Molecular breeding method for genetic variation of eggshell strength anti-aging of laying hens based on c12orf4 gene eqtl and application

Abstract

This invention belongs to the field of molecular breeding technology and provides a method based on... C12orf4 Molecular breeding methods and applications for genetic variations of the eQTL gene in eggshell strength and anti-aging resistance. This invention utilizes molecular marker combinations... C12orf4 Gene expression levels have a regulatory role; the molecular marker combination is located at Chr1:72995066, Chr1:73250348, and Chr1:73614612 loci in the chicken genome, respectively. This invention, through multidimensional association analysis integrating genomic, transcriptomic, and phenotypic data, identified and constructed a key gene that can prospectively and systematically reflect the process of tissue functional decline in laying hens. C12orf4 The molecular marker combinations provided by this invention offer a new technical means for the accurate assessment of aging-related traits in laying hens and for molecular breeding, and have significant application value and industrial significance.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology, specifically involving... C12orf4 Molecular breeding methods and applications of genetic variations in eggshell strength and anti-aging resistance caused by the eQTL gene. Background Technology

[0002] While pursuing high egg production performance, modern egg-laying hen breeding also faces the problem of tissue function decline that intensifies with the extension of the laying cycle. Achieving an ultra-long laying cycle has become a core direction for future development. However, achieving this goal not only depends on the improvement of breeding technology, but also requires the establishment of systematic technical support in areas such as precision nutrition, egg quality maintenance, pullet rearing, and immune control, while simultaneously ensuring the stable optimization of survival rate and feed conversion rate.

[0003] With the continuous extension of the laying period, laying hens experience progressive structural and functional degeneration in multiple organs (such as the liver, reproductive tract, and intestines), including lipid metabolism disorders, accumulation of oxidative stress, reproductive system decline, and decreased immune function. This systemic aging not only affects the health and welfare of laying hens in later production stages but also directly leads to problems such as declining egg production, reduced egg quality, and increased mortality, becoming a significant bottleneck restricting the improvement of industry efficiency.

[0004] Currently, the industry's assessment of the degree of tissue decline in laying hens mainly relies on external phenotypes in the later stages of production (such as changes in the laying curve, eggshell quality, and body condition scores) or post-slaughter histopathological examinations, lacking objective molecular indicators that can accurately reflect the internal physiological state in an early stage. This deficiency severely limits the efficiency of genetic improvement for complex traits such as ultra-long laying cycles.

[0005] With the development of multi-omics technologies, molecular marker-assisted selection has gradually become an important means of livestock and poultry genetic breeding. However, existing studies often focus on local genetic loci directly related to a single productive trait, lacking a set of molecular markers that can systematically, across tissues, and dynamically reflect the process of physiological decline, making it difficult to meet the needs of precise breeding targeting the overall functional state of the organism.

[0006] Therefore, there is an urgent need to provide a molecular marker and breeding method that can reflect the decline of laying hen tissue function in an early, accurate and systematic way, in order to solve the problems of existing technologies lacking objective molecular indicators, making it difficult to accurately assess the aging status of laying hens, and making it impossible to efficiently carry out genetic improvement of eggshell strength and anti-aging traits. Summary of the Invention

[0007] The purpose of this invention is to provide a method based on C12orf4 Molecular breeding methods and applications for genetic variations of the eQTL gene in eggshell strength and anti-aging resistance. The gene identified in this invention... C12orf4The regulatory sites Chr1:72995066, Chr1:73250348 and Chr1:73614612 can prospectively and systematically reflect the process of tissue function decline in laying hens, providing a new technical means for the accurate assessment of aging-related traits in laying hens and molecular breeding, and have important application value and industrial significance.

[0008] This invention provides the application of reagents for identifying molecular marker combinations in identifying eggshell strength and anti-aging properties and / or in breeding to improve eggshell strength and anti-aging traits. The molecular marker combinations... C12orf4 Gene expression levels have a regulatory effect; The molecular marker combinations are located at the chicken genome at Chr1:72995066, Chr1:73250348, and Chr1:73614612 sites, respectively.

[0009] As a preferred embodiment, the genotype of the Chr1:72995066 locus is AA, AB, or BB; The genotypes at the Chr1:73250348 locus are AA, AB, and BB. The genotypes at the Chr1:73614612 locus are AA, AB, and BB.

[0010] As a preferred embodiment, the chicken genome references the chicken Gallus_gallus-7.0 version sequence information.

