Multi-gene SNP (Single Nucleotide Polymorphism) molecular marker combination for predicting chicken first laying day age and application of multi-gene SNP molecular marker combination

Abstract

The invention discloses a polygene SNP (Single Nucleotide Polymorphism) molecular marker combination for predicting the first laying age of chickens and application of the polygene SNP molecular marker combination, and belongs to the fields of molecular genetics and agricultural animal breeding. The marker combination comprises SNP (Single Nucleotide Polymorphism) of four genes which influence gonad development and play a key expression regulation role in a reproduction endocrine network, and the SNP is specifically positioned on 273282348, 1169302032, 273273533 and 1169302490 of a reference genome GRCg7b. The invention proves that the specific genotype combination of the four SNP loci is obviously related to the early laying day age of the chicken. By simultaneously detecting the four SNP loci of an individual to be detected, the premature genetic potential of the individual can be calculated and evaluated. The multi-gene combined prediction system constructed by the invention overcomes the defect of insufficient prediction effectiveness of a single marker, and provides a reliable molecular tool for efficient and accurate breeding of precocious strains of laying hens.

Description

Technical Field

[0001] This invention belongs to the fields of molecular genetics and agricultural animal breeding, specifically involving the application of SNP molecular marker combinations in the assisted selection of chickens based on their age at laying. Background Technology

[0002] Age at first egg (AFE), a core economic trait for measuring the timing of sexual maturity and reproductive potential in laying hens, not only affects the start time of the entire laying cycle but also has a significant impact on the total annual egg production. Especially in the context of intensive breeding, early prediction and precise control of AFE have become important goals for improving breeding efficiency. However, AFE is a complex quantitative trait typically controlled by multiple genes with minor effects. Traditional phenotypic selection is highly susceptible to environmental interference and has long generation intervals. Relying solely on a single SNP marker for assisted breeding often fails to achieve the desired improvement results in actual breeding populations due to limited explanatory power of genetic variation.

[0003] In recent years, genome-wide association studies (GWAS) combined with quantitative trait locus (eQTL) analysis and causal inference (such as colocation analysis) have enabled precise localization of causal variations and functional modules that regulate traits. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the insufficient accuracy of single molecular markers in predicting complex traits, and to provide a multi-gene SNP molecular marker combination that can more accurately and efficiently predict the age of first laying in chickens and its application in assisted breeding.

[0005] The technical solution of the present invention is: the application of SNP molecular marker combination in predicting or assisting breeding of chickens at the age of first laying, wherein the SNP molecular marker combination is composed of four SNP loci: SNP1, SNP2, SNP3 and SNP4.

[0006] SNP1: Located at position 3282348 bp on chromosome 27, it exhibits C / T polymorphism, with the dominant allele being C;

[0007] SNP2: Located at position 169302032 bp on chromosome 1, it exhibits T / A polymorphism, with the dominant allele being A;

[0008] SNP3: Located at position 3273533 bp on chromosome 27, it exhibits T / C polymorphism, with the dominant allele being T;

[0009] SNP4: Located at 169302490 bp on chromosome 1, it exhibits C / G polymorphism, with the dominant allele being G;

[0010] Individuals carrying more dominant alleles at the four SNP loci had a lower age at onset of labor, with the reference genome version being GRCg7b.

[0011] Application of substances for detecting the genotypes of the four SNP loci mentioned above in the preparation of chicken age-assisted breeding kits or liquid-phase chips.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] This invention, based on large-scale yellow-feathered broiler population data, precisely identified four genes—ENSGALG00010026239, ENSGALG00010004500, ENSGALG00010026607, and ENSGALG00010004493—that play a key regulatory role in the gonadal development and reproductive endocrine network. By combining specific SNP sites affecting the expression of these four genes, a multi-gene evaluation system is constructed, which can produce a synergistic genetic improvement effect, thereby significantly improving the prediction accuracy and genetic progress of precocious puberty traits in laying hens. This provides a practical and feasible technical path for molecular convergence breeding of precocious laying hen strains. Attached Figure Description

[0014] Figure 1 GWAS co-localization analysis of the eQTL of the gene corresponding to the Chr27:3282348 SNP site with age at labor.

[0015] Figure 2 Co-location analysis of eQTL and age at onset of labor for genes corresponding to SNP sites in Chr1:169302032 using GWAS.

[0016] Figure 3 GWAS co-localization analysis of the eQTL of the gene corresponding to the Chr27:3273533 SNP site with age at labor.

[0017] Figure 4 Co-location analysis of eQTL and age at onset of labor for genes corresponding to SNP sites in Chr1:169302490 using GWAS.

[0018] Figure 5 Differences in age-at-first-time phenotype distribution among populations carrying different numbers of favorable alleles at Chr27:3282348 locus.

[0019] Figure 6 Differences in age-at-first-time phenotype distribution among populations carrying different numbers of favorable alleles at the Chr1:169302032 locus.

[0020] Figure 7Differences in age-at-first-time phenotype distribution among populations carrying different numbers of favorable alleles at the Chr27:3273533 locus.

