Application of molecular marker associated with first laying weight in genetic breeding of chicken

Abstract

The invention provides an application of a molecular marker associated with the first laying weight in chicken genetic breeding. The molecular marker associated with the first laying weight comprises at least one of ASM tag1, ASM tag2 and ASM tag3. The Ensepbl serial number of the ASMtag1 is rs13972990, the Ensepbl serial number corresponds to the 170147177th site of a positive-sense strand of a chromosome 1 of a chicken reference genome bGalGal1. Mat.broil.GRCg7b sequence disclosed in NCBI (National Center of Biotechnology Information), and a basic group at the site is C or T. The Ensepbl serial number of the ASMtag1 is rs13972990, and the Ensepbl serial number of the ASMtag1 is rs13972990. The Ensepbl serial number of ASMtag2 is rs15496977, corresponds to the 168798323 site of a positive-sense strand of a chromosome 1 of a chicken reference genome bGalGal1. Mat.broil.GRCg7b sequence disclosed in NCBI (National Center for Biotechnology Information), and belongs to a downstream sequence of a CYSLTR2 gene, and a basic group in the downstream sequence is G or C; the Ensepbl serial number of ASMtag3 is rs13552185, corresponds to the 167725378 site of a positive-sense strand of a chromosome 1 of a chicken reference genome bGalGal1. Mat.broil.GRCg7b sequence disclosed in NCBI (National Center of Biotechnology Information), and belongs to a first intron sequence of an ERICH6B gene, and a basic group in the first intron sequence is G or C. The three molecular markers are all helpful for heredity improvement of the first laying weight, and when the molecular markers are applied to genetic breeding of chickens, the first laying weight level of a laying hen group is improved, and then a laying hen variety with excellent first laying weight uniformity is obtained.

Description

Technical Field

[0001] The invention belongs to the field of animal genetic breeding and biotechnology, and particularly relates to the use of a molecular marker associated with initial laying weight in chicken genetic breeding. Background Art

[0002] The weight at the beginning of laying is a commonly used indicator for breeding of laying hens. It can be used to evaluate the sexual maturity of hens, measure the uniformity of the group, and predict egg-laying performance. Studies have shown that there is a clear lower limit for poultry weight, and hens below the threshold cannot reach sexual maturity. The beginning of laying of chickens is a complex physiological development process, which is not only regulated by external environmental factors, such as the duration of light exposure, but also affected by genetic factors. Generally speaking, only by genetically changing the frequency of related genes can lasting and cumulative progress be achieved. In recent years, the GWAS method has been extended to the analysis of the genetic structure of quantitative traits of laying hens, mainly used for the study of egg production, feed utilization efficiency and egg quality. It is rare to use the GWAS method to analyze the genetic structure of the weight at the beginning of laying of laying hens. The weight at the beginning of laying trait is regulated by micro-effect polygenes, and it is difficult for conventional breeding methods to obtain effective genetic progress. The only way is to clarify the genetic structure of the weight at the beginning of laying and improve its breeding accuracy through genomic selection, so as to achieve genetic influence on the weight at the beginning of laying.

[0003] Dongxiang green-shell laying hens are native to the area near Dongxiang County, Jiangxi Province. They are a famous local chicken breed for laying eggs in my country and a national geographical indication variety. Dongxiang green-shell laying hens are named after the green-shelled eggs they lay. They have a long history of being raised in rural areas and have many traits. Their feathers are black, white, yellow, and hemp, their skin color is black or white, and their ear lobes are yellow, white, and purple. The selected Dongxiang green-shell laying hens are used to build the Suqin green-shell laying hens and Shendan No. 6 green-shell laying hens supporting system, and they occupy an important position in the laying hen market. Systematic research on the genetic structure of the laying weight of Dongxiang green-shell laying hens is of great significance for the development and utilization of Dongxiang green-shell laying hens. However, the laying weight can only be measured when laying begins. Selecting at the time of laying will increase the cost of feeding. Therefore, it is urgent to find suitable molecular markers to achieve early selection through genetic marker-assisted breeding or genomic selection, so as to quickly obtain the genetic progress of laying weight of laying hens and achieve the purpose of improving the uniformity of laying weight of laying hens.

