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

Abstract

The application provides application of a genetic marker related to the age of laying onset in a STON2 gene in genetic breeding of laying hens, belongs to the field of animal genetic breeding and biotechnology, and the genetic marker related to the age of laying onset in the STON2 gene is AFE_tag5, which corresponds to sequence information of the 5th chromosome physical position of the bGa lGa l 1.mat.broi ler.GRCg7b version published in a chicken reference genome of NCBI, is encoded as rs315655133 in Ensemb l, belongs to a synonymous mutation in an exon region of a gene STON2, and the base is T or C. The genetic marker AFE_tag5 is helpful for exploration and utilization of local chicken gene resources, is applied to genetic breeding of the chicken, and is favorable for improving the age of laying onset and obtaining a laying hen breed with excellent performance in uniformity of the age of laying onset.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of animal genetics and biotechnology, and particularly relates to application of a genetic marker in a STON2 gene related to age at first egg in genetic breeding of laying hens. BACKGROUND

[0002] Age at first egg is a marker of sexual maturity of hens and a commonly used index for breeding of laying hens. 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 native chickens generally start laying eggs at 25 weeks of age. The age at first egg can also be used to measure population uniformity and predict egg-laying performance. Chicken egg laying is a complex physiological development process, which is not only regulated by external environmental factors such as photoperiod, but also affected by endocrine and genetic factors. Generally speaking, only by changing the frequency of related genes genetically can we obtain lasting and cumulative progress. In recent years, the GWAS method has been extended to the genetic structure analysis of laying hens for quantitative traits, mainly for egg number, feed utilization efficiency and egg quality. There are few studies on the genetic structure of age at first egg in laying hens using the GWAS method. The age at first egg is increasingly valued by breeders, but it is difficult to make genetic progress through conventional breeding methods because the trait is controlled by multiple genes with small effects. Therefore, it is necessary to clarify the genetic structure of the age at first egg to genetically affect the age at first egg.

[0003] Dongxiang green-shell laying hens are originally from the vicinity of Dongxiang County in Jiangxi Province, China, and are a famous local egg-type chicken breed in China, as well as a national geographical indication breed. Dongxiang green-shell laying hens are named for their green-shell eggs. After selection, Dongxiang green-shell laying hens are used to construct the Suqin green-shell laying hen and Shendan No. 6 green-shell laying hen complete line, and play an important role in the egg market. Systematic study of the genetic structure of the age at first egg in Dongxiang green-shell laying hens is of great significance for the development and utilization of Dongxiang green-shell laying hens. The age at first egg is a late-life trait of living beings, and can only be measured at the time of first egg, which increases the cost of feeding. Therefore, it is necessary to find suitable molecular markers to achieve early selection through marker-assisted breeding or genomic selection and quickly obtain genetic progress in the age at first egg of laying hens.

[0004] In addition, Dongxiang green-shell laying hens do not perform well in production performance due to not having undergone high-intensity chicken breeding, especially in terms of uniformity, which is difficult to meet the needs of modern egg production. In order to meet the demand for green-shell eggs in the egg market and to tap the genetic resources of local chicken breeds in China, it is necessary to find new molecular markers to improve the age at first egg of laying hen populations genetically. SUMMARY

[0005] In order to obtain a chicken breed with an earlier age of onset, the present application provides an application of a genetic marker related to the age of onset in a STON2 gene in genetic breeding of laying hens, the genetic marker AFE_tag5 related to the age of onset in the STON2 gene helps to explore and utilize local chicken genetic resources, and application of the genetic marker in genetic breeding of chickens is beneficial to shorten the age of onset and obtain a laying hen breed with excellent uniformity of the age of onset.

[0006] The present application is achieved by the following technical solutions:

[0007] The present application provides an application of a genetic marker related to the age of onset in a STON2 gene in genetic breeding of laying hens, the genetic marker AFE_tag5 related to the age of onset in the STON2 gene helps to explore and utilize local chicken genetic resources, and application of the genetic marker in genetic breeding of chickens is beneficial to shorten the age of onset and obtain a laying hen breed with excellent uniformity of the age of onset.

[0008] Based on the same inventive concept, the present application provides an early selection method of chickens for the age of onset, the early selection method comprising early selection of the age of onset of chickens based on a genotype of a genetic marker AFE_tag5;

[0009] The AFE_tag5 corresponds to a physical position of 40382979 of a chromosome No. 5 in sequence information of a chicken reference genome bGalGal1.mat.broiler.GRCg7b version published in NCBI, is encoded as rs315655133 in Ensembl, belongs to a synonymous mutation in an exon region of a gene STON2, and a base here is T or C.

