Molecular marker related to chicken curled feather character and detection reagent and application thereof

Abstract

The invention discloses a molecular marker related to chicken curled feather traits as well as a detection reagent and application of the molecular marker. According to the invention, through whole genome re-sequencing and whole genome association analysis, four SNP loci capable of distinguishing two phenotypes of curled feather and piece feather of chicken are obtained on a chromosome 34 of an Enmbl chicken reference genome CRCg7b version. The invention further provides a detection reagent and a kit for detecting the four SNP loci, the detection reagent and the kit can be applied to early-stage auxiliary selection of the curly-feather chickens, the production cost can be saved, the molecular breeding process can be accelerated, and therefore the economic benefits of local chicken breeding are improved.

Description

Technical Field

[0001] The invention relates to the technical field of poultry breeding, and in particular to molecular markers related to chicken feather curling traits and detection reagents and applications thereof. Background Art

[0002] Poultry have a variety of feather structures, showing a high degree of diversity. Feather-related traits are important appearance-related traits that affect the selection and breeding of chickens. Curly feathers are an important type of poultry feather type, which is conducive to the heat dissipation of the chicken body surface and is suitable for breeding in tropical or southern my country. It is of great significance for the breeding of heat stress-resistant chickens. In addition, curly feathers can be used as a unique outer packaging trait and breed characteristic to distinguish from other chicken breeds. Therefore, the breeding of curly-feathered chickens has important economic value.

[0003] The Guangdong Curly-Feathered Chicken has a unique curled feather appearance. It is also tolerant to roughage, has strong resistance to adversity, and is tall. It is bred in Zhanjiang, Maoming, and other places in Guangdong. It is a very popular local breed. At present, the protection and breeding of the Guangdong Curly-Feathered Chicken is in progress, but there are still few related sites and identification technologies for the curly feather trait of chickens, which makes it difficult to meet the needs of the breeding industry. Therefore, it is necessary to find more sites related to the curly feather trait, so as to establish a molecular breeding technology that can quickly and easily identify the curly feather trait. Summary of the invention

[0004] In order to overcome the above defects and shortcomings in the prior art, the present invention provides molecular markers related to the chicken feather curling trait and detection reagents and applications thereof.

[0005] The first object of the present invention is to provide an application of a molecular marker in identifying the feather traits of chickens.

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

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

[0008] A 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 seventh object of the present invention is to provide application of the above method in molecular assisted breeding of Curly-feathered Chicken.

[0012] Therefore, the present invention claims the following:

[0013] Application of a molecular marker in identifying the feather traits of chickens, wherein the molecular marker consists of four single nucleotide polymorphism sites SNP1, SNP2, SNP3 and / or SNP4:

[0014] SNP1 is located at position 2040798 on chromosome 34 of the chicken genome and is a G or A polymorphism, with GG, GA, and AA genotypes.

[0015] SNP2 is located at position 2040857 on chromosome 34 of the chicken genome and is a G or T polymorphism, with GG, GT, and TT genotypes.

[0016] SNP3 is located at position 2040864 on chromosome 34 of the chicken genome and is an A or G polymorphism, with AA, AG, and GG genotypes.

[0017] SNP4 is located at position 2040873 on chromosome 34 of the chicken genome and is a T or C polymorphism, with TT, TC, and CC genotypes.

[0018] The chicken genome is the CRCg7b version of the Ensembl database;

[0019] The feather phenotype of the individual whose SNP1 genotype is AA, whose SNP2 genotype is TT, whose SNP3 genotype is GG or whose SNP4 genotype is CC is curly feather.

[0020] The above-mentioned molecular marker detection reagent is a primer with a nucleotide sequence as shown in SEQ ID NO: 1-2.

[0021] A kit comprising the above-mentioned detection reagent.

[0022] Preferably, the kit further contains PCR amplification reagents and ddH2O.

[0023] The use of the above detection reagent and / or kit in identifying the feather traits of chickens.

[0024] The above detection reagent and / or kit is used in the preparation of chicken breeding products, wherein the breeding is molecular breeding of feather traits.

[0025] A method for identifying chicken feather traits, using the above detection reagent to detect the above molecular markers.

