Use of molecular markers associated with broiler weight in chicken genetic breeding

Abstract

The application provides a use of molecular markers associated with open production body weight in chicken genetic breeding, the molecular markers associated with open production body weight include at least one of ASM_tag1, ASM_tag2 and ASM_tag3; the Ensembl number of the ASM_tag1 is rs13972990, corresponding to the 170147177th base of the positive strand of the 1st chromosome of a chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI, and the base is C or T; the Ensembl number of the ASM_tag2 is rs15496977, corresponding to the 168798323th base of the positive strand of the 1st chromosome of a chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI, belonging to a sequence downstream of a CYSLTR2 gene, and the base is G or C; the Ensembl number of the ASM_tag3 is rs13552185, corresponding to the 167725378th base of the positive strand of the 1st chromosome of a chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI, belonging to a 1st intron sequence of an ERICH6B gene, and the base is G or C. The three molecular markers are all helpful to genetically improve open production body weight, and when applied to chicken genetic breeding, the three molecular markers are beneficial to improving the open production body weight level of a laying hen population, and further obtaining a laying hen breed with excellent open production body weight uniformity performance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of animal genetics and biotechnology, and particularly relates to the use of molecular markers associated with onset body weight in chicken genetic breeding. BACKGROUND

[0002] Onset body weight is a common index for egg-laying chicken breeding, which can be used to evaluate the sexual maturity of hens, measure the uniformity of the population, and predict the egg-laying performance. Studies have shown that there is a clear lower limit for poultry body weight, and hens below the threshold value cannot enter sexual maturity. The onset of egg production in chickens is a complex physiological development process, which is not only regulated by external environmental factors such as photoperiod, but also influenced by genetic factors. Generally speaking, only by changing the frequency of related genes from a genetic point of view can we obtain lasting and cumulative progress. In recent years, the GWAS method has been extended to the genetic structure analysis of quantitative traits in laying hens, mainly used for egg production, feed utilization efficiency and egg quality research, and less used for GWAS method to analyze the genetic structure of egg-laying chicken onset body weight. Onset body weight is regulated by multiple genes with small effects, and it is difficult to obtain effective progress in genetic breeding by conventional breeding methods. Only by clarifying the genetic structure of onset body weight and improving the accuracy of breeding through genomic selection can we genetically affect the onset body weight.

[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 and have a long history of breeding in rural areas. They have a variety of traits, including black, white, yellow, and mottled feathers, black and white skin, and yellow, white, and purple ear leaves. After selection, Dongxiang green-shell laying hens are used to construct Suqin green-shell laying hens and Shendan No. 6 green-shell laying hens, which play an important role in the egg market. Systematic study of the genetic structure of Dongxiang green-shell laying hen onset body weight is of great significance for the development and utilization of Dongxiang green-shell laying hens. However, onset body weight can only be measured at onset, and selecting at onset will increase the cost of feeding, so it is urgent to find suitable molecular markers to achieve early selection through genetic marker-assisted breeding or genomic selection to quickly obtain genetic progress in egg-laying chicken onset body weight and achieve the goal of improving the uniformity of onset egg-laying hens.

[0004] Due to the lack of high-intensity chicken breeding, Dongxiang green-shell laying hens have poor production performance, especially in terms of uniformity, which cannot 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 urgent to find new molecular markers to enrich the genetic marker library related to chicken onset body weight, so as to genetically improve the onset body weight of egg-laying hens. SUMMARY

[0005] In order to obtain an egg chicken breed with higher body weight at the beginning of production, the application provides a use of a molecular marker associated with the body weight at the beginning of production in chicken genetic breeding, the molecular markers ASM_tag1, ASM_tag2 and ASM_tag3 associated with the body weight at the beginning of production are helpful to genetically improve the body weight at the beginning of production, and application of the molecular markers to chicken genetic breeding is beneficial to improve the body weight at the beginning of production of an egg chicken population, and further obtain an egg chicken breed with excellent uniformity of the body weight at the beginning of production.

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

[0007] The application provides a use of a molecular marker associated with the body weight at the beginning of production in chicken genetic breeding, and the molecular marker associated with the body weight at the beginning of production includes at least one of ASM_tag1, ASM_tag2 and ASM_tag3.

[0008] The Ensembl number of the ASM_tag1 is rs13972990, corresponding to the 170147177th position of the positive strand of chromosome 1 in the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI, belonging to a sequence 6385bp upstream of the gene DHRS12, and the base at the position is C or T.

