Application of genetic marker related to first laying day age in STON2 gene in genetic breeding of laying hens

Abstract

The invention provides application of a genetic marker related to the first laying day age in an STON2 gene to genetic breeding of laying hens, and belongs to the technical field of animal genetic breeding and biology, the genetic marker package AFEtag5 related to the first laying day age in the STON2 gene corresponds to the 40382979 site of the physical position of a fifth chromosome of chicken reference genome bGa < l1 >. Mat.broi ler.GRCg7b version sequence information published in NCBI, Ensemb l is encoded as rs315655133, Ensemb l is encoded as rs315655133, Ensemb l is encoded as rs315655133, Ensemb l is encoded as rs315655133, Ensemb l is encoded as The mutant belongs to synonymous mutation of an exon region of a gene STON2, and a basic group is T or C. The genetic marker AFEtag5 is beneficial to mining and utilization of gene resources of local chickens, is applied to genetic breeding of the chickens, is beneficial to improvement of the first laying day age, and obtains a laying hen variety with excellent first laying day age uniformity.

Description

Technical Field

[0001] The present invention belongs to the fields of animal genetic breeding and biotechnology, and particularly relates to the application of genetic markers related to the age at first egg in the STON2 gene in the genetic breeding of laying hens. Background Art

[0002] The age at first egg is a marker of hen sexual maturity and is also a commonly used indicator for laying hen breeding. Since domestication, chickens have undergone long-term artificial selection, and the age at first egg has been significantly shortened. According to statistics by Schutz et al., commercial laying hens generally start laying eggs at 20 weeks of age, while wild red junglefowl generally start laying eggs at 25 weeks of age. The age at first egg can also be used to measure flock uniformity and predict egg production performance. The onset of egg laying in chickens is a complex physiological development process that is regulated not only by external environmental factors such as light duration but also by endocrine and genetic factors. Generally speaking, only by genetically changing the relevant gene frequencies can sustainable and cumulative progress be achieved. In recent years, the GWAS method has been extended to the analysis of the genetic architecture of quantitative traits in laying hens, mainly used for research on egg production, feed utilization efficiency, and egg quality. There are few reports on using the GWAS method to analyze the genetic architecture of the age at first egg in laying hens. The age at first egg has increasingly attracted the attention of breeders. However, this trait is regulated by minor-effect polygenes, and it is difficult to make effective genetic progress using conventional breeding methods. Only by clarifying the genetic architecture of the age at first egg can the age at first egg be genetically influenced.

[0003] Dongxiang green-shell layer chickens are native to the area near Dongxiang County, Jiangxi Province. They are a famous local egg-laying chicken breed in China and a national geographical indication variety. Dongxiang green-shell layer chickens are named for laying green-shell eggs. After selection, Dongxiang green-shell layer chickens are used to construct the matching lines of Suqin green-shell layer chickens and Shendan No. 6 green-shell layer chickens, occupying an important position in the laying hen market. Systematic research on the genetic architecture of the age at first egg in Dongxiang green-shell layer chickens is of great significance for the development and utilization of Dongxiang green-shell layer chickens. The age at first egg belongs to a late trait of living organisms and can only be measured at the time of egg laying, resulting in an increase in breeding costs. Therefore, it is urgent to find suitable molecular markers to achieve early selection through genetic marker-assisted breeding or genomic selection and quickly obtain genetic progress in the age at first egg of laying hens.

[0004] In addition, for Dongxiang green-shell layer chickens, due to the lack of high-intensity chicken breeding, their production performance is not good, especially the uniformity is difficult to meet the needs of modern laying hen production. In order to meet the market demand for green-shell eggs and to explore the gene resources of local chicken breeds in China, it is urgent to find new molecular markers to genetically improve the age at first egg of laying hen populations. Summary of the Invention

[0005] In order to obtain a chicken breed with an earlier age at first egg, the present invention provides the application of genetic markers related to the age at first egg in the STON2 gene in the genetic breeding of laying hens. The genetic marker AFE_tag5 related to the age at first egg in the STON2 gene is helpful for the exploration and utilization of local chicken gene resources. Applying it to the genetic breeding of chickens is beneficial to shortening the age at first egg and obtaining a laying hen breed with excellent uniformity in the age at first egg.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides the application of genetic markers related to the age at first egg in the STON2 gene in the genetic breeding of laying hens. The genetic markers related to the age at first egg in the STON2 gene include AFE_tag5. The AFE_tag5 corresponds to the physical position at 40382979 on chromosome 5 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI. The Ensembl code is rs315655133, which belongs to a synonymous mutation in the exon region of the gene STON2, and the base here is T or C.

