SNP (Single Nucleotide Polymorphism) site related to foot weight and foot index of mountain chicken

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) site related to foot weight and foot index of a mountain chicken, and relates to the field of poultry molecular breeding, and the SNP site comprises the following components: chr474671312, chr474671339, chr474671343, chr474671388, chr474671516, chr474671566 and chr474671617. Early individual breeding is carried out on the genome level, dependence on phenotypic information is avoided, the selection efficiency is remarkably improved, the breeding process is accelerated, meanwhile, the feeding cost is reduced, and good application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to the field of poultry molecular breeding, and mainly to the field of SNP sites related to mountain chicken foot weight and foot index. Background Art

[0002] Chicken feet, a common poultry by-product, hold significant commercial value globally, particularly in Asia. In the food processing sector, chicken feet are made into ready-to-eat snacks like pickled pepper chicken feet and braised dishes, as well as hot pot and tea restaurant dishes. Their low cost and high profit margins make them a popular ingredient in the catering and pre-prepared meal industries. Furthermore, chicken feet are rich in collagen, which can be extracted and used in health and beauty products, further increasing their added value. Consumer demand is strong in Asian markets (such as China, South Korea, and Southeast Asia), while sales in Europe and the United States are primarily through Chinese-speaking channels. Driven by trends toward health and flavor innovation, the chicken feet industry continues to grow, but the industry must remain vigilant regarding challenges such as food safety and international competition. Overall, chicken feet, due to their affordability and wide range of applications, hold a key position in the food industry and cross-border trade.

[0003] Traditional breeding methods rely on phenotypic measurements, which are time-consuming and susceptible to environmental influences. Existing SNP markers are mostly focused on egg production or growth traits, but lack molecular markers specific to the foot characteristics of mountain chickens. Summary of the Invention

[0004] In response to the shortcomings of the above-mentioned existing technologies, the present invention proposes a SNP site related to mountain chicken foot weight and foot index, which can be used for early individual selection at the genome level, avoiding dependence on phenotypic information, significantly improving selection efficiency, accelerating the breeding process, and reducing breeding costs, which has good application prospects.

[0005] To achieve the above object, the technical solution of the present invention is: the SNP sites include the following: chr4_74671312, chr4_74671339, chr4_74671343, chr4_74671388, chr4_74671516, chr4_74671566 and chr4_74671617.

[0006] Preferably, chr4_74671312 is a G or A polymorphism.

[0007] Preferably, chr4_74671339 is T or G polymorphism.

[0008] Preferably, chr4_74671343 is a C or T polymorphism.

[0009] Preferably, chr4_74671388 is a G or A polymorphism.

[0010] Preferably, chr4_74671516 is a G or C polymorphism.

[0011] Preferably, chr4_74671566 is T or C polymorphic.

[0012] Preferably, chr4_74671617 is a G or A polymorphism.

[0013] Preferably, the foot weight and foot index shape select the SNP 1 site AA dominant genotype, SNP 2 site GG dominant genotype, SNP 3 site TT dominant genotype, SNP 4 site AA dominant genotype, SNP 5 site CC dominant genotype, SNP 6 site TT dominant genotype and SNP 7 site AA dominant genotype.

[0014] The technical principles and beneficial effects of the present invention are as follows: This study uses genome-wide association analysis to screen mountain chickens, identifying SNP molecular markers significantly associated with foot weight and foot index. These markers, including at least one of seven SNP loci, can be applied to molecular marker-assisted breeding of mountain chickens. The breeding method disclosed in this invention enables early individual selection at the genomic level, avoiding reliance on phenotypic information, significantly improving selection efficiency, accelerating the breeding process, and reducing breeding costs, demonstrating promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only seven of the drawings of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 This is a diagram showing the statistical analysis of the foot weight index trait of 120-day-old Chengkou mountain chickens; Figure 2 Schematic diagram of SNPs significantly associated with FW and FI traits; Figure 3 Schematic diagram of Manhattan plot and QQ plot of FW and FI GWAS; Figure 4 Schematic diagram of linkage disequilibrium analysis and haplotype construction of SNPs significantly associated with FW and FI on chromosome 4; Figure 5 Schematic diagram of the effects of SNPs and genotypes on FW traits; Figure 6 Schematic diagram of the effects of SNPs and genotypes on FI traits; Figure 7Schematic diagram of association analysis between haplotype combinations and traits; DETAILED DESCRIPTION

[0017] The technical solutions of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. It is apparent that the embodiments described are merely preferred embodiments of the present invention, not all embodiments. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention. Example

[0018] The present invention provides SNPs (chr4_74671312, chr4_74671339, chr4_74671343, chr4_74671388, chr4_74671516, chr4_74671566, chr4_74671617) for molecular breeding of foot weight and foot index of Chengkou mountain chickens.

[0019] SNP 1 corresponds to position 74671312 of the positive strand of chromosome 4 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg6a version published in NCBI, which is a G or A polymorphism.

