SNP loci associated with growth performance of Qingyuan Silkie chicken and their application

Abstract

The present invention relates to the field of animal breeding technology, and in particular to SNP sites related to the growth performance of Qingyuan Silkie chickens and their applications. The present invention uses whole-genome association analysis to screen the growth performance of Qingyuan Silkie chickens, and obtains SNP molecular markers significantly correlated with growth performance, including at least one of 8 SNP sites, which can be applied to molecular marker-assisted breeding of Qingyuan Silkie chickens. The breeding method disclosed in the present invention performs early individual selection at the genomic level, avoids dependence on phenotypic information, significantly improves selection efficiency, accelerates the breeding process, and reduces feeding costs, which has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of animal breeding, and in particular to SNP sites related to the growth performance of Qingyuan Silkie chickens and applications thereof. Background Art

[0002] Growth performance is one of the key traits used to measure the profitability of Qingyuan Silkie chicken farming. Indicators include weight per week, daily weight gain, and daily feed intake. These traits are directly related to the chicken's growth rate and production efficiency. Improving growth performance not only accelerates the production cycle and increases economic benefits, but also plays a positive role in resource utilization efficiency. Growth performance is a complex economic trait, and improving traits such as weight per week and daily weight gain through genetic selection has become a key area of industrial development. Focusing on developing molecular markers based on growth performance-related indicators for use in genetic selection is of great significance for improving the production efficiency of Qingyuan Silkie chickens and promoting industrial upgrading.

[0003] Traditional breeding relies primarily on experience-based artificial selection of phenotypes. This is time-consuming and lacks in-depth genomic information for long-term, high-precision selection. With the continuous development of high-throughput sequencing technology and genomic association study methods, genome-wide association studies (GWAS) have become a new method for efficiently mining genetic variation associated with growth performance and discovering relevant candidate genes. This provides a theoretical basis for formulating molecular breeding programs for poultry growth performance, and combined with locus detection technology, genomic selection can be used to improve target traits. However, no studies using GWAS have been conducted to investigate genetic variation associated with growth performance in Qingyuan Silkie chickens and to discover relevant candidate genes. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide SNP sites related to the growth performance of Qingyuan Silkie chickens and applications thereof.

[0005] The object of the present invention is achieved by the following technical solution: The SNP sites related to the growth performance of Qingyuan Silkie chicken include at least one of the eight SNP sites:

[0006] The SNP site chr13_5736133 is located on chromosome 13, with a physical position of 5736133 and alleles of G or A;

[0007] The SNP site chr2_97297443 is located on chromosome 2, with a physical position of 97297443 and alleles of G or A;

[0008] The SNP site chr4_85114337 is located on chromosome 4, with a physical position of 85114337 and alleles of T or A;

[0009] The SNP site chr1_169294981 is located on chromosome 1, with a physical position of 169294981 and alleles of T or G;

[0010] The SNP site chr2_97224498 is located on chromosome 2, with a physical position of 97224498 and alleles of G or T;

[0011] The SNP site chr2_97064987 is located on chromosome 2, with a physical position of 97064987 and alleles of G or A;

[0012] The SNP site chr8_21652832 is located on chromosome 8, with a physical position of 21652832 and alleles of C or T;

[0013] The SNP site chr8_21652833 is located on chromosome 8, its physical position is 21652833, and its allele is G or A.

[0014] The primer pairs were used to detect the above-mentioned SNP sites related to the growth performance of Qingyuan Silkie chickens.

[0015] As the preferred technical solution,

[0016] The nucleotide sequences of the primer pair for chr13_5736133 are shown in SEQ ID NO. 1 and SEQ ID NO. 2;

[0017] The nucleotide sequences of the primer pair for chr2_97297443 are shown in SEQ ID NO. 3 and SEQ ID NO. 4;

[0018] The nucleotide sequences of the primer pair for chr4_85114337 are shown in SEQ ID NO. 5 and SEQ ID NO. 6;

[0019] The nucleotide sequences of the primer pair for chr1_169294981 are shown in SEQ ID NO. 7 and SEQ ID NO. 8;

[0020] The nucleotide sequences of the primer pair for chr2_97224498 are shown in SEQ ID NO. 9 and SEQ ID NO. 10;

[0021] The nucleotide sequences of the primer pair for chr2_97064987 are shown in SEQ ID NO. 11 and SEQ ID NO. 12;

[0022] The nucleotide sequences of the primer pair for chr8_21652832 are shown in SEQ ID NO. 13 and SEQ ID NO. 14;

[0023] The nucleotide sequences of the primer pair for chr8_21652833 are shown in SEQ ID NO.15 and SEQ ID NO.16.

[0024] Application of the above-mentioned SNP site or the above-mentioned primer pair in Qingyuan Silkie chicken breeding.

