Pigeon individual typing detection system, kit and application thereof

Abstract

The invention relates to a pigeon individual typing detection system, a kit and application thereof. The kit comprises a pigeon individual typing detection system, the pigeon individual typing detection system comprises a primer combination and a composite amplification premix, and the primer combination comprises primers for amplifying 23 STR (short tandem repeat) microsatellite markers, bird sex detection CHD gene loci and 5 SNP (single nucleotide polymorphism) genetic markers. The pigeon individual typing detection system and the kit provided by the invention can realize single-tube detection of 29 pigeon DNA genetic markers, comprise STR genetic markers for realizing pigeon individual identification and parent-child relationship identification and single nucleotide polymorphism genetic markers for evaluating the flying ability of pigeons, and are a detection system which can be detected at present and contains the maximum number of pigeon DNA gene loci; the method has a good application prospect in the fields of pigeon identity identification, parent-child relationship identification and flight ability evaluation.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a pigeon individual typing detection system, a kit and applications thereof. Background Art

[0002] The domestication of pigeons can be traced back to thousands of years ago and is closely related to human activities. In ancient times, pigeons played an important role in human long-distance communication due to their good spatial orientation and long-distance homing ability. Nowadays, various communication networks are extremely developed, and pigeons no longer need to send messages, but have become the protagonists in racing events. The pigeon racing sport is an event officially approved by the General Administration of Sports of China, which is attracting more and more pigeon lovers and pigeon friends from all over the world to participate. Based on the high prize money of the competition, the standardization of pigeon racing events is also gradually advancing, and the fairness and authenticity of the competition process are particularly concerned. In order to ensure the transparency of individual pigeons and prevent the emergence of AB pigeons, genetic testing using STR microsatellites for identity identification has been introduced into pigeon racing events; secondly, as the pigeon racing sport has gradually become familiar to the public, pigeon lovers and pigeon friends have begun to pursue pigeons with excellent bloodlines, which has spawned a corresponding trading market. The offspring of pigeons that have achieved excellent results in the competition can often be sold at high prices in the trading market, so the use of STR microsatellites for pigeon parent-child relationship detection has also become an important application direction.

[0003] At the same time, in order to evaluate the pigeons' innate conditions such as orientation, flight speed, cardiopulmonary endurance and resistance to evil, and increase their chances of winning, SNP detection related to the pigeons' flying ability has gradually been promoted. SNP stands for Single Nucleotide Polymorphisms, which refers to the variation of a single nucleotide in the genome, including conversion, transversion, deletion and insertion. There are many methods for detecting SNP, including direct DNA sequencing, real-time fluorescence quantitative PCR, HRM high-resolution melting curve technology, mass spectrometry, SNaPshot method, etc., but their use is often limited by the cumbersome and time-consuming operation process, high material and technical requirements or cost issues. Summary of the invention

[0004] The purpose of the present invention is to disclose a pigeon individual typing detection system, a kit and its application, so as 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 object, the present invention is implemented through the following technical solutions:

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

[0007] The 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 the present invention is to provide the use of the pigeon individual typing detection system described in the first aspect of the present invention and the kit described in the second aspect of the present invention in pigeon individual identification and / or pigeon parentage identification and / or flight ability assessment.

[0009] The pigeon individual typing detection system described in the first aspect of the present invention comprises a primer combination and a composite amplification premix, wherein the primer combination comprises primers for amplifying 23 STR microsatellite markers and a bird sex detection CHD locus and 5 SNP genetic markers, wherein the STR microsatellite markers are CliμD32, CliμD19, CliμD35, CliμT02, PIGN10, PIGN26, PG6, CliμD11, Cli12, PG2, CliμC06, CliμD17, PG3, PG5, PIGN04, CliμD01, PG1, Cli02, PIGN12, PIGN15, PIGN57, CliμT13 and CliμD16, and the SNP genetic markers are 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, respectively.

