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

Abstract

The present invention relates to an LCORL 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 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 A / A genotype pigeon is higher than that of the A / T and T / T genotype individuals, and the weight of the A / T genotype pigeon is higher than that of the T / T genotype individual. By eliminating the A / T and T / T genotype individuals and retaining the A / A genotype individuals, 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; it can efficiently and quickly identify the pigeon weight trait, improve the uniformity of the pigeon group weight, provide a scientific basis for the early selection of pigeons, better serve pigeon breeding, and has economic application value and scientific research value.

Description

Technical Field

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

[0002] my country holds a dominant position in global pigeon breeding and consumption, accounting for over 80% of the global pigeon population. While my country's pigeon farming industry began relatively late, in the 1990s, it has become the fourth largest poultry industry after chickens, ducks, and geese. With economic development and rising living standards, consumers are demanding higher quality poultry products, particularly taste. Pigeon meat and eggs are high-protein, low-fat, premium ingredients. Due to their rarity, nutritional value, and health benefits, they have gradually become a mainstream consumer staple for high-quality, high-end specialty poultry eggs. However, the pigeon farming industry has long been plagued by problems such as slow growth and low feed utilization. Therefore, the use of modern molecular markers for molecular breeding of pigeons is of vital importance.

[0003] Weight and body measurements are key indicators of livestock and poultry growth and development. They not only reflect individual health and nutritional status but also provide a direct measure of production performance. Weight is a crucial parameter for assessing muscle mass, fat deposition, and overall growth and development, while body measurements (such as chest width and shank length) further reflect skeletal development and body structure. Traditional breeding methods have long generation intervals and slow results. However, the use of molecular marker-assisted selection (MAM) allows for earlier selection, shortening generation intervals and accelerating breeding progress.

[0004] The LCORL gene (Ligand Dependent Nuclear Receptor Corepressor Like) encodes a protein that is widely expressed in humans and a variety of livestock. The transcriptional regulator encoded by this gene may influence the expression of downstream genes by participating in nuclear receptor signaling pathways, thereby playing an important role in biological processes such as spermatogenesis and skeletal development. Early studies focused on the gene's association with tumor development. Recent studies have found that genetic variation in the LCORL gene is significantly associated with growth traits (such as weight, height, skeletal development, and body size) in livestock, suggesting its key function in growth and development regulation. The NCAPG gene is found adjacent to the LCORL gene in the genomes of multiple animals, and is therefore often collectively referred to as the NCAPG-LCORL locus. The NCAPG-LCORL locus is not only associated with height in humans but has also been shown to be significantly associated with growth and body size traits in a variety of livestock, including pigs, cattle, sheep, and dogs. Studies have shown that the NCAPG gene is significantly associated with chicken tibia length, tibia mass, femur length, femur area, and shank length, and that the NCAPG-LCORL locus is significantly associated with chicken foot weight. Studies on the body weight traits of Chinese indigenous chicken breeds have shown that the NCAPG-LCORL locus is significantly associated with the body weight traits of indigenous chicken breeds. Summary of the Invention

[0005] The purpose of the present invention is to address the defects of the existing technology, propose a LCORL gene molecular marker related to pigeon weight traits 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 LCORL gene molecular markers significantly correlated with pigeon weight traits through whole-genome association analysis, providing new gene and molecular marker resources for pigeon weight trait selection and breeding.

[0007] The present invention solves the technical problem through the following technical solution: first, a molecular marker of the LCORL gene related to pigeon weight is provided. The nucleotide sequences of the SNP primers corresponding to the molecular marker are shown in SEQ ID NO: 1 and SEQ ID NO: 2. The molecular marker is located at base 17439739 on chromosome 4 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 T. The molecular marker is located at base 175 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 409 bp in length and contains base 17439739 of pigeon chromosome 4;

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

[0011] The third step is to determine the SNP molecular marker genotype of base 17439739 on chromosome 4 based on the sequencing peak graph.

[0012] The deoxyribonucleotide sequence of the molecular marker primer pair described in the first step is:

[0013] Upstream primer: 5'-TGACTGGCTGGGTAGATGTG-3' (SEQ ID NO: 1)

[0014] Downstream primer: 5'-TCAGGTGCTTCTCTGCTCAA-3' (SEQ ID NO: 2)

[0015] The reaction system is based on 50 μl.