[0011] As a preferred embodiment, the C12orf4 The gene's NCBI accession number is 419041.

[0012] The present invention also provides a product for identifying the strength and anti-aging properties of eggshells, the product comprising substances for detecting Chr1:72995066, Chr1:73250348 and Chr1:73614612 sites.

[0013] As a preferred embodiment, the substance includes one or more of the following: reagents for nucleic acid extraction and amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, or reagents for sequencing.

[0014] As a preferred embodiment, the substance includes primers for detecting Chr1:72995066, Chr1:73250348 and Chr1:73614612 sites.

[0015] This invention also provides a method for screening laying hens with low eggshell strength degradation rate, comprising the following steps: Genomic DNA was extracted from the laying hens to be tested, and genotyping was performed at three genetic variation loci: Chr1:72995066, Chr1:73250348, and Chr1:73614612. Laying hens carrying the AB genotype at Chr1:72995066, the AA genotype at Chr1:73250348, and the AB genotype at Chr1:73614612 were classified as laying hens with low eggshell strength degradation rates.

[0016] As a preferred embodiment, the identification method includes identification using the Axiom™ Genome-Wide Chicken Genotyping Array genotyping chip.

[0017] This invention also provides a marker-assisted breeding method for improving eggshell strength and anti-aging traits, comprising the following steps: Using the screening method described above, genotypes were identified at three genetic variation loci: Chr1:72995066, Chr1:73250348, and Chr1:73614612. Laying hens carrying the AB genotype at Chr1:72995066, the AA genotype at Chr1:73250348, and the AB genotype at Chr1:73614612 were selected for breeding, resulting in laying hens with improved eggshell strength and anti-aging traits.

[0018] Beneficial effects: This invention provides the application of reagents for identifying molecular marker combinations in identifying eggshell strength and anti-aging properties and / or in breeding to improve eggshell strength and anti-aging traits. The molecular marker combinations can influence... C12orf4 The gene expression levels were specifically located at Chr1:72995066, Chr1:73250348, and Chr1:73614612 loci in the chicken genome. This invention, through multidimensional association analysis integrating genomic, transcriptomic, and phenotypic data, identified and constructed a key gene that can prospectively and systematically reflect the process of tissue functional decline in laying hens. C12orf4 The invention also includes its regulatory sites Chr1:72995066, Chr1:73250348, and Chr1:73614612. The molecular markers provided by this invention offer a new technical means for the accurate assessment of aging-related traits in laying hens and for molecular breeding, possessing significant application value and industrial significance.

[0019] Compared with the prior art, the present invention also has the following beneficial effects: 1. Effectively slows down the target gene C12orf4Senescent-related decline in expression levels. Compared to unverified individuals, laying hens selected to carry superior genotype combinations (AB type at Chr1:72995066, AA type at Chr1:73250348, and AB type at Chr1:73614612) exhibited lower expression levels. C12orf4 The decline in gene expression during the aging process of laying hens will be significantly suppressed. The stable maintenance of this gene expression level is directly related to the slowing down of the decline in eggshell formation function.

[0020] 2. Significantly reduces the rate of decline in eggshell strength during aging. Because... C12orf4 Gene expression stability is positively correlated with eggshell strength. Therefore, the eggshell strength of the laying hen population bred using the marker-assisted selection method of this invention exhibits a more stable downward trend during aging. Specifically, within the same feeding period, the eggshell strength of the selected laying hens is significantly better than that of the ordinary population, and the rate of decline in eggshell strength slows down significantly with increasing age.

[0021] 3. Extend the economic cycle of high-quality egg production. By maintaining... C12orf4 By ensuring stable gene expression, this invention can significantly extend the high-quality egg production cycle of laying hens throughout their lifespan, effectively prolonging the "maintenance period for high-quality eggs." This improvement directly reduces the rate of defective eggs and production losses caused by declining eggshell quality, thereby enhancing the overall economic benefits and sustainability of laying hen farming.

[0022] 4. This invention fully considers the interactions between the three molecular markers. When carrying the Chr1:72995066 AB type, the Chr1:73250348 AA type, and the Chr1:73614612 AB type, late-laying hens have a higher genetic predisposition. C12orf4 The expression level and eggshell strength were both measured (mean 3.605). However, when only one molecular marker was selected for breeding, the eggshell strength achieved under that molecular module could not be reached. This invention greatly avoids the drawbacks of using a single molecular marker for breeding. Breeding based on the molecular module provided in this invention can fully utilize the role of molecular markers, alleviating the declining trend of eggshell strength in the later stages of egg production. It has significant economic and scientific research value and represents an optimization and upgrade of molecular marker technology.