[0004] Since Dongxiang green-shell laying hens have not undergone intensive breeding, their production performance is poor, especially the uniformity is difficult to meet the needs of modern laying hen production. In order to meet the demand for green-shell eggs in the egg market and to tap into the genetic resources of local chicken breeds in my country, it is urgent to find new molecular markers and enrich the genetic marker library related to the initial laying weight of chickens, so as to genetically improve the initial laying weight level of laying hens. Summary of the invention

[0005] In order to obtain a laying hen breed with a higher body weight at first laying, the present invention provides the use of molecular markers associated with body weight at first laying in chicken genetic breeding. The molecular markers ASM_tag1, ASM_tag2, and ASM_tag3 associated with body weight at first laying contribute to genetically improving the body weight at first laying. Applying them to chicken genetic breeding is beneficial to improving the body weight level at first laying of the laying hen population, and thus obtaining a laying hen breed with excellent uniformity in body weight at first laying.

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

[0007] The present invention provides the use of molecular markers associated with body weight at first laying in chicken genetic breeding, and the molecular markers associated with body weight at first laying include at least one of ASM_tag1, ASM_tag2, and ASM_tag3;

[0008] The Ensembl ID of the ASM_tag1 is rs13972990, corresponding to the 170,147,177th position on the sense strand of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI, which is 6385 bp upstream of the gene DHRS12, and the base here is C or T;

[0009] The Ensembl ID of the ASM_tag2 is rs15496977, corresponding to the 168,798,323rd position on the sense strand of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI, which is the downstream sequence of the CYSLTR2 gene, and the base here is G or C;

[0010] The Ensembl ID of the ASM_tag3 is rs13552185, corresponding to the 167,725,378th position on the sense strand of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI, which is the intron 1 sequence of the ERICH6B gene, and the base here is G or C.

[0011] Based on the same inventive concept, the present invention provides an early screening method for the body weight trait at first laying in chickens. The early screening method includes early selection of the body weight trait at first laying in chickens based on the genotype of at least one of the molecular markers ASM_tag1, ASM_tag2, and ASM_tag3;

[0012] The Ensembl ID of ASM_tag1 is rs13972990, corresponding to the 170147177th position on the plus strand of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI. It is located 6385 bp upstream of the gene DHRS12, and the base here is C or T;

[0013] The Ensembl ID of ASM_tag2 is rs15496977, corresponding to the 168798323rd position on the plus strand of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI. It is located in the downstream sequence of the CYSLTR2 gene, and the base here is G or C;

[0014] The Ensembl ID of ASM_tag3 is rs13552185, corresponding to the 167725378th position on the plus strand of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI. It is located in the first intron sequence of the ERICH6B gene, and the base here is G or C.

[0015] Further, the early screening method specifically includes:

[0016] Detecting the genotype of at least one of the molecular markers ASM_tag1, ASM_tag2, and ASM_tag3 in the chicken to be tested;

[0017] Based on the genotype of at least one of ASM_tag1, ASM_tag2, and ASM_tag3, early selection is performed on the age at first egg body weight trait of the chicken to be tested;

[0018] Among them, the age at first egg body weight of individuals with the TT genotype of ASM_tag1 is higher than that of individuals with the TC genotype, and the age at first egg body weight of individuals with the TC genotype is higher than that of individuals with the CC genotype;

[0019] The age at first egg body weight of individuals with the CC genotype of ASM_tag2 is higher than that of individuals with the CG genotype, and the age at first egg body weight of individuals with the CG genotype is higher than that of individuals with the GG genotype;

[0020] The age at first egg body weight of individuals with the CC genotype of ASM_tag3 is higher than that of individuals with the CG genotype, and the age at first egg body weight of individuals with the CG genotype is higher than that of individuals with the GG genotype.