[0010] The pigeon individual typing detection system is a composite amplification detection system, which detects the STR microsatellite markers and CHD loci by designing typing primers in the flanking conservative regions of the locus core sequence, and designs ARMS typing primers to match different alleles of SNP genetic markers respectively. The lactate dehydrogenase gene (LDHA) is involved in aerobic and anaerobic metabolism, and determines the endurance, recovery and aerobic capacity of muscles. The single nucleotide polymorphism g.2582481G>A in this gene is closely related to the behavioral performance of racing pigeons, among which the A allele is more common in top racing pigeons; the dopamine receptor D4 gene (DRD4) is also considered to be closely related to the racing performance of racing pigeons. g.129954C>T and g.129456C>T are two very potential single nucleotide polymorphism sites, and their combined genotype CTCT is usually associated with stronger competition performance, while the CCCC genotype usually indicates a lower level of competition ability; myostatin (MSTN) is considered to be one of the most powerful negative regulators of muscle growth, and plays an important role in muscle development. The single nucleotide polymorphism 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 mutation will lead to muscle gain, which will further lead to improved athletic performance. Therefore, individuals carrying the T allele usually have abundant muscle mass and a higher 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 at longer distances because β-keratin is a key protein for the normal development of feathers, and the mutation from T to G in g.710T>G may lead to changes in keratin structure, thereby affecting feather quality and competition performance.

[0011] In some embodiments of the first aspect of the present invention, the 5' end of at least one of the upstream and downstream primers targeting the same STR microsatellite marker or the CHD locus or the SNP genetic marker is labeled with a fluorescent dye, and the fluorescent dye is 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 markers, the CHD loci and the SNP genetic markers 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 composite amplification detection system can be reduced by adjusting the final concentration of each primer to an appropriate range. The specific concentrations are shown in Table 1.

[0014] In some embodiments of the first aspect of the present invention, specific primer groupings 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 of pigeon genetic markers.

[0017]

[0018]

[0019]

[0020] In some embodiments of the first aspect of the present invention, the composite amplification premix includes one or more of a PCR reaction buffer, dNTPs, a 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 comprises an allele typing ladder. The allele typing ladder is prepared by diluting all recombinant plasmids containing allele inserts in a single locus to 1 ng / μL and then mixing them evenly, performing PCR amplification using fluorescent primers of the locus, and recording the average peak height of all alleles of the locus; converting the average peak height of the ladder of the single locus into a volume ratio to prepare the PCR products of the mixed locus, and purifying them by phenol-chloroform method.

[0023] The application direction provided by the third aspect of the present invention is to use 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 the present invention can realize single-tube detection of 29 pigeon DNA genetic markers, including detection of STR genetic markers for realizing pigeon individual identification and parent-offspring relationship identification and detection of single nucleotide polymorphism genetic markers for evaluating pigeon flight ability. It is the detection system with the largest number of pigeon DNA loci that can be detected at present, and has good application prospects in the fields of pigeon identity identification, parent-offspring relationship identification and flight ability evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

[0031] Figure 6 It is a comparison chart of the amplification procedure of the composite amplification detection system optimized for the CliμD11 site in Example 3;

[0032] Figure 7 It is the detection spectrum of the composite amplification detection system for pigeon positive reference DNA in Example 3;

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

[0034] Fig. 9 is the species-specific validation map in Example 5;

[0035] Fig.10 is a bar graph of the sensitivity verification results in Example 6;

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

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

[0038] Fig.13 It is the parental pigeon detection spectrum for kinship identification in Example 9;

[0039] Fig.14 It is the young pigeon detection spectrum for kinship identification in Example 9. DETAILED DESCRIPTION

[0040] The molecular biology experimental methods not specifically described in the following examples were all performed with reference to the Molecular Cloning Experiment Guide (3rd edition) or in accordance with the methods and product instructions; the biological materials described in the methods, unless otherwise specified, can be obtained from commercial channels.

[0041] Example 1: Site selection.

[0042] By collecting racing pigeon feather samples nationwide, a genetic polymorphism survey of the racing pigeon population was conducted on 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 helix protein binding gene (CHD) of pigeon was selected as the sex identification locus, and the difference in the length of its introns on chromosomes Z and W was used for identification.

[0044] Lactate dehydrogenase A (LDHA), dopamine D4 receptor (DRD4), muscle regulation (MSTN) and feather keratin (F-KER) are four congenital genetic genes that have a significant impact on the flying ability of pigeons. Among them, the lactate dehydrogenase gene (LDHA) is involved in aerobic and anaerobic metabolism, which determines the endurance, recovery and aerobic capacity of muscles. The single nucleotide polymorphism g.2582481G>A in this gene is closely related to the behavioral performance of racing pigeons, among which the A allele is more common in top racing pigeons; the dopamine receptor D4 gene (DRD4) is also considered to be closely related to the performance of racing pigeons. g.129954C>T and g.129456C>T are two very potential single nucleotide polymorphism sites. Their combined genotype CTCT is usually associated with stronger competition performance, while the CCCC genotype usually indicates a lower level of competition ability; myostatin (MSTN) is considered to be one of the most powerful negative regulators of muscle growth. The single nucleotide polymorphism g.66493737C>T in this gene plays an important role in meat development. The single nucleotide polymorphism 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 will lead to muscle gain, which will further lead to improved athletic performance. Therefore, individuals carrying the T allele usually have abundant muscle mass and a higher 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 at longer distances because β-keratin is a key protein for the normal development of feathers, and the mutation from T to G in g.710T>G may lead to changes in keratin structure, thereby affecting feather quality and competition performance. Therefore, the system of the present invention selects lactate dehydrogenase (LDHA) gene g.2582481G>A, dopamine receptor D4 (DRD4) gene g.129954C>T and g.129456C>T, myostatin (MSTN) gene g.66493737C>T and feather keratin (F-KER) gene g.710T>G as 5 SNP genetic markers for evaluating pigeon flight ability.

