A SNP molecular marker related to comb tooth number trait of broiler chickens, primer thereof and application thereof

Abstract

The application belongs to the technical field of biomolecular markers, and particularly relates to a SNP molecular marker related to the crest tooth number character of broiler chickens, a primer thereof and application. Through crest tooth number character related phenotype determination and whole genome resequencing of broiler chicken individuals, whole genome association analysis is carried out, the Chr7:391959 site mined has extremely high genetic correlation with the crest tooth number, the significance reaches 2.90*10 ‑7 , and the phenotype variance explained respectively reaches 1.75%, the genetic effect is very significant, so it can be effectively used for molecular marker assisted selection breeding of the crest tooth number character of broiler chickens, and individuals with the TT genotype of the molecular marker Chr7_391959 site are selected as breeding hens, so that the genetic selection process of the crest tooth number character of yellow feather broiler chickens can be accelerated, individuals are directly selected at the genome level in the early stage, breeding efficiency is improved, breeding cost is reduced, and the industrial competitiveness of yellow feather broiler chickens is effectively improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomolecular markers, and particularly relates to a SNP molecular marker related to the comb tooth number trait of broiler chickens, a primer thereof and application thereof. BACKGROUND

[0002] China is a major producer of poultry consumption and production. However, in recent years, due to the frequent occurrence of poultry diseases, a large number of live poultry markets have been closed, which poses a challenge to yellow-feathered broilers that are sold in the form of live poultry and rely on feather color as the main identification feature. After switching to the marketing of chilled chicken, the original identification mark will no longer exist. Under this background of transformation, the comb tooth number, as a trait with significant phenotypic variation, has become an important appearance feature and genetic marker for yellow-feathered broilers in the process of changing from live poultry to fresh chicken.

[0003] The comb tooth number is not only the most significant and important appearance marker in the second sex characteristic of chickens, but also an effective heat dissipating 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 population with large comb tooth number has an earlier age at first laying than the population with small comb tooth number; in Qingyuan chickens, large comb tooth number is significantly positively correlated with body weight in the early growth stage. In addition, there is a high correlation (correlation coefficient 0.98) between the comb tooth number of the parent generation and that of the offspring, indicating that the comb tooth number trait has potential value in the auxiliary breeding of sexual precocity and early growth body weight of poultry.

[0004] At present, techniques based on single or multiple SNPs (single nucleotide polymorphism) sites as molecular markers for the auxiliary selection of growth traits have been widely applied in poultry breeding, significantly improving breeding efficiency and shortening the breeding cycle. However, although these techniques are relatively mature, the whole-genome association analysis research on the comb tooth number trait of yellow-feathered broilers is still relatively scarce, and most of the sites have low significance levels.

[0005] Therefore, the development of comb tooth number trait-related molecular markers with significant genetic effects is of great significance for the molecular marker-assisted selection of related traits such as sexual precocity and early growth body weight. This will help to accelerate the genetic selection process of the comb tooth number trait of yellow-feathered broilers, improve the production application value, and effectively enhance the competitiveness of the yellow-feathered broiler industry. SUMMARY

[0006] In view of the above problems, the purpose of the present application is to provide a SNP molecular marker related to the comb tooth number trait of broiler chickens, a primer thereof and application thereof.

[0007] The technical content of the present application is as follows:

[0008] The application provides a SNP molecular marker related to a meat chicken comb tooth number character, the SNP molecular marker comprises a T / C mutation site at GRCg7b 391959 on a 7.0 reference genome of a chicken 7th chromosome, and is Chr7_391959.

[0009] The SNP site has a base mutation of T / C, and different genotypes of TT, TC and CC are generated.

[0010] When the genotype of Chr7_391959 is TT, the chicken comb tooth number is large.

[0011] When the genotype of Chr7_391959 is TC, the chicken comb tooth number is medium.

[0012] When the genotype of Chr7_391959 is CC, the chicken comb tooth number is small.

[0013] The application further provides a primer set for detecting the above SNP molecular marker, and the primer set comprises:

[0014] an upstream primer with the sequence of F: 5'-AGCCCGCATAGTCAACAATCT-3';

[0015] a downstream primer with the sequence of R: 5'-AGATTCGAACACCGTGCAAC-3'.

[0016] The application further provides a kit comprising the above primer set.

[0017] The application further provides application of the above SNP molecular marker, the above primer set or the above kit in early screening of a meat chicken comb tooth number character.

