Pigeon individual typing detection system, reagent kit and its application

Abstract

This invention relates to a pigeon individual typing detection system, kit, and their applications. The pigeon individual typing detection system includes primer combinations and multiplex amplification premixes. The primer combinations include primers for amplifying 23 STR microsatellite markers, the CHD locus for sex detection in birds, and 5 SNP genetic markers. The pigeon individual typing detection system and kit provided by this invention can detect 29 pigeon DNA genetic markers in a single tube, including STR genetic markers for pigeon identification and parentage determination, and single nucleotide polymorphism genetic markers for assessing pigeon flight ability. It is currently the detection system capable of detecting the largest number of pigeon DNA loci and has promising application prospects in pigeon identification, parentage determination, and flight ability assessment.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, and in particular to a pigeon individual typing detection system, reagent kit, and its application. Background Technology

[0002] The domestication of pigeons dates back thousands of years and is closely related to human activities. In ancient times, pigeons played a vital role in long-distance communication due to their excellent spatial orientation and homing ability. Today, with highly developed communication networks, pigeons no longer need to deliver messages but have become the main characters in racing events. Pigeon racing is an officially approved sport by the General Administration of Sport of China and is attracting increasing participation from pigeon enthusiasts and fanciers worldwide. Due to the high prize money, the standardization of pigeon racing events is gradually improving, with fairness and authenticity being particularly important. To ensure transparency and prevent the emergence of hybrid pigeons (AB pigeons), genetic testing using STR microsatellites for identification has been introduced into pigeon racing events. Secondly, as pigeon racing becomes more widely known, enthusiasts and fanciers are increasingly seeking pigeons with superior bloodlines, leading to the development of a corresponding trading market. The offspring of pigeons that achieve excellent results in races often fetch high prices in the market, making STR microsatellite testing for pigeon parentage an important application.

[0003] Meanwhile, in order to assess the innate conditions of pigeons, such as orientation ability, flight speed, cardiopulmonary endurance, and resistance to adverse conditions, and to increase their chances of winning, SNP testing related to pigeon flight ability has gradually been promoted. SNP stands for Single Nucleotide Polymorphism, which refers to variations in a single nucleotide in the genome, including transitions, transversions, deletions, and insertions. There are many methods for detecting SNPs, including direct DNA sequencing, real-time quantitative PCR, HRM high-resolution melting curve technology, mass spectrometry, and SNaPshot method, but their use is often limited by cumbersome procedures, long operating times, high requirements for material collection and technology, or cost issues. Summary of the Invention

[0004] The purpose of this invention is to disclose a pigeon individual typing detection system, reagent kit and its application, in order to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The first aspect of this invention is to provide a pigeon individual typing detection system.

[0007] A second aspect of the present invention is to provide a kit comprising the pigeon individual typing detection system described in the first aspect of the present invention.

[0008] The third aspect of this invention is to provide the application of the pigeon individual typing detection system described in the first aspect of this invention and the reagent kit described in the second aspect of this invention in pigeon individual identification and / or pigeon parentage identification and / or flight ability assessment.

[0009] The pigeon individual typing detection system of the first aspect of the present invention includes primer combinations and multiplex amplification premixes. The primer combinations include primers for amplifying 23 STR microsatellite markers, the CHD locus for sex detection in birds, and 5 SNP genetic markers. The STR microsatellite markers are CliμD32, CliμD19, CliμD35, CliμT02, PIGN10, PIGN26, PG6, CliμD11, Cli12, PG2, CliμC06, and CliμD17. PG3, PG5, PIGN04, CliμD01, PG1, Cli02, PIGN12, PIGN15, PIGN57, CliμT13, and CliμD16, respectively, with the SNP genetic markers being g.2582481G>A of the lactate dehydrogenase gene, g.129954C>T and g.129456C>T of the dopamine receptor D4 gene, g.66493737C>T of the myostatin gene, and g.710T>G of the feather keratin gene.

[0010] The pigeon individual genotyping system is a multiplex amplification detection system. Genotyping primers are designed in conserved regions flanking the core sequence of the locus to detect the STR microsatellite markers and CHD locus. ARMS genotyping primers are designed to match different alleles of the SNP genetic markers. Lactate dehydrogenase (LDHA) is involved in aerobic and anaerobic metabolism, determining muscle endurance, recovery, and aerobic capacity. The single nucleotide polymorphism g.2582481G>A in this gene is closely related to the behavioral performance of racing pigeons, with the A allele being more common in top-level racing pigeons. The dopamine receptor D4 gene (DRD4) is also considered to be closely related to racing performance. g.129954C>T and g.129456C>T are two promising single nucleotide polymorphism sites. The combined genotype CTCT is usually associated with strong racing performance, while the CCCC genotype usually indicates a lower level of racing ability. Myostatin (MSTN) is considered one of the most powerful negative regulators of muscle growth, playing a crucial role in muscle development. The β-keratin gene plays a crucial role in the development of racing pigeons. The single nucleotide polymorphism (SNP) g.66493737C>T in this gene is a marker that can be used to analyze the muscle traits of racing pigeons. The loss of myostatin (MSTN) caused by the mutation leads to an increase in muscle mass, which will further improve athletic performance. Therefore, individuals carrying the T allele usually have abundant muscle mass and a high body mass to muscle mass ratio. For the single nucleotide polymorphism g.710T>G in the feather keratin (F-KER) gene, pigeons with the TT genotype perform best over longer distances because β-keratin is a key protein for normal feather development. The mutation from T to G in g.710T>G may lead to changes in keratin structure, thereby affecting feather quality and racing performance.