[0045] Example 2: Design of primers.

[0046] When designing multiple composite amplification primers, it is necessary to consider the interaction between primers of each locus, and the similar physical properties and reaction kinetics between different primers, so as to ensure the balance requirements of each locus in the amplification system. At the same time, the ARMS-PCR primers for SNP genetic markers are designed for the nucleic acid sequences of different alleles, so that the 3' terminal nucleotides of the primers match different allele base types respectively, and 1-2 mismatched bases are introduced near the 3' end region of the ARMS typing primer to further improve the specificity of the reaction, and the length difference of the two genotyping amplification products is distinguished and identified. The construction difficulty of the composite amplification detection system is directly related to the number of loci contained in the system and the complexity of the reaction system. With the increase of the number of primers in the system, the reaction kinetics will become more and more complicated, and the primers of different loci are also very likely to interact and interfere with each other, so the requirements for primer sequences will be more stringent than conventional PCR. In the actual work process, it is necessary to design a large number of primer sequences for complex testing and explore the appropriate concentration ratio between primers in the composite amplification system to ensure that more loci can be detected simultaneously without reducing the detection specificity and sensitivity.

[0047] The designed primer combination is first tested for single-plex PCR amplification of STR microsatellite markers for amplification efficiency and specificity screening, and the typing accuracy and efficiency of ARMS-PCR primers are tested for SNP genetic markers. In particular, a common primer and two typing primers are added to the ARMS-PCR amplification system for SNP genetic markers at the same time, and the genomes of different genotypes are used as templates for amplification reactions. The amplification products of the corresponding genotypes are qualified; when the amplification template is a heterozygote, two corresponding amplification products can be obtained; if the ARMS primers cross-react with non-corresponding gene templates, it is necessary to readjust the primer sequence or introduce the number or type of mismatched bases to obtain a more ideal differentiation effect. The different genotyping DNA templates used for primer testing can be collected through sanger sequencing results or commissioned to Shanghai Sangon Biotechnology Co., Ltd. to be synthesized by artificial means. The final primer screening map of the 5 SNP genetic markers is shown below. Figure 1 shown.

[0048] After the primer single amplification test is completed, the primer pairs of single genetic markers are combined one by one into the composite amplification detection system to verify the effect; if during the construction of the composite amplification detection system, there are problems such as mismatch with other primers causing mixed peaks or low efficiency due to inhibition by other primers, the primer sequence needs to be adjusted. Therefore, the construction process of the entire composite amplification detection system needs to be completed in stages through multiple experiments. Because the interaction relationship between dozens of primers in the multiple amplification system is intricate and cannot be effectively predicted in advance, a primer combination with good effect can only be obtained through repeated testing and adjustment.

[0049] During the construction of single amplification test and composite amplification detection system, there were many cases where the effect of designed primers was significantly lower than expected.

[0050] The single amplification test takes the primer design of DRD4-1 (g.129954C>T) site as an example: the first ARMS primer sequence is 5'-TACCTTACGAGCGGTGACA T T-3' (SEQ ID No: 64). Since the amplification effect of this primer on the TT typing DNA template was significantly lower than expected, the reason why the amplification efficiency of the ARMS primer was not ideal was analyzed. It may be caused by the excessive influence of the introduced mismatched base on the primer efficiency. Therefore, it is considered to move the mismatch position to the fourth base at the 3' end, that is, the second primer sequence is: 5'-TACCTTACGAGCGGTGA G AAT-3' (SEQ ID No: 53). The amplification results of the two sets of primers are shown in Figure 2 As shown, the peaks can be induced normally after the mismatched base positions are modified.

