A molecular marker of MSTN gene related to pigeon weight traits and its application

Abstract

The present invention relates to an MSTN gene molecular marker related to pigeon weight traits and its application, and belongs to the field of biotechnology. The present invention uses the genomic DNA of the pigeon to be tested as a template, adopts SNP primers to perform PCR amplification on it, and then performs Sanger sequencing and SNP molecular marker genotyping on the PCR amplification product, thereby selecting the pigeon weight trait. The weight of the pigeon with G / G genotype is higher than that of the individuals with A / G and A / A genotypes, and the weight of the pigeon with A / G genotype is higher than that of the individuals with A / A genotype. By eliminating the individuals with A / G and A / A genotypes and retaining the individuals with G / G genotype, the beneficial effect is that the molecular marker can be used as a genetic marker for pigeon breeding to select pigeons with larger and more uniform weight; the pigeon weight trait can be identified efficiently and quickly, and the uniformity of the weight of the pigeon group can be improved, providing a scientific basis for the early selection of pigeons, and better serving pigeon breeding.

Description

Technical Field

[0001] The invention relates to an MSTN gene molecular marker related to pigeon weight traits and application thereof, belonging to the field of biotechnology. Background Art

[0002] Weight is a key growth trait in pigeons. Conventional breeding techniques, however, have long generation cycles and slow progress, making precise selection difficult. With the rapid development of modern biotechnology, molecular marker-assisted breeding has gained widespread application in the selection of new plant and animal varieties. Combined with conventional breeding techniques, it can improve the accuracy of target trait selection, enable earlier selection, accelerate breeding progress, reduce breeding costs, and improve breeding efficiency. However, currently, there are few molecular markers associated with pigeon weight.

[0003] The MSTN gene (myostatin gene), a member of the transforming growth factor-β (TGF-β) superfamily, encodes the MSTN protein, a key negative regulator of skeletal muscle growth. Studies have found that natural loss-of-function mutations in MSTN in cattle result in a "double-muscle" phenotype, leading to a significant increase in muscle mass. Interference with the MSTN gene promotes the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells, confirming the biological function of MSTN interference in regulating muscle development and differentiation. These studies demonstrate that the MSTN gene plays a crucial regulatory role in muscle growth and development. Summary of the Invention

[0004] The purpose of the present invention is to address the defects of the existing technology, propose an MSTN gene molecular marker related to pigeon weight traits and its application, and improve the breeding efficiency of pigeons.

[0005] The present invention records the weight traits of pigeons, performs SNP genotyping using whole-genome resequencing technology, and obtains related MSTN gene molecular markers through whole-genome association analysis, providing new gene and molecular marker resources for the weight trait selection and breeding of pigeons.

[0006] The present invention solves the technical problem through the following technical solution: First, a molecular marker primer for the MSTN gene associated with pigeon 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 base 16,694,595 on chromosome 6 of the pigeon reference genome Cliv_NAU_1.0 version (National Genome Science Data Center https: / / ngdc.cncb.ac.cn / gwh, accession number GWHFCQQ00000000.1), with the base mutation being A or G. The molecular marker is located at base 156 as shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0007] The present invention further provides an application of the above-mentioned molecular marker primers for detecting SNP genotypes related to pigeon weight traits, and the detection method comprises the following steps:

[0008] The first step is to provide a pigeon DNA sample to be tested, and perform PCR amplification using a molecular marker primer pair to obtain an amplified product. The amplified product is 352 bp in length and contains base 16694595 of pigeon chromosome 6;

[0009] Step 2: Sanger sequencing of the PCR product;

[0010] The third step is to determine the SNP molecular marker genotype at base 16694595 of chromosome 6 based on the sequencing peak graph.

[0011] The sequence of the pigeon DNA specific primer pair described in the first step is:

[0012] Upstream primer: 5'-GTGAGTACTGTTTGCCATGC-3' (SEQ ID NO: 1)

[0013] Downstream primer: 5'-GGCTCATGTCGTCAAACCC-3' (SEQ ID NO: 2)

[0014] The reaction system is 50 μl.

[0015] 50 ng of pigeon DNA to be tested

[0016] Accurate Taq DNA Polymerase 1.25 IU

[0017] 5 μl 10X PCR reaction buffer containing Mg2+

[0018] 10mM dNTPs 1μl

[0019] 10μM upstream primer F 1μl

[0020] 10μM downstream primer R 1μl

[0021] Add sterile water to 50 μl;

[0022] The reaction conditions for the 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, and extension at 72°C for 60 sec, for a total of 30 cycles; extension at 72°C for 5 min; and storage at 4°C. The nucleotide sequence of the amplified product was as shown in SEQ ID NO:3 or SEQ ID NO:4. The amplified product was 352 bp in length and contained base 16,694,595 of pigeon chromosome 6.

