A SNP molecular marker associated with the average feeding time of chickens and its application

Abstract

The present invention belongs to the technical field of livestock and poultry genetic markers and animal genetic breeding, and specifically relates to a SNP molecular marker related to the average feeding time of chickens and its application. The SNP molecular markers are located at 1,035,978bp and 1,035,986bp of chicken chromosome 2, respectively. The candidate genes are <h2 style=";text-align:left;direction:ltr">GIMAP7 Gene. The SNP molecular markers associated with average feeding time per chicken provided by the present invention provide key genetic information for broiler genetic breeding, facilitating the precise selection of populations with excellent average feeding time traits during broiler breeding, improving broiler feeding efficiency and production performance, and enhancing the economic benefits of the broiler industry. The discovery of SNP molecular markers associated with average feeding time per chicken also provides an important research foundation for in-depth studies of the genetic mechanisms of chicken feeding behavior, helping to promote the development of livestock and poultry genetic breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of livestock and poultry genetic markers and animal genetic breeding, and particularly relates to a SNP molecular marker associated with the average feeding time of chickens and an application thereof. Background Art

[0002] Feeding behavior generally has moderate heritability, with specific levels varying by breed and rearing conditions. Selecting for feeding behavior has the potential to improve poultry production management and increase feeding efficiency, which is of great significance. Furthermore, feeding behavior is associated with animal health and can serve as an important indicator for disease detection. However, current research on the genetic mechanisms of chicken feeding behavior traits is insufficient, especially the lack of genetic markers associated with specific feeding behavior traits, such as average feeding duration per feed (FDV, seconds per feed). This has limited the precision of broiler genetic breeding efforts and hindered the need to improve the production efficiency of high-quality broiler breeds.

[0003] Existing technologies are insufficient in studying the genetic mechanisms of feeding behavior in chickens, making it difficult to identify genetic markers closely associated with specific feeding behavior traits. Traditional research methods are inaccurate in identifying genes and variant loci that influence feeding behavior, failing to provide sufficient theoretical support and effective genetic markers for broiler genetic breeding. This results in a lack of precise guidance for selecting broiler breeds with superior feeding behavior traits, hindering the production efficiency and economic benefits of the broiler industry.

[0004] The present invention aims to solve this problem by accurately identifying SNP molecular markers related to the average feeding time per chicken, providing strong support for broiler genetic breeding. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a SNP molecular marker related to the average feeding time of chickens and its application.

[0006] The present invention provides a SNP molecular marker associated with the average feeding time of chickens, wherein the SNP molecular marker includes one or more of the following:

[0007] 1) The SNP molecular marker corresponds to the 885,500 bp position on chromosome 2 of the chicken reference genome GRCg6a version sequence information published by NCBI. The polymorphism of the SNP molecular marker is G or A. The SNP molecular marker is located in the intron region of the SMARCC1 gene on chicken chromosome 2. The SNP molecular marker number is rs3384943691;

[0008] 2) The SNP molecular marker corresponds to the 1,035,978 bp on chromosome 2 of the chicken reference genome version GRCg6a sequence information published by NCBI. The polymorphism of the SNP molecular marker is T or C. The SNP molecular marker is located in the upstream region of the GIMAP7 gene on chicken chromosome 2;

[0009] 3) The SNP molecular marker corresponds to bp 1,035,986 on chromosome 2 of the chicken reference genome GRCg6a version sequence information published by NCBI. The polymorphism of the SNP molecular marker is C or A. The SNP molecular marker is located in the upstream region of the GIMAP7 gene on chicken chromosome 2;

[0010] 4) The SNP molecular marker corresponds to bp 26,404,634 on chromosome 7 of the chicken reference genome GRCg6a version sequence information published by NCBI. The SNP molecular marker polymorphism is C or T. The SNP molecular marker is located in the intron region of the SLC15A2 gene on chicken chromosome 7. The SNP molecular marker number is rs731344589;

[0011] 5) The SNP molecular marker corresponds to the 26,429,746 bp on chromosome 7 of the chicken reference genome GRCg6a version sequence information published in NCBI. The polymorphism of the SNP molecular marker is A or G. The SNP molecular marker has an interaction effect with the target gene HSPBAP1 of the enhancer on chicken chromosome 7. The SNP molecular marker number is rs739957878.

