CN118389709B - A molecular marker of FAM124A gene related to chicken body weight traits and its application - Google Patents

Abstract

The invention relates to a FAM124A gene molecular marker related to chicken weight traits and application thereof, and belongs to the technical field of biology. The molecular marker is positioned at 171073681 base of chicken reference genome GRCg a version 1 chromosome, has C/T polymorphism and is positioned near FAM124A gene; the molecular marker has three genotypes of C/C, C/T and T/T, the weight of the T/T genotype chicken at the 8 th week is higher than that of the C/T genotype chicken and the C/C genotype chicken, and the weight of the C/T genotype chicken at the 8 th week is higher than that of the C/C genotype chicken. The molecular marker can be applied to breeding individuals of yellow-feather broilers with the weight of 8 weeks, and can improve the uniformity of the weight of the yellow-feather broilers with the weight of 8 weeks; can be applied to early selection of yellow-feather broilers, is beneficial to accelerating breeding progress, improving breeding accuracy and reducing breeding cost.

Description

A molecular marker of FAM124A gene related to chicken body weight traits and its application

Technical Field

The invention relates to a FAM124A gene molecular marker related to chicken body weight traits and application thereof, belonging to the field of biotechnology.

Background Art

Compared with fast-growing large white-feathered broilers, high-quality yellow-feathered broilers have better meat flavor, and have the advantages of tolerance to roughage, disease resistance and strong adaptability, which are more popular with consumers. However, the long breeding cycle and slow growth rate of high-quality yellow-feathered broilers result in high production costs, which limits the development of the high-quality yellow-feathered broiler industry. The 8th week of age is the critical period for the selection and retention of yellow-feathered broilers. The body weight at the 8th week of age is significantly positively correlated with the weight at the beginning of laying. In addition, it also affects the age at the beginning of laying, the weight of the first egg, and the egg production during the entire laying cycle of the group, and is one of the breeding indicators. Generally speaking, as the weight increases at the 8th week of age, the age at the beginning of laying will be delayed, and the weight of the first egg will also be positively correlated. Excessive or too small weight at the 8th week of age will affect the quality of the breeding eggs, thereby affecting breeding. When the rearing chickens reach or exceed the standard weight, they will start laying in time; individuals whose weight at the 8th week of age is lower than the standard weight may start laying late, the peak of egg laying will be postponed, and the egg production rate and survival rate will decrease. The laying weight can only be measured at the beginning of laying. If the breeders are selected at the beginning of laying, it will cause a huge waste of feeding costs. The consistency of the laying time of high-quality broiler breeders can be controlled by selecting the weight size and uniformity at the 8th week of age before laying.

Single nucleotide polymorphism (SNP) refers to the variation of a single nucleotide in the genome. It can be stably inherited by offspring during the transmission of genetic material and is easy to detect. Therefore, it can be used as a molecular marker for population selection and seed conservation. Through marker-assisted selection, early selection can be achieved to achieve the goal of uniform weight.

The FAM124A (family with sequence similarity 124 member A) gene is a member of the FAM family. Many studies have reported that FAM family genes are associated with body weight, such as FAM184A. TWAS results showed that FAM124A was significantly associated with body weight, initial laying weight, weight gain, leg muscle and breast muscle weight, indicating that this gene may be an important candidate gene for regulating chicken body weight.

Summary of the invention

The purpose of the present invention is to address the defects of the prior art, propose a FAM124A gene molecular marker related to chicken body weight traits and its application, so as to improve the breeding efficiency of chickens.

The present invention measures the body weight trait of yellow-feathered broiler chickens at the 8th week of age, uses whole genome resequencing technology to perform SNP genotyping, and obtains significantly correlated FAM124A gene molecular markers through whole genome association analysis screening, thereby providing new gene and molecular marker resources for the breeding of chickens' 8th week of age body weight trait.

The present invention solves the technical problem through the following technical scheme: a FAM124A gene molecular marker significantly correlated with the weight trait of chickens at the 8th week of age is provided, the SNP molecular marker is located at the 171073681st base of chromosome 1 of the chicken reference genome GRCg6a version 1, there is a C/T polymorphism, and the sequence is the 142nd base shown in SEQ ID NO:3 or SEQ ID NO:4; the deoxyribonucleotide sequence of the chicken DNA specific primer pair required for the molecular marker detection is shown in SEQ ID NO:1 and SEQ ID NO:2.

The present invention further provides an application of the above-mentioned FAM124A gene molecular marker primers, including for detecting SNP genotypes related to the weight of chickens at the 8th week of age. Specifically, it is applied to a method for detecting FAM124A gene molecular markers related to the weight trait of chickens at the 8th week of age by PCR amplification combined with Sanger sequencing, and the detection method comprises the following steps:

The first step is to provide a chicken DNA sample to be tested, and perform PCR amplification using a DNA-specific primer pair to obtain an amplified product;

The second step is to perform Sanger sequencing on the PCR products.