[0018] The application further provides application of the above SNP molecular marker, the above primer set or the above kit in meat chicken comb tooth number character assisted selection breeding, through detection of the SNP molecular marker, when the genotype of Chr7_391959 is TT, the chicken comb tooth number is large; when the genotype of Chr7_391959 is TC, the chicken comb tooth number is medium; when the genotype of Chr7_391959 is CC, the chicken comb tooth number is small; and individuals with the genotype of Chr7_391959 being TT are selected as breeding hens, which is helpful for genetic progress of meat chicken comb tooth number selection.

[0019] The application provides a method for meat chicken comb tooth number character assisted selection breeding, comprising the following steps:

[0020] 1) taking DNA of a chicken to be detected as a template, and performing PCR amplification by using the above primer set to obtain an amplification product;

[0021] The reaction system of the PCR amplification is: 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 adding enzyme-free sterile water to a total reaction system of 50 μL;

[0022] The reaction program of the PCR amplification is: 94℃ for 30s; 98℃ for 10s, 60℃ for 30s, 72℃ for 1min, 35 cycles; 72℃ for 2min;

[0023] 2) The amplification product is subjected to Sanger sequencing to obtain the genotype of the SNP molecular marker;

[0024] 3) The comb number trait of the to-be-detected broiler chicken is judged according to the genotype;

[0025] When the genotype of Chr7_391959 is TT, the comb number of the chicken is large;

[0026] When the genotype of Chr7_391959 is TC, the comb number of the chicken is medium;

[0027] When the genotype of Chr7_391959 is CC, the comb number of the chicken is small;

[0028] Directly selecting the individual with the genotype of TT of Chr7_391959 as a breeder is helpful for the genetic process of the comb number selection of the broiler chicken.

[0029] The beneficial effects of the present application are as follows:

[0030] The SNP molecular marker related to the comb number trait of the broiler chicken in the present application is a SNP site located at Chr7_391959 of the chicken 7.0 reference genome GRCg7b, which has a base mutation of T / C, appears different genotypes of TT, TC and CC, has extremely high genetic correlation with the comb number, and the significance reaches 2.90x10 -7 , and the explained phenotypic variance reaches 1.75%, the genetic effect is very significant, so it can be used to accurately judge the comb number trait of the broiler chicken, early selection and assisted selection breeding are carried out on the comb number trait of the broiler chicken by determining the genotype of the SNP, the individual with the genotype of TT of the SNP molecular marker Chr7_391959 is selected as a breeder, the genetic selection process of the comb number trait of the yellow broiler chicken is accelerated, the individual is directly selected at the genome level in the early stage, the breeding efficiency is improved, the breeding cost is reduced, and the industrial competitiveness of the yellow broiler chicken is effectively improved. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1Figure 1 is a genome-wide association analysis chart of the number of crown teeth at 500 days of age for a population of 1496 pure line Qingyuan Game hen females;

[0032] Figure 2 Figure 4 is a genotyping chart of the molecular marker site Chr7:391959 (where Y represents a T / C mutation);

[0033] Figure 3 Figure 5 is a Kruskal-Wallis test analysis chart of the number of crown teeth at 500 days of age for the 1496 pure line Qingyuan Game hen females and the three genotypes of the Chr7:391959 site. DETAILED DESCRIPTION

[0034] The application will be described in further detail below with specific implementation examples and accompanying drawings. It should be understood that these examples are only used to illustrate the application and are not used to limit the protection scope of the application. After reading the application, those skilled in the art can make various equivalent modifications to the application, which fall within the scope of the appended claims.

[0035] Unless otherwise specified, all raw materials and reagents of the application are conventional market raw materials and reagents.

[0036] Example 1

[0037] Positioning of the SNP molecular marker site Chr7:391959 (GRCg7b) related to the number of crown teeth in broilers

[0038] 1) Select a population of 1501 pure line Qingyuan Game hen females, carry out phenotypic determination of the number of crown teeth at 500 days of age, and collect blood samples from each individual to carry out whole genome resequencing (average sequencing depth >10x). After excluding 5 individuals with a site deletion rate greater than 0.05, 1496 individual genomic data were retained, and VCFtools was used for population SNP quality control with the following quality control conditions: “--not-chr W --not-chr Z --min-alleles 2 --max-alleles 2 --maf 0.05 --max-missing 0.95”, obtaining 11807487 high-quality autosomal SNPs; using Plink 1.9 to perform LD-pruning (parameter “--indep-pairwise 50 5 0.5”) on the autosomal SNPs, retaining 2568220 low-linkage autosomal SNPs for whole genome association analysis;

[0039] 2) Based on 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). Simultaneously, the proportion of phenotypic variance explained (PVE) for each SNP was estimated; the result was 3.8937 × 10⁻⁶. -7 (Bonferroni correction; 1 / 2568220) is the genome-wide potential significance threshold.

