A molecular marker combination for determining chicken feather color and a method for its application in breeding

Abstract

The present application belongs to the technical field of biological breeding, and relates to a chicken feather color related molecular marker combination, primers, and a method and application for determining feather color, breeding and / or producing specific feather color chickens. The present application provides a chicken feather color related molecular marker combination, which comprises an MC1R gene, a PMEL17 gene, an SLC45A2 gene, a TYR gene and a SOX10 gene. The present application solves the problem that the existing single marker molecular breeding is not widely applicable, and according to the combination mode of various genes listed in the present application, a target feather color chicken complete set can be quickly and effectively bred.

Description

Technical Field

[0001] This invention belongs to the field of biological breeding technology, specifically relating to a combination of molecular markers related to chicken feather color, primers, and methods and applications for determining feather color, breeding and / or producing chickens with specific feather colors. Background Technology

[0002] While chicken production performance is not affected by feather color, people generally prefer chickens with colored feathers. Furthermore, feather color is easily identifiable and can serve as a genetic marker for breeding. Feather color-linked genes can be used to breed sex-differentiated poultry lines based on feather color. Therefore, for breeding high-quality chickens in China, feather color is an important selection indicator with significant economic value.

[0003] Because feather color traits in chickens are controlled by numerous genes, feather color selection is one of the key challenges in poultry breeding. However, with advancements in modern biotechnology and extensive research into feather pigmentation at the molecular and cell biological levels, many feather color-related genes and causative mutation sites have been identified. These include the melanocortin receptor 1 (MC1R) gene encoding the melanin diffusion site (E), the premelanosome protein 17 (PMEL17) gene encoding the dominant white locus (I), the tyrosinase (TYR) gene encoding the recessive white locus (c), and the sex-determining region Y-box 10 (SOX10) gene encoding the dark brown locus (DB). To date, identified feather color-related gene variations include insertion / deletion mutations in amino acid sequences; missense mutations in amino acid sequences; frameshift mutations caused by base insertions or deletions; variations in non-coding regions of genes; and alternative splicing variations caused by exogenous sequence insertions. In summary, feather color formation is a complex and delicate process, precisely regulated by numerous genes and multiple signaling pathways. Molecular breeding methods utilizing these genetic variations are frequently used to develop breeding lines for specific feather colors in chickens. However, previous techniques often focused only on the role of a single genetic variation in breeding line development. Summary of the Invention

[0004] The purpose of this invention is to provide a combination of molecular markers related to chicken feather color, primers, and methods and applications for determining feather color, breeding, and / or producing chickens with specific feather colors. This invention solves the problem that existing molecular breeding methods using only a single marker lack broad applicability. Based on the various gene combination patterns listed in this invention, chicken breeding lines with the target feather color can be rapidly and effectively bred.

[0005] This invention provides a combination of chicken feather color-related molecular markers, which include the MC1R gene, PMEL17 gene, SLC45A2 gene, TYR gene, and SOX10 gene.

[0006] This invention also provides the application of reagents for detecting chicken feather color-related molecular markers in breeding and / or producing chickens and chicken breeding lines with specific feather colors or in determining chicken feather color. The chicken feather color-related molecular markers include the MC1R gene, PMEL17 gene, SLC45A2 gene, TYR gene, and SOX10 gene.

[0007] Preferably, the parent chickens used in the breeding and / or production process include White Leghorn chickens.

[0008] Preferably, the reagent includes primers; the primers include primers MC1R-F and MC1R-R for detecting the MC1R gene and primer PMEL17-F for detecting the PMEL17 gene. 2 and PMEL17-R 2 Primers for detecting the SLC45A2 gene: SLC45A2-ex4F and SLC45A2-ex4R; primers for detecting the TYR gene: TYR-F1, TYR-R and TYR-F2; and primers for detecting the SOX10 gene: SOX10-1F, SOX10-1R and SOX10-6R.

[0009] The nucleotide sequence of MC1R-F is shown in SEQ ID NO.1, and the nucleotide sequence of MC1R-R is shown in SEQ ID NO.2; PMEL17-F 2 The nucleotide sequence is shown in SEQ ID NO.3, namely PMEL17-R. 2 The nucleotide sequences of the following are shown in SEQ ID NO.4; the nucleotide sequences of the SLC45A2-ex4F are shown in SEQ ID NO.5, the nucleotide sequences of the SLC45A2-ex4R are shown in SEQ ID NO.6; the nucleotide sequences of the TYR-F1 are shown in SEQ ID NO.7, the nucleotide sequences of the TYR-R are shown in SEQ ID NO.8, the nucleotide sequences of the TYR-F2 are shown in SEQ ID NO.9; the nucleotide sequences of the SOX10-1F are shown in SEQ ID NO.10, the nucleotide sequences of the SOX10-1R are shown in SEQ ID NO.11, and the nucleotide sequences of the SOX10-6R are shown in SEQ ID NO.12.

[0010] This invention also provides a set of primers for detecting chicken feather color-related molecular markers, including primers MC1R-F and MC1R-R for detecting the MC1R gene and primer PMEL17-F for detecting the PMEL17 gene. 2 and PMEL17-R 2 Primers for detecting the SLC45A2 gene: SLC45A2-ex4F and SLC45A2-ex4R; primers for detecting the TYR gene: TYR-F1, TYR-R and TYR-F2; and primers for detecting the SOX10 gene: SOX10-1F, SOX10-1R and SOX10-6R.

[0011] The nucleotide sequence of MC1R-F is shown in SEQ ID NO.1, and the nucleotide sequence of MC1R-R is shown in SEQ ID NO.2; PMEL17-F 2 The nucleotide sequence is shown in SEQ ID NO.3, namely PMEL17-R. 2 The nucleotide sequences of the following are shown in SEQ ID NO.4; the nucleotide sequences of the SLC45A2-ex4F are shown in SEQ ID NO.5, the nucleotide sequences of the SLC45A2-ex4R are shown in SEQ ID NO.6; the nucleotide sequences of the TYR-F1 are shown in SEQ ID NO.7, the nucleotide sequences of the TYR-R are shown in SEQ ID NO.8, the nucleotide sequences of the TYR-F2 are shown in SEQ ID NO.9; the nucleotide sequences of the SOX10-1F are shown in SEQ ID NO.10, the nucleotide sequences of the SOX10-1R are shown in SEQ ID NO.11, and the nucleotide sequences of the SOX10-6R are shown in SEQ ID NO.12.

