Application of SNP (Single Nucleotide Polymorphism) marker in evaluating flight ability of racing pigeons, detection system and method for predicting comprehensive flight ability of racing pigeons

Abstract

The invention discloses application of an SNP (Single Nucleotide Polymorphism) marker in evaluating the flight ability of racing pigeons, a detection system and a method for predicting the comprehensive flight ability of racing pigeons. According to the invention, only three fluorescent quantitative PCR reaction systems are needed to realize typing detection of nine SNP loci of eight genes, melting curve data are directly converted into genotypes through a program, and the result interpretation is simple, convenient and rapid. In addition, an evaluation method for quantifying the comprehensive flight ability of the racing pigeons is further established after score assignment according to genotype results, more accurate and systematic evaluation and prediction of the flight ability of the racing pigeons are achieved, and the accuracy reaches up to 80% or above.

Description

Technical Field

[0001] The present invention relates to the field of gene detection technology, and in particular to the application of SNP markers in evaluating the flying ability of racing pigeons, a detection system and a method for predicting the comprehensive flying ability of racing pigeons. Background Art

[0002] The racial pedigree (genetic genes) of racing pigeons has always been recognized as a key factor in determining their flying ability. Serine protein kinase gene (CASK), cryptochrome gene (CRY1), dopamine receptor D4 gene (DRD4), feather keratin gene (F-KER), glutathione reductase (GSR), lactate deoxygenase gene A (LDHA), low-density lipoprotein receptor-related protein 8 (LRP8) and muscle generation regulatory gene (MSTN) have been confirmed to be genetic factors affecting the flying ability of racing pigeons. Serine protein kinase (CASK) is a multifunctional protein that participates in the development of neural tissue and cell signal transduction, affecting the intellectual development of pigeons; cryptochrome gene (CRY1) is related to the ability to sense magnetic fields; dopamine D4 receptor (DRD4) is related to the homing ability of racing pigeons. The stronger the homing ability of racing pigeons, the better the flying ability; feather keratin (F-KER) affects the structure and strength of feathers, reducing air resistance to increase speed during flight; glutathione reductase (GSR) plays a key role in the orientation ability of pigeons; and lactate Dehydrogenase A (LDHA) is an important factor in the lactic acid metabolism cycle. When racing pigeons fly long distances, a large amount of lactic acid will accumulate in the body. The faster the enzyme metabolism, the less fatigued they are, and therefore the better their endurance. The low-density lipoprotein receptor-related protein 8 (LRP8) gene may be involved in the establishment and maintenance of neural circuits related to cognitive ability, which is crucial for the navigation and homing ability of racing pigeons. The muscle regulatory gene (MSTN) mainly controls the amount of muscle cells and muscle fiber bundles generated. Good muscle development is congenital, and the effect can be more obvious after acquired training. Therefore, the typing results of these 8 genes can be used as reference indicators for racing pigeon breeding and selection.

[0003] In the prior art, a genetic testing kit for racing pigeons has been developed, but the typing results based on individual genes can only qualitatively determine the potential of racing pigeons in one or some aspects, but cannot accurately predict the overall ability of racing pigeons, which lacks effective guidance for the selection and breeding of excellent racing pigeons. Therefore, it is urgent to develop a method that can more comprehensively and accurately predict the comprehensive flight ability of racing pigeons. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides the application of SNP markers in evaluating the flying ability of racing pigeons, a detection system and a method for predicting the comprehensive flying ability of racing pigeons.

[0005] The first object of the present invention is to provide an application of SNP markers in evaluating the flying ability of racing pigeons.

[0006] The second object of the present invention is to provide a reagent for detecting the SNP marker for use in evaluating the flying ability of racing pigeons.

[0007] The third object of the present invention is to provide a primer composition for detecting the SNP marker.

[0008] The fourth object of the present invention is to provide a primer-probe combination for detecting the SNP marker.

[0009] The fifth object of the present invention is to provide a fluorescent quantitative PCR detection system for racing pigeon genotyping.

[0010] A sixth object of the present invention is to provide a method for predicting the comprehensive flying ability of racing pigeons.

[0011] A seventh object of the present invention is to provide a system for predicting the comprehensive flying ability of racing pigeons.