[0025] As a preferred technical solution, it includes the following steps:

[0026] S1. Extract genomic DNA from the Qingyuan Ma chicken.

[0027] S2. The genomic DNA from step S1 was used as a template and amplified using the above primer pairs;

[0028] S3. Sequencing is performed based on the amplification results, and genotyping of the Qingyuan Ma chickens to be tested is performed to identify the genotype of the Qingyuan Ma chickens to be tested, and Qingyuan Ma chickens with genotypes having a high frequency of genes for positive effect loci related to average daily weight gain per week and / or a high frequency of genes for negative effect loci related to metabolic weight per week and / or a low frequency of genes for negative effect loci related to weight per week and average daily weight per week are selected.

[0029] As a preferred technical solution, the effector gene with low frequency of negative effect loci for weight per week and average daily gain per week is:

[0030] The effect allele of chr13_5736133 is G, the effect allele of chr2_97297443 is G, the effect allele of chr4_85114337 is T, the effect allele of chr2_97224498 is G, the effect allele of chr2_97064987 is G, and the effect allele of chr8_21652832 is C.

[0031] As a preferred technical solution, the effect gene with a high frequency of the negative effect site gene related to age-related metabolic weight is: the chr1_169294981 effect allele is T.

[0032] As a preferred technical solution, the effect gene with a high frequency of the positive effect locus gene for average daily gain per week is:

[0033] The effect allele of chr8_21652833 is G.

[0034] As the preferred technical solution, the Qingyuan Silkie chicken breed with good growth performance is cultivated.

[0035] The growth performance described in the present invention includes metabolic weight per week, body weight per week and average daily gain per week.

[0036] The present invention has the following advantages: It uses genome-wide association analysis to screen the growth performance of Qingyuan Silkie chickens, obtaining SNP molecular markers significantly associated with these traits, including at least one of eight SNP loci, which can be applied to molecular marker-assisted breeding of Qingyuan Silkie chickens. The breeding method disclosed in the present 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, thus promising promising applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a Manhattan plot of the genome-wide effect SNPs for the weight at 12 weeks of age, weight at 17 weeks of age, metabolic weight at 12 weeks of age, and average daily gain from 12 to 17 weeks of age of Qingyuan Silkie chickens in Example 1; wherein the horizontal axis represents the chromosome number; and the vertical axis represents the -logP value. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments. The scope of protection of the present invention is not limited to the following description.

[0039] The animals, instruments, reagents, and kits used in the following examples can all be purchased commercially.

[0040] Example 1:

[0041] 1. Collection of experimental population and phenotypic data:

[0042] A total of 633 healthy Qingyuan Ma roosters were selected and housed in individual cages at a breeding base according to the Production Technology Department's "Broiler Management Standards" and the "Poultry Production Technology Department Standards." After a two-week acclimatization period, the experiment began at 12 weeks of age and ended at 17 weeks. They were fasted for 12 hours before being weighed, and their body weight and feed intake were recorded regularly at weeks 12, 14, and 17. In the later stage, the average daily feed intake values for each period (average daily feed intake from 12 weeks to 14 weeks of age, average daily feed intake from 14 weeks to 17 weeks of age, average daily feed intake from 12 weeks to 17 weeks of age), average daily weight gain for each period (average daily weight gain from 12 weeks to 14 weeks of age, average daily weight gain from 14 weeks to 17 weeks of age, average daily weight gain from 12 weeks to 17 weeks of age) and mid-term metabolic weight for each period (metabolic weight at 12 weeks of age, metabolic weight at 14 weeks of age, metabolic weight at 17 weeks of age) were obtained by calculation. The mid-term metabolic weight was calculated as the 0.75th power of the body weight.

[0043] 2. Phenotypic data collation and analysis:

[0044] All phenotypic data collected in step 1 were statistically analyzed, including minimum, maximum, mean, standard deviation, and coefficient of variation (CV), and the results are shown in Table 1 ;

[0045] Table 1: Statistical analysis of growth performance phenotype data of Qingyuan Silkie chicken

[0046]

[0047] 3. DNA Extraction and Sequencing:

[0048] Blood was collected from the experimental chickens at 17 weeks of age, and genomic DNA was extracted. The blood was then sent to Shenzhen BGI Genomics Co., Ltd. for whole-genome resequencing. The sequencing platform was Illumina PE150, and the resequencing depth was above 10×. To ensure data quality, preliminary quality control of the FASTQ files was performed, and paired-end sequencing files were merged using fastp v0.23.4.

[0049] 4. Genomic data comparison and variation detection:

[0050] The sequencing data were aligned, analyzed, sorted, deduplicated, and converted to the correct format using the bioinformatics analysis software BWA v0.7.17 and SAMtools v1.18. The reference genome was the seventh edition of the chicken reference genome (bGalGal1.mat.broiler.GRCg7b). Subsequently, GATK v4.4 was used for variant detection and merging to generate population genotype VCF files. Finally, Beagle v5.4 was used to impute genotypes to address missing genotypes.