[0016] 50 ng of pigeon DNA to be tested

[0017] Accurate Taq DNA Polymerase 1.25 IU

[0018] 5 μl 10X PCR reaction buffer containing Mg2+

[0019] 10mM dNTPs 1μl

[0020] 10 μM upstream primer F 1 μl

[0021] 10μM downstream primer R 1μl

[0022] Add sterile water to 50 μl;

[0023] 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 409 bp in length and contained base 17,439,739 of pigeon chromosome 4.

[0024] In the third step, the judgment standard is that the weight of pigeons with the A / A genotype at the SNP site is higher than that of individuals with the A / T genotype, and the weight of individuals with the A / T genotype is higher than that of individuals with the T / T genotype.

[0025] The present invention detects the genotype of pigeon weight traits by LCORL gene molecular markers, and obtains that the weight of pigeons with A / A genotype is higher than that of individuals with A / T genotype and T / T genotype, and the weight of pigeons with A / T genotype is higher than that of individuals with T / T genotype. By using the genomic DNA of the pigeon to be tested as a template, adopting specific primers to carry out PCR amplification, and then subjecting the PCR amplification product to Sanger sequencing and SNP molecular marker genotyping, the genotype based on the SNP molecular marker can be used to select the pigeon weight trait. For example, in breeding, it is necessary to cultivate meat pigeon varieties with higher weight. By eliminating T / T genotype individuals and retaining A / T and A / A genotype individuals, the beneficial effect is that the pigeon weight trait can be selected early, the accuracy of seed selection is improved, the breeding progress is accelerated, the breeding cost is saved, the breeding efficiency is improved, the breeding is better served, and the breeding of pigeons has great economic application and scientific research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the result of genome-wide association analysis of pigeon weight traits.

[0027] Figure 2 These are the Sanger sequencing results of the PCR amplification products of the three genotypes. DETAILED DESCRIPTION

[0028] The pigeon breeds used in the following examples are all commercially available and will not be described in detail.

[0029] Example

[0030] In this example, the weight traits of Danish Silver King Pigeon were recorded, SNP genotyping was performed using whole genome resequencing technology, and related LCORL gene molecular markers were obtained through whole genome association analysis. The results are as follows: Figure 1 shown.

[0031] This example uses the following experiments to identify and apply the LCORL gene molecular marker related to pigeon weight traits

[0032] Phenotyping and genotyping

[0033] (1) Experimental materials and body weight phenotype determination

[0034] 146 pairs of adult American Silver King pigeons of the same 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.

[0035] (2) Genomic DNA extraction

[0036] 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.

[0037] (3) PCR amplification

[0038] The fragment containing the 17439739 base site of chromosome 4 was amplified using the genomic DNA extracted as a template.

[0039] Upstream primer: 5'-TGACTGGCTGGGTAGATGTG-3' (SEQ ID NO: 1)

[0040] Downstream primer: 5'-TCAGGTGCTTCTCTGCTCAA-3' (SEQ ID NO: 2)

[0041] The system is as follows

[0042] 50 ng of pigeon DNA to be tested

[0043] Accurate Taq DNA Polymerase 1.25 IU

[0044] 10X PCR reaction buffer (containing Mg2+) 5μl

[0045] 10mM dNTPs 1μl

[0046] 10 μM upstream primer F 1 μl

[0047] 10μM downstream primer R 1μl

[0048] Add sterile water to 50 μl;

[0049] 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.

[0050] (4) Sanger sequencing and genotyping

[0051] The PCR products of each sample were subjected to Sanger sequencing, and the sequencing peak diagram was as follows: Figure 2 As shown, the genotyping data of the SNP molecular marker at base 17439739 on chromosome 4 of the pigeon reference genome Cliv_NAU_1.0 version were obtained.

[0052] 2. Correlation Analysis

[0053] A correlation analysis was conducted on 292 150-week-old American Silver King pigeons (half male and half female) with clearly documented weight phenotypes. Statistical analysis was performed using the ANOVA function in GraphPad Prism 9 statistical software, selecting the pairwise mean comparison mode for the genotype and weight traits of the experimental pigeon population. P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference. The results showed that weight differed significantly among the three genotypes (P < 0.01). The average weight of pigeons with the A / A genotype was 561.5 g, significantly higher than that of the A / T genotype (511.0 g) and the T / T genotype (453.3 g) (P < 0.05). Pigeons with the A / T genotype weighed more than those with the T / T genotype (P > 0.05). The results showed that the locus at base 17,439,739 on chromosome 4 of the pigeon reference genome, Cliv_NAU_1.0, was significantly associated with the pigeon weight phenotype. Based on actual breeding goals, individuals with the A / A 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.

