MSTN (myostatin) gene molecular marker related to pigeon body weight character and application of MSTN gene molecular marker

Abstract

The invention relates to an MSTN (myostatin) gene molecular marker related to a pigeon body weight character and application of the MSTN gene molecular marker, and belongs to the technical field of biology. According to the invention, genome DNA of a to-be-detected pigeon is taken as a template, PCR amplification is carried out on the to-be-detected pigeon by using an SNP primer, then Sanger sequencing and SNP molecular marker genotyping are carried out on a PCR amplification product, and then pigeon body weight traits are selected. The weight of G / G genotype pigeons is higher than that of A / G and A / A genotype individuals, and the weight of A / G genotype pigeons is higher than that of A / A genotype individuals. By eliminating A / G and A / A genotype individuals and retaining G / G genotype individuals, the molecular marker has the beneficial effects that the molecular marker is used as a genetic marker for breeding pigeons, and the pigeons with large weight and uniformity are bred; the method can efficiently and quickly identify the weight traits of the pigeons, improves the uniformity of the weight of pigeons, provides a scientific basis for the early breeding of the pigeons, and better serves the breeding of the pigeons.

Description

Technical Field

[0001] The present invention relates to an MSTN gene molecular marker related to the body weight trait of pigeons and its application, belonging to the field of biotechnology. Background Art

[0002] Body weight is one of the important growth traits of pigeons. Due to the long generation cycle and slow progress of conventional breeding techniques, it is difficult to accurately select and breed. With the rapid development of modern biotechnology, molecular marker-assisted breeding technology has been widely applied in the field of breeding new varieties of animals and plants. Combining with conventional breeding techniques can improve the accuracy of selecting target traits, achieve early selection, accelerate the breeding progress, save breeding costs, and improve breeding efficiency. However, there are currently few molecular markers related to the body weight trait of pigeons.

[0003] The MSTN gene (Myostatin gene), also known as the myostatin gene, is a member of the transforming growth factor-β (TGF-β) superfamily. The MSTN protein encoded by it is a key negative regulator of skeletal muscle growth. Studies have found that a natural functional deletion mutation of MSTN in cattle forms a "double-muscled" phenotype, resulting in a significant increase in muscle mass. Interfering with the MSTN gene can promote the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells, verifying the biological function of interfering with the MSTN gene in regulating muscle development and differentiation. The above studies indicate that the MSTN gene plays an important regulatory role in the process of muscle growth and development. Summary of the Invention

[0004] The object of the present invention is to propose an MSTN gene molecular marker related to the body weight trait of pigeons and its application to improve the breeding efficiency of pigeons in view of the defects existing in the prior art.

[0005] The present invention records the body weight trait of pigeons, uses whole-genome resequencing technology for SNP genotyping, and screens out relevant MSTN gene molecular markers through genome-wide association analysis, providing new gene and molecular marker resources for the selection and breeding of the body weight trait of pigeons.

[0006] The present invention solves the technical problem through the following technical solutions: First, a pair of MSTN gene molecular marker primers related to pigeon body weight is provided. The nucleotide sequences of the SNP primers corresponding to the molecular markers are shown in SEQ ID NO:1 and SEQ ID NO:2. The molecular marker is located at the 16,694,595th base of chromosome 6 of the pigeon reference genome Cliv_NAU_1.0 version (National Genomics Data Center, https: / / ngdc.cncb.ac.cn / gwh, accession number GWHFCQQ00000000.1), and the base mutation is A or G. The molecular marker is located at the 156th base shown in SEQ ID NO:3 or SEQ ID NO:4.

[0007] The present invention further provides the application of the above-mentioned molecular marker primers for detecting the SNP genotypes related to pigeon body weight traits. The detection method includes the following steps: First step: Provide a DNA sample of the pigeon to be tested, and perform PCR amplification with the molecular marker primer pair to obtain an amplification product. The length of the amplification product is 352 bp and contains the 16,694,595th base of pigeon chromosome 6. Second step: Perform Sanger sequencing on the PCR product. Third step: Judge the SNP molecular marker genotype of the 16,694,595th base of chromosome 6 according to the sequencing peak map.