[0021] Further, the detection of the genotype of at least one of the molecular markers ASM_tag1, ASM_tag2, and ASM_tag3 in the chicken to be tested specifically includes:

[0022] Genotype of at least one of the molecular markers ASM_tag1, ASM_tag2 and ASM_tag3 in the chicken to be tested is detected. Among them, the method for detecting the genotype of ASM_tag1 is as follows:

[0023] Using Pr_asm1f and Pr_asm1r as primers, PCR amplification is performed on the genomic DNA of the chicken to be tested;

[0024] The PCR amplification product is sequenced to obtain the genotype at position 170147177 on the sense strand of chromosome 1 of the chicken to be tested;

[0025] The method for detecting the genotype of ASM_tag2 is as follows:

[0026] Using Pr_asm2f and Pr_asm2r as primers, PCR amplification is performed on the genomic DNA of the chicken to be tested;

[0027] The PCR amplification product is sequenced to obtain the genotype at position 168798323 on the sense strand of chromosome 1 of the chicken to be tested;

[0028] The method for detecting the genotype of ASM_tag3 is as follows:

[0029] Using Pr_asm3f and Pr_asm3r as primers, PCR amplification is performed on the genomic DNA of the chicken to be tested;

[0030] The PCR amplification product is sequenced to obtain the genotype at position 167725378 on the sense strand of chromosome 1 of the chicken to be tested;

[0031] Among them, the nucleotide sequence of Pr_asm1f is as shown in SEQ ID NO.1, and the nucleotide sequence of Pr_asm1r is as shown in SEQ ID NO.2; the nucleotide sequence of Pr_asm2f is as shown in SEQ ID NO.3, and the nucleotide sequence of Pr_asm2r is as shown in SEQ ID NO.4; the nucleotide sequence of Pr_asm3f is as shown in SEQ ID NO.5, and the nucleotide sequence of Pr_asm3r is as shown in SEQ ID NO.6.

[0032] Preferably, the breeds of the chickens to be tested include Dongxiang Green-shelled Chickens and / or White Leghorns.

[0033] Based on the same inventive concept, the present invention provides detection primers for molecular markers associated with the body weight at first egg laying, and the detection primers include primers for detecting at least one of ASM_tag1, ASM_tag2, and ASM_tag3. Among them, the primers for detecting ASM_tag1 include Pr_asm1f and Pr_asm1r, the primers for detecting ASM_tag2 include Pr_asm2f and Pr_asm2r, and the primers for detecting ASM_tag3 include Pr_asm3f and Pr_asm3r;

[0034] The nucleotide sequence of Pr_asm1f is shown in SEQ ID NO.1, and the nucleotide sequence of Pr_asm1r is shown in SEQ ID NO.2; the nucleotide sequence of Pr_asm2f is shown in SEQ ID NO.3, and the nucleotide sequence of Pr_asm2r is shown in SEQ ID NO.4; the nucleotide sequence of Pr_asm3f is shown in SEQ ID NO.5, and the nucleotide sequence of Pr_asm3r is shown in SEQ ID NO.6.

[0035] Based on the same inventive concept, the present invention provides the application of the detection primers for molecular markers associated with the body weight at first egg laying in chicken genetic breeding.

[0036] Based on the same inventive concept, the present invention provides a kit, and the kit contains the above-mentioned detection primers for molecular markers associated with the body weight at first egg laying.

[0037] Based on the same inventive concept, the present invention provides the application of the above-mentioned kit in chicken genetic breeding.

[0038] Based on the same inventive concept, the present invention further provides the application of the molecular markers associated with the body weight at first egg laying in predicting the body weight at first egg laying of chickens, and the molecular markers associated with the body weight at first egg laying include at least one of ASM_tag1, ASM_tag2, and ASM_tag3;

[0039] The Ensembl number of ASM_tag1 is rs13972990, corresponding to the 170147177th position on the sense strand of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI, which is 6385 bp upstream of the gene DHRS12, and the base here is C or T;

[0040] The Ensembl ID of ASM_tag2 is rs15496977, corresponding to the 168,798,323rd position on the plus strand of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI. It belongs to the downstream sequence of the CYSLTR2 gene, and the base here is G or C;

[0041] The Ensembl ID of ASM_tag3 is rs13552185, corresponding to the 167,725,378th position on the plus strand of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI. It belongs to the first intron sequence of the ERICH6B gene, and the base here is G or C.

