SNP (Single Nucleotide Polymorphism) molecular marker related to weight of Beijing fatty chicken and application

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker related to the weight of Beijing fatty chicken and application. The SNP molecular marker has G / A base mutation at 75bp, 655bp and 498bp of the No.4 chromosome of Beijing fatty chicken; the version number of a Beijing fatty chicken reference genome where the SNP molecular marker is located is GRCg6a. The SNP molecular marker provided by the invention can be used as a molecular marker for identifying the weight character of Beijing fatty chicken. The molecular marker provided by the invention is not limited by the age, sex and the like of Beijing fatty chicken, can be used for breeding high-weight dominant Beijing fatty chicken individuals, and greatly accelerates the breeding process of Beijing fatty chicken varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology and genetic breeding, in particular to a SNP molecular marker related to the body weight of Beijing oil chicken and application thereof. BACKGROUND

[0002] At present, the genetic identification of chicken body weight traits mainly relies on the following methods: (1) Phenotypic selection: direct selection based on body weight, but it is greatly affected by environmental factors and has a long breeding cycle (requiring multi-generation data); (2) QTL positioning: positioning the chromosome region through linkage analysis, but the resolution is low; (3) SNP chip typing: such as 600K chip, which has high cost and relies on fixed sites, and has poor flexibility; (4) Simplified genome sequencing: which requires complex data analysis and is difficult to popularize at the grassroots level.

[0003] The above identification methods mainly have the following disadvantages: 1) Low efficiency: traditional methods require multi-generation phenotypic data, which takes 3-5 years; 2) High cost: the unit price of chip typing is high, which is not suitable for large-scale screening; 3) Insufficient marker density: the existing body weight-related SNPs lack targeted detection schemes.

[0004] Existing researches are mostly focused on ordinary chicken breeds, and there is a lack of specific researches on Beijing oil chicken, which is an important economic breed. The control of body weight of Beijing oil chicken is of great significance to its breeding efficiency, and therefore a specially designed detection method is needed to meet the demand for genetic improvement.

[0005] Existing detection methods are usually suitable for specific economic traits or chicken breeds, and are difficult to meet the specific needs of Beijing oil chicken body weight control. SUMMARY

[0006] The purpose of the present application is to provide a SNP molecular marker related to the body weight of Beijing oil chicken and application thereof.

[0007] In order to achieve the purpose of the present application, in the first aspect, the present application provides a SNP molecular marker related to the body weight of Beijing oil chicken, which contains a nucleotide sequence with polymorphism of G / A at position 300 of the sequence shown in SEQ ID NO: 3 (n is g or a).

[0008] The genotype of the site with the polymorphism is AA, which corresponds to the high body weight dominant type, the genotype is GG, which corresponds to the low body weight type, and the genotype is AG, which corresponds to the intermediate type.

[0009] In the second aspect, the present application provides primers for amplifying the SNP molecular marker, including forward primers and reverse primers with sequences shown in SEQ ID NO: 1-2.

[0010] In the third aspect, the present application provides detection reagents or kits containing the primers.

[0011] In a fourth aspect, the present application provides use of the SNP molecular marker, the primer, the detection reagent or the kit in identifying the body weight trait of Beijing fowl.

[0012] In a fifth aspect, the present application provides use of the SNP molecular marker, the primer, the detection reagent or the kit in early prediction of the high body weight trait of Beijing fowl.

[0013] In a sixth aspect, the present application provides use of the SNP molecular marker, the primer, the detection reagent or the kit in genetic improvement of Beijing fowl breed.

[0014] In a seventh aspect, the present application provides a method for identifying the body weight trait of Beijing fowl, comprising the following steps: 1) detecting the genotype of the polymorphic site of the SNP molecular marker in the Beijing fowl to be tested; 2) judging the body weight trait of the Beijing fowl to be tested according to the genotype obtained in step 1).

[0015] Further, step 1) comprises extracting the genomic DNA of the Beijing fowl to be tested, performing PCR amplification by using the primers shown in SEQ ID NO: 1-2, and analyzing the PCR amplification product.

[0016] The specific judging method of step 2) is that if the genotype is AA, the Beijing fowl to be tested is of high body weight advantage type, if the genotype is GG, it corresponds to low body weight type, and if the genotype is AG, it corresponds to intermediate type.

[0017] By means of the above technical solution, the present application has at least the following advantages and beneficial effects: (1) High detection precision: the target SNP site base is directly read by one generation sequencing (Sanger method), the typing accuracy is > 99.9%, and false positive / negative problems caused by probe hybridization or fluorescence signal interference are avoided.

[0018] (2) Significant cost reduction: the present application uses ordinary PCR technology, without the need for additional equipment or expensive reagents, thereby reducing experimental costs, and the cost of single sample detection is < 30 yuan (including primer, reagent and sequencing cost), at the same time, the experimental operation is simple and no special equipment needs to be purchased (ordinary PCR instrument can be used), which is suitable for ordinary farms or small laboratories, and has wide popularization potential.

