SNP (Single Nucleotide Polymorphism) molecular marker related to egg laying character of chicken and application thereof

Abstract

The invention belongs to the technical field of molecular markers, and particularly relates to an SNP (Single Nucleotide Polymorphism) molecular marker related to egg laying traits of chickens and application of the SNP molecular marker. The SNP molecular marker is a single nucleotide polymorphism site in a chicken GTF2A1 gene, and the single nucleotide polymorphism site comprises a site 1 and / or a site 2; the site 1 is located at the 40334089bp site of the chromosome 5, the mutation base is G / C, the site 2 is located at the 40341114bp site of the chromosome 5, and the mutation base is A / G. The SNP molecular marker can accurately show the related characters of eggs, such as the number of eggs at the first laying day age and the number of eggs at the 300-day age.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular markers, and in particular relates to SNP molecular markers related to chicken egg-laying traits and uses thereof. Background Art

[0002] Egg production in breeder chickens is a core economic trait in poultry genetics and breeding. Its genetic optimization directly determines the reproductive efficiency of breeder chickens and the sustainable development of the industry. Using modern genomics technology to analyze the genetic characteristics of breeder chickens, screen key genetic markers, and achieve precision breeding is an important technical approach to improving reproductive performance.

[0003] The GTF2A1 (General Transcription Factor IIA Subunit 1) gene is a key factor in eukaryotic transcriptional regulation. The protein it encodes participates in RNA polymerase II transcription initiation and promoter clearance, and stabilizes DNA binding through interaction with TATA-binding protein (TBP), playing a central role in regulating gene expression. Its functions are not limited to transcriptional regulation but also involve the regulation of germ cell development and reproductive system function. Studies have shown that variants in the GTF2A1 gene are specifically expressed in germ cells and may affect reproductive performance by regulating germ cell differentiation. The association between its methylation levels and ovarian cancer further suggests its potential role in reproductive system function.

[0004] In the study of animal reproductive traits, the GTF2A1 gene has been confirmed to be related to the reproductive performance of species such as sheep and chickens: in sheep, its g.89505005G>A site polymorphism is significantly associated with the number of lambs born, and individuals carrying the A allele have a higher number of lambs; in chickens, genome-wide association analysis (GWAS) showed that the GTF2A1 gene is significantly associated with the number of eggs produced, and it is speculated that it may affect egg production performance by regulating ovarian and uterine function.

[0005] Although the reproductive trait regulatory function of the GTF2A1 gene in species such as sheep has been partially revealed, and its association with egg production has been preliminarily found in chickens, there has been no systematic report on the single nucleotide polymorphisms (SNPs) of the GTF2A1 gene in chickens and their specific effects on egg production traits, and there is still a technical gap in the screening and application of related molecular markers. Summary of the Invention

[0006] Based on this, the present invention provides a SNP molecular marker related to chicken egg-laying traits and its use.

[0007] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0008] On the one hand, the present invention provides a SNP molecular marker related to chicken egg-laying traits, the SNP molecular marker is a single nucleotide polymorphism site in the chicken GTF2A1 gene, the single nucleotide polymorphism site includes site 1 and / or site 2; site 1 is located at the 40334089bp position of chromosome 5, and the polymorphism is G / C, and site 2 is located at the 40341114bp position of chromosome 5, and the polymorphism is A / G.

[0009] Another aspect of the present invention provides a primer pair for amplifying the above-mentioned SNP molecular marker.

[0010] Preferably, the primer pair sequences for amplifying the SNP molecular marker comprising site 1 are shown as SEQ ID NO:1 and SEQ ID NO:2, and the primer pair sequences for amplifying the SNP molecular marker comprising site 2 are shown as SEQ ID NO:3 and SEQ ID NO:4.

[0011] In another aspect, the present invention provides a detection reagent for detecting SNP molecular markers, wherein the detection reagent includes the above-mentioned primer pair.

[0012] In another aspect, the present invention provides a use of the above-mentioned SNP molecular marker related to chicken egg-laying traits, or the above-mentioned primer, or the above-mentioned detection reagent, the use being selected from any one of the following 1) to 8):

[0013] 1) Prediction of chicken egg production traits;

[0014] 2) preparing reagents for predicting egg production traits in chickens;

[0015] 3) Screening or identifying chicken strains that have characteristics of early onset of laying and / or high egg production at 300 days of age;

[0016] 4) preparing reagents for screening or identifying chicken strains characterized by early onset of laying and / or high egg production at 300 days of age;

[0017] 5) Breeding for egg-laying traits in chickens;

[0018] 6) Molecular marker-assisted breeding related to egg production traits in chickens;

[0019] 7) Breed improvement related to egg-laying traits of chickens;

[0020] 8) GTF2A1 genotyping;

[0021] Among them, for the SNP molecular marker containing site 1, the genotype is GG, which corresponds to the traits of early onset of egg laying and high egg production at 300 days of age;

[0022] For the SNP molecular marker containing site 2, the genotype is AA, corresponding to the traits of early onset of egg laying and high egg production at 300 days of age;

[0023] For the haplotype combination, the genotype is GGAA, which corresponds to the traits of early age at first laying and high egg number at 300 days of age.

