SNP (Single Nucleotide Polymorphism) molecular marker related to strength of egg shell of white coming aviation, primer pair and application of SNP molecular marker

Abstract

The invention relates to the technical field of animal husbandry genetic breeding, in particular to an SNP (Single Nucleotide Polymorphism) molecular marker and a primer pair related to the strength of an egg shell of white coming aviation and application of the SNP molecular marker and the primer pair. Compared with the prior art, the white leghorns with the ultra-long egg producing period are subjected to whole genome association analysis, SNP sites related to the eggshell strength character are screened out, then the SNP sites rs733980419 which reach the whole genome significant level at the age of 60 weeks are screened out through single-site GWAS analysis, the SNP molecular marker is developed on the basis of the screened SNP sites, and the SNP molecular marker has the advantages that the SNP sites related to the eggshell strength character are obtained; experiments prove that the SNP molecular marker is closely related to the eggshell strength, early molecular screening is realized, and the breeding efficiency is improved. Experiments prove that the SNP molecular marker and the primer pair provided by the invention can be used for early identification or screening of eggshell strength traits of chicken flocks and auxiliary breeding of chicken strains with high eggshell strength traits, and the eggshell breakage rate in the later period is reduced.

Description

Technical Field

[0001] This invention relates to the field of animal genetics and breeding technology, and in particular to an SNP molecular marker, primer pair and its application related to the shell strength of white lambs. Background Technology

[0002] Eggshell strength is an important indicator for evaluating egg quality, directly affecting the efficiency of egg transportation, storage, and hatching. As a high-producing egg-laying breed, the eggshell strength of the White Leghorn chicken is significantly influenced by genetic factors. Currently, eggshell strength is mainly assessed through phenotypic testing (such as using an eggshell strength meter), but this method is time-consuming, costly, and cannot facilitate early genetic screening.

[0003] Previous studies have reported several candidate genes associated with eggshell strength, such as the RYR2 and TRAM2 genes. These genes are mainly involved in calcium ion signaling pathways and cell membrane transport. For example, in Leghorn chickens, GWAS analysis revealed that eggshell strength is associated with SNP loci on chromosome 3, but these loci are mostly potential associations and have not been developed into practical molecular markers. Furthermore, existing methods for identifying eggshell strength have the following problems:

[0004] 1) Eggshell strength phenotypic determination depends on the later laying period (>60 weeks of age), which makes early screening impossible and results in low breeding efficiency; 2) There is a lack of molecular markers closely related to the eggshell strength of White Leghorn eggs, which makes molecular-assisted breeding impossible; 3) Existing GWAS results are mostly based on single-week analysis, ignoring the dynamic changes during the ultra-long laying period (35-100 weeks of age), resulting in incomplete association loci.

[0005] Therefore, there is an urgent need to find a molecular marker that can effectively identify eggshell strength. Summary of the Invention

[0006] The purpose of this invention is to provide a SNP molecular marker, primer pair, and their applications related to the shell strength of White Leghorn eggs, in order to solve the problems existing in the prior art. This invention provides an SNP molecular marker related to the shell strength of White Leghorn eggs, enabling molecular-level screening; and based on this SNP molecular marker, a dedicated primer pair has been developed to improve detection efficiency, enabling its application in the early identification, screening, and assisted breeding of eggshell strength traits.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides an SNP molecular marker associated with the shell strength (ESS) trait of white guinea pig eggs. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, and a mutation of G or A is present at the 47th base of the sequence.

[0009] The present invention provides primer pairs for amplifying the above-mentioned SNP molecular markers, wherein the primer pairs include an upstream primer with a nucleotide sequence as shown in SEQ ID NO.2, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.3, and an extension primer with a nucleotide sequence as shown in SEQ ID NO.4.

[0010] This invention provides the application of the above-mentioned primer pair in the preparation of products for identifying the strength of white guinea pig eggshells.

[0011] This invention provides a product for identifying the strength of white guinea pig eggshells, the product comprising the aforementioned primer pair.

[0012] This invention provides the application of the above-mentioned primer pairs or the above-mentioned products in identifying the strength of white guinea pig eggshells.

[0013] This invention provides a method for determining the strength of white leggy eggshells, comprising the following steps:

[0014] Using the DNA of the chicken sample to be tested as a template, genotyping was performed using the primer pairs mentioned above; based on the genotyping results, the eggshell strength trait of the chicken sample to be tested was determined.

