Application of a SNP genetic marker for chicken tibia calcium content based on the KCND2 gene in chicken genetic breeding

Abstract

This invention provides an application of a chicken tibia calcium content SNP genetic marker based on the KCND2 gene in chicken genetic breeding, belonging to the technical field of chicken genetic breeding and livestock SNP genetic markers. The SNP genetic marker includes TCC_mark1, which corresponds to the 24,298,494th position on chromosome 1 in the NCBI-published chicken reference genome bGalGal1.mat.broiler.GRCg7b version sequence information, belonging to the upstream sequence of the KCND2 gene, where the base is C or T. This SNP genetic marker helps to genetically improve chicken bone quality, solve the problem of osteoporosis in the later stages of egg production, and can be used for genomic selection or marker-assisted breeding in chickens.

Description

Technical Field

[0001] This invention belongs to the field of chicken genetic breeding and livestock SNP genetic marker technology, and specifically relates to the application of a chicken tibia calcium content SNP genetic marker based on the KCND2 gene in chicken genetic breeding. Background Technology

[0002] In modern egg-laying hen farming, bone calcium content is a core trait affecting hen welfare, egg production performance, and farming efficiency, directly determining the skeletal health of hens during the laying period and the economic benefits of farming. During the laying period, hens continuously extract calcium from their bones for eggshell formation; the calcium deposited in the eggshells of a commercial laying hen throughout its lifetime is approximately equal to its body weight. Imbalance in bone calcium metabolism is a core contributing factor to keel fractures and a high incidence of osteoporosis in the later stages of laying. The incidence of osteoporosis in caged laying hens reaches 20-35%, not only reducing egg production and increasing culling rates but also seriously affecting animal welfare and causing significant economic losses. Imbalance in bone calcium metabolism is the result of the synergistic effects of multiple factors, including genetics, physiology, nutrition, and environment. The core issue is the disruption of the dynamic balance of bone calcium deposition and mobilization. Genetic factors are the core intrinsic factors determining bone calcium content in laying hens and regulating the balance of bone calcium metabolism.

[0003] Bone calcium content is a trait with low to moderate heritability (h 2 (≈0.18–0.41), traditional phenotypic selection requires post-slaughter testing, which is costly, destructive, and unable to achieve early selection before egg production, failing to meet the early selection needs of large-scale laying hen breeding. Therefore, it is necessary to screen out stable and universal SNP genetic marker combinations associated with tibia calcium content in laying hens, focusing on functional variations of osteogenic genes, overcoming the shortcomings of existing markers' poor applicability across laying hen breeds, adapting to the breeding needs of different breeds / strains of laying hens, and specifically improving the imbalance of osteogenic calcium metabolism during the laying period in laying hens from a genetic level. Summary of the Invention

[0004] To address the problem of calcium metabolism imbalance in laying hens, this invention provides an application of a chicken tibia calcium content SNP genetic marker based on the KCND2 gene in chicken genetic breeding. The SNP genetic marker helps to genetically improve chicken bone quality, solve the problem of osteoporosis in the later stages of egg production, and can be used for genomic selection or molecular marker-assisted breeding of chickens.

[0005] This invention is achieved through the following technical solution: This invention provides an application of a chicken tibia calcium content SNP genetic marker based on the KCND2 gene in chicken genetic breeding. The SNP genetic marker includes TCC_mark1, which corresponds to the 24298494th position on chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version sequence information published by NCBI, belonging to the upstream sequence of the KCND2 gene, where the base is C or T.

[0006] Based on the same inventive concept, the present invention provides an early selection method for the trait of calcium content in chicken tibia, the early selection method comprising early selection of the trait of calcium content in chicken tibia based on the genotype of the SNP genetic marker TCC_mark1; The TCC_mark1 corresponds to the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published by NCBI, which is located at position 24298494 on chromosome 1, belonging to the upstream sequence of gene KCND2, and the base here is C or T.

[0007] Optionally, the early selection method specifically includes: The genotype of TCC_mark1 in the genome of the chicken to be tested was detected; Early selection of the tibia calcium content trait in the test chickens based on the genotype of TCC_mark1; Among them, the bone calcium content of individuals with the CC genotype of TCC_mark1 is less than that of individuals with the CT genotype, and the bone calcium content of individuals with the CT genotype is less than that of individuals with the TT genotype.