[0024] In another aspect, the present invention provides a method for breeding chickens with early onset of laying and high egg production at 300 days of age, characterized in that the method comprises: selecting individuals with early onset of laying and high egg production at 300 days of age for breeding as female parents for the next generation of breeding based on SNP molecular markers;

[0025] The SNP molecular markers are single nucleotide polymorphism sites in the chicken GTF2A1 gene, including site 1 and / or site 2; site 1 is located at position 40334089 on chromosome 5, with a mutation base of G / C; site 2 is located at position 40341114 on chromosome 5, with a mutation base of A / G;

[0026] Among them, for the SNP molecular marker containing site 1, the genotype is GG, which corresponds to the traits of early onset of egg laying and high egg production at 300 days of age;

[0027] For the SNP molecular marker containing site 2, the genotype is AA, corresponding to the traits of early onset of egg laying and high egg production at 300 days of age;

[0028] For the haplotype combination, the genotype is GGAA, which corresponds to the traits of early age at first laying and high egg number at 300 days of age.

[0029] The beneficial effects of the present invention include:

[0030] (1) g.40334089G>C locus (located at position 40334089bp on chromosome 5, with polymorphism of G / C) The GG genotype individuals had the earliest age at first laying eggs, which was significantly earlier than the CC and CG genotype individuals (P<0.05). The GG genotype individuals had the highest number of eggs laid at 300 days, which was significantly higher than the CC and CG gene individuals (P<0.05). The CC genotype individuals had the heaviest egg weight at 300 days, which was significantly higher than the CG and GG individuals (P<0.05). The GG genotype individuals had the lightest egg weight at 300 days.

[0031] (2) The age of first laying of individuals with AA genotype at the g.40341114A>G locus (located at 40341114bp on chromosome 5) was significantly earlier than that of individuals with GA and GG genotypes (P<0.05). The number of eggs produced by individuals with AA genotype at 300 days of age was significantly higher than that of individuals with GA and GG genotypes (P<0.05).

[0032] (3) The number of eggs produced by individuals with the haplotype combination H1H1 (GGAA) at 300 days of age was the highest, which was significantly higher than that of individuals with the haplotype combinations H2H1 (CGGA), H2H2 (CCGG), H2H3 (CGGG) and H3H1 (GGGA) at 300 days of age (P<0.05). Although the difference in the number of eggs produced by individuals with the haplotype combination H3H3 at 300 days of age was not significant, the number of eggs produced by individuals with the haplotype combination H3H3 at 300 days of age was 7.37 more on average than that of individuals with the haplotype combination H3H3 (GGGG). The age at first spawning of individuals with different haplotype combinations was significantly different. The age at first spawning of individuals with haplotype combination H3H3 (GGGG) was significantly earlier than that of individuals with haplotype combinations H2H1 (CGGA) and H2H2 (CCGG) (P<0.05). There was no significant difference in age at first spawning between individuals with haplotype combinations H1H1 (GGAA), H2H3 (CGGG), and H3H1 (GGCA). However, the age at first spawning of individuals with haplotype combination H3H3 (GGGG) was 1.98, 6.67, and 5.79 days earlier than that of individuals with haplotype combinations H1H1 (GGAA), H2H3 (CGGG), and H3H1 (GGCA), respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the first generation sequence map of two SNP sites in the GTF2A1 gene;

[0034] Figure 2 The location of GTF2A1 gene in chromosome and the direction of transcription initiation

[0035] Figure 3 The mass spectrometry typing diagrams of two SNP sites of GTF2A1 gene; among them, (a) is SNP1, g.40334089G>C, NOcall(1), CC(27), CG(118), GG(257), CC=0.02, CG=0.31, GG=0.67; (b) is SNP2, g.40341114A>G, NOcall(2), GG(29), GA(166), AA(187), GG=0.08, GA=0.43, AA=0.49;

[0036] Figure 4 Linkage disequilibrium analysis of two SNP sites in the GTF2A1 gene; (a) is the linkage map, D'=1.0, R 2 =0.508, LOD=57.58, the numerical value indicates the degree of linkage; (b) is haplotype analysis, the numerical value indicates the frequency of unit type. DETAILED DESCRIPTION

[0037] The examples given are for better explaining the present invention, but the content of the present invention is not limited to the given examples only. Therefore, those skilled in the art's non-essential improvements and adjustments to the implementation schemes based on the above-mentioned invention content still fall within the protection scope of the present invention.