[0051] The construction of the multiplex amplification detection system takes the primer pair for verifying PIGN10 as an example: when the STR microsatellite marker multiplex amplification system was constructed to 22 multiplexes, it was observed that the PIGN10 primers that originally performed well suddenly decreased in efficiency and could not obtain the target product of normal amplification. After repeated experiments, it was inferred that this phenomenon may be due to the inhibition caused by the excessively high concentration of some primers in the system, resulting in multiple samples missing peaks at the PIGN10 site (see Figure 3 ). After repeatedly reducing the concentrations of multiple genetic marker primers with high peaks, such as CliμD32, PG6, and CliμD17, it was found that the amplification efficiency of the PIGN10 locus was significantly improved (see Figure 4 ).

[0052] In order to avoid the overlap of the peak positions of the amplified products of different loci and further ensure the accuracy of the typing results of all loci in the composite amplification detection system, reasonable grouping and arrangement were carried out on the basis of fully considering the length range of the amplified product fragments of each locus. The primer combination finally confirmed was to divide all loci into five groups, and different fluorescent markers were used between the groups; among them, the primers for specific amplification of CliμD32, CliμD19, CliμD35, CliμT02, PIGN10 and PIGN26 loci were integrated into the first group, and the fluorescent dye marker was 6-FAM; the primers for specific amplification of FKER (g.710T>G), PG6, CliμD11, DRD4-2 (g.129456C>T), Cli12, PG2 and CHD loci were integrated into the second group, and the fluorescent dye marker was HEX; the primers for specific amplification of CliμC06, CliμD17, LDHA (g.2582481G>A ), PG3, PG5, PIGN04 and CliμD01 loci were integrated into the third group, and the fluorescent dye marker was TAMRA; the primers for specific amplification of PG1, Cli02, PIGN12 loci were integrated into the fourth group, and the fluorescent dye marker was ALEXA568; the primers for specific amplification of PIGN15, MSTN (g.66493737C>T), PIGN57, CliμT13, DRD4-1 (g.129954C>T) and CliμD16 loci were integrated into the fifth group; the fluorescent dye marker was ALEXA594; the orange fluorescent ALEXA633 was selected as the internal molecular weight standard as the fluorescent dye marker, as the sixth fluorescent dye. The specific loci are arranged as follows Figure 5 shown.

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

[0054] Through multiple experiments, the optimal reaction conditions in the composite amplification system are determined, such as the primer concentration, template amount, annealing temperature, extension temperature and time of the amplification program, etc., to ensure the site balance and detection accuracy of the composite amplification detection system. The total volume of the optimal multiple amplification reaction system finally determined is 25μL, including 2.5×PCR Master Mix 10.0μL, Primers Mix 5.0μL, 0.5~2.0ng of the DNA template to be tested, and the remainder is supplemented by sterile water. The 2.5×PCRMaster Mix contains PCR reaction buffer, MgCl2, dNTPs, DNA polymerase, etc. The DNA template to be tested is genomic DNA extracted from pigeon feathers or blood samples, which can be extracted by commercial silica gel column method or magnetic bead method kit, or extracted by chelex-100 method commonly used in forensic medicine.

[0055] The final designed primer combination is shown in Table 1.

[0056] Improper amplification program design can also lead to poor peak shapes of some genetic markers. Taking CliμD11 as an example, during the test, it was found that the peak shape of this locus had double shoulders, which directly affected the typing judgment. Therefore, we wanted to optimize the PCR amplification program and set the final extension temperature and time to "72℃ for 10 minutes", "65℃ for 30 minutes" and "68℃ for 60 minutes" to conduct experiments. The experimental results are as follows: Figure 6 As shown, when the final extension condition is "72°C for 10 minutes", the peak shape is poor; when the final extension condition is "65°C for 30 minutes", the amplification efficiency is low and the Stutter peak phenomenon is serious; and when the final extension condition is set to "68°C for 60 minutes", the amplification efficiency and peak shape of the locus are relatively good, so the parameters of 68°C 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 program

[0059]

[0060] From the collected pigeon feather samples, a single pigeon genomic DNA with good amplification effect, complete STR genotype spectrum and all known loci typing through Sanger sequencing was selected as the positive reference DNA.

[0061] After PCR amplification is completed, take 1L of PCR product and mix it with 9.5L of deionized formamide and 0.5L of molecular weight internal standard; after denaturation at 95℃ for 3 minutes and cooling in an ice bath for 3 minutes, capillary electrophoresis detection can be performed on a 3130XL or 3500 series genetic analyzer, and genotyping analysis can be performed using GeneMapper ID-X analysis software. The detection spectrum of the composite amplification detection system is shown in Figure 7 shown.

[0062] Example 4: Preparation of allelic ladder.