[0008] The sequences of the pigeon DNA specific primer pair in the first step are as follows: Forward primer: 5’-GTGAGTACTGTTTGCCATGC-3’ (SEQ ID NO:1) Reverse primer: 5’-GGCTCATGTCGTCAAACCC-3’ (SEQ ID NO:2) Taking 50 μl as the reaction system, the system is DNA of the pigeon to be tested 50 ng Accurate Taq DNA polymerase 1.25 IU 10X PCR reaction buffer containing Mg2+ 5 μl 10 mM dNTPs 1 μl 10 μM forward primer F 1 μl 10 μM reverse primer R 1 μl Sterile water Make up to 50 μl; The reaction conditions for PCR amplification were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 60 sec, for a total of 30 cycles; extension at 72°C for 5 min; storage at 4°C; the nucleotide sequence of the amplification product was as shown in SEQ ID NO:3 or SEQ ID NO:4, the length of the amplification product was 352 bp, and it contained the base at position 16694595 on chromosome 6 of pigeons.

[0009] In the third step described above, the judgment criterion was that the body weight of pigeons with the G / G genotype at the SNP locus was higher than that of individuals with the A / G genotype, and the body weight of individuals with the A / G genotype was higher than that of individuals with the A / A genotype.

[0010] The present invention detected the genotype of the body weight trait of pigeons through the MSTN gene molecular marker, and obtained that the body weight of pigeons with the G / G genotype was higher than that of individuals with the A / G genotype and the A / A genotype, and the body weight of pigeons with the A / G genotype was higher than that of individuals with the A / A genotype. By using the genomic DNA of the pigeon to be tested as a template, specific primer pairs were used for PCR amplification, and then the PCR amplification products were subjected to Sanger sequencing and SNP genotyping. Based on the genotype of this SNP molecular marker, the selection of the body weight trait of pigeons can be realized. For example, in breeding, if it is necessary to cultivate a meat pigeon variety with a higher body weight, individuals with the A / A genotype can be eliminated, and individuals with the A / G and G / G genotypes can be retained. The beneficial effect is that early selection of the body weight trait of pigeons can be realized, the accuracy of seed selection can be improved, the breeding progress can be accelerated, the breeding cost can be saved, the breeding efficiency can be improved, and better service can be provided for the breeding of pigeons. Description of the Drawings

[0011] Figure 1 is the Manhattan plot of the genome-wide association analysis of pigeon body weight.

[0012] Figure 2 is the Sanger sequencing result of the PCR amplification products of the three genotypes. Detailed Embodiments

[0013] In the following examples, the pigeon varieties used were all commercially available and will not be elaborated.

[0014] Example 1 In this example, the body weights of 150-week-old pigeons of three varieties, namely Danish Silver King Pigeons, Taishen Pigeons, and American Silver King Pigeons, were measured. Whole-genome SNP genotyping was carried out using the second-generation sequencing technology, and MSTN gene molecular markers significantly related to the 150-week-old body weight were screened through genome-wide association analysis. The results were as Figure 1 shown.

[0015] In this example, the following experiments were carried out to identify and apply the MSTN gene molecular marker related to pigeon body weight.

[0016] 1. Phenotypic determination and genotypic detection (1) Experimental materials and body weight phenotypic determination Select 389 pairs of breeding pigeons, including Danish Silver King pigeons, Taishen pigeons, and American Silver King pigeons. The numbers of the three breeds are close. They are raised under the same feeding conditions, with free access to food and water throughout the process. When the breeding pigeons reach 150 weeks of age and finish laying eggs, fast them for 12 h and then record the weight of each pigeon as the phenotypic data of pigeon body weight.

[0017] (2)Genomic DNA extraction Take the tissue samples obtained in the first step and extract genomic DNA using an Omega brand tissue genomic DNA extraction kit. The specific method refers to the standard operation procedure provided by the Omega brand.

[0018] (3)PCR amplification Using the extracted genomic DNA as a template, amplify the fragment containing the base site at position 16694595 on chromosome 6.