The third step is to determine the molecular marker genotype of the target site based on the sequencing results of the second step.

In the first step of the above method, the deoxyribonucleotide sequence of the chicken DNA specific primer pair is

Upstream primer: 5’-CAGAACCTCCAAGTCCTTCTCA-3’ (SEQ ID NO: 1)

The downstream primer is composed of 5'-CAGGGAAGAAGACAAAGCAAGC-3 (SEQ ID NO: 2). The amplified product is 343 bp in length and includes the 171073681st base on chicken chromosome 1.

The final concentration of the reaction system is calculated based on 25 μl:

Chicken DNA to be tested 50 ng

2 x Accurate Taq Master Mix 12.5 μl

Upstream primer 1 μl

Downstream primer 1 μl

Add sterile water to make up to 25 μl.

The reaction conditions of the PCR amplification are: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 60 seconds, for a total of 30 cycles; extension at 72°C for 2 minutes; and storage at 20°C.

The nucleotide sequence of the amplified product is shown in SEQ ID NO:3 or SEQ ID NO:4.

In the third step, the judgment criterion is that the weight of chickens with T/T genotype at the 8th week of age is higher than that of individuals with C/T genotype and C/C genotype, and the weight of chickens with C/T genotype at the 8th week of age is higher than that of individuals with C/C genotype.

The present invention has identified for the first time the FAM124A gene molecular marker associated with the weight trait of chickens at the 8th week of age. Based on the genotype of the specific SNP molecular marker, it is found that the weight of chickens with T/T genotype at the 8th week of age is higher than that of individuals with C/T genotype and C/C genotype, and the weight of chickens with C/T genotype at the 8th week of age is higher than that of individuals with C/C genotype. By using the genomic DNA of the chicken to be tested as a template, using a specific primer pair for PCR amplification, and then performing Sanger sequencing and SNP genotyping on the PCR amplification product, the genotype of the SNP molecular marker can be used to select the weight trait of chickens at the 8th week of age. In breeding, according to the breeding goals, specific individuals of the T/T genotype, C/C genotype or C/T genotype are retained, and the beneficial effect is that the weight trait of yellow-feathered broilers at the 8th week of age can be efficiently and quickly identified, providing a scientific basis for the early selection and breeding of yellow-feathered broilers. In addition, the detection method disclosed in the present invention is simple and easy to operate, and can be carried out in the laboratory.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a Manhattan plot of the GWAS analysis of body weight traits at 8 weeks of age.

FIG. 2 is a graph showing the correlation between the body weight at 8 weeks of age and the body weight at the beginning of calving.

FIG. 3 is the Sanger sequencing results of the PCR amplification products of the three genotypes.

FIG. 4 is a phenotypic distribution diagram of individuals with three different genotypes of the FAM124A gene molecular marker.

DETAILED DESCRIPTION

The following embodiments are applicable to the breeding of yellow-feathered broiler chickens.

Example

In this example, the body weight of yellow-feathered broiler Suqin No. 3 L-line hens at 8 weeks of age was measured, SNP genotyping was performed using whole genome resequencing technology, and the FAM124A gene molecular marker significantly correlated with the body weight trait at 8 weeks of age was screened through whole genome association analysis. The results are shown in Figure 1, and the body weight at 8 weeks of age is highly correlated with the body weight at the beginning of laying, as shown in Figure 2.

In this example, the following experiments were performed to identify and apply the FAM124A gene molecular marker associated with the chicken body weight trait.

1. Phenotype Collection and Genotype Analysis

(1) Experimental Materials

630 Suqin No. 3 yellow-feathered broiler L-line hens were selected as experimental animals and raised under the same feeding conditions with free access to food and water throughout the process. At the 8th week of age, the weight of each chicken was measured as the phenotypic data of the weight trait at the 8th week of age.

(2) Extraction of genomic DNA

Blood was collected from the subwing vein of the individual to be tested, lysed after anticoagulation, digested with proteinase K, extracted with saturated sodium chloride method, dissolved in TE and stored at -20℃.

(3) PCR amplification

Using the genomic DNA extracted as above as a template, a fragment containing the 171073681st base of chromosome 1 was amplified.

Upstream primer: 5’-CAGAACCTCCAAGTCCTTCTCA-3’ (SEQ ID NO: 1)

Downstream primer: 5’-CAGGGAAGAAGACAAAGCAAGC-3’ (SEQ ID NO: 2)

The final concentration of the reaction system (25 μl) is:

DNA to be tested 50 ng

2 x Accurate Taq Master Mix 12.5 μl

Upstream primer 1 μl

Downstream primer 1 μl

Add sterile water to make up to 25 μl.