[0063] The allele information of different genetic markers was collected from samples of pigeon populations, and DNA from pigeon feathers was extracted for common PCR amplification. The products were identified by agarose gel electrophoresis and the target DNA fragments were recovered using a gel purification kit, which was connected to the pMD18-T vector and transformed into DH-5α Escherichia coli competent cells. After blue-white screening, colony PCR was used to select and verify the cloned strains that were successfully transferred into the target allele fragments for cultivation, purified by plasmid mini-extraction kit, and sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing verification. The genetic marker alleles were named according to the sequence information obtained.

[0064] All recombinant plasmids containing allele insertion fragments in a single locus were diluted to 1 ng / μL and mixed in equal proportions. The fluorescent primers of the locus were used for PCR amplification, and the average peak height of all alleles in the locus was recorded. The PCR products of the mixed loci were prepared according to the volume ratio converted according to the average peak height of the ladder of the single locus, and purified by phenol-chloroform method. The typing map of the obtained allele standard is as follows: Figure 8 shown.

[0065] Example 5: Verification of the specificity of the composite amplification detection system.

[0066] Prepare genomic DNA of different species such as pig, cattle, sheep, fish, rabbit, mouse, dog, cat, Candida albicans, Escherichia coli, human genome standard 9947A, and human genome standard 9948, dilute to 1.0 ng / μL, and perform detection according to the optimized composite amplification detection system and electrophoresis method in Example 2. The results are as follows Fig. 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 composite amplification detection system.

[0068] Ultrapure water was used to perform gradient dilution of the pigeon positive reference DNA, and PCR amplification was performed on the gradient diluted pigeon DNA template (2ng, 1ng, 0.5ng, 0.25ng, 0.125ng, 0.0625ng, 0.03125ng and 0.015625ng). The sensitivity of the composite detection system was evaluated by analyzing the detection results under different DNA template amounts. The detection was performed according to the composite amplification detection system and electrophoresis method optimized in Example 2. The statistical results of the peaks of each gene locus are shown in Figure 2. Fig.10When the template amount is higher than or equal to 0.0625ng, all genetic markers can be effectively genotyped, and the genetic marker detection rate is 100%; when the template amount is further reduced to 0.03125ng, some genetic markers begin to lose peaks, and the genetic marker detection rate drops to 89.66%; therefore, the effective sensitivity of the newly constructed amplification system for detecting pigeon DNA samples is 0.0625ng.

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

[0070] In order to further verify the individual identification and paternity identification capabilities of the composite amplification detection system of the present invention in domestic racing pigeon competitions, a total of 266 feather samples from domestic public loft racing pigeons were collected, and DNA typing detection was performed according to the experimental conditions provided in Example 2, and forensic parameters such as the allele frequency (AF), individual identification rate (DP), and exclusion probability (PE) of 23 STR microsatellite markers were calculated, as shown in Table 3. Among them, according to the calculation formula of the cumulative individual identification probability (TDP):

[0071] TDP=1-(1-PD1)(1-PD2)…(1-PDN),

[0072] The TDP value is 1-7.28336987007997 e-19 According to the calculation formula of cumulative non-paternity 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-paternity exclusion probability.

[0073] Table 3 Statistics of genetic parameters of 23 STR microsatellite markers in domestic racing pigeon population

[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 feather part covering the tail feathers on the pigeon's back or tail, grab a feather with your fingers and gently pull out the feather tube 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: Use scissors to cut the transparent part of the quill into a 1.5 mL centrifuge tube, add 50 μL Chelex-100 and 10 μL 20 mg / mL proteinase K, shake for 15 seconds to mix the sample, and then briefly centrifuge for 10 seconds. Incubate in a metal bath at 56°C for 30 minutes, shake thoroughly, and then briefly centrifuge. Incubate in a metal bath at 98°C for 10 minutes, shake for 15 seconds to mix the sample, and 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 2.5×PCR Master Mix, 5.0 μL primer combination mixture, 0.5-2.0 ng DNA template to be tested, and the balance was made up by sterile water. The amplification program was: 95°C for 2 minutes; 94°C for 30 seconds, 58°C for 60 seconds, 72°C for 50 seconds, repeated 31 cycles; 68°C for 60 minutes.

[0081] Capillary electrophoresis detection of PCR products: 12.0 μL deionized formamide was mixed with 0.5 μL AGCU Marker SIZ-500, 1.0 μL amplification product or InDel Ladder standard was added, mixed and centrifuged at 3000 rpm for 1 minute, denatured at 95 °C for 3 minutes, and then ice-bathed for 3 minutes, and then electrophoresed and detected. ID-X analyzes the results, and the typing spectrum is as follows Fig.11 shown.

