locus.
[0042] Table 5. Information on 6 molecular marker sites
[0043] .
[0044] IV. 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. First, condition factor was 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 AA gene at the chr11:6173666 locus is 4.15% more dominant than the AG gene; therefore, individuals with the AA gene at this locus are individuals with the dominant genotype.
[0047] The AA gene at the chr11:6752389 locus is 3.95% more dominant than the AT gene; therefore, individuals with the AA gene at this locus are individuals with the dominant genotype.
[0048] The TT gene at the chr11:6557587 locus is 9.17% higher than that of AA and 4.69% higher than that of TA; therefore, individuals with the TT gene at this locus are the dominant genotype.
[0049] The TT gene at the chr11:6163141 locus is 9.69% higher than that of CC and 4.24% higher than that of TC; therefore, individuals with the TT gene at this locus are the dominant genotype.
[0050] The TT gene at the chr11:6193220 locus is 3.79% higher than the TC gene; therefore, individuals with the TT gene at this locus are the dominant genotype.
[0051] The TT gene at the chr11:6730072 locus is 4.36% higher than the TC gene; therefore, individuals with the TT gene at this locus are the dominant genotype.
[0052] See Table 7 for specific locus and phenotypic data;
[0053] Table 6. Validation group phenotypes
[0054] ;
[0055] Table 7. Loci and Phenotypes
[0056] .
[0057] Different combinations of SNP loci have different effects on the conditionality trait, 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 chr11:6173666, chr11:6752389, chr11:6557587, chr11:6163141, chr11:6193220, and chr11:6730072.
[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 condition factor in 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 fatness of turbot at position 101 of the molecular marker, the allele of the site is A or G, and the molecular marker is denoted as chr11:6173666. 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 fatness of turbot. The allele of this site is A or T. The molecular marker is denoted as chr11:6752389. 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 fatness of turbot. The allele of this site is T or A. The molecular marker is denoted as chr11:6557587. 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 fatness of turbot, and the allele of this site is T or C; the molecular marker is denoted as chr11:6163141. 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 fatness of turbot, and the allele of this site is T or C; the molecular marker is denoted as chr11:6193220. 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 fatness of turbot, and the allele of this site is T or C; the molecular marker is denoted as chr11:6730072.
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 primer sequences 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's fatness trait, characterized in that, The application involves selecting individuals with a dominant genotype from the corresponding molecular markers as breeding parents for the conditionality trait; the dominant genotype individuals are those with the AA gene at the chr11:6173666 locus. Individuals carrying the AA gene at the chr11:6752389 locus are the dominant genotype individuals; Individuals with the TT gene at the chr11:6557587 locus are the dominant genotype individuals; Individuals carrying the TT gene at the chr11:6163141 locus are the dominant genotype individuals; Individuals carrying the TT gene at the chr11:6193220 locus are the dominant genotype individuals; Individuals with the TT gene at the chr11:6730072 locus are the dominant genotype.
4. The application of the primers described in claim 2 in the breeding of turbot's fatness 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 the conditionality trait; the dominant genotype individuals were those with the AA gene at the chr11:6173666 locus. Individuals carrying the AA gene at the chr11:6752389 locus are the dominant genotype individuals; Individuals with the TT gene at the chr11:6557587 locus are the dominant genotype individuals; Individuals carrying the TT gene at the chr11:6163141 locus are the dominant genotype individuals; Individuals carrying the TT gene at the chr11:6193220 locus are the dominant genotype individuals; Individuals with the TT gene at the chr11:6730072 locus are the dominant genotype.