[0038] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. Unless having an obviously different meaning in the context, the expressions in singular form include those in plural form. As used herein, it should be understood that terms such as "comprising", "having", "containing" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility of the existence or addition of one or more other features, numbers, operations, components, parts, elements, materials or their combinations. As used herein, depending on the situation, " / " can be interpreted as "and" or "or".

[0039] In the present invention, Sanger sequencing was performed on the mRNA region of the female line of Suqin No. 4, and 2 SNP sites were found, namely the G→C mutation at the g.40334089G>C site and the A→G mutation at the g.40341114A>G site. According to the classification of SNPs, the g.40334089G>C site is a transversion-type mutation, and the g.40341114A>G site is a transition-type mutation and a synonymous mutation. Both of these 2 SNP sites have 3 genotypes, and the results of the Hardy-Weinberg equilibrium test show that both sites are in the Hardy-Weinberg equilibrium state. This indicates that the experimental population of the female line of Suqin No. 4 has not been affected by natural selection, mutation, migration or genetic drift during the long-term breeding process, and there is no inbreeding during the generation expansion. The genetic richness of the population is directly proportional to its genetic diversity, and the latter is usually measured by indicators such as expected heterozygosity (He) and polymorphic information content (PIC). Both the g.40334089G>C site and the g.40341114A>G site are moderately polymorphic (0.25 < He < 0.5, PIC < 0.5), indicating that the breeding population in this experiment has abundant genetic variations for selection and utilization. The offspring of generation expansion have higher genetic diversity, can produce stronger heterosis, have stronger ability to adapt to environmental changes, can reduce the risk of inbreeding depression, and have greater selection potential.

[0040] In addition, the present invention combines Sanger sequencing and Sequenom mass spectrometry sequencing, and finds that both of the 2 SNP sites, namely the g.40334089G>C site and the g.40341114A>G site, in the mRNA region of the female line of Suqin No. 4 are significantly correlated with the age at first laying and the number of eggs laid at 300 days of age.

[0041] In addition, gene mutations can occur in multiple functional regions of the genome, including untranslated regions (UTRs), coding regions, and introns. Mutations at different sites affect gene expression, protein function, or regulatory networks through different molecular mechanisms, leading to phenotypic variation. The present invention discovered two SNP sites, one located in the 5'UTR (g.40334089G>C) and the other located in the exon 3 region (g.40341114A>G). Although the G→A mutation in the 5'UTR did not cause changes in regulatory elements, the A→G mutation in the exon 3 region was a synonymous mutation and did not cause amino acid changes. However, the 5'-UTR mutation can interfere with the assembly of the mRNA translation initiation complex, and the translation of the exon region may also interfere with abnormal protein synthesis. Therefore, mutations at these two sites are significantly correlated with the age of first laying and the number of eggs produced at 300 days of age.

[0042] In addition, protein-protein interactions are often mediated by short linear motifs in one protein and structural domains in another, leading to changes in cellular function. In this study, MEME analysis of the GTF2A1 gene revealed that the A→G mutation at the g.40341114A>G SNP site enhanced the motif's binding to the transcription factor Myod1 and weakened its binding to the transcription factors REST and PPARG. Individuals with the AA genotype at this site began laying eggs at an earlier age and had higher egg production at 300 days of age, suggesting that the binding of the motif to REST and PPARG regulates GTF2A1 gene expression, thereby improving egg production.

[0043] In addition, phenotypic variation is mediated by single-site mutations or multi-SNP linkage effects. By locating common genetic loci through linkage disequilibrium analysis, we can further reveal the multi-site interactions of non-allelic genes, thereby systematically analyzing the genetic basis of phenotypic variation. This study found that two SNPs sites in the GTF2A1 gene, g.40334089G>C and g.40341114A>G, were in strong linkage disequilibrium (|D'|=1.0, R 2 =0.508 and LOD =57.58). Mating pattern, population mixing, and selection pressure jointly regulate the construction and maintenance of SNP linkage disequilibrium within a population. The Suqin 4 maternal line used in this experiment had been bred for six generations, resulting in strong selection pressure, which may have led to strong linkage between the two SNP loci.

[0044] Based on this, the present invention provides a SNP molecular marker related to chicken egg-laying traits and its use, and the specific technical solution is as follows:

[0045] In the first aspect, an embodiment of the present invention provides a SNP molecular marker related to chicken egg-laying traits, the SNP molecular marker being a single nucleotide polymorphism site in the chicken GTF2A1 gene, the single nucleotide polymorphism site including site 1 and / or site 2; site 1 being located at position 40334089bp on chromosome 5, with a polymorphism of G / C, and site 2 being located at position 40341114bp on chromosome 5, with a polymorphism of A / G.

[0046] In a second aspect, an embodiment of the present invention provides a primer pair for amplifying the above-mentioned SNP molecular marker.

[0047] It should be noted that the primer pairs that can amplify the above-mentioned SNP molecular markers all meet the requirements of the present invention, and the design of the primer pairs can be designed according to conventional methods in the art.