[0082] Sanger sequencing was performed simultaneously on the five single nucleotide polymorphism sites of the sample, and the detection results of this technical solution were compared with the Sanger sequencing results (such as Fig.12 ), the two are consistent.

[0083] (2)Evaluation results.

[0084] Given that spatial orientation, muscle strength, stress resistance and flight endurance are important factors that determine pigeon performance or competition results. The polymorphism of the DRD4 gene affects the homing orientation ability of pigeon flight competitions. Pigeons with the genotype CTCT have extremely strong performance in flight competitions, especially in short-distance competitions. The LDHA gene, as a candidate gene for pigeon competition performance, participates in aerobic and anaerobic metabolism, and determines muscle endurance, recovery and aerobic capacity. The A genotype is mostly found in top racing pigeons. F-KER is a gene for pigeon wing indicators. In long-distance competitions, pigeons carrying the TT genotype show better competition performance than pigeons carrying the GG genotype. MSTN is a negative regulator of skeletal muscle growth and development. The MSTN gene is a muscle indicator of pigeons. Pigeons with the CC genotype have obvious advantages in flight performance.

[0085] From the test results of this individual, the polymorphism site of DRD4 gene is TT / CC, the polymorphism site of LDHA gene is GG, the polymorphism site of F-KER gene is GT, and the polymorphism site of MSTN gene is CT. The ability is rated from 1 to 5, and the analysis conclusion is:

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

[0087] Example 9: Paternity testing case.

[0088] The parent-child relationship of the parent pigeons and the young pigeons was identified. The sample type was the back or tail feathers. The DNA extraction and amplification system conditions were the same as those in Example 8. The amplified products were detected by the capillary electrophoresis detection and analysis system and then analyzed by the data analysis software. ID-X analyzes the results. Fig.13 , Fig.14 They are the typing patterns of parent pigeons and young pigeons respectively; the specific typing results are shown in Table 4.

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

[0090]

[0091] The results showed that according to Mendel's law of inheritance, for the 29 pigeon STR loci, one allele on the young pigeon STR loci came from the parent pigeon, that is, one allele of the young pigeon was the same as the allele of the parent pigeon, and the parent-child relationship between the two was not ruled out, proving that the parent-child relationship identification results of the detection system of the present invention were consistent with the actual situation.

[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 present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A pigeon individual typing detection system, characterized in that: The invention comprises a primer combination, wherein the primer combination comprises primers for amplifying 23 STR microsatellite markers and a CHD locus for bird sex detection and 5 SNP genetic markers, wherein the STR microsatellite markers are CliμD32, CliμD19, CliμD35, CliμT02, PIGN10, PIGN26, PG6, CliμD11, Cli12, PG2, CliμC06, CliμD17, PG3, PG5, PIGN0 4. CliμD01, PG1, Cli02, PIGN12, PIGN15, PIGN57, CliμT13 and CliμD16, the SNP genetic markers are 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.

2. The pigeon individual typing detection system according to claim 1, characterized in that: The nucleotide sequences of primers for amplifying CliμD32 are shown in SEQ ID No: 1 and SEQ ID No: 2, the nucleotide sequences of primers for amplifying CliμD19 are shown in SEQ ID No: 3 and SEQ ID No: 4, the nucleotide sequences of primers for amplifying CliμD35 are shown in SEQ ID No: 5 and SEQ ID No: 6, the nucleotide sequences of primers for amplifying CliμT02 are shown in SEQ ID No: 7 and SEQ ID No: 8, the nucleotide sequences of primers for amplifying PIGN10 are shown in SEQ ID No: 9 and SEQ ID No: 10, the nucleotide sequences of primers for amplifying PIGN26 are shown in SEQ ID No: 11 and SEQ ID No: 12, the nucleotide sequences of primers for amplifying PG6 are shown in SEQ ID No: 13 and SEQ ID No: 14, the nucleotide sequences of primers for amplifying CliμD11 are shown in SEQ ID No: 15 and SEQ ID No: 16, the nucleotide sequences of primers for amplifying Cli12 are shown in SEQ ID No: 17 and SEQ ID No: 18, and the nucleotide sequences of primers for amplifying PG2 are shown in SEQ ID No:

