IGF2BP3 gene molecular marker primer related to pigeon weight traits and its application

Abstract

The present invention relates to a molecular marker primer for the IGF2BP3 gene related to the weight trait of pigeons and its application, belonging to the field of biotechnology. The present invention uses the genomic DNA of the pigeon to be tested as a template, adopts specific 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 the breeding of pigeons.

Description

Technical Field

[0001] The invention relates to an IGF2BP3 gene molecular marker primer related to pigeon weight traits and application thereof, belonging to the field of biotechnology. Background Art

[0002] Weight is crucial in pigeon breeding and production, and is one of the key traits used in pigeon breed improvement and the development of new breeds (complementary lines). Conventional breeding methods, however, are characterized by long generation cycles and slow progress, making precise selection difficult. With the rapid development of modern biotechnology, molecular marker-assisted breeding (MABS) has gained widespread application in the breeding of new animal and plant varieties. Combined with conventional breeding techniques, this technology can improve the accuracy of target trait selection, enable earlier selection, significantly accelerate breeding progress, reduce breeding costs, and improve breeding efficiency. However, relatively few molecular markers are currently available for pigeon weight.

[0003] IGF2BP3 (Insulin-like growth factor 2 mRNA-binding protein 3) is an RNA-binding protein and a member of the IGF2BP family. It plays a key role in regulating mRNA stability, localization, and translation. IGF2BP3 and the IGF2 signaling pathway it regulates play a crucial role in body weight development in mammals (humans, mice, and pigs), poultry (chicken), and fish (carp).

[0004] GPNMB (Glycoprotein Non-Metastatic Melanoma Protein B), downstream of IGF2BP3, is a transmembrane protein belonging to the melanoma-associated antigen family (MAMPs). It plays a key role in various physiological and pathological processes, including cell proliferation, differentiation, immune regulation and metabolic regulation. Studies have also reported that this gene can affect abdominal fat deposition in chickens. Summary of the Invention

[0005] The purpose of the present invention is to address the defects of the existing technology, propose a molecular marker primer for the IGF2BP3 gene related to the pigeon weight trait and its application, and improve the breeding efficiency of pigeons.

[0006] The present invention records the weight traits of pigeons, performs SNP genotyping using whole-genome resequencing technology, and obtains related IGF2BP3 gene molecular markers through whole-genome association analysis, providing new gene and molecular marker resources for the breeding of pigeons with weight traits.

[0007] The present invention solves the technical problem through the following technical solution: First, a molecular marker of the IGF2BP3 gene related to pigeon weight is provided. The molecular marker is located at base 129,265,153 on chromosome 2 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), and the base mutation is A or G. The nucleotide sequences of the molecular marker primers are shown in SEQ ID NO:1 and SEQ ID NO:2, and the molecular marker is located at base 210 as shown in SEQ ID NO:3 or SEQ ID NO:4.

[0008] 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:

[0009] 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 425 bp in length and contains bases 129, 265, 153 of pigeon chromosome 2;

[0010] Step 2: Sanger sequencing of the PCR product;

[0011] The third step is to determine the SNP molecular marker genotype of base 129265153 of chromosome 2 based on the sequencing peak graph.

[0012] The deoxyribonucleotide sequence of the pigeon DNA specific primer pair described in the first step is: Upstream primer: 5'-GCAGCTCCAGTTGTGTCTTC-3' (SEQ ID NO: 1)

[0013] Downstream primer: 5'- CAGACTCCCACTGCTCTAGG-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 425 bp in length and contained bases 129, 265, 153 of pigeon chromosome 2.

[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 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 traits by using the IGF2BP3 gene molecular marker, and concludes that the weight of pigeons with the G / G genotype is higher than that of individuals with the A / G and A / A genotypes, and that the weight of pigeons with the A / G genotype is higher than that of individuals with the A / A genotype. By using the genomic DNA of the pigeon to be tested as a template, using specific primer pairs to perform PCR amplification, and then subjecting the PCR amplification product to Sanger sequencing and SNP genotyping, the genotype based on the SNP molecular marker can be used to select pigeon weight traits. For example, in breeding, it is necessary to cultivate meat pigeon varieties with higher weight. By eliminating individuals with the A / A genotype and retaining individuals with the A / G and G / G genotypes, the beneficial effect is that early selection of pigeon weight traits can be achieved, thereby improving the accuracy of seed selection, accelerating the breeding progress, saving breeding costs, improving breeding efficiency, and better serving pigeon breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the result of genome-wide association analysis of pigeon weight traits.

[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 traits of Danish Silver King Pigeons were recorded, SNP genotyping was performed using whole genome resequencing technology, and related IGF2BP3 gene molecular markers were obtained through whole genome association analysis. The results are as follows: Figure 1 shown.

[0030] This example uses the following experiments to identify and apply the IGF2BP3 gene molecular marker related to pigeon weight traits

[0031] 1. Phenotypic and genotypic testing

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

[0033] A total of 294 adult Danish Silver King pigeons of uniform age were selected as experimental animals and maintained under identical conditions with free access to food and water. They were reared according to a natural incubation model until 150 weeks of age. After laying eggs, the pigeons were fasted for 12 hours and weighed to provide phenotypic data for weight.

[0034] (2) Genomic DNA extraction

[0035] Blood was collected from the subwing vein of the above pigeons and stored in EDTA anticoagulant, and genomic DNA was extracted using the Omiga blood extraction kit.

[0036] (3) PCR amplification

[0037] The fragment containing the 129265153rd base site of chromosome 2 was amplified using the genomic DNA extracted as a template.