[0012] In order to achieve the above object, the present invention is implemented by the following scheme:

[0013] The use of SNP markers in evaluating the flying ability of racing pigeons, wherein the SNP markers are in the following SNP sites 1 to 9:

[0014] SNP site 1 is located at base 98990834 on chromosome 1 of the rock pigeon where the CASK gene is located, which is G or A;

[0015] SNP site 2 is located at bases 153427839 to 153427840 on chromosome 1 of the rock pigeon where the CRY1 gene is located, and is AG, AA, AT, or TT;

[0016] SNP site 3 is located at base 68012131 on chromosome 5 of the rock pigeon where the DRD4a gene is located, and is C or T;

[0017] SNP site 4 is located at base 68011633 on chromosome 5 of the rock pigeon where the DRD4b gene is located, and is C or T;

[0018] SNP site 5 is located at base 5545394 on chromosome 23 of the rock pigeon where the KER gene is located, and is either T or G;

[0019] SNP site 6 is located at base 77827982 on chromosome 4 of the rock pigeon where the GSR gene is located, and is C or T;

[0020] SNP site 7 is located at base 53,833,494 on chromosome 5 of the rock pigeon where the LDHA gene is located, and is either G or A;

[0021] SNP site 8 is located at base 27,429,922 on chromosome 7 of the rock pigeon where the MSTN gene is located, and is C or T;

[0022] SNP site 9 is located at base 14737124 on chromosome 8 of the rock pigeon where the LRP8 gene is located, and is G or T;

[0023] The number of the chromosome 1 of the rock pigeon in NCBI is NC_088602.1; the number of the chromosome 4 of the rock pigeon in NCBI is NC_088605.1; the number of the chromosome 5 of the rock pigeon in NCBI is NC_088606.1; the number of the chromosome 7 of the rock pigeon in NCBI is NC_088608.1; the number of the chromosome 8 of the rock pigeon in NCBI is NC_088609.1; the number of the chromosome 23 of the rock pigeon in NCBI is NC_088624.1.

[0024] Application of a reagent for detecting the SNP marker in evaluating the flying ability of racing pigeons.

[0025] A primer composition for detecting the SNP marker, comprising primer sets 1 to 3; wherein primer set 1 comprises primers having nucleotide sequences as shown in SEQ ID NOs. 1 to 2, nucleotide sequences as shown in SEQ ID NOs. 4 to 5, and nucleotide sequences as shown in SEQ ID NOs. 7 to 8; primer set 2 comprises primers having nucleotide sequences as shown in SEQ ID NOs. 10 to 11, nucleotide sequences as shown in SEQ ID NOs. 13 to 14, and nucleotide sequences as shown in SEQ ID NOs. 16 to 17; and primer set 3 comprises primers having nucleotide sequences as shown in SEQ ID NOs. 19 to 20, nucleotide sequences as shown in SEQ ID NOs. 22 to 23, and nucleotide sequences as shown in SEQ ID NOs. 25 to 26.

[0026] Primers whose nucleotide sequences are shown in SEQ ID NOs.1 to 2 are used to detect the SNP site 1; primers whose nucleotide sequences are shown in SEQ ID NOs.4 to 5 are used to detect the SNP site 2; primers whose nucleotide sequences are shown in SEQ ID NOs.7 to 8 are used to detect the SNP site 3; primers whose nucleotide sequences are shown in SEQ ID NOs.10 to 11 are used to detect the SNP site 4; primers whose nucleotide sequences are shown in SEQ ID NOs.13 to 14 are used to detect the SNP site 5; primers whose nucleotide sequences are shown in SEQ ID NOs.16 to 17 are used to detect the SNP site 6; primers whose nucleotide sequences are shown in SEQ ID NOs.19 to 20 are used to detect the SNP site 7; primers whose nucleotide sequences are shown in SEQ ID NOs.22 to 23 are used to detect the SNP site 8; primers whose nucleotide sequences are shown in SEQ ID NOs.25 to 26 are used to detect the SNP site 9.

[0027] A primer-probe combination for detecting the SNP marker comprises the primer combination and a probe.

[0028] Preferably, the probe includes probe groups 1 to 3; wherein, probe group 1 includes probes with nucleotide sequences as shown in SEQ ID NO.3, nucleotide sequences as shown in SEQ ID NO.6 and nucleotide sequences as shown in SEQ ID NO.9; probe group 2 includes probes with nucleotide sequences as shown in SEQ ID NO.12, nucleotide sequences as shown in SEQ ID NO.15 and nucleotide sequences as shown in SEQ ID NO.18; probe group 3 includes probes with nucleotide sequences as shown in SEQ ID NO.21, nucleotide sequences as shown in SEQ ID NO.24 and nucleotide sequences as shown in SEQ ID NO.27.