[0019] Forward primer: 5’-GTGAGTACTGTTTGCCATGC-3’ (SEQ ID NO:1) Reverse primer: 5’-GGCTCATGTCGTCAAACCC-3’ (SEQ ID NO:2) The final concentration of the reaction system (25 μl) is as follows: DNA of the pigeon to be tested 50 ng Accurate Taq DNA polymerase 1.25 IU 10X PCR reaction buffer containing Mg2+ 5 μl 10 mM dNTPs 1 μl 10 μM forward primer F 1 μl 10 μM reverse primer R 1 μl Sterile water Make up to 50 μl; The reaction conditions for PCR amplification are: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 sec, annealing at 55 °C for 30 sec, extension at 72 °C for 60 sec, for a total of 30 cycles; extension at 72 °C for 2 min; store at 4 °C; take 10 μl for agarose detection. The amplified product with a single target band length of 352 bp is obtained, and it contains the SNP molecular marker at the base site at position 16694595 on pigeon chromosome 6. The nucleotide sequence of the amplified product is shown in SEQ ID NO:3 or SEQ ID NO:4.

[0020] (4)Sanger sequencing and genotyping The PCR products of each sample were subjected to Sanger sequencing, and the sequencing peak maps of different genotypes are as Figure 2 shown.

[0021] 2. Correlation analysis A total of 389 pigeon individuals with clear phenotypic records of body weight at 150 weeks of age were selected for correlation analysis. The ANOVA test function of the statistical plotting software GraphPad Prism 9 was used for statistical testing. The average comparison mode between pairs was selected to statistically test the genotypes and body weight traits of the experimental pigeon population. P<0.05 indicates a significant difference. The results are shown in Tables 1 and 2. The body weights of pigeons with three genotypes were significantly different (P<0.05). The average body weight of G / G genotype individuals was 639.2 g, which was higher than that of A / G genotype individuals (586.9 g, P<0.05) and A / A genotype individuals (558.8 g, P<0.05). The body weight of A / G genotype individuals was higher than that of A / A genotype individuals (P<0.05). The results indicate that the locus at the 16,694,595th base of chromosome 6 in pigeons is significantly associated with the phenotypic traits of pigeon body weight. According to the actual breeding goals, G / G genotype individuals can be selected to breed pigeons with larger body weights, improve the overall body weight and uniformity, and improve the breeding efficiency.

[0022] Genotype Number / each Body weight at 150 weeks of age / g CV / % G / G 19 <![CDATA[639.2±79.3 a > 12.4 A / G 158 <![CDATA[586.9±71.0 b > 12.1 A / A 601 <![CDATA[558.8±60.8 c > 10.9 Note: Data in the same column with the same superscript letter indicate no significant difference, while different superscript letters indicate significant difference (P<0.05).

[0023] Genotype Number of male pigeons / each Body weight at 150 weeks of age / g Number of female pigeons / each Body weight at 150 weeks of age / g G / G 16 <![CDATA[648.6±83.05 a > 3 <![CDATA[589.3±20.01 ab <!-- 3 -->]]> A / G 80 <![CDATA[601.2±70.23 b > 78 <![CDATA[572.3±69.29 b > A / A 299 <![CDATA[567.6±59.71 c > 302 <![CDATA[550.1±60.76 a > Note: Data in the same column with the same superscript letter indicate no significant difference, while different superscript letters indicate significant difference (P<0.05).

[0024] Example 2 Gene frequencies of different breeds 1. Blood sample collection Blood samples of 15 strains / breeds such as Danish Silver King pigeons, Taishen pigeons, and American Silver King pigeons were collected using the wing vein blood collection method and stored at -20°C for later use.

[0025] 2. Genomic DNA extraction The tissue samples obtained in the first step were taken, and genomic DNA was extracted using an Omega brand tissue genomic DNA extraction kit. The specific method was referred to the standard operation procedure provided by the Omega brand.

[0026] 3. Genotype detection Using the genomic DNA obtained in the second step as a template, PCR amplification and Sanger sequencing were performed with a primer pair consisting of the F nucleotide sequence (SEQ ID NO: 1) and the R nucleotide sequence (SEQ ID NO: 2) to obtain the G / G genotype, A / G genotype, and G / G genotype of the individual.