[0019] (3) The body weight of AA genotype individuals is 0.294 kg higher than that of GG type at 38 weeks of age, the superior breed of chickens can be screened in advance, the breeding cycle is shortened by more than 50%, and the feed cost is reduced by about 20%.

[0020] (Four) By detecting the G / A SNP site related to the body weight of Beijing oil chicken, the application provides an important tool for genetic improvement of chickens, and helps to screen individuals carrying favorable alleles, thereby improving breeding efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 For the high body weight homozygous GG type individual of Beijing oil chicken in the preferred embodiment of the application.

[0022] Figure 2 For the low body weight homozygous AA type individual of Beijing oil chicken in the preferred embodiment of the application.

[0023] Figure 3 For the intermediate body weight heterozygous AG type individual of Beijing oil chicken in the preferred embodiment of the application. DETAILED DESCRIPTION

[0024] The application aims at the certain limitations of the existing SNP detection method in detection efficiency, specificity and application range, especially the lack of special research on the body weight control of Beijing oil chicken. Therefore, the application develops a primer for detecting the G / A SNP site related to the body weight of Beijing oil chicken, and designs a primer with high efficiency and good specificity, which overcomes the problems of high cost, poor universality and complex operation of the prior art, and provides an innovative solution for efficient breeding of local chicken breeds.

[0025] The application adopts the following technical solutions: 1. Development of specific SNP marker SNP site: located at 75,655,498 bp of chromosome 4 of chicken genome GRCg6a version 4, which is G / A polymorphism.

[0026] Phenotype association: the average body weight of AA genotype individual is 0.294 kg (p=1.25x10 -14 ) higher than that of GG type, which can explain 7.55% of phenotype variation.

[0027] 2. Establishment of efficient detection method Primer design: 545 bp fragment containing SNP site is amplified to ensure covering upstream and downstream regulatory regions.

[0028] First generation sequencing: the target SNP site genotype (G / A) is directly read by Sanger sequencing, the base type is judged by sequencing peak chart, the accuracy is >99.9%, and the false positive risk caused by chip hybridization or probe design is avoided.

[0029] 3. Breeding application Genotype determination: AA is the high body weight dominant type, GG is the low body weight type, and AG is the intermediate type.

[0030] Early screening: screening at the chick stage, shortening the breeding cycle by more than 50%.

[0031] The following examples are used to illustrate the present application but not to limit the scope of the present application. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art, and the raw materials used are commercially available.

[0032] Example 1 Development of SNP molecular markers related to the body weight of Beijing Fatty Chickens I. Sample collection and phenotype determination Experimental population: Beijing Fatty Chicken pure line breeding population selected from Beijing Fatty Chicken Resource Preservation Farm of Beijing Academy of Agriculture and Forestry Sciences, all individuals were bred under standardized feeding conditions.

[0033] Phenotype determination: 224 female Beijing Fatty Chickens at 38 weeks of age were determined for body weight traits.

[0034] Sample collection: blood was collected from the subclavian vein, 2 mL of whole blood was taken from each chicken, and was divided into EDTA anticoagulant tubes and stored at -20°C for standby use.

[0035] II. Whole genome sequencing and variation detection 1. DNA extraction and library construction: Genomic DNA was extracted using FlaPure animal blood DNA extraction kit (Genesand Biotech).

[0036] 2. Library construction and sequencing: DNBSEQ-T7 platform (Huada) generated 150 bp double-end sequencing data with an average depth of 6x.

[0037] 3. Data quality control and variation detection: 4. Raw data filtering: fastp software was used to remove low-quality reads.

[0038] 5. Sequence alignment: reference genome GRCg6a, BWA-MEM algorithm alignment generated BAM file.

[0039] 6. Variation detection (GATK v4.4.0): joint variation detection (GVCF merging) generated the original VCF file.

[0040] 7. SNP screening criteria: QD≥2.0, QUAL≥30, FS≤60, MQ≥40, ReadPosRankSum≥-8.0.

[0041] 8. PLINK quality control: remove sites with MAF<5%, genotype missing rate>2%, finally retain 5,630,768 SNPs.

[0042] The GEMMA software was used for whole genome association analysis of the SNP data to obtain a significant peak on chromosome 4, and it was found that the highest site explained 7.55% of the phenotypic variation, so the highest site was selected as the SNP marker.

[0043] Example 2 Primer design and application for detecting SNP molecular markers related to the body weight of Beijing oil chicken I. The following primers were designed and synthesized: Forward primer (SEQ ID NO: 1): 5'-TGTGTTTTGTGCTCTTTGGC-3' Reverse primer (SEQ ID NO: 2): 5'-AGAGACTGCATGCCTCACTT-3' Amplification region: Targeting the upstream and downstream regions of the 75,655,498 bp SNP site on chicken chromosome 4, the amplification length is 545 bp (GRCg6a genome version).

[0044] II. The method for identifying the body weight traits of Beijing oil chicken using the above designed primers is as follows: 1. Genomic DNA extraction Collect chicken wing vein blood, and use the Jinsha high-efficiency animal tissue / cell / blood genomic DNA extraction kit DE712-50, and the specific steps are as follows: (1) Take 5-20 μL of fresh or frozen anticoagulated blood sample, add buffer GA1 to make up to 200 μL.

