Application of SNP genetic markers affecting egg-yolk ratio in chicken genetics and breeding

Abstract

The invention provides an application of a SNP genetic marker affecting an egg yolk ratio in chicken genetic breeding, belonging to the fields of chicken genetic breeding and biotechnology. The SNP genetic marker affecting the egg yolk ratio includes YR_tag22, and the YR_tag22 corresponds to the physical position 2608650 of chromosome 22 of the chicken reference genome bGalGal 1.mat.broi ler.GRCg7b version sequence information published in NCBI, and belongs to the 11th intron sequence of gene ADAM9, where the base is G or A. The SNP genetic marker helps to genetically improve the egg yolk ratio of 60-week-old chickens, and is applied to the genetic breeding of chickens, which is conducive to improving the egg yolk ratio and obtaining egg-laying chicken varieties with a high egg yolk ratio.

Description

Technical Field

[0001] The invention belongs to the field of chicken genetic breeding and biotechnology, and particularly relates to the application of a SNP genetic marker affecting the egg yolk ratio in chicken genetic breeding. Background Art

[0002] The egg market has put forward new requirements for egg quality, and more and more consumers prefer table eggs with a high yolk ratio. In order to cultivate laying hens that meet market demand, it is urgent to conduct research on the genetic structure of yolk ratio to obtain genetic markers associated with yolk ratio. Yolk ratio changes regularly with age and is a quantitative trait that is affected by both genetics and the environment. The application of genome-wide association analysis (GWAS) to analyze the genetic structure of quantitative traits is a current popular trend. The advantages of GWAS analysis are: 1) the detected markers are more polymorphic and more evenly distributed; 2) the detected marker technology has high repeatability; 3) the analysis is relatively simple and fast. In recent years, the GWAS method has been extended to the genetic analysis of poultry quantitative traits, mainly used for the study of egg production, body weight, feed utilization efficiency and egg quality. There is no research literature on the application of GWAS method to analyze the genetic law of yolk ratio. The yolk ratio trait is regulated by multiple genes with micro-effects, and it is difficult to make effective genetic progress using conventional breeding methods. The only way to meet consumer demand is to clarify the genetic structure of the yolk ratio, improve its breeding accuracy through genomic selection, and obtain new chicken breeds or supporting lines that can produce high yolk ratios. Summary of the invention

[0003] In order to solve the problem of lack of SNP genetic markers that affect the egg yolk ratio, the present invention provides the application of SNP genetic markers that affect the egg yolk ratio in chicken genetic breeding. The SNP genetic marker helps to genetically improve the egg yolk ratio of 60-week-old chickens. Applying it to the genetic breeding of chickens is beneficial to improving the egg yolk ratio and obtaining laying hen varieties with a high egg yolk ratio.

[0004] The present invention is achieved through the following technical solutions:

[0005] The present invention provides an application of a SNP genetic marker that affects the egg yolk ratio in chicken genetic breeding. The SNP genetic marker that affects the egg yolk ratio includes YR_tag22, and the YR_tag22 corresponds to the physical position 2608650 of chromosome 22 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version sequence information published in NCBI, and belongs to the 11th intron sequence of the gene ADAM9, where the base is G or A.

[0006] Based on the same inventive concept, the present invention provides an early selection method for the egg yolk ratio trait, the early selection method comprising performing early selection on the egg yolk ratio trait based on the genotype of the SNP genetic marker YR_tag22;

[0007] The YR_tag22 corresponds to the physical position 2608650 of chromosome 22 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version sequence information published in NCBI, and belongs to the intron sequence of the gene ADAM9, where the base is G or A.

[0008] Furthermore, the early selection method specifically includes:

[0009] Detect the genotype of the chicken YR_tag22 to be tested;

[0010] Performing early selection on the egg yolk ratio trait to be tested based on the genotype of YR_tag22;

[0011] Among them, the yolk ratio of the GG genotype individual of YR_tag22 at 60 weeks of age is greater than that of the GA genotype individual at 60 weeks of age, and the yolk ratio of the GA genotype individual at 60 weeks of age is greater than that of the AA genotype individual at 60 weeks of age.