[0012] The genotypes of the SNP molecular markers described in the present invention and the corresponding average feeding duration traits are as follows:

[0013] 1) According to the above-mentioned SNP molecular marker, the average feeding time per feeding of the group with the GG genotype in the SNP molecular marker was longer than that of the groups with the GA and AA genotypes;

[0014] 2) According to the above-mentioned SNP molecular markers, the average feeding duration per feeding in the group with the TT genotype in the SNP molecular markers was longer than that in the groups with the TC and CC genotypes;

[0015] 3) According to the above-mentioned SNP molecular markers, the average feeding duration per feeding in the group with CC genotype in the SNP molecular markers was longer than that in the groups with CA and AA genotypes;

[0016] 4) According to the above-mentioned SNP molecular markers, the average feeding duration per feeding in the group with CC genotype in the SNP molecular markers was longer than that in the groups with CT and TT genotypes;

[0017] 5) According to the above-mentioned SNP molecular marker, the average feeding time per feeding of the group with the AA genotype in the SNP molecular marker was longer than that of the groups with the AG and GG genotypes.

[0018] The present invention provides a method for identifying the average feeding time of chickens, wherein the selection method comprises the following steps:

[0019] 1) detecting the SNP molecular marker in the genome of the chicken to be tested;

[0020] 2) The chickens to be tested are identified based on the genotype of the SNP molecular marker and the corresponding average feeding duration trait.

[0021] Specifically, the feeding duration traits of different chickens identified by the above method are used for further breeding analysis.

[0022] The present invention provides a SNP molecular marker associated with the average feeding time of chickens. Specifically, the SNP molecular marker located at 885,500bp on chromosome 2 of chicken is predicted to be an enhancer element in the cerebral cortex, hypothalamus and lung tissue of chickens. The enhancer element may be related to SMARCC1 Gene expression is associated with higher expression in chicken retina, thymus, and testis compared to other tissues SMARCC1 High gene expression.

[0023] Specifically, the SNP molecular marker located at 26,404,634bp on chicken chromosome 2 is predicted to be an enhancer element in the cerebral cortex, hypothalamus and cerebellum, and the enhancer element may be related to SLC15A2 Gene expression is related to the chicken hypothalamus and brain compared with other tissues. SLC15A 2 genes were highly expressed.

[0024] Specifically, the SNP molecular marker located at 885,500bp on chicken chromosome 2 and the SNP molecular marker located at 26,404,634bp on chicken chromosome 7 may be regulated by SMARCC1 Genes and SLC15A 2 gene expression, thereby affecting the chicken's neural network and hormone signals, and ultimately regulating the chicken's feeding behavior (FDV).

[0025] Application of the SNP molecular marker of the present invention in any of the following:

[0026] 1) Assisted breeding of broiler breeds;

[0027] 2) Improvement of broiler chicken germplasm resources;

[0028] 3) Differentiate broiler breeds with different average feeding durations.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This study successfully identified SNP molecular markers and candidate genes associated with the FDV trait in chicken feeding behavior, providing critical genetic information for broiler breeding. By screening and utilizing these SNP molecular markers, it is hoped that the precise selection of broiler chickens with excellent FDV traits can be achieved during broiler breeding, improving broiler feeding efficiency and production performance, and ultimately increasing the economic benefits of the broiler industry. Furthermore, the discovery of these SNP molecular markers provides an important research foundation for in-depth study of the genetic mechanisms of chicken feeding behavior, helping to advance the field of livestock and poultry genetic breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 These are the SNPs-GWAS Manhattan plot and QQ plot of the chicken FDV trait of the present invention; A is the SNPs-GWAS Manhattan plot of the chicken FDV trait, and B is the QQ plot of the SNPs-GWAS analysis of the chicken FDV trait.