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

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

[0040] The reaction system is based on 50 μl and is as follows:

[0041] 50 ng of pigeon DNA to be tested

[0042] Accurate Taq DNA Polymerase 1.25 IU

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

[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 are: 94°C pre-denaturation for 5 minutes; 94°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 72°C extension for 60 seconds, for a total of 27 cycles; 72°C extension for 5 minutes; and storage at 4°C. The 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 were as follows: Figure 2 As shown, the genotyping data of the SNP molecular marker at base 129265153 of chromosome 2 of the pigeon reference genome Cliv_NAU_1.0 version were obtained.

[0051] 2. Correlation Analysis

[0052] A correlation analysis was conducted on 294 Danish Silver King pigeons (half male and half female) aged 150 weeks with clearly documented weight phenotypes. Statistical analysis was performed using the ANOVA function in GraphPad Prism 9 statistical software, using a pairwise comparison of mean values. P < 0.05 indicated significant differences, and P < 0.01 indicated extremely significant differences. The results showed that weight differences among the three genotypes were extremely significant (P < 0.01). The average weight of pigeons with the G / G genotype was 685.5 g, significantly higher than that of the A / G genotype (635.1 g) and the A / A genotype (577.5 g) (P < 0.05). Pigeons with the A / G genotype weighed more than those with the A / A genotype (P < 0.05). The results showed that the locus at base 129,265,153 on chromosome 2 in the pigeon reference genome Cliv_NAU_1.0 was significantly associated with the pigeon weight phenotype. Based on actual breeding goals, individuals with the G / G genotype can be selected to breed heavier pigeons, improve overall weight and uniformity, and enhance breeding efficiency. The data are shown in Tables 1 and 2.

[0053] genotype Number of individuals 150-week-old body weight / g CV / % G / G 11 <![CDATA[685.5±63.8 a ]]> 9.3 A / G 52 <![CDATA[635.1±63.6 b ]]> 10.0 A / A 231 <![CDATA[577.5±59.9 c ]]> 10.4 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).

[0054] genotype Number of male pigeons 150-week-old body weight / g Number of female pigeons 150-week-old body weight / g G / G 8 <![CDATA[684.9±60.4 a ]]> 3 <![CDATA[687.4±87.2 a ]]> A / G 25 <![CDATA[644.0±64.5 a ]]> 27 <![CDATA[626.9±62.9 a ]]> A / A 115 <![CDATA[594.1±63.7 b ]]> 116 <![CDATA[561.1±51.3 b ]]> 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] Example 2

[0056] Gene frequencies in different breeds

[0057] 1. Blood Sample Collection

[0058] Blood samples were collected from six heavier meat pigeon breeds, including Danish Silver King, American Silver King, Thai Deep Pigeon, White Feather King Pigeon, Yellow Cardinal Pigeon, and Gray Feather King Pigeon; two medium-weight domestic local breeds, Tarim Pigeon and Shiqi Pigeon; and five lighter ornamental pigeon breeds, including Lady Pigeon, Angel Pigeon, Taihu Dove, Hibiscus Pigeon, and Fantail Pigeon, using the subwing vein blood collection method and stored at -20℃ for future use.

[0059] 2. Extraction of Genomic DNA

[0060] 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.

[0061] 3. Genotype detection

[0062] 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 A / A genotype, A / G genotype, and G / G genotype.

[0063] 4. Results Analysis

[0064] Danish Silver King, American Silver King, Taishen, and White King are all popular meat pigeon breeds in the market. After selective breeding for weight, the G allele frequency accounts for a certain proportion in their populations. However, ornamental pigeon breeds such as Lady, Angel, Hibiscus, and Taihu Dot, which have not been bred for weight, have lower G allele frequencies in their populations than these meat pigeon breeds. These results confirm that this SNP locus can be used as a molecular marker for pigeon weight.

[0065] variety A allele frequency G allele frequency Number of individuals Danish Silver King Pigeon 0.87 0.13 50 Thai Deep Pigeon 0.99 0.01 50 American Silver King Pigeon 0.97 0.03 50 Yellow Cardinal Pigeon 0.92 0.08 23 Gray King Pigeon 0.90 0.10 18 white feathered king pigeon 0.85 0.15 9 Shiqi Pigeon 0.97 0.03 36 Tarim pigeon 1.00 0.00 28 Taihu Point Pigeon 1.00 0.00 15 Hibiscus Pigeon 1.00 0.00 15 Lady Pigeon 1.00 0.00 16 fantail pigeon 1.00 0.00 10 Angel Pigeon 1.00 0.00 18

[0066] 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 molecular marker primer for the IGF2BP3 gene associated with pigeon weight traits, characterized in that: The SNP site detected by the molecular marker primer is located at base 129265153 of chromosome 2 of the pigeon reference genome Cliv_NAU_1.0 version, and the base is A or G. The nucleotide sequence of the molecular marker primer is shown in SEQ ID NO: 1 and SEQ ID NO:

2.

2. The application of the IGF2BP3 gene molecular marker primers relevant to pigeon body weight traits according to claim 1, characterized in that: The molecular marker primers are used for pigeon weight trait detection, 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 molecular marker primers shown in SEQ ID NO: 1-2 to obtain an amplified product. The amplified product is 425 bp in length and contains bases 129, 265, 153 of pigeon chromosome 2; Step 2: Sanger sequencing of the PCR product; The third step is to determine the genotype of the SNP molecular marker at base 129265153 of chromosome 2 based on the sequencing results of the second step.

3. The application of the IGF2BP3 gene molecular marker primers relevant to pigeon body 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 10 μM upstream primer F 1 μl 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 application of the IGF2BP3 gene molecular marker primers relevant to pigeon body weight traits according to claim 2, characterized in that: 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 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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