SNP (Single Nucleotide Polymorphism) molecular marker related to chicken qualified egg rate as well as primer and application of SNP molecular marker

Abstract

The invention belongs to the technical field of biomolecular markers, and particularly relates to an SNP (Single Nucleotide Polymorphism) molecular marker related to a chicken qualified egg rate as well as a primer and application of the SNP molecular marker. The SNP molecular marker is located at the 10389047 G / A mutation site on the No.9 chromosome of a chicken 7.0 reference genome and is Chr9: 10389047, AA, GA and GG genotypes appear, the SNP molecular marker has the strongest genetic correlation with the qualified egg rate of 300 days old, A allele individuals correspond to higher qualified egg rate, the SNP molecular marker belongs to qualified egg rate dominant alleles, and the SNP molecular marker can be used for detecting the qualified egg rate of the chicken. By determining the genotype of the SNP, early selection and auxiliary selective breeding are carried out on the qualified egg rate of a chicken individual at the age of 300 days, and in breeding of yellow feather broilers, the individual can be directly bred at the genome level at the early stage by detecting the SNP marker, directionally selecting AA genotype breeding hens and reserving breeds and improving the genetic progress of selection of the qualified egg rate of 300-day-old eggs, so that the breeding efficiency of the yellow feather broilers is improved. The breeding efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological molecular markers, and in particular relates to a SNP molecular marker related to the qualified egg rate of chickens, a primer thereof and an application thereof. Background Art

[0002] The qualified egg rate of chickens is an important indicator for evaluating the reproductive performance of chickens, and its determination is also one of the key links in the production management and genetic improvement of breeder chickens. Yellow-feathered broilers, which are bred from local breeds in my country or from the combination of excellent local breeds in my country with commercial breeds with high production performance abroad, are my country's national chickens. Their annual output accounts for about 25%-30% of the total output of broilers in my country, and are an important part of my country's broiler industry. Compared with white-feathered broilers, yellow-feathered broilers have richer population genetic diversity and phenotypic diversity due to their shorter breeding time and lower artificial selection intensity, but they also have a larger population phenotypic coefficient of variation and lower production performance, and the qualified egg rate is one of them. The determination of the qualified egg rate trait of chickens usually requires statistics of all breeding eggs within 43 weeks of age (300 days of age) or 66 weeks of age, which is very time-consuming and labor-intensive. Therefore, it is very important to develop qualified egg rate molecular markers with significant genetic effects for yellow-feathered broilers for auxiliary selection, which is very important for accelerating the genetic improvement of the qualified egg rate trait of yellow-feathered broilers in my country and enhancing the overall competitiveness of the seed industry of yellow-feathered broilers.

[0003] In the prior art, patent CN202011539023.1 discloses a molecular marker method for detecting the qualified rate of goose breeding eggs. SNP molecular markers are developed for the qualified egg rate of poultry as marker-assisted selection, but its application scenario is in the goose population, and it is disclosed that the qualified egg rate determination involved is the 35-60 week period of 209 Sichuan white geese. Genetically, the qualified egg rate is a quantitative trait controlled by micro-effect polygenes. At present, the qualified egg rate in the yellow-feathered broiler industry is mainly determined by the number of qualified eggs within 300 days of age or 66 weeks of age. The phenotypic determination cycle is very long, so it is easily affected by environmental factors. The use of molecular markers to assist in the selection of qualified egg rates can effectively improve the genetic selection progress of this trait. At present, there are many technologies related to the selection of other traits based on single or multiple (haplotype) SNPs sites as molecular markers in chickens, but molecular marker technologies with significant genetic correlation with qualified egg rates have not been reported. Summary of the invention

[0004] In view of the above problems, the object of the present invention is to provide a SNP molecular marker related to the qualified egg rate of chickens, a primer thereof and an application thereof.

[0005] The technical contents of the present invention are as follows:

[0006] The present invention provides a SNP molecular marker related to the qualified egg rate of chickens, wherein the SNP molecular marker is located at the 10389047th G / A mutation site on chromosome 9 of the chicken 7.0 reference genome, which is Chr9:10389047 (rs317959148);

[0007] The SNP molecular marker has a base mutation of G / A (the reverse complementary base is C / T), resulting in different genotypes of AA, GA and GG;

[0008] When the genotype of Chr9:10389047 is AA (the reverse complementary base is TT), the qualified egg rate of the chicken at 300 days of age is high;

[0009] When the genotype of Chr9:10389047 was GA (the reverse complementary base was CT), the qualified egg rate of the chicken at 300 days of age was moderate;

[0010] When the genotype of Chr9:10389047 is GG (the reverse complementary base is CC), the qualified egg rate of individual chickens at 300 days of age is low.