[0032] Figure 2 Figure 2 is a genotyping box plot of the five SNP molecular markers for the chicken FDV trait of the present invention; ...

[0033] Figure 3 For the present invention SMARCC1 Genes and SLC15A2 Gene regulatory element prediction map; A is SMARCC1 Gene regulatory element prediction map, B is SLC15A2 Gene regulatory element prediction map.

[0034] Figure 4 For the present invention SMARCC1 Distribution of gene expression levels in different chicken tissues.

[0035] Figure 5 For the present invention SLC15A2 Distribution of gene expression levels in different chicken tissues. DETAILED DESCRIPTION

[0036] The following is a technical solution of the present invention, a SNP molecular marker related to the average feeding time of chickens and its application, further illustrated by specific examples.

[0037] Example 1

[0038] 1. Experimental population and feeding management

[0039] A total of 205 healthy yellow-feathered broiler chickens from the same batch were selected. After hatching, they were housed flat on the floor in a semi-open shed with fresh sawdust bedding in the pens. They were fed a corn-soybean meal diet and were not given antibiotics. The birds were fitted with radio frequency identification (RFID) chip anklets, and the feeders were equipped with RFID chip antennas to automatically record individual information, entry and exit times, and feed trough weight changes. The trial lasted from 56 to 76 days of age.

[0040] 2. Sample Collection

[0041] At 76 days of age, approximately 1 mL of whole blood was collected aseptically from the sub-wing vein of chickens in a centrifuge tube containing ACD anticoagulant, mixed, and stored at -20°C for subsequent genomic DNA extraction. Fecal samples were also collected, and intestinal contents from the duodenum, jejunum, ileum, and cecum were dissected and rapidly frozen in liquid nitrogen before being stored at -80°C.

[0042] 3. DNA Extraction and Sequencing

[0043] Host genomic DNA was extracted using the TIANGEN Blood Genomic DNA Extraction Kit (DP318) and, after a series of processing steps, assessed for concentration and quality using NanoDrop. For library construction, DNA samples were ultrasonically fragmented and end-repaired. Paired-end 150 bp sequencing was performed using an Illumina HiSeq 2500 System at a sequencing depth of 10×. The chicken reference genome, version GRCg6a, published by NCBI, was used for subsequent analysis.

[0044] 4. Genetic variation detection

[0045] SNPs were detected using the GATK v.4.2.0.0 toolkit based on the processed BAM files. The HaplotypeCaller module was used to extract variant data and generate gVCF files. After merging the gVCF files for all samples, genotyping was performed using the GenotypeGVCFs module. Biallelic SNP variants were extracted using the SelectVariants module. Strict quality control was performed using VariantFiltration. SNP quality control criteria included QD > 10.0, MQ > 10.0, FS < 60.0, MQRankSum > -12.5, and ReadPosRankSum > -8.0. Further screening was performed using PLINK v.1.9 to ensure that the sample and SNP call rates met quality standards (sample call rate > 90% and SNP call rate > 95%). Genotype data were imputed using Beaglev.4.0 software, and secondary quality control was performed.

[0046] 5. Genome-wide association analysis of average feeding duration per chicken

[0047] Genome-wide association analysis was performed using the univariate linear mixed model (ULMM) in GEMMA v.0.98.5 software. The feeding behavior trait, mean feeding time per visit (FDV, seconds per visit), was used as the research object, and the model was y = Wα + xβ + u + , y is the FDV phenotype vector of broiler chickens, W is the covariate matrix, x is the genotype, β is the gene effect size, u is the random effect, The error is 0. A kinship matrix was constructed based on SNPs. The first four principal components (PCs) based on the SNP data were selected as covariates for model correction. A Bonferroni-adjusted genome-wide significance threshold was set to -log10(0.05 / 1,535,191)=7.488, and a potential significance threshold was set to -log10(1 / 1,535,191)=6.186. After identifying significant or potentially significant variants associated with FDV through GWAS analysis, SnpEff was used to annotate and identify candidate genes using the chicken reference genome file. The regulatory elements and candidate gene expression levels associated with the variants were analyzed using data from the UCSC Genome Browser and the ChickenGTEx website.