[0012] This invention also provides a method for determining the color of chicken feathers, comprising the following steps:

[0013] Using the primers described in the above technical solution, the MC1R gene, PMEL17 gene, SLC45A2 gene, TYR gene, and SOX10 gene were amplified by PCR. Based on the size of the PCR amplification products and the genotype of each gene, the chicken feather color was determined according to the judgment method in the table below:

[0014]

[0015] Preferably, each 50 μL of the PCR amplification reaction system for the MC1R gene comprises: PCR SuperMix (+dye) 25 μL, MC1R-F 1 μL, MC1R-R 1 μL, genomic DNA 6.25 μL and ddH2O 16.75 μL;

[0016] Each 20 μL reaction system for PCR amplification of the PMEL17 gene includes: 10 μL of 2×Taq PCR Mix, PMEL17-F 2 0.3μL, PMEL17-R 2 0.3 μL, 1 μL of genomic DNA, and 8.4 μL of ddH2O;

[0017] Each 50 μL reaction volume for PCR amplification of the SLC45A2 gene includes: PCR SuperMix (+dye) 25 μL, SLC45A2-ex4F 1 μL, SLC45A2-ex4R 1 μL, genomic DNA 6.25 μL and ddH2O 16.75 μL;

[0018] Each 20 μL reaction system for PCR amplification of the TYR gene includes: PCR SuperMix (+dye) 10 μL, TYR-F1 or TYR-F 0.4 μL, TYR-R 0.4 μL, genomic DNA 2.5 μL and ddH2O 6.7 μL;

[0019] Each 20 μL reaction system for PCR amplification of the SOX10 gene includes: PCR SuperMix (+dye) 10 μL, SOX10-1F 0.4 μL, SOX10-1R or SOX10-6R 0.4 μL, genomic DNA 2.5 μL and ddH2O 6.7 μL.

[0020] Preferably, the reaction conditions for PCR amplification of the MC1R gene include: 98℃, 2 min; 98℃, 10 s, 60℃, 30 s, 72℃, 1 min, 35 cycles; 72℃, 5 min;

[0021] The reaction conditions for PCR amplification of the PMEL17 gene included: 95℃ for 5 min; 95℃ for 30 s, 67℃ for 30 s, 72℃ for 30 s, 34 cycles; 72℃ for 7 min.

[0022] The reaction conditions for PCR amplification of the SLC45A2 gene were as follows: 94℃, 5 min; 94℃, 30 s, 60℃, 30 s, 72℃, 15 s, 35 cycles; 72℃, 10 min.

[0023] The reaction conditions for PCR amplification of the TYR gene are as follows: TYR-F1 / TYR-R: 95℃, 3 min; 95℃, 30 s, 62℃, 30 s, 72℃, 1 min, 35 cycles; 72℃, 5 min; or TYR-F2 / TYR-R: 95℃, 3 min; 95℃, 30 s, 60℃, 30 s, 72℃, 1 min, 35 cycles; 72℃, 5 min.

[0024] The reaction conditions for PCR amplification of the SOX10 gene included: 94℃, 5 min; 94℃, 30 s, 60℃, 30 s, 72℃, 1 min, 30 cycles; 72℃, 10 min.

[0025] The present invention also provides a method for breeding and / or producing hybrid offspring chickens with a specific feather color, comprising the following steps:

[0026] The target feather color of the hybrid offspring is determined. Based on the genetic variation combination pattern corresponding to different feather colors of chickens in the method described above, the genotype of the specific genetic variation of the parents is inferred. The chicken parents are purified according to the genotype of the specific genetic variation of the parents. The purified chicken parents are then used for hybridization to obtain hybrid offspring with specific feather colors.

[0027] Preferably, the matching mode for producing hybrid offspring chickens with specific feather color by genotypic combinations of specific genetic variations of the parents is shown in the table below:

[0028]

[0029]

[0030] Where DB*N / DB*N represents the homozygous wild type; DB*DB / DB*DB represents the dark brown homozygous type; and DB*DB / DB*N represents the dark brown heterozygous type.

[0031] This invention provides a molecular marker combination related to chicken feather color. Using this molecular marker combination, chicken feather color can be determined, and the breeding and production of chickens with specific feather colors can be achieved. Crossbreeding white Leghorn chickens with other colored-feathered chickens (red or black-feathered) is a common method for breeding laying hen breeding lines. However, the feather color of the offspring is not fixed, exhibiting different phenotypes such as white feathers, red feathers, red and white, and white feathers with black spots. This invention provides a molecular marker combination related to chicken feather color, listing the genotypes of five genes corresponding to offspring with specific feather colors, as well as all available crossbreeding combinations. It provides a practical molecular breeding method for breeding feather-related breeding lines. This invention, by detecting the genetic variation combinations of the MC1R, PMEL17, SLC45A2, TYR, and SOX10 genes in the paternal and maternal chickens, can quickly determine the feather color of offspring. It can also determine the genotypes of each gene in the parents based on the target feather color of the hybrid offspring. By purifying specific genetic variations of these genes in the parents, breeding lines with specific feather colors can be cultivated. In other words, breeders can purify and select the genotypes of each gene in the parent chickens based on the gene variation combinations corresponding to the target feather color of the hybrid offspring, thereby cultivating a laying hen breeding line that meets the expected specifications. This invention provides the feather colors corresponding to the above-mentioned different combinations of genetic variations in hybrid offspring. Existing technologies focus on the influence of single gene variations on feather color, neglecting the interactions between genes. Often, a single marker applicable in one hybrid breeding combination is no longer applicable in other similar hybrid combinations because the determining role of the genetic variation combination patterns (interactions) of each gene on feather color has not been clarified. This invention solves the problem that existing molecular breeding methods using only single markers lack broad applicability. Based on the various gene combination patterns listed in this invention, chicken breeding lines with the target feather color can be cultivated quickly and effectively. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The image shows a gel electrophoresis diagram of the MC1R gene PCR product provided by this invention.

[0034] Figure 2 This is a first-generation sequencing peak diagram of the MC1R gene PCR product provided by the present invention;

[0035] Figure 3 The image shows the detection results of the PMEL17 gene insertion mutation provided by this invention.

[0036] Figure 4This is a gel electrophoresis image of the SLC45A2 gene PCR product provided by the present invention;

[0037] Figure 5 This is a first-generation sequencing peak diagram of the SLC45A2 gene PCR product provided by the present invention;

[0038] Figure 6 The image shows the detection results of the 8.3kb deletion mutation in the SOX10 gene of chickens provided by this invention.

[0039] Figure 7 The image shows the detection results of the 7.7kb insertion mutation in the TYR gene of chickens provided by this invention.