[0023] In summary, this invention provides a novel molecular marker-assisted selection tool for laying hen breeding, which has significant application value in the field of laying hen genetics and breeding, especially in improving the anti-aging ability of laying hen populations, optimizing eggshell quality, and extending the high-laying cycle, and has broad industrialization prospects. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0025] Figure 1 The results of multi-time transcriptome analysis for locating key genes are shown in the following diagrams: A is a volcano plot of Deseq2 analysis in the hypothalamus, ovary, and pituitary gland; B is a bar chart showing the number of genes exhibiting sustained and significant upregulation or downregulation trends in the hypothalamus, ovary, and pituitary gland at 50, 70, and 100 weeks; C is a bar chart of GO functional enrichment analysis of differentially expressed genes in multiple tissues; D is a bar chart based on ovarian... C12orf4 Line graph showing changes in egg production performance after grouping by gene expression level; Figure 2 for C12orf4 A graph showing the changes in gene expression at different ages in laying hens; where A represents... C12orf4 Gene expression curves at different ages (50 weeks, 70 weeks, 100 weeks) in laying hens; B represents... C12orf4 Line graph showing the relationship between gene expression levels in different tissues and eggshell strength (ESS). Figure 3 for C12orf4 Figure showing the results of the molecular module combinatorial effect analysis of genes; where A represents... C12orf4 A) Plot showing the difference in gene expression levels in laying hen populations; B) Plot showing the difference in the effect of different molecular marker combinations on eggshell strength; C) Plot showing the difference in the effect of different molecular module combinations. C12orf4 Plot showing the difference in gene expression levels; D represents... C12orf4 Correlation analysis of gene expression levels and eggshell strength (ESS); In the accompanying diagram of the instruction manual, "ESS" represents eggshell strength. Detailed Implementation

[0026] This invention provides the application of reagents for identifying molecular marker combinations in identifying eggshell strength and anti-aging properties and / or in breeding to improve eggshell strength and anti-aging traits. The molecular marker combinations can all regulate... C12orf4 Gene expression levels; as one implementation method, genes C12orf4 The Ensembl ID is ENSGALG00000017289; the NCBI ID is 419041. The gene described in this invention... C12orf4 It is a key gene associated with aging in laying hens, reflecting the decline in tissue function and closely related to eggshell strength.

[0027] The present invention has discovered genes. C12orf4 During the laying hen production process (from 50 weeks of age to 100 weeks of age), a significant trend of decreasing expression levels was observed, and C12orf4The eggshell strength ESS in the high-expression group was significantly higher than that in the low-expression group, and this phenomenon was consistent across multiple tissues, with high gene expression in the hypothalamus, liver, and uterus. C12orf4 Individuals with higher eggshell strength also have higher eggshell strength.

[0028] The molecular marker combinations described in this invention are located at the Chr1:72995066, Chr1:73250348, and Chr1:73614612 loci in the chicken genome. As one embodiment, the genotypes at the Chr1:72995066 locus are AA, AB, and BB; the genotypes at the Chr1:73250348 locus are AA, AB, and BB; and the genotypes at the Chr1:73614612 locus are AA, AB, and BB. As another embodiment, the chicken genome uses the chicken Gallus_gallus-7.0 sequence information as a reference. This invention demonstrates that individuals carrying the AB genotype at the Chr1:72995066 locus, the AA genotype at the Chr1:73250348 locus, and the AB genotype at the Chr1:73614612 locus represent a superior genotype combination with high anti-aging potential. These individuals with superior genotypes are preferentially retained as parents and can be used to establish the core breeding population for the next generation. Embodiments of this invention demonstrate that loci Chr1:72995066, Chr1:73250348, and Chr1:73614612 mitigate gene [transmission / gain]. C12orf4 The rate of expression decreases during the later stages of egg production, thereby mitigating the negative impact of decreased eggshell strength with increasing age.

[0029] This invention also provides a product for identifying the anti-aging properties of eggshell strength, the product comprising substances for detecting Chr1:72995066, Chr1:73250348, and Chr1:73614612 loci. The substances of this invention include one or more of the following: reagents for nucleic acid extraction and amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, or reagents for sequencing. The substances of this invention also include primers for detecting Chr1:72995066, Chr1:73250348, and Chr1:73614612 loci.