[0048] 6. Test results

[0049] (1) Detection of potential significant sites and candidate genes of SNP molecular markers associated with chicken FDV traits

[0050] The SNPs-GWAS Manhattan plot and QQ plot of chicken FDV trait are shown in Figure 2. Figure 1 As shown in (A and B), SNP-GWAS analysis based on SNP genetic markers identified five potential significant loci for SNP molecular markers on chicken chromosomes 2 and 7, and annotated three candidate genes, as follows:

[0051] The SNP molecular marker polymorphism at 885,500bp on chicken chromosome 2 is G or A. The SNP molecular marker is located on chicken chromosome 2. SMARCC1 Intron region of the gene, the SNP molecular marker number is rs3384943691;

[0052] The SNP molecular marker polymorphism at 1,035,978bp on chicken chromosome 2 is T or C. The SNP molecular marker is located on chicken chromosome 2. GIMAP7 upstream region of a gene;

[0053] The SNP molecular marker polymorphism at 1,035,986bp on chicken chromosome 2 is C or A. The SNP molecular marker is located on chicken chromosome 2. GIMAP7 upstream region of a gene;

[0054] The SNP molecular marker polymorphism at 26,404,634bp on chicken chromosome 7 is C or T. The SNP molecular marker is located on chicken chromosome 7. SLC15A2 Intron region of the gene, the SNP molecular marker number is rs731344589;

[0055] The SNP molecular marker polymorphism at the 26,429,746 bp on chicken chromosome 7 is A or G, and the SNP molecular marker is associated with the target gene of the enhancer on chicken chromosome 7. HSPBAP1 There is an interaction effect, and the SNP molecular marker number is rs739957878.

[0056] Figure 1 A shows the results of genome-wide association analysis based on SNPs molecular markers, with the horizontal axis being the chromosome position and the vertical axis being -log10 ( p ) value, intuitively presenting the significant distribution of SNP molecular marker sites related to FDV traits across the entire genome, quickly locating potential SNP molecular marker sites on chromosomes 2 and 7, and annotating potential SNP molecular marker sites to 3 candidate genes ( SMARCC1 Gene 、SLC15A2 Genes and GIMAP7 Gene). Figure 1 B shows the QQ diagram based on SNPs-GWAS analysis, with the horizontal axis being p The vertical axis is the theoretical expected distribution of the value, and the vertical axis is the actual observed value. p The observed value area fits the theoretical distribution, indicating that the model correction is effective and verifies Figure 1 The robustness of the analysis method in A.

[0057] The experimental results showed that the SNP molecular markers related to the FDV trait in chicken feeding behavior were located on chromosomes 2 and 7 of chickens. Specifically, the SNP molecular markers related to the FDV trait were located at 885,500bp, 1,035,978bp, and 1,035,986bp of chromosome 2 of chickens and at 26,404,634bp and 26,429,746bp of chromosome 7 of chickens. SMARCC1 Gene 、SLC15A2 Genes and GIMAP7 These three candidate genes.

[0058] (2) Genotyping of potential significant sites of SNP molecular markers associated with chicken FDV traits and SMARCC1 Gene, SLC15A2 Gene regulatory element prediction

[0059] The genotyping box plot of the potential significant sites of the five SNP molecular markers for chicken FDV traits is shown in the figure. Figure 2 As shown in (AE), the x-axis represents different genotype groups, the y-axis represents the FDV value (unit: seconds / time), the horizontal line in the center of the colored box represents the median FDV value of the corresponding genotype group, and the red dot represents the average FDV value of the corresponding genotype group.