[0054] genotype Number of individuals 150-week-old body weight / g CV / % A / A 233 <![CDATA[561.5±60.5 a ]]> 10.8 A / T 56 <![CDATA[511.0±66.2 b ]]> 13.0 T / T 3 <![CDATA[453.3±38.7 b ]]> 8.6 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 A / A 96 <![CDATA[569.8±55.9 a ]]> 94 <![CDATA[553.0±64.1 a ]]> A / T 25 <![CDATA[528.1±79.9 b ]]> 21 <![CDATA[490.6±38.7 b ]]> T / T 1 484.5 2 <![CDATA[437.4±39.1 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).

[0056] Gene frequencies in different breeds

[0057] 1. Blood Sample Collection

[0058] Blood samples were collected using the subwing vein blood collection method from eight heavier introduced meat pigeon strains (species), including Danish Silver King, American Silver King, Thai Deep Pigeon, Yellow Cardinal Pigeon, and Gray Feather King Pigeon; two medium-weight local strains (species), Shiqi Pigeon and Tarim Pigeon; and five lighter ornamental pigeon strains (species), including Fantail Pigeon, Angel Pigeon, Hibiscus Pigeon, Taihu Dove, and Lady Pigeon. The samples were 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 of 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 / T genotype, and T / T genotype.

[0063] 4. Results Analysis

[0064] The Danish Silver King, American Silver King, Thai Deep, Gray King, and European meat pigeons are all popular meat pigeon breeds in the Chinese market. After undergoing selective breeding for weight, the T allele frequency is at a disadvantage within their populations. The Tarim pigeon, a native breed of Xinjiang, my country, has undergone relatively low levels of selective breeding and has a higher T allele frequency than other introduced meat pigeon breeds. However, the three ornamental pigeon breeds—the Hibiscus pigeon, the Fantail pigeon, and the Lady pigeon—have not been bred for weight and are relatively small. Their populations have a much higher T allele frequency than commercially bred breeds (see Table 3). These results confirm that this SNP locus can serve as a molecular marker for pigeon weight.

[0065] variety A allele frequency T allele frequency Number of individuals Danish Silver King Pigeon 0.97 0.03 50 Thai Deep Pigeon 0.97 0.03 50 American Silver King Pigeon 0.89 0.11 50 Yellow Cardinal Pigeon 0.82 0.18 11 Gray King Pigeon 0.97 0.03 18 European pigeon 0.90 0.10 9 White Canu pigeon 0.95 0.05 9 Deep King Pigeon 0.90 0.10 9 Shiqi Pigeon 0.91 0.09 36 Tarim pigeon 0.45 0.55 28 Taihu Point Pigeon 0.88 0.13 15 Hibiscus Pigeon 0.19 0.81 15 Lady Pigeon 0.68 0.32 16 fantail pigeon 0.36 0.64 10 Angel Pigeon 0.87 0.13 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 primer for the LCORL gene molecule marker relevant to pigeon body weight traits, characterized in that: The molecular marker SNP site is located at base 17439739 on chromosome 4 of the pigeon reference genome Cliv_NAU_1.0 version, and the base mutation is A or T. The nucleotide sequences of the SNP primers corresponding to the molecular marker are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

2. The application of the primer of the LCORL gene molecule marker relevant to pigeon body weight according to claim 1, wherein: The SNP primers are used for weight trait detection of 150-week-old pigeons, 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 409 bp in length and contains base 17439739 of pigeon chromosome 4; Step 2: Sanger sequencing of the PCR product; The third step is to determine the genotype of the SNP molecular marker at base 17439739 of chromosome 4 based on the sequencing results of the second step.

3. according to the application of the primer of the LCORL gene molecule marker relevant to pigeon body weight trait of claim 2, it is characterized in that: The final volume of the reaction system in the first step 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. according to the application of the primer of the LCORL gene molecule marker relevant to pigeon body weight trait of claim 2, it is characterized in that: The judgment standard in the third step is that the weight of pigeons with A / A genotype at the SNP site is higher than that of individuals with A / T genotype and T / T genotype, and the weight of individuals with A / T genotype is higher than that of individuals with T / T genotype.

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