Kit for pigeon individual identification and paternity identification typing and application
By introducing 16 new STR loci and the sex identification gene CHD into pigeons, and combining multiplex PCR and capillary electrophoresis, the shortcomings of pigeon STR typing technology have been overcome, enabling efficient and accurate identification of individual pigeons and paternity, and supporting the superior breeding of racing pigeon breeds.
Patent Information
- Application Number
- CN202511423141.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2025-11-18
AI Technical Summary
Existing STR typing technology for pigeons suffers from problems such as insufficient number of loci, high typing difficulty, and high typing error rate, which makes it difficult to identify individual pigeons and determine parentage.
Using 16 novel tetranucleotide and pentanucleotide repeat STR sites, combined with multiplex PCR and capillary electrophoresis, specific primer combinations were designed for pigeon individual identification and paternity testing, with the sex identification gene CHD used for auxiliary identification.
It achieves accurate identification of individual pigeons and reliable identification of kinship, provides stable identification of genetic advantages, and supports the breeding of superior racing pigeon breeds.
Smart Images
Figure CN120967009A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the technical field of gene detection, specifically to a kit and its application for pigeon individual identification and paternity typing. Background Technology
[0002] Pigeons, as birds with extremely high competitive and economic value, have a long history of domestication and racing worldwide. With their exceptional homing ability, endurance in flight, and precise directional awareness, they have become core participants in the world of pigeon racing. Related competitions have not only formed a mature industrial system but have also spawned a vast market for breeding, training, and trading. In the pigeon racing industry, breeding superior breeds is key to improving racing performance. Top racing pigeons often possess stable genetic advantages, and their bloodline purity and kinship directly affect the competitive potential of their offspring. Therefore, "pedigree" has become one of the core indicators for measuring the value of racing pigeons.
[0003] Currently, individual identification and paternity testing in pigeons mainly rely on two types of technologies: First, physical identification systems such as leg band numbers, which are easily tampered with or counterfeited; or feather tags, which are easily affected by feather replacement and cannot achieve lifelong identity binding. Second, molecular marker technologies, the most widely used of which is microsatellite (STR) genotyping, which requires the combined detection of multiple STR loci (usually 13-19) and has relatively high technical requirements. Besides microsatellite (STR) genotyping, SNP genotyping is also widely used, generally utilizing gene chips to perform genotyping detection on dozens or hundreds of SNP loci, but this is relatively expensive.
[0004] Although STR typing technology has been successfully applied in model organisms such as humans and mice, STR detection in pigeons still faces three major technical barriers: (1) Insufficient number of sites: Currently, only more than 20 STR sites have been reported in the literature, which makes it difficult to distinguish complex individuals; (2) Nearly half of the existing STR sites are dinucleotide repeat sites: On the one hand, the inherent defects of dinucleotide repeat sites (the presence of severe stutter peaks) greatly increase the difficulty of typing; on the other hand, it is relatively difficult to produce the corresponding allele ladder for dinucleotide repeat sites; (3) There is crosstalk between fluorescence channels in capillary electrophoresis, which easily leads to typing errors, especially the pull-up peak caused by dinucleotide repeat sites.
[0005] Therefore, in order to make up for the shortcomings of pigeon individual identification and paternity identification technologies, it is urgent to develop new STR loci. Summary of the Invention
[0006] To overcome the aforementioned defects and shortcomings in the existing technology, the present invention provides a kit and its application for pigeon individual identification and paternity testing.
[0007] The first objective of this invention is to provide the application of STR molecular markers in pigeon individual identification and paternity typing.
[0008] The second objective of this invention is to provide the application of the above-mentioned STR molecular marker detection reagent in pigeon individual identification and paternity typing.
[0009] A third objective of this invention is to provide the application of the above-mentioned STR molecular marker detection reagent in the preparation of products for pigeon individual identification and paternity typing.
[0010] The fourth objective of this invention is to provide a kit for pigeon individual identification and paternity testing.
[0011] The fifth objective of this invention is to provide a method for pigeon individual identification and paternity testing.
