Molecular marker pmel17 gene for blackening of breast muscle of mushan chicken and genetic marker method

Abstract

The present application belongs to the field of molecular biology technology and breeding of Muxuan black-bone chickens, and particularly relates to a PMEL17 gene for breast muscle blackness of Muxuan black-bone chickens and a genetic marker method thereof. The nucleotide sequence of the gene is shown as SEQ ID NO. 1. The marker method is to design a primer pair, take the genomic DNA of Muxuan black-bone chickens as a template, perform PCR amplification, obtain a sequence shown as SEQ ID NO. 4, identify whether the sequence has a mutation, and analyze the correlation between the mutation and the breast muscle blackness of Muxuan black-bone chickens. The screened gene is a gene related to the breast muscle blackness of Muxuan black-bone chickens, and further screening of a SNP related to the trait at a 553th site of the PMEL17 gene sequence can be used as a genetic marker for auxiliary selection of Muxuan black-bone chickens. Through the genetic marker, Muxuan black-bone chickens with more melanin deposition and darker color can be selected and bred, which has important significance and application value for the genetic breeding of Muxuan black-bone chickens.

Description

Technical Field

[0001] This invention belongs to the field of molecular biotechnology and breeding of Muchuan black-bone chicken, specifically involving the PMEL17 gene, a molecular marker for the blackness of the pectoral muscle of Muchuan black-bone chicken, and its genetic marker method. Background Technology

[0002] Muchuan Silkie Chicken is an important breed of Silkie chicken in my country. This breed is known for its strong disease resistance, high survival rate, and high market acceptance, making it widely raised and easy to promote. However, during the generational breeding process, some individuals have experienced separation of their blackness, resulting in incomplete and impure blackness within the flock, which poses challenges to the preservation and breeding of Silkie chickens.

[0003] With the rapid development of biotechnology, molecular marker-based breeding techniques for Silkie chickens have emerged. Zhao Jingying's research found that the MC1R gene SNPc.-244T>C and SNPc.-460G>A can serve as molecular markers for selecting the blackness of Wuding Silkie chickens; G. Wang discovered that the MITF gene SNPc.-638T>c can be used to mark and assist in the selection of skin color in Silkie chickens; YU.S. et al. found that the ASIP gene SNPc.-1826A>T can serve as a molecular marker for selecting the blackness of Silkie chickens; Peng Canyang discovered that the MLPH gene SNPc.-1411C>T and the PMEL17 genes SNPc.-1843G>C, -2812C>T, and -2794G>A can serve as molecular markers for selecting the blackness of Xuefeng Silkie chickens. Although several genes related to melanin deposition have been identified as molecular markers, their applicability to melanin deposition in Muchuan Silkie chickens remains to be investigated.

[0004] Melanin synthesis and packaging occur within melanosomes. The pre-melanosomal protein 17 (PMEL17) gene encodes a melanocyte-specific transmembrane glycoprotein that helps maintain the structure of the pre-melanosome in melanocytes. In vertebrates, loss of PMEL17 function leads to the loss of melanin pigment in the skin, hair, and eyes. Tilapia lacking PMEL17 are golden yellow, while the normal wild type is black; a stop codon mutation in the mouse PMEL17 gene produces a silver-haired phenotype; a p.Arg618Cys mutation in the horse PMEL17 gene also produces a silver-haired phenotype; a p.R618C mutation in the chicken PMEL17 gene produces a reddish-brown feather phenotype, and a 9bp insertion in exon 10 results in a white feather phenotype. This demonstrates that the function of the PMEL17 gene is closely related to melanin. A search revealed no reports on the PMEL17 gene in the Muchuan Silkie chicken. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the PMEL17 gene, a molecular marker for the black-boned chicken breast muscle, and a genetic marker method.

[0006] The objective of this invention is achieved through the following technical solution: a molecular marker gene for the blackness of the breast muscle of Muchuan black-boned chicken, wherein the gene is the PMEL17 gene, and the nucleotide sequence is shown in SEQ ID NO.1.

[0007]

[0008]

[0009]

[0010] Furthermore, the PMEL17 gene amplification product SEQ ID NO.4 has a G / A base mutation at position 553.

[0011] A genetic marker method for PMEL17 gene, a molecular marker for the blackness of the breast muscle of Muchuan black-boned chicken, includes the following steps:

[0012] S1. Amplification: Primer pairs were designed to cover exons 4 to 6 of the PMEL17 gene. Using the genomic DNA of Muchuan black-boned chicken as a template, PCR amplification was performed to obtain the sequence shown in SEQ ID NO.4.

[0013] SEQ ID NO.4:

[0014]

[0015] S2. Identification: Identify whether there is a mutation in the bases at position 553 of the gene.

[0016] Furthermore, the nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3.

[0017] SEQ ID NO.2: 5'-ATCGCATCCCCGTCCCTTATC-3' (forward).

[0018] SEQ ID NO.3: 5'-GGACCCAACCATCCCCTTGC-3' (reverse).

[0019] Furthermore, the PCR amplification system is as follows: 50 μL reaction system: 25 μL of 2×T5 Super PCR mix (Basic), 2 μL each of forward and reverse primers (10 μmol / L), 1 μL of DNA template, and 20 μL of ultrapure water; PCR amplification reaction program: 35 PCR cycles after pre-denaturation at 98℃ for 3 min, with parameters as follows: 98℃ for 10 s, 62.2℃ for 10 s, 72℃ for 1 min, and finally 72℃ for 5 min.

