SNP (Single Nucleotide Polymorphism) marker related to white feather broiler breeder first laying day age character and application of SNP marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) marker related to a white feather broiler breeder laying start age character and application thereof. The four SNP markers provided by the invention have obvious influence on the white feather broiler chicken first-laying day-age character, can obviously improve the prediction accuracy of genome selection, are beneficial to acceleration of genetic improvement of the white feather broiler chicken first-laying day-age character, can be used for genetic breeding, and are simple to operate and reliable in result.

Description

Technical Field

[0001] The present invention relates to the fields of genetic breeding and molecular biology, and in particular to a SNP marker associated with the age at which white-feathered broiler breeders lay eggs and an application thereof. Background Art

[0002] Chicken is the most consumed meat in the world and the second most consumed in my country. It is an important source of high-quality protein in human production and life. Among them, white-feathered broiler breeders are characterized by fast growth rate and high feed conversion efficiency. However, compared with the carefully selected meat production traits, the egg production traits of white-feathered broiler breeders are still relatively less reported in genetic and breeding research. The egg production traits of white-feathered broiler breeders are of great significance to actual production. By selecting breeders with excellent egg production traits, the production performance of offspring can be improved and the breeding process can be accelerated. In addition, egg production traits are also one of the important indicators for evaluating the quality of breeder chickens, which can reflect the health status and production potential of breeder chickens. At present, there are still few research reports on the age of white-feathered broiler breeders.

[0003] Age at first laying is one of the indicators directly related to egg production traits. This trait is determined by genetic factors. Early related studies have shown that the estimated heritability of age at first laying ranges from 0.20 to 0.56 in different strains. It is a complex quantitative trait regulated by micro-effect polygenes, and its physiological process involves the entire hypothalamic-pituitary-gonadal axis. Gonadotropin-releasing hormone (GnRH) is the beginning of a cascade reaction. GnRH binds to its receptor, stimulating the synthesis and secretion of gonadotropins, thereby inducing gonadal steroid production, ultimately leading to follicular development, ovulation, and egg production in hens. Controlling the start of laying in chickens at an appropriate age is crucial for increasing egg production.

[0004] With the advancement of high-throughput sequencing technology and the continuous development of genome mining techniques, GWAS have been widely used to identify major genes for economic traits in livestock and poultry. Currently, several candidate genes influencing traits related to feed conversion have been discovered in chickens. Age at first laying is a quantitative trait, often regulated by multiple genes. Therefore, by identifying key SNP markers through genome-wide association analysis and fine-mapping analysis based on large sample populations, combined with genomic selection techniques, we can accelerate the selection of white broiler breeders for age at first laying.

[0005] Genomic selection (GS) technology is currently a widely used breeding method in livestock and poultry. Its essence is to estimate the effect value of each individual marker using genome-wide genetic markers. The genomic estimated breeding value (GEBV) is then accumulated to derive the genomic estimated breeding value (GEBV), which is then used to sort and select individuals. Currently, the G-matrix calculation method proposed by Vanraden is the most widely used. However, in practice, only a small number of SNPs have a significant effect on the target trait, while most traits are regulated by multiple genes and loci with minor effects. Therefore, Zhang et al. developed a new method, GA-BLUP, to estimate GEBV by integrating a small number of significant SNPs to construct a weighted G-matrix, which significantly improves prediction accuracy.

[0006] In summary, by studying the genetic markers of the age at first laying through GWAS analysis, and using SNP markers and genomic selection to assist in the breeding of white-feathered broiler breeders, production efficiency and benefits can be improved, providing a reference basis for future breeding work, and accelerating the improvement process of independently bred varieties. Summary of the Invention

[0007] The present invention aims to provide a SNP marker related to the trait of the laying age of white-feathered broiler breeders and an application thereof.

[0008] To achieve the purpose of the present invention, in a first aspect, the present invention provides SNP markers associated with the age at start of laying of white-feathered broiler breeders, selected from one or more of markers rs431896560, rs16207893, rs314315024, and rs14303789.