[0010] Further, the early selection method specifically comprises:

[0011] detection of a genotype of a genetic marker AFE_tag5 of a to-be-tested chicken;

[0012] early selection of the age of onset of the to-be-tested chicken based on the genotype of the AFE_tag5;

[0013] The age of onset of an individual with a TT genotype of the AFE_tag5 is earlier than the age of onset of individuals with TC and CC genotypes.

[0014] Further, the detection of the genotype of the genetic marker AFE_tag5 of the to-be-tested chicken specifically comprises:

[0015] PCR amplification is carried out on the genomic DNA of the to-be-tested chicken by using Pr_afe5_1f and Pr_afe5_1r as primers;

[0016] The PCR amplification product is sequenced to obtain the genotype of the chicken at the physical position 40382979 of chromosome 5;

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

[0018] Further, the to-be-tested chicken breeds include Dongxiang green shell laying hens and / or white lai heng chickens.

[0019] Based on the same inventive concept, the present application provides detection primers of the genetic marker in the STON2 gene related to the age of first egg laying, which include Pr_afe5_1f and Pr_afe5_1r, the nucleotide sequence of the Pr_afe5_1f is shown in SEQ ID NO. 1, and the nucleotide sequence of the Pr_afe5_1r is shown in SEQ ID NO. 2.

[0020] The genetic marker in the STON2 gene related to the age of first egg laying includes AFE_tag5, which corresponds to the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version of the physical position 40382979 of chromosome 5 published in NCBI, the Ensembl code is rs315655133, which belongs to the synonymous mutation of the exon region of the gene STON2, and the base here is T or C.

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

[0022] Based on the same inventive concept, the present application provides a kit, which contains the detection primers of the genetic marker in the STON2 gene related to the age of first egg laying.

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

[0024] Based on the same inventive concept, the application provides application of the genetic marker related to the age of onset of laying in the STON2 gene in predicting the age of onset of laying of chickens, wherein the genetic marker related to the age of onset of laying in the STON2 gene comprises AFE_tag5, the AFE_tag5 corresponds to the sequence information of the 40,038,297th 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, and the base is T or C.

[0025] The one or more technical solutions in the embodiments of the application have at least the following technical effects or advantages:

[0026] The application of the genetic marker related to the age of onset of laying in the STON2 gene in the genetic breeding of laying hens, the genetic marker related to the age of onset of laying in the STON2 gene comprises AFE_tag5, and the population with the superior genotype TT of AFE_tag5 has an earlier age of onset of laying; AFE_tag5 is helpful to improve the age of onset of laying of the population of laying hens from the genetic aspect, and the application of AFE_tag5 in the genetic breeding of chickens is beneficial to improving the uniformity of the age of onset of laying of the population of laying hens and meeting the needs of modern chicken breeding for green-shell egg chicken varieties. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the application, the following will briefly introduce the drawings needed in the embodiment description. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0028] Figure 1 The Manhattan plot for GWAS analysis of the age of onset of laying of the resource population in Embodiment 2 of the application;

[0029] Figure 2 The QQ plot for GWAS analysis of the age of onset of laying of the resource population in Embodiment 2 of the application;

[0030] Figure 3 The box plot of the age of onset of laying of individuals with different genotypes in Embodiment 3 of the application. DETAILED DESCRIPTION

[0031] The advantages and various effects of the present application will be more clearly presented hereinafter in conjunction with specific embodiments and examples. Those skilled in the art should understand that these embodiments and examples are used to illustrate the present application, rather than limit the present application. Throughout the specification, unless otherwise specifically indicated, 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 meanings as generally understood by those skilled in the art to which the present application belongs. If there is a contradiction, the present specification takes priority.

[0032] Unless otherwise specifically indicated, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by existing methods.

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

[0034] Example 1

[0035] Resource population construction

[0036] In order to analyze the genetic structure of the egg laying trait, a laying hen resource population was constructed according to the F2 design. Local chicken breed Dongxiang Green Shell Laying Hen and breeding variety Bailaihang chicken were used as parents, F1 generation was obtained by forward and reverse crossing, and 1029 chicks were hatched. Then F1 was used as parent to breed F2 generation, and 1989 F2 generation chicks were obtained. Pedigree information was recorded. The test chickens were individually numbered on the wing and were raised in single cages in a fully enclosed chicken house, and the chickens were artificially lighted for 16 hours during the laying period. The chickens were cooled by fans and wet curtains. Routine immunization was carried out according to the immunization program formulated by Jiangsu Poultry Research Institute. The feed came from COFCO, and the composition of the laying hen feed included 16.5% crude protein and 11511 kJ / kg feed metabolizable energy. The chickens were free to eat during the laying period, and the water was supplied by a nipple waterer, and the feed was supplied by a cart-type feeder, and the manure was cleaned by a chicken manure conveyor belt. The age at first egg of each test chicken was recorded.