[0009] The Ensembl number of the ASM_tag2 is rs15496977, corresponding to the 168798323th position of the positive strand of chromosome 1 in the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI, belonging to a sequence downstream of the CYSLTR2 gene, and the base at the position is G or C.

[0010] The Ensembl number of the ASM_tag3 is rs13552185, corresponding to the 167725378th position of the positive strand of chromosome 1 in the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI, belonging to a sequence of the 1st intron of the ERICH6B gene, and the base at the position is G or C.

[0011] Based on the same inventive concept, the application provides an early screening method for a chicken on a body weight at the beginning of production, and the early screening method includes early selection of the chicken on the body weight at the beginning of production based on genotypes of at least one of the molecular markers ASM_tag1, ASM_tag2 and ASM_tag3.

[0012] The Ensembl number of the ASM_tag1 is rs13972990, corresponding to the sequence of the positive strand of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b published in NCBI, which is 170147177, and is 6385bp upstream of the gene DHRS12, and the base is C or T;

[0013] The Ensembl number of the ASM_tag2 is rs15496977, corresponding to the sequence of the positive strand of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b published in NCBI, which is 168798323, and is the downstream sequence of the CYSLTR2 gene, and the base is G or C;

[0014] The Ensembl number of the ASM_tag3 is rs13552185, corresponding to the sequence of the positive strand of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b published in NCBI, which is 167725378, and is the first intron sequence of the ERICH6B gene, and the base is G or C.

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

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

[0017] early selecting the egg-laying weight trait of the to-be-tested chicken based on the genotype of at least one of the ASM_tag1, ASM_tag2 and ASM_tag3;

[0018] The TT genotype individual of the ASM_tag1 has a higher egg-laying weight than the TC genotype individual, and the TC genotype individual has a higher egg-laying weight than the CC genotype individual.

[0019] The CC genotype individual of the ASM_tag2 has a higher egg-laying weight than the CG genotype individual, and the CG genotype individual has a higher egg-laying weight than the GG genotype individual.

[0020] The CC genotype individual of the ASM_tag3 has a higher egg-laying weight than the CG genotype individual, and the CG genotype individual has a higher egg-laying weight than the GG genotype individual.

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

[0022] detecting the genotype of at least one of the chicken molecular markers ASM_tag1, ASM_tag2 and ASM_tag3 to be tested, wherein the method for detecting the genotype of the ASM_tag1 is as follows:

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

[0024] sequencing the PCR amplification product to obtain the genotype of the 170147177th position of the positive strand of chromosome 1 of the chicken to be tested;

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

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

[0027] sequencing the PCR amplification product to obtain the genotype of the 168798323th position of the positive strand of chromosome 1 of the chicken to be tested;

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

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

[0030] sequencing the PCR amplification product to obtain the genotype of the 167725378th position of the positive strand of chromosome 1 of the chicken to be tested;

[0031] The nucleotide sequence of the Pr_asm1f is shown in SEQ ID NO. 1, and the nucleotide sequence of the Pr_asm1r 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.

[0032] Preferably, the breed of the chicken to be tested includes Dongxiang Green Shell Egg Chicken and / or Bailaihang Chicken.

[0033] Based on the same inventive concept, the present application provides detection primers of molecular markers associated with the body weight of a cock, wherein the detection primers comprise primers for detecting at least one of ASM_tag1, ASM_tag2 and ASM_tag3, wherein the primers for detecting ASM_tag1 comprise Pr_asm1f and Pr_asm1r, the primers for detecting ASM_tag2 comprise Pr_asm2f and Pr_asm2r, and the primers for detecting ASM_tag3 comprise Pr_asm3f and Pr_asm3r.

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

[0035] Based on the same inventive concept, the present application provides application of the detection primers of molecular markers associated with the body weight of a cock in chicken genetic breeding.

[0036] Based on the same inventive concept, the present application provides a kit comprising the detection primers of molecular markers associated with the body weight of a cock.

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

[0038] Based on the same inventive concept, the present application further provides application of the molecular markers associated with the body weight of a cock in predicting the body weight of a cock, wherein the molecular markers associated with the body weight of a cock comprise at least one of ASM_tag1, ASM_tag2 and ASM_tag3.

[0039] The Ensembl number of the ASM_tag1 is rs13972990, which corresponds to the 170147177th position of the positive strand of chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI, and is located at 6385bp upstream of the gene DHRS12, wherein the base at this position is C or T.

