SNP (Single Nucleotide Polymorphism) molecular marker for influencing 210-day-old laying number of chicken by influencing R3HCC1L gene expression and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker for influencing the 210-day-old laying number of chicken by influencing R3HCC1L gene expression and application thereof, and belongs to the fields of molecular genetics and agricultural animal breeding, the SNP site is located at the position of a chicken No.6 chromosome 22973843bp, the reference genome version is GRCg7b, the basic group at the position has T / C polymorphism, and the SNP molecular marker is used for identifying the laying number of the chicken at the 210-day-old laying number of the chicken at the 210-day-old laying number of the chicken at the 210-day-old laying number of the chicken at the 2973843bp. The individual with the genotype of TT or CT at the position has more egg laying number than the individual with the genotype of CC at the age of 210 days. The invention constructs the SNP molecular marker obviously related to the mRNA expression level of the R3HCC1L gene, and the SNP molecular marker can be used for prediction of the number of laying hens at the age of 210 days and molecular marker-assisted selection, thereby providing a feasible and efficient technical path for poultry breeding practice.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of molecular genetics and agricultural animal breeding, and particularly relates to a molecular genetic marker affecting 210-day egg number of chicken by affecting the expression of R3HCC1L gene and application thereof. BACKGROUND

[0002] With the continuous progress of China's livestock genetic breeding technology, the egg industry plays an important role in ensuring animal protein supply and promoting rural economic development. As one of the core economic traits for measuring the production capacity of laying hens, egg production performance directly affects the cost of feed per unit, breeding efficiency and market supply stability, and is an important index in the breeding of laying hens. In production practice, 210-day egg number is often used as an important evaluation parameter for measuring the level of early peak and sustained egg production. Although traditional family selection and phenotypic selection have achieved certain results in improving egg production performance, it is difficult to further achieve precise and efficient selection due to environmental interference, long determination period and low breeding efficiency. Therefore, it is urgent to identify and apply functional genes and genetic markers closely related to egg production performance by means of molecular breeding to accelerate the breeding process of excellent germplasm.

[0003] Among the numerous candidate genes, R3HCC1L (R3H contains C-terminal class 1 domain protein) has been found to be involved in cell proliferation, differentiation and tissue function maintenance and other biological processes as an important factor in RNA processing and gene expression regulation. Recent studies suggest that R3HCC1L has certain expression specificity in the reproductive system of poultry, and its expression level may affect ovarian function and follicular development rhythm, thereby indirectly regulating egg production performance. The genetic variation of R3HCC1L is significantly related to 210-day egg number, suggesting that this gene may be an important molecular regulatory factor affecting the formation of early egg production peak. Therefore, mining regulatory SNP sites around the R3HCC1L gene and evaluating their effects on 210-day egg number are expected to provide new molecular targets for the breeding of laying hens. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a SNP site significantly related to 210-day egg number of chicken and its application in molecular marker-assisted breeding.

[0005] The technical solution of the present application is the application of a substance for detecting SNP site polymorphism or genotype in the assisted breeding of 210-day egg number of chicken. The SNP site is located at position 22973843bp on chicken chromosome 6, and the reference genome version is GRCg7b. The base at this position has T / C polymorphism. The genotype of the SNP site is detected using the substance, and individuals with TT or CT genotype at this position have higher 210-day egg number than individuals with CC genotype.

[0006] The application of a substance for detecting SNP site polymorphism or genotype in the preparation of a chicken 210-day egg number trait assisted selection kit, the SNP site is located at the position of 22973843bp of chicken chromosome 6, the reference genome version is GRCg7b, the base at this position has T / C polymorphism, and the individual with TT or CT genotype at this position has more egg number at 210 days than the individual with CC genotype.

[0007] A chicken 210-day egg number trait molecular assisted selection method, the genotype of the 22973843bp position of chromosome 6 of the chicken sample to be detected is detected, the reference genome version is GRCg7b, and the individual with TT or CT genotype at this position has higher 210-day egg number than the individual with CC genotype.

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

[0009] The application constructs an SNP molecular marker which is significantly related to the mRNA expression level of R3HCC1L gene, and can be used for chicken 210-day egg number prediction and molecular marker assisted selection, and provides a feasible and efficient technical path for poultry breeding practice. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 The R3HCC1L eQTL is co-located with the chicken 210-day egg number GWAS.

[0011] Figure 2 The 210-day egg number of the three genotypes of the SNP site is compared. DETAILED DESCRIPTION

[0012] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified.

[0013] Example 1: Mining of SNP molecular markers

[0014] Low-depth whole genome resequencing and 210-day egg number phenotype collection are performed on a white-feathered laying hen population (a total of 12,952 individuals), and combined with whole genome association analysis (GWAS), a mutation signal significantly associated with chicken 210-day egg number is detected at the Chr6:22973843 site (reference genome version GRCg7b), and the allelic substitution effect of the mutant allele T relative to the reference allele C is 0.8119 (a positive effect value indicates an increase in egg number, and a negative effect value indicates a decrease in egg number). The reference allele of the site is C, the mutant allele is T, and the mutant gene frequency is 0.398291.Figure 2 ).

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

[0016] Mutation position Reference / mutant allele Mutant gene frequency Mutation effect Chr6:22973843 C / T 0.398291 0.8119 pieces

[0017] Further eQTL analysis showed that this SNP significantly affected the mRNA expression level of the R3HCC1L gene: the T allele significantly upregulated R3HCC1L expression. Colocalization analysis revealed that this site was highly colocalized with the quantitative trait locus (eQTL) for R3HCC1L gene expression. Figure 1 The posterior probability (PPH4) was as high as 0.9922215, which is significantly higher than the threshold (generally 0.8) that is usually considered to have evidence of colocalization. This indicates that this site is likely to affect the egg production performance of chickens at 210 days of age by regulating the expression level of the R3HCC1L gene.

[0018] Example 2: Detection of SNP molecular markers

[0019] Genotypes at loci were detected using the PCR-RFLP method.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] Example 3: Application of SNP molecular markers

[0027] This molecular marker was used in chicken breeding populations for assisted selection to increase the number of eggs laid by hens at 210 days.

[0028] (1) The genotype of the SNP locus in chicken samples was tested. Individuals with the genotype TT or TC at this location had more eggs laid in 210 days than those with the CC genotype.

[0029] (2) The substance used to detect the genotype of this site can be prepared into a kit for the selection of the number of eggs laid by chickens in 210 days.

Claims

1. Application of a substance for detecting SNP site polymorphism or genotype in assisted breeding of egg production at 210 days of age in chickens. The SNP site is located at 22973843 bp on chicken chromosome 6, with a reference genome version of GRCg7b. The bases at this site exhibit T / C polymorphism. The substance is used to detect the genotype of the SNP site. Individuals with the genotype TT or CT at this site have a higher egg production at 210 days of age than individuals with the genotype CC.

2. Application of substances that detect SNP site polymorphism or genotype in the preparation of a chicken egg production trait-assisted breeding kit at 210 days of age. The SNP site is located at position 22973843bp on chicken chromosome 6, with the reference genome version being GRCg7b. The bases at this position exhibit T / C polymorphism. Individuals with the genotype TT or CT at this position have a higher egg production number at 210 days of age than individuals with the CC genotype.

3. A method for molecular-assisted breeding of chickens at 210 days of age for the trait of egg production, characterized in that, The genotype at the 22973843bp position on chromosome 6 of the chicken samples to be tested was analyzed. The reference genome version was GRCg7b. Individuals with the TT or CT genotype at this position had a higher number of eggs laid at 210 days of age than individuals with the CC genotype.

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