[0012] This invention claims protection for the following: The application of STR molecular markers in pigeon individual identification and paternity identification typing, wherein the STR molecular markers include PGT8, PGP11, PGP6, PGP10, PGP12, PGP3, PGP5, PGT6, PGT4, PGT5, PGP4, PGP7, PGP9, PGT1, PGP8 and PGP1; The repeating unit of the STR molecular marker PGT8 is ATGG, located at positions 10208638 to 10208791 of the sequence shown in NC_088620.1; The repeating unit of the STR molecular marker PGP11 is TATTC, located at positions 58224420 to 58224756 of the sequence shown in NC_088606.1; The repeating unit of the STR molecular marker PGP6 is AAAGA, located at positions 12839178 to 12839632 of the sequence shown in NC_088608.1; The repeating unit of the STR molecular marker PGP10 is TTCTA, located at positions 10719524 to 10719680 of the sequence shown in NC_088609.1; The repeating unit of the STR molecular marker PGP12 is TTCTA, located at positions 21428537 to 21428787 of the sequence shown in NC_088605.1; The repeating unit of the STR molecular marker PGP3 is TATTC, located at positions 11513596 to 11513945 of the sequence shown in NC_088606.1; The repeating unit of the STR molecular marker PGP5 is TATTC, located at positions 37221956 to 37222449 of the sequence shown in NC_088607.1; The repeating unit of the STR molecular marker PGT6 is AGAT, located at positions 3019445 to 3019560 of the sequence shown in NC_088607.1; The repeating unit of the STR molecular marker PGT4 is GATG, located at positions 33835866 to 33836050 of the sequence shown in NC_088606.1; The repeating unit of the STR molecular marker PGT5 is TCTA, located at positions 8810287 to 8810546 of the sequence shown in NC_088607.1; The repeating unit of the STR molecular marker PGP4 is TTCTA, located at positions 46995247 to 46995602 of the sequence shown in NC_088606.1; The repeating unit of the STR molecular marker PGP7 is TTCTA, located at positions 30378420 to 30378868 in the sequence shown in NC_088608.1; The repeating unit of the STR molecular marker PGP9 is TATTC, located at positions 30402000 to 30402180 of the sequence shown in NC_088607.1; The repeating unit of the STR molecular marker PGT1 is GATG, located at positions 1242898 to 1243148 of the sequence shown in NC_088605.1; The repeating unit of the STR molecular marker PGP8 is TCTAT, located at positions 73256–73601 of the sequence shown in NC_088610.1; The repeating unit of the STR molecular marker PGP1 is ATTCT, located at positions 260969–261457 of the sequence shown in NC_088602.1.
[0013] The application of the above-mentioned STR molecular marker detection reagents in pigeon individual identification and paternity typing.
[0014] The above-mentioned STR molecular marker detection reagents are used in the preparation of products for pigeon individual identification and paternity typing.
[0015] As one feasible approach, the detection reagent for the STR molecular marker PGT8 is a primer with a nucleotide sequence as shown in SEQ ID NO: 1-2; The detection reagent for the STR molecular marker PGP11 is a primer with a nucleotide sequence as shown in SEQ ID NO: 5-6; The detection reagent for the STR molecular marker PGP6 is a primer with a nucleotide sequence as shown in SEQ ID NO: 7-8; The detection reagent for the STR molecular marker PGP10 is a primer with a nucleotide sequence as shown in SEQ ID NO: 9-10; The detection reagent for the STR molecular marker PGP12 is a primer with a nucleotide sequence as shown in SEQ ID NO: 11-12; The detection reagent for the STR molecular marker PGP3 is a primer with a nucleotide sequence as shown in SEQ ID NO: 13-14; The detection reagent for the STR molecular marker PGP5 is a primer with a nucleotide sequence as shown in SEQ ID NO: 15-16; The detection reagent for the STR molecular marker PGT6 is a primer with a nucleotide sequence as shown in SEQ ID NO: 17-18; The detection reagent for the STR molecular marker PGT4 is a primer with a nucleotide sequence as shown in SEQ ID NO: 19-20; The detection reagent for the STR molecular marker PGT5 is a primer with a nucleotide sequence as shown in SEQ ID NO: 21-22; The detection reagent for the STR molecular marker PGP4 is a primer with a nucleotide sequence as shown in SEQ ID NO: 23-24; The detection reagent for the STR molecular marker PGP7 is a primer with a nucleotide sequence as shown in SEQ ID NO: 25-26; The detection reagent for the STR molecular marker PGP9 is a primer with a nucleotide sequence as shown in SEQ ID NO: 27-28; The detection reagent for the STR molecular marker PGT1 is a primer with a nucleotide sequence as shown in SEQ ID NO: 29-30; The detection reagent for the STR molecular marker PGP8 is a primer with a nucleotide sequence as shown in SEQ ID NO: 31-32; The detection reagent for the STR molecular marker PGP1 is a primer with a nucleotide sequence as shown in SEQ ID NO: 33-34.