[0040] Figure 8 Genotyping diagram of MC1R, SLC45A2, PMEL17, TYR, and SOX10 genes in the hybrid offspring of roosters (white feathers) and hens (red feathers) provided by this invention;

[0041] Figure 9 Genotyping diagram of MC1R, SLC45A2, PMEL17, TYR, and SOX10 genes in hybrid offspring hens (white feathers) provided by this invention;

[0042] Figure 10 Genotyping diagram of the MC1R, SLC45A2, PMEL17, TYR, and SOX10 genes for red and white feathered chickens provided by this invention. Detailed Implementation

[0043] This invention provides a combination of molecular markers related to chicken feather color, comprising the MC1R gene, PMEL17 gene, SLC45A2 gene, TYR gene, and SOX10 gene. The above-mentioned molecular marker combination is associated with chicken feather color. Specifically, the MC1R gene may or may not have an A / G single nucleotide mutation at position 427; the PMEL17 gene may or may not have a 9 bp insertion mutation in exon 10; the SLC45A2 gene may or may not have a C / A single nucleotide mutation at position 1111 in exon 4; the TYR gene may or may not have a 7.7 kb insertion sequence in intron 4; and the SOX10 gene may or may not have an 8.3 kb genomic DNA deletion 14 kb upstream of the transcription start site. By detecting these mutations, chicken feather color can be determined, or the production of chickens with specific feather colors and breeding lines can be guided.

[0044] This invention provides reagents for detecting chicken feather color-related molecular markers in the breeding and / or production of chickens and chicken breeding lines with specific feather colors, or in the determination of chicken feather color. The chicken feather color-related molecular markers include the MC1R gene, PMEL17 gene, SLC45A2 gene, TYR gene, and SOX10 gene. The molecular markers of this invention include the MC1R gene, PMEL17 gene, SLC45A2 gene, TYR gene, and SOX10 gene. This invention can determine chicken feather color or guide the production of chickens and breeding lines with specific feather colors by detecting whether an A / G single nucleotide mutation occurs at position 427 of the MC1R gene; whether a 9 bp insertion mutation is present or absent in exon 10 of the PMEL17 gene; whether a C / A single nucleotide mutation occurs at position 1111 of exon 4 of the SLC45A2 gene; whether a 7.7 kb insertion sequence is present or absent in intron 4 of the TYR gene; and whether an 8.3 kb genomic DNA deletion is present or absent 14 kb upstream of the transcription start site of the SOX10 gene. In this invention, the parent chickens used in the breeding and / or production process preferably include White Leghorn chickens.

[0045] The silver (S) gene, located on sex chromosome Z, is a sex-linked feather color gene locus. This locus contains the silver allele (S), the wild-type gold (S) allele, and the sex-linked imperfect albinism (Sal) allele, with the silver allele being dominant relative to the other two alleles. The silver allele (S*S) in chicken flocks is associated with the transition of exon 4 (1111C>A) (Leu347Met) of the SLC45A2 gene, which affects the transmembrane region 7 of the protein. Because roosters have a ZZ chromosome composition, their silver genotype is homozygous or heterozygous; while hens have a ZW chromosome composition, they are either silver hemizygous or gold hemizygous. The distribution of the SLC45A2 gene C / A alleles in different chicken groups includes the gold homozygous CC (Z s Z s Z s W), Silver Feather Pure Hybrid Type AA(Z) S Z S Z S W), Silver Feather Hybrid CA(Z) S Z s ).

[0046] In chickens, the melanin extension site (E) controlling feather color encodes the MC1R gene, located on chromosome 11. The chicken MC1R gene is a single-exon gene without introns, encoding 314 amino acids, and the encoded protein has seven transmembrane domains. MC1R is the smallest member of the G protein-coupled receptor family and plays a crucial role in melanin production. Studies have found that poultry feather color phenotypes are mainly regulated by different alleles at the MC1R site, with different MC1R genotypes corresponding to different feather colors. The MC1R gene contains the 427A>G mutation, including a GG homozygous type, a GA heterozygous type, and an AA homozygous type.

[0047] The PMEL17 gene in chickens is considered a candidate gene for the dominant non-red feather trait, namely I, the dominant white gene (I), which is incompletely dominant relative to the wild-type allele (i). Kerje et al. performed linkage analysis on a cross between red junglefowl and white leghorn chickens, and the results showed that the dominant white trait was caused by a 9 bp insertion mutation in exon 10 of the PMEL17 gene, which added 3 amino acids to the transmembrane domain of the PMEL17 gene. In the population of this invention, the non-dominant white homozygous genotype is represented as ii; the dominant white homozygous genotype as II; and the dominant white heterozygous genotype as Ii.

[0048] In addition to the wild-type allele, the recessive white feather locus (C) also includes the mutated recessive white feather allele (C0). c ) and autosomal albino allele (C A Several chicken breeds, including the White Locker and Silkie chickens, carry the recessive white feather gene. A Homozygous individuals at the locus exhibited partial albinism, while recessive albinism homozygous individuals displayed a white-feathered phenotype. The study identified the gene encoding the C locus as the tyrosinase (TYR) gene. The chicken TYR gene is located on chromosome 1. An insertion mutation exists in the fourth intron of the chicken TYR gene, specifically the insertion of a complete 7.7kb sequence associated with avian leukosis. In this invention's population, the recessive white-feathered homozygous type is represented as C*C / C*C; the homozygous wild type as C*N / C*N; and the heterozygous wild type as C*N / C*C.

[0049] The darkbrown (DB) gene locus is located on chromosome 1 of the chicken. The darkbrown (DB) locus in chickens can reduce the expression of eumelanin in feathers at specific locations on the wings and increase the expression of pheomelanin. There is a pair of alleles at the darkbrown gene locus: wild-type (DB*N) and darkbrown-type (DB*DB). Gunnarsson et al. found that an 8.3kb deletion of genomic DNA 14kb upstream of the SOX10 gene transcription start site is the mutation that causes the darkbrown feather phenotype in chickens. In the population of this invention, the homozygous darkbrown type is represented as DB*DB / DB*DB; the homozygous wild-type as DB*N / DB*N; and the heterozygous darkbrown type as DB*DB / DB*N.

[0050] This invention can quickly determine the feather color of offspring by detecting the genetic variation combinations of the MC1R, PMEL17, SLC45A2, TYR, and SOX10 genes in the paternal and maternal chickens. It can also determine the genotypes that each gene in the parents should possess based on the target feather color of the hybrid offspring. By purifying the specific genetic variations of the above genes in the parents, it is possible to breed a breeding line that produces a specific feather color.

[0051] In this invention, the reagent includes primers; the primers include primers MC1R-F and MC1R-R for detecting the MC1R gene, and primer PMEL17-F for detecting the PMEL17 gene. 2 and PMEL17-R 2 Primers for detecting the SLC45A2 gene: SLC45A2-ex4F and SLC45A2-ex4R; primers for detecting the TYR gene: TYR-F1, TYR-R and TYR-F2; and primers for detecting the SOX10 gene: SOX10-1F, SOX10-1R and SOX10-6R.