[0008] Optionally, the detection of the TCC_mark1 genotype in the genome of the chicken to be tested specifically includes: Using Pr_TCC1f and Pr_TCC1r as primers, PCR amplification was performed on the genomic DNA of the chickens to be tested; The PCR amplification products were sequenced to obtain the genotype at position 24298494 on the positive strand of chromosome 1 of the chicken to be tested; The nucleotide sequence of Pr_TCC1f is shown in SEQ ID NO.1, and the nucleotide sequence of Pr_TCC1r is shown in SEQ ID NO.2.

[0009] Based on the same inventive concept, the present invention provides primers for detecting SNP genetic markers related to calcium content in chicken tibias, the primers comprising Pr_TCC1f and Pr_TCC1r, the nucleotide sequence of Pr_TCC1f being shown in SEQ ID NO.1 and the nucleotide sequence of Pr_TCC1r being shown in SEQ ID NO.2.

[0010] Based on the same inventive concept, this invention provides the application of primers for detecting SNP genetic markers related to calcium content in chicken tibias in chicken genetic breeding.

[0011] Based on the same inventive concept, the present invention provides a kit for detecting SNP genetic markers related to calcium content in chicken tibia, the kit comprising the primers described above for detecting SNP genetic markers related to calcium content in chicken tibia.

[0012] Based on the same inventive concept, this invention provides a kit for detecting SNP genetic markers related to calcium content in chicken tibias, and its application in chicken genetic breeding.

[0013] Based on the same inventive concept, this invention also provides the application of SNP genetic markers related to chicken tibia calcium content in predicting bone traits in the later stages of chicken egg production. The SNP genetic markers include TCC_mark1, which corresponds to the 24298494th position on chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version sequence information published by NCBI, belonging to the upstream sequence of gene KCND2, where the base is C or T.

[0014] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: This invention relates to the application of a chicken tibia calcium content SNP genetic marker based on the KCND2 gene in chicken genetic breeding. The SNP genetic marker is TCC_mark1. The dominant genotype of this SNP marker can improve the balance of chicken bone calcium metabolism, which helps to improve chicken bone quality genetically. Increasing the frequency of the TT allele of the TCC_mark1 marker can increase the calcium content of chicken tibia. Applying it to the genetic breeding of laying hens is beneficial for screening chicken breeds with high bone quality, solving the problem of osteoporosis in the later stage of egg production, increasing the calcium content of chicken tibia, and improving the physical condition of laying hens. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is an example of linkage disequilibrium analysis of the KCND2 gene related to bone calcium content in Example 1 of the present invention; Figure 2 This is a GWAS analysis QQ plot of bone calcium content in the resource population of Example 1 of the present invention; Figure 3 This is a comparison chart of bone calcium content for different genotypes in Example 2 of the present invention. Detailed Implementation

[0017] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0018] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0019] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0020] The overall concept of this invention is as follows: Bone calcium content is a trait with low to moderate heritability (h 2 (≈0.18–0.41), traditional phenotypic selection requires post-slaughter testing, which is costly, destructive, and unable to achieve early selection before egg production, failing to meet the early selection needs of large-scale laying hen breeding. Therefore, it is necessary to screen out stable and universal SNP genetic marker combinations associated with tibia calcium content in laying hens, focusing on functional variations of osteogenic genes, overcoming the shortcomings of existing markers' poor applicability across laying hen breeds, adapting to the breeding needs of different breeds / strains of laying hens, and specifically improving the imbalance of osteogenic calcium metabolism during the laying period in laying hens from a genetic level.

[0021] Based on this, this invention utilizes high-density gene chip genotyping technology and GWAS analysis to discover the genetic marker TCC_mark1, which is closely associated with tibial calcium content in chickens at 72 weeks of age, near the KCND2 gene on chromosome 1. This genetic marker can help improve bone quality in chickens genetically, addressing osteoporosis in the later stages of egg production, and can be used for genomic selection or marker-assisted selection in chickens.

[0022] The following will describe in detail the application of a chicken tibia calcium content SNP genetic marker based on the KCND2 gene in chicken genetic breeding, in conjunction with embodiments and experimental data.