[0029] The probe with a nucleotide sequence as shown in SEQ ID NO.3 is used to detect the SNP site 1; the probe with a nucleotide sequence as shown in SEQ ID NO.6 is used to detect the SNP site 2; the probe with a nucleotide sequence as shown in SEQ ID NO.9 is used to detect the SNP site 3; the probe with a nucleotide sequence as shown in SEQ ID NO.12 is used to detect the SNP site 4; the probe with a nucleotide sequence as shown in SEQ ID NO.15 is used to detect the SNP site 5; the probe with a nucleotide sequence as shown in SEQ ID NO.18 is used to detect the SNP site 6; the probe with a nucleotide sequence as shown in SEQ ID NO.21 is used to detect the SNP site 7; the probe with a nucleotide sequence as shown in SEQ ID NO.24 is used to detect the SNP site 8; the probe with a nucleotide sequence as shown in SEQ ID NO.27 is used to detect the SNP site 9.

[0030] More preferably, the probes in each probe group are provided with different fluorescent reporter groups at their 5' ends and fluorescent quencher groups at their 3' ends.

[0031] Further preferably, in the probe set 1, the 5' ends of the probes having the nucleotide sequence shown in SEQ ID NO.3, the nucleotide sequence shown in SEQ ID NO.6 and the nucleotide sequence shown in SEQ ID NO.9 are provided with FAM, HEX and ROX respectively.

[0032] Further preferably, in the probe set 2, the 5' ends of the probes having the nucleotide sequence shown in SEQ ID NO.12, the nucleotide sequence shown in SEQ ID NO.15 and the nucleotide sequence shown in SEQ ID NO.18 are provided with FAM, HEX and ROX respectively.

[0033] Further preferably, in the probe group 3, the 5' ends of the probes having the nucleotide sequence shown in SEQ ID NO.21, the nucleotide sequence shown in SEQ ID NO.24 and the nucleotide sequence shown in SEQ ID NO.27 are provided with FAM, ROX and HEX respectively.

[0034] Further preferably, in the probe set 1, the 3' end of the probes with nucleotide sequence as shown in SEQ ID NO.3 and SEQ ID NO.6 is provided with BHQ1, and the 3' end of the probe with nucleotide sequence as shown in SEQ ID NO.9 is provided with BHQ2.

[0035] Further preferably, in the probe set 2, the 3' end of the probes with nucleotide sequence as shown in SEQ ID NO.12 and SEQ ID NO.15 is provided with BHQ1, and the 3' end of the probe with nucleotide sequence as shown in SEQ ID NO.18 is provided with BHQ2.

[0036] Further preferably, in the probe set 3, the probes with nucleotide sequences as shown in SEQ ID NO.21 and SEQ ID NO.27 have BHQ1 at their 3' ends, and the probes with nucleotide sequences as shown in SEQ ID NO.24 have BHQ2 at their 3' ends.

[0037] Further preferably, in the probe, the base corresponding to the SNP site detected by the probe is provided with a locked nucleic acid modification.

[0038] More preferably, the probe having a nucleotide sequence as shown in SEQ ID NO. 3 has a locked nucleic acid modification at the 13th base in the 5'→3' direction.

[0039] More preferably, the probe having a nucleotide sequence as shown in SEQ ID NO. 6 has a locked nucleic acid modification at the 13th base in the 5'→3' direction.

[0040] More preferably, the probe having a nucleotide sequence as shown in SEQ ID NO. 15 has a locked nucleic acid modification at the 13th base in the 5'→3' direction.

[0041] More preferably, the probe having a nucleotide sequence as shown in SEQ ID NO. 18 has a locked nucleic acid modification at the 13th base in the 5'→3' direction.

[0042] More preferably, the probe having a nucleotide sequence as shown in SEQ ID NO. 21 has a locked nucleic acid modification at the 12th base in the 5'→3' direction.

[0043] More preferably, the probe having a nucleotide sequence as shown in SEQ ID NO. 24 has a locked nucleic acid modification at the 19th base in the 5'→3' direction.

[0044] More preferably, the probe having a nucleotide sequence as shown in SEQ ID NO. 27 has a locked nucleic acid modification at the 12th base in the 5'→3' direction.

[0045] A fluorescent quantitative PCR detection system for racing pigeon genotyping comprises reaction systems 1 to 3; wherein the reaction system 1 comprises the primer set 1, the reaction system 2 comprises the primer set 2, and the reaction system 3 comprises the primer set 3.

