Application of SNP marker in evaluating flight ability of racing pigeon, detection system and method for predicting comprehensive flight ability of racing pigeon

Abstract

The application discloses application of SNP markers in evaluating flight capacity of racing pigeons, a detection system and a method for predicting comprehensive flight capacity of racing pigeons. The application realizes typing detection of 8 genes and 9 SNP sites through only 3 fluorescent quantitative PCR reaction systems, and converts melting curve data into genotypes through a program, so that result interpretation is simple, rapid, and the like. In addition, according to the genotype result, an evaluation method for quantifying comprehensive flight capacity of racing pigeons is further established after scoring, so that more accurate and systematic evaluation and prediction of the flight capacity of racing pigeons are realized, and the accuracy is as high as 80% or above.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene detection, in particular to the application of SNP markers in evaluating the flight ability of racing pigeons, a detection system and a method for predicting the comprehensive flight ability of racing pigeons. BACKGROUND

[0002] The racial pedigree (genetic genes) of racing pigeons has always been considered as a key factor determining their flight ability. Serine protein kinase gene (CASK), cryptochrome gene (CRY1), dopamine receptor D4 gene (DRD4), feather keratin gene (F-KER), glutathione reductase (GSR), lactate dehydrogenase gene A (LDHA), low density lipoprotein receptor-related protein 8 (LRP8) and muscle generation regulation gene (MSTN) have been proven to be genetic factors affecting the flight ability of racing pigeons. Serine protein kinase (CASK) is a multifunctional protein involved in neural tissue development and cell signal transduction, affecting the intelligence development of pigeons; cryptochrome gene (CRY1) is related to the magnetic field sensing ability; dopamine D4 receptor (DRD4) is related to the homing ability of racing pigeons, and the stronger the homing ability, the better the flight ability; feather keratin (F-KER) affects the structure and strength of feathers, reducing air resistance to improve speed during flight; glutathione reductase (GSR) plays a key role in the directional ability of pigeons; lactate dehydrogenase A (LDHA) is an important factor in the lactate metabolism cycle, and when racing pigeons fly long distances, a large amount of lactic acid will accumulate in the body, and the faster the enzyme metabolism, the less likely it is to fatigue, so the endurance is better; 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; muscle regulation gene (MSTN) mainly controls the amount of muscle cell and muscle fiber bundle generation, and good muscle development is congenital, and the effect can be more prominent after training. Therefore, the typing results of the 8 genes can be used as a reference index for the breeding and selection of racing pigeons.

[0003] A racing pigeon gene detection kit has been developed in the prior art, but the typing results of individual genes can only qualitatively determine the potential of racing pigeons in certain aspects, and cannot accurately predict the overall ability of racing pigeons, which lacks effective guidance for the breeding of excellent racing pigeons. Therefore, it is urgent to develop a method for more comprehensive and accurate prediction of the comprehensive flight ability of racing pigeons. SUMMARY

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

[0005] The first object of the present application is to provide an application of SNP marker in evaluating flight ability of racing pigeon.

[0006] The second object of the present application is to provide an application of reagent for detecting the SNP marker in evaluating flight ability of racing pigeon.

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

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

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

[0010] The sixth object of the present application is to provide a method for predicting comprehensive flight ability of racing pigeon.

[0011] The seventh object of the present application is to provide a system for predicting comprehensive flight ability of racing pigeon.

[0012] In order to achieve the above objects, the present application is implemented by the following solutions:

[0013] The application of SNP marker in evaluating flight ability of racing pigeon, wherein the SNP marker is one of SNP sites 1-9 as follows:

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

[0015] SNP site 2 is located at base 153427839-153427840 of CRY1 gene on chromosome 1 of pet rock pigeon, and is AG, AA, AT or TT;

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

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

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

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

[0020] The SNP site 7 is located at the base 53833494 of the 5th chromosome of the rock pigeon, and is G or A.

[0021] The SNP site 8 is located at the base 27429922 of the 7th chromosome of the rock pigeon, and is C or T.

[0022] The SNP site 9 is located at the base 14737124 of the 8th chromosome of the rock pigeon, and is G or T.

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

[0024] The reagent for detecting the SNP marker is used for evaluating the flight ability of racing pigeons.