[0012] Further, the detection of the genotype of the chicken YR_tag22 to be tested specifically includes:

[0013] The genomic DNA of the chicken to be tested was amplified by PCR using Pr_yr1f and Pr_yr1r as primers;

[0014] The PCR amplification product was sequenced to obtain the genotype of position 2608650 of the positive strand of chromosome 22 of the tested chicken;

[0015] Among them, the nucleotide sequence of the Pr_yr1f is shown as SEQ ID NO.1, and the nucleotide sequence of the Pr_yr1r is shown as SEQ ID NO.2.

[0016] Furthermore, the breeds of chickens to be tested include Dongxiang green-shell laying hens and / or White Leghorn chickens.

[0017] Based on the same inventive concept, the present invention provides primers for detecting SNP genetic markers that affect the egg yolk ratio, the primers include Pr_yr1f and Pr_yr1r, the nucleotide sequence of Pr_yr1f is shown in SEQ ID NO.1, and the nucleotide sequence of Pr_yr1r is shown in SEQ ID NO.2;

[0018] The SNP genetic markers affecting the egg yolk ratio include YR_tag22, which corresponds to the physical position 2608650 of chromosome 22 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version sequence information published in NCBI, and belongs to the 11th intron sequence of the gene ADAM9, where the base is G or A.

[0019] Based on the same inventive concept, the present invention provides the use of primers for detecting SNP genetic markers affecting egg yolk ratio in chicken genetic breeding.

[0020] Based on the same inventive concept, the present invention provides a kit comprising primers Pr_yr1f and Pr_yr1r.

[0021] Based on the same inventive concept, the present invention provides the use of the above-mentioned kit in chicken genetic breeding.

[0022] Based on the same inventive concept, the present invention also provides the application of SNP genetic markers that affect the egg yolk ratio in predicting the egg yolk ratio of 60-week-old eggs. The SNP genetic markers that affect the egg yolk ratio include YR_tag22, and the YR_tag22 corresponds to the physical position 2608650 of chromosome 22 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version sequence information published in NCBI, and belongs to the 11th intron sequence of the gene ADAM9, where the base is G or A.

[0023] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0024] The present invention discloses an application of a SNP genetic marker affecting the egg yolk ratio in chicken genetic breeding. The SNP genetic marker affecting the egg yolk ratio is YR_tag22. Individuals with the dominant genotype GG of YR_tag22 have a higher egg yolk ratio. Applying the SNP genetic marker to chicken genetic breeding helps to genetically improve the egg yolk ratio, thereby improving egg quality and facilitating the acquisition of laying hens with a high egg yolk ratio. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 This is the Manhattan plot of the GWAS analysis of the yolk ratio of the resource population in Example 2 of the present invention;

[0027] Figure 2 This is a QQ graph of the GWAS analysis of the yolk ratio of the resource population in Example 2 of the present invention;

[0028] Figure 3 This is a box plot of the yolk ratio of individuals with different genotypes in Example 3 of the present invention. DETAILED DESCRIPTION

[0029] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0030] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0031] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0032] The application of the SNP genetic marker affecting the egg yolk ratio in chicken genetic breeding will be described in detail below in combination with the embodiments and experimental data.

[0033] Example 1

[0034] Resource group building

[0035] The laying hen resource group was constructed according to the F2 design, with Dongxiang green-shell laying hens and white Leghorn chickens as parents, and the F1 and F2 generations were obtained by positive and negative crosses. The experimental chickens were raised in single cages in a fully enclosed chicken house, with artificial light supplementation for 16 hours during the laying period, and cooling with fans and wet curtains. Routine immunization was carried out according to the immunization program formulated by the Jiangsu Poultry Research Institute. The feed came from COFCO, and the laying hen feed components included 16.5% crude protein and 11511 kJ / kg feed metabolizable energy. During the laying period, the chickens were fed freely, with water supplied by nipple drinkers, feed supplied by driving feeders, and manure removed by chicken manure conveyor belts. At 60 weeks of age, the yolk weight and egg weight were weighed with an electronic balance, and the yolk weight divided by the egg weight was the yolk ratio.