[0020] SNP 2 corresponds to position 74671339 of the positive strand of chromosome 4 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg6a version published in NCBI, which is a T or G polymorphism.

[0021] SNP 3 corresponds to position 74671343 of the positive strand of chromosome 4 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg6a version published in NCBI, which is a C or T polymorphism.

[0022] SNP 4 corresponds to position 74671388 of the positive strand of chromosome 4 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg6a version published in NCBI, which is a G or A polymorphism.

[0023] SNP 5 corresponds to position 74671516 of the positive strand of chromosome 4 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg6a version published in NCBI, which is a G or C polymorphism.

[0024] SNP 6 corresponds to position 74671566 of the positive strand of chromosome 4 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg6a version published in NCBI, which is a T or C polymorphism.

[0025] SNP 7 corresponds to position 74671617 of the positive strand of chromosome 4 in the sequence information of the chicken reference genome bGalGal1.mat.broiler.GRCg6a version published in NCBI, which is a G or A polymorphism.

[0026] Determine the genotype of the chicken to be bred, the genotype of the above-mentioned SNP molecular marker combination. For the FW and FI traits, select the AA dominant genotype at SNP 1 site, the GG dominant genotype at SNP 2 site, the TT dominant genotype at SNP 3 site, the AA dominant genotype at SNP 4 site, the CC dominant genotype at SNP 5 site, the TT dominant genotype at SNP 6 site, and the AA dominant genotype at SNP 7 site.

[0027] As a local breed, Chengkou mountain chicken needs to maintain its excellent foot structure (adapted to the mountain environment) through marker-assisted selection. To improve breeding efficiency, it is necessary to develop SNP markers directly associated with foot weight (FW) and foot index (FI=foot weight / body weight).

[0028] By identifying SNP molecular markers significantly associated with foot weight (FW) and foot index (FI) traits in Chengkou mountain chickens, this study addresses core issues in traditional poultry breeding, including long selection cycles, inaccurate phenotypic measurements, and significant environmental interference. This technology accurately predicts foot developmental potential through genotyping at the chick stage, significantly shortening the selection cycle and improving selection accuracy. It also helps improve the adaptability of mountain chickens and optimize broiler processing efficiency, providing a highly effective tool for molecular marker-assisted breeding.

[0029] 1. Experimental Population and Phenotypic Data Collection 502 healthy 120-day-old Chengkou mountain chicken roosters were selected as research subjects. The experimental chickens were raised at the Chengkou Mountain Chicken Genetic Resources Research Institute in Chengkou County, Chongqing. The experimental chickens were all raised in single cages, with free access to food and water, sufficient light, and basically the same feeding and management conditions. When the experimental chickens were fed to 120 days of age, they were slaughtered according to the slaughtering method, and the foot weight and foot index were measured. The measurement method refers to the agricultural industry standard of the People's Republic of China NY / T283-2020 "Terminology and Measurement Statistical Methods for Poultry Production Performance" 2. Phenotypic Data Collation and Analysis Statistical analysis of traits Note: Abb.: abbreviation; Min: minimum value; Max: maximum value; Mean: mean; SD: standard deviation; SE: standard error; Var: variance; CV: coefficient of variation.

[0030] Phenotypic data were collected and processed using Microsoft Excel 2016MSO (v2411 Build 16.0.18227.20082). IBM SPSS Statistics 27, R (v4.4.2), and RStudio (v2024.09.1) software were used to filter the data, remove extreme values ​​and outliers, and calculate the descriptive statistics of each trait, including minimum (Min), maximum (Max), mean (Mean), standard deviation (SD), standard error (SE), variance (Var), and coefficient of variation (CV). Correlation analysis was also performed for each trait.

[0031] 3. DNA Extraction and Sequencing Before slaughter, 2 mL of blood was collected from the wing vein of the experimental chicken using an anticoagulant tube containing EDTA anticoagulant. After blood collection, the anticoagulant tube was shaken up and down to fully mix the blood and anticoagulant. The blood was then transported to the laboratory with the help of ice packs and stored at −20°C until use.

[0032] After DNA was extracted from the sample tissue using the magnetic bead method, the concentration of the DNA sample was detected using a Qubit fluorescence quantifier, and the integrity of the DNA sample was detected by 1% agarose gel electrophoresis. Samples that passed quality control were used for library preparation.

[0033] DNA samples were fragmented by enzyme digestion, and sequencing adapters were ligated to the fragmented DNA via PCR. The ligation products were purified using magnetic beads. The concentration of the purified products was measured using a Qubit fluorescence quantifier, and samples with qualified concentrations were used for subsequent reactions.

[0034] The linear library is denatured into single strands and then circularized. After digesting the uncircularized linear DNA molecules, a single-stranded circular library is obtained. The concentration of the single-stranded circular library is measured using a Qubit fluorescence quantification instrument. If the concentration is within the acceptable range, the subsequent reaction is carried out.