[0048] In some specific examples, the primer pair sequences for amplifying the SNP molecular marker containing site 1 are shown as SEQ ID NO:1 and SEQ ID NO:2, and the primer pair sequences for amplifying the SNP molecular marker containing site 2 are shown as SEQ ID NO:3 and SEQ ID NO:4.

[0049] In a third aspect, an embodiment of the present invention provides a detection reagent for detecting SNP molecular markers, wherein the detection reagent includes the above-mentioned primer pair.

[0050] It should be noted that the detection reagent in the present invention generally refers to the form of a reagent or a kit. In addition, the forms of the reagent and the kit are well known in the art.

[0051] In a fourth aspect, an embodiment of the present invention provides a use of the above-mentioned SNP molecular marker related to chicken egg-laying traits, or the above-mentioned primer, or the above-mentioned detection reagent, the use being selected from any one of the following 1)-8):

[0052] 1) Prediction of chicken egg production traits;

[0053] 2) preparing reagents for predicting egg production traits in chickens;

[0054] 3) Screening or identifying chicken strains that have characteristics of early onset of laying and / or high egg production at 300 days of age;

[0055] 4) preparing reagents for screening or identifying chicken strains characterized by early onset of laying and / or high egg production at 300 days of age;

[0056] 5) Breeding for egg-laying traits in chickens;

[0057] 6) Molecular marker-assisted breeding related to egg production traits in chickens;

[0058] 7) Breed improvement related to egg-laying traits of chickens;

[0059] 8) GTF2A1 genotyping;

[0060] Among them, for the SNP molecular marker containing site 1, the genotype is GG, which corresponds to the traits of early onset of egg laying and high egg production at 300 days of age;

[0061] For the SNP molecular marker containing site 2, the genotype is AA, corresponding to the traits of early onset of egg laying and high egg production at 300 days of age;

[0062] For the haplotype combination, the genotype is GGAA, which corresponds to the traits of early age at first laying and high egg number at 300 days of age.

[0063] In some specific examples, in the above-mentioned use, the chicken breed is Suqin No. 4.

[0064] In a fifth aspect, an embodiment of the present invention provides a method for breeding chickens with an early onset of laying and a high egg production at 300 days of age, characterized in that the method comprises: selecting individuals with an early onset of laying and a high egg production at 300 days of age for breeding as female parents for the next generation of breeding based on SNP molecular markers;

[0065] The SNP molecular markers are single nucleotide polymorphism sites in the chicken GTF2A1 gene, including site 1 and / or site 2; site 1 is located at position 40334089 on chromosome 5, with a mutation base of G / C; site 2 is located at position 40341114 on chromosome 5, with a mutation base of A / G;

[0066] Among them, for the SNP molecular marker containing site 1, the genotype is GG, which corresponds to the traits of early onset of egg laying and high egg production at 300 days of age;

[0067] For the SNP molecular marker containing site 2, the genotype is AA, corresponding to the traits of early onset of egg laying and high egg production at 300 days of age;

[0068] For the haplotype combination, the genotype is GGAA, which corresponds to the traits of early age at first laying and high egg number at 300 days of age.

[0069] In some specific examples, in the above method, the chicken breed is Suqin No. 4.

[0070] It should be noted that the GG genotype individuals at the g.40334089G>C site (located at position 40334089bp on chromosome 5, with the mutant base being G / C) had the earliest age at first laying eggs, which was significantly earlier than the age at first laying eggs of the CC and CG genotype individuals (P<0.05). The GG genotype individuals had the highest number of eggs laid at 300 days, which was significantly higher than the number of eggs laid at 300 days of age of the CC and CG gene individuals (P<0.05). The CC genotype individuals had the heaviest egg weight at 300 days of age, which was significantly higher than the egg weight of the CG and GG individuals (P<0.05). The GG genotype individuals had the lightest egg weight at 300 days of age. The age of first laying of AA genotype individuals with g.40341114A>G site (located at position 40341114bp on chromosome 5, with the mutant base being A / G) was significantly earlier than that of GA and GG genotype individuals (P<0.05), and the number of eggs laid at 300 days of age of AA genotype individuals was significantly higher than that of GA and GG genotype individuals (P<0.05). The number of eggs produced by individuals with the haplotype combination H1H1 (GGAA) at 300 days of age was the highest, which was significantly higher than the number of eggs produced by individuals with the haplotype combinations H2H1 (CGGA), H2H2 (CCGG), H2H3 (CGGG) and H3H1 (GGGA) at 300 days of age (P<0.05). Although the difference in the number of eggs produced by individuals with the haplotype combination H3H3 (GGGG) at 300 days of age was not significant, the number of eggs produced by individuals with the haplotype combination H3H3 (GGGG) at 300 days of age was 7.37 more on average. The age at first laying of individuals with different haplotype combinations was significantly different. The age at first laying of individuals with haplotype combination H3H3 (GGGG) was significantly earlier than that of individuals with haplotype combinations H2H1 (CGGA) and H2H2 (CCGG). Although the age at first laying of individuals with haplotype combination H1H1 (GGAA), H2H3 (CGGG) and H3H1 (GGCA) was not significantly different, the age at first laying of individuals with haplotype combination H1H1 (GGAA), H2H3 (CGGG) and H3H1 (GGCA) was on average 1.98, 6.67 and 5.79 days earlier than that of individuals with haplotype combination H1H1 (GGAA), H2H3 (CGGG) and H3H1 (GGCA), respectively.

