SNP (Single Nucleotide Polymorphism) molecular marker related to crown tooth number character of broiler chicken 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 the crown tooth number character of broiler chicken as well as a primer and application of the SNP molecular marker. Through phenotypic determination and whole genome re-sequencing related to crown tooth number characters of broiler individuals and whole genome association analysis, the excavated Chr7: 391959 locus and the crown tooth number have extremely high genetic correlation, the significance reaches 2.90 * 10 <-7 >, phenotypic variation variances capable of being explained reach 1.75%, the genetic effect is very remarkable, and the method has the advantages that the method is simple and convenient to operate, and the method is suitable for popularization and application. Therefore, the molecular marker can be effectively used for molecular marker-assisted selective breeding of the crown tooth number character of the broiler chicken, individuals with the molecular marker Chr7391959 site as the TT genotype are selected as breeding hens, the genetic selection process of the crown tooth number character of the yellow feather broiler chicken can be accelerated, the individuals are directly bred on the genome level in the early stage, the breeding efficiency is improved, and the breeding cost is reduced. The industrial competitiveness of the yellow feather broilers is effectively 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 trait of the number of denticles in broiler chickens, a primer thereof and an application thereof. Background Art

[0002] China is a major producer and consumer of poultry meat, and the national chicken is the Chinese chicken, which has the advantages of delicious meat and strong resistance to adversity. However, in recent years, a large number of live poultry markets have been closed due to frequent poultry diseases, posing a challenge to yellow-feathered broilers, which are sold as live poultry and rely on coat color as the main identification feature. After the chicken is put on the market as chilled chicken, its original identification mark will no longer exist. In this transitional context, the number of comb teeth, as a trait with significant phenotypic variation, has become an important appearance feature and genetic marker for yellow-feathered broilers in the process of going from live poultry to fresh market.

[0003] The number of comb teeth is not only the most significant and important external sign of the secondary sexual characteristics of chickens, but also an effective heat dissipation organ and an important reference for measuring sexual maturity. It is closely related to reproductive traits and production performance. For example, in Xianghuang chickens, the group with a large number of comb teeth starts laying eggs earlier than the group with a small number of comb teeth; in Qingyuan Ma chickens, the number of large comb teeth is significantly positively correlated with the early growth weight of chickens. In addition, there is a high correlation between the number of comb teeth in the parent generation and the number of comb teeth in the offspring (correlation coefficient 0.98), indicating that the comb tooth number trait has potential value in auxiliary breeding for precocious puberty and early growth weight in poultry.

[0004] At present, the technology of selecting growth traits based on single or multiple SNPs (single nucleotide polymorphism) sites as molecular markers has been widely used in poultry breeding, significantly improving breeding efficiency and shortening the breeding cycle. However, although these technologies are relatively mature, there is still a lack of genome-wide association analysis studies on the crown tooth number trait of yellow-feathered broiler chickens, and the significance level of most sites is low.

[0005] Therefore, the development of crown tooth number trait-related molecular markers with significant genetic effects is of great significance for achieving molecular marker-assisted selection of related traits such as precocious puberty and early growth weight. This will help accelerate the genetic selection process of the crown tooth number trait of yellow-feathered broiler chickens, improve the production application value, and effectively enhance the competitiveness of the yellow-feathered broiler industry. Summary of the invention

[0006] In view of the above problems, the object of the present invention is to provide a SNP molecular marker related to the trait of the number of dentitions in broiler chickens, and primers and applications thereof.

[0007] The technical contents of the present invention are as follows: The present invention provides a SNP molecular marker associated with the trait of the number of crown teeth in broiler chickens, wherein the SNP molecular marker includes a T / C mutation site at position 391959 of GRCg7b on chromosome 7 of the chicken 7.0 reference genome, which is Chr7_391959; The SNP site has a T / C base mutation, resulting in different genotypes of TT, TC and CC; When the genotype of Chr7_391959 is TT, the number of comb teeth is large; When the genotype of Chr7_391959 was TC, the number of teeth in the comb was medium; When the genotype of Chr7_391959 is CC, the number of comb teeth is small.

