Molecular marker related to egg shell bubble hole number and total perimeter and application thereof

Abstract

The invention discloses a molecular marker related to egg shell bubble hole number and total perimeter and application thereof, and belongs to the technical field of gene engineering. Eight SNP loci related to the number of eggshell bubble holes and the total perimeter are obtained through screening, the SNP molecular marker is developed by utilizing the screened SNP loci, and through experimental verification, the eight SNP molecular markers are closely related to the number of eggshell bubble holes and the total perimeter, and can be used for early identification or screening of chicken flocks eggshell bubble hole traits; and auxiliary breeding of chicken strains with specific eggshell bubble hole characters. According to the invention, variation sites and genes related to eggshell bubble pore formation are disclosed, theoretical support is provided for deep research of an eggshell pore genetic regulation mechanism, and reference and direction are provided for further improvement of eggshell quality.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a molecular marker related to the number and total perimeter of eggshell gas bubble holes and an application thereof. Background Art

[0002] The eggshell contains about 95.0% calcium carbonate, 3.5% organic matrix and a small amount of magnesium carbonate, calcium phosphate, etc. It is formed in the chicken uterus and the biomineralization process is accompanied by the continuous transformation of amorphous calcium carbonate into calcite crystals. According to the differences in the morphology of the calcite crystals that make up the eggshell, the eggshell can be structurally divided into a papillae layer, a palisade layer, and a vertical crystal layer. These mineralized layers contain different types of pores, which are divided into three categories according to their size: micron-level pores, submicroscopic bubble pores, and nanometer-level mesopores. Among them, the research on pores is the most extensive and in-depth. The average pore diameter is 10μm-20μm, and the average number is 1.35 / mm 2 , and the number of pores at the blunt end is about twice that of the sharp end. Most pores penetrate the mineralized layers of the eggshell. However, there are relatively few studies on bubble pores. Existing studies have directly measured the size of bubble pores by X-ray micro-computed tomography, and concluded that most of the pores are about 250nm in diameter, and the total voids occupy about 1 / 3 of the total volume of the mineralized layer. In terms of distribution, the number of bubble pores is relatively evenly distributed at each end of the eggshell, but there are large differences between different mineralized layers, among which the palisade layer has the most, followed by the papilla layer, and the vertical crystal layer has the least. Mesopores are interstitial voids located between the vertical crystal layer and the cuticle, with a size of less than 10nm. Pore formation originates from the incomplete fusion of the core gap of the papilla in the early stage of eggshell formation and its columnar extension in the palisade layer, while the cause of the formation of bubble pores has not been reported, but in the study of eggshells of species such as American ostriches, anteaters, emus, storks and various raptors, similar structures of mesh pore systems have been reported, indicating that bubble pores are a ubiquitous pore.

[0003] Eggshell pores are an important indicator of eggshell quality. This special pore structure and the eggshell together ensure gas exchange inside and outside the shell and prevent external bacteria from invading the embryo. Although stomata have been considered the main pathway for gas conduction for a long time, subsequent gas conduction experiments have found that bubble pores may play an actual dominant role. Nevertheless, the relationship between stomata and bubble pores has not yet been clarified, and the root cause of pore formation is not yet clear. The pores may be affected by multiple factors such as environment, nutrition and genetics during the formation process, but there is currently no clear report on the influencing mechanism. This has limited the improvement of eggshell quality and the breeding of new laying hens with excellent eggshell quality. Therefore, it is very necessary to study the genes and molecular mechanisms that affect bubble pore-related traits. Summary of the invention

[0004] The purpose of the present invention is to provide molecular markers related to the number and total perimeter of eggshell bubble holes and their applications to solve the problems existing in the above-mentioned prior art. The present invention screened and obtained 8 SNP sites related to the number and total perimeter of eggshell bubble holes, and developed SNP molecular markers using the screened SNP sites. Through experimental verification, the 8 SNP molecular markers are closely related to the number and total perimeter of eggshell bubble holes, which can be used for early identification or screening of eggshell bubble hole traits in chicken flocks, as well as auxiliary breeding of chicken strains with specific eggshell bubble hole traits. The present invention reveals the variant sites and genes related to the formation of eggshell bubble holes, provides theoretical support for the in-depth study of the genetic regulation mechanism of eggshell pores, and also provides reference and direction for further improving eggshell quality.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides molecular markers related to eggshell bubble hole traits, wherein the molecular markers include molecular marker rs71872696 related to the quantitative trait of eggshell bubble holes and molecular markers rs24644081, rs24614443, rs22339582, rs22339589, rs22351868, rs24673198 and rs25170406 related to the total perimeter trait of eggshell bubble holes;

