Molecular marker associated with chicken rumpless trait, reagent for detecting the same and application thereof

Abstract

The application discloses a molecular marker related to a chicken curly feather trait, a detection reagent and application thereof. Through whole genome resequencing and whole genome association analysis, four SNP sites capable of distinguishing two phenotypes of curly feather and vane feather of chickens are obtained on chromosome 34 of an Ensembl chicken reference genome GRCg7b version 34. The application further provides detection reagents and kits for detecting the four SNP sites, which can be applied to early assisted selection of curly feather chickens, can save production cost and accelerate the molecular breeding process, and thus improve the economic benefits of local chicken breeding.

Description

Technical Field

[0001] This invention relates to the technical field of poultry breeding, and more specifically, to molecular markers related to the curly feather trait in chickens, their detection reagents, and applications. Background Technology

[0002] Poultry exhibit a wide variety of feather structures, demonstrating a high degree of diversity. Feather-related traits are important appearance-related traits influencing chicken selection and breeding. Curly feathers are an important type of poultry feather, facilitating heat dissipation from the chicken's body surface, making them suitable for raising in tropical regions or southern my country, and are of great significance for breeding heat-stress-resistant chicken breeds. Furthermore, curly feathers can serve as a unique outer packaging trait and breed characteristic, distinguishing chickens from other breeds; therefore, breeding curly-feathered chickens has significant economic value.

[0003] The Western Guangdong Dalmatian chicken, with its unique curly feather appearance, is also known for its hardiness, resilience, and large size. It is a popular local breed, raised in Zhanjiang and Maoming, Guangdong. Currently, conservation and breeding efforts for the Western Guangdong Dalmatian chicken are underway, but the available loci and identification techniques related to the curly feather trait are still limited, failing to meet the needs of the poultry industry. Therefore, it is necessary to identify more loci associated with the curly feather trait to establish a rapid and convenient molecular breeding technique for its identification. Summary of the Invention

[0004] To overcome the aforementioned defects and shortcomings in the existing technology, the present invention provides molecular markers related to chicken curly feather traits, their detection reagents, and applications.

[0005] The first objective of this invention is to provide an application of molecular markers in identifying feather traits in chickens.

[0006] A second objective of this invention is to provide a detection reagent for the aforementioned molecular markers.

[0007] A third objective of this invention is to provide a reagent kit.

[0008] A fourth object of the present invention is to provide the application of the above-described detection reagents and / or kits in identifying feather traits in chickens.

[0009] A fifth objective of this invention is to provide the application of the above-described detection reagents and / or kits in the preparation of chicken breeding products.

[0010] The sixth object of the present invention is to provide a method for identifying feather characteristics in chickens.

[0011] The seventh objective of this invention is to provide the application of the above-described method in molecular-assisted breeding of Dalmatian chickens.

[0012] Therefore, this invention claims protection for the following:

[0013] The application of a molecular marker in identifying feather traits in chickens, said molecular marker consisting 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. It is a G or A polymorphism and has GG, GA and AA genotypes.

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

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

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

[0018] The chicken genome is version GRCg7b of the Ensembl database;

[0019] Individuals with the genotype AA for SNP1, TT for SNP2, GG for SNP3, or CC for SNP4 exhibit a curly feather phenotype.

[0020] The above-mentioned molecular marker detection reagents are primers with nucleotide sequences as shown in SEQ ID NO: 1-2.

[0021] A kit comprising the above-described detection reagents.

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

[0023] The application of the above-mentioned test reagents and / or kits in identifying feather traits in chickens.

[0024] The above-mentioned detection reagents and / or kits are used in the preparation of chicken breeding products, wherein the breeding is molecular breeding of feather traits.

[0025] A method for identifying feather traits in chickens, using the aforementioned detection reagent to detect the aforementioned molecular markers.

[0026] As one feasible approach, the method includes the following steps:

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

[0028] S2. Use the above detection reagents to perform PCR amplification on the genomic DNA obtained in step S1, and then sequence the PCR amplification products 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 molecular markers mentioned above.