[0020] This invention has the following advantages: It discloses the PMEL17 gene, a molecular marker for the darkening of the breast muscles of the Muchuan Black-boned Chicken, and its genetic marker method. The invention screens for relevant gene sequences of the PMEL17 gene as a molecular marker for the darkening of the breast muscles of the Muchuan Black-boned Chicken, and further screens for trait-related SNPs at position 553 of the PMEL17 gene-related sequence. The PMEL17 gene SNPs screened by this invention can serve as genetic markers for auxiliary selection of Muchuan Black-boned Chickens. Through genetic marker selection, Muchuan Black-boned Chickens with greater melanin deposition and a deeper black color can be bred, which has significant meaning and application value for the genetic selection and breeding of Muchuan Black-boned Chickens. Attached Figure Description

[0021] Figure 1 This is a sequencing peak diagram. In the diagram, the arrow points to the 553 mutation site. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and embodiments. The scope of protection of the present invention is not limited to the following: Embodiment 1:

[0023] 1. By searching the literature, we identified the gene PMEL17, which is strongly associated with pectoral muscle tint, and its PCR amplification primers.

[0024] 2. The Muchuan Black-boned Chicken was raised according to the broiler feeding program. The chickens were raised from 1 day old to 35 days old. 48 healthy Muchuan Black-boned Chickens were selected for the experiment. 2 ml of blood was collected. After slaughter, the brightness (L) of the pectoral muscle was measured using a colorimeter and recorded.

[0025] 3. Genomic DNA was extracted from blood using a blood genomic DNA extraction kit (Sangon Biotech). The purity was determined using a micro-nucleic acid protein analyzer, and the integrity was checked by 1% agarose gel electrophoresis. Only samples meeting the required standards were used for the next step of the experiment.

[0026] 4. Primers used for cloning the entire sequence of exons 4 to 6 of the PMEL17 gene, as provided in relevant literature. Primer sequences:

[0027] 5'-ATCGCATCCCCGTCCCTTATC-3' (forward), SEQ ID NO:2

[0028] 5'-GGACCCAACCATCCCCTTGC-3' (reverse), SEQ ID NO:3

[0029] Synthesized by Qingke Biotechnology Co., Ltd.

[0030] 5. PCR amplification was performed using genomic DNA from Muchuan black-boned chicken as a template. The 50 μL PCR amplification reaction system consisted of: 25 μL of 2×T5 Super PCR mix (Basic), 2 μL each of forward and reverse primers (10 μmol / L), 1 μL of DNA template, and 20 μL of ultrapure water. The PCR amplification program was: 35 PCR cycles after pre-denaturation at 98℃ for 3 min, with the following parameters: 98℃ for 10 s, 62.2℃ for 10 s, 72℃ for 1 min, and a final cycle at 72℃ for 5 min.

[0031] 6. Take 2 μL of the amplification product and perform 1% agarose gel electrophoresis to check if the product fragment size matches the target fragment size. Take 1 μL and test the purity and concentration of the amplification product using a micro-volume nucleic acid and protein analyzer. After both meet the sequencing requirements, send it to Qingke Biotechnology Co., Ltd. for first-generation sequencing. After obtaining the nucleotide sequence of SEQ ID NO.4, search for SNP sites and determine the genotype. Experiments showed that the SNP is located at position 553 of SEQ ID NO.4. Figure 1

[0032] 7. Calculate the frequency of each genotype and perform correlation analysis between SPSS and pectoral muscle brightness (L).

[0033] Table 1: Genotypes and Genotype Frequencies at SNP Loci

[0034]

[0035] Different SNP genotypes of SNP.G-553A significantly affected the breast muscle L of Muchuan black-bone chicken: P = 0.023 < 0.05, r = 0.329; among them, 10.55% of individuals with the AA genotype had a lower breast muscle L than the GA genotype, and the difference reached a significant level (P = 0.018 < 0.05).

[0036] As shown in Table 1, the PMEL17 gene SNP.G-553A can be used as a molecular marker for the blackness of the breast muscle of Muchuan black-boned chicken, and the AA genotype is the dominant genotype.

[0037] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection of the present invention.

Claims

1. A method for detecting the blackness of the breast muscle of Muchuan black-boned chicken using a molecular marker, wherein the molecular marker is the SNP.G-553A site of the PMEL17 gene, and the nucleotide sequence of the PMEL17 gene is shown in SEQ ID NO.1, characterized in that... Includes the following steps: S1. Amplification: Primer pairs were designed to cover the entire sequence of exons 4 to 6 of the PMEL17 gene. Using the genomic DNA of Muchuan black-boned chicken as a template, PCR amplification was performed to obtain the sequence shown in SEQ ID NO.

4. The nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

3. S2. Identification: Identify whether there is a mutation at the 553rd position of the gene. The PMEL17 gene amplification product has a G / A base mutation at the 553rd position, in which the AA genotype is the dominant genotype.

2. The method for detecting the molecular marker of purplish-blackness in the pectoral muscle of Muchuan black-boned chicken according to claim 1, characterized in that, The PCR amplification system was as follows: 50 μL reaction system: 25 μL of 2×T5 Super PCR mix Basic, 2 μL each of forward and reverse primers (10 μmol / L), 1 μL of DNA template, and 20 μL of ultrapure water; PCR amplification reaction program: 35 PCR cycles after pre-denaturation at 98℃ for 3 min, with the following parameters: 98℃ for 10 s, 62.2℃ for 10 s, 72℃ for 1 min, and finally 72℃ for 5 min.

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