[0007] The nucleotide sequence of rs71872696 is shown in SEQ ID NO.25, and there is a mutation of A or C at base 225;

[0008] The nucleotide sequence of rs24644081 is shown in SEQ ID NO.26, and there is a G or A mutation at the 67th base;

[0009] The nucleotide sequence of rs24614443 is shown in SEQ ID NO.27, and there is a C or T mutation at the 116th base;

[0010] The nucleotide sequence of rs22339582 is shown in SEQ ID NO.28, and there is a C or T mutation at the 132nd base;

[0011] The nucleotide sequence of rs22339589 is shown in SEQ ID NO.29, and there is a C or T mutation at the 139th base;

[0012] The nucleotide sequence of rs22351868 is shown in SEQ ID NO.30, and there is a G or A mutation at the 38th base;

[0013] The nucleotide sequence of rs24673198 is shown in SEQ ID NO.31, and there is an A or T mutation at the 52nd base.

[0014] The nucleotide sequence of rs25170406 is shown in SEQ ID NO.32, and there is an A or G mutation at the 358th base.

[0015] The present invention also provides a primer combination for detecting the above molecular markers, wherein the primer combination for detecting the rs71872696 comprises an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and an extension primer as shown in SEQ ID NO.3;

[0016] The primer combination for detecting the rs24644081 includes an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and an extension primer as shown in SEQ ID NO.6;

[0017] The primer combination for detecting the rs24614443 includes an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and an extension primer as shown in SEQ ID NO.9;

[0018] The primer combination for detecting the rs22339582 includes an upstream primer as shown in SEQ ID NO.10, a downstream primer as shown in SEQ ID NO.11, and an extension primer as shown in SEQ ID NO.12;

[0019] The primer combination for detecting the rs22339589 includes an upstream primer as shown in SEQ ID NO.13, a downstream primer as shown in SEQ ID NO.14, and an extension primer as shown in SEQ ID NO.15;

[0020] The primer combination for detecting the rs22351868 includes an upstream primer as shown in SEQ ID NO.16, a downstream primer as shown in SEQ ID NO.17, and an extension primer as shown in SEQ ID NO.18;

[0021] The primer combination for detecting the rs24673198 includes an upstream primer as shown in SEQ ID NO.19, a downstream primer as shown in SEQ ID NO.20, and an extension primer as shown in SEQ ID NO.21;

[0022] The primer combination for detecting the rs25170406 includes an upstream primer as shown in SEQ ID NO.22, a downstream primer as shown in SEQ ID NO.23 and an extension primer as shown in SEQ ID NO.24.

[0023] The present invention also provides the use of the primer combination in preparing reagents, kits and chips for detecting the molecular markers.

[0024] The present invention also provides a kit for detecting the above molecular markers, wherein the kit comprises the above primer combination.

[0025] The present invention also provides the use of the above molecular marker, the above primer combination or the above kit in the early identification or screening of eggshell bubble pore traits, wherein the eggshell bubble pore traits include the number of eggshell bubble pores and the total perimeter of the eggshell bubble pores.

[0026] The present invention also provides the use of the molecular marker, the primer combination or the kit in chicken strain breeding for assisting eggshell pore traits, wherein the eggshell pore traits include the number of eggshell pores and the total perimeter of the eggshell pores.

[0027] The present invention also provides a method for early identification of egg shell bubble pore characteristics, characterized in that it comprises the following steps:

[0028] Extracting DNA from chicken samples to be tested;

[0029] Using the DNA as a template, the above primer combination is used for genotyping;

[0030] According to the genotyping results, the eggshell bubble pore characteristics of the tested chicken samples were determined.

[0031] Furthermore, the eggshell bubble pore properties include the number of eggshell bubble pores and the total circumference of the eggshell bubble pores.

[0032] Furthermore, the genotyping method is time-of-flight mass spectrometry.

[0033] Furthermore, the judging method is:

[0034] The number of eggshell pores in the chicken samples with rs71872696 genotype AA was significantly lower than that in the chicken samples with rs71872696 genotype CC.