[0036] The yolk ratio data were preliminarily screened, and after removing obviously erroneous and duplicated data, outliers were removed and organized into an Excel table. After data cleaning, 1333 records remained in the F2 generation yolk ratio data set of the resource population. Using IBM SPSS 21.0 software, QQ plot observation, kurtosis skewness statistics, and Kolmogorov-Smirnov test, it was determined that the distribution of yolk ratio data was basically unskewed, which can be used for the next GWAS analysis and analyze its genetic structure.

[0037] Example 2

[0038] GWAS analysis of egg yolk ratio at 60 weeks of age

[0039] The experimental chickens were from the laying hen resource population constructed in Example 1. About 0.5 ml of blood samples were collected from the wing vein of the experimental chickens, placed in BD anticoagulant tubes (Suzhou BD Medical Instruments Co., Ltd.), and stored at -70°C. Genomic DNA was extracted by phenol imitation method, detected by 0.8% agarose electrophoresis and ultraviolet spectrophotometry, and the DNA samples were diluted to 50±5 ng / μl after passing the test for gene chip typing.

[0040] Affymetrix GeneChip 600K Chicken Genotyping Array was used for genotyping. Data quality control was performed according to the chip manual, including: using APT software for quality control before typing; using PLINK for quality control, removing SNP markers with a detection rate lower than 0.97 and deviating from Hardy-Weinberg equilibrium; using metrics.R, SNP_filter.R and SNP, CR, and FLD information analysis to screen SNPs; and using BEAGLE for genotype filling. After quality control, 435,867 autosomal SNPs remained for subsequent analysis.

[0041] Before the genome-wide association analysis, a multidimensional principal component analysis was performed to eliminate false positives. The first five principal components were added to the genetic model as covariate parameters, and the chicken house effect model was fixed. The R script "simpleM" method was used to calculate the independent test estimate of each SNP site, and 59,308 independent markers were obtained. Bonferroni correction was used to obtain a genome-wide significant threshold of 8.43×10 -7 The recommended threshold for genome size is 1.69×10 -5 The mixed linear model was used to analyze the yolk ratio of eggs at 60 weeks of age, and the P value of the significance test of each SNPs marker was obtained. The matrix expression of the linear model is:

[0042] y=Wα+xβ+Gu+ε

[0043] Where y represents the sample phenotypic value vector; 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; ε is the residual.

[0044] After GWAS screening, YR_tag22 was obtained, which was associated with the yolk ratio at 60 weeks of age (Table 1). A genome-wide association analysis was performed on the yolk ratio of 1333 chickens at 60 weeks of age. Figure 1 , Figure 2 As shown. From the Manhattan plot, it can be seen that there is a genomic significant level marker on chromosome 22 of chicken, and there are 8 SNPs around it that exceed the recommended level of the genome, which can be used as evidence to support YR_tag22. The QQ plot further verifies that the GWAS results are reliable. From the QQ plot, it can be seen that the vast majority of SNPs are affected by genetic drift, and the SNPs at the tail of the QQ plot are affected by artificial selection. According to QQman analysis, the expansion coefficient is 1.007, indicating that there is no obvious population stratification in the yolk ratio trait of the resource population. The genetic parameters were analyzed using the genomic genetic relationship matrix, and the heritability of the yolk ratio at 60 weeks of age was 0.34±0.05, of which the YR_tag22 genetic marker can explain 2.18% of the phenotypic variance.