[0025] A primer composition for detecting the SNP marker comprises primer groups 1-3; wherein the primer group 1 comprises primers with nucleotide sequences as shown in SEQ ID NO. 1-2, nucleotide sequences as shown in SEQ ID NO. 4-5, and nucleotide sequences as shown in SEQ ID NO. 7-8; the primer group 2 comprises primers with nucleotide sequences as shown in SEQ ID NO. 10-11, nucleotide sequences as shown in SEQ ID NO. 13-14, and nucleotide sequences as shown in SEQ ID NO. 16-17; and the primer group 3 comprises primers with nucleotide sequences as shown in SEQ ID NO. 19-20, nucleotide sequences as shown in SEQ ID NO. 22-23, and nucleotide sequences as shown in SEQ ID NO. 25-26.

[0026] The primer with the nucleotide sequence as shown in SEQ ID NO. 1-2 is used for detecting the SNP site 1; the primer with the nucleotide sequence as shown in SEQ ID NO. 4-5 is used for detecting the SNP site 2; the primer with the nucleotide sequence as shown in SEQ ID NO. 7-8 is used for detecting the SNP site 3; the primer with the nucleotide sequence as shown in SEQ ID NO. 10-11 is used for detecting the SNP site 4; the primer with the nucleotide sequence as shown in SEQ ID NO. 13-14 is used for detecting the SNP site 5; the primer with the nucleotide sequence as shown in SEQ ID NO. 16-17 is used for detecting the SNP site 6; the primer with the nucleotide sequence as shown in SEQ ID NO. 19-20 is used for detecting the SNP site 7; the primer with the nucleotide sequence as shown in SEQ ID NO. 22-23 is used for detecting the SNP site 8; and the primer with the nucleotide sequence as shown in SEQ ID NO. 25-26 is used for detecting the SNP site 9.

[0027] A primer probe composition for detecting the SNP marker, comprising the primer composition and the probe.

[0028] Preferably, the probe comprises probe set 1-3; wherein the probe set 1 comprises the probe with the nucleotide sequence as shown in SEQ ID NO. 3, the nucleotide sequence as shown in SEQ ID NO. 6 and the nucleotide sequence as shown in SEQ ID NO. 9; the probe set 2 comprises the probe with the nucleotide sequence as shown in SEQ ID NO. 12, the nucleotide sequence as shown in SEQ ID NO. 15 and the nucleotide sequence as shown in SEQ ID NO. 18; and the probe set 3 comprises the probe with the nucleotide sequence as shown in SEQ ID NO. 21, the nucleotide sequence as shown in SEQ ID NO. 24 and the nucleotide sequence as shown in SEQ ID NO. 27.

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

[0030] More preferably, the 5' end of each probe in the probe set is provided with a different fluorescent reporter group, and the 3' end is provided with a fluorescent quencher group.

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

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

[0033] Further preferably, in the probe set 3, the 5' end of the probe with the nucleotide sequence as shown in SEQ ID NO. 21, the nucleotide sequence as shown in SEQ ID NO. 24 and the nucleotide sequence as shown in SEQ ID NO. 27 is respectively provided with FAM, ROX and HEX.

[0034] Further preferably, in the probe set 1, the 3' end of the probe with the nucleotide sequence as shown in SEQ ID NO. 3 and the nucleotide sequence as shown in SEQ ID NO. 6 is provided with BHQ1, and the 3' end of the probe with the 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 probe with the nucleotide sequence as shown in SEQ ID NO. 12 and the nucleotide sequence as shown in SEQ ID NO. 15 is provided with BHQ1, and the 3' end of the probe with the nucleotide sequence as shown in SEQ ID NO. 18 is provided with BHQ2.

[0036] Further preferably, in the probe set 3, the 3' end of the probe with the nucleotide sequence as shown in SEQ ID NO. 21 and the nucleotide sequence as shown in SEQ ID NO. 27 is provided with BHQ1, and the 3' end of the probe with the nucleotide sequence as shown in SEQ ID NO. 24 is provided with BHQ2.

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

[0038] Further preferably, the 13th base in the 5'→3' direction of the probe with the nucleotide sequence as shown in SEQ ID NO. 3 is provided with a locked nucleic acid modification.

[0039] Further preferably, the 13th base in the 5'→3' direction of the probe with the nucleotide sequence as shown in SEQ ID NO. 6 is provided with a locked nucleic acid modification.

[0040] Further preferably, the 13th base in the 5'→3' direction of the probe with the nucleotide sequence as shown in SEQ ID NO. 15 is provided with a locked nucleic acid modification.