[0035] The total perimeter of eggshell bubble holes of the tested chicken samples with rs24644081 genotype GG was significantly lower than that of the tested chicken samples with genotype AA;

[0036] The total perimeter of eggshell pores of the tested chicken samples with rs24614443 genotype CC was significantly lower than that of the tested chicken samples with genotype TT.

[0037] The total perimeter of eggshell bubble holes of the tested chicken samples with rs22339582 genotype CC was significantly lower than that of the tested chicken samples with genotype TT;

[0038] The total perimeter of eggshell bubble holes of the tested chicken samples with rs22339589 genotype CC was significantly lower than that of the tested chicken samples with genotype TT;

[0039] The total perimeter of eggshell pores of the tested chicken samples with rs22351868 genotype GG was significantly lower than that of the tested chicken samples with genotype AA;

[0040] The total perimeter of eggshell pores of the tested chicken samples with rs24673198 genotype AA was significantly lower than that of the tested chicken samples with rs24673198 genotype TT.

[0041] The total circumference of eggshell bubble holes of the tested chicken samples with rs25170406 genotype AA was significantly lower than that of the tested chicken samples with rs25170406 genotype GG.

[0042] The present invention discloses the following technical effects:

[0043] The present invention studies the genetic parameters of the indicators related to the eggshell bubble hole trait, and screens the candidate genes and variant sites that regulate its formation, and finally screens out 8 SNP sites, which are distributed on chromosomes 2 and 4 and involve 5 genes. The present invention uses the screened SNP sites to develop SNP molecular markers. Through experimental verification, the 8 SNP molecular markers are closely related to the number and total perimeter of eggshell bubble holes, which can be used for early identification or screening of eggshell bubble hole traits in chicken flocks, and can be used to assist the breeding of chicken strains with specific eggshell bubble hole traits. The present invention reveals the variant sites and genes related to the formation of eggshell bubble holes, provides theoretical support for the in-depth study of the genetic regulation mechanism of eggshell pores, and also provides reference and direction for further improving eggshell quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1 Operational workflow for the SNP flight mass spectrometry assay. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] Example 1 Screening of molecular markers related to the number of eggshell pores and the total perimeter of pores

[0052] 1. Test materials

[0053] Dwarf brown-shell laying hens (DWL) that have been bred for 7 generations were selected as the experimental group and purchased from Hengshui Rongde Poultry Breeding Co., Ltd. in Hebei Province. Due to the presence of the Dwarf gene, DWL has a feed conversion rate about 15% higher than that of ordinary laying hens, and has a strong resistance to diseases such as Marek's. When the experiment was carried out, DWL had been bred to the 7th generation. From the age of 18 weeks to the end of the experiment, all laying hens were raised in the same fully enclosed chicken house, individual cages, automatic mechanical ventilation, and maintained the same lighting program (16L:8D), feed and drinking water.

[0054] 2. Test methods

[0055] About 1000 DWL of the 7th generation were selected. Each hen came from a different family. The sample eggs were collected for 1 day. Only one egg was collected from each hen. The sample eggs were marked with the corresponding individual number. A total of 961 eggs with no damage, clean surface and smooth surface were collected. 80 eggs were randomly selected from the collected eggs to measure the indicators related to eggshell bubble holes. The indicators related to eggshell bubble holes include the number of bubble holes (QBP) and the total perimeter of bubble holes (TPBP). The determination method is: wash the egg white attached to the eggshell with deionized water, take two pieces of 0.3×0.3cm at the blunt and sharp ends of the eggshell sample, respectively. 2 The large and small eggshells were observed and the bubble pore related indicators were measured using a scanning electron microscope (reference: "Traits of Eggshells and Shell Membranes of Translucent Eggs").

[0056] Maternal blood samples corresponding to 80 randomly selected eggshell samples were collected from the subwing venous sinus using sodium heparin anticoagulation blood collection tubes. Genomic DNA of the blood samples was extracted according to the manufacturer's product instructions. The sample concentration and OD260 / OD280 values ​​were tested using a spectrophotometer to ensure that the OD260 / OD280 values ​​were within the range of 1.8-2.0. After all the samples were of qualified quality, they were resequenced, and the resequencing results were subjected to genotyping and GWAS analysis to screen out significant SNP sites associated with the eggshell bubble pore trait. Finally, linkage disequilibrium analysis was used to screen out candidate genes associated with the eggshell bubble pore trait.