[0035] The single-stranded circular DNA molecule replicates through rolling circles, forming a DNA nanoball (DNB) containing over 300 copies. DNB concentration is measured using a Qubit fluorometer, and subsequent reactions proceed only if the concentration meets the criteria.

[0036] All individuals underwent low-depth sequencing, and data quality was assessed using FastQC (v0.12.1) software. Low-quality data were filtered using Trimmomatic (v0.36) software, with a 4-base window and an average base quality of no less than 15. Sequencing data were aligned to the reference genome (Gallus6) using BWA (v0.7.17) on an FPGA-accelerated GTX-one computing platform.

[0037] BaseVar uses algorithms based on maximum likelihood and multiple likelihood ratio tests to identify polymorphic SNPs and their population frequencies from low-depth population data. The algorithm detects bi-allelic, tri-allelic, and tetra-allelic SNPs. The filtering criteria for BaseVar (v0.0.1.3) are: DP > 1.5 IQR, EAF > 0.01.

[0038] Using the STITCH method, the final variant set can be obtained according to the conditions of minimum allele frequency MAF>0.01, typing score INFO_SCORE>0.4, and callrate>0.95, thereby obtaining the genotyping results.

[0039] Twenty individuals underwent 20× deep sequencing, and data quality was assessed using FastQC (v0.12.1). The reference genome was indexed using Samtools (v1.17), BWA (v0.7.17), and Picard (v2.18.29) on a FPGA-accelerated GTX-one computing platform. Sequencing data were aligned to the reference genome (Gallus6) using BWA (v0.7.17). After alignment, the bam files were sorted and indexed using Samtools (v1.17). Variant detection and quality control were performed.

[0040] GATK (v4.2.2.0) software was used for SNP detection to obtain a gvcf file for each individual. Joint calling was then performed on all samples to obtain a chromosome-specific vcf file.

[0041] The obtained vcf was hard filtered based on FS > 30 and QD < 2. Further quality control was performed using vcftools, and the final genotyping results were obtained based on the minor allele frequency (MAF) > 0.01.

[0042] GWAS analysis was performed using GEMMA software, and the model was LMM (mixed linear model). The Wald test statistic was used to evaluate the significance of the association between SNPs and phenotypes, and the Bonferroni correction was used to adjust the association analysis results. P = 0.05 / N or 1 / N to calculate significance and potential association thresholds for genome-wide analysis, where P Bonferroni-corrected p-values ​​indicating significant or potential associations across the genome P value, and N represents the number of independent SNPs obtained from the population structure analysis.

[0043] In the production and breeding of Chengkou mountain chickens, individual selection is carried out on the tested population according to the results of genotyping, and Chengkou mountain chickens with a genotype with a low frequency of genes at negative effect sites related to foot weight and foot index, or Chengkou mountain chickens with a high frequency of genes at positive effect sites are selected. That is, the frequency of genes at negative effect sites is gradually reduced, and the frequency of genes at positive effect sites is increased. For example, the gene frequency of the effect allele G at the effect site 4_74671312 is reduced, and the gene frequency of the effect allele G at the effect site 4_74671339 is increased, so as to improve the foot weight and foot index of Chengkou mountain chickens.

[0044] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A SNP site associated with foot weight and foot index of mountain chicken, characterized in that: The SNP sites include the following: chr4_74671312, chr4_74671339, chr4_74671343, chr4_74671388, chr4_74671516, chr4_74671566 and chr4_74671617.

2. The SNP locus associated with foot weight and foot index of mountain chicken according to claim 1, characterized in that: chr4_74671312 is polymorphic to G or A.

3. The SNP site associated with foot weight and foot index of mountain chicken according to claim 1, characterized in that: chr4_74671339 is T or G polymorphic.

4. The SNP locus associated with foot weight and foot index of mountain chicken according to claim 1, characterized in that: chr4_74671343 is polymorphic with C or T.

5. The SNP site associated with foot weight and foot index of mountain chicken according to claim 1, characterized in that: chr4_74671388 is polymorphic to G or A.

6. The SNP locus associated with foot weight and foot index of mountain chicken according to claim 1, characterized in that: chr4_74671516 is polymorphic to G or C.

7. The SNP locus associated with foot weight and foot index of mountain chicken according to claim 1, characterized in that: chr4_74671566 is polymorphic to T or C.

8. The SNP locus associated with foot weight and foot index of mountain chicken according to claim 1, characterized in that: chr4_74671617 is polymorphic to G or A.

9. The SNP locus associated with foot weight and foot index of mountain chicken according to claim 1, characterized in that: The dominant genotypes of SNP 1 site AA, SNP 2 site GG, SNP 3 site TT, SNP 4 site AA, SNP 5 site CC, SNP 6 site TT and SNP 7 site AA were selected for foot weight and foot index shape.

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