[0041] Further preferably, the 13th base in the 5'→3' direction of the probe with the nucleotide sequence as shown in SEQ ID NO. 18 is provided with a locked nucleic acid modification.

[0042] Further preferably, the 12th base in the 5'→3' direction of the probe with the nucleotide sequence as shown in SEQ ID NO. 21 is provided with a locked nucleic acid modification.

[0043] Further preferably, the 19th base in the 5'→3' direction of the probe with the nucleotide sequence as shown in SEQ ID NO. 24 is provided with a locked nucleic acid modification.

[0044] Further preferably, the 12th base in the 5'→3' direction of the probe with the nucleotide sequence as shown in SEQ ID NO. 27 is provided with a locked nucleic acid modification.

[0045] A fluorescent quantitative PCR detection system for pigeon genotyping, comprising reaction systems 1-3; 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 system 1-3 further comprises a probe, and the probe comprises a probe set 1-3; wherein the probe set 1 comprises probes with nucleotide sequences as shown in SEQ ID NO. 3, SEQ ID NO. 6 and SEQ ID NO. 9; the probe set 2 comprises probes with nucleotide sequences as shown in SEQ ID NO. 12, SEQ ID NO. 15 and SEQ ID NO. 18; and the probe set 3 comprises probes with nucleotide sequences as shown in SEQ ID NO. 21, SEQ ID NO. 24 and SEQ ID NO. 27.

[0047] The reaction system 1 comprises the probe set 1, the reaction system 2 comprises the probe set 2, and the reaction system 3 comprises the probe set 3.

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

[0049] Preferably, the reaction system 1-3 further comprises a fluorescent quantitative PCR reaction reagent, including but not limited to a fluorescent quantitative PCR reaction enzyme and a fluorescent quantitative PCR reaction buffer.

[0050] Preferably, the procedure for performing fluorescent quantitative PCR amplification of the reaction system 1-3 comprises the following conditions: (1) constant temperature stage: 94-96℃, 0.5-1.5min, 1 cycle; (2) cycle stage: 94-96℃, 8-12s, 53-57℃, 28-32s, 68-72℃, 28-32s, 48-52 cycles; (3) constant temperature stage: 94-96℃, 28-32s, 43-47℃, 4-6min, 1 cycle; (4) melting stage: 45℃→90℃, 0.01-0.05℃ per 1s, continuously collecting fluorescence.

[0051] More preferably, the procedure for performing fluorescent quantitative PCR amplification of the reaction system 1-3 comprises the following conditions: (1) constant temperature stage: 95℃, 1min, 1 cycle; (2) cycle stage: 95℃, 10s, 55℃, 30s, 70℃, 30s, 50 cycles; (3) constant temperature stage: 95℃, 30s, 45℃, 5min, 1 cycle; (4) melting stage: 45℃→90℃, 0.03℃ per 1s, continuously collecting fluorescence.

[0052] A method for predicting the comprehensive flight ability of racing pigeons, comprising the following steps: detecting racing pigeons by using the fluorescent quantitative PCR detection system, genotyping and scoring the SNP marker according to the obtained melting curve data, and summing up the obtained scores to obtain the flight ability score of the racing pigeons; and determining the comprehensive flight ability of the racing pigeons according to the flight ability score in combination with a threshold value.

[0053] Preferably, the melting curve data comprises a peak type and an annealing temperature, and the criteria for the genotyping are shown in the following table:

[0054]

[0055]

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

[0057]

[0058]

[0059] The criteria for determining the comprehensive flight ability of the racing pigeons are as follows:

[0060] When the flight ability score is greater than or equal to 80, the racing pigeon is determined to have an excellent comprehensive flight ability;

[0061] When the flight ability score is greater than or equal to 70 but less than 80, the racing pigeon is determined to have an excellent comprehensive flight ability;

[0062] When the flight ability score is less than 70, the racing pigeon is determined to have an ordinary comprehensive flight 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 for acquiring the melting curve data of racing pigeons obtained by using the fluorescent quantitative PCR detection system; the storage module is used for storing the melting curve data in the data acquisition module; the analysis module takes the melting curve data acquired by the data acquisition module as input data, determines the comprehensive flight ability of the racing pigeons by executing any of the methods for predicting the comprehensive flight ability of racing pigeons, and the output module is used for outputting the determination result of the comprehensive flight ability obtained by the analysis module.