[0057] Whole genome resequencing (WGR): After DNA shearing, adapter ligation, PCR product selection and other operations, a DNA library with a size of 300-350 bp was constructed. The blood samples were then sequenced using the Illumina HiSeq PE150 platform, and a total of 1020.16 Gb of raw data was obtained. Reads were filtered using Fastp quality control, and the quality filtering criteria were as follows: a. Remove the adapter sequence; b. Remove reads with N (non-AGCT) bases greater than or equal to 5; c. Slide the window with a window size of 4 bases, and cut off the average base quality value less than 20; d. Remove reads with a length less than 75 bp or an average base quality value less than 15. The 1017.04Gb Clean Reads were randomly aligned to the red junglefowl GRCg6a reference genome using BWA, with an average alignment rate of 99.58% and an average alignment depth of 11.74×. The results were reordered according to chromosome order using SAMtools, and duplicate reads were removed using Picard's MarkDuplicates module. The analysis results of the GC content and other alignment genome results were obtained using Qualimap software. SNPs were filtered across the genome using GATK software, and the filtering parameters were based on the standards officially recommended by GATK. Finally, 9,084,960 SNP sites were obtained, and the gene annotation results were calculated using Annovar software. The resequencing clean reads have been deposited in the National Center for Biotechnology Information (NCBI) Bioproject database (https: / / www.ncbi.nlm.nih.gov / bioproject), with the accession number PRJNA1016067.

[0058] Genotyping: Genotyping is performed using time-of-flight mass spectrometry. The operating procedures are as follows: Figure 1 As shown. The first genotyping used 1.5% agarose gel electrophoresis to evaluate the quality of the extracted DNA, and the DNA concentration was detected by a spectrophotometer. The final DNA concentration was 30 ng / μL. The SNP locus gene sequence was obtained using the Ensembl database (http: / / asia.ensembl.org). Two PCR primers and one extension primer were designed for each SNP for PCR amplification. Finally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used on the MassARRAYiPLEX platform for genotyping. The operation process of time-of-flight mass spectrometry is as follows Figure 1 The specific operation steps of time-of-flight mass spectrometry are as follows:

[0059] (1) PCR reaction: The overall reaction system is shown in Table 1, and the reaction procedure is shown in Table 2.

[0060] Table 1 Reaction system

[0061] Reactant Name Volume (μL) <![CDATA[dddH2O]]> 1.8 10*buffer 0.5 <![CDATA[Mg 2+ ]]> 0.4 dNTP 0.1 Hotstar 0.2 FPrimer / RPrimer 1 DNA samples to be tested 1(30ng) Total 5

[0062] Table 2 Reaction procedure

[0063]

[0064] (2) SAP enzyme digestion reaction: The SAP enzyme Mix system is shown in Table 3, and the SAP enzyme digestion procedure is shown in Table 4.

[0065] Table 3 SAP enzyme Mix system

[0066] Reactant Name Volume (μL) <![CDATA[dddH2O]]> 1.53*460 SAPBuffer 0.17*460 SAPEnzyme 0.3*460 Total 2*460

[0067] Table 4 SAP enzyme Mix system

[0068] 37℃ 40min 85℃ 5min 25℃ ∞

[0069] (3) Single base extension reaction: The single base extension reaction Mix system is shown in Table 5, and the single base extension reaction procedure is shown in Table 6.

[0070] Table 5 Single base extension reaction Mix system

[0071]

[0072]

[0073] Table 6 Single base extension reaction program

[0074]

[0075] (4) Resin purification

[0076] Add 16 μL of triple distilled water to the 384-well plate of the reaction product, centrifuge at 2000 rpm for 3 min; add resin, perform resin purification reaction on an inverted shaker for 35 min, and desalt; after the reaction is completed, centrifuge at 2000 rpm for 3 min. Spot the desalted sample on the sample target and allow it to crystallize naturally.

[0077] Genome-wide association study (GWAS): A mixed linear model (MLM) was used to conduct association analysis of eggshell bubble hole traits. In order to increase the accuracy of the results, the population genetic structure and individual kinship matrix were added to the model. The model used is as follows:

[0078] Y=Xα+Zβ+Wμ+e

[0079] y: phenotypic trait, X: indicator matrix of trait, α: estimated parameter of fixed effect, Z: indicator matrix of SNP, β: effect of SNP; W: indicator matrix of individual difference, μ: predicted random individual, e: random residual, subject to e-(0, δ2e). The Kinship matrix was calculated using EMMAX software, and the first four PCA components were selected as covariates to perform association analysis on a large number of variant sites and the experimental target traits. The Manhattan plot and quantile-quantile (QQ) plot of GWAS results were drawn using the "gap" package in R software. The genome-wide significant P threshold of 5.5×10 was determined using Bonferron correction. -9 (0.05 / 9,084,960), the potential P threshold is 1.1×10 -7 (1 / 9,084,960).