[0011] The present invention also provides a primer set for detecting the above-mentioned SNP molecular marker, the primer set comprising:

[0012] The upstream primer had the sequence F: 5′-GTGCTACGATGCCCCTAGAT-3′;

[0013] The downstream primer has the sequence R: 5'-GGCCTACTGCTTGGACTTGT-3'.

[0014] The present invention also provides a kit, which comprises the above primer set.

[0015] The present invention also provides the use of the SNP molecular marker, the primer set or the kit in early screening of qualified chicken eggs.

[0016] The present invention also provides an application of the above-mentioned SNP molecular marker, the above-mentioned primer set or the above-mentioned kit in selective breeding for improving the qualified egg rate of 300-day-old chickens. Through the detection of the SNP molecular marker, when the genotype of Chr9:10389047 is AA (the reverse complementary base is TT), the qualified egg rate of the 300-day-old chicken individual is high; when the genotype of Chr9:10389047 is GA (the reverse complementary base is CT), the qualified egg rate of the 300-day-old chicken individual is medium; when the genotype of Chr9:10389047 is GG (the reverse complementary base is CC), the qualified egg rate of the 300-day-old chicken individual is low; and directional selection of individuals with the genotype of Chr9:10389047 being AA (the reverse complementary base is TT) as breeding chickens is helpful to improve the genetic progress of the selection of the qualified egg rate of 300-day-old chickens.

[0017] The present invention provides a method for selective breeding of chickens with improved egg quality rate at 300 days of age, comprising the following steps:

[0018] 1) Using the DNA of the chicken to be tested as a template, PCR amplification is performed using the above primer set to obtain an amplified product;

[0019] The reaction system of the PCR amplification is: 500 ng of genomic DNA, 25 μL of 2X Pro Taq Master Mix (dyeplus), 1 μL of 0.2 μM upstream primer, 1 μL of 0.2 μM downstream primer, and enzyme-free sterile water is added to a total reaction system of 50 μL;

[0020] The reaction program of the PCR amplification is: 94°C for 30s; 98°C for 10s, 59°C for 30s, 72°C for 1min, 35 cycles; 72°C for 2min;

[0021] 2) performing Sanger sequencing on the amplified product to obtain the genotype of the SNP molecular marker;

[0022] 3) Determine the qualified egg rate of the 300-day-old chickens to be tested based on the genotype;

[0023] When the genotype of Chr9:10389047 is AA (the reverse complementary base is TT), the qualified egg rate of the chicken individual at 300 days of age is high;

[0024] When the genotype of Chr9:10389047 was GA (the reverse complementary base was CT), the qualified egg rate of the chicken at 300 days of age was moderate;

[0025] When the genotype of Chr9:10389047 is GG (the reverse complementary base is CC), the qualified egg rate of individual chickens at 300 days of age is low.

[0026] Therefore, directional selection of individuals with AA genotype of Chr9:10389047 as breeding hens will help improve the genetic progress of selecting qualified egg production rate of 300-day-old chickens.

[0027] The beneficial effects of the present invention are as follows:

[0028] The SNP molecular marker related to the qualified egg rate of chickens of the present invention is a SNP molecular marker located at Chr9:10389047 of the chicken 7.0 reference genome GRCg7b, which has a G / A base mutation and different genotypes of AA, GA and GG. The qualified egg rate at 300 days of age has the strongest genetic correlation, and its significance reaches 2.50678×10 -7 The genetic effect is very significant. In the phenotype of qualified egg rate at 300 days of age, the genotype AA group (99.81±0.62)> genotype GA group (99.68%±0.76)> genotype GG group (99.05±2.45). The A allele individuals correspond to a higher qualified egg rate, which is a dominant allele for qualified egg rate. By determining the genotype of this SNP, early selection and auxiliary selection breeding can be carried out for the qualified egg rate of chicken individuals at 300 days of age. In the breeding of yellow-feathered broilers, the genetic progress of the selection of qualified egg rate at 300 days of age can be improved by detecting this SNP marker and selecting AA genotype breeding chickens for seed retention. Individuals can be selected directly at the genome level in the early stage, breeding efficiency can be improved, breeding costs can be reduced, and the industrial competitiveness of yellow-feathered broilers can be effectively improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The whole genome association analysis was conducted on the qualified egg rate of 1496 pure Qingyuan Ma chicken hens at 300 days of age (the percentage of deformed, sandy shell, double yolk and small eggs was counted, and soft-shell eggs were not included);

[0030] Figure 2 is the PCR reverse primer to amplify the full length of the sequence;

