SNP site related to growth performance of Qingyuan partridge chicken and application thereof

Abstract

The invention relates to the technical field of animal breeding, in particular to SNP loci related to growth performance of Qingyuan partridge chickens and application of the SNP loci. The growth performance of the Qingyuan partridge chickens is screened through whole genome association analysis, the SNP molecular marker remarkably related to the growth performance is obtained, and the SNP molecular marker comprises at least one of eight SNP loci and can be applied to molecular marker-assisted breeding of the Qingyuan partridge chickens. According to the breeding method disclosed by the invention, 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 the breeding method has a good application prospect.

Description

Technical Field

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

[0002] Growth performance is one of the important traits to measure the benefits of Qingyuan Silkie chicken farming, and its indicators include body weight per week, daily weight gain, daily feed intake, etc. These traits are directly related to the growth rate and production efficiency of chickens. Improving growth performance can not only speed up the production cycle and improve economic benefits, but also play a positive role in resource utilization efficiency. Growth performance is a complex economic trait. How to improve traits such as body weight per week and daily weight gain through genetic breeding has become an important direction for industrial development. Focusing on the development of molecular markers based on growth performance-related indicators for use in genetic breeding of traits is of great significance to improving the production efficiency of Qingyuan Silkie chickens and promoting industrial upgrading.

[0003] Traditional breeding mainly relies on experience to artificially select phenotypes. Artificial breeding is time-consuming and does not go deep into genomic information for long-term, high-precision selection. With the continuous development of high-throughput sequencing technology and genome association research methods, genome-wide association analysis (GWAS) can be used as a new method to efficiently mine genetic variation related to growth performance and discover related candidate genes, providing a theoretical basis for the formulation of molecular breeding plans for poultry growth performance, and combining site detection technology to achieve genomic selection to improve target traits. After searching, no research has been conducted using GWAS to study genetic variation related to growth performance of Qingyuan Silkie chicken and discover related 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 Ma chicken and applications thereof.

[0005] The object of the present invention is achieved by the following technical scheme: The SNP loci related to the growth performance of Qingyuan Ma chicken include at least one of the 8 SNP loci: The SNP locus chr13_5736133 is located on chromosome 13, its physical position is 5736133, and its allele is G or A; The SNP site chr2_97297443 is located on chromosome 2, its physical position is 97297443, and its allele is G or A; The SNP site chr4_85114337 is located on chromosome 4, its physical position is 85114337, and its alleles are T or A; The SNP site chr1_169294981 is located on chromosome 1, its physical position is 169294981, and its allele is T or G; The SNP locus chr2_97224498 is located on chromosome 2, its physical position is 97224498, and its allele is G or T; The SNP locus chr2_97064987 is located on chromosome 2, its physical position is 97064987, and its allele is G or A; The SNP locus chr8_21652832 is located on chromosome 8, its physical position is 21652832, and its allele is C or T; The SNP site chr8_21652833 is located on chromosome 8, its physical position is 21652833, and its allele is G or A.

[0006] The primer pairs were used to detect the above-mentioned SNP sites related to the growth performance of Qingyuan Ma chicken.

[0007] As a preferred technical solution, The nucleotide sequences of the primer pair of chr13_5736133 are shown in SEQ ID NO.1 and SEQ ID NO.2; The nucleotide sequences of the primer pair of chr2_97297443 are shown in SEQ ID NO.3 and SEQ ID NO.4; The nucleotide sequences of the primer pair of chr4_85114337 are shown in SEQ ID NO.5 and SEQ ID NO.6; The nucleotide sequences of the primer pair of chr1_169294981 are shown in SEQ ID NO.7 and SEQ ID NO.8; The nucleotide sequences of the primer pair of chr2_97224498 are shown in SEQ ID NO.9 and SEQ ID NO.10; The nucleotide sequences of the primer pair of chr2_97064987 are shown in SEQ ID NO.11 and SEQ ID NO.12; The nucleotide sequences of the primer pair of chr8_21652832 are shown in SEQ ID NO.13 and SEQ ID NO.14; The nucleotide sequences of the primer pair of chr8_21652833 are shown in SEQ ID NO.15 and SEQ ID NO.16.

[0008] Application of the above-mentioned SNP site or the above-mentioned primer pair in Qingyuan Ma chicken breeding.

[0009] As a preferred technical solution, it includes the following steps: S1. Extracting genomic DNA of the Qingyuan Ma chicken to be tested; S2. Using the genomic DNA of step S1 as a template, amplification is performed using the above primer pair; S3. Sequencing is performed according to the amplification results, and genotyping is performed on the Qingyuan Ma chicken to be tested, so as to identify the genotype of the Qingyuan Ma chicken to be tested, and Qingyuan Ma chicken with a genotype with a high frequency of genes for the positive effect site related to the average daily weight per week and / or a high frequency of genes for the negative effect site related to the metabolic weight per week and / or a low frequency of genes for the negative effect site related to the body weight per week and the average daily weight per week is selected.

