Detection reagent and method for identifying feather traits of chickens and application

Abstract

The invention discloses a detection reagent and method for identifying feather traits of chickens and application. It is found that when the feather phenotype of a chicken is a curled feather, an InDel site exists at 2040242-2040255bp of a chromosome 34 of an Ensembl chicken reference genome CRCg7b version, the deleted nucleotide sequence is shown as SEQ ID NO: 1, the site is a homozygous deletion type, and meanwhile, the SNP site is g.2040608Ggt; and the genotype of A is AA type. The combination of the two sites can be applied to early auxiliary selection of the curled chickens. When the detection reagent is used for detecting the two sites, the feather phenotype of the chicken can be quickly, effectively and accurately predicted, so that the breeding time is shortened, and the breeding process is accelerated.

Description

Technical Field

[0001] The present invention relates to the technical field of poultry breeding, and in particular to a detection reagent, method and application for identifying the feather traits of chickens. Background Art

[0002] The Western Guangdong Curly-Feathered Chicken, also known as the Kirin Chicken, is a high-quality native chicken breed originating from the Western Guangdong region of Guangdong Province, China, with a history of more than 840 years. This chicken breed is named for its unique curly feathers, partially exposed skin, good heat dissipation performance, strong adaptability and good growth performance. The unique curly feather trait of the Curly-Feathered Chicken is conducive to the heat dissipation of its body surface, and is suitable for growing in the high temperature and high humidity environment and climate conditions in the Western Guangdong region. It has strong stress resistance and is of great significance for breeding chicken breeds resistant to heat stress. In addition, as a local fine breed, the Western Guangdong Curly-Feathered Chicken is not only an important economic animal in the local area, but also a part of culture and diet, and is deeply loved by the people. The protection and breeding of the Western Guangdong Curly-Feathered Chicken is underway to prevent the loss of genetic resources. Therefore, it is necessary to establish a molecular technology that can quickly and easily identify the curly feather trait.

[0003] SNP refers to a change in the base of a single nucleotide in the DNA sequence, while InDel refers to the insertion or deletion of one or more bases in the DNA sequence. These variations may affect the regulatory mechanism of gene function and expression. InDel and SNP are the most common genetic variations in organisms and the most commonly used molecular markers. They are huge in number, have a high degree of whole-genome coverage, and have the characteristics of high diversity and strong genetic stability. Therefore, they are widely used in marker-assisted selection in molecular breeding. However, there are still few relevant sites and identification technologies for the curly feather trait of chickens, which is difficult to meet the needs of the breeding industry. Summary of the invention

[0004] In order to overcome the above-mentioned defects and shortcomings in the prior art, the present invention provides a detection reagent, method and application for identifying the feather traits of chicken.

[0005] The first object of the present invention is to provide a composition for use in identifying feather traits of chickens.

[0006] The second object of the present invention is to provide a detection reagent for the above-mentioned composition.

[0007] The third object of the present invention is to provide a kit.

[0008] The fourth object of the present invention is to provide the use of the above-mentioned detection reagent and / or kit in identifying the feather traits of chickens.

[0009] A fifth object of the present invention is to provide the use of the above-mentioned detection reagent and / or kit in the preparation of chicken breeding products.

[0010] A sixth object of the present invention is to provide a method for identifying feather traits of chickens.

[0011] The present invention claims the following:

[0012] A use of a composition in identifying chicken feather traits, the composition comprising an InDel site and a SNP site;

[0013] The InDel site is located at 2040242-2040255 bp on chromosome 34 of the chicken genome, and the deleted nucleotide sequence is shown in SEQ ID NO: 1;

[0014] The SNP site is located at position 2040608 on chromosome 34 of the chicken genome, is a G or A polymorphism, and has GG and AA genotypes;

[0015] The chicken genome is the CRCg7b version of the Ensembl database;

[0016] The InDel site is a homozygous deletion type, and the feather phenotype of the individual whose genotype of the SNP site is AA is curly feathers;

[0017] The InDel site is a homozygous deletion type, and the feather phenotype of the individual whose genotype of the SNP site is GG is a piece feather;

[0018] The feather phenotype of an individual whose InDel site is homozygous wild type or heterozygous is a piece feather.