[0011] In some embodiments of the first aspect of the present invention, at least one of the upstream and downstream primers targeting the same STR microsatellite marker, the CHD locus, or the SNP genetic marker has a fluorescent dye labeled at its 5' end, the fluorescent dye being selected from 6-FAM, HEX, SUM, LYN, PUR, TAMRA, ALEXA568, ALEXA594, ROX, VIC, PET, NED, or TAZ.

[0012] In some embodiments of the first aspect of the present invention, the nucleotide sequences of the primers for detecting the STR microsatellite marker, the CHD locus, and the SNP genetic marker are shown in Table 1.

[0013] In some embodiments of the first aspect of the present invention, the mutual influence of each primer in the multiplex amplification detection system can be reduced by adjusting the final concentration of each primer to a suitable range, as shown in Table 1.

[0014] In some embodiments of the first aspect of the present invention, specific primer groups are shown in Table 1.

[0015] In some embodiments of the first aspect of the present invention, the types of fluorescent dyes selected for specific groups are shown in Table 1.

[0016] Table 1. Primer sequence information for pigeon genetic markers.

[0017]

[0018]

[0019]

[0020] In some embodiments of the first aspect of the present invention, the multiplex amplification premix comprises one or more of PCR reaction buffer, dNTPs, DNA polymerase and MgCl2.

[0021] The kit described in the second aspect of the present invention comprises the above-mentioned pigeon individual typing detection system.

[0022] In some embodiments of the second aspect of the present invention, the kit further includes allele typing standards. The allele typing standards are prepared by diluting all recombinant plasmids containing allele inserts within a single locus to 1 ng / μL, mixing them thoroughly, performing PCR amplification using fluorescent primers for that locus, and recording the average peak height of all alleles at that locus; adjusting the PCR products of the mixed locus according to the volume ratio calculated based on the average peak height of the ladder at a single locus, and purifying them using the phenol-chloroform method.

[0023] The third aspect of this invention provides an application direction of using the pigeon individual typing detection system or the kit for pigeon individual identification and / or pigeon parentage identification and / or pigeon flight ability assessment.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The pigeon individual typing detection system and kit provided by this invention can detect 29 pigeon DNA genetic markers in a single tube, including STR genetic markers for pigeon individual identification and parentage determination, as well as single nucleotide polymorphism genetic markers for assessing pigeon flight ability. It is currently the detection system that can detect the largest number of pigeon DNA loci, and has good application prospects in the fields of pigeon identification, parentage determination and flight ability assessment. Attached Figure Description

[0026] Figure 1 This is the ARMS primer screening map for the five single nucleotide polymorphism genetic markers in Example 2;

[0027] Figure 2 This is a comparative spectrum of primer mismatches designed for the DRD4-1 (g.129954C>T) site in Example 2;

[0028] Figure 3 This is the detection spectrum before adjusting the primer concentration for the PIGN10 site in Example 2;

[0029] Figure 4 This is the detection spectrum after adjusting the primer concentration for the PIGN10 site in Example 2;

[0030] Figure 5 This is a diagram showing the genetic marker arrangement of the multiplex amplification detection system in Example 2;

[0031] Figure 6 This is a comparison graph of the amplification procedure of the optimized multiplex amplification detection system for the CliμD11 site in Example 3;

[0032] Figure 7 This is the detection pattern of pigeon positive reference DNA by the multiplex amplification detection system in Example 3;

[0033] Figure 8 This is the typing diagram of the allelic lag plasmid described in Example 4;

[0034] Figure 9 This is the species-specific verification map in Example 5;

[0035] Figure 10 This is a bar chart of the sensitivity verification results in Example 6;

[0036] Figure 11 This is the gene detection map of the pigeon feather sample in Example 8;

[0037] Figure 12 This is the Sanger sequencing map of the five single nucleotide polymorphisms in the pigeon feather sample from Example 8;

[0038] Figure 13 This is the parent pigeon detection spectrum used in Example 9 for kinship identification;

[0039] Figure 14 This is a pheasant test pattern for kinship identification in Example 9. Detailed Implementation

[0040] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed in accordance with Molecular Cloning: A Laboratory Manual (3rd Edition) or the method and product instructions. Unless otherwise specified, the biological materials used in these methods are commercially available.

[0041] Example 1: Site selection.

[0042] By collecting pigeon feather samples nationwide, a genetic polymorphism survey of pigeon populations was conducted targeting selectable pigeon STR loci, and the following STR microsatellite markers were identified: CliμD32, CliμD19, CliμD35, CliμT02, PIGN10, PIGN26, PG6, CliμD11, Cli12, PG2, CliμC06, CliμD17, PG3, PG5, PIGN04, CliμD01, PG1, Cli02, PIGN12, PIGN15, PIGN57, CliμT13, and CliμD16.