[0023] Example 1 GWAS analysis of calcium content in chicken tibia Using Dongxiang Green-shelled Chickens and White Leghorn Chickens as parents, F1 and F2 generations were obtained through reciprocal crosses. The experimental chickens were housed individually in cages, with mechanized feeding and manure removal, and received routine immunizations. A slaughter trial was conducted at 72 weeks of age (late egg production), and tibiae were collected. Bone calcium content was determined by potassium permanganate titration. The bone calcium content data underwent preliminary screening, removing obviously erroneous data, missing wing numbers, and outliers, and was then compiled into a table. After data cleaning, the F2 generation bone calcium dataset contained 383 records.

[0024] Approximately 1 ml of blood was collected from the wing vein of the experimental chicken using a disposable syringe and placed into a BD anticoagulant tube (Suzhou BD Medical Instrument Co., Ltd.) and stored at -70℃. Genomic DNA was extracted from the blood sample and detected by 0.8% agarose gel electrophoresis and ultraviolet spectrophotometry. After passing the detection, the DNA sample was diluted to 50±5 ng / μl for gene chip typing.

[0025] Using Affymetrix gene chip from Affymetrix Axiom Genotyping was performed using a 600K Chicken Genotyping Array. Data quality control was conducted according to the array's instruction manual, including: pre-genotyping quality control using APT software; PLINK quality control to remove SNPs with a detection rate below 0.97 and those deviating from Hardy-Weinberg equilibrium; SNP screening using metrics.R, SNP_filter.R, and SNP CR and FLD information analysis; and genotyping using BEAGLE. After quality control, 435,243 SNPs and 383 samples remained for subsequent analysis.

[0026] Prior to genome-wide association analysis (GWAS), multidimensional principal component analysis (PCA) was performed to eliminate false positives. The first five principal components were added to the genetic model as covariate parameters. Independent tests for each SNP were estimated using plink analysis, yielding 20,449 independent markers. Bonferroni correction was used to determine the genomic significance threshold as 2.445 × 10⁻⁶. -6 The suggested threshold for genome sequencing is 4.89 × 10⁻⁶. -5 Bone calcium content was analyzed using a mixed linear model, and the p-values ​​for the significance test of each SNP were obtained. The matrix expression of the linear model is as follows:

[0027] Where y represents the phenotypic vector of bone calcium content in the sample; W represents the covariance matrix; α is the intercept vector; x is the genotype vector of the marker; u is the random effects vector; and ε is the residual.

[0028] A genome-wide association analysis was performed on the calcium content of the tibia of 383 chickens, and the results are as follows: Figure 1 , Figure 2 As shown. Linkage disequilibrium analysis of the KCND2 gene, which is related to bone calcium content, revealed that AX-75411853 is a significant SNP located at position 24298494 on chicken chromosome 1. The QQ plot further validated the reliability of the GWAS results.

[0029] The obtained genomic significance markers are shown in Table 1: Table 1. Genetic markers related to calcium content in chicken tibia. ; The physical location of the marker chromosome is referenced to the whole chicken genome (bGalGal1.mat.broiler.GRCg7b).

[0030] Example 2 1. Post-GWAS analysis of candidate genes Linkage disequilibrium analysis was performed on the candidate genes obtained in Example 1. Plink software was used to extract SNP variation information surrounding the genetic markers and the corresponding physical locations of the markers from the microarray genotyping dataset. Haploview software was used to determine the haplotype fragment size of the candidate QTLs according to the solid spine of LD criterion. Figure 1 As shown, the haplotype fragment containing TCC_mark1 is 29kb in length. Further, the candidate gene was converted into Ensembl code, and gene set enrichment and KEGG analyses were performed using GeneDio's cloud analytics tool. Enrichment analysis results indicated that the candidate gene KCND2 is involved in membrane potential regulation and is related to metabolic processes, environmental information processing, and cellular life activities. KEGG analysis showed that although the KCND2 gene product does not directly transport calcium ions, it determines the amount of calcium ions entering the cell by precisely controlling the voltage gradient.

[0031] 2. Detection and validation of genetic markers The SNP genetic markers screened in Example 1 were used to perform candidate gene association analysis on the Dongxiang Green-shelled Egg Chicken-Leihun Chicken resource population. The specific steps are as follows: 1) PCR primers: DNA template sequence information was downloaded from the NCBI website, and PCR amplification primers were designed using Primer Premier 6.0 software. Primer information is shown in Table 2. PCR primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0032] Table 2. Amplification primers and amplification conditions used for detecting genetic markers of calcium content in chicken tibia. ; 2) Genomic DNA extraction: Genomic DNA was extracted from 383 blood samples using the CTAB method. After passing the tests by UV spectrophotometer and agarose electrophoresis, PCR amplification was performed.