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

[0065] The application only needs 3 fluorescent quantitative PCR reaction systems to realize the genotyping detection of 8 genes and 9 SNP sites, and the melting curve data is directly converted into genotypes through a program, so that the result interpretation is simple and fast. In addition, according to the genotype results, an evaluation method for quantifying the comprehensive flight ability of racing pigeons is further established after scoring, so that the flight ability of racing pigeons is more accurately and systematically evaluated and predicted, and the AUC is as high as more than 80%. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1 The schematic diagram is set for the fluorescence detection channel of the PCR detection project.

[0067] Figure 2 The melting curve is for reaction system 1.

[0068] Figure 3 The melting curve is for reaction system 2.

[0069] Figure 4 The melting curve is for reaction system 3.

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

[0071] The application will be further described in detail below in combination with the drawings and specific embodiments of the specification. The embodiments are only used to explain the application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

[0072] Example 1: Detection method of racing pigeon genotyping

[0073] 1. Racing pigeon feather follicle genomic DNA extraction:

[0074] The feathers of the racing pigeons to be tested were collected, and the hair follicle part (length 2-3 cm) was cut and subjected to proteinase K digestion at 56℃ for 2h. The digested sample was subjected to commercial kit extraction to obtain genomic DNA as a template for subsequent detection.

[0075] 2. Detection of racing pigeon genotyping

[0076] (1) Detection target

[0077] The present application takes 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 dehydrogenase gene A (LDHA), low density lipoprotein receptor related protein 8 (LRP8) and muscle generation regulation gene (MSTN)) as detection targets, and uses fluorescence quantitative PCR (TaqMan probe-melting curve method) for determination of typing. The information of SNP sites is shown in Table 1, and the sequences of primers and probes used are shown in Table 2.

[0078] Table 1 Related information of 9 SNP sites

[0079]

[0080]

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

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

[0083]

[0084]

[0085] Note: The base marked in the probe sequence of SNP site 2 in the table is The base marked in the probe sequence of SNP site 2 in the table is

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

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

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

[0089] Table 3: Fluorescence quantitative PCR detection reaction system

[0090]

[0091] Each sample to be tested in 200 μL PCR tube prepared above 3 reaction system, each reaction system total volume of 20 μL, after mixing thoroughly, centrifugation after standby. Operation should avoid the generation of bubbles and ensure that the PCR tube wall no liquid adhesion.

[0092] (3) PCR detection

[0093] The prepared reaction system is transferred to the sample groove of the macro stone PCR instrument, and the sample order is recorded. According to the information shown in Figure 1 , set the fluorescence detection channel of the PCR detection project, select FAM channel to detect SNP site 1 located in CASK gene, SNP site 4 located in DRD4b gene and SNP site 7 located in LDHA gene, select HEX channel to detect SNP site 2 located in CRY1 gene, SNP site 5 located in F-KER gene and SNP site 9 located in LRP8 gene, select ROX channel to detect SNP site 3 located in DRD4a gene, SNP site 6 located in GSR gene and SNP site 8 located in MSTN gene.

[0094] According to the information shown in Table 4, set the PCR reaction program, the temperature of the hot cover is 105℃, and the liquid volume is 20 μL. After amplification, discard the PCR tube. Strictly prohibit opening the PCR tube to prevent contamination.

[0095] In the matching "SLAN full-automatic medical PCR analysis system 8.2.2", select experimental analysis, select the experimental hole where the sample is placed in the hole plate selector, and select "1 (FAM channel), 2 (HEX channel), 3 (ROX channel)" in the channel selection to analyze the melting curve of the sample to be tested.

[0096] Table 4 Fluorescence quantitative PCR detection reaction program

[0097]

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

[0099] 1) Negative quality control (H2O) has no melting curve peak; 2) Positive quality control (identified by Sanger sequencing) has target melting curve peak.

[0100] The above two requirements must be met at the same time in the same detection, otherwise the detection is invalid and needs to be retested.

[0101] The abnormal result judgment standard and processing method of fluorescence quantitative PCR detection are as follows:

[0102] 1) If one of the detection sites of a certain sample to be tested has no typical melting peak, it indicates that the detection signal of the site is abnormal; 2) If three detection sites of a certain sample to be tested have no typical melting peak, it indicates that the detection signal of the sample is abnormal.

[0103] If any one of the above two items exists in the sample to be tested, it is considered that the detection result is invalid, and the sample to be tested needs to be retested or reextracted for nucleic acid detection.