[0015] Preferably, when the genotype of the molecular marker is AA, the eggshell strength of the chicken sample being tested is the highest; when the genotype is GA, the eggshell strength of the chicken sample being tested is in the middle; and when the genotype is GG, the eggshell strength of the chicken sample being tested is the lowest.

[0016] This invention provides the application of the above-mentioned primer pairs or the above-mentioned products in screening eggs with high shell strength.

[0017] This invention provides the application of the above-described primer pairs or products in the preparation of products for screening eggs with high shell strength.

[0018] This invention provides the application of the above-mentioned primer pairs or the above-mentioned products in chicken assisted breeding.

[0019] The present invention discloses the following technical effects:

[0020] Compared with existing technologies, this invention utilizes genome-wide association analysis (GWAS) on Leghorn chickens with an extended laying period (35-100 weeks of age) to screen for SNP loci associated with eggshell strength (ESS). Subsequent single-site GWAS analysis identified the SNP locus rs733980419 (near the ANP32A gene), which reaches genome-wide significance at 60 weeks of age. Based on this identified SNP locus, an SNP molecular marker was developed. Experimental verification showed that this SNP molecular marker is closely related to eggshell strength (P-value 8.60E-07), enabling early (chick stage) molecular screening and improving breeding efficiency. Experimental verification demonstrates that the SNP molecular marker and primer pairs provided by this invention can be used for early identification or screening of eggshell strength traits in chicken flocks, as well as for assisted breeding of chicken strains with high eggshell strength traits, reducing later eggshell breakage rates. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 GWAS Manhattan plot for eggshell strength at 60wk;

[0023] Figure 2 A QQ image showing the eggshell strength of 60wk.

[0024] Figure 3 This is the operational procedure for SNP time-of-flight mass spectrometry experiments. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Example 1: Screening of molecular markers related to shell strength traits of white legion eggs

[0031] 1. Test materials

[0032] Two thousand 15 generations of purebred White Leghorn chickens, provided by a poultry breeding company in Hebei Province, were selected as the experimental subjects. These chickens were raised in the same fully enclosed chicken house from 18 weeks of age, individually in cages, with automatic mechanical ventilation, and maintained with the same light program (16L:8D), feed, and water conditions. During the experiment, eggshell strength (ESS) and other egg quality indicators were measured for each chicken at 35 weeks of age (wk), 40wk, 50wk, 60wk, 70wk, 80wk, 90wk, and 100wk. Eggshell strength was measured using an eggshell strength meter (model: ESTG-01; Israel Aoke Co., Ltd., Israel), and the unit is N / cm². 2 Specific measurement method: Place a fresh egg on the platform of the strength tester and apply vertical pressure until the eggshell cracks, recording the maximum pressure value. Collect all eggs at each week of age for measurement.

[0033] At 40 weeks of age, blood was collected from the subpterygdal veins of all individuals in the population using heparin-anticoagulated blood collection tubes. The collected blood samples were then stored at -20°C. Genomic DNA was extracted from the blood samples using a whole blood genomic DNA extraction kit (Beijing Tiangen Biotech Co., Ltd.), and the extracted DNA was stored at -80°C.

[0034] 2. Test methods

[0035] (1) Genomic DNA extraction and quality testing

[0036] Genomic DNA was extracted from blood samples using the Tiangen Blood DNA Extraction Kit (Tiangen Biotech, Beijing). The specific steps are as follows:

[0037] a. Take 200 μL of anticoagulated blood, add 400 μL of lysis buffer (Buffer GA), and mix thoroughly;

[0038] b. Add 20 μL of proteinase K (20 mg / mL) and digest in a water bath at 56°C for 1 hour;

[0039] c. Add 200 μL Buffer GB, mix thoroughly, and incubate in a 56°C water bath for 10 min;

[0040] d. Add 200 μL of anhydrous ethanol, mix well, transfer to the adsorption column, and centrifuge at 12000 rpm for 30 s;

[0041] e. Add 500 μL of Buffer GD and PW elution buffer once each, and centrifuge at 12000 rpm for 30 s;

[0042] f. Add 50 μL of TE buffer, let stand at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min to collect DNA.

[0043] DNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher), ensuring a concentration >50 ng / μL and OD0.05. 260 / OD 280 The value should be in the range of 1.8-2.0. Qualified samples are used for subsequent genotyping.

[0044] (2) Genotyping

[0045] Genotyping of the genomic DNA of all individuals was performed using the Illumina platform's 50K Jingxin No. 1 chicken gene chip. Specific procedures:

[0046] a. The genotype data were quality controlled using Plink 1.90 software. SNPs with a deletion rate higher than 5%, deviation from Hardy-Weinberg equilibrium P < 10 × E⁻⁶, and minor allele frequency less than 0.05 were removed, resulting in 26,689 SNP data.