[0040] The Ensembl number of the ASM_tag2 is rs15496977, corresponding to the 168798323th position of the positive strand of the chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI, belonging to the sequence downstream of the CYSLTR2 gene, and the base here is G or C;

[0041] The Ensembl number of the ASM_tag3 is rs13552185, corresponding to the 167725378th position of the positive strand of the chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b sequence published in NCBI, belonging to the 1st intron sequence of the ERICH6B gene, and the base here is G or C.

[0042] The one or more technical solutions in the embodiment of the present application have at least the following technical effects or advantages:

[0043] The application relates to the use of molecular markers associated with the initial production body weight in chicken genetic breeding, and the molecular markers associated with the initial production body weight include ASM_tag1, ASM_tag2 and ASM_tag3, which are all located on the chromosome 1 of the chicken, and the ASM_tag1, ASM_tag2 and ASM_tag3 are all significantly associated with the initial production body weight of the chicken, and the population with a favorable genotype has a higher level of initial production body weight, the application of the ASM_tag1, ASM_tag2 and ASM_tag3 in the genetic breeding of laying hens helps to improve the initial production body weight level of the laying hen population from the genetic aspect, and further helps to obtain a laying hen breed with excellent initial production body weight uniformity performance and to accelerate the breeding process. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0045] Figure 1 The Manhattan plot for the GWAS analysis of the initial production body weight of the resource population in the embodiment 2 of the present application;

[0046] Figure 2 The QQ plot for the GWAS analysis of the initial production body weight of the resource population in the embodiment 2 of the present application;

[0047] Figure 3 The box plot of the initial production body weight of the individual with different genotypes in the embodiment 3-5 of the present application. DETAILED DESCRIPTION

[0048] 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.

[0049] Throughout this specification, unless otherwise specifically indicated otherwise, the terms used herein are understood to have the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. If there is a conflict between the present specification and the prior art, the present specification takes precedence.

[0050] Unless otherwise specifically indicated otherwise, 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.

[0051] The use of the molecular marker associated with the body weight of the chicken in the genetic breeding of the chicken will be described in detail below in conjunction with the examples and experimental data.

[0052] Example 1

[0053] Resource population construction

[0054] In order to analyze the genetic structure of egg traits, an egg chicken resource population was constructed according to the F2 design. The local chicken breed Dongxiang green shell egg chicken and the breeding variety Bailaihang chicken were used as parents, and F1 generation was obtained by forward and reverse crossing, and then F2 generation was bred by using F1 as parents. Pedigree information was recorded. The test chickens were individually numbered with wing numbers and were raised in single cages in a fully enclosed chicken house. Artificial light was provided for 16 hours during the laying period, and air conditioning and wet curtains were used for cooling. Routine immunization was carried out according to the immunization program formulated by Jiangsu Poultry Research Institute. The feed came from COFCO Corporation, and the composition of the laying chicken feed included 16.5% crude protein and 11511 kJ / kg feed metabolizable energy. The laying chickens were free to eat, and the water was supplied by a nipple waterer, and the feed was supplied by a feeding machine, and the manure was cleaned by a chicken manure conveyor belt. The onset weight was determined according to the industry standard of the Ministry of Agriculture and Rural Affairs of the People's Republic of China.

[0055] After preliminary screening of the onset weight data, removing obviously incorrect and repeated data, removing outliers, and arranging into an excel table form. After data cleaning, the resource population F2 generation onset weight data set remained 1512 records, which were used for the next GWAS analysis and to analyze the genetic structure.

[0056] Example 2

[0057] Onset weight GWAS analysis

[0058] 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. Genomic DNA was extracted, and after 0.8% agarose electrophoresis detection and ultraviolet spectrophotometric detection, the DNA sample was diluted to 50±5 ng / μl for gene chip typing.

[0059] Genotyping was performed using the 600K Chicken Genotyping Array of Affymetrix. Genotyping was performed using the 600K Chicken Genotyping Array of Affymetrix.

[0060] 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 body weight, and the significance test P value of each SNP marker was obtained. The matrix expression of the linear model is

[0061] y = Wα + xβ + Gu + ε

[0062] where y represents the sample phenotype value vector; W represents the covariance matrix; α 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 construction, u is the random effect vector (here it is breeding value); and ε is the residual.