[0030] This invention also provides a method for screening laying hens with low eggshell strength degradation rate, comprising the following steps: extracting genomic DNA from the laying hens to be tested, and performing genotyping on three genetic variation sites: Chr1:72995066, Chr1:73250348, and Chr1:73614612; laying hens carrying the AB genotype at Chr1:72995066, the AA genotype at Chr1:73250348, and the AB genotype at Chr1:73614612 are laying hens with low eggshell strength degradation rate.

[0031] This invention does not specifically limit the method for extracting genomic DNA from laying hens; any method commonly used in the art can be employed. The sample from the laying hen can be venous blood or a feather sample with feather follicles, for subsequent genomic DNA extraction. In a specific embodiment of this invention, the chicken sample is obtained by venous blood collection, treated with an anticoagulant, followed by lysis and protease digestion, and then the individual genomic DNA data is extracted using the phenol-chloroform method.

[0032] This invention does not specifically limit the method for genotyping; any method commonly used in the field can be employed. In a specific embodiment of this invention, the genotype of an individual is detected using the Axiom™ Genome-Wide Chicken Genotyping Array genotyping chip. Finally, referring to the chicken genome Gallus_gallus-7.0 version sequence information, molecular biology techniques are used to identify the genotypes of the three genetic variation sites (Chr1:72995066, Chr1:73250348, and Chr1:73614612).

[0033] This invention also provides a marker-assisted breeding method for improving eggshell strength and anti-aging traits, comprising the following steps: Using the screening method described above, genotypes were identified at three genetic variation loci: Chr1:72995066, Chr1:73250348, and Chr1:73614612. Laying hens carrying the AB genotype at Chr1:72995066, the AA genotype at Chr1:73250348, and the AB genotype at Chr1:73614612 were selected for breeding, resulting in laying hens with improved eggshell strength and anti-aging traits.

[0034] During the breeding process, individuals carrying the AB genotype at Chr1:72995066, the AA genotype at Chr1:73250348, and the AB genotype at Chr1:73614612 were identified as individuals with superior genotype combinations possessing high anti-aging potential. In breeding decisions, these individuals with superior genotypes were prioritized as parents to form the core breeding population for the next generation. Individuals not carrying this superior combination were either culled or used as a control group, depending on the breeding program. By subjecting the core breeding population to the above-mentioned cyclical treatment, targeted genetic improvement of the decline in eggshell strength in the later stages of egg production was gradually achieved, ultimately enhancing the production sustainability of the laying hen population.

[0035] The marker-assisted breeding method of the present invention can improve the eggshell strength and anti-aging ability of laying hens during the aging process, thereby extending the economic cycle of high-quality egg production.

[0036] To further illustrate the present invention, the following description, in conjunction with embodiments, explains the invention based on... C12orf4 The molecular breeding methods and applications of the genetic variation of the eQTL gene in eggshell strength and anti-aging are described in detail, but they should not be construed as limiting the scope of protection of this invention.

[0037] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0038] Example 1: Identification process of loci Chr1:72995066, Chr1:73250348 and Chr1:73614612 in locus screening test I. Sample Collection To locate the genetic information related to changes in eggshell strength during the later stages of egg production in laying hens, representative individuals were collected at 50, 70, and 100 weeks of gestation, with 6, 6, and 12 individuals collected at each time point, respectively. Samples were collected from three key tissues: the hypothalamus, ovary, and pituitary gland.

[0039] To identify locus information related to changes in eggshell strength during the later stages of egg production in laying hens, blood samples were collected from 246 individuals at 100 weeks of age for genomic analysis. Simultaneously, hypothalamic, ovarian, and pituitary tissues were collected from 246 individuals to obtain transcriptome data.

[0040] II. Gene Expression Analysis and Multi-omics Association Analysis 1. Quantitative analysis of gene expression Total RNA was extracted from each tissue, RNA-seq libraries were constructed, and high-throughput sequencing was performed. High-quality sequencing sequences were aligned to the chicken reference genome (Gallus_gallus-7.0) using STAR software, and gene expression was counted using featureCounts. The results were then uniformly converted to TPM values ​​as a standard indicator of gene expression levels.