[0071] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0072] In the following example, the experiment was conducted at the Yizheng base of the Jiangsu Poultry Science Research Institute. Suqin No. 4 maternal line (M line) hens were housed in individual cages according to breeder breeding management conditions. After they began laying, their weight at first laying, egg weight at first laying, average egg weight at 300 days of age, and number of eggs laid at 300 days of age were recorded and statistically analyzed. From a large flock of hens, 384 were randomly selected, and 1 ml of blood was drawn from the subwing vein. Blood was added to 0.3 ml of anticoagulant (5 mg / ml heparin sodium) and stored at -20°C.

[0073] In the following examples, Taq DNA polymerase, dNTPs, and blood / cell / tissue genomic DNA extraction kit (model: DP318) were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. UV spectrophotometer: NanoDrop 2000 (ThermoFisher Scientific).

[0074] In the following example, the genomic DNA extraction method includes: extracting genomic DNA from chicken whole blood using a blood / cell / tissue genomic DNA extraction kit, detecting DNA quality and concentration using a UV spectrophotometer (NanoDrop 2000), and diluting the DNA to 50 ng / μL.

[0075] Example 1 SNPs site determination

[0076] (1) Primer design

[0077] Sanger sequencing primers Six pairs of primers were designed in the chicken GTF2A1 gene mRNA region using Primer5.0 software based on the DNA sequence of the chicken GTF2A1 gene published in GenBank (GenBank accession number: NC_052536.1). The primer information is shown in Table 1. The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0078] Table 1 Related information of first-generation sequencing primers in the exon region of GTF2A1 gene

[0079]

[0080]

[0081] In addition, the MassARRAY SNP primer set was designed based on the chicken genome (version number: bGalGal1.mat.broiler.GRCg7b) sequence at the 40334089 bp site and the 40341114 bp site of the chromosome 5 genome sequence of the M line hen determined by Sanger sequencing using the primer design software Assay design 3.1 of Sequen om Company. The primer information is shown in Table 2. The primers were synthesized by Jinweizhi Biotechnology (Beijing) Co., Ltd.

[0082] Table 2 Primer information for SNP sites in the exon region of GTF2A1 gene

[0083]

[0084]

[0085] (2) PCR amplification, first-generation sequencing, and SNP site determination

[0086] 1 μL of DNA sample from each of the 384 individuals was placed in a 5 mL sterile centrifuge tube and mixed thoroughly. The sample was then used as a DNA template for PCR amplification using first-generation sequencing primers. The amplification system (25 μL) consisted of 2.5 μL of 10× PCR buffer (containing Mg2+), 1.0 μL of each 10 μmol / L upstream and downstream primer, 2.0 μL of 2 mmol / L dNTPs, 0.5 μL of 5.0 U / μL Taq DNA polymerase, 1.0 μL of 50 ng / μL DNA template, and ultrapure water to 25 μL. PCR amplification conditions included initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing for 15 s (see Table 1 for annealing temperature), and extension at 72°C for 30 s. Finally, the sample was extended at 72°C for 10 min and stored at 4°C. The PCR products were detected by 1.5% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing using an ABI3730 sequencer. The sequencing results were analyzed using DNAstar software to determine the SNPs sites.

[0087] (III) Sequenom MassARRAY-SNPs site determination

[0088] Multiplex PCR (primers see Table 2) was used to amplify DNA fragments containing multiple target sites. Shrimp alkaline phosphatase (SAP) was used to purify and remove excess primers and dNTPs in the multiplex PCR reaction system. Single-base extension reactions were performed using dideoxynucleotides (ddNTPs) as substrates in the presence of iPLEX enzyme using extension primers designed for each target site (primers see Table 2). The extension products were diluted threefold, desalted on a resin, and transferred to a 384-well SpectroCHIP chip for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) on a Sequenom mass spectrometer. Different alleles at the same target site generated distinct detection peaks due to their different molecular weights. Mass spectrometry peaks were detected using Typer 4.0 software, and the SNP genotype at each site was determined based on the different detection peak sizes.