[0043] The chromosome helical protein binding gene (CHD) of pigeons was selected as the sex identification locus, and the difference in the length of its introns on the Z and W chromosomes was used for identification.

[0044] Lactate dehydrogenase A (LDHA), dopamine D4 receptor (DRD4), muscle regulation (MSTN), and feather keratin (F-KER) are four innate genes that significantly influence the flight ability of pigeons. Among them, the lactate dehydrogenase gene (LDHA) is involved in aerobic and anaerobic metabolism, determining muscle endurance, recovery, and aerobic capacity. The single nucleotide polymorphism (SNP) g.2582481G>A in this gene is closely related to the behavioral performance of racing pigeons, with the A allele being more common in top-level racing pigeons. The dopamine receptor D4 gene (DRD4) is also considered to be closely related to racing performance. g.129954C>T and g.129456C>T are two promising SNP sites; their combined genotype CTCT is usually associated with strong racing performance, while the CCCC genotype usually indicates a lower level of racing ability. Myostatin (MSTN) is considered one of the most powerful negative regulators of muscle growth, playing a crucial role in muscle development. The β-keratin gene plays a crucial role in muscle development. The single nucleotide polymorphism (SNP) g.66493737C>T in this gene is a marker that can be used to analyze the muscle traits of racing pigeons. The loss of myostatin (MSTN) caused by the mutation leads to an increase in muscle mass, which in turn leads to improved athletic performance. Therefore, individuals carrying the T allele usually have abundant muscle mass and a high body mass to muscle mass ratio. For the single nucleotide polymorphism g.710T>G in the feather keratin (F-KER) gene, pigeons with the TT genotype perform best over longer distances because β-keratin is a key protein for normal feather development. The mutation from T to G in g.710T>G may lead to changes in keratin structure, thereby affecting feather quality and racing performance. Therefore, the present invention selects the lactate dehydrogenase (LDHA) gene g.2582481G>A, dopamine receptor D4 (DRD4) genes g.129954C>T and g.129456C>T, myostatin (MSTN) gene g.66493737C>T, and feather keratin (F-KER) gene g.710T>G as five SNP genetic markers for evaluating pigeon flight ability.

[0045] Example 2: Primer design.

[0046] When designing primers for multiplex amplification, it is crucial to consider the interactions between primers at different loci, as well as the similar physical properties and reaction kinetics among different primers, to ensure the balance of each locus in the amplification system. Furthermore, ARMS-PCR primers for SNP genetic markers involve designing two ARMS genotyping primers for each allele's nucleic acid sequence, ensuring that the 3' terminal nucleotides of the primers match different allele base types. Introducing 1-2 mismatched bases near the 3' end of the ARMS genotyping primer further enhances the specificity of the reaction. The two genotyping amplification products are then distinguished based on their length difference. The difficulty in constructing the multiplex amplification detection system is directly related to the number of loci included and the complexity of the reaction system. As the number of primers in the system increases, the reaction kinetics become more complex, and primers at different loci are more prone to mutual influence and interference. Therefore, the requirements for primer sequences are more stringent than for conventional PCR. In practical work, it is necessary to design a large number of primer sequences, conduct complex tests, and explore the appropriate concentration ratio between primers in the multiplex amplification system in order to ensure that more loci can be detected simultaneously without reducing detection specificity and sensitivity.

[0047] The designed primer combinations were first screened for amplification efficiency and specificity using singleton PCR amplification tests against STR microsatellite markers, and for genotyping accuracy and efficiency tests using ARMS-PCR primers against SNP genetic markers. Specifically, the ARMS-PCR amplification system for SNP genetic markers included one common primer and two genotyping primers, amplifying the genomes of different genotypes as templates. Obtaining amplification products corresponding to the genotype was considered acceptable. When the template was heterozygous, two corresponding amplification products were obtained. If cross-reactions occurred between the ARMS primers and non-corresponding gene templates, the primer sequences needed to be readjusted, or the number or type of mismatched bases needed to be introduced to achieve a better distinguishing effect. Different genotyping DNA templates used for primer testing could be collected from Sanger sequencing results or synthesized artificially by Shanghai Sangon Biotech Co., Ltd. The final primer screening map for the five SNP genetic markers is shown below. Figure 1 As shown.

[0048] After primer single-amplification testing is completed, primer pairs for individual genetic markers are sequentially combined into the multiplex amplification detection system for efficacy verification. If mismatches with other primers causing heterogeneous peaks or inefficiency due to suppression by other primers are encountered during the construction of the multiplex amplification detection system, the primer sequences need to be adjusted. Therefore, the entire construction process of the multiplex amplification detection system requires multiple experiments in stages. Because the interactions between dozens of primers in the multiplex amplification system are complex and cannot be effectively predicted in advance, only through repeated testing and adjustments can effective primer combinations be obtained.

[0049] During the construction of single-amplification and multiplex amplification detection systems, the designed primers repeatedly failed to achieve the expected results.