[0041] 2. Transcriptome sequencing experiment process RNA from the total sample was isolated and purified using TRIzol (Thermofisher, 15596018) according to the manufacturer's instructions. The quantity and purity of the total RNA were then quality controlled using a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA), and RNA integrity was tested using a Bioanalyzer 2100 (Agilent, CA, USA); concentration >50 ng / μL, RIN value >7.0, and total RNA >1 μg met the requirements for downstream experiments. PolyA-containing mRNA was specifically captured using oligo(dT) magnetic beads (Dynabeads Oligo (dT), cat.25-61005, Thermo Fisher, USA) through two rounds of purification. The captured mRNA was fragmented at high temperature using a magnesium fragmentation reagent (NEBNextR 12340ES97, Frag / Prime Buffer) (NEBNextR Magnesium RNAFragmentation Module, cat. E6150S, USA) at 94℃ for 5 min. The fragmented RNA was then used to synthesize cDNA using reverse transcriptase (Invitrogen SuperScript™ II Reverse Transcriptase, cat. 1896649, CA, USA). Then, E. coli DNA polymerase I (NEB, cat.m0209, USA) and RNase H (NEB, cat.m0297, USA) were used to synthesize double-stranded DNA and RNA, converting the complex double strands into DNA double strands. Simultaneously, dUTP Solution (Thermo Fisher, cat.R0133, CA, USA) was incorporated into the double strands to blunt the ends, and an A base was added to each end to allow ligation with adapters ending in T bases. Magnetic beads were used to screen and purify the fragments by size. After screening and purification, PCR amplification was performed, with a cycle of 98℃ for 1 min pre-denaturation, 98℃ for 10 s denaturation, 60℃ for 30 s annealing, and 72℃ for 30 s extension, repeated 14 times, followed by a final extension at 72℃ for 5 min. In PCR amplification, the characteristic of high-fidelity DNA polymerase that only amplifies cDNA chains without U bases is utilized to achieve chain specificity, resulting in a library with a fragment size of 300bp±50bp (chain-specific library).Finally, paired-end sequencing was performed using an Illumina Novaseq™ 6000 (LC Bio Technology CO.,Ltd. Hangzhou, China) according to standard operating procedures in PE150 sequencing mode.

[0042] 3. Locating key genes through cross-time transcriptome analysis Using raw transcriptome counts data from the hypothalamus, ovary, and pituitary gland of laying hens at 50 and 100 weeks, differential expression analysis was performed in the R environment using the DESeq2 package (v1.38.3). Before analysis, genes were screened based on their expression trends at the three time points (50, 70, and 100 weeks), retaining only those showing a sustained upregulation or downregulation trend for subsequent differential analysis. Based on this, the significantly upregulated and significantly downregulated gene sets in each tissue were located using the hypothalamus, ovary, and pituitary transcriptome data from 50 and 100 weeks. The Benjamini-Hochberg p-value was corrected for the false discovery rate (FDR), and transcripts with an FDR < 0.05 and |log2 fold change| > 1 were defined as differentially expressed genes. Finally, gene sets showing a sustained increase or decrease in expression in each tissue at the three time points (50, 70, and 100 weeks) were obtained.

[0043] This invention utilizes Venn diagram analysis to screen gene sets that exhibit significant changes across multiple tissues. Functional enrichment analysis was conducted to explore biological pathways associated with aging-related differentially expressed genes (DEGs). The GO database (http: / / www.geneontology.org / ) categorizes gene function into three domains: molecular function, biological process, and cellular component, and annotation analysis was performed using the ClusterProfiler package (v4.6.2) in R. Simultaneously, the KEGG database (http: / / www.genome.jp / kegg / ) was used to comprehensively analyze relevant information about biological systems from systemic function, genomic, and chemical perspectives.

[0044] This invention utilizes the average egg production water content at multiple time points from 41 to 95 weeks to ultimately pinpoint the key gene. C12orf4 .

[0045] 4. Multi-omics association analysis This invention utilizes transcriptomic and genomic data from 246 individuals to identify gene-locus associations through eQTL mapping analysis. First, the quality-controlled genotype data was converted into a numerical matrix of an additive genetic model (0 / 1 / 2) using PLINK software, and then arranged into a genotype matrix with single nucleotide polymorphisms (SNPs) as rows and individual samples as columns. Simultaneously, the FPKM expression matrix obtained from transcriptomic sequencing was cleaned and standardized, gene nomenclature was unified, and transcriptomic sample numbers were accurately mapped to their corresponding genomic individual numbers. To ensure strict matching of multi-omics data, samples lacking corresponding expression information in the genotype data were removed, resulting in a matching matrix with a completely consistent sample number and arrangement order for subsequent analysis.