[0009] Among them, marker rs431896560 contains the nucleotide sequence with the polymorphism of G / A at 6694125bp on chicken chromosome 19;

[0010] Marker rs16207893 contains the nucleotide sequence with a T / C polymorphism at bp 3265988 on chicken chromosome 27;

[0011] Marker rs314315024 contains a nucleotide sequence with a G / A polymorphism at bp 3266655 on chicken chromosome 27;

[0012] Marker rs14303789 contains a nucleotide sequence with polymorphism A / C at 3278202 bp on chicken chromosome 27.

[0013] Furthermore, if the genotype of the polymorphic site contained in marker rs431896560 is GG, it indicates that the individual has a late calving age; if the genotype is GA, it indicates that the individual has a medium calving age; if the genotype is AA, it indicates that the individual has an early calving age;

[0014] The genotype of the polymorphic site contained in marker rs16207893 is TT, indicating that the individual has a late calving age; if the genotype is TC, it indicates that the individual has an intermediate calving age; if the genotype is CC, it indicates that the individual has an early calving age;

[0015] The genotype of the polymorphic site contained in marker rs314315024 is GG, indicating that the individual has a late calving age; if the genotype is GA, it indicates that the individual has an intermediate calving age; if the genotype is AA, it indicates that the individual has an early calving age;

[0016] The genotype of the polymorphic site contained in marker rs14303789 is AA, indicating that the individual has a late age of calving; if the genotype is AC, it indicates that the individual has a medium age of calving; if the genotype is CC, it indicates that the individual has an early age of calving.

[0017] In a second aspect, the present invention provides primers for amplifying the SNP markers, wherein the upstream and downstream of the amplified marker rs431896560 are shown in SEQ ID NOs: 1-2, the upstream and downstream of the amplified marker rs16207893 are shown in SEQ ID NOs: 3-4, the upstream and downstream of the amplified marker rs314315024 are shown in SEQ ID NOs: 5-6, and the upstream and downstream of the amplified marker rs14303789 are shown in SEQ ID NOs: 7-8.

[0018] In a third aspect, the present invention provides a detection reagent or kit containing the primers.

[0019] In a fourth aspect, the present invention provides a method for identifying and breeding early-onset or late-onset white-feathered broiler breeder lines, comprising:

[0020] 1) Extracting total DNA from white-feathered broiler breeder chickens to be tested;

[0021] 2) using the DNA as a template and performing PCR amplification using the primers;

[0022] 3) Analyze the PCR amplification products.

[0023] Preferably, the PCR reaction system is: 12.5 μL of 2×Master mix, 1.5 μL of DNA template, 1 μL each of 10 pmol / μL upstream and downstream primers, and 9 μL of ddH2O.

[0024] Preferably, the PCR reaction program is: 94°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, for a total of 35 cycles; 72°C for 5 min.

[0025] Furthermore, step 3) includes sequencing the amplified product and determining the following based on the sequencing results:

[0026] The genotype of the polymorphic site contained in marker rs431896560 is GG, indicating that the individual has a late calving age; if the genotype is GA, it indicates that the individual has an intermediate calving age; if the genotype is AA, it indicates that the individual has an early calving age;

[0027] The genotype of the polymorphic site contained in marker rs16207893 is TT, indicating that the individual has a late calving age; if the genotype is TC, it indicates that the individual has an intermediate calving age; if the genotype is CC, it indicates that the individual has an early calving age;

[0028] The genotype of the polymorphic site contained in marker rs314315024 is GG, indicating that the individual has a late calving age; if the genotype is GA, it indicates that the individual has an intermediate calving age; if the genotype is AA, it indicates that the individual has an early calving age;

[0029] The genotype of the polymorphic site contained in marker rs14303789 is AA, indicating that the individual has a late age of calving; if the genotype is AC, it indicates that the individual has a medium age of calving; if the genotype is CC, it indicates that the individual has an early age of calving.

[0030] The forward sequencing images of the three genotypes of rs431896560 are shown in Figure 1 , the sequencing diagram of the three genotypes of rs16207893 locus is shown in Figure 2 , the sequencing diagram of the three genotypes of rs314315024 locus is shown in Figure 3 , the sequencing diagram of the three genotypes of rs14303789 locus is shown in Figure 4 .