[0019] The detection reagent of the above-mentioned composition, the detection reagent of the InDel site is a primer with a nucleotide sequence as shown in SEQ ID NO: 2-3, and the detection reagent of the SNP site is a primer with a nucleotide sequence as shown in SEQ ID NO: 4-5.

[0020] A kit comprising the above-mentioned detection reagent.

[0021] Preferably, the kit further contains PCR amplification reagents and ddH2O.

[0022] The use of the above detection reagent and / or kit in identifying the feather traits of chickens.

[0023] Application of the above detection reagent and / or kit in the preparation of chicken breeding products.

[0024] A method for identifying the feather characteristics of chickens, using the above detection reagent to detect the above composition.

[0025] As an practicable manner, the method comprises the following steps:

[0026] S1. Extract genomic DNA of the sample to be tested;

[0027] S2. Performing PCR amplification on the genomic DNA obtained in step S1 using the above-mentioned detection reagent, and then sequencing the PCR amplification product to obtain sequencing data;

[0028] S3. Analyze the sequencing data obtained in step S2, and determine the feather characteristics of the chicken to be tested based on the above-mentioned composition.

[0029] Preferably, in step S1, the chicken is the western Guangdong curly-feather chicken, dwarf curly-feather chicken, Qibainong chicken, dwarf chicken, Wenchang chicken, Huaixiang chicken, Qingyuan Ma chicken, bearded chicken, or the hybrid offspring with the western Guangdong curly-feather chicken, dwarf curly-feather chicken, Qibainong chicken, dwarf chicken, Wenchang chicken, Huaixiang chicken, Qingyuan Ma chicken, and bearded chicken as parents.

[0030] The InDel site is a homozygous deletion type, the genotype of the SNP site is AA, and the chicken's feather phenotype is curly feathers;

[0031] The InDel site is a homozygous deletion type, the genotype of the SNP site is GG, and the feather phenotype of the chicken is a piece feather;

[0032] The InDel site is a homozygous wild type or a heterozygous type, and the feather phenotype of the chicken is a piece feather.

[0033] As an practicable manner, in step S2, the sequencing is Sanger sequencing.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention discloses a detection reagent, method and application for identifying the feather traits of chickens. The present invention finds that there is an InDel site at 2040242-2040255bp of chromosome 34 of the Ensembl chicken reference genome CRCg7b version, and the missing nucleotide sequence is shown in SEQ ID NO: 1. When the InDel site is a homozygous wild type or a heterozygous type, the feather phenotype of the chicken is a piece feather; when the InDel site is a homozygous deletion type, the feather phenotype of an individual with a genotype of AA type at the SNP site g.2040608G>A is a curly feather, and the feather phenotype of an individual with a genotype of GG at the SNP site g.2040608G>A is a piece feather. The combination of these two sites can be applied to the early auxiliary selection of curly-feathered chickens. The detection reagent of the present invention is used to detect these two sites, and the feather phenotype of the chicken can be predicted quickly, effectively and accurately, thereby shortening the breeding time and accelerating the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1Manhattan plot and QQ-plot of InDel sites related to chicken curly feather traits.

[0037] Figure 2 This is the Sanger sequencing result of different genotypes of the site 2040241bp (g.2040242_2040255delGACCCTGTGACACC) on chromosome 34 of the Ensembl chicken reference genome (CRCg7b version); A: homozygous deletion type individual, B: homozygous wild-type individual, C: heterozygous individual.