[0046] By analyzing the genes involved C12orf 4. Relevant sites were screened, and the sites that have regulatory effects were finally obtained: Chr1:72995066, Chr1:73250348 and Chr1:73614612.

[0047] III. Results Analysis This invention located 842, 1490, and 768 genes with continuously increasing and significant changes in the hypothalamus, ovary, and pituitary gland, respectively, and 1130, 1714, and 1532 genes with continuously decreasing and significant changes in the hypothalamus, ovary, and pituitary gland, respectively. Figure 1 China A and Figure 1 (B). GO enrichment results showed that during the process of tissue functional decline, multiple tissues exhibited drastic changes related to translation function (B). Figure 1 (C). By dividing the gene expression level in ovarian tissue into two groups, high (H) and low (L), this invention ultimately located the gene related to changes in egg production levels from 41 to 95 weeks. C12orf4 ( Figure 1 (D).

[0048] Subsequently, this invention utilizes genomic data, combined with cross-time transcriptome, 246 individual transcriptome and genome eQTL mapping analysis, to finally locate the specific information of key genes and regulatory sites (Table 1).

[0049] Table 1 Genes C12orf4 eQTL mapping analysis results

[0050] Example 2: Genes C12orf4 Analysis of trends expressed in different production cycles I. Sample Collection To verify the invention proposed C12orf4To investigate gene expression dynamics across different production cycles, representative individuals were collected at three time points: 50, 70, and 100 weeks of age, with sample sizes of 6, 6, and 12 individuals at each time point, respectively. Tissue samples were collected from three key tissues: hypothalamus, liver, and uterus.

[0051] II. Gene Expression Detection Total RNA was extracted from each tissue, RNA-seq libraries were constructed, and high-throughput sequencing was performed. High-quality sequencing sequences were aligned to the chicken reference genome (Gallus_gallus-7.0) using STAR software, and gene expression was counted using featureCounts. The results were then uniformly converted to TPM values ​​as a standard indicator of gene expression levels.

[0052] Expression data normalized to TPM (transcripts per million) were used to plot the expression trends of key genes in different tissues using the ggplot2 package in R, visually demonstrating the dynamic expression patterns of these genes as they change with age (50, 70, and 100 weeks). ggplot2 was also used to plot the phenotypic changes of two groups of individuals at different production cycles, thus visually demonstrating the potential regulatory effects of differential expression of target genes on production phenotypic characteristics.

[0053] III. Results Analysis Transcriptome analysis of the hypothalamus, liver, and uterus revealed genes C12orf4 During the laying hen production process (from 50 weeks of age to 100 weeks of age), there was a clear trend of decreasing expression levels. Figure 2 (A). At the same time, according to C12orf4 Gene expression levels were used to divide the population into high-expression and low-expression groups. The eggshell strength of the two groups during the production cycle was then compared and displayed. It was found that the eggshell strength ESS of the high-expression group was significantly higher than that of the low-expression group. Figure 2 (B). Furthermore, this phenomenon exhibits multi-organ consistency, with high gene expression in the hypothalamus, liver, and uterus. C12orf4 Individuals with these characteristics also have higher eggshell strength. This indicates that the genes... C12orf4 These are genes related to changes in eggshell strength during the later stages of egg production in laying hens.

[0054] Example 3: Effect validation based on genotyping at sites Chr1:72995066, Chr1:73250348, and Chr1:73614612 I. Sample Collection Blood samples were obtained by collecting blood from the wing veins of 246 individuals, and the eggshell strength ESS phenotypic data of this population during the production process were recorded.

[0055] II. Genotyping at Target Locus 1. Quantitative analysis of gene expression Total RNA was extracted from each tissue, RNA-seq libraries were constructed, and high-throughput sequencing was performed. High-quality sequencing sequences were aligned to the chicken reference genome (Gallus_gallus-7.0) using STAR software, and gene expression was counted using featureCounts. The results were then uniformly converted to TPM values ​​as a standard indicator of gene expression levels.

[0056] Based on TPM data, the expression trends of key genes in different tissues were plotted using the ggplot2 package in R, visually demonstrating their expression patterns as the individual grows older. The population was divided into high-expression and low-expression groups based on the target gene expression values. The phenotypic trends of the two groups were then plotted using ggplot2 throughout different production cycles, visually demonstrating the potential effect of gene expression levels on phenotype.