[0008] Based on the same inventive concept, the present invention provides an early selection method for the trait of age at first egg in chickens. The early selection method includes early selection of the trait of age at first egg in chickens based on the genotype of the genetic marker AFE_tag5;

[0009] The AFE_tag5 corresponds to the physical position at 40382979 on chromosome 5 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI. The Ensembl code is rs315655133, which belongs to a synonymous mutation in the exon region of the gene STON2, and the base here is T or C.

[0010] Furthermore, the early selection method specifically includes:

[0011] Detecting the genotype of the genetic marker AFE_tag5 in the chicken to be tested;

[0012] Based on the genotype of the AFE_tag5, early selection of the trait of age at first egg in the chicken to be tested is carried out;

[0013] Among them, the individuals with the TT genotype of the AFE_tag5 have an earlier age at first egg than those with the TC and CC genotypes.

[0014] Furthermore, the detection of the genotype of the genetic marker AFE_tag5 in the chicken to be tested specifically includes:

[0015] Using Pr_afe5_1f and Pr_afe5_1r as primers, perform PCR amplification on the genomic DNA of the chicken to be tested;

[0016] Sequence the PCR amplification product to obtain the genotype at the 40,382,979th position of the physical location of chromosome 5 of the chicken to be tested;

[0017] Among them, the nucleotide sequence of Pr_afe5_1f is as shown in SEQ ID NO.1, and the nucleotide sequence of Pr_afe5_1r is as shown in SEQ ID NO.2.

[0018] Furthermore, the breeds of the chickens to be tested include Dongxiang green-shell layer chickens and / or White Leghorn chickens.

[0019] Based on the same inventive concept, the present invention provides detection primers for genetic markers related to age at first egg in the STON2 gene. The detection primers include Pr_afe5_1f and Pr_afe5_1r. The nucleotide sequence of Pr_afe5_1f is as shown in SEQ ID NO.1, and the nucleotide sequence of Pr_afe5_1r is as shown in SEQ ID NO.2;

[0020] The genetic markers related to age at first egg in the STON2 gene include AFE_tag5. AFE_tag5 corresponds to the sequence information at the 40,382,979th position of the physical location of chromosome 5 in the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI. The Ensembl code is rs315655133, which belongs to a synonymous mutation in the exon region of the gene STON2, and the base here is T or C.

[0021] Based on the same inventive concept, the present invention provides the application of the detection primers for genetic markers related to age at first egg in the STON2 gene in chicken genetic breeding.

[0022] Based on the same inventive concept, the present invention provides a kit, and the kit contains the above-mentioned detection primers for genetic markers related to age at first egg in the STON2 gene.

[0023] Based on the same inventive concept, the present invention provides the application of the above-mentioned kit in chicken genetic breeding.

[0024] Based on the same inventive concept, the present invention provides the application of genetic markers related to the age at first egg in the STON2 gene in predicting the age at first egg of chickens. The genetic markers related to the age at first egg in the STON2 gene include AFE_tag5. The AFE_tag5 corresponds to the physical position of the 5th chromosome at the 40382979th position in the chicken reference genome bGalGal1.mat.broiler.GRCg7b version sequence information published in NCBI, and the Ensembl code is rs315655133, which belongs to a synonymous mutation in the exon region of the gene STON2, and the base here is T or C.