[0050] Taking the primer design for the DRD4-1 (g.129954C>T) site as an example for single-amplification assays: The first ARMS primer sequence is 5'-TACCTTACGAGCGGTGACA T T-3' (SEQ ID No: 64). Since the amplification efficiency of this primer against the TT genotyping DNA template was significantly lower than expected, the reason for the unsatisfactory amplification efficiency of this ARMS primer was likely due to the excessive influence of the introduced mismatched bases. Therefore, it was considered to move the mismatch position to the fourth base at the 3' end, resulting in the second primer sequence: 5'-TACCTTACGAGCGGTGA G AAT-3' (SEQ ID No: 53). The amplification results of the first and last primer sets are as follows: Figure 2 As shown, peaks were observed normally after the positions of the mismatched bases were modified.

[0051] The construction of the multiplex amplification detection system was used to verify the PIGN10 primer pair as an example: When the STR microsatellite labeling multiplex amplification system was built up to 22 layers, the efficiency of the previously well-performing PIGN10 primers suddenly decreased, and the target product could not be amplified normally. After repeated experiments, it was inferred that this phenomenon might be due to the inhibitory effect caused by the excessively high concentration of certain primers in the system, resulting in the loss of peaks at the PIGN10 site in multiple samples (see...). Figure 3 After repeatedly lowering the concentrations of several high-emergence genetic marker primers, such as CliμD32, PG6, and CliμD17, the amplification efficiency of the PIGN10 locus was significantly improved (see [link to article]). Figure 4 ).

[0052] To avoid overlapping peak positions of amplification products from different loci and further ensure the accuracy of genotyping results for all loci in the multiplex amplification detection system, a reasonable grouping and arrangement was implemented based on a full consideration of the length range of amplification product fragments from each locus. The final confirmed primer combinations divided all loci into five groups, each group using different fluorescent labels. Primers specifically amplifying CliμD32, CliμD19, CliμD35, CliμT02, PIGN10, and PIGN26 loci were integrated into the first group, labeled with 6-FAM fluorescent dye. Primers specifically amplifying FKER (g. 710T>G), PG6, CliμD11, DRD4-2 (g. 129456C>T), Cli12, PG2, and CHD loci were integrated into the second group, labeled with HEX fluorescent dye. Primers specifically amplifying CliμC06, CliμD17, and LDHA (g. 2582481G>A) were integrated into the third group. Primers for specific amplification of loci PG1, Cli02, and PIGN12 were integrated into the third group, with TAMRA as the fluorescent dye label. Primers for specific amplification of loci PG1, Cli02, and PIGN12 were integrated into the fourth group, with ALEXA568 as the fluorescent dye label. Primers for specific amplification of loci PIGN15, MSTN (g. 66493737C>T), PIGN57, CliμT13, DRD4-1 (g. 129954C>T), and CliμD16 were integrated into the fifth group, with ALEXA594 as the fluorescent dye label. The molecular weight internal standard was selected as the orange fluorescent dye ALEXA633, serving as the sixth fluorescent dye. The specific locus arrangement is as follows: Figure 5 As shown.

[0053] Example 3: Optimization of the multiplex amplification detection system.

[0054] Optimal reaction conditions for the multiplex amplification system were determined through multiple experiments, including primer concentration, template amount, annealing temperature, extension temperature, and time in the amplification program, ensuring site uniformity and detection accuracy. The final determined optimal reaction volume for multiplex amplification was 25 μL, including 10.0 μL of 2.5×PCR Master Mix, 5.0 μL of Primers Mix, 0.5–2.0 ng of the DNA template to be tested, with the remainder made up with sterile water. The 2.5×PCR Master Mix contained PCR reaction buffer, MgCl2, dNTPs, DNA polymerase, etc. The DNA template to be tested was genomic DNA extracted from pigeon feathers or blood samples, which could be obtained using commercial silica gel column or magnetic bead extraction kits, or through the Chelex-100 method commonly used in forensic medicine.

[0055] The final primer combinations obtained are shown in Table 1.

[0056] Improper amplification program design can also lead to poor peak shapes for some genetic markers. Taking CliμD11 as an example, during testing, a double-shoulder peak was observed at the gene locus, directly affecting genotyping. Therefore, the PCR amplification program was optimized by setting the final extension temperature and time to "72℃ for 10 minutes," "65℃ for 30 minutes," and "68℃ for 60 minutes," respectively. The experimental results are as follows... Figure 6 As shown, when the final extension condition is "72℃ for 10 minutes", the peak shape is poor; when the final extension condition is "65℃ for 30 minutes", the amplification efficiency is low and the stuttering phenomenon is severe; while when the final extension condition is set to "68℃ for 60 minutes", the amplification efficiency and peak shape of this locus are relatively good. Therefore, the parameters of 68℃ and 60 minutes are selected as the final extension program.

[0057] The optimized multiplex amplification program is shown in Table 2.

[0058] Table 2. Multiplex Amplification Procedure

[0059]

[0060] One single pigeon genomic DNA sample with good amplification results, complete STR genotype profile, and known genotypes at all loci through Sanger sequencing was selected from the collected pigeon feather samples as a positive reference DNA.

[0061] After PCR amplification, 1 L of PCR product was mixed thoroughly with 9.5 L of deionized formamide and 0.5 L of molecular weight internal standard. After denaturation at 95°C for 3 minutes and cooling in an ice bath for 3 minutes, the mixture was ready for capillary electrophoresis detection on a 3130XL or 3500 series genetic analyzer. Genotyping analysis was then performed using GeneMapper ID-X software. The detection pattern of the multiplex amplification detection system is shown below. Figure 7 As shown.