[0027] 4. Result Analysis White - feather lines (White King Pigeon, White Carneau Pigeon, Deep King Pigeon, European Meat Pigeon), Danish Silver King Pigeon, American Silver King Pigeon, Taishen Pigeon, etc. are all pigeon meat lines introduced into China. Their body weight traits have been selectively bred to a certain extent, and the G allele frequency has occupied a certain proportion in the population. There is also a certain proportion of G alleles in the populations of the native Chinese breeds, Shiqi Pigeon and Tarim Pigeon. However, in the populations of ornamental pigeon breeds such as Lady Pigeon, Angel Pigeon, and Taihu Spotted Pigeon, which have not been selectively bred for body weight traits, the G allele frequency is lower than that of the above - mentioned meat pigeon breeds, and there is almost no G allele. These results can all confirm that this SNP locus can be used as a molecular marker for the later body weight traits of pigeons.

[0028] Variety A allele frequency G allele frequency Number of individuals Danish Silver King Pigeon 0.87 0.13 50 Taishen Pigeon 0.96 0.04 50 American Silver King Pigeon 0.85 0.15 50 Yellow Carneau Pigeon 0.76 0.24 23 Deep King Pigeon 0.70 0.30 9 Grey Feather King Pigeon 0.83 0.17 18 White Feather King Pigeon 0.80 0.20 9 European Meat Pigeon 0.50 0.50 9 White Carneau Pigeon 0.55 0.45 9 Shiqi Pigeon 0.80 0.20 36 Tarim Pigeon 0.90 0.10 28 Taihu Spotted Pigeon 1.00 0.00 15 Furong Pigeon 0.88 0.12 15 Lady Pigeon 1.00 0.00 16 Fantail Pigeon 1.00 0.00 10 In addition to the above - mentioned embodiments, the present invention may have other embodiments. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope required by the present invention.

Claims

1. A molecular marker primer for the MSTN gene related to pigeon weight traits, characterized in that: The nucleotide sequences of the molecular marker primers are shown in SEQ ID NO: 1 and SEQ ID NO:

2. The SNP site is located at the 16694595th base on chromosome 6 of the pigeon reference genome Cliv_NAU_1.0 version, and the base mutates to A or G.

2. The application of the MSTN gene molecular marker primers related to pigeon body weight traits according to claim 1, characterized in that: The SNP primers are used for the detection of pigeon weight traits, and the detection method comprises the following steps: The first step is to perform PCR amplification on the pigeon DNA sample to be tested using the SNP primers shown in SEQ ID NO: 1-2 to obtain an amplification product, wherein the amplification product is 352 bp in length and contains the 16694595th base of pigeon chromosome 6; Step 2: Sanger sequencing of PCR products; Step 3: Determine the genotype of the SNP molecular marker at base 16694595 of chromosome 6 based on the sequencing results of the second step.

3. The application of the MSTN gene molecular marker primers relevant to pigeon body weight traits according to claim 2, characterized in that: The reaction system is 50 μl. Pigeon DNA to be tested 50 ng Accurate Taq DNA Polymerase 1.25 IU 5 μl 10X PCR reaction buffer containing Mg2+ 10mM dNTPs 1μl 10μM Upstream Primer F 1μl 10μM downstream primer R 1μl Make up to 50 μl with sterile water; The reaction conditions of the PCR amplification are: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 60 seconds, for a total of 30 cycles; extension at 72°C for 5 minutes; storage at 4°C; the nucleotide sequence of the amplified product is shown in SEQ ID NO:3 or SEQ ID NO:

4.

4. The application of the MSTN gene molecule marker primers relevant to pigeon body weight traits according to claim 2, characterized in that: The judgment criterion of the third step is that the weight of pigeons with the G / G genotype at the SNP site is higher than that of individuals with the A / G genotype and the A / A genotype, and the weight of individuals with the A / G genotype is higher than that of individuals with the A / A genotype.

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