[0021] Figure 8 Differences in age-at-first-time phenotype distribution among populations carrying different numbers of favorable alleles at the Chr1:169302490 locus. Detailed Implementation

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial channels.

[0023] Example 1: Mining of Multi-Gene SNP Molecular Marker Combinations

[0024] High-density genomic variation detection was performed on a population of 22,328 yellow-feathered broiler chickens. Combined with strict age-onset phenotypic records, genome-wide association analysis (GWAS) and tissue-specific transcriptome eQTL colocalization analysis were used to precisely identify four causal variation sites with significant allelic substitution effects on chromosomes 1 and 27. Figures 1-8 The reference genome version for this location is GRCg7b.

[0025] Table 1. Basic information on multi-gene genetic marker loci

[0026] mutation location Reference / Mutant Alleles Mutant gene frequency Mutation effect Chr27:3282348 C / T 0.3599 -1.475 Chr1:169302032 T / A 0.3151 0.809 Chr27:3273533 T / C 0.3978 -1.547 Chr1:169302490 C / G 0.3229 0.779

[0027] Example 2: Combined detection of multiple gene SNP sites

[0028] Genotypes at loci were detected using the PCR-RFLP method.

[0029] Sample collection: Collect blood or other usable tissues from the sample chicken population, ensuring that the samples are uncontaminated, and label each sample with a number.

[0030] DNA extraction: Genomic DNA was extracted from the collected samples using commercial DNA extraction kits (such as Tiangen, QIAGEN, etc.) to ensure that the DNA quality met the requirements for PCR amplification.

[0031] Primer design: Design specific primers corresponding to the target SNP site. Online tools (such as Primer3, Primer-BLAST, etc.) are usually used to design primers to ensure that the primers can effectively amplify the target region.

[0032] PCR amplification: PCR amplification is performed using well-designed primers to ensure that the SNP sites of each sample are amplified under appropriate temperature and time conditions.

[0033] PCR product purification: After the PCR reaction is completed, primers, dNTPs and other impurities in the reaction system are removed using a DNA purification kit to obtain purified PCR products.

[0034] Genotyping: Using appropriate detection methods, such as restriction fragment length polymorphism (RFLP), high-resolution melting curve analysis (HRM), Sanger sequencing, etc., genotyping of PCR products is performed to determine the genotype of each sample at the target SNP site.

[0035] Example 3: Application of genetic scoring and multi-gene aggregation breeding

[0036] In practical breeding operations, a strategy of introducing multi-gene aggregation genetic scoring is adopted:

[0037] 1. Obtain high-throughput genotyping results for 4 SNP loci (Chr27:3282348, Chr1:169302032, Chr27:3273533, Chr1:169302490) for each individual in the candidate breeder chicken population.

[0038] 2. Count the total number of "precocious favorable alleles" carried by an individual (since each SNP locus is diploid, 4 loci can contribute a maximum of 8 favorable alleles), and construct a polygenic genetic scoring system of 0–8 points. Based on the scoring results, candidate breeder chickens are divided into high-performing groups (6–8), medium-performing groups (4–5), and low-performing groups (0–3), and this score is used to assist in the decision-making of breeder chicken selection, mating, and culling, so as to achieve rapid genetic stratification of breeder chickens in the early stage.

[0039] To verify the application value of this method in actual breeding, the genetic scores of the current generation of candidate breeding chickens were used as the basis for selection, and the age at first egg production in their next generation was counted. The results are shown in Table 2.

[0040] Table 2. Statistical results of age at onset in offspring populations of different genetic score groups.

[0041] Grouping Sample size AFE_mean std High (6-8) 2376 171.1001684 11.21750812 Mid(4-5) 1727 172.5993052 11.09509927 Low(0-3) 720 174.3111111 10.76156499

[0042] Screening is based on the number of favorable alleles carried, prioritizing high-scoring individuals carrying a high number of favorable alleles (e.g., homozygous or heterozygous individuals carrying 6-8 favorable alleles) and accelerating the elimination of individuals carrying unfavorable alleles. Joint evaluation of these four loci can overcome the bottleneck of single-gene selection and achieve rapid genetic improvement of the age-to-laying trait.

Claims

1. Application of SNP molecular marker combinations in predicting or assisting breeding of chickens at the age of first laying, wherein the SNP molecular marker combinations consist of four SNP loci: SNP1, SNP2, SNP3, and SNP4. SNP1: Located at position 3282348 bp on chromosome 27, it exhibits C / T polymorphism, with the dominant allele being C; SNP2: Located at position 169302032 bp on chromosome 1, it exhibits T / A polymorphism, with the dominant allele being A; SNP3: Located at position 3273533 bp on chromosome 27, it exhibits T / C polymorphism, with the dominant allele being T; SNP4: Located at 169302490 bp on chromosome 1, it exhibits C / G polymorphism, with the dominant allele being G; Individuals carrying more dominant alleles at the four SNP loci had a lower age at onset of labor, with the reference genome version being GRCg7b.

2. The application of the substance for detecting the genotypes of the four SNP loci described in claim 1 in the preparation of a chicken age-assisted breeding kit or liquid-phase chip.

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