[0025] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0026] The application of the genetic markers related to the age at first egg in the STON2 gene of the present invention in the genetic breeding of laying hens. The genetic markers related to the age at first egg in the STON2 gene include AFE_tag5. The population with the advantageous genotype TT has an earlier age at first egg. AFE_tag5 helps to genetically improve the age at first egg of the laying hen population. Applying it to the genetic breeding of chickens is beneficial to improving the uniformity of the age at first egg of the laying hen population and meeting the needs of modern chicken farming production for the green-shelled egg chicken breed. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to these drawings.

[0028] Figure 1 It is the Manhattan plot of the GWAS analysis of the age at first egg of the resource population in Example 2 of the present invention;

[0029] Figure 2 It is the QQ plot of the GWAS analysis of the age at first egg of the resource population in Example 2 of the present invention;

[0030] Figure 3 It is the box plot of the age at first egg of individuals with different genotypes in Example 3 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention will be specifically described below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are for illustrating the present invention, rather than limiting the present invention. Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of any contradiction, this specification shall prevail.

[0032] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, equipment, etc. used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0033] The application of genetic markers related to age at first egg in the STON2 gene of the present application in the genetic breeding of laying hens will be described in detail below in conjunction with examples and experimental data.

[0034] Example 1

[0035] Construction of resource population

[0036] To analyze the genetic structure of the age at first egg trait, a laying hen resource population was constructed according to the F2 design. The local chicken breed Dongxiang Green-shelled Laying Hens and the selected breed White Leghorns were used as parents respectively, and the F1 generation was obtained through reciprocal crosses, with 1029 chicks hatched. Then, the F1 generation was used as parents to breed the F2 generation, and 1989 F2 generation chicks were obtained. The pedigree information was recorded. The experimental chickens were individually tagged with wing numbers and raised in single cages in a fully enclosed chicken house. During the laying period, artificial supplementary lighting was provided for 16 hours, and the temperature was reduced by fans and wet curtains. Routine immunization was carried out according to the immunization program formulated by the Jiangsu Institute of Poultry Sciences. The feed was from COFCO. The feed components for laying hens included 16.5% crude protein and 11511 kJ / kg feed metabolic energy. During the laying period, the chickens had free access to food and water was supplied by nipple drinkers, feed was supplied by a traveling feeder, and chicken manure was removed by a manure conveyor belt. The age at first egg of each experimental chicken was recorded.

[0037] The age at first egg data was preliminarily screened. After removing the obviously incorrect and duplicate data, the outliers were removed and organized into an excel table form. After data cleaning, one-way analysis of variance was used to statistically analyze the effects of generations and breeds on the experimental data, and the year and chicken house effects were determined as factors included in the fixed effects. The WOMBAT software was used to construct a genetic relationship matrix based on the pedigree data, calculate the heritability and breeding value. The breeding value was used as pseudo-phenotype data for the next GWAS analysis and to analyze the genetic structure of the age at first egg.

[0038] Example 2

[0039] GWAS analysis of age at first egg

[0040] The experimental chickens were adult hens of the F2 generation of the laying hen resource population constructed in Example 1. Approximately 0.5 ml of blood samples were collected from the wing veins of the experimental chickens and placed into BD anticoagulant tubes (BD Medical Devices (Suzhou) Co., Ltd.) and stored at -70°C. Genomic DNA was extracted, and after detection by 0.8% agarose gel electrophoresis and ultraviolet spectrophotometry, the DNA samples were diluted to 50 ± 5 ng / μl after passing the tests for subsequent genotyping using gene chips.

[0041] Genotyping was performed using the Affymetrix gene chip 600K Chicken Genotyping Array. Quality control of the data was carried out according to the chip instruction manual, mainly including: performing quality control before genotyping using APT software; performing quality control using PLINK to remove SNPs with a detection rate lower than 0.97 and to eliminate SNP markers deviating from the Hardy-Weinberg equilibrium; screening SNPs using metrics.R, SNP_filter.R, and SNP, CR, and FLD information analysis; and performing genotype imputation using BEAGLE. After quality control, 435,867 autosomal SNPs remained for subsequent analysis.