[0010] As a preferred technical solution, the effect gene with low frequency of negative effect loci for weight per week and average daily weight gain per week is: The chr13_5736133 effect allele is G, the chr2_97297443 effect allele is G, the chr4_85114337 effect allele is T, the chr2_97224498 effect allele is G, the chr2_97064987 effect allele is G, and the chr8_21652832 effect allele is C.

[0011] 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 effect allele of chr1_169294981 is T.

[0012] As a preferred technical solution, the effect gene with high frequency of the positive effect locus gene for average daily weight gain per week is: The effect allele of chr8_21652833 is G.

[0013] As the preferred technical solution, the Qingyuan Ma chicken variety with good growth performance is cultivated.

[0014] The growth performance described in the present invention includes metabolic weight per week, body weight per week and average daily weight gain per week.

[0015] The present invention has the following advantages: the present invention uses whole genome association analysis to screen the growth performance of Qingyuan Ma chicken, obtains SNP molecular markers significantly associated with the trait, including at least one of the 8 SNP sites, which can be applied to molecular marker-assisted breeding of Qingyuan Ma chicken. The breeding method disclosed in the present invention performs early individual breeding at the genome level, avoids dependence on phenotypic information, significantly improves selection efficiency, speeds up the breeding process, and reduces feeding costs, which has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1This is a Manhattan plot of the genome-wide effect SNPs of Qingyuan Ma chickens at 12 weeks of age, 17 weeks of age, metabolic weight at 12 weeks of age, and average daily weight gain from 12 to 17 weeks of age in Example 1; wherein the horizontal axis represents the chromosome number; and the vertical axis represents the -logP value. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with the accompanying drawings and embodiments, and the protection scope of the present invention is not limited to the following descriptions; The animals, instruments, reagents and kits used in the following examples can all be purchased from commercial sources.

[0018] Embodiment 1: 1. Collection of experimental population and phenotypic data: 633 healthy Qingyuan Ma roosters were selected and raised in individual cages at the breeding base according to the "Broiler Management Standards" and "Poultry Production Technology Department Standards" of the Production Technology Department. After two weeks of environmental adaptation, the experiment started at 12 weeks of age and ended at 17 weeks of age. The roosters were fasted for 12 hours before weighing, and the weight and feed intake of each individual were recorded regularly in the 12th, 14th and 17th weeks. In the later stage, the average daily feed intake of 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 of 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 of 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.

[0019] 2. Phenotypic data collation and analysis: 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 ; Table 1: Statistical analysis of growth performance phenotype data of Qingyuan Ma chicken

[0020] 3. DNA extraction and sequencing: Blood was collected from the experimental chickens at 17 weeks of age and genomic DNA was extracted. The blood was sent to Shenzhen BGI Co., Ltd. for whole genome resequencing. The sequencing platform was Illumina PE150, and the depth of resequencing was above 10×. To ensure data quality, preliminary quality control was performed on the FASTQ files, and fastp v0.23.4 was used to merge the paired-end sequencing files.

[0021] 4. Genome data comparison and variation detection: The genomic data obtained by sequencing were aligned, analyzed, sorted, deduplicated, and converted to different formats 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 to detect mutations and merge to obtain the population genotype VCF file. Finally, beagle v5.4 was used to infer the genotype to solve the problem of missing genotypes.

[0022] 5. SNP quality control and filtering: The population VCF files were converted to binary format using Plink v1.90b5.3, and filtering parameters were set to retain SNPs with a missing genotype rate of the smallest allele less than 0.1, a smallest 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.

[0023] 6. Genome-wide association analysis: 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 is as follows:

[0024] Y is the phenotypic vector, Xβ is the population structure effect, To test the marker effect, For polygenic effects, is the residual effect. K The kinship matrix inferred for the markers.

[0025] The results are as follows Figure 1 The figure shows the Manhattan plot of the whole gene association analysis for 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.

[0026] 7. Screening and extraction of SNP molecular marker sites related to growth performance traits: 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.

[0027] Table 2: Information of 23 molecular marker sites

[0028] 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.

[0029] Example 2: Breeding of Qingyuan Ma chicken S1. Extracting genomic DNA of Qingyuan Ma chicken to be tested; the same as step 3 in Example 1; S2. Using the genomic DNA from step S1 as a template, amplify using the primer pairs described in Table 3. The total volume of the PCR reaction system 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 mixture (2.5 mM each), 1 μL each of upstream primer and downstream primer (10 μM), 0.25 μL of Taq DNA polymerase (5 U / μL), and make up to 25 μL with sterile deionized water.

[0030] Design of PCR amplification primers for each molecular marker site: 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.

[0031] Table 3: Primer information of 23 molecular markers

[0032] S3. Perform genotyping on the Qingyuan Ma chicken to be tested, thereby identifying 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.

[0033] S31. Identification of molecular marker loci genotypes: 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.