[0045] Table 1 YR_tag22 genetic markers associated with egg-yolk ratio

[0046]

[0047] Wherein: the physical position of the marker chromosome refers to the whole chicken genome (bGalGal1.mat.broiler.GRCg7b).

[0048] Example 3

[0049] Detection and validation of genetic markers

[0050] The above SNP genetic markers were used to conduct candidate gene association analysis on the Dongxiang green-shell laying hen-white Leghorn chicken resource population. The specific steps are as follows:

[0051] 1) PCR primers: DNA template sequence information was downloaded from the NCBI website, and PCR amplification primers were designed using Primer Premier 6.0 software. The primer information is shown in Table 2. PCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0052] Table 2 Amplification primers used to detect genetic markers of yolk ratio in 60-week-old chickens

[0053]

[0054] 2) Genomic DNA extraction: Genomic DNA of 1333 blood samples was extracted by phenol imitation method, and PCR amplification was performed after passing the ultraviolet spectrophotometer and agarose electrophoresis tests.

[0055] 3) PCR amplification process:

[0056] ① Reaction system: 10 μl system includes 50 ng DNA template of identification material, 10 ng of forward and reverse primers, 5 μl 2×power Taq MasterMix, and the remaining volume is made up with ultrapure water.

[0057] ② Reaction procedure: first, denature at 94°C for 30 s, anneal at 54.8°C for 30 s, and extend at 72°C for 30 s, for a total of 5 cycles; then denature at 94°C for 30 s, anneal at 54.8°C for 30 s, and extend at 72°C for 30 s, for a total of 30 cycles; extend at 72°C for 5 min and store at 4°C.

[0058] 4) The amplified product is sent to a sequencing company for sequence polymorphism detection.

[0059] The sequence of the amplified fragment is as follows:

[0060] >YR_tag22

[0061]

[0062]

[0063] In the sequence, the ones marked with [] are mutation sites, those in brackets are allele variations, and the primer sequences are shown in bold at the beginning and end of the sequence.

[0064] 5) Association analysis: The subjects were all given genotypes and 60-week-old egg yolk ratios, and then a significance test was performed. Figure 3 As shown, the average yolk ratio of GG genotype individuals at 60 weeks of age is 0.324, the average yolk ratio of GA genotype individuals at 60 weeks of age is 0.319, and the average yolk ratio of AA genotype individuals at 60 weeks of age is 0.313. By using genotyping technology, increasing the frequency of the G allele can improve the yolk quality.

[0065] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0066] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall 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 equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of SNP genetic markers affecting egg yolk ratio in chicken genetic breeding, characterized in that: The SNP genetic marker affecting the egg yolk ratio includes YR_tag22, which corresponds to the physical position 2608650 of chromosome 22 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b version sequence information published in NCBI, and belongs to the 11th intron sequence of the gene ADAM9, where the base is G or A; Among them, the 60-week-old yolk ratio of the GG genotype individual of YR_tag22 is greater than the 60-week-old yolk ratio of the GA genotype individual, and the 60-week-old yolk ratio of the GA genotype individual is greater than the 60-week-old yolk ratio of the AA genotype individual; The chicken breeds include Dongxiang green-shell laying hens and / or white Leghorn chickens; The chicken genetic breeding is the genetic breeding of chickens regarding the yolk ratio trait.

2. An early selection method for egg yolk ratio trait, characterized in that: The early selection method comprises performing early selection on the egg yolk ratio trait based on the genotype of the SNP genetic marker YR_tag22; The YR_tag22 corresponds to the physical position 2608650 of chromosome 22 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b version sequence information published in NCBI, and belongs to the intron sequence of the gene ADAM9, where the base is G or A; Among them, the 60-week-old yolk ratio of the GG genotype individual of YR_tag22 is greater than the 60-week-old yolk ratio of the GA genotype individual, and the 60-week-old yolk ratio of the GA genotype individual is greater than the 60-week-old yolk ratio of the AA genotype individual; The chicken breeds include Dongxiang green-shell laying hens and / or white Leghorn chickens.