[0023] In the third step, the judgment standard 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 weight of individuals with the A / G genotype is higher than that of individuals with the A / A genotype.

[0024] The present invention detects the genotype of pigeon weight trait by MSTN gene molecular marker, and obtains that the weight of pigeon with G / G genotype is higher than that of individuals with A / G genotype and A / A genotype, and the weight of pigeon with A / G genotype is higher than that of individuals with A / A genotype. By taking the genomic DNA of pigeon to be tested as template, adopting specific primer to carry out PCR amplification, then carrying out Sanger sequencing and SNP genotyping of PCR amplification product, the genotype based on this SNP molecular marker can realize selecting pigeon weight trait. For example, in breeding, it is necessary to cultivate meat pigeon varieties with higher body weight, and by eliminating A / A genotype individuals, retaining A / G and G / G genotype individuals, its beneficial effect is that pigeon weight trait can be carried out early selection, improves the accuracy of seed selection, accelerates breeding progress, saves breeding cost, improves breeding efficiency, and better serves the breeding of pigeon. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the Manhattan plot of the genome-wide association analysis of pigeon body weight.

[0026] Figure 2 These are the Sanger sequencing results of the PCR amplification products of the three genotypes. DETAILED DESCRIPTION

[0027] The pigeon breeds used in the following examples are all commercially available and will not be described in detail.

[0028] Example 1

[0029] In this example, the weight of three breeds of Danish Silver King pigeon, Thai Deep pigeon, and American Silver King pigeon at 150 weeks of age was measured. Whole-genome SNP genotyping was performed using second-generation sequencing technology. The MSTN gene molecular markers significantly associated with weight at 150 weeks of age were screened through genome-wide association analysis. The results are as follows: Figure 1 shown.

[0030] This example identifies and applies the MSTN gene molecular markers related to pigeon weight through the following experiments.

[0031] 1. Phenotypic and genotypic testing

[0032] (1) Experimental materials and body weight phenotype determination

[0033] A total of 389 pairs of breeding pigeons were selected, including Danish Silver King Pigeon, Thai Deep Pigeon and American Silver King Pigeon. The three breeds were of similar number and raised under the same feeding conditions with free access to food and water throughout the whole process. When the breeding pigeons were 150 weeks old, the weight of each pigeon was recorded after fasting for 12 hours after laying eggs, which was used as the phenotypic data of pigeon weight.

[0034] (2) Extraction of genomic DNA

[0035] Take the tissue sample obtained in step 1 and use the Omega brand tissue genomic DNA extraction kit to extract genomic DNA. The specific method refers to the standard operating procedure provided by the Omega brand.

[0036] (3) PCR amplification

[0037] The fragment containing the 16694595th base site of chromosome 6 was amplified using the genomic DNA extracted as a template.

[0038] Upstream primer: 5'-GTGAGTACTGTTTGCCATGC-3' (SEQ ID NO: 1)

[0039] Downstream primer: 5'-GGCTCATGTCGTCAAACCC-3' (SEQ ID NO: 2)

[0040] The final concentration of the reaction system (25 μl) is:

[0041] 50 ng of pigeon DNA to be tested

[0042] Accurate Taq DNA Polymerase 1.25 IU

[0043] 5 μl 10X PCR reaction buffer containing Mg2+

[0044] 10mM dNTPs 1μl

[0045] 10μM upstream primer F 1μl

[0046] 10μM downstream primer R 1μl

[0047] Add sterile water to 50 μl;

[0048] The PCR amplification reaction conditions were as follows: 94°C pre-denaturation for 5 minutes; 30 cycles of 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, and 72°C extension for 60 seconds; 72°C extension for 2 minutes; and storage at 4°C. A 10 μl sample was used for agarose gel analysis, yielding a single target band of 352 bp containing the SNP marker 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.

[0049] (4) Sanger sequencing and genotyping

[0050] The PCR products of each sample were subjected to Sanger sequencing, and the sequencing peaks of different genotypes were as follows: Figure 2 shown.