[0052] The nucleotide sequence of MC1R-F is shown in SEQ ID NO.1, and the nucleotide sequence of MC1R-R is shown in SEQ ID NO.2; PMEL17-F 2 The nucleotide sequence is shown in SEQ ID NO.3, namely PMEL17-R. 2The nucleotide sequences of the following are shown in SEQ ID NO.4; the nucleotide sequences of the SLC45A2-ex4F are shown in SEQ ID NO.5, the nucleotide sequences of the SLC45A2-ex4R are shown in SEQ ID NO.6; the nucleotide sequences of the TYR-F1 are shown in SEQ ID NO.7, the nucleotide sequences of the TYR-R are shown in SEQ ID NO.8, the nucleotide sequences of the TYR-F2 are shown in SEQ ID NO.9; the nucleotide sequences of the SOX10-1F are shown in SEQ ID NO.10, the nucleotide sequences of the SOX10-1R are shown in SEQ ID NO.11, and the nucleotide sequences of the SOX10-6R are shown in SEQ ID NO.12.

[0053] This invention also provides a set of primers for detecting chicken feather color-related molecular markers, including primers MC1R-F and MC1R-R for detecting the MC1R gene and primer PMEL17-F for detecting the PMEL17 gene. 2 and PMEL17-R 2 Primers for detecting the SLC45A2 gene: SLC45A2-ex4F and SLC45A2-ex4R; primers for detecting the TYR gene: TYR-F1, TYR-R and TYR-F2; and primers for detecting the SOX10 gene: SOX10-1F, SOX10-1R and SOX10-6R.

[0054] The nucleotide sequence of MC1R-F is shown in SEQ ID NO.1, and the nucleotide sequence of MC1R-R is shown in SEQ ID NO.2; PMEL17-F 2 The nucleotide sequence is shown in SEQ ID NO.3, namely PMEL17-R. 2 The nucleotide sequences of the following are shown in SEQ ID NO.4; the nucleotide sequences of the SLC45A2-ex4F are shown in SEQ ID NO.5, the nucleotide sequences of the SLC45A2-ex4R are shown in SEQ ID NO.6; the nucleotide sequences of the TYR-F1 are shown in SEQ ID NO.7, the nucleotide sequences of the TYR-R are shown in SEQ ID NO.8, the nucleotide sequences of the TYR-F2 are shown in SEQ ID NO.9; the nucleotide sequences of the SOX10-1F are shown in SEQ ID NO.10, the nucleotide sequences of the SOX10-1R are shown in SEQ ID NO.11, and the nucleotide sequences of the SOX10-6R are shown in SEQ ID NO.12.

[0055] This invention also provides a method for determining the color of chicken feathers, comprising the following steps:

[0056] Using the primers described in the above technical solution, the MC1R gene, PMEL17 gene, SLC45A2 gene, TYR gene, and SOX10 gene were amplified by PCR. Based on the size of the PCR amplification products and the genotype of each gene, the chicken feather color was determined according to the judgment method in the table below:

[0057]

[0058] Where DB*N / DB*N represents the homozygous wild type; DB*DB / DB*DB represents the dark brown homozygous type; and DB*DB / DB*N represents the dark brown heterozygous type.

[0059] In this invention, the parent chickens in the determination method preferably include White Leghorn chickens.

[0060] In this invention, the preferred method for determining the phenotype is as follows: the silver feather gene (S) and the recessive white homozygous gene (cc) are absent, and the genotype of the dark brown gene (SOX10) is homozygous wild type (DB*N / DB*N). When the MC1R gene mutates to the GG type, the red feather phenotype is exhibited regardless of the presence or absence of the dominant white gene (I). When the MC1R gene at position 427 is of the GA / AA type, the white feather phenotype is exhibited under the influence of the dominant white gene, and the black feather phenotype is exhibited when the dominant white gene is absent.

[0061] There is no silver feather gene (S) or recessive white homozygous gene (cc), and the genotype of dark brown gene (SOX10) is dark brown heterozygous (DB*DB / DB*N). When the 427 site of MC1R gene is GA type, a red and white feather color phenotype appears under the influence of dominant white gene (I).

[0062] When I and S are present at the same time, regardless of whether the MC1R gene 427 site is GG type or GA / AA type, the white feather phenotype is observed.

[0063] When cc (recessive white) is present, regardless of whether the MC1R gene 427 position is GG type or GA / AA type, it is a white feather phenotype;

[0064] The chromosome composition of roosters is ZZ, and that of hens is ZW.

[0065] Taking the MC1R gene as an example, existing detection methods can select the 427th locus of the MC1R gene in White Leghorn chickens as GG, allowing them to be crossed with red-feathered chickens such as the Loch Ness Red to produce red-feathered offspring. However, this method is not applicable to all crosses between White Leghorn and Loch Ness Red strains. This is because current technology does not consider the genotypes of other genes such as SOX10, PMEL17, and SLC45A2. This invention comprehensively considers the interactions between the genetic variations of various genes. Under the premise that the MC1R gene at position 427 is GG, if the SOX10 genotype is DB*N / DB*N (wild-type homozygous), the SLC45A2 genotype is ss (recessive homozygous), and the TYR gene is CC or Cc (at least one copy of the dominant gene), then the hybrid offspring will have red feathers. If other genes remain unchanged, and the SOX10 genotype is DB*DB / DB*N (heterozygous), then the hybrid offspring will have red and white feathers. If other genes remain unchanged, and the TYR gene is cc (recessive homozygous) or the SLC45A2 genotype is SS or Ss (at least one copy of the dominant gene), then the hybrid offspring will have white feathers.

[0066] In this invention, the PCR amplification reaction system for the MC1R gene preferably comprises, per 50 μL: PCR SuperMix (+dye) 25 μL, MC1R-F 1 μL, MC1R-R 1 μL, genomic DNA 6.25 μL and ddH2O 16.75 μL;

[0067] The optimal reaction system for PCR amplification of the PMEL17 gene is as follows: 10 μL of 2×Taq PCR Mix and PMEL17-F per 20 μL. 2 0.3μL, PMEL17-R 2 0.3 μL, 1 μL of genomic DNA, and 8.4 μL of ddH2O;

[0068] The optimal reaction system for PCR amplification of the SLC45A2 gene includes, per 50 μL: PCR SuperMix (+dye) 25 μL, SLC45A2-ex4F 1 μL, SLC45A2-ex4R 1 μL, genomic DNA 6.25 μL and ddH2O 16.75 μL;

[0069] The optimal reaction system for PCR amplification of the TYR gene is as follows: (20 μL) PCR SuperMix (+dye) 10 μL, TYR-F1 or TYR-F 0.4 μL, TYR-R 0.4 μL, genomic DNA 2.5 μL and ddH2O 6.7 μL;

[0070] The optimal reaction system for PCR amplification of the SOX10 gene comprises, per 20 μL: PCR SuperMix (+dye) 10 μL, SOX10-1F 0.4 μL, SOX10-1R or SOX10-6R 0.4 μL, genomic DNA 2.5 μL and ddH2O 6.7 μL.