[0089] Among them, each reaction system is as follows:

[0090] Multiplex PCR reaction system: 972.5 μL of HPLC-grade water, 331.25 μL of 10× Buffer, 172.25 μL of 25 mM MgCl₂, 53 μL of 25 mM dNTPs, 530 μL of 0.5 μM upstream and downstream primer mix, 106 μL of 5 U / μL Taq polymerase. After mixing, add 4 μL / well of the mixture to a 384-well plate. Then, add 1 μL of 10 ng / μL DNA template to each well. Multiplex PCR amplification protocol: 94°C initial denaturation for 2 min; 94°C denaturation for 20 s, 56°C annealing for 30 s, 72°C extension for 60 s, for a total of 45 cycles of denaturation, annealing, and extension; 72°C extension for 3 min.

[0091] SAP digestion system: 810.9 μL of HPLC-grade water, 90.1 μL of 10× Buffer, 159 μL of 1.7 U / μL SAP, mix well, and add 24 μL / well to a 384-well plate. SAP reaction procedure: 37°C for 40 min, 85°C for 5 min;

[0092] Extension reaction system: 400.2 μL of HPLC-grade water, 106 μL of 10× iPLEX Buffer plus, 106 μL of iPLEX stop buffer, 426.1 μL of 1.0 μM primer, 21.7 μg of iPLEX enzyme. After mixing, add 2 μL / well to a 384-well plate. Extension reaction protocol: initial denaturation at 94°C for 30 seconds; one cycle of denaturation at 94°C for 5 seconds, annealing at 52°C for 5 seconds, extension at 80°C for 5 seconds, annealing and extension for 5 cycles, followed by 40 cycles of denaturation, annealing and extension; and extension at 72°C for 3 minutes.

[0093] (IV) Statistical analysis

[0094] The gene frequency, genotype frequency, heterozygosity (He), polymorphic information content (PIC), and effective number of alleles (Ne) at the GTF2A1 gene g.40334089G>C and g.40341114A>G loci in Suqin No. 4 maternal line (M line) hens were analyzed. The Hardy-Weinberg test was performed using the chi-squared (χ2) test. Linkage disequilibrium between the two SNPs was analyzed online.

[0095] Univariate analysis of variance was performed to analyze the association between genotypes and egg-laying traits such as age at first laying, weight at first laying, egg weight at first laying, egg weight at 300 days of age, and egg number at 300 days of age in Suqin No. 4 hens (M line). All data are presented as mean ± standard deviation. Fixed factors included different SNP marker genotypes, and dependent variables included age at first laying, weight at first laying, egg weight at first laying, egg weight at 300 days of age, and egg number at 300 days of age. LSD multiple comparisons were performed to determine the significance of differences in age at first laying, weight at first laying, egg weight at first laying, average egg weight at 300 days of age, and egg number at 300 days of age between different marker genotypes. P < 0.05 indicated a significant difference.

[0096] The MEME website (https: / / meme-suite.org) was used to analyze the conserved motif of the short sequence pattern at the GTF2A1 gene mutation site, and the Tomtom plug-in was used to analyze the transcription factor binding region of the motif at the GTF2A1 gene SNP site.

[13] , the GOMo plug-in was used to predict the function of the GTF2A1 gene SNP site motif.

[0097] (V) Results

[0098] (1) Sequencing and sequence analysis

[0099] The amplification products of 384 mixed samples using primers P1 to P6 were sequenced, and the results showed that one site each of the 5'UTR and exon3 of the GTF2A1 gene was mutated ( Figure 1 The position of the GTF2A1 gene SNP site corresponding to the whole genome can be located through the European Bioinformatics Institute Ensembl database "http: / / asia.ensembl.org / index.html?redirect=no". SNP1 is located at the 40334089bp position (5'UTR region) of the genomic sequence of chromosome 5 based on the chicken genome (version number: bGalGal1.mat.broiler.GRCg7b), where a G→C mutation occurs. Analysis found that the transcription start of the GTF2A1 gene is reversed ( Figure 2 ), so the mutation of the g.40334089G>C site in the 5'UTR region did not cause changes in the regulatory elements. SNP2 was located at the 40341114bp site (exon3 region) of the chromosome 5 genome sequence based on the chicken genome (version number: bGalGal1.mat.broiler.GRCg7b), and an A→G mutation occurred. Analysis found that the mutation of the g.40341114A>G site in the exon3 region was a synonymous mutation and did not cause amino acid changes.

[0100] (2) Mass spectrometry analysis of GTF2A1 gene SNP sites

[0101] Mass spectrometry analysis diagrams of different genotypes of 2 SNP loci of the GTF2A1 gene in 384 M-line hens are shown in Figure 3 , Sequenom Genotyping by SNP technology found that there were three genotypes, GG, GC, and CC, at the g.40334089G>C locus of the GTF2A1 gene in M-line hens ( Figure 3 (a)), with a detection rate of 99.5% (382 detected). There were three genotypes, AA, AG, and GG, at the g.40341114A>G locus of the GTF2A1 gene in M-line hens ( Figure 3 (b)), with a detection rate of 99.7% (383 detected).