[0031] In a fifth aspect, the present invention provides any of the following applications of the SNP marker or a detection reagent thereof:

[0032] (1) Used for early prediction of early or late laying white-feathered broiler breeders;

[0033] (2) Used for molecular marker-assisted breeding of white-feathered broiler breeders.

[0034] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0035] The four SNP markers provided by the present invention have a significant effect on the age at which white-feathered broiler breeders lay eggs, can significantly improve the prediction accuracy of genomic selection, and are conducive to accelerating the genetic improvement of the age at which white-feathered broiler breeders lay eggs. At the same time, they can be used for genetic breeding with simple operation and reliable results. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the forward sequencing diagram of the three genotypes of the rs431896560 site provided by the present invention.

[0037] Figure 2 The three genotype sequencing maps of the rs16207893 site provided by the present invention.

[0038] Figure 3 The three genotype sequencing maps of the rs314315024 site provided by the present invention.

[0039] Figure 4 The three genotype sequencing maps of the rs14303789 site provided by the present invention.

[0040] Figure 5 This is the result of GWAS association analysis of the paternal age at first calving trait in a preferred embodiment of the present invention.

[0041] Figure 6 This is the result of GWAS association analysis of maternal age at first calving traits in a preferred embodiment of the present invention.

[0042] Figure 7 This is the result of the meta-association analysis of the paternal and maternal age at first calving traits in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0043] The present invention aims to provide a SNP molecular marker related to the age at which white-feathered broiler breeders lay eggs and its application, which can improve the prediction accuracy of genomic selection and help accelerate the genetic improvement of the age at which white-feathered broiler breeders lay eggs.

[0044] The present invention adopts the following technical solutions:

[0045] The present invention provides a significant SNP marker associated with the age at first laying of chickens, wherein the SNP marker is selected from any one or more of rs431896560, rs16207893, rs314315024, and rs14303789.

[0046] Detailed information of the SNP markers is shown in Table 1.

[0047] Table 1 Location information of four key SNP sites

[0048] SNP ID chromosome Location Allele rs431896560 19 6694125 G / A rs16207893 27 3265988 T / C rs314315024 27 3266655 G / A rs14303789 27 3278202 A / C

[0049] The above loci have been reported to be associated with the age at start of laying in white-feathered broiler breeders (GRCg7b reference genome), providing new molecular markers for improving the age at start of laying traits in white-feathered broiler breeders.

[0050] According to the SNP marker related to the age at which white-feathered broiler breeder chickens start laying, the genotype of the polymorphic site contained in the SNP marker rs431896560 is a GG genotype, indicating that the individual starts laying at a later age; if the genotype is a GA genotype, it indicates that the individual has a medium-level age at which the individual starts laying; if the genotype is an AA genotype, it indicates that the individual starts laying at an earlier age.

[0051] The genotype of the polymorphic site contained in the SNP marker rs16207893 is TT genotype, indicating that the individual has a late age of calving; if the genotype is TC genotype, it indicates that the individual has a medium age of calving; if the genotype is CC genotype, it indicates that the individual has an early age of calving.

[0052] The genotype of the polymorphic site contained in the SNP marker rs314315024 is GG genotype, indicating that the individual has a late age of calving; if the genotype is GA genotype, it indicates that the individual has a medium age of calving; if the genotype is AA genotype, it indicates that the individual has an early age of calving.

[0053] The genotype of the polymorphic site contained in the SNP marker rs14303789 is AA genotype, indicating that the individual has a late age of calving; if the genotype is AC genotype, it indicates that the individual has a medium age of calving; if the genotype is CC genotype, it indicates that the individual has an early age of calving.

[0054] The present invention also provides a primer for amplifying the SNP marker associated with the trait of the age at which white-feathered broiler breeders begin laying.

[0055] Preferably, the primers for amplifying marker rs431896560 are shown in SEQ ID NO: 1-2.

[0056] Preferably, the primers for amplifying marker rs16207893 are shown in SEQ ID NO: 3-4.