[0071] In this invention, the preferred reaction conditions for PCR amplification of the MC1R gene include: 98℃, 2 min; 98℃, 10 s, 60℃, 30 s, 72℃, 1 min, 35 cycles; 72℃, 5 min.

[0072] The preferred reaction conditions for PCR amplification of the PMEL17 gene include: 95℃, 5 min; 95℃, 30 s, 67℃, 30 s, 72℃, 30 s, 34 cycles; 72℃, 7 min.

[0073] The preferred reaction conditions for PCR amplification of the SLC45A2 gene are: 94℃, 5 min; 94℃, 30 s, 60℃, 30 s, 72℃, 15 s, 35 cycles; 72℃, 10 min.

[0074] The preferred reaction conditions for PCR amplification of the TYR gene include: TYR-F1 / TYR-R: 95℃, 3 min; 95℃, 30 s, 62℃, 30 s, 72℃, 1 min, 35 cycles; 72℃, 5 min; or TYR-F2 / TYR-R: 95℃, 3 min; 95℃, 30 s, 60℃, 30 s, 72℃, 1 min, 35 cycles; 72℃, 5 min.

[0075] The preferred reaction conditions for PCR amplification of the SOX10 gene include: 94℃, 5 min; 94℃, 30 s, 60℃, 30 s, 72℃, 1 min, 30 cycles; 72℃, 10 min.

[0076] The present invention also provides a method for breeding and / or producing hybrid offspring chickens with a specific feather color, comprising the following steps:

[0077] The target feather color of the hybrid offspring is determined. Based on the genetic variation combination pattern corresponding to different feather colors of chickens in the method described above, the genotype of the specific genetic variation of the parents is inferred. The chicken parents are purified according to the genotype of the specific genetic variation of the parents. The purified chicken parents are then used for hybridization to obtain hybrid offspring with specific feather colors.

[0078] In this invention, the parent chickens used in the breeding and / or production process preferably include White Leghorn chickens. In this invention, the White Leghorn chicken is preferably used as the maternal parent. This invention lists the genotypes of five genes corresponding to offspring with specific plumage colors, as well as all available hybridization combinations. It provides a practical molecular breeding method for breeding plumage-related breeding lines.

[0079] This invention produces hybrid offspring with specific genotypes by combining different paternal and maternal genotypes in order to obtain offspring with the desired plumage color.

[0080] In this invention, the matching mode for producing hybrid offspring chickens with specific feather color by genotype combinations of specific genetic variations in the parents is shown in the table below:

[0081]

[0082]

[0083] Where DB*N / DB*N represents the homozygous wild type; DB*DB / DB*DB represents the dark brown homozygous type; and DB*DB / DB*N represents the dark brown heterozygous type. The " / " in other positions in the table indicates an "or" relationship, such as CC / Cc representing CC or Cc. Compared with the judgment method described in the above technical solutions, the method for breeding and / or producing chickens of specific feather colors in this invention increases the number of paternal and maternal genotypes required for offspring with different feather colors, thus achieving the purpose of breeding a matching line.

[0084] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a combination of molecular markers related to chicken feather color, primers, and methods and applications for determining feather color, breeding and / or producing chickens with specific feather colors, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0085] Example 1

[0086] The methods for detecting genetic variations in the MC1R, PMEL17, SLC45A2, TYR, and SOX10 genes are as follows:

[0087] 1. Collect blood samples

[0088] First, blood was collected from the subwing vein of each chicken, and heparin lithium vacuum anticoagulant tubes were used for anticoagulation. The blood was then placed in a -20°C freezer for subsequent genomic DNA extraction.

[0089] 2. Genomic DNA extraction

[0090] Genomic DNA was extracted from the above samples using a blood genomic DNA extraction kit, as follows:

[0091] (1) Add the amount of anhydrous ethanol specified in the kit to buffer GD and wash buffer PW, and set aside;

[0092] (2) Take 10 μL of anticoagulated blood from chickens, add 190 μL of buffer GA, and then perform the following lysis steps;

[0093] (3) Add 20 μL of Proteinase K solution and mix well;

[0094] (4) Add 200 μL buffer GB, mix thoroughly by inverting, and place at 70°C for 3 hours. The solution should become clear at this time.

[0095] (5) Add 200 μL of anhydrous ethanol and use a vortex shaker to mix thoroughly for 15 seconds;

[0096] (6) Place the adsorption column CB3 into the collection tube, add the solution obtained in the previous step into the adsorption column, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0097] (7) Add 500 μL of buffer GD (anhydrous ethanol has been added according to the amount specified in the kit) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

[0098] (8) Add 600 μL of washing buffer PW (anhydrous ethanol has been added according to the amount specified in the kit) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

[0099] (9) Repeat step (7);

[0100] (10) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any remaining rinsing liquid.

[0101] (11) Transfer the adsorption column CB3 into a clean 1.5 mL centrifuge tube, add 60 μL of elution buffer TE to the middle of the adsorption membrane, let it stand at room temperature for 2-5 min, centrifuge at 12,000 rpm for 2 min, and collect the solution into a 1.5 mL centrifuge tube.

[0102] (12) Concentration determination: Place the DNA sample on ice, and add 1 μL of each sample sequentially to the NanoDrop 2000 nucleic acid analyzer to determine the DNA concentration and purity. If the OD... 260 / OD 280 A ratio between 1.8 and 2.0 indicates good DNA quality.

[0103] The extracted genomic DNA was used as a template for subsequent PCR amplification reactions.

[0104] 3. Genotyping

[0105] 3.1. MC1R Genotyping

[0106] The primer sequences for the MC1R gene are shown in Table 1:

[0107] Table 1 MC1R gene primers

[0108]

[0109] The PCR reaction system for the MC1R gene is shown in Table 2.

[0110] Table 2 PCR reaction system

[0111]

[0112] The PCR amplification program for the MC1R gene was as follows: 98℃, 2 min; 98℃, 10 s, 60℃, 30 s, 72℃, 1 min, 35 cycles; 72℃, 5 min.

[0113] Primer pair MC1R-F / MC1R-R amplified the MC1R gene, and the amplified product fragment size was 846bp (see gel electrophoresis image of MC1R gene PCR product). Figure 1 (As shown), it contains the red feather-causing mutation site;

[0114] Use SnapGene software to view the sequencing results (MC1R gene PCR product first-generation sequencing peak diagram as shown in the image). Figure 2 As shown in the figure, the genotypes of each SNP site of the MC1R gene were identified based on the sequencing results.

[0115] 3.2. PMEL17 Genotyping

[0116] The primer sequences for the PMEL17 gene are shown in Table 3.