[0063] After GWAS screening, ASM_tag1 associated with laying body weight was obtained (Table 1). Whole genome association analysis was performed on the laying body weight of 1512 chickens, and the results are shown in Figure 1 , Figure 2 Figure 1 ​The Manhattan plot shows that there are significant levels of markers on chromosome 1, with 539 SNPs exceeding the genome-wide significant level and 284 SNPs exceeding the genome-wide suggestive level, which can be used as evidence to support the ASM_tag1. The QQ plot further verifies the reliability of the GWAS results. From the Figure 2 The QQ plot shows that most of the 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 factor is calculated to be 0.949. By analyzing the genetic parameters using the pedigree genetic relationship matrix, the heritability of the body weight at first egg is obtained to be 0.686±0.038, wherein 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 the body weight at first egg

[0065]

[0066] Wherein, the marker chromosome physical position refers to the chicken whole genome (bGalGal1.mat.broiler.GRCg7b).

[0067] Example 3

[0068] Detection and verification of the genetic marker ASM_tag1

[0069] 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:

[0070] 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 Sheng Wu Bioengineering (Shanghai) Co., Ltd.

[0071] Table 2 Amplification primers for detecting the chicken body weight genetic marker ASM_tag1 at first egg

[0072]

[0073] 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.

[0074] 3) PCR amplification process:

[0075] ①Reaction system: 10 μl system includes 50 ng of identification material DNA template, 10 ng of forward and reverse primers, 5 μl of 2 x power Taq MasterMix, and the rest is supplemented with ultrapure water.

[0076] ②Reaction procedure: first denaturation at 94℃ for 30 s, annealing at 52.7℃ for 30 s, extension at 72℃ for 30 s, for a total of 5 cycles; then denaturation at 94℃ for 30 s, annealing at 52.7℃ for 30 s, extension at 72℃ for 30 s, for a total of 30 cycles; extension at 72℃ for 5 min, and storage at 4℃.

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

[0082] 5) Association analysis: the test individuals all have genotypes and open production body weight, and then significance test is performed. The analysis result is as shown in Figure 3 The open production body weight of the CC genotype individual is 1100.52±108.86 g, the open production body weight of the CT genotype individual is 1171.80±113.65 g, and the open production body weight of the TT genotype individual is 1236.77±124.63 g. The single factor variance analysis shows that the open production body weight of the three genotypes is significantly different, and pairwise comparison further shows that the open production body weight is significantly different. By improving the T allele frequency through genotyping technology, the open production body weight of the laying hen can be significantly improved.

[0083] Example 4

[0084] Detection and verification of genetic marker ASM_tag2

[0085] The SNP genetic marker is used for candidate gene association analysis on Dongxiang green shell egg chicken- Bailaihang chicken resource population. The specific operation steps are as follows:

[0086] 1) PCR primer: the DNA template sequence information is downloaded from the NCBI website, the PCR amplification primer is designed by primer premier 6.0 software, the primer information is shown in Table 3. The PCR primer is synthesized by Sheng Wu Bioengineering (Shanghai) Co., Ltd.

[0087] Table 3 Amplification primers for detecting chicken open production body weight genetic marker ASM_tag2

[0088]

[0089] 2) Genomic DNA extraction: 1512 blood samples were extracted by phenol method, and the genomic DNA was detected by ultraviolet spectrophotometer and agarose electrophoresis. After passing the detection, the genomic DNA was amplified by PCR.

[0090] 3) PCR amplification process:

[0091] ① Reaction system: 10 μl system includes 50 ng of identification material DNA template, 10 ng of forward and reverse primers, 5 μl of 2x power Taq MasterMix, and the remaining volume is supplemented with ultrapure water.

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

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

[0094] The amplified fragment sequence is as follows:

[0095] > ASM_tag2

[0096]

[0097] In the sequence, the mutation site is marked in [], the allele variation is in the parentheses, and the primer sequence is shown in bold and underlined at the beginning and end of the sequence.

[0098] 5) Association analysis: the test individuals have genotypes and open birth weight, and then significance test is performed. The analysis results are shown in Figure 3 , the open birth weight of CC (0.35) genotype individuals is 1230.97 ± 115.23 g, the open birth weight of CG (0.46) genotype individuals is 1166.43 ± 117.66 g, and the open birth weight of GG (0.19) genotype individuals is 1099.51 ± 110.77 g. Single factor variance analysis shows that the open birth weights corresponding to the three genotypes are significantly different, and pairwise comparison further shows that the open birth weight is significantly different. Through genotyping technology, the C allele frequency can be improved, which can significantly improve the open birth weight of laying hens.