The reaction conditions of PCR amplification are: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30sec, 60℃ annealing for 30sec, 72℃ extension for 60sec, 30 cycles in total; 72℃ extension for 2min; 20℃ storage; 10μl is taken for agarose detection, and a single target band with a length of 343bp is amplified, which contains the 171073681st base of chicken chromosome 1. The nucleotide sequence of the amplified product is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

(4) Sequencing verification

The PCR products of each sample were subjected to Sanger sequencing, and the sequencing peak graph is shown in FIG3 .

2. Correlation Analysis

403 Suqin No. 3 L-line hens with clear records of weight phenotype at the 8th week of age were selected for correlation analysis. The ANOVA test function of R 4.2 software was used for statistical test, and the pairwise mean comparison mode was selected to perform statistical test on the genotype and weight trait at the 8th week of age of the test chickens. P<0.05 indicated significant differences, and P<0.01 indicated extremely significant differences. The results are shown in Table 1 and Figure 4. Among the tested individuals, there were 32 C/C genotypes, 162 C/T genotypes, and 209 T/T genotypes. The weight of the three genotypes at the 8th week of age was significantly different (P<0.05). The average weight of the T/T genotype individuals at the 8th week of age was 753.48g, which was 34.34g higher than that of the C/T genotype individuals (P<0.05) and 62.82g higher than that of the C/C genotype individuals (P<0.05). The average weight of C/T genotype individuals at 8 weeks of age was 719.14g, which was 28.48g higher than that of C/C genotype individuals (P<0.05). The results showed that the chicken FAM124A gene molecular marker was significantly correlated with the phenotype of chicken weight at 8 weeks of age. According to the actual breeding goals, T/T genotype individuals can be selected to improve the weight of high-quality broiler breeders at 8 weeks of age, improve the overall uniformity of weight at 8 weeks of age, and improve breeding efficiency.

Table 1. Association analysis between FAM124A gene molecular markers and body weight traits at 8 weeks of age

Note: Data in the same column with the same letters on their shoulders indicate no significant difference, while data with different letters on their shoulders indicate significant difference (P<0.05).

In addition to the above implementations, the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims (6)

1. An application of primers for a FAM124A gene molecular marker related to a chicken weight trait in detecting a chicken weight trait, characterized in that: it includes a molecular detection for detecting a weight trait related to a yellow-feathered broiler Suqin No. 3 hen at 8 weeks of age, wherein the molecular marker is located at base 171073681 of chromosome 1 of the chicken reference genome GRCg6a version, the base is C or T, and the genotype is C/C, C/T and T/T; the deoxyribonucleotide sequences of the chicken DNA-specific primer pairs required for molecular marker detection are shown in SEQ ID NO: 1 and SEQ ID NO: 2. 2. The use of primers for the FAM124A gene molecular marker associated with the chicken weight trait according to claim 1 in detecting the chicken weight trait, characterized in that the detection method comprises the following steps: The first step is to perform PCR amplification on the chicken DNA sample to be tested using a chicken DNA specific primer pair to obtain an amplified product, wherein the chicken DNA sample to be tested contains a SNP molecular marker at the 171073681st base of chicken chromosome 1; The second step is to perform Sanger sequencing on the amplified product; The third step is to determine the FAM124A gene molecular marker genotype of the target site based on the sequencing results of the second step. 3. The use of primers for the FAM124A gene molecular marker related to the chicken weight trait according to claim 2 in detecting the chicken weight trait, characterized in that: in the first step, the chicken DNA-specific primer pair consists of an upstream primer SEQ ID NO: 1 and a downstream primer SEQ ID NO: 2, and the amplified product is 329 bp in length and includes the 171073681st base on chicken chromosome 1. 4. The use of primers for the FAM124A gene molecular marker associated with the chicken weight trait according to claim 3 in detecting the chicken weight trait, characterized in that: in the first step, the final concentration of the reaction system is calculated as 25 μl: Chicken DNA to be tested 50 ng 2 x Accurate Taq Master Mix 12.5μl Upstream primer 1 μl Downstream primer 1 μl Sterile water to make up to 25 μl, The reaction conditions of the PCR amplification are: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 60 seconds, for a total of 30 cycles; extension at 72°C for 2 minutes; and storage at 20°C. 5. Use of the primers of the FAM124A gene molecular marker related to the chicken weight trait according to claim 4 in detecting the chicken weight trait, characterized in that the nucleotide sequence of the amplified product is shown as SEQ ID NO: 3 or SEQ ID NO: 4. 6. The use of primers of the FAM124A gene molecular marker associated with the chicken weight trait according to claim 4 in detecting the chicken weight trait is characterized in that: in the third step, the judgment criterion is that the weight of the chicken with T/T genotype at the 8th week of age is higher than that of the chicken with C/T genotype and the chicken with C/C genotype, and the weight of the chicken with C/T genotype at the 8th week of age is higher than that of the chicken with C/C genotype.