[0080] Linkage disequilibrium analysis (LD): Based on the significant SNP sites of each trait, all genes within the 50 kb region before and after the site were queried through NCBI. A series of linkage disequilibrium analyses were performed using the algorithm in Haploview software version 4.2 to detect regions that may be linked to the significant sites.

[0081] 3. Results

[0082] GWAS finally screened out 9 genome-wide significant SNP sites associated with the number and perimeter of bubble holes. Further, according to the elimination criteria, all SNP sites with individual genotype detection rates less than 95% and minor allele frequencies less than 5% were eliminated, and 8 SNP sites were obtained, all of which were polymorphic SNPs with allele frequencies >5% and genotype detection rates >95%. The information of the 8 SNP sites, PCR primers and extension primers are shown in Table 7.

[0083] Table 7 SNP loci and primer information

[0084]

[0085]

[0086] Through linkage disequilibrium analysis, the regions that may be linked to the significant SNP sites were detected. The candidate genes or specific gene positions related to the bubble hole traits in the regions are shown in Table 8. It can be seen that only one SNP site on chromosome 2 is located in the CDH10 gene, which is significantly related to the number of bubble holes; SPOCK3, AADAT, RAPGEF2, and FSTL5 genes are significantly related to the total perimeter of the bubble holes. Finally, AADAT, CDH10, RAPGEF2, FSTL5, and SPOCK3 were considered candidate genes.

[0087] Table 8 SNP sites and annotated gene information

[0088]

[0089] Example 2 Development of SNP Molecular Markers

[0090] SNP molecular markers were developed based on the SNP sites screened in Example 1. The molecular marker information is shown in Table 9.

[0091] Table 9 SNP molecular markers

[0092]

[0093]

[0094] 49 eggs were randomly selected from eggs laid on the same day in the 7th generation DWL to measure the number of bubble holes (QBP) and the total perimeter of bubble holes (TPBP). Blood samples from the mothers corresponding to each egg were collected, genomic DNA was extracted, and PCR primers and extension primers of the 8 molecular markers shown in Table 7 were used to perform typing tests on the DNA of the 49 maternal samples. The test results are shown in Table 10. It can be seen that the number of bubble holes of genotype AA of rs71872696 is significantly lower than that of genotype CC; the total perimeter of bubble holes of genotype GG of rs24644081 is significantly lower than that of genotype AA; the total perimeter of bubble holes of genotype CC of rs24614443 is significantly lower than that of genotype TT; the total perimeter of bubble holes of genotype CC of rs22339582 is significantly lower than that of genotype TT; the total perimeter of bubble holes of genotype CC of rs22339589 is significantly lower than that of genotype TT; the total perimeter of bubble holes of genotype GG of rs22351868 is significantly lower than that of genotype AA; the total perimeter of bubble holes of genotype AA of rs24673198 is significantly lower than that of genotype TT; the total perimeter of bubble holes of genotype AA of rs25170406 is significantly lower than that of genotype GG. It shows that the SNP molecular marker developed by the present invention can be used to identify eggshell bubble hole-related traits, and then used for chicken breeding.

[0095] Table 10 Statistical results of genotype and phenotype values

[0096]

[0097] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A molecular marker related to eggshell pore characteristics, characterized in that: The molecular markers include the molecular marker rs71872696 related to the quantitative trait of eggshell bubble holes and the molecular markers rs24644081, rs24614443, rs22339582, rs22339589, rs22351868, rs24673198 and rs25170406 related to the total perimeter trait of eggshell bubble holes; The nucleotide sequence of rs71872696 is shown in SEQ ID NO.25, and there is a mutation of A or C at base 225; The nucleotide sequence of rs24644081 is shown in SEQ ID NO.26, and there is a G or A mutation at the 67th base; The nucleotide sequence of rs24614443 is shown in SEQ ID NO.27, and there is a C or T mutation at the 116th base; The nucleotide sequence of rs22339582 is shown in SEQ ID NO.28, and there is a C or T mutation at the 132nd base; The nucleotide sequence of rs22339589 is shown in SEQ ID NO.29, and there is a C or T mutation at the 139th base; The nucleotide sequence of rs22351868 is shown in SEQ ID NO.30, and there is a G or A mutation at the 38th base; The nucleotide sequence of rs24673198 is shown in SEQ ID NO.31, and there is an A or T mutation at the 52nd base. The nucleotide sequence of rs25170406 is shown in SEQ ID NO.32, and there is an A or G mutation at the 358th base.