[0016] Preferably, the primer composition further comprises a detection primer for the sex identification gene CHD, wherein the detection primer for the sex identification gene CHD is a primer with a nucleotide sequence as shown in SEQ ID NO: 3-4.
[0017] More preferably, the primer composition comprises primer sets 1 to 4; Primer set 1 contains primers with nucleotide sequences as shown in SEQ ID NO: 1-8; Primer set 2 contains primers with nucleotide sequences as shown in SEQ ID NO: 9-16; Primer set 3 contains primers with nucleotide sequences as shown in SEQ ID NO: 17-26; Primer set 4 contains primers with nucleotide sequences as shown in SEQ ID NO: 27-34; In primer sets 1 to 4, primers within the same set are labeled with the same fluorescent marker, while primers from different sets are labeled with different fluorescent markers.
[0018] A kit for pigeon individual identification and paternity testing includes detection reagents for the aforementioned STR molecular markers.
[0019] Preferably, the kit further includes detection primers for the sex identification gene CHD, wherein the detection primers for the sex identification gene CHD are primers with nucleotide sequences as shown in SEQ ID NO: 3-4.
[0020] Preferably, the kit further comprises multiplex PCR reaction reagents.
[0021] A method for pigeon individual identification and paternity testing includes the following steps: S1. Extract genomic DNA from the pigeons to be tested; S2. Perform multiplex PCR amplification on the genomic DNA obtained in step S1 using the kit described above; S3. Perform capillary electrophoresis and data analysis on the PCR products.
[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a kit and its application for pigeon individual identification and paternity typing. The invention provides 16 new STR loci for pigeon individual identification and paternity typing, all of which are tetranucleotide and pentanucleotide repeat sites. The kit based on these new STR loci provides clear, simple, and accurate typing results, strong individual identification ability, and reliable paternity identification, thus ensuring the breeding of superior racing pigeon breeds and the identification of paternity. Attached Figure Description
[0023] Figure 1 This is a capillary electrophoresis analysis result of 16 new STR loci and CHD sex identification loci from one of the pigeon samples in Example 2.
[0024] Figure 2 This is a capillary electrophoresis analysis result of 14 new STR loci and CHD sex identification loci of a pigeon (father) from one family in Example 4.
[0025] Figure 3 This is a capillary electrophoresis analysis result of 14 new STR loci and CHD sex identification loci of a female pigeon from one family in Example 4.
[0026] Figure 4 This is a capillary electrophoresis analysis result of 14 new STR loci and CHD sex identification loci of pigeons (offspring) from one family in Example 4.
[0027] Figure 5 The image shows the capillary electrophoresis results of 14 STR sites and CHD sex identification sites when using 1 ng of pigeon genomic DNA as a template.
[0028] Figure 6 The image shows the capillary electrophoresis results of 14 STR sites and CHD sex identification sites using 0.1 ng of pigeon genomic DNA as a template.
[0029] Figure 7 The image shows the capillary electrophoresis results of 14 STR sites and CHD sex identification sites using 0.01 ng of pigeon genomic DNA as a template. Detailed Implementation
[0030] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.
[0031] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.
[0032] Example 1: Screening and determination of STR loci for individual pigeon identification and paternity testing New STR loci meeting the following criteria were identified by screening the pigeon reference genome sequence using bioinformatics methods: (1) All are tetranucleotide and pentanucleotide repeat sites; (2) The number of repetitions is between 12 and 30; Sixteen new STR loci were ultimately selected for primer design, and the information of the sixteen new STR loci is shown in Table 1.