[0047] b. Use BEAGLE v4.0 software to populate the processed genotype data.

[0048] (3) Genome-wide association analysis (GWAS)

[0049] Association analysis of eggshell strength trait was performed using a mixed linear model (MLM). The MLM model was used for screening, with the following model: Y = Xα + Zβ + μ + e. TASSEL v5.0 software was used to perform association analysis on genotype and phenotype data. Bonferroni correction was used to calculate the genomic significance and latent significance thresholds, which were 1.87 × 10⁻⁶. -6 and 3.75×10 -5 Use the "gap" package in R software to draw Manhattan diagrams and QQ diagrams.

[0050] 3. Results

[0051] This example demonstrates a genome-wide association analysis (GWAS) of eggshell strength in eggs aged 35-100 weeks. The results showed that the SNP locus selected in this example—rs733980419—exhibited the strongest association signal at 60 weeks of age and reached genome-wide significance (P < 1.87 × 10⁻⁶). -6 ).in, Figure 1 and Figure 2 The GWAS analysis results (Manhattan plot and QQ plot) of eggshell strength at 60 weeks of age are shown. Detailed site information is shown in Table 1.

[0052] Table 1. Information on SNP sites related to eggshell strength (ESS)

[0053]

[0054] Note: Genome version number is GRCg6a; effect size unit is "N / cm". 2 Although the PVE level is low, it reaches a significant level across the entire genome and has breeding value.

[0055] Example 2: Development and Validation of SNP Molecular Markers

[0056] 1. Molecular marker development

[0057] The SNP sites in Table 1 selected in Example 1 were obtained from the genome with version number GRCg6a, i.e., using GRCg6a coordinates. To ensure the specificity and timeliness of the detection primers, the rs733980419 site was mapped to the latest reference genome GRCg7b, and SNP molecular markers were developed based on the sequence of the latest reference genome.

[0058] The nucleotide sequence of the rs733980419 molecular marker is shown in SEQ ID NO.1, and there is a G or A mutation at the 47th base of the sequence.

[0059] Based on the SNP molecular markers above, primers were designed to detect the rs733980419 molecular marker. The primer pair is as follows: upstream primer with nucleotide sequence as shown in SEQ ID NO.2, downstream primer with nucleotide sequence as shown in SEQ ID NO.3, and extension primer with nucleotide sequence as shown in SEQ ID NO.4.

[0060] Table 2 SNP molecular markers

[0061]

[0062] Table 3 SNP sites and their primer information

[0063]

[0064] 2. Test methods

[0065] DNA was extracted from the chickens of Example 1 (using the same method as in Example 1). Genotyping was performed using time-of-flight mass spectrometry, as follows: Figure 3 As shown. Specific steps:

[0066] (1) PCR reaction: The reaction system is shown in Table 4, and the procedure is shown in Table 5.

[0067] Table 4 PCR reaction system

[0068]

[0069] Table 5 PCR reaction procedure

[0070]

[0071] (2) SAP enzyme digestion: The digestion system is shown in Table 6, and the procedure is shown in Table 7.

[0072] Table 6 SAP enzyme digestion system

[0073]

[0074] Table 7 SAP Enzyme Digestion Procedure

[0075]

[0076] (3) Single base extension reaction: The extension system is shown in Table 8, and the procedure is shown in Table 9.

[0077] Table 8 Single-base extension reaction system

[0078]

[0079] Table 9. Single-base extension reaction procedure

[0080]

[0081] (4) Resin purification: Add 16 μL of triple-distilled water to the reaction product and centrifuge at 2000 rpm for 3 min; add resin and reverse shake for 35 min; centrifuge at 2000 rpm for 3 min. Spot the sample onto the target, allow it to crystallize naturally, and then analyze it using the MassARRAY system.

[0082] 3. Results

[0083] (1) Genotyping detection: Using DNA samples from 2,577 white Leghorn chickens in Example 1, the SNP site rs733980419 was genotyped using the primer pairs and time-of-flight mass spectrometry shown in Table 3 of this example.

[0084] Of the 2,577 samples, 2,560 valid genotype data were obtained, with a genotyping success rate of 99.34%. Three genotypes were detected: GG, GA, and AA.