2. A primer combination for detecting the molecular marker according to claim 1, characterized in that: The primer combination for detecting the rs71872696 includes an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and an extension primer as shown in SEQ ID NO.3; The primer combination for detecting the rs24644081 includes an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and an extension primer as shown in SEQ ID NO.6; The primer combination for detecting the rs24614443 includes an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and an extension primer as shown in SEQ ID NO.9; The primer combination for detecting the rs22339582 includes an upstream primer as shown in SEQ ID NO.10, a downstream primer as shown in SEQ ID NO.11, and an extension primer as shown in SEQ ID NO.12; The primer combination for detecting the rs22339589 includes an upstream primer as shown in SEQ ID NO.13, a downstream primer as shown in SEQ ID NO.14, and an extension primer as shown in SEQ ID NO.15; The primer combination for detecting the rs22351868 includes an upstream primer as shown in SEQ ID NO.16, a downstream primer as shown in SEQ ID NO.17, and an extension primer as shown in SEQ ID NO.18; The primer combination for detecting the rs24673198 includes an upstream primer as shown in SEQ ID NO.19, a downstream primer as shown in SEQ ID NO.20, and an extension primer as shown in SEQ ID NO.21; The primer combination for detecting the rs25170406 includes an upstream primer as shown in SEQ ID NO.22, a downstream primer as shown in SEQ ID NO.23 and an extension primer as shown in SEQ ID NO.

24.

3. Use of the primer combination described in claim 2 in the preparation of reagents, kits and chips for detecting the molecular markers described in claim 1.

4. A kit for detecting the molecular marker according to claim 1, characterized in that: The kit comprises the primer combination according to claim 2.

5. Use of the molecular marker according to claim 1, the primer combination according to claim 2 or the kit according to claim 4 in early identification or screening of egg shell pore properties, characterized in that: The eggshell bubble pore properties include the number of eggshell bubble pores and the total perimeter of the eggshell bubble pores.

6. Use of the molecular marker according to claim 1, the primer combination according to claim 2 or the kit according to claim 4 in assisting the breeding of chicken lines for eggshell bubble pore traits, characterized in that: The eggshell bubble pore properties include the number of eggshell bubble pores and the total perimeter of the eggshell bubble pores.

7. A method for early identification of eggshell pore properties, characterized in that: The steps include: Extracting DNA from chicken samples to be tested; Using the DNA as a template, the primer combination of claim 2 is used for genotyping; According to the genotyping results, the eggshell bubble pore characteristics of the tested chicken samples were determined.

8. The method according to claim 7, characterized in that The eggshell bubble pore properties include the number of eggshell bubble pores and the total perimeter of the eggshell bubble pores.

9. The method according to claim 7, characterized in that: The genotyping method is time-of-flight mass spectrometry.

10. The method according to claim 7, characterized in that The judgment method is: The number of eggshell pores of the tested chicken samples with rs71872696 genotype AA was significantly lower than that of the tested chicken samples with genotype CC; the total perimeter of eggshell pores of the tested chicken samples with rs24644081 genotype GG was significantly lower than that of the tested chicken samples with genotype AA; The total perimeter of eggshell pores of the tested chicken samples with rs24614443 genotype CC was significantly lower than that of the tested chicken samples with genotype TT. The total perimeter of eggshell bubble holes of the tested chicken samples with rs22339582 genotype CC was significantly lower than that of the tested chicken samples with genotype TT; The total perimeter of eggshell bubble holes of the tested chicken samples with rs22339589 genotype CC was significantly lower than that of the tested chicken samples with genotype TT; The total perimeter of eggshell pores of the tested chicken samples with rs22351868 genotype GG was significantly lower than that of the tested chicken samples with genotype AA; The total perimeter of eggshell pores of the tested chicken samples with rs24673198 genotype AA was significantly lower than that of the tested chicken samples with rs24673198 genotype TT. The total circumference of eggshell bubble holes of the tested chicken samples with rs25170406 genotype AA was significantly lower than that of the tested chicken samples with rs25170406 genotype GG.

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