[0042] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0043] The use of the molecular markers associated with the age at first egg weight in chicken genetic breeding. The molecular markers associated with the age at first egg weight include ASM_tag1, ASM_tag2, and ASM_tag3, all of which are located on chicken chromosome 1. ASM_tag1, ASM_tag2, and ASM_tag3 are all significantly associated with the age at first egg weight trait in chickens. The favorable genotype population has a higher level of age at first egg weight. Applying ASM_tag1, ASM_tag2, and ASM_tag3 to the genetic breeding of laying hens helps to genetically improve the age at first egg weight level of the laying hen population, and then obtain a laying hen breed with excellent uniformity in age at first egg weight, and can also accelerate the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] 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 drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0045] Figure 1 It is the Manhattan plot of the GWAS analysis of the age at first egg weight of the resource population in Example 2 of the present invention;

[0046] Figure 2 It is the QQ plot of the GWAS analysis of the age at first egg weight of the resource population in Example 2 of the present invention;

[0047] Figure 3 It is the box plot of the age at first egg weight of individuals with different genotypes in Examples 3 - 5 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0048] In the following, the present invention will be specifically described in combination 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 used to illustrate the present invention, rather than to limit the present invention.

[0049] 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 meanings as those generally understood by those skilled in the art to which the present invention pertains. In case of contradiction, this specification shall prevail.

[0050] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0051] Next, the use of the molecular markers associated with the age at first egg weight in chicken genetic breeding of the present application will be described in detail in combination with examples and experimental data.

[0052] Example 1

[0053] Construction of the resource population

[0054] To analyze the genetic architecture of egg production traits, 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. The F1 generation was obtained through reciprocal crosses, and then the F2 generation was bred with the F1 as parents. 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 weight was measured according to the industry standard of the Ministry of Agriculture of the People's Republic of China.

[0055] The data of the age at first egg weight were initially screened. After removing the obviously incorrect and duplicate data, the outliers were removed and the data were organized in the form of an excel table. After data cleaning, the dataset of the age at first egg weight of the F2 generation in the resource population remained 1512 records, which were used for the next GWAS analysis and to analyze its genetic architecture.

[0056] Example 2

[0057] GWAS analysis of the age at first egg weight

[0058] The experimental chickens were adult hens from 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 anticoagulation tubes (BD Medical Devices (Suzhou) Co., Ltd.) and stored at -70°C. Genomic DNA was extracted and detected by 0.8% agarose gel electrophoresis and ultraviolet spectrophotometry. After passing the tests, the DNA samples were diluted to 50 ± 5 ng / μl for genotyping using gene chips.

[0059] 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 the APT software; performing quality control using PLINK, removing SNPs with a detection rate lower than 0.97 and removing SNP markers that deviated 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.

[0060] Before performing the genome-wide association study (GWAS), 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 of 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 body weight at first egg production, and the significance test P values of each SNPs marker were obtained. The matrix expression of the linear model was,

[0061] y = Wα + xβ + Gu + ε

[0062] where y represents the vector of sample phenotypic values; 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.

[0063] After GWAS screening, ASM_tag1 associated with the body weight at first egg production was obtained (Table 1). Genome-wide association analysis was performed on the body weight at first egg production of 1,512 chickens, and the results are as Figure 1 、 Figure 2 shown. As Figure 1(Manhattan plot) shows that there are markers at the genome-wide significant level on chicken chromosome 1. There are 539 SNPs exceeding the genome-wide significant level and 284 SNPs exceeding the genome-recommended level around it, which can be used as evidence to support ASM_tag1. 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 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 0.949. Using the pedigree genetic relationship matrix to analyze genetic parameters, the heritability of age at first egg body weight was obtained as 0.686 ± 0.038. Among them, the ASM_tag1 genetic marker can explain 11.6% of the phenotypic variance, the ASM_tag2 genetic marker can explain 9.7% of the phenotypic variance, and the ASM_tag3 genetic marker can explain 4.0% of the phenotypic variance.

[0064] Table 1 Genetic markers related to age at first egg body weight

[0065]

[0066] Among them: The physical position of the marked chromosome refers to the chicken whole genome (bGalGal1.mat.broiler.GRCg7b).

[0067] Example 3

[0068] Detection and verification of genetic marker ASM_tag1

[0069] Perform candidate gene association analysis on the Dongxiang Green-shelled Chicken - White Leghorn chicken resource population using the above SNP genetic markers. The specific operation steps are as follows:

[0070] 1) PCR primers: Download the DNA template sequence information from the NCBI website, and design PCR amplification primers using primer premier 6.0 software. The primer information is shown in Table 2. The PCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0071] Table 2 Amplification primers used to detect the genetic marker ASM_tag1 of chicken age at first egg body weight

[0072]

[0073] 2) Genomic DNA extraction: Extract genomic DNA from 1512 blood samples by the phenol-chloroform method. After passing the detection by ultraviolet spectrophotometer and agarose gel electrophoresis, perform PCR amplification.