[0008] The present invention also provides a primer set for detecting the above-mentioned SNP molecular marker, the primer set comprising: The upstream primer had the sequence F: 5′-AGCCCGCATAGTCAACAATCT-3′; The downstream primer sequence is R: 5'-AGATTCGAACACCGTGCAAC-3'.

[0009] The present invention also provides a kit, which comprises the above primer set.

[0010] The present invention also provides the use of the SNP molecular marker, the primer set or the kit in early screening of the trait of the number of comb teeth in broilers.

[0011] 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 assisted selection breeding of broiler comb teeth number traits. Through the detection of the SNP molecular marker, when the genotype of Chr7_391959 is TT, the number of comb teeth is large; when the genotype of Chr7_391959 is TC, the number of comb teeth is medium; when the genotype of Chr7_391959 is CC, the number of comb teeth is small; and individuals with the genotype of Chr7_391959 being TT are directional selected as breeding chickens, which is conducive to the genetic process of broiler comb teeth number selection.

[0012] The present invention provides a method for assisted selection breeding of broiler comb tooth number traits, comprising the following steps: 1) Using the DNA of the chicken to be tested as a template, perform PCR amplification using the above primer set to obtain an amplified product; 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 the total reaction system of 50 μL; The reaction program of the PCR amplification was: 94°C for 30s; 98°C for 10s, 60°C for 30s, 72°C for 1min, 35 cycles; 72°C for 2min; 2) performing Sanger sequencing on the amplified product to obtain the genotype of the SNP molecular marker; 3) Determine the crown tooth number trait of the broiler to be tested based on the genotype; When the genotype of Chr7_391959 is TT, the number of comb teeth is large; When the genotype of Chr7_391959 was TC, the number of teeth in the comb was medium; When the genotype of Chr7_391959 was CC, the number of comb teeth was small; Directed selection of individuals with the genotype of Chr7_391959 as TT as breeder chickens will contribute to the genetic process of selection for the number of crown teeth in broiler chickens.

[0013] The beneficial effects of the present invention are as follows: The SNP molecular marker related to the crown tooth number trait of broiler chickens of the present invention is a SNP site located at Chr7_391959 of the chicken 7.0 reference genome GRCg7b, which has a T / C base mutation, and has different genotypes of TT, TC and CC, and has a very high genetic correlation with the crown tooth number, and its significance reaches 2.90×10 -7 , and the explained phenotypic variation variance reached 1.75% respectively. The genetic effect is very significant. Therefore, it can be used to accurately identify the crown number trait of broiler chickens. By determining the genotype of this SNP, early selection and assisted selection breeding for the crown number trait of broiler chickens can be carried out, and individuals with the TT genotype at the molecular marker Chr7_391959 locus are selected as breeding chickens. This can accelerate the genetic selection process of the crown number trait of yellow-feathered broilers, directly select individuals at the genome level in the early stage, improve breeding efficiency, reduce breeding costs, and effectively enhance the industrial competitiveness of yellow-feathered broilers. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a genome-wide association analysis of the crown tooth number of 1496 pure Qingyuan Ma chicken hens at 500 days of age; Figure 2 It is the genotyping diagram of the molecular marker site Chr7:391959 (where Y represents T / C mutation); Figure 3 This is a Kruskal-Wallis test analysis of the crown tooth number and three genotyping of Chr7:391959 loci in a group of 1496 purebred Qingyuan Ma chicken hens at 500 days of age. DETAILED DESCRIPTION

[0015] 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.