[0117] Table 3 Primers for PMEL17 gene

[0118]

[0119] The PCR reaction system for the PMEL17 gene is shown in Table 4.

[0120] Table 4 PCR reaction system

[0121]

[0122] The PCR amplification program for the PMEL17 gene is as follows: 95℃, 5 min; 95℃, 30 s, 67℃, 30 s, 72℃, 30 s, 34 cycles; 72℃, 7 min.

[0123] The PCR amplification product of the PMEL17 target fragment was digested by RFLP: 10 μL of PCR product was added to 2 μL of 10×NEBuffer (with restriction enzyme), 0.5 μL of BsrBI restriction enzyme, and 1 μL of 2.5 mM MgCl2 aqueous solution. Then, ultrapure water was added to a final volume of 20 μL. After thorough mixing, the mixture was briefly centrifuged and incubated at 37°C for 4 h. The PMEL17 gene restriction enzyme digestion reaction system is shown in Table 5.

[0124] Table 5 RFLP Enzyme Digestion Reaction System

[0125]

[0126] After the reaction, the enzyme digestion products were electrophoresed in a 3% agarose gel in 1×TAE at 100V for 1.5h. Finally, the dominant white genotype was identified using a gel imaging system.

[0127] The genotyping criteria are as follows: When there is no 9bp insertion mutation in exon 10 of the PMEL17 gene sequence of the chicken genomic DNA being tested, the amplified target fragment length is 219bp; while when there is a 9bp insertion mutation, the amplified target fragment length is 228bp. For dominant white-feathered chickens, due to the 9bp insertion mutation, a BsrBI recognition sequence is formed on the DNA sequence, which can cut the 228bp sequence into 186bp and 42bp fragments. Due to the efficiency of agarose gel electrophoresis, the 42bp band may not be visible on the gel image because it is too short. Therefore, on the gel electrophoresis image, dominant white homozygous II genotype individuals show one 186bp band; dominant white heterozygous Ii genotype individuals show two bands, 219bp and 186bp; and non-dominant white homozygous ii genotype individuals show one 219bp band (the detection results of the PMEL17 gene insertion mutation are shown in the figure below). Figure 3 As shown; where A: M: Marker; 1-4: PMEL17 gene digested with BsrB I restriction enzyme shows a 186bp band, representing dominant homozygous genotype II; 5: PMEL17 gene digested with BsrB I restriction enzyme shows a 219bp band, representing non-dominant homozygous genotype ii. B: M: Marker; 1-6: PMEL17 gene digested with BsrB I restriction enzyme shows two bands, 219bp and 186bp, representing dominant heterozygous genotype Ii).

[0128] 3.3. SLC45A2 Genotyping

[0129] The primer sequences for the SLC45A2 gene are shown in Table 6:

[0130] Table 6 Primers for SLC45A2 gene

[0131]

[0132] The PCR reaction system for the SLC45A2 gene is shown in Table 7.

[0133] Table 7 PCR Reaction System

[0134]

[0135] The SLC45A2 gene PCR amplification program is as follows: 94℃, 5 min; 94℃, 30 s, 60℃, 30 s, 72℃, 15 s, 35 cycles; 72℃, 10 min.

[0136] Primer pair SLC45A2-ex4F / SLC45A2-ex4R amplifies a 104bp fragment on exon 4 of the SLC45A2 gene. The target fragment also contains an SNP mutation site: 1111C>A.

[0137] Use SnapGene software to view sequencing results and identify the gold and silver feather genotypes based on the results. Perform first-generation sequencing analysis on exon 4 of the SLC45A2 gene in the chicken flock. If only a single C base peak appears at position 1111 of exon 4 of the SLC45A2 gene (including the C / A alleles), the chicken is C homozygous, meaning the rooster is a gold feather homozygote. s Z s The hen is a Z-type hemizygote with golden feathers. s W; The presence of only an A single base peak indicates a homozygous A genotype, meaning the rooster is a homozygous for the silver feather gene. Z S Z S The hen is a hemizygote of the silver feather gene (Z). S W); the appearance of C and A double base peaks indicates that the rooster is a heterozygote for the golden feather gene. Z S Z s . Figure 4 This is a gel electrophoresis image of the SLC45A2 gene PCR product. Figure 5 This is a first-generation sequencing peak diagram of the SLC45A2 gene PCR product.

[0138] 3.4. SOX10 Genotyping

[0139] The primer sequences for the SOX10 gene are shown in Table 8:

[0140] Table 8. Primers for the SOX10 gene

[0141]

[0142] The SOX10 gene PCR reaction system is shown in Table 9.

[0143] Table 9 PCR Reaction System

[0144]

[0145] The SOX10 gene PCR amplification program is (1F / 1R; 1F / 6R): 94℃, 5min; 94℃, 30s, 60℃, 30s, 72℃, 1min, 30 cycles; 72℃, 10min.

[0146] Primers SOX10-1F and SOX10-6R are both located outside the 8.3kb deletion region, while SOX10-1R is located inside the 8.3kb deletion region. If SOX10-1F and SOX10-1R amplify a 611bp target band, while SOX10-1F and SOX10-6R do not amplify any product, the individual is a wild-type homozygote (genotype DB*N / DB*N). If SOX10-1F and SOX10-6R amplify a 1257bp target band, while SOX10-1F and SOX10-1R do not amplify any product, the individual is a dark brown homozygote (genotype DB*DB / DB*DB). If both the 611bp and 1257bp target fragments are present, the individual is a dark brown heterozygote (genotype DB*DB / DB*N).

[0147] Figure 6 The image shows the detection results of the 8.3kb deletion mutation in the SOX10 gene in chicken flocks; among them, 1-12 and 13-24 are the PCR amplification results of 12 samples using SOX10-1F and SOX10-1R and SOX10-1F and SOX10-6R, respectively.

[0148] 3.5. TYR Genotyping

[0149] The primer sequences for the TYR gene are shown in Table 10:

[0150] Table 10 TYR gene primers

[0151]

[0152]

[0153] The PCR reaction system for the TYR gene is shown in Table 11.

[0154] Table 11 PCR reaction system

[0155]

[0156] The PCR amplification procedure for the TYR gene is as follows:

[0157] F1 / R: 95℃, 3min; 95℃, 30s, 62℃, 30s, 72℃, 1min, 35 cycles; 72℃, 5min.

[0158] F2 / R: 95℃, 3min; 95℃, 30s, 60℃, 30s, 72℃, 1min, 35 cycles; 72℃, 5min.