[0062] Example 4: Preparation of allele typing standards.

[0063] Allelic information of different genetic markers was collected from pigeon population samples. Pigeon feather DNA was extracted and amplified by conventional PCR. After identification by agarose gel electrophoresis, the target DNA fragment was recovered using a gel purification kit, ligated into the pMD18-T vector, and transformed into DH-5α competent E. coli cells. After blue-white screening, colony PCR was used to select and verify clones successfully transformed with the target allele fragment. These clones were then cultured, purified using a plasmid miniprep kit, and sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing verification. The genetic marker alleles were named based on the obtained sequence information.

[0064] Recombinant plasmids containing allele inserts within a single locus were diluted to 1 ng / μL and mixed in an equal proportion. PCR amplification was performed using the fluorescent primers for that locus, and the average peak height of all alleles at that locus was recorded. The PCR products from the mixed locus were then prepared according to the volume ratio calculated from the average ladder peak height of each locus and purified using the phenol-chloroform method. The resulting allele standard genotyping map is shown below. Figure 8 As shown.

[0065] Example 5: Specificity verification of the multiplex amplification detection system.

[0066] Genomic DNA from different species, including pigs, cattle, sheep, fish, rabbits, mice, dogs, cats, Candida albicans, Escherichia coli, and human genome standards 9947A and 9948, was prepared and diluted to 1.0 ng / μL. The DNA was then analyzed using the optimized multiplex amplification detection system and electrophoresis method described in Example 2. The results are as follows: Figure 9 As shown, no specific DNA peaks appeared for the genomic DNA templates of the above genera, indicating that the detection system has good species specificity.

[0067] Example 6: Sensitivity verification of the multiplex amplification detection system.

[0068] Pigeon positive reference DNA was serially diluted with ultrapure water. PCR amplification was performed on the serially diluted pigeon DNA templates (2 ng, 1 ng, 0.5 ng, 0.25 ng, 0.125 ng, 0.0625 ng, 0.03125 ng, and 0.015625 ng). The sensitivity of the multiplex detection system was evaluated by analyzing the detection results under different DNA template amounts. Detection was performed according to the optimized multiplex amplification detection system and electrophoresis method in Example 2. The peak values ​​of each locus are statistically analyzed as follows: Figure 10As shown, when the template amount is higher than or equal to 0.0625 ng, all genetic markers can be effectively genotyped, and the detection rate of genetic markers is 100%. When the template amount is further reduced to 0.03125 ng, some genetic markers begin to show peak loss, and the detection rate of genetic markers drops to 89.66%. Therefore, the effective sensitivity of the newly constructed amplification system for detecting pigeon DNA samples is 0.0625 ng.

[0069] Example 7: Statistical analysis of STR microsatellite marker genetic parameters.

[0070] To further verify the individual identification and parentage testing capabilities of the multiplex amplification detection system described in this invention in domestic pigeon racing competitions, feather samples from 266 domestic pigeon lofts were collected. DNA typing was performed according to the experimental conditions provided in Example 2, and forensic parameters such as allele frequency (AF), individual identification rate (DP), and probability of exclusion (PE) for the 23 STR microsatellite markers were calculated, as detailed in Table 3. The cumulative individual identification probability (TDP) was calculated using the following formula:

[0071] TDP=1-(1-PD1)(1-PD2)…(1-PDN),

[0072] The TDP value is calculated to be 1-7.28336987007997. e-19 According to the formula for calculating the cumulative non-parent exclusion probability (CPE), CPE = 1 - (1 - PE1)(1 - PE2)...(1 - PEN), the value of CPE is 0.999765115442638, indicating that it has a high individual recognition ability and non-parent exclusion probability.

[0073] Table 3. Statistical analysis of genetic parameters of 23 STR microsatellite markers in domestic racing pigeon populations.

[0074]

[0075]

[0076] Example 8: Detection case of individual identification and flight capability assessment.

[0077] (1) Introduce the actual case detection process of individual identification and flight ability assessment of a racing pigeon sample.

[0078] Sampling: Select the feathers covering the tail feathers on the back or tail of the pigeon. Gently pull out the quill of a feather by pulling it with your fingers along the direction of feather growth. Take three to four or more feathers and dry them at room temperature for 5 to 10 minutes.

[0079] DNA extraction: Cut the clear portion of the plume tube into a 1.5 mL centrifuge tube. Add 50 μL of Helex-100 and 10 μL of 20 mg / mL proteinase K. Vortex for 15 seconds to mix the sample, then briefly centrifuge for 10 seconds. Incubate in a metal bath at 56°C for 30 minutes, vortexing thoroughly and then briefly centrifuging. Incubate in a metal bath at 98°C for 10 minutes, vortexing for 15 seconds to mix the sample, then centrifuge at 10,000 rpm for 3 minutes. Transfer the supernatant to a new 1.5 mL centrifuge tube for later use.