[0026] As an practicable manner, the method comprises the following steps:

[0027] S1. Extract genomic DNA of the sample to be tested;

[0028] 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;

[0029] S3. Analyze the sequencing data obtained in step S2, and determine the feather traits of the chicken to be tested based on the above-mentioned molecular markers.

[0030] As an practicable manner, in step S2, the sequencing is Sanger sequencing.

[0031] Application of the above method in molecular assisted breeding of Curly-feathered chicken.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention discloses molecular markers related to the curly feather trait of chickens, detection reagents and applications thereof. The present invention obtains four SNP sites on chromosome 34 of the Ensembl chicken reference genome CRCg7b version that can distinguish the two phenotypes of curly feathers and flaked feathers of chickens through whole genome resequencing and whole genome association analysis. The present invention also provides detection reagents and kits for detecting these four SNP sites, which can be applied to the early auxiliary selection of curly feather chickens, can save production costs and accelerate the molecular breeding process, thereby improving the economic benefits of local chicken breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The flowchart of sequencing.

[0035] Figure 2 Manhattan plot and QQ-plot of InDel sites related to chicken curly feather traits.

[0036] Figure 3 The figure shows the Sanger sequencing results of different genotypes of four SNP sites. DETAILED DESCRIPTION

[0037] 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.

[0038] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0039] Example 1 Whole genome resequencing

[0040] 1. Experimental Methods

[0041] 1. Preparation of DNA Samples

[0042] Preparation of DNA samples of Guangdong Curly Feather Chicken: Blood samples were collected from 356 140-day-old Guangdong Curly Feather Chickens through the sub-wing vein and stored in a -20°C refrigerator for subsequent use. The genomic DNA was extracted from the whole blood samples of 356 Guangdong Curly Feather Chickens using the Nanjing Novozymes DNA / RNA extraction kit, and the DNA was stored in a -20°C refrigerator for subsequent resequencing and PCR amplification.

[0043] Preparation of DNA samples of other chicken breeds: Blood samples were collected from 210 chickens, including 30 chickens of each breed, Qibainong chicken, dwarf chicken, dwarf curly-feathered chicken, Wenchang chicken, Huaixiang chicken, Qingyuan Ma chicken, and bearded chicken. The Nanjing Novozymes DNA / RNA extraction kit was used to extract genomic DNA from the whole blood samples of the above chicken breeds for subsequent PCR amplification, Sanger sequencing, and genotyping verification.

[0044] 2. Whole-genome resequencing

[0045] (1) DNA library construction and whole genome sequencing

[0046] DNA samples from 356 western Guangdong curly-feathered chickens were sent to Shenzhen BGI Genomics Co., Ltd. for whole genome resequencing.

[0047] The main steps include: ① DNA sample detection; ② DNA sample shearing; ③ fragment size selection; ④ end repair, adding "A", adapter connection; ⑤ PCR reaction and product recovery; ⑥ PCR product circularization; ⑦ library detection; ⑧ sequencing on the machine. The single-stranded circular DNA molecule replicates through rolling circles to form a DNA nanoball (DNB) containing more than 300 copies. The obtained DNB is added to the mesh holes on the chip using high-density DNA nanochip technology and sequenced through combined probe anchor polymerization technology (cPAS). The specific process is as follows Figure 1 shown.

[0048] (2) Data Filtering

[0049] Data processing Some original sequences contain adapter sequences or a small amount of low-quality sequences. First, a series of data processing is performed to remove impurity data and obtain valid data. This step of analysis is completed using the filtering software SOAPnuke independently developed by BGI. The filtering parameters of SOAPnuke software are: "-n 0.001-l 10-q 0.5--adaMR 0.25--polyX 50--minReadLen 150". The filtering steps include:

[0050] ① Filter adapters: If the sequencing read matches 25.0% or more of the adapter sequence (up to 2 base mismatches are allowed), the entire read is deleted;

[0051] ② Filter read length: If the length of the sequencing read is less than 150bp, delete the entire read;

[0052] ③ Remove N: If the N content in the sequencing read accounts for 0.1% or more of the entire read, the entire read is deleted;

[0053] ④ Remove polyX: If the length of polyX (X can be A, T, G or C) in the sequencing read exceeds 50bp, the entire read is deleted;

[0054] ⑤ Filter low-quality data: If the bases with a quality value lower than 10 in the sequencing read account for 50.0% or more of the entire read, the entire read is deleted;

[0055] ⑥ Get Clean reads: The output read quality value system is set to Phred+33.