3. The early selection method for the egg yolk ratio trait according to claim 2, characterized in that: The early selection method specifically includes: Detect the genotype of the chicken YR_tag22 to be tested; Early selection is performed on the egg yolk ratio trait to be tested based on the genotype of YR_tag22.

4. The early selection method for yolk ratio trait according to claim 3, characterized in that: The detection of the genotype of the chicken YR_tag22 to be tested specifically comprises: The genomic DNA of the chicken to be tested was amplified by PCR using Pr_yr1 f and Pr_yr1 r as primers; The PCR amplification product was sequenced to obtain the genotype of position 2608650 of the positive strand of chromosome 22 of the tested chicken; Among them, the nucleotide sequence of Pr_yr1 f is shown as SEQ ID NO.1, and the nucleotide sequence of Pr_yr1 r is shown as SEQ ID NO.

2.

5. Application of primers for detecting SNP genetic markers affecting egg yolk ratio in chicken genetic breeding, characterized in that: The primers include Pr_yr1 f and Pr_yr1 r, the nucleotide sequence of Pr_yr1 f is shown in SEQ ID NO.1, and the nucleotide sequence of Pr_yr1 r is shown in SEQ ID NO.2; The SNP genetic marker affecting the egg yolk ratio includes YR_tag22, which corresponds to the physical position 2608650 of chromosome 22 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b version sequence information published in NCBI, and belongs to the 11th intron sequence of the gene ADAM9, where the base is G or A; Among them, the 60-week-old yolk ratio of the GG genotype individual of YR_tag22 is greater than the 60-week-old yolk ratio of the GA genotype individual, and the 60-week-old yolk ratio of the GA genotype individual is greater than the 60-week-old yolk ratio of the AA genotype individual; The chicken breeds include Dongxiang green-shell laying hens and / or white Leghorn chickens; The chicken genetic breeding is the genetic breeding of chickens regarding the yolk ratio trait.

6. Application of a kit in chicken genetic breeding, characterized in that: The kit comprises primers for detecting SNP genetic markers affecting egg yolk ratio, the primers comprising Pr_yr1 f and Pr_yr1 r, the nucleotide sequence of Pr_yr1 f is shown in SEQ ID NO.1, and the nucleotide sequence of Pr_yr1 r is shown in SEQ ID NO.2; The SNP genetic marker affecting the egg yolk ratio includes YR_tag22, which corresponds to the physical position 2608650 of chromosome 22 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b version sequence information published in NCBI, and belongs to the 11th intron sequence of the gene ADAM9, where the base is G or A; Among them, the 60-week-old yolk ratio of the GG genotype individual of YR_tag22 is greater than the 60-week-old yolk ratio of the GA genotype individual, and the 60-week-old yolk ratio of the GA genotype individual is greater than the 60-week-old yolk ratio of the AA genotype individual; The chicken breeds include Dongxiang green-shell laying hens and / or white Leghorn chickens; The chicken genetic breeding is the genetic breeding of chickens regarding the yolk ratio trait.

7. Application of SNP genetic markers affecting egg yolk ratio in predicting the egg yolk ratio of 60-week-old eggs, characterized in that: The SNP genetic marker affecting the egg yolk ratio includes YR_tag22, which corresponds to the physical position 2608650 of chromosome 22 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b version sequence information published in NCBI, and belongs to the 11th intron sequence of the gene ADAM9, where the base is G or A; Among them, the 60-week-old yolk ratio of the GG genotype individual of YR_tag22 is greater than the 60-week-old yolk ratio of the GA genotype individual, and the 60-week-old yolk ratio of the GA genotype individual is greater than the 60-week-old yolk ratio of the AA genotype individual; The chicken breeds include Dongxiang green-shell laying hens and / or white Leghorn chickens.

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