IGF2BP3 gene molecular marker primer related to pigeon body weight character and application of IGF2BP3 gene molecular marker primer

Abstract

The invention relates to an IGF2BP3 gene molecular marker primer related to pigeon weight traits and application of the IGF2BP3 gene molecular marker primer, 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, a specific primer is adopted to carry out PCR amplification on the genome DNA, then a PCR amplification product is subjected to Sanger sequencing and SNP molecular marker genotyping, and then pigeon body weight character selection is carried out. 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 IGF2BP3 gene molecular marker primer related to the body weight trait of pigeons and its application, belonging to the field of biotechnology. Background Art

[0002] The body weight trait is very important in pigeon breeding and production, and is also one of the main traits for pigeon breed improvement and new variety (matching line) cultivation. Due to the long generation cycle and slow progress of conventional breeding, it is difficult to accurately select. With the rapid development of modern biotechnology, molecular marker-assisted breeding technology has been widely applied in the field of new variety breeding of animals and plants. Combining with conventional breeding technology can improve the accuracy of target trait selection, achieve early selection, greatly accelerate the breeding progress, save breeding costs, and improve breeding efficiency. However, there are few molecular markers related to the pigeon body weight trait.

[0003] IGF2BP3 (Insulin-like growth factor 2 mRNA-binding protein 3) is an RNA-binding protein and belongs to the IGF2BP family members, playing a key role in mRNA stability, localization and translational regulation. IGF2BP3 and its regulated IGF2 signaling pathway play key roles in the body weight development of mammals (humans, mice, pigs), birds (chickens) and fish (carps).

[0004] GPNMB (Glycoprotein Non-Metastatic Melanoma Protein B) downstream of IGF2BP3 is a transmembrane protein and belongs to the melanoma-associated antigen family (MAMPs), playing a key role in a variety of physiological and pathological processes, including cell proliferation, differentiation, immune regulation and metabolic regulation. There are also studies reporting that this gene can affect the abdominal fat deposition of chickens. Summary of the Invention

[0005] The purpose of the present invention is to propose an IGF2BP3 gene molecular marker primer related to the pigeon body weight trait and its application to improve the breeding efficiency of pigeons in view of the defects existing in the prior art.

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

[0007] The present invention solves the technical problems through the following technical solutions: First, a molecular marker of the IGF2BP3 gene related to pigeon body weight is provided. The molecular marker is located at the 129265153rd base of chromosome 2 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 nucleotide sequences of the primers of the molecular marker are shown in SEQ ID NO:1 and SEQ ID NO:2, and the molecular marker is located at the 210th base shown in SEQ ID NO:3 or SEQ ID NO:4.

[0008] The present invention further provides the application of the above molecular marker primers for the detection of 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 425 bp and contains the 129265153rd base of pigeon chromosome 2. Second step: Perform Sanger sequencing on the PCR product. Third step: Judge the SNP molecular marker genotype of the 129265153rd base of chromosome 2 according to the sequencing peak map.

[0009] The deoxyribonucleotide sequences of the pigeon DNA specific primer pair in the first step are: Forward primer: 5’-GCAGCTCCAGTTGTGTCTTC-3’ (SEQ ID NO:1) Reverse primer: 5’- CAGACTCCCACTGCTCTAGG-3’ (SEQ ID NO:2) The reaction system is calculated based on 50 μl, and 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 425 bp, and it contained the base at position 129265153 on chromosome 2 of pigeons.

[0010] 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 pigeons 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.

[0011] The present invention detected the genotype of the body weight trait of pigeons through the IGF2BP3 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 achieved. For example, in breeding, if it is necessary to breed meat pigeon varieties with 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

[0012] Figure 1 is the result of the genome-wide association analysis of the body weight trait of pigeons.

[0013] Figure 2 is the Sanger sequencing result of the PCR amplification products of three genotypes. Detailed Embodiments

[0014] In the following examples, the pigeon varieties used were all commercially available and will not be elaborated.

[0015] Example 1 In this example, by recording the body weight trait of Danish Silver King pigeons and using the whole-genome resequencing technology for SNP genotyping, relevant IGF2BP3 gene molecular markers were screened through genome-wide association analysis, and the results were as Figure 1 shown.

[0016] In this example, the following experiments were carried out to identify and apply the IGF2BP3 gene molecular marker related to the body weight trait of pigeons 1. Phenotype determination and genotype detection (1)Experimental materials and body weight phenotype determination Two hundred and ninety-four adult breeding pigeons of the same age, of the Danish Silver King variety, were selected as experimental animals and raised under the same feeding conditions. Throughout the process, they had free access to food and water. They were raised according to the natural incubation pattern until 150 weeks of age. After the breeding pigeons laid their eggs, they were fasted for 12 h, and their body weights were measured as phenotypic data for body weight traits.

[0017] (2)Genomic DNA extraction The above pigeons were bled from the wing vein and stored in EDTA anticoagulant. Genomic DNA was extracted using an Omiga blood extraction kit.

[0018] (3)PCR amplification Using the genomic DNA extracted above as a template, a fragment containing the base site at position 129,265,153 on chromosome 2 was amplified.