[0034] S32. Breeding: In the production and breeding of Qingyuan Ma chicken, individual selection is performed on the tested population according to the genotyping results, and Qingyuan Ma chicken with a low genotype of the gene frequency of the negative effect locus related to the weight at week-age and the average daily weight gain at week-age, or Qingyuan Ma chicken with a high gene frequency of the positive effect locus is selected, that is, the gene frequency of the negative effect locus is gradually reduced, and the gene frequency of the positive effect locus is increased, such as reducing the gene frequency of the effect allele G on the negative effect locus chr13_5736133 and increasing the gene frequency of the effect allele G on the positive effect locus chr8_21652833, so as to improve the growth performance of Qingyuan Ma chicken; Qingyuan Ma chicken with a high genotype of the gene frequency of the negative effect locus related to the metabolic weight at week-age is selected, that is, gradually increasing the gene frequency of the negative effect locus, such as increasing the gene frequency of the effect allele T on the negative effect locus chr1_169294981, so as to reduce the metabolic weight of Qingyuan Ma chicken. Thereby achieving the improvement of the growth performance of the offspring flock and accelerating the breeding progress.

[0035] Specifically, the effect genes with low frequency of negative effect loci related to weight per week and average daily weight gain per week are: The chr13_5736133 effect allele is G, the chr2_97297443 effect allele is G, the chr4_85114337 effect allele is T, the chr2_97224498 effect allele is G, the chr2_97064987 effect allele is G, and the chr8_21652832 effect allele is C.

[0036] Effect genes with high frequency of positive effect loci for average daily weight gain per week: The effect allele of chr8_21652833 is G.

[0037] Effect genes with high frequency of negative effect loci for age-related metabolic weight: The effect allele of chr1_169294981 is T.

[0038] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims

1. The SNP loci associated with the growth performance of Qingyuan Ma chicken are characterized by: Includes at least one of the following 8 SNP loci: The SNP locus chr13_5736133 is located on chromosome 13, its physical position is 5736133, and its allele is G or A; The SNP locus chr2_97297443 is located on chromosome 2, its physical position is 97297443, and its allele is G or A; The SNP locus chr4_85114337 is located on chromosome 4, its physical position is 85114337, and its alleles are T or A; The SNP site chr1_169294981 is located on chromosome 1, its physical position is 169294981, and its allele is T or G; The SNP locus chr2_97224498 is located on chromosome 2, its physical position is 97224498, and its allele is G or T; The SNP locus chr2_97064987 is located on chromosome 2, its physical position is 97064987, and its allele is G or A; The SNP locus chr8_21652832 is located on chromosome 8, its physical position is 21652832, and its allele is C or T; The SNP site chr8_21652833 is located on chromosome 8, its physical position is 21652833, and its allele is G or A.

2. The detection primer pair is characterized in that: Detect the SNP loci related to the growth performance of Qingyuan Ma chicken as described in claim 1.

3. The detection primer pair according to claim 2, characterized in that: The nucleotide sequences of the primer pair of chr13_5736133 are shown in SEQ ID NO.1 and SEQ ID NO.2; The nucleotide sequences of the primer pair of chr2_97297443 are shown in SEQ ID NO.3 and SEQ ID NO.4; The nucleotide sequences of the primer pair of chr4_85114337 are shown in SEQ ID NO.5 and SEQ ID NO.6; The nucleotide sequences of the primer pair of chr1_169294981 are shown in SEQ ID NO.7 and SEQ ID NO.8; The nucleotide sequences of the primer pair of chr2_97224498 are shown in SEQ ID NO.9 and SEQ ID NO.10; The nucleotide sequences of the primer pair of chr2_97064987 are shown in SEQ ID NO.11 and SEQ ID NO.12; The nucleotide sequences of the primer pair of chr8_21652832 are shown in SEQ ID NO.13 and SEQ ID NO.14; The nucleotide sequences of the primer pair of chr8_21652833 are shown in SEQ ID NO.15 and SEQ ID NO.

16.

4. Use of the SNP site described in claim 1 or the primer pair described in claim 2 or 3 in breeding Qingyuan Ma chicken.

5. The use according to claim 4, characterized in that: It includes the following steps: S1. Extracting genomic DNA of the Qingyuan Ma chicken to be tested; S2. Using the genomic DNA of step S1 as a template, amplifying using the primer pair according to claim 3; S3. Sequencing is performed according to the amplification results, and genotyping is performed on the Qingyuan Ma chicken to be tested, so as to identify the genotype of the Qingyuan Ma chicken to be tested, and Qingyuan Ma chicken with a genotype with a high frequency of genes for the positive effect site related to the average daily weight per week and / or a high frequency of genes for the negative effect site related to the metabolic weight per week and / or a low frequency of genes for the negative effect site related to the body weight per week and the average daily weight per week is selected.

6. The use according to claim 5, characterized in that: The effect genes with low gene frequencies of the negative effect loci related to weekly body weight and weekly average daily weight gain are: chr13_5736133 effect allele is G, chr2_97297443 effect allele is G, chr4_85114337 effect allele is T, chr2_97224498 effect allele is G, chr2_97064987 effect allele is G, and chr8_21652832 effect allele is C.

7. The use according to claim 5, characterized in that: The effect gene with a high frequency of the negative effect locus gene related to age-related metabolic weight is: the effect allele of chr1_169294981 is T.

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

9. The use according to claim 4, characterized in that: Cultivate the Qingyuan Ma chicken breed with good growth performance.

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