[0051] 5. SNP quality control and filtration:

[0052] Population VCF files were converted to binary format using Plink v1.90b5.3. Filtering parameters were set to retain SNPs with a missing genotype ratio of the minor allele less than 0.1, a minor allele frequency greater than 0.05, and a Hardy-Weinberg equilibrium test P value greater than 0.000001. After screening, a total of 11,442,036 SNPs from 663 chickens were retained for subsequent genome-wide association analysis.

[0053] 6. Genome-wide association analysis:

[0054] GEMMA v0.98.1 was used to build a kinship matrix for the genome-wide association analysis using a mixed linear model in conjunction with phenotypic traits. The model was as follows:

[0055]

[0056] Y is the phenotypic vector, Xβ is the population structure effect, To test the marker effect, It is a polygenic effect. is the residual effect. K The kinship matrix inferred for the markers.

[0057] The results are as follows Figure 1 The figure shows the Manhattan plot obtained from the whole gene association analysis of growth performance related traits. Figure 1 It can be seen that: 1 significant molecular marker site was identified in the body weight at 12 weeks of age; 2 significant molecular marker sites were identified in the body weight at 17 weeks of age; 1 significant molecular marker site was identified in the metabolic body weight at 12 weeks of age; in addition, 4 significant molecular marker sites were identified in the average daily weight gain from 12 to 17 weeks of age.

[0058] 7. Screening and extraction of SNP molecular marker sites related to growth performance traits:

[0059] The Bonferroni method was used to determine the significant threshold line at the genome level. and the recommended threshold line is Finally, 8 SNP molecular marker sites were obtained, and the information of these sites is shown in Table 2.

[0060] Table 2: Information of 23 molecular marker sites

[0061]

[0062] In the table, R stands for A or G, Y stands for C or T / U, K stands for G or T / U, and W stands for A or T / U.

[0063] Example 2: Breeding of Qingyuan Silkie Chicken

[0064] S1 extract the genomic DNA of the tested Qingyuan Ma chicken; specific as in Step 3 of Example 1;

[0065] S2. Using the genomic DNA from step S1 as a template, perform amplification using the primer pairs described in Table 3. The total PCR reaction volume is 25 μL, including 1-2 μL of genomic DNA template (10-100 ng / μL), 2.5 μL of 10× PCR buffer, 2 μL of dNTP mix (2.5 mM each), 1 μL each of the upstream and downstream primers (10 μM), 0.25 μL of Taq DNA polymerase (5 U / μL), and make up to 25 μL with sterile deionized water.

[0066] Design of PCR amplification primers for each molecular marker site:

[0067] The DNA template sequence information was downloaded from the NCBI website, and PCR amplification primers were designed using Primer Premier 5. The primer information is shown in Table 3.

[0068] Table 3: Primer information of 23 molecular markers

[0069]

[0070] S3. Genotyping the Qingyuan Ma chicken to be tested to identify the genotype of the Qingyuan Ma chicken to be tested, and selecting Qingyuan Ma chicken with a genotype having a low frequency of the gene at the positive effect site and / or a high frequency of the gene at the negative effect site.

[0071] S31. Identification of molecular marker genotypes:

[0072] After PCR amplification, the obtained products were sent to a biological company for first-generation sequencing. The sequencing results of each site were matched to the sequence of the base fragments before and after the SNP molecular marker given in Table 2. The marked area in brackets is the mutation site area, so as to determine the genotype of each molecular marker site.

[0073] S32. Breeding:

[0074] In the production and breeding of Qingyuan Ma chickens, individual selection is conducted based on genotyping results within the tested population. Chickens with low genotypes associated with negative effect loci related to weight at age and average daily gain at age, or with high genotypes associated with positive effect loci, are selected. This approach involves gradually decreasing the frequency of negative effect loci and increasing the frequency of positive effect loci. For example, the frequency of the effect allele G at the negative effect locus chr13_5736133 is decreased, while the frequency of the effect allele G at the positive effect locus chr8_21652833 is increased. This improves the growth performance of Qingyuan Ma chickens. Furthermore, chickens with high genotypes associated with metabolic weight at age are selected. This approach involves gradually increasing the frequency of negative effect loci, such as the effect allele T at the negative effect locus chr1_169294981, to reduce metabolic weight. This improves the growth performance of future generations and accelerates breeding progress.

[0075] Specifically, the effect genes with low frequencies of negative effect loci related to weight per week and average daily gain per week are:

[0076] The effect allele of chr13_5736133 is G, the effect allele of chr2_97297443 is G, the effect allele of chr4_85114337 is T, the effect allele of chr2_97224498 is G, the effect allele of chr2_97064987 is G, and the effect allele of chr8_21652832 is C.