[0102] (3) Population genetics analysis of SNP loci of the GTF2A1 gene

[0103] According to the genotyping results of the g.40334089G>C locus and the g.40341114A>G locus of the GTF2A1 gene, the genotype frequencies and allele frequencies of the two loci in M-line hens were statistically analyzed (see Table 3). The results showed that there were three genotypes in M-line hens at the g.40334089G>C locus, namely CC, CG, and GG genotypes, with the GG genotype being the main one, and the G allele was significantly higher than the C allele; there were three genotypes in M-line hens at the g.40341114A>G locus, namely AA, GA, and GG genotypes, with the AA genotype being the main one, and the A allele frequency was significantly higher than the G allele frequency.

[0104] Table 3 Allele frequencies and genotype frequencies of exon 2 of the GTF2A1 gene

[0105]

[0106] In addition, the results of the Hardy-Weinberg equilibrium test are shown in Table 4, χ 2 The test results showed that both the g.40334089G>C locus and the g.40341114A>G locus were in Hardy-Weinberg equilibrium (P>0.05). For the g.40334089G>C locus and the g.40341114A>G locus, 0.25<He<0.5 and PIC<0.5, indicating moderate polymorphism.

[0107] Table 4 Hardy-Weinberg equilibrium test and population genetics parameters of 2 SNP loci

[0108]

[0109] (4) Linkage disequilibrium and haplotype analysis

[0110] The results of linkage disequilibrium analysis are shown in Figure 4 , according to the classic threshold standard (|D'|>0.80 and R 2 >0.33), and there is a high degree of linkage disequilibrium between SNPs. In this experiment, the two SNPs sites of GTF2A1 gene g.40334089G>C and g.40341114A>G have |D'| = 1.0 Figure 4 -(a)) indicates that the two loci are in complete linkage disequilibrium, R 2 =0.508( Figure 4 (a)) indicates that the two loci are in high linkage disequilibrium. Morton proposed LOD>3.0 as the significance standard for linkage analysis (P<0.0001). In this experiment, LOD=57.58 ( Figure 4 (a)), indicating that the possibility of linkage between the two loci is extremely high, which effectively reduces false positives. Figure 4 (b) It can be seen that the strong linkage between SNP1 and SNP2 produces three haplotypes, namely H1:GA, H2:CG and H3:GG, with haplotype frequencies of GA:0.707, CG:0.174 and GG:0.119 respectively.

[0111] The genotypes and genotype frequencies after the g.40334089G>C and g.40341114A>G haplotype combinations are shown in Table 5. There are 6 haplotype combinations in total, the dominant haplotype combinations are GGAA and CGGA, and the three haplotype combinations of CCGA, CCAA, and CGAA were not detected.

[0112] Table 5 Haplotype combinations, genotypes and genotype frequencies of two SNP sites in the GTF2A1 gene

[0113] Unit type combination genotype Number of individuals Genotype frequency H1H1 GGAA 187 0.49 H2H1 CGGA 100 0.26 H2H2 CCGG 8 0.02 H2H3 CGGG 17 0.04 H3H1 GGGA 66 0.17 H3H3 GGGG 4 0.01

[0114] (5) Association analysis between GTF2A1 gene single-site polymorphism and maternal egg-laying traits of Suqin 4

[0115] Association analysis was conducted between the two polymorphic sites (g.40334089G>C and g.40341114A>G) of the GTF2A1 gene in the Suqin No. 4 maternal population and egg production traits. The results are shown in Table 6. Significant differences in egg production traits were observed between the genotypes at the two SNP sites (g.40334089G>C and g.40341114A>G). Individuals with the GG genotype at the g.40334089G>C site had the earliest age at onset of egg production, significantly earlier than those with the CC and CG genotypes (P<0.05). Individuals with the GG genotype had the highest egg production at 300 days, significantly higher than those with the CC and CG genotypes (P<0.05). Individuals with the CC genotype had the heaviest egg weight at 300 days, significantly higher than those with the CG and GG genotypes (P<0.05). Individuals with the GG genotype had the lightest egg weight at 300 days. The age of first laying of individuals with AA genotype at g.40341114A>G locus was significantly earlier than that of individuals with GA and GG genotypes (P<0.05), and the number of eggs laid at 300 days of age of individuals with AA genotype was significantly higher than that of individuals with GA and GG genotypes (P<0.05).

[0116] Table 6 Association analysis between different genotypes of two SNP sites of GTF2A1 gene and egg production traits

[0117]

[0118] Note: There was no significant difference between the mean values ​​of the same site with the same letter shoulder (P>0.05), while there was a significant difference between the mean values ​​of the same site with different letter shoulders (P<0.05).

