SNP (Single Nucleotide Polymorphism) molecular marker related to TRIM66 gene expression and application of SNP molecular marker in auxiliary breeding of 210-day egg laying number of chicken

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to TRIM66 gene expression and application of the SNP molecular marker in 210-day egg laying number assisted breeding of chickens, and belongs to the fields of molecular genetics and agricultural animal breeding, an SNP site is located at the position 9403550 of a chicken chromosome 5, a reference genome version is GRCg7b, the SNP molecular marker has G / A polymorphism, and the SNP molecular marker has a nucleotide sequence shown in SEQ ID NO.1. Compared with other genotypes, the individual with the genotype AA at the position has a higher 210-day egg laying number. According to the invention, a molecular genetic marker system which is remarkably related to the TRIM66 expression level and can be used for early prediction and selection of the 210-day laying number character of the chicken is constructed, and a feasible technical path is provided for breeding practice of high-yield laying hens. The method has important significance for developing efficient laying hen molecular marker-assisted breeding.

Description

Technical Field

[0001] This invention relates to the fields of molecular genetics and agricultural animal breeding, specifically to a molecular genetic marker that affects the number of eggs laid in chickens at 210 days by influencing the expression of the TRIM66 gene and its application, applicable to molecular marker-assisted breeding of various chicken breeds. Background Technology

[0002] Egg production count, a key economic trait for measuring laying hen productivity, directly impacts the economic value of commercial laying hens and the efficiency of breeding selection, making it one of the key target traits in laying hen breeding. Especially in the early stages of breeding stock, the cumulative egg production count over 210 days is often used as an important indicator of early reproductive performance and laying potential. Although traditional family selection and phenotypic selection have achieved some success in improving laying performance, they suffer from low efficiency and long selection cycles due to the low heritability of laying trait, significant environmental influences, and long testing periods. Therefore, it is urgent to utilize molecular breeding techniques to identify and apply functional genes and molecular genetic markers closely related to laying regulation, accelerating the screening and genetic improvement of high-quality laying hen germplasm resources.

[0003] Among numerous candidate genes, TRIM66, as a transcriptional regulator and chromatin remodeling-related protein, has been found in recent years to be closely related to reproductive development, ovarian function, and hormone regulation in mammals and poultry. Previous studies have shown that TRIM66 is actively expressed in chicken ovaries and reproductive endocrine tissues, and may affect egg production rhythm and quantity by regulating gonadotropin pathways and follicle development. Further quantitative trait locus (eQTL) studies have revealed a significant positive correlation between TRIM66 expression levels and the egg production number phenotype, suggesting that this gene may play a key role in regulating chicken egg production performance. Therefore, the discovery and validation of regulatory SNPs around the TRIM66 gene, and the assessment of its impact on 210-day egg production in chickens, are expected to provide new genetic targets for molecular-assisted breeding of high-producing egg-laying chicken breeds. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide an SNP molecular marker that is significantly correlated with the number of eggs laid by chickens at 210 days and its application in assisted breeding.

[0005] The technical solution of this invention is: the application of substances that detect SNP site polymorphism or genotype in assisted breeding of chickens with egg production number at 210 days. The SNP site is located at 9403550bp on chicken chromosome 5, with a reference genome version of GRCg7b, and has G / A polymorphism. Individuals with genotype AA or AG at this location have a higher egg production number at 210 days than those with genotype GG.

[0006] The application of substances that detect SNP site polymorphism or genotype in the preparation of a chicken 210-day egg production trait-assisted breeding kit. The SNP site is located at position 9403550bp on chicken chromosome 5, with a reference genome version of GRCg7b, and has G / A polymorphism. Individuals with genotype AA or AG at this position have a higher 210-day egg production number than those with genotype GG.

[0007] A method for breeding chickens with a high egg production trait at 210 days involves detecting the genotype at the 9403550bp location on chromosome 5 of the chicken samples to be tested, with the reference genome version being GRCg7b. Individuals with the genotype AA or AG at this location have a higher egg production trait at 210 days than those with the GG genotype.

[0008] Compared with the prior art, the present invention has the following beneficial effects:

[0009] This invention constructs a molecular genetic marker system that is significantly correlated with TRIM66 expression levels and can be used for early prediction and selection of egg production at 210 days in chickens, providing a practical technical approach for high-yielding egg-laying hen breeding. It is of great significance for conducting efficient molecular marker-assisted breeding of egg-laying hens. Attached Figure Description

[0010] Figure 1 TRIM66 eQTL is co-located with GWAS (Genomic Value Interchange) for the number of eggs laid by chickens at 210 days.

