CN118166111A

CN118166111A - Application of SNP genetic marker related to egg short diameter in ATP7B gene in chicken genetic breeding

Info

Publication number: CN118166111A
Application number: CN202410243720.4A
Authority: CN
Inventors: 童海兵; 郭军; 曲亮; 邵丹; 王强; 李永峰; 窦套存; 马猛
Original assignee: Jiangsu Institute Poultry Sciences
Current assignee: Jiangsu Institute Poultry Sciences
Priority date: 2024-03-04
Filing date: 2024-03-04
Publication date: 2024-06-11
Anticipated expiration: 2044-03-04
Also published as: CN118166111B

Abstract

The invention provides an application of SNP genetic markers associated with egg short paths in ATP7B genes in chicken genetic breeding, and belongs to the technical fields of livestock and poultry genetic markers and chicken genetic breeding, wherein the SNP genetic markers associated with egg short paths in the ATP7B genes comprise EB_at7b, the SNP number is rs318027552, the EB_at7b corresponds to the 170198448 th bit of chromosome 1 of version sequence information bGa lGa l 1.mat.broi ler.GRCg7b published in NCBI, and the SNP genetic markers are positioned at the downstream of gene ATP7B, and the base is G or A. The SNP genetic marker is beneficial to improving the egg short diameter genetically, and is applied to the genetic breeding of chickens, so that the improvement of the egg short diameter is facilitated, and the egg variety with longer egg short diameter is obtained.

Description

Application of SNP genetic marker related to egg short diameter in ATP7B gene in chicken genetic breeding

Technical Field

The invention belongs to the technical fields of livestock and poultry genetic markers and chicken genetic breeding, and particularly relates to application of SNP genetic markers associated with egg short diameters in ATP7B genes in chicken genetic breeding.

Background

Egg size is a key factor influencing purchasing decisions of consumers, is a main index of egg grading, and is a main selection character of layer breeding. The indexes for measuring the egg size mainly comprise: weight, egg minor diameter and egg major diameter. With the increasing wide application of machine vision technology in egg analysis, european and American layer chicken breeding companies pay more attention to the breeding of short diameters of eggs. If the short diameter of the eggs can be genetically increased, the defective egg rate can be reduced.

Disclosure of Invention

In order to genetically improve the size of the egg short diameter, the invention provides application of SNP genetic markers associated with the egg short diameter in an ATP7B gene in chicken genetic breeding, and the SNP genetic markers are beneficial to the genetic improvement of the egg short diameter, and can be applied to the genetic breeding of chickens, so that the improvement of the egg short diameter is facilitated, and the egg variety with longer egg short diameter is obtained.

The invention is realized by the following technical scheme:

The invention provides an application of SNP genetic markers associated with egg short diameters in ATP7B genes in chicken genetic breeding, wherein the SNP genetic markers associated with egg short diameters in the ATP7B genes comprise EB_atp7b, the SNP number is rs318027552, the EB_atp7b corresponds to 170198448 th bit of chromosome sense strand 1 of chicken reference genome bGalGal1.Mat. Broiler. GRCg7b version sequence information published in NCBI, and is positioned at the downstream of gene ATP7B, and the base is G or A.

Based on the same inventive concept, the invention provides an early selection method of egg short diameter characters, which comprises the steps of early selecting the egg short diameter characters based on the genotype of an SNP genetic marker EB_at7b;

the eb_at7b corresponds to chromosome 1 sense strand 170198448 of version sequence information brgalgal 1.Mat. B version of chicken reference genome, published in NCBI, downstream of gene ATP7B, where the base is G or a.

Further, the early selection method specifically includes:

Detecting the genotype of the chicken EB_at7b to be detected;

Early selecting the short diameter character of the chicken eggs to be tested based on the genotype of the EB_at7b;

Wherein, the egg short diameter of the GG genotype individual of EB_at7b is larger than that of the GA genotype individual, and the egg short diameter of the GA genotype individual is larger than that of the AA genotype individual.