[0046] Preferably, the reaction systems 1 to 3 further include probes, and the probes include probe groups 1 to 3; wherein the probe group 1 includes probes having nucleotide sequences as shown in SEQ ID NO.3, nucleotide sequences as shown in SEQ ID NO.6, and nucleotide sequences as shown in SEQ ID NO.9; the probe group 2 includes probes having nucleotide sequences as shown in SEQ ID NO.12, nucleotide sequences as shown in SEQ ID NO.15, and nucleotide sequences as shown in SEQ ID NO.18; the probe group 3 includes probes having nucleotide sequences as shown in SEQ ID NO.21, nucleotide sequences as shown in SEQ ID NO.24, and nucleotide sequences as shown in SEQ ID NO.27;

[0047] The reaction system 1 includes the probe group 1, the reaction system 2 includes the probe group 2, and the reaction system 3 includes the probe group 3.

[0048] The reaction system 1 is used to detect the SNP site 1, SNP site 2 and SNP site 3, the reaction system 2 is used to detect the SNP site 4, SNP site 5 and SNP site 6, and the reaction system 3 is used to detect the SNP site 7, SNP site 8 and SNP site 9.

[0049] Preferably, the reaction systems 1 to 3 further include fluorescent quantitative PCR reaction reagents, including but not limited to fluorescent quantitative PCR reaction enzyme and fluorescent quantitative PCR reaction buffer.

[0050] Preferably, the program of the reaction systems 1 to 3 for performing fluorescence quantitative PCR amplification includes the following conditions: (1) constant temperature stage: 94 to 96°C, 0.5 to 1.5 min, 1 cycle; (2) cycle stage: 94 to 96°C, 8 to 12 s, 53 to 57°C, 28 to 32 s, 68 to 72°C, 28 to 32 s, 48 ​​to 52 cycles; (3) constant temperature stage: 94 to 96°C, 28 to 32 s, 43 to 47°C, 4 to 6 min, 1 cycle; (4) melting stage: 45°C → 90°C, increasing by 0.01 to 0.05°C every 1 s, and continuously collecting fluorescence.

[0051] More preferably, the program of the reaction systems 1 to 3 for performing fluorescence quantitative PCR amplification includes the following conditions: (1) constant temperature stage: 95°C, 1 min, 1 cycle; (2) cycle stage: 95°C, 10 s, 55°C, 30 s, 70°C, 30 s, 50 cycles; (3) constant temperature stage: 95°C, 30 s, 45°C, 5 min, 1 cycle; (4) melting stage: 45°C→90°C, increasing by 0.03°C every 1 s, and continuously collecting fluorescence.

[0052] A method for predicting the comprehensive flight ability of racing pigeons comprises the following steps: detecting racing pigeons with the fluorescent quantitative PCR detection system, genotyping and scoring the SNP markers according to the obtained melting curve data, the sum of the obtained scores being the flight ability score of the racing pigeons; and judging the comprehensive flight ability of the racing pigeons according to the flight ability score combined with a threshold.

[0053] Preferably, the melting curve data includes peak shape and annealing temperature, and the criteria for genotype determination are shown in the following table:

[0054]

[0055]

[0056] More preferably, the scoring criteria are as shown in the following table:

[0057]

[0058]

[0059] The judging criteria of the comprehensive flying ability of the racing pigeon are as follows:

[0060] When the flying ability score is ≥80 points, the racing pigeon is judged to have excellent comprehensive flying ability;

[0061] When 70 points ≤ flying ability score < 80 points, the racing pigeon is judged to have excellent comprehensive flying ability;

[0062] When the flying ability score is <70 points, the racing pigeon is judged to have average comprehensive flying ability.

[0063] A system for predicting the comprehensive flight ability of racing pigeons, comprising a data acquisition module, a storage module, an analysis module and an output module; the data acquisition module is used to obtain the melting curve data of the racing pigeon obtained by the fluorescent quantitative PCR detection system; the storage module is used to store the melting curve data in the data acquisition module; the analysis module uses the melting curve data obtained by the data acquisition module as input data, executes any method for predicting the comprehensive flight ability of the racing pigeon, and determines the comprehensive flight ability of the racing pigeon; the output module is used to output the judgment result of the comprehensive flight ability obtained by the analysis module.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] The present invention only needs 3 fluorescent quantitative PCR reaction systems to achieve typing detection of 9 SNP sites in 8 genes, and directly converts the melting curve data into genotypes through a program, and the result interpretation is simple and fast. In addition, a method for evaluating the comprehensive flight ability of racing pigeons is further established after scoring according to the genotype results, achieving a more accurate and systematic evaluation and prediction of the flying ability of racing pigeons, with an AUC of more than 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 Schematic diagram of the fluorescence detection channel settings for PCR detection projects.