[0030] As one feasible approach, in step S2, the sequencing is Sanger sequencing.

[0031] The above methods are applied in molecular-assisted breeding of Dalmatian chickens.

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

[0033] This invention discloses molecular markers associated with the curly feather trait in chickens, along with their detection reagents and applications. Through whole-genome resequencing and genome-wide association analysis, this invention identified four SNP loci on chromosome 34 of the Ensembl chicken reference genome (GRCg7b version) that can distinguish between the curly and flat-feather phenotypes in chickens. This invention also provides detection reagents and kits for these four SNP loci, which can be applied to early-stage auxiliary selection in curly-feathered chickens, saving production costs and accelerating the molecular breeding process, thereby improving the economic benefits of breeding local chicken breeds. Attached Figure Description

[0034] Figure 1 This is a sequencing flowchart.

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

[0036] Figure 3 The image shows the Sanger sequencing results for different genotypes at four SNP loci. Detailed Implementation

[0037] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

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

[0039] Example 1: Whole Genome Resequencing

[0040] I. Experimental Methods

[0041] 1. DNA sample preparation

[0042] Preparation of DNA samples from the Guangdong West Dalmatian: Blood samples were collected from 356 140-day-old Guangdong West Dalmatian chickens. Blood was collected from the subwing vein and stored at -20℃ for subsequent use. Genomic DNA was extracted from the whole blood samples of the 356 Guangdong West Dalmatian chickens using the Nanjing Novizan DNA / RNA extraction kit. The DNA was stored at -20℃ for subsequent resequencing and PCR amplification.

[0043] Preparation of DNA samples from other chicken breeds: Blood samples were collected from 210 chickens, including 30 each of the following breeds: Qibainong chicken, Dwarf chicken, Dwarf curly-feathered chicken, Wenchang chicken, Huaixiang chicken, Qingyuan Ma chicken, and Bearded chicken. Genomic DNA was extracted from the whole blood samples of the above chicken breeds using the Nanjing Novizan DNA / RNA extraction kit 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 Dalmatian chickens in western Guangdong were sent to BGI Genomics Co., Ltd. in Shenzhen for whole-genome resequencing.

[0047] The main steps include: ① DNA sample detection; ② DNA sample fragmentation; ③ Fragment size selection; ④ End repair, addition of "A", and adapter ligation; ⑤ PCR reaction and product recovery; ⑥ PCR product circularization; ⑦ Library detection; ⑧ Sequencing. Single-stranded circular DNA molecules replicate through rolling circles to form a DNA nanosphere (DNB) containing more than 300 copies. The obtained DNB is then added to the mesh wells on a high-density DNA nanochip using high-density DNA nanochip technology, and sequencing is performed using combined probe-anchored polymerization (cPAS) technology. The specific process is as follows: Figure 1 As shown.

[0048] (2) Data Filtering

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

[0050] ① Filtering adapters: If the sequencing read matches 25.0% or more of the adapter sequence (maximum of 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, the entire read is deleted;

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

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

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

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

[0056] (3) Obtaining data

[0057] Dalmatian feather data: Paired-end sequencing data from 356 Dalmatian chicken samples from western Guangdong were aligned to the Ensembl chicken reference genome (GRCg7b 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: SNP and InDel VCF files from the resequencing data of 60 Qibainong chickens in the early stage of the laboratory, and raw fastq data of 25 Qiandongnan Xiaoxiang chickens published in the NCBI database, were processed by the laboratory's server platform to obtain SNP and InDel VCF files.

[0059] 3. Genome-wide association analysis

[0060] Using the SNP and InDel data mentioned above, genome-wide association analysis (GWAS) was performed using emmax software to associate the phenotypes of tufted feathers and tufted feathers. Manhattan plots and QQplots were generated to screen for variant sites that exceeded the threshold. The most significant P-value was used as the criterion to select the most suitable candidate sites for analysis.