[0060] The results of population analysis showed that the number of wild homozygous populations in the five SNP molecular markers associated with the chicken FDV trait was greater in the sample group, and the FDV values ​​of the wild homozygous population were significantly higher than those of other genotype populations. In addition, the FDV values ​​of the wild homozygous population and other genotype populations in the five SNP molecular markers associated with the chicken FDV trait were significantly different. Among them, the FDV values ​​of the wild homozygous population and other genotype populations in the four SNP molecular markers located at 885,500bp, 1,035,978bp, 1,035,986bp on chicken chromosome 2 and 26,404,634bp on chromosome 7 were extremely significantly different ( p < 0.001).

[0061] The above-mentioned wild homozygous population refers to the population with a genotype of GG in the SNP molecular marker located at 885,500bp on chicken chromosome 2, the population with a genotype of TT in the SNP molecular marker located at 1,035,978bp on chicken chromosome 2, the population with a genotype of CC in the SNP molecular marker located at 1,035,986bp on chicken chromosome 2, the population with a genotype of CC in the SNP molecular marker located at 26,404,634bp on chicken chromosome 7, and the population with a genotype of AA in the SNP molecular marker located at 26,429,746bp on chicken chromosome 7 in the same breed of this scheme; the above-mentioned other genotype populations refer to the population whose genotypes at the corresponding specific sites on the chromosomes in the five SNP molecular markers in this scheme do not belong to the wild homozygous type.

[0062] Specifically, a SNP molecular marker located at 885,500bp on chicken chromosome 2, wherein the average feeding time per feeding of the group with the genotype GG in the SNP molecular marker is longer than that of the groups with the genotypes GA and AA;

[0063] A SNP molecular marker located at 1,035,978 bp on chicken chromosome 2, wherein the average feeding duration per feeding in a group with a genotype of TT is greater than that in groups with genotypes of TC and CC;

[0064] A SNP molecular marker located at bp 1,035,986 on chicken chromosome 2, wherein the average feeding duration per feeding in a group with a CC genotype is longer than that in groups with CA and AA genotypes;

[0065] A SNP molecular marker located at bp 26,404,634 on chicken chromosome 7, wherein the average feeding duration per feeding in a group with a CC genotype is longer than that in groups with CT and TT genotypes;

[0066] A SNP molecular marker located at 26,429,746 bp on chicken chromosome 7, wherein the average feeding time per feeding of a group with genotype AA is longer than that of groups with genotypes AG and GG.

[0067] pass Figure 2 (AE) The intuitive comparison of the differences in FDV values ​​among different genotype groups also illustrates the association between genotype and FDV traits.

[0068] The above experimental results show that five SNP molecular markers located at 885,500bp, 1,035,978bp, and 1,035,986bp on chromosome 2 and at 26,404,634bp and 26,429,746bp on chromosome 7 are associated with the chicken FDV trait. Specifically, chickens with wild homozygous genotypes at these five SNP molecular marker sites have longer feeding time per session, and the FDV value of the wild homozygous group is significantly higher than that of the other genotype groups.

[0069] SMARCC1 Genes and SLC15A The regulatory element prediction diagram of 2 genes is as follows Figure 3 As shown in A and B, where Figure 3 The yellow regions shown represent enhancer regions. Figure 3 A shows SMARCC 1. Distribution of regulatory elements in the gene region. The experimental results showed that the SNP molecular marker located at 885,500bp on chromosome 2 of chicken was predicted to be an enhancer element in the chicken's cerebral cortex, hypothalamus and lung tissues. Figure 3 B shows SLC15A 2. Distribution of regulatory elements in gene regions. Experimental results showed that the SNP molecular marker located at 26,404,634bp of chicken chromosome 7 was predicted to be an enhancer element in the chicken cerebral cortex, hypothalamus and cerebellum.