[0057] Preferably, the primers for amplifying marker rs314315024 are shown in SEQ ID NO: 5-6.

[0058] Preferably, the primers for amplifying marker rs14303789 are shown in SEQ ID NOs: 7-8.

[0059] The present invention also provides a kit comprising the primers.

[0060] The present invention also provides application of the SNP molecular marker, the primer or the kit in breeding white-feather broiler breeders.

[0061] The present invention also provides the use of the SNP molecular marker, the primer or the kit in screening or breeding early-onset or late-onset broiler strains.

[0062] The present invention also provides a method for estimating GEBV based on integrating significant SNPs associated with age-onset traits, and selecting lines that start laying earlier or later. The method includes obtaining genomic DNA from chicken blood; performing genome-wide SNP profiling; and using an improved method to estimate GEBV, selecting individuals with high GEBV for breeding. If sequencing of the four loci is unsuccessful using other sequencing techniques, PCR with specific primers followed by sequencing or detection with a detection kit can be used.

[0063] The present invention also provides a method for determining significant SNP markers based on whole genome resequencing or PCR amplification and sequencing using the following specific primers.

[0064] Chicken genomic DNA was used as a template and PCR amplification and sequencing were performed using the upstream and downstream primers listed in Table 2.

[0065] Table 2 Primer sequences for amplification of the four sites

[0066]

[0067] The PCR reaction system is calculated based on 25 μl:

[0068]

[0069]

[0070] The PCR reaction conditions were as follows: 94°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, for a total of 35 cycles; 72°C for 5 min.

[0071] According to the sequencing results, the genotypes of the four SNP sites were counted.

[0072] The present invention also provides a method for selecting an earlier-onset and / or later-onset white-feathered broiler breeder strain, comprising the following steps:

[0073] 1) Obtain genomic DNA from white-feathered broiler breeder chicken samples and perform genome-wide SNP typing;

[0074] 2) obtain genotyping data of four SNP markers rs431896560, rs16207893, rs314315024, and rs14303789 for all samples;

[0075] 3) Estimating the whole genome breeding value of the genotyping data in step 2) and selecting individuals with better breeding values.

[0076] According to the method for selecting an earlier-laying and / or later-laying chicken strain, the method for estimating the whole genome breeding value in step 3) includes:

[0077] (1) G matrix construction and heritability estimation:

[0078] Using ASreml software, the whole genome SNPs without the four SNP markers and the G matrix based on the four SNPs, namely G0 and G SNP G is calculated as follows SNP Correction to G0 level:

[0079]

[0080] in, G after correction SNP Matrix, G SNP For the matrix constructed for the four SNPs, the calculation formulas for a and b are:

[0081] Avg(diag(Gsnp))*b+a=Avg(diag(G1))

[0082] Avg(offdiag(Gsnp)*b+a=Avg(offdiag(G1)

[0083] Set G1 and The relative weight formula is:

[0084]

[0085] In the formula, G2 represents the weight G matrix, G1 and See the above formula, the weight coefficient c is calculated as follows:

[0086]

[0087] and are the heritabilities of age at first laying trait estimated based on the whole genome SNPs after removing the four SNPs and the four SNPs, respectively. The heritabilities were estimated by ASReml v4.1 software;

[0088] (2) Estimation of genomic breeding values:

[0089] The breeding value of the first day of calving was estimated using the single-trait animal model of the constrained maximum likelihood method in ASReml v4.1 software. The model is as follows:

[0090] y=Xb+Za+e

[0091] Where y represents the phenotype vector, b represents the fixed effect vector, including batch and sex, a represents the random additive genetic effect vector and e represents the random residual effect vector, X and Z represent the correlation matrices of fixed effects and random additive genetic effects, respectively;

[0092] The random vector variance matrix is ​​as follows:

[0093]

[0094] in, and represent additive genetic variance and residual environmental variance, respectively; G2 is the integration matrix that assigns specific weights to the four SNPs; and I represents the identity matrix.

[0095] According to the SNP molecular marker, the primer, the kit, the application or the method for selecting an earlier or later-blooming line, the chicken is a white-feathered broiler breeder.