[0067] Figure 2 This is the melting curve of reaction system 1.

[0068] Figure 3 This is the melting curve of reaction system 2.

[0069] Figure 4 This is the melting curve of reaction system 3.

[0070] Figure 5 ROC curve for the method of predicting comprehensive flying ability of racing pigeons. DETAILED DESCRIPTION

[0071] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0072] Example 1 Detection method for racing pigeon genotyping

[0073] 1. Extraction of genomic DNA from racing pigeon feather follicles:

[0074] Feathers of the racing pigeons to be tested were collected, and the hair follicles (length 2-3 cm) were cut for proteinase K digestion at 56°C for 2 h. The digested samples were extracted using a commercial kit to obtain genomic DNA as a template for subsequent testing.

[0075] 2. Detection of racing pigeon genotyping

[0076] (1) Detection target

[0077] The present invention uses 9 SNP sites of 8 genes (serine protein kinase gene (CASK), cryptochrome gene (CRY 1), dopamine receptor D4 gene (DRD4), feather keratin gene (F-KER), glutathione reductase (GSR), lactate deoxygenase gene A (LDHA), low-density lipoprotein receptor-related protein 8 (LRP8) and muscle generation regulatory gene (MSTN)) as detection targets, and adopts fluorescent quantitative PCR (TaqMan probe-melting curve method) to determine typing. The information of the SNP site is shown in Table 1, and the sequences of the primers and probes used are shown in Table 2.

[0078] Table 1 Relevant information of 9 SNP loci

[0079]

[0080]

[0081] Note: The number of chromosome 1 of rock pigeon in NCBI is NC_088602.1, the number of chromosome 4 of rock pigeon in NCBI is NC_088605.1, the number of chromosome 5 of rock pigeon in NCBI is NC_088606.1, the number of chromosome 7 of rock pigeon in NCBI is NC_088608.1, the number of chromosome 8 of rock pigeon in NCBI is NC_088609.1, and the number of chromosome 23 of rock pigeon in NCBI is NC_088624.1.

[0082] Table 2 Sequence information of detection primers and probes

[0083]

[0084]

[0085] Note: The probe sequence of SNP site 2 in the table is marked with a box The marked bases indicate the bases corresponding to the SNP site; / iXNA_N / indicates that the base N is the base corresponding to the SNP site and there is a locked nucleic acid modification here.

[0086] (2) Preparation of fluorescence quantitative PCR reaction system

[0087] The method provided in this embodiment is divided into three reaction systems for fluorescence quantitative PCR detection, wherein reaction system 1 detects SNP site 1 located at the CASK gene, SNP site 2 located at the CRY1 gene, and SNP site 3 located at the DRD4a gene; reaction system 2 detects SNP site 4 located at the DRD4b gene, SNP site 5 located at the F-KER gene, and SNP site 6 located at the GSR gene; reaction system 3 detects SNP site 7 located at the LDHA gene, SNP site 8 located at the MSTN gene, and SNP site 9 located at the LRP8 gene.

[0088] The composition ratios of each reaction system are shown in Table 3. In terms of working concentration, the primer-probe mixture of reaction system 1 includes 400 nM of CASK-R (SEQ ID NO.1), CRY1-R (SEQ ID NO.4), and DRD4a-R (SEQ ID NO.7), 200 nM of CASK-P (SEQ ID NO.3), CRY1-P (SEQ ID NO.6), and DRD4a-P (SEQ ID NO.9), and 100 nM of CASK-F (SEQ ID NO.2), CRY1-F (SEQ ID NO.5), and DRD4a-F (SEQ ID NO.8); the primer-probe mixture of reaction system 2 includes 400 nM of DRD4b-R (SEQ ID NO.11), KER-R (SEQ ID NO.14), and GSR-R (SEQ ID NO.17), and 200 nM of DRD4b-P (SEQ ID NO.12), KER-P (SEQ ID NO.13), and GSR-R (SEQ ID NO.14). NO.15) and GSR-P (SEQ ID NO.18), 100 nM of DRD4b-F (SEQ ID NO.10), KER-F (SEQ ID NO.13), GSR-F (SEQ ID NO.16); the primer-probe mixture of reaction system 3 contained 400 nM of LDHA-R (SEQ ID NO.20), MSTN-R (SEQ ID NO.23), LRP8-R (SEQ ID NO.26), 200 nM of LDHA-P (SEQ ID NO.21), MSTN-P (SEQ ID NO.24) and LRP 8-P (SEQ ID NO.27), 100 nM of LDHA-F (SEQ ID NO.19), MSTN-F (SEQ ID NO.22), LRP8-F (SEQ ID NO.25).