[0038] Figure 3 This is the Sanger sequencing result of different genotypes at the 2040608bp (g.2040608G>A) site on chromosome 34 of the Ensembl chicken reference genome (CRCg7b version); A: AA type individual, B: GG type individual. DETAILED DESCRIPTION

[0039] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0040] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0041] Example 1 Whole genome resequencing

[0042] 1. Experimental Methods

[0043] 1. Preparation of DNA Samples

[0044] The whole genome DNA of the blood of the Guangdong Curly-feathered Chicken was extracted by the phenol-chloroform method, and the quality and concentration of the DNA were tested using a Nanodrop One spectrophotometer. The A260 / 280 ratio was 1.8-2.0, and the A260 / 230 ratio was 1.7-1.9, which was considered qualified. The qualified DNA samples were uniformly diluted to 50 ng / μL.

[0045] 2. Whole-genome resequencing

[0046] (1) DNA library construction and whole genome sequencing

[0047] The DNA samples were sheared using a Covaris ultrasonic instrument, and short DNA fragments that met the length requirements were obtained by adjusting the shearing parameters. The sheared samples were selected using magnetic beads so that the sample bands were concentrated around 300-400 bp; the amount of purified DNA samples was detected using a Qubit fluorescence quantifier.

[0048] Prepare the reaction system, repair the double-stranded DNA end, add the A base at the 3' end, prepare the adapter connection reaction system, connect the adapter to the DNA. Prepare the PCR reaction system, set the reaction program, and amplify the connection product. The amplified product is screened for fragments using magnetic beads.

[0049] After the PCR product is denatured into a single strand, a cyclization reaction system is prepared, and after being fully mixed, the reaction is allowed to react at an appropriate temperature for a certain period of time to obtain a single-stranded circular product. After digesting the uncyclized linear DNA molecules, the final library is obtained. The single-stranded circular DNA molecules are replicated through rolling circles to form a DNA nanoball (DNB) containing more than 300 copies. The obtained DNBs are added to the mesh holes on the chip using high-density DNA nanochip technology. Sequencing is performed using the combined probe anchor polymerization technology.

[0050] (2) Data Filtering

[0051] The original data were processed and analyzed using SOAPnuke, a filtering software independently developed by BGI. The filtering parameters of SOAPnuke software were as follows: "-n 0.001-l 10-q 0.5--adaMR 0.25--polyX 50--minReadLen 150", and valid data were obtained.

[0052] (3) Obtaining data

[0053] Dalmatian chicken data: The paired-end sequencing data were aligned to the Ensembl chicken reference genome (CRCg7b version), and the alignment rate of the sequencing data exceeded 99%, which can be used for subsequent analysis. Finally, a VCF file containing SNP and InDel data was obtained through variant detection, filtering and annotation steps for subsequent analysis.

[0054] Feathered chicken (normal feather) data: VCF files of SNPs and InDels derived from the laboratory's previous resequencing data of 60 Qibainong chickens and the fastq raw data of 25 Qiandongnan small fragrant chickens published in the NCBI database. The VCF files of SNPs and InDels were processed by the laboratory's server platform.

[0055] (4) Genome-wide association analysis

[0056] The VCF file was converted to tped format using plink software, and the data was filtered using the command "--maf 0.05--geno 0.1". The emmax-kin-intel64 program in the emmax software package was used to calculate the kinship matrix. The emmax-intel64 program in the emmax software package was used to combine the phenotypic data and the kinship matrix for GWAS analysis. Manhattan plots and QQplots were drawn using R language for GWAS results.

[0057] 2. Experimental Results

[0058] GWAS analysis results Figure 1 As shown, the GWAS results showed that there were a large number of InDel sites on chromosome 34 that were significantly associated with the curly feather trait, among which the InDel site (g.2040242_2040255delGACCCTGTGACACC) at 2040241bp on chromosome 34 of the chicken reference genome (CRCg7b version) in Ensembl was one of the sites with the strongest association, and the resequencing data showed that all curly-feathered chickens were homozygous deletion type, while the site in the above-mentioned flat-feathered chicken resequencing data was homozygous wild type or heterozygous, indicating that this molecular marker is very likely to assist in the selection of the curly feather trait.