[0080] The total volume of the amplification system was 25.0 μL, including 10.0 μL of 2.5×PCR Master Mix, 5.0 μL of primer combination mixture, 0.5–2.0 ng of DNA template to be tested, and the remainder was made up with sterile water. The amplification program was as follows: 95℃ for 2 minutes; 94℃ for 30 seconds, 58℃ for 60 seconds, 72℃ for 50 seconds, repeated for 31 cycles; 68℃ for 60 minutes.

[0081] Capillary electrophoresis detection of PCR products: Mix 12.0 μL of deionized formamide with 0.5 μL of AGCU Marker SIZ-500, add 1.0 μL of amplification product or InDel Ladder standard, mix well, centrifuge at 3000 rpm for 1 minute, denature at 95℃ for 3 minutes, then incubate on ice for 3 minutes, and detect by electrophoresis. ID-X analyzes the results, and the genotyping map is as follows: Figure 11 As shown.

[0082] Sanger sequencing was performed simultaneously on five single nucleotide polymorphism sites in this sample. The results of this technical approach were compared with the results of Sanger sequencing (e.g., Figure 12 As shown in the figure, the two are consistent.

[0083] (2) Evaluation results.

[0084] Given that spatial orientation, muscle strength, stress resistance, and flight endurance are crucial factors determining a pigeon's performance or racing results, the polymorphism of the DRD4 gene influences a pigeon's homing orientation ability in flight competitions. Pigeons with the CTCT genotype exhibit exceptional performance, especially in short-distance races. The LDHA gene, a candidate gene for pigeon racing performance, participates in aerobic and anaerobic metabolism, determining muscle endurance, recovery, and aerobic capacity; the A genotype is prevalent among top-level racing pigeons. F-KER is a gene used to assess pigeon feather performance; in long-distance races, pigeons carrying the TT genotype demonstrate better racing performance than those carrying the GG genotype. MSTN is a negative regulator of skeletal muscle growth and development; the MSTN gene, used as a muscle indicator, shows that pigeons with the CC genotype have a significant advantage in flight performance.

[0085] Based on the test results of this individual, the DRD4 gene polymorphism genotype was TT / CC, the LDHA gene polymorphism genotype was GG, the F-KER gene polymorphism genotype was GT, and the MSTN gene polymorphism genotype was CT. Each ability was rated on a scale of 1 to 5. The analysis concluded that:

[0086] "Their homing ability (4 points) is very strong, their wing index (3 points) and muscle index (3 points) are at an average level, and their endurance index (1 point) is relatively weak. The possibility of achieving excellent results in the competition is low."

[0087] Example 9: Case of paternity testing.

[0088] Phylogenetic identification was performed on parent pigeons and their offspring with known parent-child relationships. Samples were taken from the back or tail feather tubes. DNA extraction and amplification conditions were the same as in Example 8. Amplified products were analyzed using a capillary electrophoresis detection and analysis system, and then analyzed using data analysis software. ID-X analyzes the results. Figure 13 , Figure 14 The following are the genotyping charts for parent pigeons and young pigeons; the specific genotyping results are shown in Table 4.

[0089] Table 4. Genetic marker typing of parent pigeons and young pigeons in parentage testing cases.

[0090]

[0091] The results showed that, according to Mendel's laws of inheritance, for each of the 29 pigeon STR loci, each young pigeon has one allele that comes from its parent pigeon. In other words, each young pigeon has one allele that is the same as the parent pigeon's allele, which does not rule out the possibility of a parent-child relationship. This proves that the parentage identification results of the detection system described in this invention are consistent with reality.