Further, the detecting the genotype of the chicken EB_at7b to be detected specifically comprises:

Carrying out PCR amplification on genomic DNA of the chicken to be detected by taking PEB_A7f and PEB_A7r as primers;

sequencing the PCR amplification product to obtain the 170198448 th genotype of the chromosome 1 sense strand of the chicken to be detected;

The nucleotide sequence of PEB_A7f is shown as SEQ ID NO.1, and the nucleotide sequence of PEB_A7r is shown as SEQ ID NO. 2.

Further, the varieties of the chickens to be tested comprise Dongxiang green-shell laying hens and/or white-legged chickens.

Based on the same inventive concept, the invention provides a primer for detecting SNP genetic markers associated with egg short diameters in an ATP7B gene, wherein the primer comprises PEB_A7f and PEB_A7r, the nucleotide sequence of the PEB_A7f is shown as SEQ ID NO.1, and the nucleotide sequence of the PEB_A7r is shown as SEQ ID NO. 2;

The SNP genetic marker associated with the chicken egg short diameter in the ATP7B gene comprises EB_atp7b, wherein the EB_atp7b corresponds to the 170198448 th chromosome sense strand of version 1 sequence information of chicken reference genome bGalGalGal1. Mat. Broiler. GRCg7b published in NCBI, and is positioned downstream of the gene ATP7B, and the base is G or A.

Based on the same inventive concept, the invention provides application of a primer for detecting SNP genetic markers associated with egg short diameters in ATP7B genes in chicken genetic breeding.

Based on the same inventive concept, the invention also provides a kit comprising primers peb_a7f and peb_a7r.

Based on the same inventive concept, the invention also provides application of the kit in genetic breeding of chickens.

One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:

the SNP genetic marker related to the egg short diameter in the ATP7B gene is EB_at7b, and the dominant genotype GG individual of the EB_at7b has larger egg short diameter, so that the SNP genetic marker related to the egg short diameter in the ATP7B gene is applied to the genetic breeding of chickens, the egg short diameter is improved genetically, the egg quality is improved, and the egg variety with larger egg short diameter is obtained.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is an egg short diameter characteristic curve constructed based on a stochastic regression model;

FIG. 2 is a Manhattan diagram of an egg short-diameter-associated molecular marker;

FIG. 3 is a QQ chart of an egg short-diameter related molecular marker;

FIG. 4 is an analysis of the association of EB_at7b genetic markers with egg short diameter.

Detailed Description

The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.

Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.

The whole idea of the invention is as follows:

The short diameter of the egg belongs to the character of function value and can be repeatedly measured for many times. The analysis of the functional value traits by using a stochastic regression model, which describes the target traits in the form of regression curves by constructing a genetic covariance equation, is a current epidemic trend. Wherein the first eigenvalue of the genetic effect represents the intercept and is also the first principal component for determining the short diameter; the second characteristic value of the genetic effect represents the slope of the regression curve, namely the speed and direction of the dynamic change of the short diameter; the third eigenvalue of the genetic effect represents a quadratic coefficient, and is usually ignored in genetic evaluation due to small occupation. Therefore, the applicant believes that the egg minor diameter dynamic change rule is analyzed by using a random regression model, and the first characteristic value of the additive genetic matrix is selected, so that the egg minor diameter can be changed.

In recent years, genome-wide association analysis (Genome-wide associat ion stud ies, GWAS) has become a powerful tool for analyzing quantitative trait genetic structures of livestock and poultry. The correlation between the base variation and the phenotype value is researched by using the GWAS method, so that the molecular marker affecting the economic character can be identified, and the method is particularly suitable for the characters which are difficult to measure, the characters in the late stage of life, the restriction characters and the slaughter characters. If the egg short-diameter molecular markers can be screened through whole genome association analysis, the rapid acquisition of genetic progress through molecular marker-assisted breeding or whole genome selection is facilitated.