[0099] Example 5

[0100] Detection and verification of genetic marker ASM_tag3

[0101] The SNP genetic marker is applied to Dongxiang Green-shell Laying Hen- Bailaihang chicken resource population for candidate gene association analysis. The specific operation steps are as follows:

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

[0103] Table 4 Amplification primers for detecting chicken onset weight genetic marker ASM_tag3

[0104]

[0105] 2) Genomic DNA extraction: the genomic DNA of 1512 blood samples is extracted by phenol chloroform method, and after detection by ultraviolet spectrophotometer and agarose electrophoresis, PCR amplification is carried out.

[0106] 3) PCR amplification process:

[0107] 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.

[0108] ② Reaction program: first 94℃ denaturation for 30s, 55.7℃ annealing for 30s, 72℃ extension for 30s, a total of 5 cycles; then 94℃ denaturation for 30s, 55.7℃ annealing for 30s, 72℃ extension for 30s, a total of 30 cycles; 72℃ extension for 5 min, 4℃ preservation.

[0109] 4) The amplification product is 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 mutation site marked [] is the allele variation in the brackets, and the primer sequence is shown in bold and underlined at the beginning and end of the sequence.

[0115] 5) Association analysis: the test individuals have genotypes and onset body weight, and then significant test is carried out. The analysis results are as follows Figure 3As shown, the body weight of the CC (0.46) genotype individual is 1215.14±121.80g, the body weight of the CG (0.41) genotype individual is 1156.94±113.99g, and the body weight of the GG (0.13) genotype individual is 1095.79±114.90g. Single factor variance analysis shows that the body weight of the three genotypes is significantly different, and pairwise comparison further shows that the body weight of the three genotypes is significantly different. Through genotyping technology, the C allele frequency can be improved, and the body weight of the laying hen at the beginning of laying can be significantly improved.

[0116] Finally, it should also be noted that the terms "comprising", "containing" or any other variant thereof are intended to cover non-exclusive inclusions, so that a process, method, article or apparatus that includes a list of elements not only includes those elements, but also includes other elements not explicitly listed, or inherent to such a process, method, article or apparatus.

[0117] Although preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0118] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. Use of a detection reagent for a molecular marker associated with the onset of weight in chicken genetic breeding, characterized in that, The molecular marker associated with the body weight at the onset of laying is at least one of ASM_tag1, ASM_tag2 and ASM_tag3; The Ensembl number of the ASM_tag1 is rs13972990, corresponding to the 170147177th position of the positive strand of chromosome 1 in the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, which is 6385bp upstream of the gene DHRS12 and the base is C or T; The Ensembl number of the ASM_tag2 is rs15496977, corresponding to the 168798323th position of the positive strand of chromosome 1 in the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, which is a downstream sequence of the CYSLTR2 gene and the base is G or C; The Ensembl number of the ASM_tag3 is rs13552185, corresponding to the 167725378th position of the positive strand of chromosome 1 in the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, which is a 1st intron sequence of the ERICH6B gene and the base is G or C; The chicken breed is Dongxiang Green Shell Egg Chicken and / or Bailaihang Chicken; The chicken genetic breeding is the genetic breeding of the chicken on the body weight at the onset of laying.

2. Use according to claim 1, characterized in that, The detection reagent of the molecular marker associated with the body weight at the onset of laying includes primers for detecting at least one of the ASM_tag1, the ASM_tag2 and the ASM_tag3, wherein the primers for detecting the ASM_tag1 include Pr_asm1 f and Pr_asm1 r, the primers for detecting the ASM_tag2 include Pr_asm2f and Pr_asm2r, and the primers for detecting the ASM_tag3 include Pr_asm3f and Pr_asm3r; 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.