[0085] (2) Population allele and genotype frequencies: In a population of 2,560 chickens, the frequency of allele G at the rs733980419 locus was 85.7%, and the frequency of allele A was 14.3%, which was basically consistent with the MAF (0.144) in the GWAS analysis in Example 1.

[0086] The distribution of the three genotypes is as follows:

[0087] GG type: 1879 (73.4%); GA type: 631 (24.6%); AA type: 50 (2.0%).

[0088] (3) Genotype and phenotype association verification: To verify the association between the SNP marker and eggshell strength (ESS), the three genotypes (GG, GA, AA) were analyzed in association with the ESS phenotype values ​​measured at 60 weeks of age in Example 1.

[0089] The results are shown in Table 10. Eggshell strength showed extremely significant differences among the three genotypes (P<0.001).

[0090] Table 10 Association analysis between rs733980419 genotype and eggshell strength at 60 weeks of age.

[0091]

[0092] Note: The total effective sample size is 2560 (original sample size 2577, 17 samples with failed genotyping were removed).

[0093] Data were analyzed using ANOVA. The differences between different genotypes were extremely significant. Different capital letters in the superscript of the data in the same column indicate extremely significant differences (P<0.01).

[0094] Example 3: Application of Molecular Markers in Early Identification of Eggshell Strength

[0095] 1. Experimental animals and early sampling: To verify the application value of the molecular markers of the present invention in early screening, 500 chicks (1 day old) were randomly selected from the offspring of the chicken flock described in Example 1 (i.e., the 16th generation White Leghorn chickens) as the verification group.

[0096] Genomic DNA was extracted from the wing veins of chicks using the same method as in Example 1.

[0097] 2. Early genotyping and prediction: Using the primer pair (SEQ ID NO.2-4) developed in Example 2 and time-of-flight mass spectrometry, the DNA of these 500 chicks was genotyped at the rs733980419 locus.

[0098] Based on genotype, they were divided into three prediction groups:

[0099] Predicted high ESS group: Genotype AA;

[0100] Predicted ESS group: genotype GA;

[0101] Predicted low ESS group: Genotype GG.

[0102] 3. Feeding and post-feedback phenotyping: All 500 chickens were fed under the same standard conditions (same feeding and management as in Example 1) until they reached 60 weeks of age, and their eggshell strength (ESS) phenotypic values ​​were measured uniformly.

[0103] 4. Results: The early genotyping results of 500 chicks are compared with the actual ESS phenotypes measured at 60 weeks of age, as shown in Table 11. As can be seen from Table 11, the genotyping results performed on chicks at 1 day of age using the rs733980419 molecular marker and primer pair provided by this invention highly match the actual eggshell strength phenotypes (high, medium, and low) at 60 weeks of age.

[0104] Table 11 Comparison of early genotype predictions with actual ESS phenotypes at 60 weeks of age

[0105]

[0106] Note: Data were analyzed using ANOVA. The differences between different genotypes were extremely significant. Different capital letters in the superscript of the data in the same column indicate extremely significant differences (P<0.01).

[0107] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A SNP molecular marker associated with the shell strength trait of white guinea pig eggs, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, and a mutation of G or A is present at the 47th base of the sequence.

2. A primer pair for amplifying the SNP molecular marker of claim 1, characterized in that, The primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.2, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.3, and an extension primer with a nucleotide sequence as shown in SEQ ID NO.

4.

3. The application of the primer pair according to claim 2 in the preparation of a product for identifying the strength of white guinea pig eggshells.

4. A product for determining the strength of white guinea pig eggshells, characterized in that, The product comprises the primer pair as described in claim 2.

5. The application of the primer pair of claim 2 or the product of claim 4 in identifying the strength of white guinea pig eggshells.

6. A method for determining the strength of white guinea pig eggshells, characterized in that, Includes the following steps: Using the DNA of the chicken sample to be tested as a template, genotyping was performed using the primer pair described in claim 2; based on the genotyping results, the eggshell strength trait of the chicken sample to be tested was determined.

7. The method according to claim 6, characterized in that, When the genotype of the molecular marker is AA, the eggshell strength of the chicken sample being tested is the highest; when the genotype is GA, the eggshell strength of the chicken sample being tested is in the middle; and when the genotype is GG, the eggshell strength of the chicken sample being tested is the lowest.

8. The application of the primer pair of claim 2 or the product of claim 4 in screening eggs with high shell strength.

9. The use of the primer pair of claim 2 or the product of claim 4 in the preparation of a product for screening eggs with high shell strength.

10. The application of the primer pair of claim 2 or the product of claim 4 in chicken assisted breeding.

Images