[0056] (3) Obtaining data

[0057] Curly feather data: The paired-end sequencing data of 356 samples of Curly Feather Chicken in western Guangdong were aligned to the Ensembl chicken reference genome (CRCg7b version), which is now a chromosome-level genome with high assembly quality. Finally, a VCF file containing SNP and InDel data was obtained through variant detection, filtering and annotation.

[0058] Feather (normal feather) data: VCF files of SNP and InDel of the laboratory's early resequencing data of 60 Qibainong chickens and the fastq raw data of 25 Qiandongnan small fragrant chickens published in the NCBI database, and the VCF files of SNP and InDel obtained by processing on the laboratory's server platform.

[0059] 3. Genome-wide association analysis

[0060] Using the above SNP and InDel data, emmax software was used to perform genome-wide association analysis (GWAS) to associate the curled feather and leaf feather phenotypes, make Manhattan plots and QQplots, screen the variant sites that exceed the threshold line, and select the most suitable candidate sites for analysis based on the most significant P value.

[0061] The steps of GWAS analysis are as follows:

[0062] ① Use plink software to convert the VCF file into tped format, and use the "--maf 0.05--geno 0.1" command to filter the data.

[0063] ② Phenotypic data preparation: mark each individual, mark the curly feather trait as 0 and the non-curly feather trait as 1.

[0064] ③ Use the emmax-kin-intel64 program in the emmax software package to calculate the kinship matrix.

[0065] ④ Use the emmax-intel64 program in the emmax software package to combine the above phenotypic data and kinship matrix for GWAS analysis.

[0066] ⑤ Use R language to draw Manhattan plot and QQplot plot of GWAS results.

[0067] 2. Experimental Results

[0068] The sequencing results showed that there were a large number of significant sites on chromosomes 34 and Z, with chromosome 34 having a very high peak. A total of 473 significant SNPs were found on chromosome 34 ( Figure 2 A), the first 30 P values ​​were selected for statistics (Table 1).

[0069] Table 1 GWAS detected the top 30 SNP sites with significant P values ​​on chromosome 34

[0070]

[0071]

[0072] Example 2 PCR amplification and Sanger sequencing

[0073] 1. Experimental Methods

[0074] 1. Primer Design

[0075] According to the sites and P values ​​in Table 1 in Example 1, the mutation site at position 2040798 on chromosome 34 was selected as an auxiliary candidate site for verification in the SNP mutation site. This site has a point mutation of G>A (g.2040798G>A). It can be found from Table 1 that there is another mutation site at position 2040864 (g.2040864A>G) near this site.

[0076] Specific amplification primers were designed for sites 2040798 and 2040864 on chromosome 34. The primer sequences are shown in Table 2.

[0077] Table 2 Primer sequences

[0078]

[0079] 2. PCR Amplification and Sequencing

[0080] 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;

[0081] Table 3 Samples used for PCR validation

[0082] variety Quantity (pcs) Western Guangdong Curly-Feathered Chicken 90 Bantam Chicken 30 Qibainong Chicken 30 Wenchang Chicken 30 Nostalgic Chicken 30 Bearded Chicken 30 Qingyuan Chicken 30

[0083] PCR reaction system: 2×Es Taq MasterMix 25μL, upstream primer (10μmol·L -1 )2μL, downstream primer (10μmol·L -1 )2μL, DNA template 1μL, add ddH2O to 50μL.

[0084] PCR reaction program: 94℃ pre-denaturation for 2min; 94℃ denaturation for 30s, 62℃ annealing for 30s, 72℃ extension for 30s, 32 cycles; 72℃ extension for 2min, 12℃∞. PCR products were stored at 4℃ and used for subsequent sequencing.

[0085] The PCR amplification product was tested by 1.5% agarose gel electrophoresis (w / v). If the electrophoresis band was consistent with the target band, the PCR product was sent to the company for unidirectional Sanger sequencing. The genotyping of the candidate loci was statistically analyzed based on the sequencing results.

[0086] 2. Experimental Results

[0087] In the sequencing results, in addition to the two sites g.2040798G>A and g.2040864A>G in the GWAS analysis, two new SNP sites were found, namely g.2040857G>T and g.2040873T>C. The results of the two new SNP sites are consistent with the sites obtained by GWAS analysis and are also located in the intron region of chromosome 34 of the Ensembl chicken reference genome (CRCg7b version). The phenotypic results of these four sites were subsequently counted.