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

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

[0063] ②Phenographic data preparation: Each individual was labeled, with the curly feather trait labeled as 0 and the non-curly feather trait labeled as 1.

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

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

[0066] ⑤ Use R language to draw Manhattan plots and QQplots of the GWAS results.

[0067] II. Experimental Results

[0068] Sequencing results showed a large number of significant sites on chromosomes 34 and Z, with chromosome 34 exhibiting a particularly high peak. A total of 473 significant SNPs were found on chromosome 34. Figure 2 In A), the top 30 p-values ​​were selected for statistical analysis (Table 1).

[0069] Table 1 lists the top 30 SNP sites on chromosome 34 with significant p-values ​​detected by GWAS.

[0070]

[0071]

[0072] Example 2: PCR Amplification and Sanger Sequencing

[0073] I. Experimental Methods

[0074] 1. Design primers

[0075] Based on the loci and P-values ​​in Table 1 of Example 1, a variant locus at position 2040798 on chromosome 34 was selected as an auxiliary candidate locus for verification. This locus contains a point mutation of G>A (g. 2040798G>A). Table 1 shows that there is another variant locus at position 2040864 nearby (g. 2040864A>G).

[0076] Specific amplification primers were designed for loci 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] DNA samples from Dalmatian and Elm-feathered chickens were validated using PCR amplification primers. The samples used for PCR validation are shown in Table 1.

[0081] Table 3 Samples used for PCR validation

[0082] variety Quantity (pieces) Western Guangdong Dwarf Chicken 90 dwarf chicks 30 700 Chicken 30 Wenchang Chicken 30 Huaixiang Chicken 30 Bearded Chicken 30 Qingyuan Ma Chicken 30

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

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

[0085] The PCR amplification products were analyzed by 1.5% agarose gel electrophoresis (w / v). If the electrophoretic bands matched the target band size, the obtained PCR products were sent to the company for one-way Sanger sequencing. Genotyping of candidate loci was then analyzed based on the sequencing results.

[0086] II. Experimental Results

[0087] In the sequencing results, in addition to the two loci g.2040798G>A and g.2040864A>G identified in the GWAS analysis, two new SNP loci were discovered: g.2040857G>T and g.2040873T>C. These two new SNP loci are consistent with those obtained from the GWAS analysis and are also located in an intron region on chromosome 34 of the Ensembl chicken reference genome (GRCg7b version). The phenotypic results of these four loci were subsequently analyzed.

[0088] like Figure 3 As shown in the diagram, the sequencing results of the four SNP loci were all classified into three genotypes: wild-type, heterozygous, and mutant. Statistical analysis of all individual results at these loci, as shown in Table 4, reveals that at each locus, all individuals in the Yuexi Dalmatian chicken exhibited the mutant phenotype, while other non-Dalmatian chickens showed either the wild-type or heterozygous phenotype. Therefore, these four SNP loci can be used to clearly distinguish the Yuexi Dalmatian chicken from other non-Dalmatian chickens.

[0089] Table 4. Genotyping statistics of the four SNP loci

[0090]

[0091] Example 3: A method for identifying feather traits in chickens.

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

[0093] 2. Using the DNA extracted from the chicken sample in step 1 as a template, perform PCR amplification:

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

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

[0096] 3. Perform agarose gel electrophoresis on the PCR amplification products obtained in step 2. If the electrophoretic band is the same size as the target band and is clear and bright, then sequence the PCR amplification products obtained in step 2. Compare the sequencing results with chromosome 34 of the Ensembl chicken reference genome (GRCg7b version) and perform genotyping.

[0097] 4. Result Judgment

[0098] SNP1 is located at position 2040798 on chromosome 34 of the Ensembl chicken reference genome (GRCg7b version), and is a G or A polymorphism, with GG, GA and AA genotypes.

[0099] SNP2 is located at position 2040857 on chromosome 34 of the Ensembl chicken reference genome (GRCg7b 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 (GRCg7b version), and is an A or G polymorphism, with AA, AG and GG genotypes.