[0074] 3) PCR amplification process:

[0075] ①Reaction system: The 10 μl system includes 50 ng of the identification material DNA template, 10 ng each of the forward and reverse primers, 5 μL of 2×power Taq MasterMix, and the remaining volume is made up with ultrapure water.

[0076] ②Reaction program: First, denature at 94 °C for 30 s, anneal at 52.7 °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 52.7 °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.

[0077] 4) The amplified product is sent to a sequencing company for sequence polymorphism detection.

[0078] The amplified fragment sequence is as follows:

[0079] >ASM_tag1

[0080]

[0081] 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.

[0082] 5) Association analysis: All the tested individuals have genotypes and the body weight at first egg production, and then a significance test is carried out. The analysis results are as Figure 3 shown. The body weight at first egg production of individuals with the CC genotype is 1100.52 ± 108.86 g, that of individuals with the CT genotype is 1171.80 ± 113.65 g, and that of individuals with the TT genotype is 1236.77 ± 124.63 g. One-way ANOVA shows that there are significant differences in the body weight at first egg production corresponding to the three genotypes, and pairwise comparisons further show that there are significant differences in the body weight at first egg production. By using the gene typing technology to increase the frequency of the T allele, the body weight at first egg production of laying hens can be significantly improved.

[0083] Example 4

[0084] Detection and verification of the genetic marker ASM_tag2

[0085] The above SNP genetic marker is used for candidate gene association analysis in the Dongxiang blue-shelled chicken - White Leghorn chicken resource population. The specific operation steps are as follows:

[0086] 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 3. The PCR primers are synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0087] Table 3 Amplification primers used for detecting the genetic marker ASM_tag2 of the body weight at first egg production in chickens

[0088]

[0089] 2) Genomic DNA extraction: Genomic DNA of 1512 blood samples was extracted by the phenol-chloroform method. After being qualified by ultraviolet spectrophotometer detection and agarose gel electrophoresis detection, PCR amplification was carried out.

[0090] 3) PCR amplification process:

[0091] ① Reaction system: The 10 μl system included 50 ng of DNA template of the identification material, 10 ng each of the forward and reverse primers, 5 μL of 2×power Taq MasterMix, and the remaining volume was made up with ultrapure water.

[0092] ② Reaction program: First, denaturation at 94°C for 30 s, annealing at 55.2°C for 30 s, extension at 72°C for 30 s, for a total of 5 cycles; then denaturation at 94°C for 30 s, annealing at 55.2°C for 30 s, extension at 72°C for 30 s, for a total of 30 cycles; extension at 72°C for 5 min, and preservation at 4°C.

[0093] 4) The amplified product was sent to a sequencing company for sequence polymorphism detection.

[0094] The sequence of the amplified fragment is as follows:

[0095] >ASM_tag2

[0096]

[0097] In the sequence, the sites marked with [] 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.

[0098] 5) Association analysis: All the tested individuals had genotypes and body weights at first egg production, and then significant tests were carried out. The analysis results are as Figure 3 shown. The body weight at first egg production of individuals with the CC (0.35) genotype was 1230.97 ± 115.23 g, that of individuals with the CG (0.46) genotype was 1166.43 ± 117.66 g, and that of individuals with the GG (0.19) genotype was 1099.51 ± 110.77 g. One-way ANOVA showed that there were significant differences in the body weights at first egg production corresponding to the three genotypes, and pairwise comparisons further showed significant differences in the body weights at first egg production. By using the gene typing technology to increase the frequency of the C allele, the body weight at first egg production of laying hens can be significantly improved.