[0088] like Figure 3 As shown in Figure 4, the sequencing results of the four SNP sites are all divided into three genotypes, wild type, heterozygous type and mutant type. The statistics of all individual results of these sites are shown in Table 4. It can be found that in each site, all the curly-feathered chickens in western Guangdong are mutants, while the phenotypes of other non-curly-feathered chickens are wild type or heterozygous. Therefore, these four SNP sites can be used to clearly distinguish the curly-feathered chickens in western Guangdong from other non-curly-feathered chickens.

[0089] Table 4 Genotyping statistics of 4 SNP loci

[0090]

[0091] Example 3 A method for identifying the feather traits of chickens

[0092] 1. Extract DNA from the chicken sample to be tested.

[0093] 2. Use the DNA of the chicken sample to be tested extracted in step 1 as a template for PCR amplification:

[0094] PCR reaction system: 25 μL of 2×Es Taq MasterMix, 2 μL of each primer with nucleotide sequence as shown in SEQ ID NO: 1-2, 1 μL of DNA template, add ddH2O to 50 μL.

[0095] PCR reaction program: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 30 s, 32 cycles; extension at 72°C for 2 min.

[0096] 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.

[0097] 4. Result judgment

[0098] SNP1 is located at position 2040798 on chromosome 34 of the Ensembl chicken reference genome (CRCg7b version) and is a G or A polymorphic, with GG, GA, and AA genotypes;

[0099] SNP2 is located at position 2040857 on chromosome 34 of the Ensembl chicken reference genome (CRCg7b version) and is a G or T polymorphism, with GG, GT, and TT genotypes.

[0100] SNP3 is located at position 2040864 on chromosome 34 of the Ensembl chicken reference genome (CRCg7b version) and is an A or G polymorphic, with AA, AG, and GG genotypes present;

[0101] SNP4 is located at position 2040873 on chromosome 34 of the Ensembl chicken reference genome (CRCg7b version) and is a T or C polymorphism, with TT, TC, and CC genotypes;

[0102] The individuals whose genotype of SNP1 is AA, whose genotype of SNP2 is TT, whose genotype of SNP3 is GG or whose genotype of SNP4 is CC are curly-feathered chickens, otherwise they are flat-feathered chickens.

[0103] Example 4 A kit for identifying chicken feather traits

[0104] 1. Composition

[0105] The nucleotide sequences are shown in SEQ ID NO: 1-2, primers, 2×Es Taq MasterMix and ddH2O.

[0106] 2. Usage

[0107] The detection and result interpretation were performed according to Example 3.

[0108] 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. An application of a molecular marker in identifying the feather traits of chickens, characterized in that: The molecular marker consists of four single nucleotide polymorphism sites SNP1, SNP2, SNP3 and / or SNP4: SNP1 is located at position 2040798 on chromosome 34 of the chicken genome and is a G or A polymorphism, with GG, GA, and AA genotypes. SNP2 is located at position 2040857 on chromosome 34 of the chicken genome and is a G or T polymorphism, with GG, GT, and TT genotypes. SNP3 is located at position 2040864 on chromosome 34 of the chicken genome and is an A or G polymorphism, with AA, AG, and GG genotypes. SNP4 is located at position 2040873 on chromosome 34 of the chicken genome and is a T or C polymorphism, with TT, TC, and CC genotypes. The chicken genome is the CRCg7b version of the Ensembl database; The feather phenotype of the individual whose SNP1 genotype is AA, whose SNP2 genotype is TT, whose SNP3 genotype is GG or whose SNP4 genotype is CC is curly feather.

2. The molecular marker detection reagent according to claim 1, characterized in that: The detection reagent is a primer with a nucleotide sequence as shown in SEQ ID NO: 1-2.

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 molecular marker described in claim 1 is detected using the detection reagent described in claim 2.

8. The method according to claim 7, characterized in that The following steps are involved: S1. Extract genomic DNA from the chicken 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, According to the molecular markers described in claim 1, the feather traits of the chicken to be tested are determined.

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

10. Use of the method according to any one of claims 7 to 9 in molecular assisted breeding of Dalmatian chicken.

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