Based on the method, the high-density chip is used for genotyping, then the whole genome association analysis is carried out, and the SNP genetic marker associated with the egg short diameter is obtained, wherein the marker is positioned at the downstream of the gene ATP7B, and the dominant genotype GG individuals named EB_at7b and EB_at7b have larger egg short diameters, so that the method can be applied to the genetic breeding of chickens and is beneficial to the genetic promotion of the egg short diameters. Compared with the association analysis of candidate genes, the GWAS effectively solves the problem of population layering, and the positioning result is more reliable; compared with QTL linkage analysis, the GWAS marker has high density, can analyze rare and low-frequency variation, can analyze the genetic structure of complex characters, and can identify novel variation. The application of the high and new technology is a precondition for ensuring that we get reliable genetic markers.

The application of the SNP genetic markers associated with the minor axis of chicken in the ATP7B gene of the present application in chicken genetic breeding will be described in detail with reference to examples and experimental data.

Example 1

Random regression analysis yields pseudo-phenotype data:

Test data were collected from a resource community of laying hens in the poultry science institute of Jiangsu province. In short, F1 generation and F2 generation are obtained by taking Dongxiang green-shell laying hens and white-legged chickens as parents through forward and backward crossing. The test chickens are raised in a single cage with the wing numbers as marks. The feed is taken freely in the egg laying period, and the nipple drinking bowl is used for supplying water. The feed component for the laying hens comprises 16.5% of crude protein and 11 511kJ/kg of feed metabolizable energy. The chicken house is cooled by a fan and a wet curtain, and is mechanically fed and cleaned. Conventional immunization was performed according to immunization program established by poultry research in Jiangsu province. The test chickens measured the short diameter of the eggs at the age of 32 weeks to 72 weeks, each individual measured at least 2 eggs, and the longest diameter of the middle of the eggs was measured by an electronic vernier caliper on the same day as the eggs were collected.

And (3) carrying out primary screening on the pedigree records and the production data, removing obvious error and repeated data, removing outliers, and finishing the outliers into exce l table forms. After data cleaning, the resource population egg short diameter data 20584 are collected. And determining the variety, batch and henhouse as fixed effects through single-factor analysis of variance. Then, WOMBAT software is used for analyzing the egg short diameter variance component and genetic parameters of the resource group. The genetic model formula is as follows:

Wherein y _ikl is the short diameter phenotype value of the ith batch of the first Zhou Lingdi K eggs; HY _i is a batch fixation effect; b _m is the mth fixed regression coefficient; a _km is the additive effect mth random regression coefficient; p _km is the mth random regression coefficient of the permanent environmental effect; z _klm is the embedded Legendre polynomial covariate; e _ikl is the residual effect.

The invention adopts AIC criterion and BIC criterion to select the most suitable statistical model for describing the egg short diameter and compares the statistical model, so as to improve the accuracy of data analysis. Through model comparison analysis, the additive genetic effect in the egg short-diameter random regression model is preferably embedded into a 3-order Legendre polynomial, the permanent environmental effect is preferably embedded into a 3-order Legendre polynomial, and the fixed regression term is preferably embedded into a 2-order Legendre polynomial. The residual was heterogenized and split into 9 levels.

The eigenvalue and eigenvector of the additive genetic matrix are obtained by R calculation. To obtain the eigenvalue, Λe, E is calculated as the eigenvector and Λ is the legendre polynomial coefficient matrix. The characteristic curve corresponding to the egg short diameter additive genetic effect of the laying hen resource group is shown in figure 1, the whole course of the additive genetic 1 st characteristic curve is positive, and the 1 st characteristic value accounts for 96.76%. The 2 nd characteristic curve shows a declining trend with the increase of the week, and is negative after 53.5 weeks, and the 2 nd characteristic value accounts for 3%. The characteristic equation shows that the 1 st characteristic value of the additive genetic coefficient matrix is a main factor influencing the breeding value, namely the main index of the short diameter of the bred eggs, and the short diameter of the eggs can be improved by selecting the characteristic value. Thus, the present invention lists the 1 st eigenvalue as pseudo-phenotype data for GWAS analysis.