[0057] III. Results Analysis This invention discovers C12orf4 Gene expression levels vary considerably within a population; some individuals maintain high gene expression levels at 100 weeks of age, while others experience a rapid decline, indicating that genetic factors play a significant role in changes in gene expression levels. Figure 3 (A)

[0058] By analyzing the genotype combinations of three molecular markers Chr1:72995066, Chr1:73250348, and Chr1:73614612, it was found that individuals carrying the following genotype combinations... C12orf4 The combination showed high gene expression levels and eggshell strength ESS: the AB type at Chr1:72995066, the AA type at Chr1:73250348, and the AB type at Chr1:73614612 had an average ESS of 3.605, which was the best among all combinations. Figure 3 B, Figure 3 C and Figure 3 (D).

[0059] Based on the above results, loci Chr1:72995066, Chr1:73250348, and Chr1:73614612 slow down the gene... C12orf4 The expression rate of these three genetic variation sites decreases during the later stages of egg production, thereby mitigating the negative impact of declining eggshell strength with age. Using marker-assisted selection for these three genetic variation sites holds promise for significantly improving the anti-aging ability of eggshell strength, thus enhancing the overall production durability of the population.

[0060] Example 4: Validation of the association between genotyping at loci Chr1:72995066, Chr1:73250348, and Chr1:73614612 and eggshell strength in another population. I. Sample Collection Blood samples were obtained by collecting blood from the wing veins of 563 individuals, and the eggshell strength ESS phenotype data of this population were recorded at 90 weeks.

[0061] Chicken samples were collected using a wing vein blood collection method. After anticoagulation with an anticoagulant, the samples were lysed, digested with a protease, and genomic DNA was extracted using the phenol-chloroform method. The extracted DNA was dissolved in sterile double-distilled water and stored for later use. The blood collection method and phenol-chloroform extraction technique described herein are standard procedures in the field.

[0062] II. Genotyping and Analysis of Target Loci 1. Genotyping process Using extracted genomic DNA as a template, whole-genome SNP genotyping was performed on each experimental individual using the Axiom™ Genome-Wide Chicken Genotyping Array (product catalog number: 902148) manufactured by Axiom Technologies, Inc., USA. This included the loci Chr1:72995066, Chr1:73250348, and Chr1:73614612.

[0063] A genotype is the combination of two alleles at the same gene locus. Usually, a gene locus has only two alleles, so there are usually three genotypes: "homozygous type I", "homozygous type II", and "heterozygous type".

[0064] Genotyping of the three genetic variation sites (Chr1:72995066, Chr1:73250348, and Chr1:73614612) was performed using molecular biology techniques. The chicken reference genome Gallus_gallus-7.0 version sequence information was publicly available on the NCBI website.

[0065] 2. Correlation analysis between key site combinations and eggshell strength at 90 weeks Using R language, we statistically analyzed different combinations of the loci Chr1:72995066, Chr1:73250348 and Chr1:73614612, and calculated the mean eggshell strength for each group.

[0066] III. Results Analysis After grouping the 563 samples, it was found that 106 individuals carried the Chr1:72995066 locus AB type, the Chr1:73250348 locus AA type, and the Chr1:73614612 locus AB type, with a mean eggshell strength of 2.88 at 90 weeks (Table 2). This means that this SNP combination is also suitable for improving eggshell strength in the later stages of egg production in another population.

[0067] Table 2. Correlation verification between key site combinations and eggshell strength at 90 weeks.

[0068] Comparative Example: Comparison of Predictive Intensities between Single-Site and Three-Site Combinations I. Sample Collection Blood samples were collected from the wing veins of 246 individuals, and eggshell strength ESS phenotypic data of this population during the production process were recorded.

[0069] Chicken samples were collected using a wing vein blood collection method. After anticoagulation with an anticoagulant, the samples were lysed, digested with a protease, and genomic DNA was extracted using the phenol-chloroform method. The extracted DNA was dissolved in sterile double-distilled water and stored for later use. The blood collection method and phenol-chloroform extraction technique described herein are standard procedures in the field.