[0016] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0017] Example 1 Location of SNP molecular marker locus Chr7:391959 (GRCg7b) associated with the number of denticles in broiler chickens 1) A total of 1501 purebred Qingyuan Ma chicken hens were selected for phenotypic determination of crown tooth number at 500 days of age. Blood samples were collected from each individual for whole genome resequencing (average sequencing depth>10×). After removing 5 individuals with individual locus deletion rate greater than 0.05, 1496 individual genome data were retained and population SNP quality control was performed using VCFtools. The quality control conditions were: "--not-chr W --not-chr Z --min-alleles 2 --max-alleles 2 --maf 0.05 --max-missing 0.95", and 11807487 high-quality autosomal SNPs were obtained. Plink 1.9 was used to perform LD-pruning on autosomal SNPs (parameter "--indep-pairwise 50 5 0.5"), and 2568220 low-linked autosomal SNPs were retained for whole genome association analysis. 2) Based on the high-quality autosomal SNPs after LD-pruning, a genome-wide association analysis was performed on the number of crown teeth at 500 days of age using a mixed linear model of GCTA (PC1-PC10 as quantitative covariates), and the phenotypic variance explained (PVE) of each SNP was estimated. The PVE was 3.8937×10 -7 (Bonferroni correction; 1 / 2568220) is the genome-wide potential significance threshold.

[0018] The results are as follows Figure 1As shown in the figure, the upper figure is a QQ plot (Quantile-quantile plot), which represents the degree of fit between the actual observed value and the expected value; the lower figure is a Manhattan plot, which represents the correlation between all SNP sites on the autosomes of the whole genome (chromosomes 1-39) and the number of crown teeth at 500 days of age, where the blue dotted line is the potential significance threshold of the whole genome (-log10 (1 / 2568220) = 6.4096), and the significance reaches 2.90×10 -7 The variance of phenotypic variation that can be explained reached 1.75%. The genetic effect of this site is a typical additive effect. The site is located in the ORMDL1 gene region (Chr7:391676-392764; GRCg7b).

[0019] Example 2 Design and synthesis of primers for detecting SNP molecular markers associated with the number of denticles in broiler chickens NCBI Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) was used to design upstream and downstream primers for amplification of site Chr7:391959 (GRCg7b), as follows: The upstream primer sequences were: F: 5′-AGCCCGCATAGTCAACAATCT-3′; The downstream primer sequences were: R: 5′-AGATTCGAACACCGTGCAAC-3′; The upstream primers and downstream primers were commissioned to be synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0020] Example 3 Application of SNP molecular markers associated with the number of broiler comb teeth trait in assisted selection breeding to improve the number of broiler comb teeth trait The method of assisted selection breeding includes the following: 1) Extract blood samples from individual Qingyuan Ma chickens to be tested, and extract genomic DNA using the phenol-chloroform method; 2) 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 Acre Biotechnology Co., Ltd. (Changsha, China), and the reaction system and reaction conditions are shown in Tables 2 and 3 below, respectively.

[0021] Table 2 PCR reaction system *1

[0022] *1: The reaction system needs to be prepared on ice, and then the prepared reaction solution is placed in a PCR instrument for reaction; *2: When using 2X Pro Taq Master Mix (dye plus) for the first time, centrifuge it before use to avoid enzyme loss. *3: Normally, the recommended template addition amount is no more than 500 ng; the template amount can be adjusted according to actual needs; *4: Primers are usually used at a final concentration of 0.2μM, which can be adjusted within the range of 0.2~1.0μM based on experimental results.

[0023] Table 3 PCR reaction conditions

[0024] 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. Figure 2 As shown, the 1001th base (shaded base Y; T / C mutation) is the site Chr7:391959 (GRCg7b)-SNP marker; the genotyping of the site Chr7:391959 (GRCg7b) is as follows Figure 2 As shown, during Sanger sequencing, the red and blue peaks of the TC heterozygote may overlap, making it difficult to judge by the color of the peaks, but its overall peak height is only half of that of the TT or CC homozygote.

[0025] 3) The genotypes of the locus Chr7:391959 corresponding to the number of crown teeth of yellow-feathered broilers at 500 days of age were counted. The results are as follows Figure 3 As shown. When the individual Sanger sequencing peak was TT, the number of crown teeth of the individual at 500 days of age was 5.40±0.90, which was too many. When the individual Sanger sequencing peak was TC, the number of crown teeth of the individual at 500 days of age was 5.54±0.93, which was medium. When the individual Sanger sequencing peak was CC, the number of crown teeth of the individual at 500 days of age was 5.64±0.87, which was too few. The p value of the Kruskal-Wallis test between the three groups was 4.10×10 -3 , genotype TT group> genotype TC group> genotype CC group, T allele individuals correspond to more crown teeth, which is a dominant allele. Therefore, in the breeding of yellow-feathered broilers, the genetic process of crown teeth number selection at 500 days of age can be accelerated by detecting this SNP marker and selectively selecting TT genotype individuals as breeders, which can also indirectly improve the uniformity of the crown teeth number of yellow-feathered broilers at 500 days of age.