[0033] Table 1 Information on 16 new STR loci
[0034]
[0035] Example 2 Genotyping based on multiplex fluorescent PCR capillary electrophoresis I. Experimental Methods Feathers were collected from 1050 unrelated racing pigeons. Feather follicles (2-3 cm in length) were cut and digested with lysis buffer and proteinase K at 56°C for 2 h. Genomic DNA was extracted from the digested samples using a kit.
[0036] Using the extracted genomic DNA as a template, the STR sites in Table 1 of Example 1 were combined and primers were designed for multiplex PCR amplification (Table 2). In addition, a sex identification site CHD was added for sex determination.
[0037] Table 2 Primer sequences for each STR locus
[0038]
[0039] The multiplex PCR amplification system is shown in Table 3. The primer mixture is obtained by mixing the primers in Table 2. The specific concentrations and amounts of each primer are shown in Table 4.
[0040] Table 3 Multiplex PCR amplification system
[0041] Table 4 Primer concentrations and dosages
[0042]
[0043] The multiplex PCR reaction procedure is shown in Table 5.
[0044] Table 5 Multiplex PCR reaction procedure
[0045] Multiplex PCR amplification products were analyzed using an ABI 3500 DNA analyzer, with LIZ500 used as an internal standard to determine fragment sizes. The analysis results were imported into Genemapper 6 software to read the fragment size of each STR locus. The fragment sizes and occurrence frequencies of STR loci from 1050 unrelated pigeons were statistically analyzed and calculated.
[0046] In order to determine the allele type, PCR amplification of single STR loci was performed on some pigeons, and the amplification products were subjected to Sanger sequencing to identify the number of repeats of short tandem repeat sequences corresponding to the size of each fragment at the 16 STR loci.
[0047] II. Experimental Results The actual fragment size and occurrence frequency of STR loci in 1050 unrelated pigeons are statistically analyzed and calculated as shown in Tables 6 and 7 (the fragment size values in these tables are obtained by rounding the values read by the software).
[0048] Table 6. Statistics on STR locus fragment size, occurrence frequency, and occurrence number of over 1000 unrelated pigeons.
[0049]
[0050]
[0051]
[0052]
[0053]
[0054] Table 7. Actual fragment size range of 16 new STR loci and sex identification loci
[0055] Taking the determination of the allele type of one pigeon as an example, the capillary electrophoretic analysis of its 16 new STR loci and CHD sex identification loci is as follows: Figure 1 As shown, the pigeon's PGP11 locus is homozygous, containing only a 321.85 bp fragment. Sanger sequencing revealed that it contains 12 TATTC repeats, therefore, the allele type is 12.
[0056] Based on the Sanger sequencing results, after converting fragment size into allele type, the allele types and frequencies of the 16 new STR loci are shown in Table 8.
[0057] Table 8 Allele types and frequencies at STR loci
[0058]
[0059]
[0060]
[0061] Example 3: Discriminative power, exclusion probability, and cumulative discriminative power and cumulative exclusion probability of STR loci I. Experimental Methods Based on the allele types and corresponding frequencies of the 16 new STR loci in Example 2, the matching probability (Pm), the discrimination power of a single locus (Pd), the cumulative discrimination power (CPd), the exclusion probability (PE), and the combined exclusion probability (CPE) were calculated.
[0062] According to the literature report (PMID: 18041042), the formula for calculating the matching probability (Pm) is:
[0063] In the formula, P K Represents genotype frequency.
[0064] The formula for calculating the discrimination power (Pd) of a single site is: Pd = 1 - Pm; The formula for calculating cumulative discrimination power (CPd) is:
[0065] The formula for calculating the probability of exclusion (PE) is: PE = H 2 (1-(1-H)H 2 ), where H is the degree of heterozygosity.
[0066] Formula for calculating the joint exclusion probability (CPE):
[0067] II. Experimental Results The discriminative power and exclusion probability of the 16 new STR loci, as well as the cumulative discriminative power and cumulative exclusion probability, are shown in Table 9. The results show that the cumulative discriminative power of the 16 new STR loci of this invention is 0.999999999999999999999999908, and the joint exclusion probability is 0.999999999720944.