[0070] II. Genotyping at Target Locus 1. Genotyping process Using extracted genomic DNA as a template, whole-genome SNP genotyping was performed on each experimental individual using the Axiom™ Genome-Wide Chicken Genotyping Array (product catalog number: 902148) manufactured by Axiom Technologies, Inc., USA. This included the loci Chr1:72995066, Chr1:73250348, and Chr1:73614612.

[0071] 2. Statistical analysis of the mean eggshell strength of different groups after genotyping. III. Results Analysis Current technologies can use molecular markers for breeding, and the results of group breeding targeting a single locus are shown in Table 3. The average eggshell strength of each group is lower than 3.605 after screening with three-locus combinations. This means that molecular breeding targeting only a single marker does not have broad applicability, nor can it maximize the development of the breeding potential of that locus.

[0072] This invention fully considers the interactions between three molecular markers. When carrying the Chr1:72995066 AB type, the Chr1:73250348 AA type, and the Chr1:73614612 AB type, late-laying hens have a higher genetic predisposition. C12orf4 Expression level and eggshell strength (mean 3.605); however, if only one molecular marker is selected for breeding, the eggshell strength of that molecular module cannot be achieved.

[0073] Table 3. Mean eggshell strength of individuals with different genotypes

[0074] In summary, this invention largely avoids the drawbacks of using a single molecular marker for breeding. Breeding based on the molecular modules provided in this invention can fully utilize the role of molecular markers, alleviate the declining trend of eggshell strength in the later stages of egg production, and has great economic and scientific research value. It represents an optimization and upgrade of molecular marker technology.

[0075] Therefore, this invention, by integrating genomic, transcriptomic, and phenotypic data to conduct multidimensional association analysis, identifies and constructs a key gene that can prospectively and systematically reflect the process of tissue function decline in laying hens. C12orf4 The invention also includes its regulatory sites Chr1:72995066, Chr1:73250348, and Chr1:73614612. The molecular markers provided by this invention offer a new technical means for the accurate assessment of aging-related traits in laying hens and for molecular breeding, possessing significant application value and industrial significance.

[0076] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of reagents for identifying molecular marker combinations in identifying eggshell strength and anti-aging properties and / or in breeding to improve eggshell strength and anti-aging traits, characterized in that, The molecular marker combination pair C12orf4 Gene expression levels have a regulatory effect; The molecular marker combinations are located at the chicken genome at Chr1:72995066, Chr1:73250348, and Chr1:73614612 sites, respectively.

2. The application according to claim 1, characterized in that, The genotypes at the Chr1:72995066 locus are AA, AB, and BB. The genotypes at the Chr1:73250348 locus are AA, AB, and BB. The genotypes at the Chr1:73614612 locus are AA, AB, and BB.

3. The application according to claim 1, characterized in that, The chicken genome references the chicken Gallus_gallus-7.0 version sequence information.

4. The application according to claim 1, characterized in that, The C12orf4 The gene's NCBI accession number is 419041.

5. A product for identifying the strength and anti-aging properties of eggshells, characterized in that, The product includes substances that detect Chr1:72995066, Chr1:73250348 and Chr1:73614612 sites.

6. The product according to claim 5, characterized in that, The substance includes one or more of the following: reagents for nucleic acid extraction and amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, or reagents for sequencing.

7. The substance according to claim 5, characterized in that, The substance includes primers for detecting Chr1:72995066, Chr1:73250348 and Chr1:73614612 sites.

8. A method for screening laying hens with low eggshell strength degradation rate, characterized in that, Includes the following steps: Genomic DNA was extracted from the laying hens to be tested, and genotyping was performed at three genetic variation loci: Chr1:72995066, Chr1:73250348, and Chr1:73614612. Laying hens carrying the AB genotype at Chr1:72995066, the AA genotype at Chr1:73250348, and the AB genotype at Chr1:73614612 were classified as laying hens with low eggshell strength degradation rates.

9. The screening method according to claim 8, characterized in that, The identification method includes identification using the Axiom™ Genome-Wide Chicken Genotyping Array genotyping chip.

10. A marker-assisted breeding method for improving eggshell strength and anti-aging traits, characterized in that, Includes the following steps: Using the screening method described in claim 8 or 9, genotypes of the three genetic variation loci Chr1:72995066, Chr1:73250348, and Chr1:73614612 are identified. Laying hens carrying the AB genotype at Chr1:72995066, the AA genotype at Chr1:73250348, and the AB genotype at Chr1:73614612 are bred to obtain laying hens with improved eggshell strength and anti-aging traits.

Images