[0040] The results are as follows Figure 1 As shown, the top figure is a Quantile-Quantile plot, representing the degree of fit between the actual observed values ​​and the expected values; the bottom figure is a Manhattan plot, representing the correlation between all SNP loci on autosomes (chromosomes 1-39) and the number of crown teeth at 500 days of age. The blue dashed line represents the potential significance threshold for the whole genome (-log10(1 / 2568220) = 6.4096), and the significance reached 2.90 × 10⁻⁶. -7 The variance of phenotypic variation that can be explained reaches 1.75%. The genetic effect of this locus is a typical additive effect. This locus is located in the ORMDL1 gene region (Chr7:391676-392764; GRCg7b).

[0041] Example 2

[0042] Design and synthesis of primers for detecting SNP molecular markers associated with the number of comb teeth in broilers

[0043] The upstream and downstream primers for amplifying the Chr7:391959 (GRCg7b) site were designed using NCBI Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ), as follows:

[0044] The upstream primer sequence is: F: 5'-AGCCCGCATAGTCAACAATCT-3';

[0045] The downstream primer sequence is: R: 5'-AGATTCGAACACCGTGCAAC-3';

[0046] The upstream and downstream primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0047] Example 3

[0048] Application of SNP molecular markers associated with the number of comb teeth in broiler chickens in assisted selection breeding to improve the number of comb teeth in broilers

[0049] The method of assisted selection breeding comprises the following steps:

[0050] 1) Extract the blood sample of the individual to be tested of Qingyuan chicken, and extract the genomic DNA by phenol-chloroform method;

[0051] 2) Perform PCR amplification on the genomic DNA of the chicken to be tested by using the above primers; the PCR reagents, method and reaction program are selected from Aikewei Biological Engineering Co., Ltd. (Changsha, China), and the reaction system and reaction conditions are shown in Tables 2 and 3, respectively.

[0052] Table 2 PCR reaction system *1

[0053]

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

[0055] *2: When the product 2X Pro Taq Master Mix (dye plus) is used for the first time, it is centrifuged first and then used to avoid loss of enzyme amount;

[0056] *3: Generally, it is recommended that the template addition amount is not more than 500 ng; the template amount can be adjusted according to actual needs;

[0057] *4: The primer usually uses a final concentration of 0.2 μM, which can be adjusted within the range of 0.2-1.0 μM according to experimental results.

[0058] Table 3 PCR reaction conditions

[0059]

[0060] After the PCR amplification reaction program is completed, the product is stored at 4°C for standby, and is sent to Shengwo Biological Engineering (Shanghai) Co., Ltd. for Sanger sequencing. The PCR amplification sequence mutation site is shown in Figure 2 , wherein the 1001th base (shadow base Y; T / C mutation) is the SNP marker of site Chr7:391959 (GRCg7b); the genotyping of site Chr7:391959 (GRCg7b) is shown in Figure 2 , and the red and blue wave peaks of TC heterozygotes may overlap, and it is difficult to judge by wave peak color, but the overall wave peak height is only half of that of TT or CC homozygotes.

[0061] 3) The genotypes of site Chr7:391959 corresponding to the beak teeth number of yellow-feathered broilers at 500 days of age are counted respectively, and the results are shown in Figure 3When the individual Sanger sequencing peak chart is TT, the 500-day crown tooth number of the individual is 5.40±0.90, which is more. When the individual Sanger sequencing peak is TC, the 500-day crown tooth number of the individual is 5.54±0.93, which is moderate. When the individual Sanger sequencing peak is CC, the 500-day crown tooth number of the individual is 5.64±0.87, which is less. The p value of the Kruskal-Wallis test between the three groups is 4.10*10 -3 , the genotype TT population> genotype TC population> genotype CC population, the T allele individual corresponds to more crown tooth number, which belongs to the dominant allele. Therefore, in yellow-feathered broiler breeding, the TT genotype individual can be selected as a breeding chicken through detection of the SNP marker, so as to accelerate the genetic progress of the 500-day crown tooth number selection, and also indirectly improve the population uniformity of the 500-day crown tooth number of the yellow-feathered broiler.