[0119] (6) Association analysis between haplotype combinations and egg-laying traits of Suqin 4 maternal line

[0120] The association analysis between the g.40334089G>C and g.40341114A>G haplotype combinations and the egg-laying traits of the maternal line of Suqin No. 4 was performed, and the results are shown in Table 7. The number of eggs laid at 300 days of age among individuals with different haplotype combinations was significantly different. The number of eggs laid at 300 days of age by individuals with the haplotype combination H1H1, i.e., the GGAA genotype, was the highest, significantly higher than the number of eggs laid at 300 days of age by individuals with the haplotype combinations H2H1 (CGGA), H2H2 (CCGG), H2H3 (CGGG), and H3H1 (GGGA). Although the difference in the number of eggs laid at 300 days of age between individuals with the haplotype combination H3H3 (GGGG) was not significant, the number of eggs laid at 300 days of age was 7.37 more than that of individuals with the haplotype combination H3H3 (GGGG). The age at first laying of individuals with different haplotype combinations was significantly different. The age at first laying of individuals with haplotype combination H3H3 (GGGG) was significantly earlier than that of individuals with haplotype combinations H2H1 (CGGA) and H2H2 (CCGG). Although the age at first laying of individuals with haplotype combination H1H1 (GGAA), H2H3 (CGGG) and H3H1 (GGCA) was not significantly different, the age at first laying of individuals with haplotype combination H1H1 (GGAA), H2H3 (CGGG) and H3H1 (GGCA) was on average 1.98, 6.67 and 5.79 days earlier than that of individuals with haplotype combination H1H1 (GGAA), H2H3 (CGGG) and H3H1 (GGCA), respectively.

[0121] Table 7 Association analysis between GTF2A1 gene haplotype combinations and egg production traits

[0122]

[0123]

[0124] Note: There was no significant difference between the mean values ​​of the same site with the same letter shoulder (P>0.05), while there was a significant difference between the mean values ​​of the same site with different letter shoulders (P<0.05).

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A SNP molecular marker associated with egg-laying traits in chickens, characterized in that: The SNP molecular marker is a single nucleotide polymorphism site in the chicken GTF2A1 gene, and the single nucleotide polymorphism site includes site 1 and / or site 2; site 1 is located at position 40334089bp on chromosome 5, and the mutant base is G / C; site 2 is located at position 40341114bp on chromosome 5, and the mutant base is A / G.

2. A primer pair for amplifying the SNP molecular marker according to claim 1.

3. The primer pair according to claim 2, characterized in that The primer pair sequences for amplifying the SNP molecular marker comprising site 1 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the primer pair sequences for amplifying the SNP molecular marker comprising site 2 are shown in SEQ ID NO: 3 and SEQ ID NO:

4.

4. A detection reagent for detecting SNP molecular markers, characterized in that: The detection reagent comprises the primer pair according to claim 2 or 3.

5. Use of the SNP molecular marker associated with chicken egg-laying traits according to claim 1, the primer according to claim 2 or 3, or the detection reagent according to claim 4, wherein the use is selected from any one of the following 1) to 8): 1) Prediction of chicken egg production traits; 2) preparing reagents for predicting egg production traits in chickens; 3) Screening or identifying chicken strains that have characteristics of early onset of laying and / or high egg production at 300 days of age; 4) preparing reagents for screening or identifying chicken strains characterized by early onset of laying and / or high egg production at 300 days of age; 5) Breeding for egg-laying traits in chickens; 6) Molecular marker-assisted breeding related to egg production traits in chickens; 7) Breed improvement related to egg-laying traits of chickens; 8) GTF2A1 genotyping; Among them, for the SNP molecular marker containing site 1, the genotype is GG, which corresponds to the traits of early onset of egg laying and high egg production at 300 days of age; For the SNP molecular marker containing site 2, the genotype is AA, corresponding to the traits of early onset of egg laying and high egg production at 300 days of age; For the haplotype combination, the genotype is GGAA, which corresponds to the traits of early age at first laying and high egg number at 300 days of age.

6. A method for breeding chickens with early onset of laying and high egg production at 300 days of age, characterized in that: The method includes: selecting individuals with early egg-laying age and high egg production at 300 days of age for breeding with female parents for the next generation based on SNP molecular markers; The SNP molecular markers are single nucleotide polymorphism sites in the chicken GTF2A1 gene, including site 1 and / or site 2; site 1 is located at position 40334089 on chromosome 5, with a mutation base of G / C; site 2 is located at position 40341114 on chromosome 5, with a mutation base of A / G; Among them, for the SNP molecular marker containing site 1, the genotype is GG, which corresponds to the egg trait of early onset of laying and high egg production at 300 days of age; For the SNP molecular marker containing site 2, the genotype is AA, which corresponds to the egg trait of early onset of laying and high egg production at 300 days of age; For the haplotype combination, the genotype is GGAA, which corresponds to the egg traits of early onset of laying and high egg production at 300 days of age.

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