[0104] (4) Genotype determination of SNP sites

[0105] According to the peak type and annealing temperature (Tm) of the obtained melting curve, the genotype of each SNP site is determined according to the information shown in Table 5.

[0106] Table 5 Genotype determination criteria of each SNP site

[0107]

[0108]

[0109] The pigeon feather sample with genotypes of SNP site 1 AA, SNP site 2 AGTT, and SNP site 3 CC is identified by Sanger sequencing, and its melting curve peak type diagram is shown in Figure 2 The pigeon feather sample with genotypes of SNP site 4 CC, SNP site 5 GT, and SNP site 6 CT is identified by Sanger sequencing, and its melting curve peak type diagram is shown in Figure 3 The pigeon feather sample with genotypes of SNP site 7 AG, SNP site 8 CC, and SNP site 9 TT is identified by Sanger sequencing, and its melting curve peak type diagram is shown in Figure 4 The melting curve peak type diagrams 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 application can accurately and quickly determine the genotype corresponding to each SNP site according to the peak type and Tm of the melting curve.

[0111] Example 2 A method for predicting the comprehensive flight ability of racing pigeons

[0112] 1. Genotype determination and scoring

[0113] The sample to be tested is detected by the method of Example 1, the genotype of each SNP site is determined according to the peak type and annealing temperature (Tm) of the obtained melting curve, and the genotype determination result is recorded. Subsequently, the standard shown in Table 7 is combined to score, and the scoring result is recorded.

[0114] Table 7 SNP site genotype scoring criteria

[0115]

[0116]

[0117] 2. Method for determining comprehensive flight ability

[0118] The sum of the scores of the 8 scoring items (i.e. items 1-8 in Table 6) is calculated, i.e. the flight ability score of the pigeon to be tested is obtained, and the comprehensive flight ability of the racing pigeon is determined according to the following criteria:

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

[0120] When 70 points ≤ flight ability score < 80 points, the racing pigeon is determined to have good comprehensive flight ability;

[0121] When the flight ability score is < 70 points, the racing pigeon is determined to have ordinary comprehensive flight ability.

[0122] Evaluation of a method for predicting the comprehensive flight ability of a racing pigeon

[0123] 1. Prediction of comprehensive flight ability

[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, and the results of the genotypes of the SNP sites and the flight ability scores are shown in Table 8.

[0125] 2. Flight ability test

[0126] In this example, the flight ability of the above-mentioned 158 racing pigeons was also tested, and the test method used a single distance release scheme: in Xinxiang, Henan, the racing pigeons were taken to a unified release site to perform a 500 km middle-distance race, and the total time for each racing pigeon to fly back was recorded to calculate the flight performance (speed: m / min). Racing pigeons with high speed usually mean that they have faster flight speed and better flight ability. In this test, a total of 110 racing pigeons successfully returned home, and the ranking of the top 79 racing pigeons was calculated according to the flight performance, and the results are shown in Table 8.

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

[0128]

[0129]

[0130]

[0131]

[0132]

[0133] 3. Accuracy evaluation 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 and those that failed to successfully return home were defined as "poor". In combination with the flight ability scores, an 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] The ROC curve is shown in Figure 5 , and the AUC is more than 80%. This indicates that the prediction method based on the 9 SNP sites has a good prediction effect on the comprehensive flight ability of racing pigeons, high accuracy, and can achieve precise evaluation of the flight ability of racing pigeons.

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

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

[0138] The data acquisition module is used to acquire the melting curve data obtained by fluorescence 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 takes the melting curve data acquired by the data acquisition module as input data, executes the method shown in Example 2, and judges the comprehensive flight ability of racing pigeons.