[0096] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples were performed according to conventional experimental conditions, such as those in Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions.

[0097] Example 1 Identification of candidate intervals affecting the age at start of laying of white-feathered broiler breeders based on GWAS analysis

[0098] 1. Experimental animals and phenotypic determination

[0099] In this embodiment, 4784 maternal groups of white-feathered broiler breeders and 2058 paternal groups were used as experimental materials. All experimental individuals were placed in the laying house at the age of 140 days and raised in single cages according to the broiler breeder breeding standards (NY / T33-2004). The paternal group weighed 3.1 kg at 45 days of age, and the maternal group weighed 2.5 kg at 42 days of age. The feed conversion rate of the two strains from 28 days to 42 days of age can reach below 1.70. Blood samples were collected from the brachial vein of each chicken for sequencing. The method for determining the age of first laying is to use a barcode scanner to record the laying time when the first egg is produced, and the age of first laying is calculated by recording the time of the first egg of each chicken. At the same time, the phenotypic quality control of the age of first laying is carried out, and individuals that deviate from the mean value by more than 3 times the standard deviation are eliminated.

[0100] The descriptive statistical results of the age at first laying of 6842 white-feathered broiler breeder chickens in both paternal and maternal groups are shown in Table 3.

[0101] Table 3 Statistical results of age at first spawning

[0102]

[0103] 2. GWAS analysis of the age at onset of laying in white-feathered broiler breeders

[0104] A total of 6,842 broiler chicken paternal and maternal individuals were genotyped using a 55K SNP genotyping array developed by the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences (see CN111225986A, completed by Beijing Compson Biotechnology Co., Ltd.). First, quality control of 54,130 SNPs from 6,842 individuals was performed using PLINK (V1.9) (https: / / www.cog-genomics.org / plink / 1.9 / ) software. SNPs with a minor allele frequency (MAF) ≥ 5% and a SNP call rate ≥ 90%, as well as SNPs with a single call rate ≥ 90%, were retained. Ultimately, 42,262 SNP loci on chromosomes 1 to 28 and chromosome Z were retained for subsequent analysis. After eliminating genotypic variants, missing loci were genotyped and filled using Beagle 5.2 software.

[0105] Whole-genome sequencing was performed on 150 randomly selected individuals from two lines using the Illumina NovaSeq 6000 platform. The latest GRCg7b reference genome was used, and each library was sequenced at a depth of at least 10X. Quality control was performed using PLINK (V1.9) software, with criteria of individual call rate ≥90%, SNP call rate ≥90%, and MAF ≥0.01. After screening, a total of 11,220,611 SNP markers from the 150 sequenced individuals were retained for subsequent analysis.

[0106] We used Beagle 5.2 software to impute the chicken 55K genotyping data to the whole genome sequencing (WGS) level. This analysis used a reference panel consisting of 150 whole genome sequenced individuals and a target panel consisting of 6,482 55K SNP genotyped individuals. Because the reference panel cannot have missing genotypes, we first imputed the resequencing data using the default parameters of Beagle 5.2 software. Then, the 55K SNP chip data were imputed to the resequencing level using the default parameters. The squared correlation coefficient (R) between the imputed genotypes and the true genotypes provided by Beagle 5.2 was used. 2 ) to evaluate the accuracy of genotype estimation. To ensure the accuracy of the estimation, BCFtools-1.16 software was used to perform strict quality control on each SNP, retaining the estimated allele R 2 SNPs with MAF ≥ 0.9 and MAF ≥ 0.05 were selected. After quality control, we calculated the allele R at the chromosome level for the two lines. 2 , average R 2 The value was ≥ 0.96. Finally, a total of 7,795,273 SNPs and 6,842 individuals were used in the subsequent GWAS.

[0107] GWAS analysis of age at first calving was performed using a single-trait mixed linear model (MLM) in GCTA software, with the first three principal components used as covariates. Given the large number of regions of linkage disequilibrium between SNPs, the Bonferroni correction was overly conservative. Therefore, independent testing of SNPs was performed using plink software --indep-pairwise 25 5 0.2, with an r² threshold of 0.2. The genome-wide significant P value threshold was adjusted based on the number of independent SNPs.