[0089] Table 3 Fluorescence quantitative PCR detection reaction system

[0090]

[0091] For each sample to be tested, prepare the above three reaction systems in a 200 μL PCR tube. The total volume of each reaction system is 20 μL. After thorough mixing, centrifuge for a short time and set aside. Avoid bubbles during the operation and ensure that there is no liquid adhering to the wall of the PCR tube.

[0092] (3) PCR detection

[0093] Transfer the prepared reaction systems to the corresponding positions of the sample slots of the Macrostone PCR instrument and record the sample order. Figure 1 The information shown sets the fluorescence detection channel of the PCR detection project, selects the FAM channel to detect SNP site 1 located at the CASK gene, SNP site 4 located at the DRD4b gene, and SNP site 7 located at the LDHA gene, selects the HEX channel to detect SNP site 2 located at the CRY1 gene, SNP site 5 located at the F-KER gene, and SNP site 9 located at the LRP8 gene, and selects the ROX channel to detect SNP site 3 located at the DRD4a gene, SNP site 6 located at the GSR gene, and SNP site 8 located at the MSTN gene.

[0094] Set the PCR reaction program according to the information shown in Table 4, with the hot cover temperature at 105°C and the liquid volume at 20 μL. After the amplification is completed, discard the PCR tube. It is strictly forbidden to open the PCR tube to prevent contamination.

[0095] Select experimental analysis in the supporting "SLAN Fully Automatic Medical PCR Analysis System 8.2.2", select the experimental wells where the samples are placed in the well plate selector, and select "1 (FAM channel), 2 (HEX channel), 3 (ROX channel)" in the channel selection to perform melting curve analysis on the samples to be tested.

[0096] Table 4 Fluorescence quantitative PCR detection reaction procedure

[0097]

[0098] The quality control standards for fluorescent quantitative PCR detection are as follows:

[0099] 1) The negative quality control (H2O) has no melting curve peak; 2) The positive quality control (racing pigeon gDNA identified by Sanger sequencing) has the target melting curve peak.

[0100] The above two requirements must be met at the same time in the same test, otherwise the test will be invalid and need to be repeated.

[0101] The criteria and treatment methods for abnormal results of fluorescent quantitative PCR detection are as follows:

[0102] 1) If one of the detection sites of a certain sample to be tested does not have a typical melting peak, it indicates that the detection signal of the site is abnormal; 2) If all three detection sites of a certain sample to be tested do not have a typical melting peak, it indicates that the detection signal of the sample is abnormal.

[0103] If any of the above two items are present in the sample to be tested, the test result will be deemed invalid and the sample to be tested needs to be re-tested or the nucleic acid needs to be re-extracted before testing.

[0104] (4) Genotyping of SNP loci

[0105] The genotype of each SNP site was determined based on the peak shape and annealing temperature (Tm) of the obtained melting curve according to the information shown in Table 5.

[0106] Table 5 Genotype discrimination criteria for each SNP locus

[0107]

[0108]

[0109] The melting curve peak of the racing pigeon feather sample identified by Sanger sequencing as AA at SNP site 1, AGTT at SNP site 2, and CC at SNP site 3 is shown in the figure below. Figure 2 The melting curve peak of the racing pigeon feather sample identified by Sanger sequencing as CC at SNP site 4, GT at SNP site 5, and CT at SNP site 6 is shown in the figure below. Figure 3 The melting curve peak of the racing pigeon feather sample identified by Sanger sequencing as SNP site 7 genotype AG, SNP site 8 genotype CC, SNP site 9 genotype TT is shown in the figure. Figure 4 The melting curve peaks of the remaining genotypes of each SNP site are also consistent with the corresponding Sanger sequencing results.

[0110] The above results show that the detection method established by the present invention can accurately and quickly identify the genotype corresponding to each SNP site based on the peak shape and Tm of the melting curve.