[0101] SNP4 is located at position 2040873 on chromosome 34 of the Ensembl chicken reference genome (GRCg7b version), and is a T or C polymorphism, with TT, TC and CC genotypes.

[0102] Individuals with the genotype AA for SNP1, TT for SNP2, GG for SNP3, or CC for SNP4 are Dalmatian chickens; otherwise, they are unicorn chickens.

[0103] Example 4: A kit for identifying feather traits in chickens

[0104] I. Composition

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

[0106] II. Usage Method

[0107] The detection and result interpretation were carried out in accordance with Example 3.

[0108] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a molecular marker detection reagent in identifying feather traits in chickens, characterized in that, The molecular markers consist of single nucleotide polymorphism sites SNP1, SNP2, SNP3, and / or SNP4: SNP1 is located at position 2040798 on chromosome 34 of the chicken genome. It is a G or A polymorphism and has GG, GA and AA genotypes. SNP2 is located at position 2040857 on chromosome 34 of the chicken genome. It is a G or T polymorphism and has GG, GT and TT genotypes. SNP3 is located at position 2040864 on chromosome 34 of the chicken genome. It is an A or G polymorphism and has AA, AG and GG genotypes. SNP4 is located at position 2040873 on chromosome 34 of the chicken genome. It is a T or C polymorphism and has TT, TC and CC genotypes. The chicken genome is version GRCg7b of the Ensembl database; Individuals with the genotype AA for SNP1, TT for SNP2, GG for SNP3, or CC for SNP4 have a curly feather phenotype. The chickens mentioned are the Western Guangdong Dwarf Chicken, Dwarf Chicken, Qibainong Chicken, Wenchang Chicken, Huaixiang Chicken, Bearded Chicken, or Qingyuan Ma Chicken.

2. The application 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. The application of a reagent kit in identifying feather traits in chickens, characterized in that, The kit contains detection reagents for molecular markers, which consist of single nucleotide polymorphism sites SNP1, SNP2, SNP3, and / or SNP4: SNP1 is located at position 2040798 on chromosome 34 of the chicken genome. It is a G or A polymorphism and has GG, GA and AA genotypes. SNP2 is located at position 2040857 on chromosome 34 of the chicken genome. It is a G or T polymorphism and has GG, GT and TT genotypes. SNP3 is located at position 2040864 on chromosome 34 of the chicken genome. It is an A or G polymorphism and has AA, AG and GG genotypes. SNP4 is located at position 2040873 on chromosome 34 of the chicken genome. It is a T or C polymorphism and has TT, TC and CC genotypes. The chicken genome is version GRCg7b of the Ensembl database; Individuals with the genotype AA for SNP1, TT for SNP2, GG for SNP3, or CC for SNP4 have a curly feather phenotype. The chickens mentioned are the Western Guangdong Dwarf Chicken, Dwarf Chicken, Qibainong Chicken, Wenchang Chicken, Huaixiang Chicken, Bearded Chicken, or Qingyuan Ma Chicken.

4. The application according to claim 3, characterized in that, The detection reagent is a primer with a nucleotide sequence as shown in SEQ ID NO: 1-2; The kit also contains PCR amplification reagents and ddH2O.

5. The application of a molecular marker detection reagent in the preparation of chicken breeding products, characterized in that, The breeding is molecular breeding for feather traits; The molecular markers consist of single nucleotide polymorphism sites SNP1, SNP2, SNP3, and / or SNP4: SNP1 is located at position 2040798 on chromosome 34 of the chicken genome. It is a G or A polymorphism and has GG, GA and AA genotypes. SNP2 is located at position 2040857 on chromosome 34 of the chicken genome. It is a G or T polymorphism and has GG, GT and TT genotypes. SNP3 is located at position 2040864 on chromosome 34 of the chicken genome. It is an A or G polymorphism and has AA, AG and GG genotypes. SNP4 is located at position 2040873 on chromosome 34 of the chicken genome. It is a T or C polymorphism and has TT, TC and CC genotypes. The chicken genome is version GRCg7b of the Ensembl database; Individuals with the genotype AA for SNP1, TT for SNP2, GG for SNP3, or CC for SNP4 have a curly feather phenotype. The chickens mentioned are the Western Guangdong Dwarf Chicken, Dwarf Chicken, Qibainong Chicken, Wenchang Chicken, Huaixiang Chicken, Bearded Chicken, or Qingyuan Ma Chicken.