[0108] GWAS analysis of the paternal and maternal age-to-calving traits showed that no significant loci associated with this trait were located in the paternal line ( Figure 5 ), the maternal line was located at four significant sites on chromosomes 7, 19, and 27, and six genes including SLC6A4 and GIP were annotated ( Figure 6 ).

[0109] Example 2 Meta-analysis to determine the important candidate loci affecting the age at first spawning trait

[0110] In order to further identify the relevant regions, a meta-analysis was conducted on all individuals in the paternal and maternal lines. The results showed that three significant QTL intervals were located on chromosomes 7, 19 and 27 ( Figure 7 These association signals were primarily localized to two large QTL intervals (GGA7: 35.63-36.25 Mb and GGA19: 9.41-9.77 Mb) and annotated to multiple genes. Significant SNPs on chromosome 27 were concentrated in a smaller region, annotated to only two genes: IGF2BP1 and GIP.

[0111] Example 3 Evaluation of the effect of genomic selection on the age at start of laying of white-feathered broiler breeders using four key SNPs

[0112] 1. Experimental population and genotyping

[0113] Three generations of 4,067 white-feathered broiler breeders were used, and their phenotypes were recorded at the start of laying. In this example, a 55K SNP array was used to obtain genome-wide SNPs and four key SNPs. SNP quality control was performed according to the following standards: individual genotype detection rate less than 90%, single SNP detection rate less than 90%, and minimum allele frequency less than 5%. Missing SNPs were genotyped using Beagle 5.0 software.

[0114] 2. G matrix construction and heritability estimation using different strategies

[0115] Using ASReml software, the G matrix based on the whole genome SNPs (excluding 4 key SNPs) and the G matrix based on 4 key SNPs, namely G0 and G SNP . G is converted into SNP Correction to G0 level:

[0116]

[0117] in, G after correction SNP Matrix, G SNP The matrix constructed for the four key SNPs. Heritability estimates were performed separately. The formulas for a and b are:

[0118] Avg(diag(Gsnp))*b+a=Avg(diag(G1))

[0119] Avg(offdiag(Gsnp)*b+a=Avg(offdiag(G1)

[0120] Set G1 and The relative weight formula is:

[0121]

[0122] In the formula, G2 represents the weight G matrix, G1 and See the above formula. The weight coefficient c is calculated as follows:

[0123]

[0124] and The heritability of age-to-laying trait was estimated based on genome-wide SNPs (excluding four key SNPs) and four key SNPs, respectively. Heritability was estimated using ASReml v4.1 software.

[0125] 3) Estimation of genomic breeding values

[0126] The breeding value of the first day of calving was estimated using the single-trait animal model of the constrained maximum likelihood method in ASReml v4.1 software. The model is as follows:

[0127] y=Xb+Za+e

[0128] where y represents the phenotype vector, b represents the fixed effect vector, including batch and sex, a represents the random additive genetic effect vector, and e represents the random residual effect vector. X and Z represent the correlation matrices for the fixed effects and random additive genetic effects, respectively.

[0129] The random vector (co)variance matrix is ​​as follows:

[0130]

[0131] in, and represent additive genetic variance and residual environmental variance, respectively; G2 is the integration matrix that assigns specific weights to the four key SNPs; I represents the identity matrix.

[0132] 4) Forecast accuracy evaluation

[0133] The prediction accuracy of the age-to-lay trait was estimated using a five-fold cross-validation method. Random numbers were generated using the caret package in R v4.1, with random missing values ​​implemented to estimate prediction accuracy. The results are shown in Table 4.

[0134] Table 4 Comparison of genomic prediction accuracy after weighting 4 SNPs

[0135]

[0136] According to the results of cross-validation tests, compared with the G matrix constructed based on whole-genome SNPs, constructing an integrated G matrix by assigning specific weights to four key loci can improve the prediction accuracy of the age-to-laying trait by 4.50%.