[0092] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A pigeon individual genotyping detection system, characterized in that, The kit comprises a primer combination comprising primers for amplifying 23 STR microsatellite markers and a bird gender detection CHD locus and 5 SNP genetic markers, the STR microsatellite markers are respectively CliμD32, CliμD19, CliμD35, CliμT02, PIGN10, PIGN26, PG6, CliμD11, Cli12, PG2, CliμC06, CliμD17, PG3, PG5, PIGN04, CliμD01, PG1, Cli02, PIGN12, PIGN15, PIGN57, CliμT13 and CliμD16, the SNP genetic markers are respectively g.2582481G>A of lactate dehydrogenase gene, g.129954C>T and g.129456C>T of dopamine receptor D4 gene, g.66493737C>T of myostatin gene and g.710T>G of feather keratin gene, the nucleotide sequences of the primers for amplifying CliμD32 are shown in SEQ ID No:1 and SEQ ID No:2, the nucleotide sequences of the primers for amplifying CliμD19 are shown in SEQ ID No:3 and SEQ ID No:4, the nucleotide sequences of the primers for amplifying CliμD35 are shown in SEQ ID No:5 and SEQ ID No:6, the nucleotide sequences of the primers for amplifying CliμT02 are shown in SEQ ID No:7 and SEQ ID No:8, the nucleotide sequences of the primers for amplifying PIGN10 are shown in SEQ ID No:9 and SEQ ID No:10, the nucleotide sequences of the primers for amplifying PIGN26 are shown in SEQ ID No:11 and SEQ ID No:12, the nucleotide sequences of the primers for amplifying PG6 are shown in SEQ ID No:13 and SEQ ID No:14, the nucleotide sequences of the primers for amplifying CliμD11 are shown in SEQ ID No:15 and SEQ ID No:16, the nucleotide sequences of the primers for amplifying Cli12 are shown in SEQ ID No:17 and SEQ ID No:18, the nucleotide sequences of the primers for amplifying PG2 are shown in SEQ ID No:19 and SEQ ID No:20, the nucleotide sequences of the primers for amplifying CHD are shown in SEQ ID No:21 and SEQ ID No:22, the nucleotide sequences of the primers for amplifying CliμC06 are shown in SEQ ID No:23 and SEQ ID No:24, the nucleotide sequences of the primers for amplifying CliμD17 are shown in SEQ ID No:25 and SEQ ID No:26, the nucleotide sequences of the primers for amplifying PG3 are shown in SEQ ID No:27 and SEQ ID No:28, the nucleotide sequences of the primers for amplifying PG5 are shown in SEQ ID No:29 and SEQ ID No:30,The nucleotide sequences of the primers for amplifying PIGN04 are shown in SEQ ID No:31 and SEQ ID No:32, the nucleotide sequences of the primers for amplifying ClilD01 are shown in SEQ ID No:33 and SEQ ID No:34, the nucleotide sequences of the primers for amplifying PG1 are shown in SEQ ID No:35 and SEQ ID No:36, the nucleotide sequences of the primers for amplifying Clil02 are shown in SEQ ID No:37 and SEQ ID No:38, the nucleotide sequences of the primers for amplifying PIGN12 are shown in SEQ ID No:39 and SEQ ID No:40, the nucleotide sequences of the primers for amplifying PIGN15 are shown in SEQ ID No:41 and SEQ ID No:42, the nucleotide sequences of the primers for amplifying PIGN57 are shown in SEQ ID No:43 and SEQ ID No:44, the nucleotide sequences of the primers for amplifying ClilT13 are shown in SEQ ID No:45 and SEQ ID No:46, the nucleotide sequences of the primers for amplifying ClilD16 are shown in SEQ ID No:47 and SEQ ID No:48, the nucleotide sequences of the primers for detecting the g.2582481 G>A typing of lactate dehydrogenase gene are shown in SEQ ID No:49 to SEQ ID No:51, the nucleotide sequences of the primers for detecting the g.129954 C>T typing of dopamine receptor D4 gene are shown in SEQ ID No:52 to SEQ ID No:54, the nucleotide sequences of the primers for detecting the g.129456 C>T typing of dopamine receptor D4 gene are shown in SEQ ID No:55 to SEQ ID No:57, the nucleotide sequences of the primers for detecting the g.66493737 C>T typing of myostatin gene are shown in SEQ ID No:58 to SEQ ID No:60,The nucleotide sequences of the primers for detecting the g.710T>G genotype of feather keratin gene are shown in SEQ ID No:61 to SEQ ID No:63; the primers with the nucleotide sequences shown in SEQ ID No:1 to SEQ ID No:12 are set as a first group; the primers with the nucleotide sequences shown in SEQ ID No:13 to SEQ ID No:22, SEQ ID No:55 to SEQ ID No:57 and SEQ ID No:61 to SEQ ID No:63 are set as a second group; the primers with the nucleotide sequences shown in SEQ ID No:23 to SEQ ID No:34 and SEQ ID No:49 to SEQ ID No:51 are set as a third group; the primers with the nucleotide sequences shown in SEQ ID No:35 to SEQ ID No:40 are set as a fourth group; the primers with the nucleotide sequences shown in SEQ ID No:41 to SEQ ID No:48, SEQ ID No:52 to SEQ ID No:54 and SEQ ID No:58 to SEQ ID No:60 are set as a fifth group, and at least one of the 5' ends of the primers in the upstream and downstream primers targeting the same STR microsatellite marker or the CHD gene locus or the SNP genetic marker is labeled with a fluorescent dye.