[0037] After preliminary screening of the age at first egg data, removing obviously incorrect and repeated data, removing outliers, and arranging into an excel table, the effects of generation and breed on the test data were statistically analyzed by one-way ANOVA, and the year and chicken house effects were included as fixed effect factors. Using pedigree data, the genetic relationship matrix was constructed by WOMBAT software, the heritability and breeding value were calculated. The breeding value was used as pseudo-phenotype data for the next GWAS analysis, and the genetic structure of the age at first egg was analyzed.

[0038] Example 2

[0039] GWAS analysis of age at first egg

[0040] The test chickens were from the F2 generation adult hens of the laying hen resource population constructed in Example 1. About 0.5 ml of blood sample was collected from the wing vein of the test chicken, placed in a BD anticoagulant tube (Suzhou Biode Medical Instrument Co., Ltd.), and stored at -70°C. The genomic DNA was extracted, detected by 0.8% agarose electrophoresis, and detected by ultraviolet spectrophotometry. After the DNA sample was diluted to 50±5 ng / μl, it was used for gene chip typing.

[0041] Genotyping was performed using the 600K Chicken Genotyping Array of Affymetrix. Genotyping was performed using the 600K Chicken Genotyping Array of Affymetrix.

[0042] Before whole genome association analysis, multidimensional principal component analysis was performed to eliminate false positives and population structure. The first five principal components were used as covariate parameters added to the genetic model, and the henhouse effect was placed in the model as a fixed effect. The "simpleM" method of R script was used to calculate the independent test estimate of each SNP site, and 59308 independent markers were obtained. Using multiple corrections, the genome significant threshold was 8.43x10 -7 , and the genome recommended threshold was 1.69x10 -5 . The mixed linear model was used to analyze the egg laying age, and the significance test P value of each SNP marker was obtained. The matrix expression of the linear model is,

[0043]

[0044] where y represents the breeding value vector of laying age; W represents the covariance matrix; a is the intercept vector; x is the genotype vector of the marker, and β is the effect value of the marker; G is the genetic relationship matrix based on the chip, u is the random effect vector (here, the breeding value); and ε is the residual.

[0045] After GWAS screening, AFE_tag5 associated with laying age was obtained (Table 1). Whole genome association analysis was performed on the laying age of 1512 chickens, and the results are shown in Figure 1 , Figure 2 Figure 1 ​As shown in the Manhattan plot, there are 19 SNPs on the periphery of chromosome 5 that exceed the genome-wide suggestive level, which can be used as evidence to support AFE_tag5. The QQ plot further verifies the reliability of the GWAS results. As shown in the QQ plot, most SNPs that do not deviate from the diagonal line are affected by genetic drift, and SNPs located at the tail of the QQ plot are affected by artificial selection. The inflation factor is calculated to be 1.016, indicating that there is no obvious population stratification in the resource population for the age at first laying trait. By analyzing the genetic parameters using the pedigree genetic relationship matrix, the heritability of the age at first laying is obtained to be 0.392 ± 0.046, and the AFE_tag5 genetic marker can explain 1.53% of the phenotypic variance. Figure 2 As shown in the QQ plot, most SNPs that do not deviate from the diagonal line are affected by genetic drift, and SNPs located at the tail of the QQ plot are affected by artificial selection. The inflation factor is calculated to be 1.016, indicating that there is no obvious population stratification in the resource population for the age at first laying trait. By analyzing the genetic parameters using the pedigree genetic relationship matrix, the heritability of the age at first laying is obtained to be 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 the age at first laying

[0047]

[0048] Wherein, the marker chromosome physical position refers to the chicken whole gene (bGall Gall 1.mat.broiler.GRCg7b).

[0049] Example 3

[0050] Detection and verification of the genetic marker AFE_tag5

[0051] The above SNP genetic marker is used for candidate gene association analysis on the Dongxiang Green Shell Laying Hen- Bailaihang Chicken resource population. The specific operation steps are as follows:

[0052] 1) PCR primer: The DNA template sequence information is downloaded from the NCBI website, and the PCR amplification primer is designed by using primer premier 6.0 software. The primer information is shown in Table 2. The PCR primer is synthesized by Shengwo Bioengineering (Shanghai) Co., Ltd.