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

[0142] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. For those skilled in the art, based on the above description and ideas, other different forms of changes or modifications can also be made, which are not required or impossible to exhaust all embodiments. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. Use of SNP markers for evaluating the flying capacity of racing pigeons, characterized in that, The SNP marker is SNP site 1-9 as follows: SNP site 1 is located at base 98990834 of the CASK gene on the rock pigeon chromosome 1, and is G or A; SNP site 2 is located at base 153427839-153427840 of the CRY1 gene on the rock pigeon chromosome 1, and is AG, AA, AT or TT; SNP site 3 is located at base 68012131 of the DRD4a gene on the rock pigeon chromosome 5, and is C or T; SNP site 4 is located at base 68011633 of the DRD4b gene on the rock pigeon chromosome 5, and is C or T; SNP site 5 is located at base 5545394 of the F-KER gene on the rock pigeon chromosome 23, and is T or G; SNP site 6 is located at base 77827982 of the GSR gene on the rock pigeon chromosome 4, and is C or T; SNP site 7 is located at base 53833494 of the LDHA gene on the rock pigeon chromosome 5, and is G or A; SNP site 8 is located at base 27429922 of the MSTN gene on the rock pigeon chromosome 7, and is C or T; SNP site 9 is located at base 14737124 of the LRP8 gene on the rock pigeon chromosome 8, and is G or T; The rock pigeon chromosome 1 in NCBI is numbered as NC_088602.1; The rock pigeon chromosome 4 in NCBI is numbered as NC_088605.1; The rock pigeon chromosome 5 in NCBI is numbered as NC_088606.1; The rock pigeon chromosome 7 in NCBI is numbered as NC_088608.1; The rock pigeon chromosome 8 in NCBI is numbered as NC_088609.1; The rock pigeon chromosome 23 in NCBI is numbered as NC_088624.

1.

2. Use of a primer composition for detecting the SNP marker as claimed in claim 1 in the evaluation of the flying capacity of racing pigeons, characterized in that, The primer composition comprises primer groups 1-3; The primer group 1 comprises primers with nucleotide sequences as shown in SEQ ID NO. 1-2, nucleotide sequences as shown in SEQ ID NO. 4-5, and nucleotide sequences as shown in SEQ ID NO. 7-8; The primer group 2 comprises primers with nucleotide sequences as shown in SEQ ID NO. 10-11, nucleotide sequences as shown in SEQ ID NO. 13-14, and nucleotide sequences as shown in SEQ ID NO. 16-17; The primer group 3 comprises primers with nucleotide sequences as shown in SEQ ID NO. 19-20, nucleotide sequences as shown in SEQ ID NO. 22-23, and nucleotide sequences as shown in SEQ ID NO. 25-26.

3. A primer probe composition for detecting the SNP marker as claimed in claim 1, characterized by, The primer composition comprises primer groups 1-3; 4. The primer probe composition of claim 3, wherein, The primer group 1 comprises primers with nucleotide sequences as shown in SEQ ID NO. 1-2, nucleotide sequences as shown in SEQ ID NO. 4-5, and nucleotide sequences as shown in SEQ ID NO. 7-8; The primer group 2 comprises primers with nucleotide sequences as shown in SEQ ID NO. 10-11, nucleotide sequences as shown in SEQ ID NO. 13-14, and nucleotide sequences as shown in SEQ ID NO. 16-17; The primer group 3 comprises primers with nucleotide sequences as shown in SEQ ID NO. 19-20, nucleotide sequences as shown in SEQ ID NO. 22-23, and nucleotide sequences as shown in SEQ ID NO. 25-26. The probe set 2 comprises 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; The probe set 3 comprises 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.

5. The primer probe composition of claim 4, wherein, The 5' end of each probe in each probe set is provided with a different fluorescent reporter group, and the 3' end is provided with a fluorescent quencher group.

6. A fluorescent quantitative PCR detection system for genotyping racing pigeons, characterized in that, The reaction system 1 comprises the primer set 1 as described in claim 2, the reaction system 2 comprises the primer set 2 as described in claim 2, and the reaction system 3 comprises the primer set 3 as described in claim 2.

7. The quantitative PCR assay system according to claim 6, wherein The probe as described in claim 3 or claim 4 is also included.

8. A method of predicting the overall flight performance of a racing pigeon, characterized in that, The method comprises the following steps: The racing pigeon is detected by the fluorescent quantitative PCR detection system as described in claim 7, the SNP marker is genotyped and scored according to the obtained melting curve data, the sum of the obtained scores is the flight ability score of the racing pigeon, and the comprehensive flight ability of the racing pigeon is determined according to the flight ability score combined with a threshold value.

9. A system for predicting the overall flight capabilities of a racing pigeon, characterized in that, The method comprises a data acquisition module, a storage module, an analysis module and an output module. The data acquisition module is used to acquire the melting curve data of the racing pigeon obtained by the fluorescent quantitative PCR detection system as described in claim 7; The storage module is used to store the melting curve data in the data acquisition module; The analysis module takes the melting curve data acquired by the data acquisition module as input data, and determines the comprehensive flight ability of the racing pigeon by executing the method as described in claim 8; The output module is used to output the determination result of the comprehensive flight ability obtained by the analysis module.

Images