[0111] Embodiment 2 A kind of method of predicting the comprehensive flying ability of racing pigeon

[0112] 1. Genotyping and scoring

[0113] The racing pigeons to be tested were detected using the method of Example 1, and the genotype of each SNP site was determined according to the peak shape and annealing temperature (Tm) of the obtained melting curve according to the information shown in Table 5, and the genotype determination results were recorded. Subsequently, the scores were assigned in combination with the standards shown in Table 7, and the scoring results were recorded.

[0114] Table 7 SNP site genotype scoring standards

[0115]

[0116]

[0117] 2. Method for determining comprehensive flight capability

[0118] The total score of the eight scoring items (i.e., items 1 to 8 in Table 6) is calculated to obtain the flying ability score of the racing pigeon to be tested. The comprehensive flying ability of the racing pigeon is determined according to the following criteria:

[0119] When the flying ability score is ≥80 points, the racing pigeon is judged to have excellent comprehensive flying ability;

[0120] When 70 points ≤ flying ability score < 80 points, the racing pigeon is judged to have excellent comprehensive flying ability;

[0121] When the flying ability score is <70 points, the racing pigeon is judged to have average comprehensive flying ability.

[0122] Example 3 Evaluation of a method for predicting the comprehensive flying ability of racing pigeons

[0123] 1. Comprehensive flight capability prediction

[0124] In this example, the comprehensive flight ability of 158 racing pigeons (age distribution: 3-5 years old; gender distribution: 75 males and 83 females) was predicted according to the method of Example 3. The genotypes of the SNP loci and the flight ability scores are shown in Table 8.

[0125] 2. Flight capability test

[0126] This embodiment also tests the flight ability of the above 158 racing pigeons. The test method adopts a single-distance release scheme: in Xinxiang, Henan, the racing pigeons are brought to a unified release for a 500-kilometer middle-distance race, and the total time for each racing pigeon to fly back is recorded, and the flight performance (speed per minute: m / min) is calculated. Racing pigeons with high speed per minute usually mean that they fly faster and have better flying ability. In this test of 158 racing pigeons, a total of 110 racing pigeons successfully returned to the nest. The ranking of the first 79 returning racing pigeons was statistically calculated based on the flight performance, and the results are shown in Table 8.

[0127] Table 8 Prediction of comprehensive flying ability and flying ability test results of 158 racing pigeons

[0128]

[0129]

[0130]

[0131]

[0132]

[0133] 3. Evaluation of the accuracy of the prediction method

[0134] The distribution of flight ability scores of 158 racing pigeons was analyzed. The first 79 racing pigeons were defined as "good", and the racing pigeons after the 79th place and those that failed to successfully return home were defined as "poor". Combined with the flight ability scores, the ROC curve was constructed and the area under the ROC curve (AUC) was calculated. The higher the AUC value, the better the prediction effect.

[0135] ROC curve Figure 5 As shown, the AUC is more than 80%, which indicates that the prediction method based on 9 SNP sites of the present invention has good prediction effect on the comprehensive flight ability of racing pigeons, high accuracy, and can achieve accurate evaluation of the flight ability of racing pigeons.

[0136] Example 4 A system for predicting the comprehensive flying ability of racing pigeons

[0137] The system for predicting the comprehensive flying ability of racing pigeons comprises a data acquisition module, a storage module, an analysis module and an output module.

[0138] Among them, the data acquisition module is used to obtain the melting curve data obtained by fluorescent quantitative PCR detection of racing pigeons.

[0139] The storage module is used to store the melting curve data in the data acquisition module.

[0140] The analysis module uses the melting curve data obtained by the data acquisition module as input data, executes the method shown in Example 2, and determines the comprehensive flying ability of the racing pigeon.

[0141] The output module is used to output the judgment results of the comprehensive flight capability obtained by the analysis module.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The application of SNP markers in evaluating the flying ability of racing pigeons is characterized by: The SNP markers are the following SNP sites 1 to 9: SNP site 1 is located at base 98990834 on chromosome 1 of the rock pigeon where the CASK gene is located, which is G or A; SNP site 2 is located at bases 153427839 to 153427840 on chromosome 1 of the rock pigeon where the CRY1 gene is located, and is AG, AA, AT, or TT; SNP site 3 is located at base 68012131 on chromosome 5 of the rock pigeon where the DRD4a gene is located, and is C or T; SNP site 4 is located at base 68011633 on chromosome 5 of the rock pigeon where the DRD4b gene is located, and is C or T; SNP site 5 is located at base 5545394 on chromosome 23 of the rock pigeon where the F-KER gene is located, and is either T or G; SNP site 6 is located at base 77827982 on chromosome 4 of the rock pigeon where the GSR gene is located, and is C or T; SNP site 7 is located at base 53,833,494 on chromosome 5 of the rock pigeon where the LDHA gene is located, and is either G or A; SNP site 8 is located at base 27,429,922 on chromosome 7 of the rock pigeon where the MSTN gene is located, and is C or T; SNP site 9 is located at base 14737124 on chromosome 8 of the rock pigeon where the LRP8 gene is located, and is G or T; The number of the rock pigeon chromosome 1 in NCBI is NC_088602.1; The number of chromosome 4 of the rock pigeon in NCBI is NC_088605.1; The number of chromosome 5 of the rock pigeon in NCBI is NC_088606.1; The number of chromosome 7 of the rock pigeon is NC_088608.1 in NCBI; The number of chromosome 8 of the rock pigeon in NCBI is NC_088609.1; The number of the rock pigeon chromosome 23 in NCBI is NC_088624.