19. The nucleotide sequences of the primers for amplifying CHD are shown in SEQ ID Nos: 19 and 20, the nucleotide sequences of the primers for amplifying CHD are shown in SEQ ID Nos: 21 and 22, the nucleotide sequences of the primers for amplifying CliμC06 are shown in SEQ ID Nos: 23 and 24, the nucleotide sequences of the primers for amplifying CliμD17 are shown in SEQ ID Nos: 25 and 26, the nucleotide sequences of the primers for amplifying PG3 are shown in SEQ ID Nos: 27 and 28, the nucleotide sequences of the primers for amplifying PG5 are shown in SEQ ID Nos: 29 and 30, the nucleotide sequences of the primers for amplifying PIGN04 are shown in SEQ ID Nos: 31 and 32, the nucleotide sequences of the primers for amplifying CliμD01 are shown in SEQ ID Nos: 33 and 34, the nucleotide sequences of the primers for amplifying PG1 are shown in SEQ ID Nos: 35 and 36, and the nucleotide sequences of the primers for amplifying Cli02 are shown in SEQ ID Nos: 37 and 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, and 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 CliμT13 are shown in SEQ ID No:45 and SEQ ID No:46, the nucleotide sequences of the primers for amplifying CliμD16 are shown in SEQ ID No:47 and SEQ ID No:48, the nucleotide sequences of the primers for detecting the g.2582481G>A typing of the 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.129954C>T typing of the 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.129456C>T typing of the 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.66493737C>T typing of the myostatin gene are shown in SEQ ID No. 58 to SEQ ID No. 60, and the nucleotide sequences of the primers for detecting the g.710T>G typing of the feather keratin gene are shown in SEQ ID No. 61 to SEQ ID No.

63.

3. The pigeon individual typing detection system according to claim 2, characterized in that: The concentration of the primer shown in SEQ ID No: 1 is 0.16 μmol / L, the concentration of the primer shown in SEQ ID No: 2 is 0.16 μmol / L, the concentration of the primer shown in SEQ ID No: 3 is 0.15 μmol / L, the concentration of the primer shown in SEQ ID No: 4 is 0.15 μmol / L, the concentration of the primer shown in SEQ ID No: 5 is 0.18 μmol / L, the concentration of the primer shown in SEQ ID No: 6 is 0.18 μmol / L, the concentration of the primer shown in SEQ ID No: 7 is 0.25 μmol / L, the concentration of the primer shown in SEQ ID No: 8 is 0.25 μmol / L, the concentration of the primer shown in SEQ ID No: 9 is 0.40 μmol / L, the concentration of the primer shown in SEQ ID No: 10 is 0.40 μmol / L, the concentration of the primer shown in SEQ ID No: 11 is 0.35 μmol / L, the concentration of the primer shown in SEQ ID No: 12 is 0.35 μmol / L, and the concentration of the primer shown in SEQ ID No: 13 is 0. The concentration of the primer shown in SEQ ID No: 13 is 0.15 μmol / L, the concentration of the primer shown in SEQ ID No: 14 is 0.15 μmol / L, the concentration of the primer shown in SEQ ID No: 15 is 0.20 μmol / L, the concentration of the primer shown in SEQ ID No: 16 is 0.20 μmol / L, the concentration of the primer shown in SEQ ID No: 17 is 0.40 μmol / L, the concentration of the primer shown in SEQ ID No: 18 is 0.40 μmol / L, the concentration of the primer shown in SEQ ID No: 19 is 0.15 μmol / L, the concentration of the primer shown in SEQ ID No: 20 is 0.15 μmol / L, the concentration of the primer shown in SEQ ID No: 21 is 0.22 μmol / L, the concentration of the primer shown in SEQ ID No: 22 is 0.22 μmol / L, the concentration of the primer shown in SEQ ID No: 23 is 0.24 μmol / L, the concentration of the primer shown in SEQ ID No: 24 is 0.24 μmol / L, and the concentration of the primer shown in SEQ ID No: 25 is 0.25 μmol / L. The concentration of the primer shown in SEQ ID No:25 is 0.50 μmol / L, the concentration of the primer shown in SEQ ID No:26 is 0.50 μmol / L, the concentration of the primer shown in SEQ ID No:27 is 0.36 μmol / L, the concentration of the primer shown in SEQ ID No:28 is 0.36 μmol / L, the concentration of the primer shown in SEQ ID No:29 is 0.18 μmol / L, the concentration of the primer shown in SEQ ID No:30 is 0.18 μmol / L, the concentration of the primer shown in SEQ ID No:31 is 0.20 μmol / L, the concentration of the primer shown in SEQ ID No:32 is 0.20 μmol / L, and the concentration of the primer shown in SEQ ID No:33 is 0.The concentration of the primer shown in SEQ ID No: 34 is 0.32 μmol / L, the concentration of the primer shown in SEQ ID No: 35 is 0.40 μmol / L, the concentration of the primer shown in SEQ ID No: 36 is 0.40 μmol / L, the concentration of the primer shown in SEQ ID No: 37 is 0.25 μmol / L, the concentration of the primer shown in SEQ ID No: 38 is 0.25 μmol / L, the concentration of the primer shown in SEQ ID No: 39 is 0.20 μmol / L, the concentration of the primer shown in SEQ ID No: 40 is 0.20 μmol / L, the concentration of the primer shown in SEQ ID No: 41 is 0.15 μmol / L, the concentration of the primer shown in SEQ ID No: 42 is 0.15 μmol / L, the concentration of the primer shown in SEQ ID No: 43 is 0.25 μmol / L, and the concentration of the primer shown in SEQ ID No: 44 is 0.25 μmol / L. The concentration of the primer shown in SEQ ID No:44 is 0.25 μmol / L, the concentration of the primer shown in SEQ ID No:45 is 0.30 μmol / L, the concentration of the primer shown in SEQ ID No:46 is 0.30 μmol / L, the concentration of the primer shown in SEQ ID No:47 is 0.25 μmol / L, the concentration of the primer shown in SEQ ID No:48 is 0.25 μmol / L, the concentration of the primer shown in SEQ ID No:49 is 0.20 μmol / L, the concentration of the primer shown in SEQ ID No:50 is 0.20 μmol / L, the concentration of the primer shown in SEQ ID No:51 is 0.40 μmol / L, the concentration of the primer shown in SEQ ID No:52 is 0.25 μmol / L, the concentration of the primer shown in SEQ ID No:53 is 0.25 μmol / L, the concentration of the primer shown in SEQ ID No:54 is 0.50 μmol / L, the concentration of the primer shown in SEQ ID No:55 is 0.20 μmol / L, and the concentration of the primer shown in SEQ ID No:56 is 0. The concentration of the primer shown in No:56 is 0.20 μmol / L, the concentration of the primer shown in SEQ ID No:57 is 0.40 μmol / L, the concentration of the primer shown in SEQ ID No:58 is 0.15 μmol / L, the concentration of the primer shown in SEQ ID No:59 is 0.15 μmol / L, the concentration of the primer shown in SEQ ID No:60 is 0.30 μmol / L, the concentration of the primer shown in SEQ ID No:61 is 0.20 μmol / L, the concentration of the primer shown in SEQ ID No:62 is 0.20 μmol / L, and the concentration of the primer shown in SEQ ID No:63 is 0.40 μmol / L. .