[0070] SMARCC1 The distribution of gene expression in different chicken tissues is shown in the figure Figure 4 This box plot is arranged in descending order according to the median expression level (TPM), showing SMARCC1 Tissue-specific expression patterns of genes. (Data source: chickenGTEx-Portal database, http: / / chickengtex.farmgtex.org / ). By checking the expression of this gene in the ChickenGTEx website, the results showed that SMARCC1 The gene is expressed in many tissues of chickens, especially in the retina, thymus, and testis compared to other tissues. SMARCC1 The gene expression level is high.

[0071] SLC15A The expression distribution of 2 genes in different chicken tissues is shown in the figure Figure 5 This box plot is arranged in descending order according to the median expression level (TPM), showing SLC15A Tissue-specific expression patterns of 2 genes. (Data source: chickenGTEx-Portal database, http: / / chickengtex.farmgtex.org / ). By checking the expression of this gene in the ChickenGTEx website, the results showed that SLC15A 2 genes are expressed in many tissues of chickens, among which the expression is higher in the chicken hypothalamus and brain than in other tissues. SMARCC1 The gene expression level is high.

[0072] Figure 3 Provides direct support for the functional mechanism of the SNP molecular marker located at 885,500bp on chicken chromosome 2 and the SNP molecular marker located at 26,404,634bp on chicken chromosome 7. Figure 4 middle SMARCC1 Genes and Figure 5 middle SLC15A The expression distribution of 2 genes in different chicken tissues showed that SMARCC1 Genes and Figure 5 middle SLC15A 2 genes are expressed in many tissues of chickens. SMARCC1 The gene is highly expressed in the retina, thymus, and testis of chickens. SLC15A The expression of 2 gene is higher in the hypothalamus and brain of chickens.

[0073] Therefore, the SNP marker located at 885,500bp on chicken chromosome 2 may regulate SMARCC1 Genes and SLC15A 2 gene expression, thereby affecting the chicken's neural network and hormone signals, and ultimately regulating the chicken's feeding behavior (FDV).

[0074] The present invention can successfully distinguish groups with different average feeding durations by utilizing five SNP molecular markers.

[0075] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for identifying the average feeding time of chickens, characterized in that: The following steps are included: 1) Detecting SNP molecular markers in the genome of yellow-feathered broiler chickens to be tested; 2) identifying the average feeding duration per feeding of the yellow-feathered broiler chickens according to the genotype of the SNP molecular marker; The SNP molecular marker is numbered rs3384943691, and the polymorphism is G or A. The yellow-feathered broiler chickens with the genotype GG in the SNP molecular marker have a longer average feeding time per session than the yellow-feathered broiler chickens with the genotypes GA and AA; or The SNP molecular marker is numbered rs731344589, and the polymorphism is C or T. The yellow-feathered broiler chickens with the CC genotype in the SNP molecular marker have a longer average feeding time per session than the yellow-feathered broiler chickens with the CT and TT genotypes; or, The SNP molecular marker is numbered rs739957878, and the polymorphism is A or G. The yellow-feathered broiler chickens with the genotype AA in the SNP molecular marker have a longer average feeding time per feeding than the yellow-feathered broiler chickens with the genotypes AG and GG.

2. Use of a reagent for detecting a SNP molecular marker associated with the average feeding time per chicken in any of the following: 1) Assisted breeding of broiler breeds; 2) Screening broiler groups with different average feeding duration traits; The SNP molecular marker is numbered rs3384943691, and the polymorphism is G or A. The yellow-feathered broiler chickens with the genotype GG in the SNP molecular marker have a longer average feeding time per session than the yellow-feathered broiler chickens with the genotypes GA and AA; or The SNP molecular marker is numbered rs731344589, and the polymorphism is C or T. The yellow-feathered broiler chickens with the CC genotype in the SNP molecular marker have a longer average feeding time per session than the yellow-feathered broiler chickens with the CT and TT genotypes; or, The SNP molecular marker is numbered rs739957878, and the polymorphism is A or G. The yellow-feathered broiler chickens with the genotype AA in the SNP molecular marker have a longer average feeding time per feeding than the yellow-feathered broiler chickens with the genotypes AG and GG.

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