[0031] Figure 3 Genotyping for locus rs317959148 (Chr9:10389047, GRCg7b). DETAILED DESCRIPTION

[0032] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0033] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0034] Example

[0035] A molecular marker related to the qualified egg rate of chickens and its application

[0036] 1. Mining of the marker-assisted selection site rs317959148 (Chr9:10389047, GRCg7b)

[0037] 1) A phenotypic determination of the qualified egg rate at 300 days of age was carried out on a group of 1501 pure Qingyuan Ma chicken hens (the proportion of deformed, sandy shelled, double yolked and small eggs was counted, and soft-shelled eggs were not included). At the same time, blood samples were collected from each individual for whole genome resequencing (average sequencing depth>10×);

[0038] After removing 5 individuals with individual site missing rates greater than 0.05, 1496 individual genome data were retained and VCFtools was used to perform population SNP quality control. The quality control conditions were: "--not-chrW--not-chrZ--min-alleles2--max-alleles2--maf0.05--max-missi ng0.95", and 11,807,487 high-quality autosomal SNPs were obtained;

[0039] Plink1.9 was used to perform LD-pruning on autosomal SNPs (parameter “--indep-pairwise5050.5”), and 2,568,220 low-linked autosomal SNPs were retained for genome-wide association analysis;

[0040] 2) Based on the high-quality autosomal SNPs after LD-pruning, a genome-wide association analysis was performed on the qualified egg rate at 300 days of age using a mixed linear model of GCTA (PC1-PC10 as quantitative covariates and batch as discrete covariate); 3.893747e-07 (Bonferroni correction; 1 / 2568220) was used as the significance threshold. The results are as follows: Figure 1 As shown, there are three SNPs that are significantly correlated with the qualified egg rate at 300 days of age; among them, the site rs317959148 (Chr9:10389047, GRCg7b) has the strongest genetic correlation with the qualified egg rate at 300 days of age, with a significance of 2.50678e-07, and the genetic effect is relatively significant.

[0041] Further analysis of the population genotype and phenotype of the site rs317959148 (Chr9:10389047, GRCg7b) revealed that the genetic effect of this site is a typical additive effect. The results are shown in Table 1, that is, in the phenotype of qualified egg rate at 300 days of age, the genotype AA group (99.81±0.62)> genotype GA group (99.68%±0.76)> genotype GG group (99.05±2.45), and the A allele individuals corresponded to a higher qualified egg rate, which was a dominant allele for qualified egg rate.

[0042] Table 1 Phenotype of qualified egg rate at 300 days of age corresponding to different genotypes of locus rs317959148 (Chr9:10389047, GRCg7b)

[0043]

[0044] Note: REF stands for reference allele and ALT stands for mutant allele.

[0045] 2. Application of molecular marker-assisted selection site rs317959148 (Chr9:10389047, GRCg7b)

[0046] 1) Extract blood samples from individual chickens to be tested, and extract genomic DNA using the phenol-chloroform method;

[0047] 2) Use NCBI Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to design upstream and downstream primers for site rs317959148 (Chr9:10389047, GRCg7b):

[0048] Upstream primer: F: 5′-GTGCTACGATGCCCCTAGAT-3′;

[0049] Downstream primer: R: 5′-GGCCTACTGCTTGGACTTGT-3′;

[0050] 3) The above primers were used to perform PCR amplification on the genomic DNA of the chickens to be tested. The PCR reagents, methods and reaction procedures were all selected from Aikerui Biotechnology Co., Ltd. (Changsha, China). The reaction system and reaction conditions are shown in Tables 2 and 3 below, respectively:

[0051] Table 2 PCR reaction system

[0052]

[0053]

[0054] *1: The reaction system needs to be prepared on ice, and then the prepared reaction solution is placed in a PCR instrument for reaction;

[0055] *2: When using 2X Pro Taq Master Mix (dye plus) for the first time, centrifuge it before use to avoid enzyme loss.

[0056] *3: Normally, the recommended template addition amount is no more than 500ng; the template dosage can be adjusted according to actual needs;

[0057] *4: Primers are usually used at a final concentration of 0.2 μM, which can be adjusted within the range of 0.2 to 1.0 μM based on experimental results.