[0159] The downstream primer TYR-R is located on exon 5 of the TYR gene, while the upstream primers TYR-F1 and TYR-F2 are located in the 7.7kb insertion region and intron 4 of the TYR gene, respectively. If only the 345bp PCR amplification fragment of primers TYR-F1 and TYR-R is generated, without the target bands of TYR-F2 and TYR-R, the individual is a recessive homozygous white-feathered genotype (cc). If only the 481bp amplification product of primers TYR-F2 and TYR-R is generated, without the target bands of TYR-F1 and TYR-R, the individual is a wild-type homozygous genotype (CC). If both the 345bp and 481bp fragments of the amplification product are present, the individual is a recessive heterozygous white-feathered genotype (Cc).

[0160] Figure 7 The image shows the detection results of the 7.7kb insertion mutation in the TYR gene of chicken flocks. Among them, 1-12 and 13-24 are the PCR amplification results of 12 samples using TYR-F1 and TYR-R and TYR-F2 and TYR-R, respectively.

[0161] After mastering the above-mentioned methods for detecting genetic variation, the genomic DNA of parent chickens can be used as a template to detect genetic variation. Only individuals that conform to the target genotype can be retained for breeding and strain purification. The purified parent breeds can then be used for breeding and production of complementary lines.

[0162] Example 2

[0163] Breeding of Red Feather Hybrid Offspring

[0164] Father's Red Island: GG ii Z s Z s CC(DB*N / DB*N) Mother White Arrival: GG II Z S W CC(DB*N / DB*N)

[0165] Genotyping was performed using the aforementioned molecular experimental methods. Through continuous selection and purification of the MC1R gene at locus 427, the dominant white gene (PMEL17), the silver feather gene (SLC45A2), the recessive white gene (TYR), and the dark brown gene (SOX10), both the paternal parent, Lodoss Red, and the maternal parent, White Ram, were homozygous for the GG genotype at MC1R gene locus 427. Simultaneously, the paternal parent, Lodoss Red, was a non-dominant white homozygous genotype ii, and the maternal parent, White Ram, was a dominant white homozygous genotype II; the paternal parent, Lodoss Red, was also a golden feather homozygous genotype Z. s Z s The mother plant, Bai Laihang, is a pure hybrid of the Silver Feather Z type. S W; and the recessive white and dark brown genes of the father, Lo Island Red, and the mother, White, are all homozygous wild-type.

[0166] GG II Z S W CC(DB*N / DB*N) genotype White Leghorn hens and GGii Z s Z s Crossbreeding Loch Ness Red roosters with the CC(DB*N / DB*N) genotype resulted in offspring where all hens had red feathers and all male chicks had non-red feathers, thus creating a red-feathered hybrid line. The resulting offspring roosters from this cross had the genotype GG Ii Z. S Z s CC(DB*N / DB*N), exhibiting a white-feathered phenotype; offspring hens have the genotype GG Ii Z. s W CC(DB*N / DB*N) exhibits a red feather phenotype. This is in line with expectations.

[0167] Figure 8 Genotyping diagram of MC1R, SLC45A2, PMEL17, TYR, and SOX10 genes in hybrid offspring roosters (white feathers) and hens (red feathers).

[0168] Example 3

[0169] Breeding of hybrid offspring with white feathers;

[0170] Father's Red Island: GG ii Z s Z s CC(DB*N / DB*N) Mother White Arrival: AAII Z S W CC(DB*N / DB*N)

[0171] Genotyping was performed using the same molecular experimental methods described above. Through continuous selection and purification of the MC1R gene at locus 427, the dominant white gene (PMEL17), the silver feather gene (SLC45A2), the recessive white gene (TYR), and the dark brown gene (SOX10), the paternal parent, Lodoss Red, and the maternal parent, White Ram, were determined to be GG homozygous and AA homozygous at locus 427 of the MC1R gene, respectively. Simultaneously, the paternal parent, Lodoss Red, was a non-dominant white homozygous genotype ii, and the maternal parent, White Ram, was a dominant white homozygous genotype II; the paternal parent, Lodoss Red, was also a golden feather homozygous genotype Z. s Z s The mother plant, Bai Laihang, is a pure hybrid of the Silver Feather Z type. S W; and the recessive white and dark brown genes of the father, Lo Island Red, and the mother, White, are all homozygous wild-type.

[0172] Will AAII Z S W CC(DB*N / DB*N) genotype White Leghorn hens and GGii Z s Z s Crossbreeding Loch Ness Red roosters with the CC(DB*N / DB*N) genotype produces offspring with non-red feathers in both male and female chicks, resulting in a white-feathered hybrid. The resulting roosters from this cross have the genotype GAIi Z. S Z s CC(DB*N / DB*N) indicates a white-feathered phenotype; the offspring hens have the genotype GAIi Z. s W CC(DB*N / DB*N) exhibits a white feather phenotype. This is in line with expectations.

[0173] Figure 9 Genotyping diagram of MC1R, SLC45A2, PMEL17, TYR, and SOX10 genes in hybrid offspring hens (white feathers).

[0174] Red and white feathers cultivation

[0175] Father's Red Island: GG ii Z s Z s CC(DB*N / DB*N) Mother White Arrival: AAII Z S W CC(DB*DB / DB*DB)

[0176] Genotyping was performed using the same molecular experimental methods described above. Through continuous selection and purification of the MC1R gene at locus 427, the dominant white gene (PMEL17), the silver feather gene (SLC45A2), the recessive white gene (TYR), and the dark brown gene (SOX10), the paternal parent, Lodoss Red, and the maternal parent, White Ram, were determined to be GG homozygous and AA homozygous at locus 427 of the MC1R gene, respectively. Simultaneously, the paternal parent, Lodoss Red, was a non-dominant white homozygous genotype ii, and the maternal parent, White Ram, was a dominant white homozygous genotype II; the paternal parent, Lodoss Red, was also a golden feather homozygous genotype Z.s Z s The mother plant, Bai Laihang, is a pure hybrid of the Silver Feather Z type. S W; and both the paternal Lo Island Red and maternal White Leghorn recessive white genes are homozygous wild-type. However, the dark brown genotype of the paternal Lo Island Red rooster is homozygous wild-type: DB*N / DB*N, while the dark brown genotype of the maternal White Leghorn hen is homozygous dark brown: DB*DB / DB*DB.

[0177] Will AA II Z S W CC(DB*DB / DB*DB) genotype White Leghorn hens and GGii Z s Z s Crossbreeding Loch Ness Red roosters with the CC(DB*N / DB*N) genotype produces offspring with all females having red and white plumage, while all male chicks have non-red plumage, resulting in a red and white plumage hybrid. The roosters resulting from this cross have the genotype GAIi Z. S Z s CC(DB*DB / DB*N), exhibiting a white-feathered phenotype; offspring hens have the genotype GA Ii Z. s W CC(DB*DB / DB*N), exhibiting red and white plumage. This is in line with expectations (see...). Figure 10 Genotyping of the MC1R, SLC45A2, PMEL17, TYR, and SOX10 genes in red-and-white and white-feathered chickens.