[0099] Example 5

[0100] Detection and verification of genetic marker ASM_tag3

[0101] Candidate gene association analysis was performed on the Dongxiang blue-shelled chicken - White Leghorn chicken resource population using the above SNP genetic markers. The specific operation steps are as follows:

[0102] 1) PCR primers: Download the DNA template sequence information from the NCBI website, design PCR amplification primers with the primer premier 6.0 software, and the primer information is shown in Table 4. The PCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0103] Table 4 Amplification primers for detecting the genetic marker ASM_tag3 of chicken age at first egg weight

[0104]

[0105] 2) Genomic DNA extraction: Extract genomic DNA from 1512 blood samples by the phenol-chloroform method. After being qualified by ultraviolet spectrophotometer detection and agarose gel electrophoresis detection, PCR amplification was carried out.

[0106] 3) PCR amplification process:

[0107] 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.

[0108] ② Reaction program: First, denature at 94 °C for 30 s, anneal at 55.7 °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 55.7 °C for 30 s, 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.

[0109] 4) The amplified products were sent to a sequencing company for sequence polymorphism detection.

[0110] The amplified fragment sequence is as follows:

[0111] >ASM_tag3

[0112]

[0113]

[0114] In the sequence, the sites marked [] are the 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.

[0115] 5) Association analysis: All the subjects had genotypes and ages at first egg weight, and then significance tests were carried out. The analysis results are as Figure 3As shown, the age at first egg weight of individuals with the CC (0.46) genotype was 1215.14 ± 121.80 g, that of individuals with the CG (0.41) genotype was 1156.94 ± 113.99 g, and that of individuals with the GG (0.13) genotype was 1095.79 ± 114.90 g. One-way analysis of variance showed that there were significant differences in the age at first egg weight corresponding to the three genotypes, and pairwise comparisons further indicated significant differences in the age at first egg weight. By genotyping technology, increasing the frequency of the C allele can significantly improve the age at first egg weight of laying hens.

[0116] Finally, it should also be noted that the term "comprising", "including" 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 not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0117] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they learn 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.

[0118] 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 is also intended to include these changes and modifications.

Claims

1. Use of molecular markers associated with body weight at first laying in chicken genetic breeding, characterized in that, The molecular markers associated with the body weight at first egg include at least one of ASM_tag1, ASM_tag2, and ASM_tag3; The Ensembl ID of ASM_tag1 is rs13972990, corresponding to the 170147177th position on the plus strand of chromosome 1 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, which is 6385 bp upstream of the gene DHRS12, and the base here is C or T; The Ensembl ID of ASM_tag2 is rs15496977, corresponding to the 168798323rd position on the plus strand of chromosome 1 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, which is the downstream sequence of the CYSLTR2 gene, and the base here is G or C; The Ensembl ID of ASM_tag3 is rs13552185, corresponding to the 167725378th position on the plus strand of chromosome 1 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, which is the intron 1 sequence of the ERICH6B gene, and the base here is G or C.

2. Early screening method for the body weight trait at the onset of lay in chickens, characterized in that, The early screening method includes early selection of the body weight at first egg trait of chickens based on the genotype of at least one of the molecular markers ASM_tag1, ASM_tag2, and ASM_tag3; The Ensembl ID of ASM_tag1 is rs13972990, corresponding to the 170147177th position on the plus strand of chromosome 1 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, which is 6385 bp upstream of the gene DHRS12, and the base here is C or T; The Ensembl ID of ASM_tag2 is rs15496977, corresponding to the 168798323rd position on the plus strand of chromosome 1 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, which is the downstream sequence of the CYSLTR2 gene, and the base here is G or C; The Ensembl ID of ASM_tag3 is rs13552185, corresponding to the 167725378th position on the plus strand of chromosome 1 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, which is the intron 1 sequence of the ERICH6B gene, and the base here is G or C.

3. The early screening method for the first egg laying body weight trait of chickens according to claim 2, characterized in that, The early screening method specifically includes: Detecting the genotype of at least one of the molecular markers ASM_tag1, ASM_tag2, and ASM_tag3 in the chicken to be tested; Early selecting the body weight at first egg trait of the chicken to be tested based on the genotype of at least one of ASM_tag1, ASM_tag2, and ASM_tag3; Among them, the onset body weight of the TT genotype individuals of the ASM_tag1 is higher than that of the TC genotype individuals, and the onset body weight of the TC genotype individuals is higher than that of the CC genotype individuals; The onset body weight of the CC genotype individuals of the ASM_tag2 is higher than that of the CG genotype individuals, and the onset body weight of the CG genotype individuals is higher than that of the GG genotype individuals; The onset body weight of the CC genotype individuals of the ASM_tag3 is higher than that of the CG genotype individuals, and the onset body weight of the CG genotype individuals is higher than that of the GG genotype individuals.