[0042] Before performing genome-wide association analysis, multi-dimensional principal component analysis was first carried out to eliminate false positives and population structure. The first five principal components were used as covariate parameters and added to the genetic model, and the chicken house effect was placed into the fixed effect of the model. The "simpleM" method in the R script was used to calculate the independent test estimates for each SNPs locus, and 59,308 independent markers were obtained. Using multiple corrections, the genome-wide significant threshold was 8.43×10 -7 and the genome-wide suggestive threshold was 1.69×10 -5 . The mixed linear model was used to analyze the age at first egg production, and the P values of the significance tests for each SNPs marker were obtained. The matrix expression of the linear model was

[0043]

[0044] where y represents the vector of breeding values for the age at first egg production; W represents the covariance matrix; α is the intercept vector; x is the genotype vector of the marker, β is the effect value of the marker; G is the genetic relationship matrix constructed based on the chip, u is the random effect vector (here it is the breeding value); and ε is the residual.

[0045] After GWAS screening, AFE_tag5 associated with the age at first egg production was obtained (Table 1). Genome-wide association analysis was performed on the age at first egg production of 1,512 chickens, and the results are as Figure 1 、 Figure 2 shown. As shown by Figure 1(Manhattan plot) shows that there are genomic significant level markers on chicken chromosome 5, and there are 19 SNPs around it exceeding the genomic recommended level, which can be used as evidence to support AFE_tag5. The QQ plot further verifies the reliability of the GWAS results. From Figure 2 (QQ plot), it can be seen that the vast majority of SNPs that do not deviate from the diagonal line are affected by genetic drift, and the SNPs located at the tail of the QQ plot are affected by artificial selection. The inflation coefficient was calculated to be 1.016, indicating that there is no obvious population stratification in the age at first egg trait of the resource population. Using the pedigree genetic relationship matrix to analyze genetic parameters, the heritability of age at first egg was obtained as 0.392 ± 0.046, and the AFE_tag5 genetic marker can explain 1.53% of the phenotypic variance.

[0046] Table 1 Genetic markers related to age at first egg

[0047]

[0048] Among them: The physical position of the marked chromosome refers to the chicken whole genome (bGa l Ga l 1.mat.bro i ler.GRCg7b).

[0049] Example 3

[0050] Detection and verification of genetic marker AFE_tag5

[0051] Using the above SNP genetic markers to perform candidate gene association analysis on the Dongxiang Green-shelled Chicken-White Leghorn chicken resource population. The specific operation steps are as follows:

[0052] 1) PCR primers: Download the DNA template sequence information from the NCBI website, and design PCR amplification primers with the primer premier 6.0 software. The primer information is shown in Table 2. The PCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0053] Table 2 Amplification primers used to detect the genetic marker AFE_tag5 of chicken age at first egg

[0054]

[0055] 2) Genomic DNA extraction: Extract the genomic DNA of 1512 blood samples by the phenol-chloroform method. After being detected by a UV spectrophotometer and qualified by agarose gel electrophoresis, PCR amplification is carried out.

[0056] 3) PCR amplification process:

[0057] ① Reaction system: The 10 μl system includes 50 ng of DNA template of the identification material, 10 ng of each forward and reverse primer, 5 μL of 2×power Taq MasterMix, and the remaining volume is made up with ultrapure water.

[0058] ② Reaction procedure: First, denature at 94°C for 30 s, anneal at 54.1°C for 30 s, extend at 72°C for 30 s, for a total of 5 cycles; then denature at 94°C for 30 s, anneal at 54.1°C for 30 s, extend at 72 degrees Celsius for 30 s, for a total of 30 cycles; extend at 72°C for 5 min and store at 4°C.

[0059] 4) The amplified product is sent to a sequencing company for sequence polymorphism detection.

[0060] The sequences of the amplified fragments are as follows:

[0061] >AFE_tag5

[0062]

[0063] In the sequence, the sites marked [] are mutation sites, and the allelic variations are in parentheses. The primer sequences are shown in bold and underlined at the beginning and end of the sequence.