[0011] Figure 2 Comparison of egg production numbers at different SNP loci and genotypes over 210 days. Detailed Implementation

[0012] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0013] Example 1: Mining of SNP Molecular Markers

[0014] Low-depth whole-genome resequencing and phenotypic collection of cumulative egg production at 210 days of age were performed on a population of broiler chickens (12,952 individuals). Combined with genome-wide association analysis (GWAS), a mutation signal significantly associated with egg production at 210 days was detected at the Chr5:9403550 locus (reference chicken genome version GRCg7b). This indicated that the allele substitution effect of mutant locus G relative to the reference locus A was -0.4996 eggs. Figure 2 ).

[0015] Table 1. Basic information on genetic marker loci

[0016] mutation location Reference / Mutant Alleles Mutant gene frequency Mutation effect Chr5:9403550 A / G 0.2519 -0.4996

[0017] Further quantitative trait locus (eQTL) analysis revealed that this SNP significantly affected the mRNA expression level of the TRIM66 gene: the G allele significantly upregulated TRIM66 expression in reproductive tissues such as the ovary. Colocalization analysis showed that this site was highly colocalized with the eQTL signal of the TRIM66 gene, with a posterior probability (PPH4) as high as 0.88056, significantly higher than the standard threshold (generally 0.8) typically considered evidence of colocalization. This indicates that this SNP is highly likely to affect the egg production performance of chickens at 210 days of age by regulating the expression level of TRIM66. Figure 1 ).

[0018] Example 2: Detection of SNP molecular markers

[0019] Sample collection: Collect blood or other usable tissues from the sample chicken population, ensuring that the samples are uncontaminated, and label each sample with a number.

[0020] DNA extraction: Genomic DNA was extracted from the collected samples using commercial DNA extraction kits (such as Tiangen, QIAGEN, etc.) to ensure that the DNA quality met the requirements for PCR amplification.

[0021] Primer design: Design specific primers corresponding to the target SNP site. Online tools (such as Primer3, Primer-BLAST, etc.) are usually used to design primers to ensure that the primers can effectively amplify the target region.

[0022] PCR amplification: PCR amplification is performed using well-designed primers to ensure that the SNP sites of each sample are amplified under appropriate temperature and time conditions.

[0023] PCR product purification: After the PCR reaction is completed, primers, dNTPs and other impurities in the reaction system are removed using a DNA purification kit to obtain purified PCR products.

[0024] Genotyping: Using appropriate detection methods, such as restriction fragment length polymorphism (RFLP), high-resolution melting curve analysis (HRM), Sanger sequencing, etc., genotyping of PCR products is performed to determine the genotype of each sample at the target SNP locus.

[0025] Example 3: Application of SNP molecular markers

[0026] This molecular marker was used in chicken breeding populations for assisted selection to improve the genetic progress efficiency of the overall 210-day egg production number.

[0027] (1) The genotype of the SNP locus in chicken samples was tested. Individuals with the genotype AA or AG at this location had a higher number of eggs laid in 210 days than those with the GG genotype.

[0028] (2) The substance used to detect the genotype at this site can be prepared into a kit for auxiliary breeding of the egg production number trait in chickens at 210 days.

Claims

1. Application of substances that detect SNP site polymorphism or genotype in assisted breeding of chicken egg production number at 210 days. The SNP site is located at 9403550bp on chicken chromosome 5, with a reference genome version of GRCg7b, and has G / A polymorphism. Individuals with genotype AA or AG at this location have a higher egg production number at 210 days than those with genotype GG.

2. Application of substances for detecting SNP site polymorphism or genotype in the preparation of a chicken 210-day egg production trait-assisted breeding kit. The SNP site is located at position 9403550bp on chicken chromosome 5, with a reference genome version of GRCg7b, and has G / A polymorphism. Individuals with genotype AA or AG at this position have a higher 210-day egg production number than those with genotype GG.

3. A method for breeding chickens with a high egg production trait of 210 days, characterized in that, The genotype at the 9403550bp position on chromosome 5 of the chicken samples to be tested was analyzed. The reference genome version was GRCg7b. Individuals with the AA or AG genotype at this position had a higher number of eggs laid at 210 days than those with the GG genotype.

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