4. The early screening method for the first egg-laying body weight trait of the chicken according to claim 3, wherein Detecting the genotype of at least one of the molecular markers ASM_tag1, ASM_tag2 and ASM_tag3 in the chicken to be tested specifically includes: Detecting the genotype of at least one of the molecular markers ASM_tag1, ASM_tag2 and ASM_tag3 in the chicken to be tested. Among them, the method for detecting the genotype of the ASM_tag1 is as follows: Using Pr_asm1 f and Pr_asm1 r as primers, performing PCR amplification on the genomic DNA of the chicken to be tested; Sequencing the PCR amplification product to obtain the genotype at the 170147177th position of the sense strand of chromosome 1 of the chicken to be tested; The method for detecting the genotype of the ASM_tag2 is as follows: Using Pr_asm2f and Pr_asm2r as primers, performing PCR amplification on the genomic DNA of the chicken to be tested; Sequencing the PCR amplification product to obtain the genotype at the 168798323rd position of the sense strand of chromosome 1 of the chicken to be tested; The method for detecting the genotype of the ASM_tag3 is as follows: Using Pr_asm3f and Pr_asm3r as primers, performing PCR amplification on the genomic DNA of the chicken to be tested; Sequencing the PCR amplification product to obtain the genotype at the 167725378th position of the sense strand of chromosome 1 of the chicken to be tested; Among them, the nucleotide sequence of the Pr_asm1 f is shown in SEQ ID NO.1, and the nucleotide sequence of the Pr_asm1 r is shown in SEQ ID NO.2; the nucleotide sequence of the Pr_asm2f is shown in SEQ ID NO.3, and the nucleotide sequence of the Pr_asm2r is shown in SEQ ID NO.4; the nucleotide sequence of the Pr_asm3f is shown in SEQ ID NO.5, and the nucleotide sequence of the Pr_asm3r is shown in SEQ ID NO.

6.

5. The early screening method for the first egg laying body weight trait of chickens according to claim 3 or 4, characterized in that, The breeds of the chickens to be tested include Dongxiang blue-shell layer chickens and / or White Leghorn chickens.

6. Detection primers for molecular markers associated with body weight at first parturition, characterized in that, The detection primers include primers for detecting at least one of ASM_tag1, ASM_tag2 and ASM_tag3. Among them, the primers for detecting ASM_tag1 include Pr_asm1f and Pr_asm1 r, the primers for detecting ASM_tag2 include Pr_asm2f and Pr_asm2r, and the primers for detecting ASM_tag3 include Pr_asm3f and Pr_asm3r; The nucleotide sequence of Pr_asm1 f is shown in SEQ ID NO.1, and the nucleotide sequence of Pr_asm1 r is shown in SEQ ID NO.2; the nucleotide sequence of Pr_asm2 f is shown in SEQ ID NO.3, and the nucleotide sequence of Pr_asm2 r is shown in SEQ ID NO.4; the nucleotide sequence of Pr_asm3 f is shown in SEQ ID NO.5, and the nucleotide sequence of Pr_asm3 r is shown in SEQ ID NO.

6.

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

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

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

10. Use of molecular markers associated with the body weight at first egg in predicting the body weight at first egg of chickens, characterized in that, The molecular marker associated with the body weight at first egg includes at least one of ASM_tag1, ASM_tag2 and ASM_tag3; The Ensembl ID of ASM_tag1 is rs13972990, corresponding to the 170147177th position on the plus strand of chromosome 1 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, which is 6385 bp upstream of the gene DHRS12, and the base here is C or T; The Ensembl ID of ASM_tag2 is rs15496977, corresponding to the 168798323rd position on the plus strand of chromosome 1 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, which is the downstream sequence of the CYSLTR2 gene, and the base here is G or C; The Ensembl ID of ASM_tag3 is rs13552185, corresponding to the 167725378th position on the plus strand of chromosome 1 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, which is the first intron sequence of the ERICH6B gene, and the base here is G or C.

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