[0137] Example 4: Breeding Method for Genomic Selection of White-Feathered Broiler Breeders for Age-to-Laying Traits by Integrating Four Key SNPs

[0138] 1. Establishment of reference population, determination of age-onset traits and genotyping

[0139] The individuals that make up the reference group must cover all existing families of the breed. A group of 2,000 to 3,000 chickens is formed as a reference group. The reference group will record individual egg production after entering the laying house. At the same time, whole blood is collected and stored in anticoagulant tubes for subsequent DNA extraction and genome variation sequencing. The reference group is subjected to whole-genome sequencing or "Beijing Core No. 1" chicken 55KSNP chip (see CN111225986A) measurement to obtain whole-genome SNPs for the next step of analysis.

[0140] 2. Establishment of test population and genotyping

[0141] The test flock is a candidate breeder flock with no documented phenotypic traits and intended for the next generation. The test flock must be related to the reference flock for no more than five generations. Blood samples should be collected from the test flock as early as possible, without compromising survival or growth, and submitted for whole-genome SNP microarray testing. Whole-genome SNP testing and quality control are then performed using the methods described above.

[0142] 3. Analysis of individual genomic estimated breeding values ​​(GEBV) of reference and candidate populations

[0143] The phenotypic value of each individual in the reference population, the genotype of each individual in the reference population's whole-genome SNP, and the whole-genotype of each individual in the test population were used. Note that 4 key SNP typings were included, with a total of 3 types of data files. This method was used to estimate the genome-estimated breeding value (GEBV).

[0144] Example 5: Direct selection of white broiler breeder chickens for age at laying using four key SNP typing

[0145] 1. Group to be selected

[0146] 300 chickens were randomly selected for testing, with a male-to-female ratio of 1:1. Blood was collected from the wing vein at around 20 days of age and stored in anticoagulant tubes at -20°C.

[0147] 2. DNA Extraction

[0148] Genomic DNA of blood samples was extracted by conventional phenol imitation method, dissolved in ultrapure water, and DNA purity and concentration were detected by agarose gel electrophoresis and NanoPhotometer-N50 spectrophotometer. The concentration was uniformly diluted to 50 ng / μL.

[0149] 3. PCR reaction and SNP typing

[0150] Primer sequences for the four key sites are shown in Table 2. The PCR reaction system consisted of 12.5 μL of 2× Master mix, 1.5 μL of DNA template, 1 μL each of 10 pmol / μL upstream and downstream primers, and 9 μL of ddH₂O, for a total of 25 μL. The prepared PCR reaction mixture was mixed and briefly centrifuged. The reaction was performed in an ABI Life ProFlex PCR instrument thermal cycler using the following program: 94°C for 5 min; 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min; and 72°C for 5 min.

[0151] Alleles of the amplified products were identified using next-generation sequencing. Based on the genotyping results, healthy male and female chickens with the desired genotypes for rs431896560, rs16207893, rs314315024, and rs14303789 were retained. A minimum of 80 males, with a male-to-female ratio of at least 1:3, were retained for breeding, and new lineages were established during peak egg-laying season.

[0152] The present invention relates to the screening of SNP markers related to the first laying age of chickens and the application of a method for integrating specific SNP markers to estimate GEBV. The invention screens four key SNP markers that affect the first laying age of chickens through methods such as genome-wide association analysis (GWAS).

[0153] Using SNP markers to construct a weighted G matrix for GEBV estimation, this allows for broiler breeding of target traits. By assigning specific weights to the G matrix constructed from these four key SNPs, an integrated G matrix was constructed, which improved the accuracy of predicting the age-to-lay trait for white-feathered broiler breeders. This improved prediction, and a breeding strategy for chicken lines with the best age-to-lay trait was developed based on this information.

[0154] The SNP markers and GEBV estimation method integrating significant SNPs provided by this invention can serve as an important reference for the selection of chickens for the age-to-lay trait and has important practical application value in genetic breeding. This method is beneficial for improving breeding progress, shortening the breeding cycle, and accelerating the improvement of independently cultivated varieties and the development of new broiler chicken lines.