4. The pigeon individual typing detection system according to claim 2 or 3, characterized in that: The 5' end of at least one of the upstream and downstream primers targeting the same STR microsatellite marker or the CHD locus or the SNP genetic marker is labeled with a fluorescent dye, and the fluorescent dye is selected from 6-FAM, HEX, SUM, LYN, PUR, TAMRA, ALEXA568, ALEXA594, ROX, VIC, PET, NED or TAZ.

5. The pigeon individual typing detection system according to claim 4, characterized in that: Primers whose nucleotide sequences are shown as SEQ ID No: 1 to SEQ ID No: 12 are set as the first group; primers whose nucleotide sequences are shown as 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 the second group; primers whose nucleotide sequences are shown as SEQ ID No: 23 to SEQ ID No: 34 and SEQ ID No: 49 to SEQ ID No: 51 are set as the third group; primers whose nucleotide sequences are shown as SEQ ID No: 35 to SEQ ID No: 40 are set as the fourth group; primers whose nucleotide sequences are shown as 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 the fifth group.

6. The pigeon individual typing detection system according to claim 5, characterized in that: The fluorescent dye selected by the first group is 6-FAM, the fluorescent dye selected by the second group is HEX, the fluorescent dye selected by the third group is TAMRA, the fluorescent dye selected by the fourth group is ALEXA568, and the fluorescent dye selected by the fifth group is ALEXA594.

7. The pigeon individual typing detection system according to claim 6, characterized in that: Also included is a composite amplification premix, which includes one or more of a PCR reaction buffer, dNTPs, a DNA polymerase, and MgCl2.

8. A kit, characterized in that: It comprises the pigeon individual typing detection system as described in any one of claims 1 to 7.

9. The kit according to claim 8, characterized in that: Allelic ladders are also included.

10. Use of the pigeon individual typing detection system according to any one of claims 1 to 7 or the kit according to any one of claims 8 to 9 in pigeon individual identification and / or pigeon parentage identification and / or pigeon flight ability assessment.

Images