[0068] Table 9. Discriminative power and exclusion probability of 16 new STR loci, and cumulative discriminative power and cumulative exclusion probability.
[0069] Example 4: Family Phylogenetic Relationship Identification I. Experimental Methods The STR loci for pedigree identification were determined based on the actual fragment size distribution in Table 7 of Example 2, combined with fluorescence channel information, and it was required that the fragment sizes of the loci within each fluorescence channel did not overlap. Since PGP3 overlapped with both the loci above and below it, and PGT5 overlapped with PGP4, PGP3 and PGT5 loci, which overlapped with the adjacent STR loci, were not included in the pedigree identification experiment. Calculations showed that the cumulative discriminative power of the 14 new STR loci after removing the two loci was still 0.9999999999999999999999670832, and the joint exclusion probability was 0.99999999530644.
[0070] The effectiveness of 14 new STR loci in paternity testing was verified through pedigree identification. Fourteen STR loci were selected and tested on pigeons (father, mother, and offspring) from one family. The testing method followed that in Example 2. The paternity index (PI) and cumulative paternity index (CPI) were calculated based on the capillary electrophoresis analysis results. The calculation formulas are as follows: Paternity Index (PI): PI = X / Y, where X is the probability that the assumed parents are the actual parents, and Y is the probability that a random individual is a parent. Cumulative Parentage Index (CPI): CPI is the product of the individual point PIs.
[0071] II. Experimental Results The capillary electrophoresis analysis results of pigeons (father, mother, and offspring) from one family are as follows: Figures 2-4 As shown in Table 10, the fragment sizes at 14 sites of the father, mother, and child were statistically analyzed based on the capillary electrophoresis results.
[0072] Table 10. Segment size statistics of 14 STR loci and 1 sex locus in pigeons (father, mother, and offspring) from one family.
[0073] Based on the above data and the allele frequencies in Table 6, the cumulative paternity index (CPI) in this family can be calculated to be 130431585093012000 (>10000). Therefore, the paternity probability is 99.99999999999999% (>99.99%), confirming their biological kinship.
[0074] Although PGP3 and PGT5 loci cannot be detected simultaneously with the other 14 STR loci in a single tube, these two loci can be detected additionally for supplementary evaluation. However, since the discriminative power of using 14 STR loci is already high enough, this invention does not require the additional detection of these two loci.
[0075] Example 5 Sensitivity Experiment I. Experimental Methods To determine the sensitivity of the detection system, multiplex PCR amplification and ABI3500 fragment analysis were performed using 1 ng, 0.1 ng, and 0.01 ng pigeon genomic DNA as templates, respectively, in accordance with Example 2.
[0076] II. Experimental Results The results are as follows Figures 5-7 As shown, the results indicate that 14 STR loci and CHD sex loci can be stably detected with only 0.1 ng of pigeon genomic DNA.
[0077] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. The application of STR molecular markers in pigeon individual identification and paternity typing, characterized in that, The STR molecular markers include PGT8, PGP11, PGP6, PGP10, PGP12, PGP3, PGP5, PGT6, PGT4, PGT5, PGP4, PGP7, PGP9, PGT1, PGP8 and PGP1; The repeating unit of the STR molecular marker PGT8 is ATGG; The repeating unit of the STR molecular marker PGP11 is TATTC; The repeating unit of the STR molecular marker PGP6 is AAAGA; The repeating unit of the STR molecular marker PGP10 is TTCTA; The repeating unit of the STR molecular marker PGP12 is TTCTA; The repeating unit of the STR molecular marker PGP3 is TATTC; The repeating unit of the STR molecular marker PGP5 is TATTC; The repeating unit of the STR molecular marker PGT6 is AGAT; The repeating unit of the STR molecular marker PGT4 is GATG; The repeating unit of the STR molecular marker PGT5 is TCTA; The repeating unit of the STR molecular marker PGP4 is TTCTA; The repeating unit of the STR molecular marker PGP7 is TTCTA; The repeating unit of the STR molecular marker PGP9 is TATTC; The repeating unit of the STR molecular marker PGT1 is GATG; The repeating unit of the STR molecular marker PGP8 is TCTAT; The repeating unit of the STR molecular marker PGP1 is ATTCT.