[0178] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for determining the plumage color of a chicken based on detecting a molecular marker associated with chicken plumage color using primers, characterized in that, Comprising the following steps: PCR amplification of the genes using the primers MC1R genes, PMEL17 genes, SLC45A2 genes, TYR genes and SOX10 genes, the chicken feather color was judged according to the size of the PCR amplification product and the genotype of each gene, according to the following judging method: ； The chicken feather color related molecular marker is MC1R gene, PMEL17 gene, SLC45A2 gene, TYR gene and SOX10 gene; The primers are for detecting MC1R the gene MC1R - F and MC1R - R, the primers for detecting PMEL17 the gene PMEL17 - F 2 and PMEL17 - R 2 , the primers for detecting SLC45A2 the gene SLC45A2 - ex4F and SLC45A2 - ex4R, the primers for detecting TYR the gene TYR - F1, TYR - R and TYR - F2, and the primers for detecting SOX10 the gene SOX10 - 1F, SOX10 - 1R and SOX10 - 6R; The MC1R The nucleotide sequence of -F is shown in SEQ ID NO.

1. MC1R The nucleotide sequence of -R is shown in SEQ ID NO. 2; PMEL17 -F 2 The nucleotide sequence is shown in SEQ ID NO.

3. PMEL17 -R 2 The nucleotide sequence is shown in SEQ ID NO.4; SLC45A2 The nucleotide sequence of -ex4F is shown in SEQ ID NO.

5. SLC45A2 The nucleotide sequence of -ex4R is shown in SEQ ID NO. 6; TYR The nucleotide sequence of -F1 is shown in SEQ ID NO.

7. TYR The nucleotide sequence of -R is shown in SEQ ID NO.

8. TYR The nucleotide sequence of -F2 is shown in SEQ ID NO. 9; SOX10 The nucleotide sequence of -1F is shown in SEQ ID NO.

10. SOX10 The nucleotide sequence of -1R is shown in SEQ ID NO.

11. SOX10 The nucleotide sequence of -6R is shown in SEQ ID NO.12; The female parent of the chicken is white Laihang chicken; The method for judging the chicken feather color is: The genotype of the silver gene S and recessive white homozygous gene cc is absent, and the genotype of the dark brown gene is SOX10 The genotype of the silver gene S and recessive white homozygous gene cc is absent, and the genotype of the dark brown gene is N / DB N, when MC1R When the gene mutation is GG type, whether the dominant white gene I exists or not, the red feather phenotype is shown; and when MC1R When the gene 427 site is GA / AA type, the white feather phenotype is caused under the action of the dominant white gene, and the black feather phenotype is shown without the action of the dominant white gene; The genotype of the bird is DB, which is a deep brown heterozygote SOX10 The genotype of the bird is DB, which is a deep brown heterozygote DB / DB N, when MC1R The genotype of the bird is DB, which is a deep brown heterozygote When I and S exist simultaneously, regardless MC1R The gene 427 site is GG type or GA / AA type, and the white feather phenotype is shown. When there is a cc, regardless MC1R The gene 427 site is GG type or GA / AA type, which is white feather phenotype; The chromosome composition of the male chicken is ZZ, and that of the female chicken is ZW.

2. The method of claim 1, wherein, PCR amplification MC1R The reaction mixture for the genes was: 2 x EasyTaq® PCR SuperMix (+ dye) 25 μL, MC1R - F 1 μL, MC1R - R 1 μL, genomic DNA 6.25 μL and ddH2O 16.75 μL, in a total volume of 50 μL; PCR amplification PMEL17 The reaction system of the gene is: 2 x Taq PCR Mix 10 μL, PMEL17 -F 2 0.3 μL, PMEL17 -R 2 0.3 μL, genomic DNA 1 μL and ddH2O 8.4 μL, total volume 20 μL; PCR amplification SLC45A2 The reaction mixture for the gene was: 2 x EasyTaq® PCR SuperMix (+dye) 25 μL, SLC45A2 - ex4F 1 μL, SLC45A2 - ex4R 1 μL, genomic DNA 6.25 μL and ddH2O 16.75 μL, in a total volume of 50 μL; PCR amplification TYR The reaction system of the gene is: 2x EasyTaq PCR SuperMix (+dye) 10 μL, TYR -F1 or TYR -F2 0.4 μL, TYR -R 0.4 μL, genomic DNA 2.5 μL and ddH2O 6.7 μL, total volume 20 μL; PCR amplification SOX10 The reaction mixture for the genes was: 2 x EasyTaq® PCR SuperMix (+dye) 10 μL, SOX10 - 1 F 0.4 μL, SOX10 - 1 R or SOX10 - 6 R 0.4 μL, genomic DNA 2.5 μL and ddH2O 6.7 μL, in a total volume of 20 μL.

3. The method of claim 1, wherein, PCR amplification MC1R The reaction conditions for the genes were: 98°C, 2 min; 98°C, 10 s, 60°C, 30 s, 72°C, 1 min, 35 cycles; 72°C, 5 min; PCR amplification PMEL17 The reaction conditions for the genes were: 95°C, 5 min; 95°C, 30 s, 67°C, 30 s, 72°C, 30 s, 34 cycles; 72°C, 7 min; PCR amplification SLC45A2 The reaction conditions for the genes were: 94°C, 5 min; 94°C, 30 s, 60°C, 30 s, 72°C, 15 s, 35 cycles; 72°C, 10 min; PCR amplification TYR The reaction conditions for the genes are: TYR -F1 / TYR -R: 95°C, 3 min; 95°C, 30 s, 62°C, 30 s, 72°C, 1 min, 35 cycles; 72°C, 5 min or TYR -F2 / TYR -R: 95°C, 3 min; 95°C, 30 s, 60°C, 30 s, 72°C, 1 min, 35 cycles; 72°C, 5 min; and PCR amplification SOX10 The reaction conditions for the genes were: 94°C, 5 min; 94°C, 30 s, 60°C, 30 s, 72°C, 1 min, 30 cycles; 72°C, 10 min.

4. A method of breeding and / or producing a specific feather color cross progeny chicken, characterized in that, Comprising the following steps: Determine the target feather color of the hybrid offspring, infer the genotype of the specific genetic variation of the parent according to the genetic variation combination mode of the genes corresponding to different feather colors of the chicken in the method of any one of claims 1-3, purify the chicken parents according to the genotype of the specific genetic variation of the parent, cross the purified chicken parents, and obtain the specific feather color hybrid offspring chicken.

5. The method of claim 4, wherein, The matching mode for producing the specific feather color hybrid offspring chicken according to the genotype combination of the specific genetic variation of the parent is shown in the following table: ； wherein DB N / DB N indicates homozygous wild type; DB DB / DB DB represents dark brown homozygous type; DB DB / DB N represents dark brown heterozygous type.

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