[0059] Example 2 Primers for amplifying InDel sites

[0060] 1. Experimental Methods

[0061] DNA samples of Yuexi curly-feathered chicken, dwarf curly-feathered chicken, Qibainong chicken, dwarf chicken, Wenchang chicken, Huaixiang chicken, Qingyuan Ma chicken and bearded chicken were used to design PCR amplification primers covering the region for the InDel (g.2040242_2040255delGACCCTGTGACACC) site at 2040241 bp on chromosome 34 of the Ensembl chicken reference genome (CRCg7b version) that was significantly associated with the curly feather trait obtained in Example 1. The primer sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3, and the amplification product size is 643 bp;

[0062] F1: 5'-GTGCTCAGAAGTCCCCCTCAACCT-3' (SEQ ID NO: 2);

[0063] R1: 5'-CCATCTCTCCACGCTCCTCT-3' (SEQ ID NO: 3).

[0064] PCR amplification primers were used to perform PCR verification on DNA samples of curly-feathered chicken and flat-feathered chicken. The samples used for PCR verification are shown in Table 1;

[0065] Table 1 Samples used for PCR validation

[0066] variety Quantity (pcs) Western Guangdong Curly-Feathered Chicken 130 Dwarf Curly Chicken 30 Qibainong Chicken 30 Bantam Chicken 30 Wenchang Chicken 30 Nostalgic Chicken 30 Qingyuan Chicken 30 Bearded Chicken 30

[0067] PCR amplification system: 2×Es Taq MasterMix 25 μL, upstream primer (10 μmol / L) 2 μL, downstream primer (10 μmol / L) 2 μL, DNA template 200 ng, add sterile double distilled water to 50 μL system.

[0068] PCR reaction program: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 61°C for 30 s, extension at 72°C for 30 s, for a total of 32 cycles; extension at 72°C for 5 min.

[0069] The PCR amplification product was tested by agarose gel electrophoresis. If the electrophoresis band was consistent with the target band in size, the PCR product was sent to the company for DNA sequence determination. The gene fragment was tested for both forward and reverse reactions. The measured sequence results were compared with the Ensembl genome sequence to obtain the corresponding gene deletion situation.

[0070] 2. Experimental Results

[0071] The sequencing results are as follows Figure 2 As shown. Among them, Figure 2 The A in the expression indicates that the site is homozygous for deletion. Figure 2 The B in the expression indicates that the site is homozygous wild type. Figure 2 The C in the sequence indicates that the locus is heterozygous. The sequencing statistical results are shown in Table 2, where all chickens with curly feather traits are homozygous deletion type; and chickens with patchy feather traits are almost all homozygous wild type and heterozygous except Huaixiang chicken.

[0072] Most of the Huaixiang chickens are homozygous wild type and heterozygous, but there are also homozygous deletion types. Therefore, another locus needs to be screened to perform auxiliary selection for this situation (between the curly-feathered chicken and the Huaixiang chicken).

[0073] Table 2 Genotyping statistics of InDel loci

[0074]

[0075]

[0076] Example 3 Primers for amplifying SNP sites

[0077] 1. Experimental Methods

[0078] A SNP site (g.2040608G>A) was found below the InDel (g.2040242_2040255delGACCCTGTGACACC) site at 2040241bp on chromosome 34 of the Ensembl chicken reference genome (CRCg7b version) obtained by screening in Example 1. This site was shown to be significantly associated with the curly feather trait in the GWAS results.

[0079] DNA samples of Yuexi Curly-feathered Chicken, Dwarf Curly-feathered Chicken, Qibainong Chicken, Dwarf Chicken, Wenchang Chicken, Huaixiang Chicken, Qingyuan Ma Chicken and Bearded Chicken were used to design PCR amplification primers covering the region for the SNP site. The primer sequences are shown in SEQ ID NO: 4 and SEQ ID NO: 5, and the amplification product size is 259 bp.