[0062] As can be seen from the above, the present application can be used for molecular marker assisted selection breeding of the broiler crown tooth number trait by 1496 500-day pure line Qingyuan chicken individuals to carry out phenotype determination and whole genome resequencing (the average sequencing depth of the individual is more than 10x) related to the crown tooth number trait, and carrying out whole genome association analysis. The Chr7:391959 site (GRCg7b) mined has a very high genetic correlation with the 500-day crown tooth number, and the significance reaches 2.90*10 -7 , the explained phenotypic variance reaches 1.75%, and the genetic effect is very significant, so it can be effectively used for molecular marker assisted selection breeding of the broiler crown tooth number trait. The individual with the TT genotype of the molecular marker Chr7_391959 site is selected as a breeding chicken, so as to accelerate the genetic selection process of the yellow-feathered broiler crown tooth number trait, directly breed individuals at the genome level in the early stage, improve the breeding efficiency, reduce the breeding cost, and effectively improve the industrial competitiveness of the yellow-feathered broiler.

[0063] The technical scheme of the present application is not limited to the above specific embodiments, and any technical modification made according to the technical scheme of the present application falls within the protection scope of the present application.

Claims

1. The application of SNP molecular markers, primer sets for detecting SNP molecular markers, or kits for detecting SNP molecular markers in the early screening of broiler chickens for the comb tooth number trait, characterized in that, The SNP molecular marker is the T / C mutation site at position 391959 of GRCg7b on chromosome 7 of the chicken 7.0 reference genome, namely Chr7_391959; The primer set includes: The upstream primer has the sequence F: 5'-AGCCCGCATAGTCAACAATCT-3'; The downstream primer has the sequence R: 5'-AGATTCGAACACCGTGCAAC-3'; The kit contains the primer set; The broiler chickens mentioned are Qingyuan Ma chickens.

2. The application of SNP molecular markers, primer sets for detecting SNP molecular markers, or kits for detecting SNP molecular markers in broiler chicken comb tooth number trait-assisted selection breeding, characterized in that... The detection of SNP molecular markers showed that when the genotype of Chr7_391959 is TT, the number of comb teeth is high; when the genotype of Chr7_391959 is TC, the number of comb teeth is moderate; and when the genotype of Chr7_391959 is CC, the number of comb teeth is low. The SNP molecular marker is the T / C mutation site at position 391959 of GRCg7b on chromosome 7 of the chicken 7.0 reference genome, namely Chr7_391959; The primer set includes: The upstream primer has the sequence F: 5'-AGCCCGCATAGTCAACAATCT-3'; The downstream primer has the sequence R: 5'-AGATTCGAACACCGTGCAAC-3'; The kit contains the primer set; The broiler chickens mentioned are Qingyuan Ma chickens.

3. The application according to claim 2, characterized in that, By detecting SNP molecular markers, targeted selection of individuals with the TT genotype of Chr7_391959 as breeding chickens can help the genetic process of selecting the number of comb teeth in broilers.

4. A method for assisted selection breeding of broiler chickens based on the trait of comb tooth number, characterized in that, Includes the following steps: 1) Using the DNA of the chicken to be tested as a template, PCR amplification was performed using a primer set to obtain the amplification product; The primer set includes: The upstream primer has the sequence F: 5'-AGCCCGCATAGTCAACAATCT-3'; The downstream primer has the sequence R: 5'-AGATTCGAACACCGTGCAAC-3'; 2) Perform Sanger sequencing on the amplified products to obtain the genotypes of the SNP molecular markers; The SNP molecular marker is the T / C mutation site at position 391959 of GRCg7b on chromosome 7 of the chicken 7.0 reference genome, namely Chr7_391959; 3) Determine the number of crown teeth in the broiler chickens to be tested based on their genotype; When the genotype of Chr7_391959 is TT, the number of comb teeth is high; When the genotype of Chr7_391959 is TC, the number of comb teeth is moderate; When the genotype of Chr7_391959 is CC, the number of comb teeth is small; Selecting individuals with the TT genotype of Chr7_391959 as breeding chickens helps the genetic process of selecting comb tooth number in broilers; The broiler chickens mentioned are Qingyuan Ma chickens.

5. The method for assisted selection breeding of broiler chickens based on the number of comb teeth trait according to claim 4, characterized in that, The PCR amplification reaction system consisted of: 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 added to a total reaction volume of 50 μL.

6. The method for assisted selection breeding of broiler chickens based on the number of comb teeth trait according to claim 4, characterized in that, The PCR amplification reaction program was as follows: 94℃ for 30s; 98℃ for 10s, 60℃ for 30s, 72℃ for 1min, 35 cycles; 72℃ for 2min.

Images