SNP molecular marker affecting 210-day egg number of chicken by affecting alternative splicing of capn6 gene and application thereof

Abstract

The application discloses a SNP molecular marker for affecting the number of 210-day egg production of chickens by affecting variable splicing of a CAPN6 gene and application thereof, belongs to the field of molecular genetics and agricultural animal breeding, and discloses that the SNP molecular marker is located at the position of 13053733 bp of chicken chromosome 4, the reference genome version is GRCg7b, has T / C polymorphism, and the individual with the genotype CC at the position has a higher number of 210-day egg production than the individual with other genotypes. The application constructs a molecular marker system which is significantly related to splicing expression of CAPN6 and can be used for early prediction and selection of the number of 210-day egg production of chickens, and provides a precise and efficient molecular technical path for breeding of laying hens. The application has important significance for promoting molecular marker assisted breeding of reproductive traits of laying hens.

Description

Technical Field

[0001] This invention belongs to the fields of molecular genetics and agricultural animal breeding, specifically involving a molecular genetic marker that affects the number of eggs laid in chickens at 210 days by influencing alternative splicing of the CAPN6 gene and its application. Background Technology

[0002] With the continuous advancement of livestock and poultry genetic breeding technology in my country, the egg-laying hen industry is playing an increasingly important role in ensuring the supply of animal protein, promoting the revitalization of the seed industry, and driving rural economic development. Egg production count, as a core economic trait of egg-laying hen production performance, directly relates to the utilization efficiency of breeding hens and the market output of eggs, and is one of the key indicators of focus in egg-laying hen genetic breeding. Especially under the goal of high-efficiency production, the cumulative egg production count over 210 days is widely used to measure early egg-laying capacity and selection potential, and is an important phenotypic basis for selecting high-producing egg-laying hen strains. Although traditional family breeding and phenotypic selection have achieved certain results in improving egg production performance, the low heritability of egg production traits, strong environmental sensitivity, and long testing cycles of traditional methods have significant limitations in terms of the speed and accuracy of genetic progress. Therefore, it is urgent to utilize molecular breeding methods to identify and apply functional genes and molecular genetic markers closely related to egg production regulation in order to accelerate the selection and optimization of superior egg-laying hen germplasm resources.

[0003] Among numerous candidate genes, CAPN6, as a member of the calmodulin family, has recently been found to possess non-protease-active structural regulatory functions, influencing poultry reproductive performance through its involvement in cytoskeleton remodeling, ovarian tissue development, and hormone regulation. Studies have shown that CAPN6 exhibits significant alternative splicing in chicken ovarian tissue, and different splice isoforms may play physiological roles in functional differentiation. Preliminary analysis shows a significant correlation between CAPN6 splice expression patterns and the cumulative egg production at 210 days of age, suggesting its potential important role in regulating egg production rhythm and ovarian activity. Therefore, further research is needed to explore...

[0004] The study of the regulatory mechanism of alternative splicing in the CAPN6 gene, including the discovery of functional SNP sites and the assessment of their impact on egg production traits, is expected to provide new functional markers and regulatory targets for molecular breeding of chicken egg production traits. Summary of the Invention

[0005] 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.

[0006] The technical solution of the present invention is as follows: a method for breeding chickens with a high number of eggs laid in 210 days, wherein the genotype at the 13053733bp position on chromosome 4 of the chicken sample to be tested is detected, with the reference genome version being GRCg7b, and individuals with the genotype CC or CT at this locus have a higher number of eggs laid in 210 days than individuals with the genotype TT.

[0007] 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 13053733bp on chicken chromosome 4, with a reference genome version of GRCg7b, and has T / C polymorphism. Individuals with genotype CC or CT at this site have a higher egg production number at 210 days than individuals with genotype TT.

[0008] 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 13053733bp on chicken chromosome 4, with a reference genome version of GRCg7b, and has T / C polymorphism. Individuals with genotype CC or CT at this site have a higher 210-day egg production number than individuals with genotype TT.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] This invention constructs a molecular marker system that is significantly correlated with CAPN6 splicing expression and can be used for early prediction and selection of egg production in chickens at 210 days, providing a precise and efficient molecular technology pathway for laying hen breeding. This is of great significance for promoting molecular marker-assisted breeding of reproductive traits in laying hens. Attached Figure Description

[0011] Figure 1 CAPN6 sQTL is co-located with GWAS, the number of eggs laid by chickens at 210 days.

[0012] Figure 2 Comparison of egg production numbers at different SNP loci and genotypes over 210 days. Detailed Implementation

[0013] 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.

[0014] Example 1: Mining of SNP Molecular Markers

[0015] Low-depth whole-genome resequencing and cumulative egg production phenotype collection at 210 days of age were performed on a white-feathered laying hen population (12,952 individuals). Combined with genome-wide association analysis (GWAS), a genetic signal significantly associated with egg production performance was detected at the Chr4:13053733 locus (reference chicken genome version GRCg7b). The analysis showed that the mutant allele T had a significant allele substitution effect compared to the reference allele C, resulting in a decrease of 0.45408 eggs in egg production. Figure 2 ).

[0016] Table 1. Basic information on genetic marker loci

[0017] mutation location Reference / Mutant Alleles Mutant gene frequency Mutation effect Chr4: 13053733 C / T 0.52584 -0.45408

[0018] Further analysis of alternative splicing quantitative trait loci (sQTLs) revealed that this SNP significantly affects the proportion of splice isoforms of the CAPN6 gene in ovarian tissue: the T allele can regulate the alternative use of specific exons / introns, leading to the production of functionally differentiated transcript structures of CAPN6, thereby altering its regulatory role in follicle development and egg production rhythm in ovarian tissue. Colocalization analysis showed a high degree of colocalization between this locus and the sQTL signal of the CAPN6 splice isoform, with a posterior probability (PPH4) as high as 0.94043, significantly higher than the threshold generally considered to be evidence of colocalization (typically 0.8). This indicates that this SNP likely affects egg production performance in chickens at 210 days of age by regulating the alternative splicing pattern of the CAPN6 gene. Figure 1 ).

[0019] Example 2

[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 improve the genetic progress efficiency of the overall 210-day egg production number.

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

[0029] (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. A method for breeding chickens with a high egg production rate at 210 days, characterized in that, The genotype at the 13053733 bp location on chromosome 4 of the chicken samples to be tested was analyzed. The reference genome version was GRCg7b. Individuals with the genotype CC or CT at this locus had a higher number of eggs laid at 210 days than individuals with the genotype TT. The breed of chickens was a white-feathered laying hen.

2. Application of substances detecting SNP site polymorphisms or genotypes in assisted breeding of chickens with egg production number at 210 days. The SNP site is located at 13053733 bp on chicken chromosome 4, with a reference genome version of GRCg7b, and exhibits T / C polymorphism. Individuals with genotype CC or CT at this site have a higher egg production number at 210 days than individuals with genotype TT. The chicken breed is a white-feathered laying hen.

3. 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 13053733 bp on chicken chromosome 4, with a reference genome version of GRCg7b, and exhibits T / C polymorphism. Individuals with the genotype CC or CT at this site have a higher 210-day egg production number than individuals with the genotype TT. The chicken breed is a white-feathered laying hen.

Images