[0080] F2: 5'-GCCCTGAAAGATGCCAAATCCAAAC-3' (SEQ ID NO: 4);

[0081] R2: 5'-GGTGAACTGAGGCTTTGGACCC-3' (SEQ ID NO: 5).

[0082] PCR amplification primers were used to perform PCR verification on DNA samples of Yuexi Curly-feathered Chicken (Curly-feathered Chicken, 30) and Huaixiang Chicken (Flake-feathered Chicken, 30). The PCR amplification system was: 2×Es Taq MasterMix 25μL, upstream primer (10μmol / L) 2μL, downstream primer (10μmol / L) 2μL, DNA template 200ng, and sterile double distilled water was added to the 50μL system.

[0083] PCR reaction program: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 61°C for 30 s, extension at 72°C for 30 s, for a total of 32 cycles; extension at 72°C for 5 min.

[0084] The PCR amplification product is tested by agarose gel electrophoresis. If the electrophoresis band is consistent with the target band in size, the PCR product is sent to the company for DNA sequence determination. The gene fragment is tested for forward and reverse reactions. The measured sequence results are compared with the Ensembl genome sequence to obtain the corresponding gene mutation situation.

[0085] 2. Experimental Results

[0086] The sequencing results are as follows Figure 3 As shown. Among them, Figure 3 The A in the genotype indicates that the locus is AA genotype. Figure 3 The B in the genotype indicates that the locus is of GG genotype. The sequencing statistical results are shown in Table 4, where all the Dalmatian chickens are of AA genotype, and all the Huaixiang chickens are of GG genotype.

[0087] Therefore, this pair of primers for amplifying the SNP site (g.2040608G>A) can clearly distinguish the curly-feathered chicken from the Huaixiang chicken (piece-feathered chicken). Therefore, the combination of the primers for amplifying the InDel site and the primers for amplifying the SNP site (g.2040608G>A) can accurately identify the feather traits of the above-mentioned different chicken breeds selected by the present invention, with a success rate and accuracy rate of 100%, which can be fully used in the actual identification of the curly feather traits of chickens.

[0088] Table 3 Genotyping statistics of SNP locus (g.2040608G>A)

[0089] variety Sanger sequencing quantity GG AA Western Guangdong Curly-Feathered Chicken 30 0 30 Nostalgic Chicken 30 30 0

[0090] Example 4 A method for identifying the feather traits of chickens

[0091] 1. Extract DNA from the chicken sample to be tested.

[0092] 2. Use the DNA of the chicken sample to be tested extracted in step 1 as a template for PCR amplification:

[0093] (1) Amplification of InDel site (g.2040242_2040255delGACCCTGTGACACC)

[0094] The PCR reaction system is: 25 μL of 2×Es Taq MasterMix, 2 μL of each primer with nucleotide sequence as shown in SEQ ID NO: 2-3, 200 ng of DNA template, and sterile double distilled water to make up to 50 μL system.

[0095] PCR reaction program: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 61°C for 30 s, extension at 72°C for 30 s, for a total of 32 cycles; extension at 72°C for 5 min.

[0096] (2) Amplification of SNP site (g.2040608G>A)

[0097] The PCR reaction system is: 25 μL of 2×Es Taq MasterMix, 2 μL of each primer with nucleotide sequence as shown in SEQ ID NO: 4-5, 200 ng of DNA template, and sterile double distilled water to make up to 50 μL system.

[0098] PCR reaction program: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 61°C for 30 s, extension at 72°C for 30 s, for a total of 32 cycles; extension at 72°C for 5 min.

[0099] 3. Perform agarose gel electrophoresis on the PCR amplification product obtained in step 2. If the electrophoresis band is consistent with the target band in size and is clear and bright, sequence the PCR amplification product obtained in step 2, and compare the sequencing result with chromosome 34 of the Ensembl chicken reference genome (CRCg7b version) and perform genotyping.