[0033] 3) PCR amplification process: ① Reaction system: The 10μl system includes 50ng of DNA template for identification, 10ng each of forward and reverse primers, 5μL of 2×power Taq MasterMix, and the remaining volume is made up with ultrapure water.

[0034] ②Reaction procedure: First, denature at 94℃ for 30s, anneal at 51.5℃ for 30s, extend at 72℃ for 30s, for a total of 5 cycles; then, denature at 94℃ for 30s, anneal at 51.5℃ for 30s, extend at 72℃ for 30s, for a total of 30 cycles; extend at 72℃ for 5min, and store at 4℃.

[0035] 4) The amplified products are sent to a sequencing company for sequence polymorphism detection.

[0036] 5) Association analysis: All subjects had genotype and bone calcium content phenotypic values, and then significance tests were performed. The analysis results are as follows: Figure 3 As shown, the bone calcium content of individuals with the TT genotype was 21.59±1.16, that of individuals with the CT genotype was 21.59±1.13, and that of individuals with the CT genotype was 20.33±1.46. Therefore, it can be concluded that TT is the preferred genotype for the genetic marker TCC_mark1, and increasing the TT genotype ratio can improve the bone health of chickens.

[0037] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0038] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0039] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a chicken tibia calcium content SNP genetic marker based on the KCND2 gene in chicken genetic breeding, characterized in that, The SNP genetic marker includes TCC_mark1, which corresponds to the 24298494th position on chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version sequence information published by NCBI. It belongs to the upstream sequence of gene KCND2, and the base here is C or T.

2. A method for early selection of the calcium content trait in chicken tibia, characterized in that, The early selection method includes early selection of chicken tibia calcium content traits based on the genotype of the SNP genetic marker TCC_mark1; The TCC_mark1 corresponds to the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version published by NCBI, which is located at position 24298494 on chromosome 1, belonging to the upstream sequence of gene KCND2, and the base here is C or T.

3. The method for early selection of chicken tibia calcium content traits according to claim 2, characterized in that, The early selection method specifically includes: The genotype of TCC_mark1 in the genome of the chicken to be tested was detected; Early selection of the tibia calcium content trait in the test chickens based on the genotype of TCC_mark1; Among them, the bone calcium content of individuals with the CC genotype of TCC_mark1 is less than that of individuals with the CT genotype, and the bone calcium content of individuals with the CT genotype is less than that of individuals with the TT genotype.

4. The method for early selection of chicken tibia calcium content traits according to claim 3, characterized in that, The detection of the TCC_mark1 genotype in the genome of the chicken to be tested specifically includes: Using Pr_TCC1f and Pr_TCC1r as primers, PCR amplification was performed on the genomic DNA of the chickens to be tested; The PCR amplification products were sequenced to obtain the genotype at position 24298494 on the positive strand of chromosome 1 of the chicken to be tested; The nucleotide sequence of Pr_TCC1f is shown in SEQ ID NO.1, and the nucleotide sequence of Pr_TCC1r is shown in SEQ ID NO.

2.

5. Primers for detecting SNP genetic markers related to calcium content in chicken tibias, characterized in that, The primers include Pr_TCC1f and Pr_TCC1r, the nucleotide sequence of which is shown in SEQ ID NO.1 and the nucleotide sequence of which is shown in SEQ ID NO.

2.

6. The application of the primers as described in claim 5 for detecting SNP genetic markers related to chicken tibia calcium content in chicken genetic breeding.

7. A kit for detecting SNP genetic markers associated with calcium content in chicken tibias, characterized in that, The kit contains the primers as described in claim 5 for detecting SNP genetic markers associated with calcium content in chicken tibias.

8. The application of the kit for detecting SNP genetic markers related to calcium content in chicken tibia as described in claim 7 in chicken genetic breeding.

9. The application of a SNP genetic marker associated with calcium content in chicken tibia in predicting bone traits during late egg production, characterized in that... The SNP genetic marker includes TCC_mark1, which corresponds to the 24298494th position on chromosome 1 of the chicken reference genome bGalGal1.mat.broiler.GRCg7b version sequence information published by NCBI. It belongs to the upstream sequence of gene KCND2, and the base here is C or T.

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