1.

2. Use of a reagent for detecting the SNP marker described in claim 1 in evaluating the flying ability of racing pigeons.

3. A primer composition for detecting the SNP marker according to claim 1, characterized in that: Includes primer sets 1 to 3; Wherein, primer set 1 comprises primers having nucleotide sequences as shown in SEQ ID NOs. 1 to 2, nucleotide sequences as shown in SEQ ID NOs. 4 to 5, and nucleotide sequences as shown in SEQ ID NOs. 7 to 8; Primer set 2 includes primers having nucleotide sequences as shown in SEQ ID NOs. 10 to 11, nucleotide sequences as shown in SEQ ID NOs. 13 to 14, and nucleotide sequences as shown in SEQ ID NOs. 16 to 17; Primer set 3 includes primers having nucleotide sequences shown in SEQ ID NOs. 19 to 20, primers having nucleotide sequences shown in SEQ ID NOs. 22 to 23, and primers having nucleotide sequences shown in SEQ ID NOs. 25 to 26.

4. A primer-probe combination for detecting the SNP marker of claim 1, characterized in that: The method comprises the primer combination and the probe according to claim 3.

5. The primer-probe combination according to claim 4, characterized in that: The probes include probe groups 1 to 3; Wherein, the probe group 1 comprises probes having a nucleotide sequence as shown in SEQ ID NO.3, a nucleotide sequence as shown in SEQ ID NO.6, and a nucleotide sequence as shown in SEQ ID NO.9; The probe set 2 comprises a probe with a nucleotide sequence as shown in SEQ ID NO.12, a nucleotide sequence as shown in SEQ ID NO.15, and a nucleotide sequence as shown in SEQ ID NO.18; The probe group 3 comprises probes having a nucleotide sequence as shown in SEQ ID NO.21, a nucleotide sequence as shown in SEQ ID NO.24, and a nucleotide sequence as shown in SEQ ID NO.

27.

6. The primer-probe combination according to claim 5, characterized in that: The 5' end of the probes in each probe group is provided with a different fluorescent reporter group, and the 3' end is provided with a fluorescent quenching group.

7. A fluorescent quantitative PCR detection system for racing pigeon genotyping, characterized in that: It comprises reaction systems 1 to 3; wherein the reaction system 1 comprises the primer set 1 described in claim 3, the reaction system 2 comprises the primer set 2 described in claim 3, and the reaction system 3 comprises the primer set 3 described in claim 3.

8. The fluorescent quantitative PCR detection system according to claim 7, characterized in that: Also included is the probe described in claim 4 or claim 5.

9. A method for predicting the comprehensive flying ability of racing pigeons, characterized in that: The following steps are involved: The racing pigeon is detected by using the fluorescent quantitative PCR detection system according to claim 8, and the SNP marker is genotyped and scored according to the obtained melting curve data, and the sum of the scores is the flight ability score of the racing pigeon; the comprehensive flight ability of the racing pigeon is determined according to the flight ability score combined with a threshold.

10. A system for predicting the comprehensive flying ability of racing pigeons, characterized in that: It includes a data acquisition module, a storage module, an analysis module and an output module; The data acquisition module is used to obtain the melting curve data of the racing pigeon obtained by the fluorescent quantitative PCR detection system according to claim 8; The storage module is used to store the melting curve data in the data acquisition module; The analysis module uses the melting curve data obtained by the data acquisition module as input data and executes the method of claim 9 to determine the comprehensive flying ability of the racing pigeon; The output module is used to output the judgment result of the comprehensive flight capability obtained by the analysis module.

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