[0053] Table 2 Amplification primers for detecting the chicken age at first laying genetic marker AFE_tag5

[0054]

[0055] 2) Genomic DNA extraction: The genomic DNA of 1512 blood samples is extracted by the phenol chloroform method, and after detection by ultraviolet spectrophotometer and agarose electrophoresis, the qualified samples are subjected to PCR amplification.

[0056] 3) PCR amplification process:

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

[0058] 2) Reaction procedure: first 94℃ denaturation for 30s, 54.1℃ annealing for 30s, 72℃ extension for 30s, for 5 cycles; then 94℃ denaturation for 30s, 54.1℃ annealing for 30s, 72℃ extension for 30s, for 30 cycles; 72℃ extension for 5min, 4℃ preservation.

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

[0060] The amplified fragment sequence is as follows:

[0061] >AFE_tag5

[0062]

[0063] In the sequence, the mutation site marked with [] is the allele variation in the parentheses, and the primer sequence is shown in bold and underlined at the beginning and end of the sequence.

[0064] 5) Association analysis: the test individuals have genotypes and age at first egg, and then significance test is performed. The results are shown in Table 2, the age at first egg of CC genotype individuals is 154.79±10.95 days, the age at first egg of TC genotype individuals is 152.28±10.21 days, and the age at first egg of TT genotype individuals is 151.35±9.85 days. Single factor variance analysis shows that the age at first egg of the three genotypes is significantly different (P<0.01). By improving the frequency of T allele through genotyping technology, the age at first egg of laying hens can be shortened; the proportion of CC genotype individuals in the current population is 52.3%, and improving the frequency of CC genotype can further improve the uniformity of the age at first egg of laying hens. Figure 3

[0065] Finally, it should be noted that the terms "comprising", "including", or any other variant thereof are intended to cover non-exclusive inclusion, so that processes, methods, articles or devices including a series of elements not only include those elements, but also include other elements not explicitly listed, or inherent to such processes, methods, articles or devices.

[0066] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to the embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to cover all changes and modifications falling within the scope of the present application.

[0067] ​Obviously, many modifications and variations of the present application are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. The application of genetic markers related to age at first laying in the STON2 gene in the genetic breeding of laying hens, characterized in that, The genetic markers related to age at first laying in the STON2 gene include AFE_tag5, which corresponds to the sequence information of chicken reference genome version bGalGal1.mat.broiler.GRCg7b published in NCBI, with a physical location of position 40382979 on chromosome 5, encoded by Ensembl as rs315655133. It belongs to a synonymous mutation in the exon region of the STON2 gene, where the base is T or C. The aforementioned laying hen genetic breeding refers to the genetic breeding of laying hens regarding the trait of age at the start of egg production.

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

3. The method for early selection of chickens regarding the age at first laying trait according to claim 2, characterized in that, The early selection method specifically includes: The genotype of the genetic marker AFE_tag5 in the chickens to be tested was determined; Early selection of the age at first laying trait in the test chickens based on the genotype of AFE_tag5; Among them, the TT genotype individuals of AFE_tag5 have an earlier age at labor than the TC and CC genotype individuals.

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

2.

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

6. Primers for detecting genetic markers related to age at onset 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 shown in SEQ ID NO.1, and the nucleotide sequence of Pr_afe5_1r is shown in SEQ ID NO.2; The genetic markers related to age at first laying in the STON2 gene include AFE_tag5, which corresponds to the sequence information of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b version published in NCBI, with a physical position of 40382979 on chromosome 5, encoded by Ensembl as rs315655133. It belongs to a synonymous mutation in the exon region of the STON2 gene, where the base is T or C.

7. The application of the detection primers as described in claim 6 in chicken genetic breeding.

8. A reagent kit, characterized in that, The kit contains the detection primers as described in claim 6.

9. The application of the kit as described in claim 8 in chicken genetic breeding.

10. The application of genetic markers related to age at first laying in the STON2 gene in predicting the age at first laying in chickens, characterized by: The genetic markers related to age at first laying in the STON2 gene include AFE_tag5, which corresponds to the sequence information of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b version published in NCBI, with a physical position of 40382979 on chromosome 5, encoded by Ensembl as rs315655133. It belongs to a synonymous mutation in the exon region of the STON2 gene, where the base is T or C.

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