Example 2

Egg short-diameter whole genome association analysis

About 2ml of blood sample was collected from the test chicken wing vein by a disposable syringe, and was placed in a BD anticoagulant tube (Bidi medical instruments Co., ltd., suzhou) and stored at 70 ℃. Genomic DNA was extracted from blood samples, and after passing the detection by 0.8% agarose electrophoresis and UV spectrophotometry, the DNA samples were diluted to 50.+ -.5 ng/. Mu.l for genotyping on a gene chip.

Chicken high-density gene chip by utilizing Eon-flying company600K Ch icken Genotyping Array for genotyping, and performing quality control of data with reference to chip specifications, mainly comprising: quality control before parting is carried out by using APT software; quality control is carried out by PLINK, and SNP markers with detection rate lower than 0.97 and deviating from Hardy temperature Berger balance are removed; screening SNP by means of meta. R, SNP_filter. R and SNP CR and FLD information analysis; genotyping was performed with BEAGLE. 435867 SNPs and 1512 samples remained after quality control were used for subsequent analysis.

The whole genome association analysis is preceded by a multidimensional principal component analysis to eliminate false positives, with the first 5 principal components being added as covariate parameters to the fixed effect of the genetic model. And calculating independent check estimation of each SNPs locus by using an R script 'simpleM' method to obtain 59308 independent markers. Correction using Bonferroni gave a genome significant threshold of 8.43×10 ^-7 and a genome recommendation threshold of 1.69×10 ^-5. The egg short-diameter pseudo-phenotype values obtained in example 1 were analyzed with a hybrid linear model to obtain individual SNPs marker significance test P values. The matrix expression of the linear model is that,

y＝Wα+xβ+u+ε

Wherein y represents a sample phenotype value vector; w represents a covariance matrix; alpha is the intercept vector; a genotype vector for x-tag; u is a random effect vector; epsilon is the residual error.

And carrying out whole genome association analysis on the characteristic coefficient 1 of the short diameter additive genetic effect of 1512 eggs, wherein the results are shown in fig. 2 and 3. From manhattan, the presence of a significant level of genomic markers on chromosome 1 of chicken specifically mapped to the ATP7B gene. The QQ map further verifies that GWAS results are reliable. Analyzing genetic parameters by using a genome genetic relation matrix to obtain the first characteristic value genetic transmission of the egg short diameter of 0.74+/-0.03, wherein the genetic markers EB_at7b shown in the table 1 can explain the phenotype variance of 3.5%. Collecting molecular markers of the selected egg short diameter data of 32-72 weeks old statistical egg short diameter:

Table 1 molecular markers at significant genomic levels

In the table: the marker chromosome physical location is referenced to the chicken whole genome (bgagal 1.Mat. Broiller. Grcg7b).

Example 3

Verification of molecular markers

And carrying out association analysis on the Dongxiang green-shell layer chicken-white legend resource group by using the SNP molecular markers. The specific operation steps are as follows:

1) PCR primer: DNA template sequence information was downloaded from NCBI website and PCR amplification primers were designed using PRIMER PREMIER software, the primer information being shown in Table 2. PCR primers were synthesized by Biotechnology (Shanghai) Co., ltd.

TABLE 2 amplification primers for detecting egg short diameter molecular markers

2) Genomic DNA extraction: extracting genome DNA of blood sample by phenol imitation method, detecting by ultraviolet spectrophotometer, and PCR amplifying after passing agarose electrophoresis detection.

3) PCR amplification process:

The reaction system: the 10. Mu.l system includes 50ng of the identification material DNA template, 0ng of each of the forward and reverse primers, 5. Mu.L of 2 XPower TAQ MASTERM ix, and the remaining volume is made up with ultrapure water.

The reaction procedure: first, denaturation at 94℃for 30s, annealing at the temperature described in Table 2 for 30s, extension at 72℃for 30s, for a total of 5 cycles; then denatured at 94℃for 30s, annealed at a temperature as described in Table 2 for 30s, and extended at 72℃for 30s for a total of 30 cycles; extending at 72 ℃ for 5min, and preserving at 4 ℃.