[0019] Forward primer: 5’-GCAGCTCCAGTTGTGTCTTC-3’ (SEQ ID NO:1) Reverse primer: 5’- CAGACTCCCACTGCTCTAGG-3’ (SEQ ID NO:2) The reaction system was calculated as 50 μl, and the system was 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 was added to make up to 50 μl; The reaction conditions for the PCR amplification were 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 27 cycles; extension at 72 °C for 5 min; and storage at 4 °C. The amplified product sequence was as 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 respectively, and the sequencing peak map was as Figure 2 shown, and the genotyping data of the SNP molecular marker at the base at position 129,265,153 on chromosome 2 of the pigeon reference genome Cliv_NAU_1.0 version was obtained.

[0021] 2. Correlation Analysis A total of 294 Danish Silver King pigeons (half male and half female) with clear phenotypic records of body weight at 150 weeks of age were selected for correlation analysis. The ANOVA test function of GraphPad Prism9 statistical software was used for statistical testing. The average value comparison mode between pairs was selected to statistically analyze the genotypes and traits of the experimental pigeon population. P<0.05 indicates a significant difference, and P<0.01 indicates a highly significant difference. The results showed that there was a highly significant difference in body weight among the three genotypes of pigeons (P <0.01). The average body weight of G / G genotype pigeons was 685.5 g, which was significantly higher than that of A / G genotype pigeons (635.1 g) and A / A genotype pigeons (577.5 g) (P<0.05). The body weight of A / G genotype pigeons was higher than that of A / A genotype pigeons (P<0.05). The results showed that the locus at the 129,265,153rd base on chromosome 2 of the pigeon reference genome version Cliv_NAU_1.0 was significantly associated with the phenotypic traits of pigeon body weight. Individuals with G / G genotype can be selected according to the actual breeding goal to breed pigeons with larger body weight, improve the overall body weight and uniformity, and improve the breeding efficiency. The data are shown in Table 1 and Table 2.

[0022] Genotype Number of individuals Body weight at 150 weeks of age / 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 superscript letter indicate no significant difference, and data with different superscript letters indicate significant difference (P<0.05).

[0023] Genotype Number of male pigeons / piece Body weight at 150 weeks of age / g Number of female pigeons / piece Body weight at 150 weeks of age / 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 superscript letter indicate no significant difference, and data with different superscript letters indicate significant difference (P<0.05).

[0024] Example 2 Gene Frequencies of Different Varieties 1. Blood Sample Collection Blood samples of 6 heavy-body meat pigeon varieties including Danish Silver King, American Silver King, Taishen pigeon, White King pigeon, Yellow Carneau pigeon, and Grey King pigeon, 2 medium-body domestic local varieties including Tarim pigeon and Shiqi pigeon, and 5 light-body ornamental pigeon varieties including Lady pigeon, Angel pigeon, Taihu spotted pigeon, Furong pigeon, and Fantail pigeon were collected using the wing vein blood sampling method and stored at -20°C for later use.

[0025] 2. Genomic DNA Extraction Taking the tissue samples obtained in the first step, genomic DNA was extracted using the tissue genomic DNA extraction kit of Omega brand. The specific method was referred to the standard operation procedure provided by 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 A / A genotype, A / G genotype, and G / G genotype of the individual.

[0027] 4. Result Analysis Danish Silver King, American Silver King, Taishen Pigeon, White King Pigeon, etc. are all relatively popular meat pigeon breeding varieties in the market. After certain breeding for their body weight traits, the G allele frequency occupies a certain proportion in the population. However, ornamental pigeon varieties such as Lady Pigeon, Angel Pigeon, Furong Pigeon, and Taihu Spotted Pigeon have not been selected for body weight traits, and the G allele frequency in the population is lower than that of the above-mentioned meat pigeon varieties. These results can confirm that this SNP locus can be used as a molecular marker for pigeon body weight traits.

[0028] Variety Allele frequency of A Allele frequency of G Number of individuals Danish Silver King Pigeon 0.87 0.13 50 Taishen Pigeon 0.99 0.01 50 American Silver King Pigeon 0.97 0.03 50 Yellow Carneau Pigeon 0.92 0.08 23 Grey Feather King Pigeon 0.90 0.10 18 White Feather King Pigeon 0.85 0.15 9 Shiqi Pigeon 0.97 0.03 36 Tarim Pigeon 1.00 0.00 28 Taihu Spotted Pigeon 1.00 0.00 15 Furong 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 In addition to the above embodiments, the present invention may have other embodiments. Any technical solutions formed by equivalent replacement or equivalent transformation fall within the protection scope required by the present invention.

Claims

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

2.

2. The application of the IGF2BP3 gene molecule marker primers related to pigeon body weight traits according to claim 1, characterized in that: The SNP 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 SNP primers shown in SEQ ID NO: 1-2 to obtain an amplification product, the amplification product is 425 bp in length and contains the 129265153rd base of pigeon chromosome 2; Step 2: Sanger sequencing of PCR products; 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 molecule marker primers related 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 IGF2BP3 gene molecule marker primers related 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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