3. A method for early selection of chickens for body weight traits at first lay, characterized in that, The early screening method includes early selection of the chicken on the body weight at the onset of laying based on the genotype of at least one of the molecular markers ASM_tag1, ASM_tag2 and ASM_tag3. The Ensembl number of the ASM_tag1 is rs13972990, which corresponds to the sequence of the positive strand of chromosome 1 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b published in NCBI, is 170147177, is located at 6385bp upstream of the gene DHRS12, and the base is C or T; The Ensembl number of the ASM_tag2 is rs15496977, which corresponds to the sequence of the positive strand of chromosome 1 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b published in NCBI, is 168798323, is located at the downstream sequence of the CYSLTR2 gene, and the base is G or C; The Ensembl number of the ASM_tag3 is rs13552185, which corresponds to the sequence of the positive strand of chromosome 1 of the chicken reference genome bGalGal 1.mat.broiler.GRCg7b published in NCBI, is 167725378, is located at the 1st intron sequence of the ERICH6B gene, and the base is G or C; The TT genotype of the ASM_tag1 has a higher body weight at the beginning of laying than the TC genotype, and the TC genotype has a higher body weight at the beginning of laying than the CC genotype; The CC genotype of the ASM_tag2 has a higher body weight at the beginning of laying than the CG genotype, and the CG genotype has a higher body weight at the beginning of laying than the GG genotype; The CC genotype of the ASM_tag3 has a higher body weight at the beginning of laying than the CG genotype, and the CG genotype has a higher body weight at the beginning of laying than the GG genotype; The chicken is Dongxiang green-shelled egg chicken and / or Bailaihang chicken.

4. The method for early selection of chickens for the egg laying body weight trait according to claim 3, wherein The early screening method specifically comprises: detecting the genotype of at least one of the molecular markers ASM_tag1, ASM_tag2 and ASM_tag3 of the to-be-tested chicken; early selecting the body weight at the beginning of laying of the to-be-tested chicken based on the genotype of at least one of the ASM_tag1, ASM_tag2 and ASM_tag3.

5. The method for early selection of chickens for the egg laying body weight trait according to claim 4, wherein The detection of the genotype of at least one of the molecular markers ASM_tag1, ASM_tag2 and ASM_tag3 of the to-be-tested chicken specifically comprises: detecting the genotype of at least one of the molecular markers ASM_tag1, ASM_tag2 and ASM_tag3 of the to-be-tested chicken, wherein the detection of the ASM_tag1 genotype is performed as follows: PCR amplification is performed on the genomic DNA of the to-be-tested chicken by using Pr_asm1 f and Pr_asm1 r as primers; sequencing is performed on the PCR amplification product to obtain the genotype of the positive strand of chromosome 1 of the to-be-tested chicken at 170147177; the detection of the ASM_tag2 genotype is performed as follows: PCR amplification is performed on the genomic DNA of the to-be-tested chicken by using Pr_asm2 f and Pr_asm2 r as primers; sequencing the PCR amplification product to obtain the genotype of the 168798323th position of the positive strand of chromosome 1 of the chicken to be tested; The method for detecting the genotype of the ASM_tag3 is as follows: PCR amplification is performed on the genomic DNA of the chicken to be tested by using Pr_asm3f and Pr_asm3r as primers; sequencing the PCR amplification product to obtain the genotype of the 167725378th position of the positive strand of chromosome 1 of the chicken to be tested; The nucleotide sequence of the Pr_asm1f is shown in SEQ ID NO. 1, and the nucleotide sequence of the Pr_asm1r 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.

6. Use of a detection reagent for a molecular marker associated with onset body weight in predicting onset body weight in a chicken, characterized in that, The molecular marker associated with the initial body weight is at least one of ASM_tag1, ASM_tag2 and ASM_tag3; The Ensembl number of the ASM_tag1 is rs13972990, which corresponds to the 170147177th position of the positive strand of chromosome 1 in the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, is located at 6385bp upstream of the gene DHRS12, and the base at this position is C or T; The Ensembl number of the ASM_tag2 is rs15496977, which corresponds to the 168798323th position of the positive strand of chromosome 1 in the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, is located in the downstream sequence of the CYSLTR2 gene, and the base at this position is G or C; The Ensembl number of the ASM_tag3 is rs13552185, which corresponds to the 167725378th position of the positive strand of chromosome 1 in the chicken reference genome bGalGal 1.mat.broiler.GRCg7b sequence published in NCBI, is located in the 1st intron sequence of the ERICH6B gene, and the base at this position is G or C; The breed of the chicken is Dongxiang Green Shell Laying Chicken and / or Bailaihang Chicken; The initial body weight of the individual with the TT genotype of the ASM_tag1 is higher than that of the individual with the TC genotype, and the initial body weight of the individual with the TC genotype is higher than that of the individual with the CC genotype; The initial body weight of the individual with the CC genotype of the ASM_tag2 is higher than that of the individual with the CG genotype, and the initial body weight of the individual with the CG genotype is higher than that of the individual with the GG genotype; The initial body weight of the individual with the CC genotype of the ASM_tag3 is higher than that of the individual with the CG genotype, and the initial body weight of the individual with the CG genotype is higher than that of the individual with the GG genotype.

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