2. The pigeon individual typing detection system according to claim 1, wherein, The concentration of the primer shown in SEQ ID No: 1 was 0.16 μmol / L, the concentration of the primer shown in SEQ ID No: 2 was 0.16 μmol / L, the concentration of the primer shown in SEQ ID No: 3 was 0.15 μmol / L, the concentration of the primer shown in SEQ ID No: 4 was 0.15 μmol / L, the concentration of the primer shown in SEQ ID No: 5 was 0.18 μmol / L, the concentration of the primer shown in SEQ ID No: 6 was 0.18 μmol / L, the concentration of the primer shown in SEQ ID No: 7 was 0.25 μmol / L, the concentration of the primer shown in SEQ ID No: 8 was 0.25 μmol / L, the concentration of the primer shown in SEQ ID No: 9 was 0.40 μmol / L, the concentration of the primer shown in SEQ ID No: 10 was 0.40 μmol / L, the concentration of the primer shown in SEQ ID No: 11 was 0.35 μmol / L, the concentration of the primer shown in SEQ ID No: 12 was 0.35 μmol / L, the concentration of the primer shown in SEQ ID No: 13 was 0.15 μmol / L, the concentration of the primer shown in SEQ ID No: 14 was 0.15 μmol / L, the concentration of the primer shown in SEQ ID No: 15 was 0.20 μmol / L, the concentration of the primer shown in SEQ ID No: 16 was 0.20 μmol / L, the concentration of the primer shown in SEQ ID No: 17 was 0.40 μmol / L, the concentration of the primer shown in SEQ ID No: 18 was 0.40 μmol / L, the concentration of the primer shown in SEQ ID No: 19 was 0.15 μmol / L, the concentration of the primer shown in SEQ ID No: 20 was 0.15 μmol / L, the concentration of the primer shown in SEQ ID No: 21 was 0.22 μmol / L, the concentration of the primer shown in SEQ ID No: 22 was 0.22 μmol / L, the concentration of the primer shown in SEQ ID No: 23 was 0.24 μmol / L, the concentration of the primer shown in SEQ ID No: 24 was 0.24 μmol / L, the concentration of the primer shown in SEQ ID No: 25 was 0.50 μmol / L, the concentration of the primer shown in SEQ ID No: 26 was 0.50 μmol / L, the concentration of the primer shown in SEQ ID No: 27 was 0.36 μmol / L, the concentration of the primer shown in SEQ ID No: 28 was 0.36 μmol / L, the concentration of the primer shown in SEQ ID No: 29 was 0.18 μmol / L, the concentration of the primer shown in SEQ ID No: 30 was 0.18 μmol / L, the concentration of the primer shown in SEQ ID No: 31 was 0.20 μmol / L, the concentration of the primer shown in SEQ ID No: 32 was 0.20 μmol / L, the concentration of the primer represented by SEQ ID No: 33 was 0.32 μmol / L, the concentration of the primer represented by SEQ ID No: 34 was 0.32 μmol / L, the concentration of the primer represented by SEQ ID No: 35 was 0.40 μmol / L, the concentration of the primer represented by SEQ ID No: 36 was 0.40 μmol / L, the concentration of the primer represented by SEQ ID No: 37 was 0.25 μmol / L, the concentration of the primer represented by SEQ ID No: 38 was 0.25 μmol / L, the concentration of the primer represented by SEQ ID No: 39 was 0.20 μmol / L, the concentration of the primer represented by SEQ ID No: 40 was 0.20 μmol / L, the concentration of the primer represented by SEQ ID No: 41 was 0.15 μmol / L, the concentration of the primer represented by SEQ ID No: 42 was 0.15 μmol / L, the concentration of the primer represented by SEQ ID No: 43 was 0.25 μmol / L, the concentration of the primer represented by SEQ ID No: 44 was 0.25 μmol / L, the concentration of the primer represented by SEQ ID No: 45 was 0.30 μmol / L, the concentration of the primer represented by SEQ ID No: 46 was 0.30 μmol / L, the concentration of the primer represented by SEQ ID No: 47 was 0.25 μmol / L, the concentration of the primer represented by SEQ ID No: 48 was 0.25 μmol / L, the concentration of the primer represented by SEQ ID No: 49 was 0.20 μmol / L, the concentration of the primer represented by SEQ ID No: 50 was 0.20 μmol / L, the concentration of the primer represented by SEQ ID No: 51 was 0.40 μmol / L, the concentration of the primer represented by SEQ ID No: 52 was 0.25 μmol / L, the concentration of the primer represented by SEQ ID No: 53 was 0.25 μmol / L, the concentration of the primer represented by SEQ ID No: 54 was 0.50 μmol / L, the concentration of the primer represented by SEQ ID No: 55 was 0.20 μmol / L, the concentration of the primer represented by SEQ ID No: 56 was 0.20 μmol / L, the concentration of the primer represented by SEQ ID No: 57 was 0.40 μmol / L, the concentration of the primer represented by SEQ ID No: 58 was 0.15 μmol / L, the concentration of the primer represented by SEQ ID No: 59 was 0.15 μmol / L, the concentration of the primer represented by SEQ ID No: 60 was 0.30 μmol / L, the concentration of the primer represented by SEQ ID No: 61 was 0.20 μmol / L, the concentration of the primer represented by SEQ ID No: 62 was 0.20 μmol / L, the concentration of the primer represented by SEQ ID No: 63 was 0.40 μmol / L.

3. The pigeon individual typing detection system according to claim 1 or 2, characterized in that, The fluorescent dye is selected from 6-FAM, HEX, TAMRA, ALEXA 568, ALEXA 594, ROX, VIC, or NED.

4. The pigeon individual typing detection system according to claim 1, wherein, The fluorescent dye selected for the first group is 6-FAM, the fluorescent dye selected for the second group is HEX, the fluorescent dye selected for the third group is TAMRA, the fluorescent dye selected for the fourth group is ALEXA 568, and the fluorescent dye selected for the fifth group is ALEXA 594.

5. The pigeon individual typing detection system according to claim 4, wherein, Also included is a multiplex amplification master mix comprising a PCR reaction buffer, dNTPs, a DNA polymerase, and MgCl2.

6. A kit characterized in that, A pigeon individual typing detection system as claimed in any one of claims 1 to 5.

7. The kit of claim 6, wherein Also included is an allelic discrimination standard.

8. Use of a pigeon individual typing detection system as claimed in any one of claims 1 to 5 or a kit as claimed in any one of claims 6 to 7 for pigeon individual identification and / or pigeon parentage determination and / or pigeon flight ability assessment.

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