[0064] 5) Association analysis: All the test individuals have genotypes and the age at first egg production, and then a significance test is carried out. The results are as Figure 3 shown. The age at first egg production of individuals with the CC genotype is 154.79 ± 10.95 days, that of individuals with the TC genotype is 152.28 ± 10.21 days, and that of individuals with the TT genotype is 151.35 ± 9.85 days. One-way ANOVA shows that the differences in the age at first egg production corresponding to the three genotypes are significant (P < 0.01). By using genotyping technology to increase the frequency of the T allele, the age at first egg production of laying hens can be shortened; the proportion of individuals with the CC genotype in the current population is 52.3%. Increasing the frequency of the CC genotype can further improve the uniformity of the age at first egg production of laying hens.

[0065] Finally, it should also be noted that the term "comprise", "include" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device.

[0066] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0067] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. Application of genetic markers related to age at first egg in STON2 gene in genetic breeding of laying hens, characterized in that, The genetic markers related to the age at first egg in the STON2 gene include AFE_tag5, and the AFE_tag5 corresponds to the physical position at 40382979 on chromosome 5 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b version sequence information published in NCBI, with the Ensembl code rs315655133. It belongs to a synonymous mutation in the exon region of the gene STON2, and the base here is T or C.

2. Early selection method for the trait of age at first egg in chickens, characterized in that, The early selection method includes early selection of the age at first egg trait of chickens based on the genotype of the genetic marker AFE_tag5. The AFE_tag5 corresponds to the physical position at 40382979 on chromosome 5 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b version sequence information published in NCBI, with the Ensembl code rs315655133. It belongs to a synonymous mutation in the exon region of the gene STON2, and the base here is T or C.

3. The early selection method for the age at first egg trait of chickens according to claim 2, characterized in that, The early selection method specifically includes: Detecting the genotype of the genetic marker AFE_tag5 of the chicken to be tested. Based on the genotype of the AFE_tag5, early selection of the age at first egg trait of the chicken to be tested is carried out. Among them, the age at first egg of individuals with the TT genotype of the AFE_tag5 is earlier than that of individuals with the TC and CC genotypes.

4. The early selection method for the first egg laying age trait of the chicken according to claim 3, characterized in that, The detection of the genotype of the genetic marker AFE_tag5 of the chicken to be tested specifically includes: Using Pr_afe5_1f and Pr_afe5_1r as primers to perform PCR amplification on the genomic DNA of the chicken to be tested. Sequencing the PCR amplification product to obtain the genotype at the physical position at 40382979 on chromosome 5 of the chicken to be tested. Among them, the nucleotide sequence of Pr_afe5_1f is as shown in SEQ ID NO.1, and the nucleotide sequence of Pr_afe5_1r is as shown in SEQ ID NO.

2.

5. The early selection method for the age at first egg trait of chickens according to claim 3 or 4, characterized in that The breeds of the chickens to be tested include Dongxiang Green-shelled Layer and / or White Leghorn.

6. Detection primers for genetic markers related to age at first egg in the STON2 gene, characterized in that, The detection primers include Pr_afe5_1f and Pr_afe5_1r. The nucleotide sequence of Pr_afe5_1f is as shown in SEQ ID NO.1, and the nucleotide sequence of Pr_afe5_1r is as shown in SEQ ID NO.

2. The genetic markers related to the age at first egg in the STON2 gene include AFE_tag5, and the AFE_tag5 corresponds to the physical position at 40382979 on chromosome 5 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b version sequence information published in NCBI, with the Ensembl code rs315655133. It belongs to a synonymous mutation in the exon region of the gene STON2, and the base here is T or C.

7. Use of the detection primers according to claim 6 in chicken genetic breeding.

8. A kit, characterized in that, The kit contains the detection primers according to claim 6.

9. Use of the kit according to claim 8 in chicken genetic breeding.

10. Application of genetic markers related to age at first egg in STON2 gene in predicting age at first egg of chickens, characterized in that, The genetic markers related to the age at first egg in the STON2 gene include AFE_tag5, and the AFE_tag5 corresponds to the physical position at the 40382979th site on chromosome 5 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b version sequence information published in NCBI, and the Ensembl code is rs315655133, which belongs to the synonymous mutation in the exon region of the gene STON2, and the base here is T or C.

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