[0045] (2) Add 20 mg / mL proteinase K solution 20 μL, mix well, then add 200 μL of buffer GA2, vortex to mix thoroughly, and incubate at 70°C for 6 h.

[0046] (3) Centrifuge briefly to remove water droplets on the inner wall of the tube cap. Add 200 μL of absolute ethanol, vortex to mix thoroughly, and centrifuge briefly to enter step (4) for column purification.

[0047] (4) Column purification: a. Centrifuge briefly, and add the solution and flocculent precipitate obtained in step (3) to the adsorption column (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm (about 13,400 ×g) for 30 s, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube; b. Add 500 μL of buffer GW1 (check whether absolute ethanol has been added before use) to the adsorption column, centrifuge at 12,000 rpm (about 13,400 ×g) for 30 s, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube; c. Add 600 μL Buffer GW2 (check if anhydrous ethanol has been added before use) to the adsorption column, centrifuge at 12,000 rpm (about 13,400 x g) for 30 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube; d. Repeat step c; e. Centrifuge at 12,000 rpm (about 13,400 x g) for 2 min, and dry at room temperature for a few minutes; f. Place the adsorption column in a new centrifuge tube (self-provided), and add 50-200 μL TE buffer or ddH2O to the middle of the adsorption membrane, and place it at room temperature for 2-5 min, centrifuge at 12,000 rpm (about 13,400 x g) for 2 min, collect the DNA solution, and store the DNA at -20°C.

[0048] 2. PCR amplification: After diluting the extracted DNA sample, use it as a PCR template for amplification with TSINGKE Master Mix (dye-free) 2x, and the components of the amplification system are shown in Table 1.

[0049] Table 1 Components of the amplification system

[0050] The amplification procedure of the above amplification system is shown in Table 2.

[0051] Table 2 Amplification procedure

[0052] Product verification: 1.5% agarose gel electrophoresis, run at 300V for 12 minutes, obtain the gel image by gel imaging instrument, and observe and compare the band length of about 545bp.

[0053] 3. First-generation Sanger sequencing of PCR products: Sequencing analysis: The raw data is analyzed by Chromas software, and the peak interpretation of the target site (chr4: 75655498 bp) is as follows: Homozygous GG type: single black peak (corresponding to G base) Figure 1 ); Homozygous AA type: single green peak (corresponding to A base) Figure 2 ); Heterozygous AG type: black and green double peaks superimposed Figure 3 ).

[0054] 4. Genotype determination and breeding decision: According to the sequencing results, the genotypes are divided: Type AA: Preferentially selected as core breeder, used for high weight line breeding; Type GG: Discarded or used as low weight control; Type AG: Selected for retention based on breeding objectives.

[0055] Although the present application has been described in detail with general descriptions and specific embodiments, modifications or improvements can be made to the present application, which will be apparent to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present application are within the scope of the present application.

Claims

1. A SNP molecular marker associated with the weight of Beijing oily chicken, characterized in that: The SNP molecular marker contains a nucleotide sequence in which the polymorphism at position 300 is G / A as shown in SEQ ID NO:

3.

2. The SNP molecular marker according to claim 1, characterized in that The genotype of the site with the polymorphism is AA, which corresponds to the high-weight dominant type; the genotype is GG, which corresponds to the low-weight type; and the genotype is AG, which corresponds to the intermediate type.

3. A primer for amplifying the SNP molecular marker according to claim 1 or 2, characterized in that: It includes a forward primer and a reverse primer whose sequences are shown in SEQ ID NO: 1-2.

4. A detection reagent or kit containing the primer according to claim 3.

5. Use of the SNP molecular marker according to claim 1 or 2, the primer according to claim 3, or the detection reagent or kit according to claim 4 in identifying the body weight trait of Beijing oily chickens.

6. Use of the SNP molecular marker according to claim 1 or 2, or the primer according to claim 3, or the detection reagent or kit according to claim 4 in the early prediction of high body weight traits in Beijing oily chicken individuals.

7. Use of the SNP molecular marker according to claim 1 or 2, the primer according to claim 3, or the detection reagent or kit according to claim 4 in genetic improvement of Beijing Oily Chicken.

8. A method for identifying the weight traits of Beijing oily chicken, characterized in that: The steps include: 1) Detecting the genotype of the polymorphic site of the SNP molecular marker according to claim 1 or 2 in a Beijing oily chicken to be tested; 2) Determine the weight trait of the Beijing oily chicken to be tested based on the genotype obtained in step 1).

9. The method according to claim 8, characterized in that Step 1) comprises: extracting genomic DNA from the Beijing oily chicken to be tested, performing PCR amplification using primers shown in SEQ ID NO: 1-2, and analyzing the PCR amplification product.

10. The method according to claim 8 or 9, characterized in that The specific judgment method of step 2) is: if the genotype is AA, the Beijing oily chicken to be tested is a high-weight dominant type; if the genotype is GG, it corresponds to a low-weight type; if the genotype is AG, it corresponds to an intermediate type.

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