[0026] In summary, the present invention conducted phenotypic determination and whole genome resequencing (average sequencing depth of individuals>10×) on 1496 500-day-old purebred Qingyuan Ma chickens, and conducted whole genome association analysis. The Chr7:391959 locus (GRCg7b) mined was highly genetically correlated with the number of crown teeth at 500 days of age, with a significance of 2.90×10 -7 , and the explained phenotypic variation variance reached 1.75%, respectively. The genetic effect is very significant. Therefore, it can be effectively used in molecular marker-assisted selection breeding of the comb tooth number trait of broiler chickens. Selecting individuals with the TT genotype at the molecular marker Chr7_391959 locus as breeding chickens can accelerate the genetic selection process of the comb tooth number trait of yellow-feathered broilers, directly select individuals at the genome level in the early stage, improve breeding efficiency, reduce breeding costs, and effectively enhance the industrial competitiveness of yellow-feathered broilers.

[0027] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A SNP molecular marker associated with the number of broiler comb teeth, characterized in that: The SNP molecular marker includes a T / C mutation site at position 391959 of GRCg7b on chromosome 7 of the chicken 7.0 reference genome, which is Chr7_391959.

2. The SNP molecular marker associated with the trait of the number of broiler comb teeth according to claim 1, characterized in that: The SNP site has a T / C base mutation, resulting in different genotypes of TT, TC and CC; When the genotype of Chr7_391959 is TT, the number of comb teeth is large; When the genotype of Chr7_391959 was TC, the number of teeth in the comb was medium; When the genotype of Chr7_391959 is CC, the number of comb teeth is small.

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′-AGCCCGCATAGTCAACAATCT-3′; The downstream primer sequence is R: 5'-AGATTCGAACACCGTGCAAC-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 the comb tooth number trait of broiler chickens.

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 assisted selection breeding for the number of dentition traits in broilers, characterized in that: Through the detection of SNP molecular markers, when the genotype of Chr7_391959 is TT, the number of comb teeth is large; when the genotype of Chr7_391959 is TC, the number of comb teeth is medium; when the genotype of Chr7_391959 is CC, the number of comb teeth is small.

7. The use according to claim 6, characterized in that: Through the detection of SNP molecular markers, individuals with the genotype of Chr7_391959 as TT were selected as breeding chickens, which would contribute to the genetic process of the selection of the number of crown teeth in broiler chickens.

8. A method for assisted selection breeding of broiler comb teeth number traits, characterized in that: The steps include: 1) Using the DNA of the chicken to be tested as a template, performing PCR amplification using the primer set of 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 crown tooth number trait of the broiler to be tested based on the genotype; When the genotype of Chr7_391959 is TT, the number of comb teeth is large; When the genotype of Chr7_391959 was TC, the number of teeth in the comb was medium; When the genotype of Chr7_391959 was CC, the number of comb teeth was small; Selecting individuals with the genotype of Chr7_391959 as TT as breeders will contribute to the genetic process of selection for the number of crown teeth in broiler chickens.

9. The method for assisted selection breeding of broiler comb teeth number traits according to claim 8, characterized in that: The reaction system for the PCR amplification was: 500 ng of genomic DNA, 25 μL of 2X Pro Taq Master Mix (dye plus), 1 μL of 0.2 μM upstream primer, 1 μL of 0.2 μM downstream primer, and enzyme-free sterile water was added to a total reaction system of 50 μL.

10. The method for assisted selection breeding of broiler comb teeth number traits according to claim 8, characterized in that: The reaction program of the PCR amplification was: 94°C for 30s; 98°C for 10s, 60°C for 30s, 72°C for 1min, 35 cycles; 72°C for 2min.

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