[0058] Table 3 PCR reaction conditions

[0059]

[0060] After the PCR amplification reaction program was completed, the product was stored at 4°C for future use and sent to Shanghai Biotech Co., Ltd. for Sanger sequencing. The reverse primer was used for single sequencing. The results were as follows: Figure 2 As shown, the total length of the sequence amplified by the PCR reverse primer is 868 bp, of which the 604th base ( Figure 2 The shaded base Y; C / T mutation, i.e., G / A mutation at the positive strand site) is the site rs317959148 (Chr9: 10389047, GRCg7b)-SNP marker;

[0061] During Sanger sequencing, the red and blue peaks of CT (complementary genotype is GA) heterozygotes may overlap, making it difficult to judge by the color of the peaks, but the overall peak height is only half of the peaks of CC (complementary genotype is GG) or TT (complementary genotype is AA) homozygotes and their adjacent base homozygotes. Figure 3 As shown, when the individual Sanger sequencing peak graph is TT (complementary genotype is AA) (a), the individual's qualified egg rate at 300 days of age is the highest; when the sequencing peak graph is CT (complementary genotype is GA) (b), the individual's qualified egg rate at 300 days of age is medium; when the sequencing peak graph is CC (complementary genotype is GG) (c), the individual's qualified egg rate is the lowest; therefore, in the breeding of yellow-feathered broilers, the genetic progress of the selection of qualified egg rate at 300 days of age can be improved by detecting this SNP marker and by directing the selection of TT genotype breeder chickens for seed retention.

[0062] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A SNP molecular marker associated with the qualified egg rate of chickens, characterized in that: The SNP molecular marker is located at the 10389047th G / A mutation site on chromosome 9 of the chicken 7.0 reference genome, which is Chr9:10389047.

2. The SNP molecular marker associated with the qualified egg rate of chicken according to claim 1, characterized in that: The SNP molecular marker has a G / A base mutation, resulting in different genotypes of AA, GA and GG; When the genotype of Chr9:10389047 was AA, the qualified egg rate of chickens at 300 days of age was high; When the genotype of Chr9:10389047 was GA, the qualified egg rate of chickens at 300 days of age was moderate; When the genotype of Chr9:10389047 is GG, the qualified egg rate of individual chickens at 300 days of age is low.

3. A primer set for detecting the SNP molecular marker according to claim 1 or 2, characterized in that: The primer set comprises: The upstream primer had the sequence F: 5′-GTGCTACGATGCCCCTAGAT-3′; The downstream primer has the sequence R: 5'-GGCCTACTGCTTGGACTTGT-3'.

4. A kit, characterized in that: It comprises the primer set according to claim 3.

5. Use of the SNP molecular marker according to claim 1 or 2, the primer set according to claim 3 or the kit according to claim 4 in early screening of qualified chicken eggs.

6. Use of the SNP molecular marker according to claim 1 or 2, the primer set according to claim 3 or the kit according to claim 4 in selective breeding to increase the qualified egg rate of 300-day-old chickens, characterized in that: Through the detection of SNP molecular markers, when the genotype of Chr9:10389047 is AA, the qualified egg rate of the chicken individual at 300 days of age is high; when the genotype of Chr9:10389047 is GA, the qualified egg rate of the chicken individual at 300 days of age is medium; when the genotype of Chr9:10389047 is GG, the qualified egg rate of the chicken individual at 300 days of age is low.

7. The use according to claim 6, characterized in that: Through the detection of SNP molecular markers, individuals with the AA genotype of Chr9:10389047 were selected as breeding hens, which will help improve the genetic progress of selecting the qualified egg production rate of 300-day-old chickens.

8. A method for selective breeding of chickens with improved egg yield at 300 days of age, characterized in that: The steps include: 1) using the DNA of the chicken to be tested as a template, and performing PCR amplification using the primer set described in claim 3 to obtain an amplified product; 2) performing Sanger sequencing on the amplified product to obtain the genotype of the SNP molecular marker; 3) Determine the qualified egg rate of the 300-day-old chickens to be tested based on the genotype; When the genotype of Chr9:10389047 was AA, the qualified egg rate of chickens at 300 days of age was high; When the genotype of Chr9:10389047 was GA, the qualified egg rate of chickens at 300 days of age was moderate; When the genotype of Chr9:10389047 is GG, the qualified egg rate of individual chickens at 300 days of age is low.

9. The method for selective breeding for improving the qualified egg rate of 300-day-old chickens according to claim 8, characterized in that: The reaction system for PCR amplification is: 500 ng genomic DNA, 25 μL 2X Pro Taq Master Mix (dye plus), 1 μL 0.2 μM upstream primer, 1 μL 0.2 μM downstream primer, and enzyme-free sterile water is added to a total reaction system of 50 μL.

10. The method for selective breeding for improving the qualified egg rate of 300-day-old chickens according to claim 8, characterized in that: The reaction program of the PCR amplification is: 94°C for 30s; 98°C for 10s, 59°C for 30s, 72°C for 1min, 35 cycles; 72°C for 2min.

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