6. The application of a reagent kit in the preparation of chicken breeding products, characterized in that, The kit contains a molecular marker detection reagent, and the breeding is molecular breeding for the feather trait; The molecular markers consist of single nucleotide polymorphism sites SNP1, SNP2, SNP3, and / or SNP4: SNP1 is located at position 2040798 on chromosome 34 of the chicken genome. It is a G or A polymorphism and has GG, GA and AA genotypes. SNP2 is located at position 2040857 on chromosome 34 of the chicken genome. It is a G or T polymorphism and has GG, GT and TT genotypes. SNP3 is located at position 2040864 on chromosome 34 of the chicken genome. It is an A or G polymorphism and has AA, AG and GG genotypes. SNP4 is located at position 2040873 on chromosome 34 of the chicken genome. It is a T or C polymorphism and has TT, TC and CC genotypes. The chicken genome is version GRCg7b of the Ensembl database; Individuals with the genotype AA for SNP1, TT for SNP2, GG for SNP3, or CC for SNP4 have a curly feather phenotype. The chickens mentioned are the Western Guangdong Dwarf Chicken, Dwarf Chicken, Qibainong Chicken, Wenchang Chicken, Huaixiang Chicken, Bearded Chicken, or Qingyuan Ma Chicken.

7. The application according to claim 6, characterized in that, The detection reagent is a primer with a nucleotide sequence as shown in SEQ ID NO: 1-2; The kit also contains PCR amplification reagents and ddH2O.

8. A method for identifying feather characteristics in chickens, characterized in that, Detect molecular markers using diagnostic reagents; The molecular markers consist of single nucleotide polymorphism sites SNP1, SNP2, SNP3, and / or SNP4: SNP1 is located at position 2040798 on chromosome 34 of the chicken genome. It is a G or A polymorphism and has GG, GA and AA genotypes. SNP2 is located at position 2040857 on chromosome 34 of the chicken genome. It is a G or T polymorphism and has GG, GT and TT genotypes. SNP3 is located at position 2040864 on chromosome 34 of the chicken genome. It is an A or G polymorphism and has AA, AG and GG genotypes. SNP4 is located at position 2040873 on chromosome 34 of the chicken genome. It is a T or C polymorphism and has TT, TC and CC genotypes. The chicken genome is version GRCg7b of the Ensembl database; Individuals with the genotype AA for SNP1, TT for SNP2, GG for SNP3, or CC for SNP4 have a curly feather phenotype. The chickens mentioned are the Western Guangdong Dwarf Chicken, Dwarf Chicken, Qibainong Chicken, Wenchang Chicken, Huaixiang Chicken, Bearded Chicken, or Qingyuan Ma Chicken; The detection reagent is a primer with a nucleotide sequence as shown in SEQ ID NO: 1-2.

9. The method according to claim 8, characterized in that, Includes the following steps: S1. Extract genomic DNA from the chicken to be tested; S2. Perform PCR amplification on the genomic DNA obtained in step S1 using detection reagents, and then sequence the PCR amplification products to obtain sequencing data; S3. Analyze the sequencing data obtained in step S2, and determine the feather traits of the chicken to be tested based on molecular markers.

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

11. The application of the method according to any one of claims 8 to 10 in molecular-assisted breeding of Dalmatian chickens in western Guangdong, characterized in that, The molecular-assisted breeding refers to molecular-assisted breeding of the feather trait.

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