2. The application of the STR molecular marker detection reagent described in claim 1 in pigeon individual identification and paternity typing.
3. The application of the STR molecular marker detection reagent described in claim 1 in the preparation of products for pigeon individual identification and paternity typing.
4. The application according to claim 2 or 3, characterized in that, The detection reagent for the STR molecular marker PGT8 is a primer with a nucleotide sequence as shown in SEQ ID NO: 1-2; The detection reagent for the STR molecular marker PGP11 is a primer with a nucleotide sequence as shown in SEQ ID NO: 5-6; The detection reagent for the STR molecular marker PGP6 is a primer with a nucleotide sequence as shown in SEQ ID NO: 7-8; The detection reagent for the STR molecular marker PGP10 is a primer with a nucleotide sequence as shown in SEQ ID NO: 9-10; The detection reagent for the STR molecular marker PGP12 is a primer with a nucleotide sequence as shown in SEQ ID NO: 11-12; The detection reagent for the STR molecular marker PGP3 is a primer with a nucleotide sequence as shown in SEQ ID NO: 13-14; The detection reagent for the STR molecular marker PGP5 is a primer with a nucleotide sequence as shown in SEQ ID NO: 15-16; The detection reagent for the STR molecular marker PGT6 is a primer with a nucleotide sequence as shown in SEQ ID NO: 17-18; The detection reagent for the STR molecular marker PGT4 is a primer with a nucleotide sequence as shown in SEQ ID NO: 19-20; The detection reagent for the STR molecular marker PGT5 is a primer with a nucleotide sequence as shown in SEQ ID NO: 21-22; The detection reagent for the STR molecular marker PGP4 is a primer with a nucleotide sequence as shown in SEQ ID NO: 23-24; The detection reagent for the STR molecular marker PGP7 is a primer with a nucleotide sequence as shown in SEQ ID NO: 25-26; The detection reagent for the STR molecular marker PGP9 is a primer with a nucleotide sequence as shown in SEQ ID NO: 27-28; The detection reagent for the STR molecular marker PGT1 is a primer with a nucleotide sequence as shown in SEQ ID NO: 29-30; The detection reagent for the STR molecular marker PGP8 is a primer with a nucleotide sequence as shown in SEQ ID NO: 31-32; The detection reagent for the STR molecular marker PGP1 is a primer with a nucleotide sequence as shown in SEQ ID NO: 33-34.
5. The application according to claim 4, characterized in that, The detection reagent also includes detection primers for the sex identification gene CHD, wherein the detection primers for the sex identification gene CHD are primers with nucleotide sequences as shown in SEQ ID NO: 3-4.
6. The application according to claim 5, characterized in that, The detection reagent includes primer sets 1 to 4; Primer set 1 contains primers with nucleotide sequences as shown in SEQ ID NO: 1-8; Primer set 2 contains primers with nucleotide sequences as shown in SEQ ID NO: 9-16; Primer set 3 contains primers with nucleotide sequences as shown in SEQ ID NO: 17-26; Primer set 4 contains primers with nucleotide sequences as shown in SEQ ID NO: 27-34; In primer sets 1 to 4, primers within the same set are labeled with the same fluorescent marker, while primers from different sets are labeled with different fluorescent markers.
7. A kit for pigeon individual identification and paternity testing, characterized in that, A detection reagent containing the STR molecular marker as described in claim 1.
8. The reagent kit according to claim 7, characterized in that, The kit also contains primers for detecting the sex-identifying gene CHD.
9. The reagent kit according to claim 8, characterized in that, The kit also includes multiplex PCR reaction reagents.
10. A method for pigeon individual identification and paternity testing, characterized in that, Includes the following steps: S1. Extract genomic DNA from the pigeons to be tested; S2. Perform multiplex PCR amplification on the genomic DNA obtained in step S1 using the kit described in any one of claims 7 to 9; S3. Perform capillary electrophoresis and data analysis on the PCR products.