[0155] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A SNP marker associated with the age at onset of laying of white-feathered broiler breeders, characterized in that: One or more selected from markers rs431896560, rs16207893, rs314315024, rs14303789; Among them, marker rs431896560 contains the nucleotide sequence with the polymorphism of G / A at 6694125bp on chicken chromosome 19; Marker rs16207893 contains the nucleotide sequence with a T / C polymorphism at bp 3265988 on chicken chromosome 27; Marker rs314315024 contains a nucleotide sequence with a G / A polymorphism at bp 3266655 on chicken chromosome 27; Marker rs14303789 contains a nucleotide sequence with polymorphism A / C at 3278202 bp on chicken chromosome 27.

2. The SNP marker according to claim 1, wherein The genotype of the polymorphic site contained in marker rs431896560 is GG, indicating that the individual has a late calving age; if the genotype is GA, it indicates that the individual has an intermediate calving age; if the genotype is AA, it indicates that the individual has an early calving age; The genotype of the polymorphic site contained in marker rs16207893 is TT, indicating that the individual has a late calving age; if the genotype is TC, it indicates that the individual has an intermediate calving age; if the genotype is CC, it indicates that the individual has an early calving age; The genotype of the polymorphic site contained in marker rs314315024 is GG, indicating that the individual has a late calving age; if the genotype is GA, it indicates that the individual has an intermediate calving age; if the genotype is AA, it indicates that the individual has an early calving age; The genotype of the polymorphic site contained in marker rs14303789 is AA, indicating that the individual has a late age of calving; if the genotype is AC, it indicates that the individual has a medium age of calving; if the genotype is CC, it indicates that the individual has an early age of calving.

3. A primer for amplifying the SNP marker according to claim 1 or 2, characterized in that: The upstream and downstream of the amplification marker rs431896560 are shown in SEQ ID NOs: 1-2, the upstream and downstream of the amplification marker rs16207893 are shown in SEQ ID NOs: 3-4, the upstream and downstream of the amplification marker rs314315024 are shown in SEQ ID NOs: 5-6, and the upstream and downstream of the amplification marker rs14303789 are shown in SEQ ID NOs: 7-8.

4. A detection reagent or kit containing the primer according to claim 3.

5. A method for identifying and breeding early-onset or late-onset white-feathered broiler breeder lines, characterized in that: include: 1) Extracting total DNA from white-feathered broiler breeder chickens to be tested; 2) using the DNA as a template and performing PCR amplification using the primers described in claim 3; 3) Analyze the PCR amplification products.

6. The method according to claim 5, characterized in that The PCR reaction system was as follows: 2× Master mix 12.5 μL, DNA template 1.5 μL, 1 μL each of 10 pmol / μL upstream and downstream primers, and ddH O added to 25 μL; The PCR reaction program was as follows: 94°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, for a total of 35 cycles; 72°C for 5 min.

7. The method according to claim 5 or 6, characterized in that Step 3) includes sequencing the amplified product and determining the following based on the sequencing results: The genotype of the polymorphic site contained in marker rs431896560 is GG, indicating that the individual has a late calving age; if the genotype is GA, it indicates that the individual has an intermediate calving age; if the genotype is AA, it indicates that the individual has an early calving age; The genotype of the polymorphic site contained in marker rs16207893 is TT, indicating that the individual has a late calving age; if the genotype is TC, it indicates that the individual has an intermediate calving age; if the genotype is CC, it indicates that the individual has an early calving age; The genotype of the polymorphic site contained in marker rs314315024 is GG, indicating that the individual has a late calving age; if the genotype is GA, it indicates that the individual has an intermediate calving age; if the genotype is AA, it indicates that the individual has an early calving age; The genotype of the polymorphic site contained in marker rs14303789 is AA, indicating that the individual has a late age of calving; if the genotype is AC, it indicates that the individual has a medium age of calving; if the genotype is CC, it indicates that the individual has an early age of calving.

8. Any of the following uses of the SNP marker or detection reagent thereof according to claim 1 or 2: (1) Used for early prediction of early or late laying white-feathered broiler breeders; (2) Used for molecular marker-assisted breeding of white-feathered broiler breeders.

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