[0051] 2. Correlation Analysis

[0052] Correlation analysis was performed on 389 pairs of pigeons at 150 weeks of age with clearly documented weight phenotypes. Statistical tests were performed using the ANOVA function in GraphPad Prism 9 statistical graphics software, using the pairwise mean comparison mode. P < 0.05 indicated significant differences between the genotypes and weight traits. The results are shown in Tables 1 and 2. Weight differences were significant among the three genotypes (P < 0.05). The mean weight of individuals with the G / G genotype was 639.2 g, higher than that of individuals with the A / G genotype (586.9 g) (P < 0.05) and the A / A genotype (558.8 g) (P < 0.05). Individuals with the A / G genotype also weighed more than those with the A / A genotype (P < 0.05). The results showed that the site at base 16694595 of chromosome 6 of pigeons was significantly associated with the pigeon weight phenotype. According to the actual breeding goals, G / G genotype individuals can be selected to breed pigeons with larger weight, improve the overall weight and uniformity, and improve breeding efficiency.

[0053] genotype Quantity / piece 150-week-old body weight / 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

[0054] Note: Data in the same column with the same letters indicate no significant difference, while data with different letters indicate significant difference (P<0.05).

[0055] genotype Number of male pigeons 150-week-old body weight / g Number of female pigeons 150-week-old body weight / 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 ]]>

[0056] Note: Data in the same column with the same letters indicate no significant difference, while data with different letters indicate significant difference (P<0.05).

[0057] Example 2

[0058] Gene frequencies in different breeds

[0059] 1. Blood Sample Collection

[0060] Blood samples from 15 strains / breeds including Danish Silver King Pigeon, Thai Deep Pigeon, and American Silver King Pigeon were collected using the subwing vein blood collection method and stored at -20℃ for future use.

[0061] 2. Extraction of Genomic DNA

[0062] Take the tissue sample obtained in step 1 and use the Omega brand tissue genomic DNA extraction kit to extract genomic DNA. The specific method refers to the standard operating procedure provided by the Omega brand.

[0063] 3. Genotype detection

[0064] Using the genomic DNA obtained in step 2 as a template, PCR amplification and Sanger sequencing were performed using 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 individual's G / G genotype, A / G genotype, and G / G genotype.

[0065] 4. Results Analysis

[0066] White-feather lines (White King, White Cano, Deep King, and European meat pigeons), Danish Silver King, American Silver King, and Thai Deep pigeons are all meat pigeon lines introduced into my country. These lines have undergone selective breeding for weight, and the G allele frequency already accounts for a significant proportion of their populations. Populations of the native Chinese breeds Shiqi and Tarim pigeons also harbor a significant proportion of the G allele. However, ornamental pigeon breeds such as the Lady and Taihu Dot pigeons, which have not been bred for weight, have lower G allele frequencies than these meat pigeon breeds, with the G allele practically absent. These results confirm the suitability of this SNP as a molecular marker for later-stage weight traits in pigeons.

[0067] variety A allele frequency G allele frequency Number of individuals Danish Silver King Pigeon 0.87 0.13 50 Thai Deep Pigeon 0.96 0.04 50 American Silver King Pigeon 0.85 0.15 50 Yellow Cardinal Pigeon 0.76 0.24 23 Deep King Pigeon 0.70 0.30 9 Gray King Pigeon 0.83 0.17 18 white feathered king pigeon 0.80 0.20 9 European pigeon 0.50 0.50 9 White Canu pigeon 0.55 0.45 9 Shiqi Pigeon 0.80 0.20 36 Tarim pigeon 0.90 0.10 28 Taihu Point Pigeon 1.00 0.00 15 Hibiscus Pigeon 0.88 0.12 15 Lady Pigeon 1.00 0.00 16 fantail pigeon 1.00 0.00 10

[0068] In addition to the above embodiments, the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the scope of protection required by the present invention.

Claims

1. A MSTN gene SNP molecular marker primer 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 base 16694595 on chromosome 6 of the pigeon reference genome Cliv_NAU_1.0 version, and the base mutation is A or G.

2. The use of the MSTN gene SNP molecular marker primers related to pigeon 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 amplified product. The amplified product is 352 bp in length and contains base 16694595 of pigeon chromosome 6. Step 2: Sanger sequencing of the PCR product; 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 SNP molecular marker primers related to pigeon weight traits according to claim 2, characterized in that: The reaction system is 50 μl. 50 ng of pigeon DNA to be tested 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 Add sterile water to 50 μl; The reaction conditions for the PCR amplification are: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 60 sec, for a total of 30 cycles; extension at 72°C for 5 min; and 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 use of the MSTN gene SNP molecular marker primers related to pigeon 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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