4) The amplified products are sent to a sequencing company for sequence polymorphism detection.

>EB_atp7b

In the sequences, the mutation sites are marked [ ], the allelic variations are in brackets, and the primer sequences are shown by thickening the head and the tail of the sequences.

5) Correlation analysis: the test individuals all had genotypes and short diameters of eggs of 32 to 72 weeks of age, and then subjected to association analysis. As shown in FIG. 4, the dominant genotypes of the molecular markers EB_at7b were GG.

And screening to obtain SNP molecular markers related to the short diameters of the eggs through random regression analysis and whole genome association analysis. Verification experiments show that the selection of the EB_atp bSNP molecular marker favorable genotype can improve the short diameter of eggs.

The primer combination can be used for breeding the short diameter character of eggs and can also be used for preparing a kit.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

The application of SNP genetic markers associated with egg short diameters in ATP7B genes in chicken genetic breeding is characterized in that the SNP genetic markers associated with egg short diameters in the ATP7B genes comprise EB_at7b, the SNP number is rs318027552, and the EB_at7b corresponds to chicken reference genome published in NCBI

BGalGal version 1.Mat. Broler. GRCg7b sequence information, position 170198448 on the sense strand of chromosome 1, downstream of gene ATP7B, where the base is G or A.
2. An early selection method of an egg short-diameter character is characterized by comprising the steps of early selecting the egg short-diameter character based on the genotype of an SNP genetic marker EB_at7b;

The eb_at7b corresponds to the 170198448 th strand of chromosome 1, version sequence information version number 1 of chicken reference genome bGalGal. Mat. Broiler. Grcg7b published in NCBI, downstream of gene ATP7B, where the base is G or a.
3. An early selection method of an egg short diameter trait according to claim 2, characterized in that the early selection method specifically comprises:

Detecting the genotype of the chicken EB_at7b to be detected;

Early selecting the short diameter character of the chicken eggs to be tested based on the genotype of the EB_at7b;

Wherein, the egg short diameter of the GG genotype individual of EB_at7b is larger than that of the GA genotype individual, and the egg short diameter of the GA genotype individual is larger than that of the AA genotype individual.
4. The method for early selection of chicken egg short diameter trait according to claim 3, wherein the detecting the genotype of chicken eb_at7b to be detected specifically comprises:

Carrying out PCR amplification on genomic DNA of the chicken to be detected by taking PEB_A7f and PEB_A7r as primers;

sequencing the PCR amplification product to obtain the 170198448 th genotype of the chromosome 1 sense strand of the chicken to be detected;

The nucleotide sequence of PEB_A7f is shown as SEQ ID NO.1, and the nucleotide sequence of PEB_A7r is shown as SEQ ID NO. 2.
5. The method for early selection of short diameter properties of chicken according to claim 3 or 4, wherein the chicken variety to be tested comprises Dongxiang green-shell layer chicken and/or white-legged chicken.
6. The primer for detecting the SNP genetic marker associated with the short diameter of the egg in the ATP7B gene is characterized by comprising PEB_A7f and PEB_A7r, wherein the nucleotide sequence of the PEB_A7f is shown as SEQ ID NO.1, and the nucleotide sequence of the PEB_A7r is shown as SEQ ID NO. 2;

The SNP genetic marker associated with the chicken egg short diameter in the ATP7B gene comprises EB_atp7b, wherein the EB_atp7b corresponds to 170198448 th bit of the chromosome 1 sense strand of chicken reference genome bGalGal. Mat. Browser. GRCg7b version sequence information published in NCBI, and is positioned downstream of the gene ATP7B, and the base is G or A.
7. Use of the primer according to claim 6 in genetic breeding of chickens.
8. A kit comprising the primer of claim 6.
9. Use of the kit according to claim 8 in genetic breeding of chickens.