[0100] 4. Result judgment

[0101] There is an InDel site at 2040242-2040255 bp on chromosome 34 of the Ensembl chicken reference genome (CRCg7b version), and the deleted nucleotide sequence is GACCCTGTGACACC (SEQ ID NO: 1), i.e., g.2040242_2040255delGACCCTGTGACACC;

[0102] There is a single nucleotide polymorphism at 2040608bp on chromosome 34 of the Ensembl chicken reference genome (CRCg7b version), which is a G or A polymorphism, and it has GG and AA genotypes;

[0103] The InDel site is a homozygous deletion type, and the feather phenotype of the individual whose genotype of the SNP site is AA is curly feathers;

[0104] The InDel site is a homozygous deletion type, and the feather phenotype of the individual whose genotype of the SNP site is GG is a piece feather;

[0105] The feather phenotype of an individual whose InDel site is homozygous wild type or heterozygous is a piece feather.

[0106] Example 5 A kit for identifying chicken feather traits

[0107] 1. Composition

[0108] Primers whose nucleotide sequences are shown in SEQ ID NOs: 2-3, primers whose nucleotide sequences are shown in SEQ ID NOs: 4-5, 2×Es Taq MasterMix and ddH2O.

[0109] 2. Usage

[0110] The detection and result interpretation were performed according to Example 4.

[0111] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of a composition in identifying the feather characteristics of chickens, characterized in that: The composition is an InDel site and a SNP site; The InDel site is located at 2040242-2040255 bp on chromosome 34 of the chicken genome, and the deleted nucleotide sequence is shown in SEQ ID NO: 1; The SNP site is located at position 2040608 on chromosome 34 of the chicken genome, is a G or A polymorphism, and has GG and AA genotypes; The chicken genome is the CRCg7b version of the Ensembl database; The InDel site is a homozygous deletion type, and the feather phenotype of the individual whose genotype of the SNP site is AA is curly feathers; The InDel site is a homozygous deletion type, and the feather phenotype of the individual whose genotype of the SNP site is GG is a piece feather; The feather phenotype of an individual whose InDel site is homozygous wild type or heterozygous is a piece feather.

2. The detection reagent of the composition according to claim 1, characterized in that: The detection reagents for the InDel sites are primers with nucleotide sequences as shown in SEQ ID NOs: 2-3, and the detection reagents for the SNP sites are primers with nucleotide sequences as shown in SEQ ID NOs: 4-5.

3. A kit, characterized in that: The kit comprises the detection reagent according to claim 2.

4. The kit according to claim 3, characterized in that The kit also contains PCR amplification reagents and ddH2O.

5. Use of the detection reagent according to claim 2 and / or the kit according to any one of claims 3 to 4 in identifying the feather traits of chickens.

6. Use of the detection reagent according to claim 2 and / or the kit according to any one of claims 3 to 4 in preparing chicken breeding products, characterized in that: The breeding is molecular breeding for feather traits.

7. A method for identifying the feather characteristics of a chicken, characterized in that: The composition of claim 1 is detected using the detection reagent of claim 2.

8. The method according to claim 7, characterized in that The following steps are involved: S1. Extract genomic DNA of the sample to be tested; S2. Performing PCR amplification on the genomic DNA obtained in step S1 using the detection reagent described in claim 2, and then sequencing the PCR amplification product to obtain sequencing data; S3. Analyze the sequencing data obtained in step S2, The composition according to claim 1 is used to determine the feather characteristics of the tested chicken.

9. The method according to claim 8, characterized in that The chicken is the western Guangdong curly-feather chicken, dwarf curly-feather chicken, Qibainong chicken, dwarf chicken, Wenchang chicken, Huaixiang chicken, Qingyuan ma chicken, and bearded chicken, or the hybrid offspring of the western Guangdong curly-feather chicken, dwarf curly-feather chicken, Qibainong chicken, dwarf chicken, Wenchang chicken, Huaixiang chicken, Qingyuan ma chicken, and bearded chicken as parents.

10. The method according to claim 8, characterized in that In step S2, the sequencing is Sanger sequencing.

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