[0077] Effect genes with high frequency of positive effect loci for average daily gain per week:

[0078] The effect allele of chr8_21652833 is G.

[0079] Effect genes with high frequency of negative effect loci related to age-related metabolic weight:

[0080] The effect allele of chr1_169294981 is T.

[0081] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, and they are all covered by the scope of protection of the present invention.

Claims

1. Use of primer pairs for detecting SNP sites associated with growth performance of Qingyuan Ma chickens in weight breeding of Qingyuan Ma chickens, wherein the reference genome for the SNP sites is the seventh edition of the chicken reference genome, and the SNP sites include at least one of the following eight SNP sites: The SNP site chr13_5736133 is located on chromosome 13, has a physical position of 5736133, and has alleles of G or A. It is associated with the phenotype of body weight in Qingyuan Ma chickens at 12 weeks of age. The nucleotide sequences of the primer pair for chr13_5736133 are shown in SEQ ID NO. 1 and SEQ ID NO.

2. The SNP site chr2_97297443 is located on chromosome 2, with a physical position of 97297443 and an allele of G or A, and is associated with the phenotype of body weight in Qingyuan Ma chickens at 17 weeks of age. The nucleotide sequences of the primer pair for chr2_97297443 are shown in SEQ ID NO. 3 and SEQ ID NO.

4. The SNP site chr4_85114337 is located on chromosome 4, with a physical position of 85114337 and alleles of T or A, and is associated with the phenotype of body weight in Qingyuan Ma chickens at 17 weeks of age. The nucleotide sequences of the primer pair for chr4_85114337 are shown in SEQ ID NO. 5 and SEQ ID NO.

6. The SNP site chr1_169294981 is located on chromosome 1, with a physical position of 169294981 and an allele of T or G, and is associated with the metabolic weight phenotype of Qingyuan Ma chickens at 12 weeks of age. The nucleotide sequences of the primer pair for chr1_169294981 are shown in SEQ ID NO. 7 and SEQ ID NO.

8. The SNP site chr2_97224498 is located on chromosome 2, has a physical position of 97224498, and has either a G or T allele. It is associated with the average daily gain phenotype of Qingyuan Ma chickens aged 12-17 weeks. The nucleotide sequences of the primer pair for chr2_97224498 are shown in SEQ ID NO. 9 and SEQ ID NO.

10. The SNP site chr2_97064987 is located on chromosome 2, has a physical position of 97064987, and has either a G or A allele. It is associated with the average daily gain phenotype of Qingyuan Ma chickens between 12 and 17 weeks of age. The nucleotide sequences of the primer pair for chr2_97064987 are shown in SEQ ID NO. 11 and SEQ ID NO.

12. The SNP site chr8_21652832 is located on chromosome 8, has a physical position of 21652832, and has a C or T allele. It is associated with the average daily gain phenotype of Qingyuan Ma chickens aged 12-17 weeks. The nucleotide sequences of the primer pair for chr8_21652832 are shown in SEQ ID NO. 13 and SEQ ID NO.

14. The SNP site chr8_21652833 is located on chromosome 8, its physical position is 21652833, the allele is G or A, and is associated with the phenotype of average daily weight gain of Qingyuan Ma chickens at 12-17 weeks of age; the nucleotide sequence of the primer pair for chr8_21652833 is shown in SEQ ID NO.15 and SEQ ID NO.

16.

2. The use according to claim 1, characterized in that It includes the following steps: S1. Extract genomic DNA from the Qingyuan Ma chicken. S2. Using the genomic DNA from step S1 as a template, amplify using primer pairs; S3. Perform sequencing based on the amplification results and perform genotyping on the Qingyuan Silkie chickens to be tested, thereby identifying the genotype of the Qingyuan Silkie chickens to be tested, and select Qingyuan Silkie chickens with a genotype having a high frequency of genes with positive effect loci related to average daily gain per week, a high frequency of genes with negative effect loci related to metabolic weight per week, or a low frequency of genes with negative effect loci related to weight per week and average daily gain per week.

3. The use according to claim 2, characterized in that The effect genes with low frequencies of the negative effect loci for weight per week are: chr13_5736133 effect allele is G, chr2_97297443 effect allele is G, and chr4_85114337 effect allele is T; the effect genes with low frequencies of the negative effect loci for average daily gain per week are: chr2_97224498 effect allele is G, chr2_97064987 effect allele is G, and chr8_21652832 effect allele is C.

4. The use according to claim 2, characterized in that The effect gene with a high frequency of the negative effect site gene related to age-related metabolic weight is: the effect allele of chr1_169294981 is T.

5. The use according to claim 2, characterized in that The effect gene with a high frequency of the positive effect locus gene for average daily weight gain per week is: the effect allele of chr8_21652833 is G.

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