Indel molecular marker related to the roll-over behavior of domestic pigeon and application thereof
By designing indel molecular markers at specific locations in the pigeon genome and performing PCR amplification and sequencing, the problem of identifying pigeon flipping behavior has been solved, thereby improving the accuracy of pigeon breeding and the efficiency of selecting ornamental breeds.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
- Filing Date
- 2026-04-27
- Publication Date
- 2026-05-29
AI Technical Summary
The lack of effective molecular markers in existing technologies for identifying pigeon flipping behavior leads to insufficient accuracy in pigeon genetic improvement and breeding selection.
An indel molecular marker is provided, located at positions 17,338,130~17,338,140 bp in the pigeon chromosome-level reference genome NC_088609.1. Primer pairs are designed for PCR amplification and sequencing analysis to detect the insertion/deletion of the indel molecular marker, which can be used to identify pigeon flipping behavior and screen for pigeon breeds with flipping ability.
It enables a rapid, simple, and low-cost test of the turning ability of domestic pigeons, improving the accuracy of domestic pigeon breeding and the efficiency of selecting ornamental breeds.
Smart Images
Figure CN122104953A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of genetic engineering and genetic breeding technology, specifically to an indel molecular marker related to the flipping behavior of domestic pigeons and its application. Background Technology
[0002] The domestication of pigeons has produced a variety of remarkable phenotypes. Through continuous and targeted artificial selection, rare traits have been repeatedly amplified and stabilized. One of the most striking traits in pigeons is the "rolling" trait. As early as the late 16th century, driven by human aesthetic preferences, breeds of pigeons were selected that involuntarily perform backflips or continuous rolls in flight or on the ground. This action can be spontaneous or a response to external stimuli. The biological basis of pigeon rolling remains controversial. Various conflicting hypotheses have failed to prove the mechanism influencing the rolling trait in domestic pigeons, and the rolling behavior itself has not been verified or identified. Therefore, there is an urgent need to develop effective strategies, such as effective genetic markers, for selective breeding of pigeons to cultivate rolling behavior.
[0003] Currently, research on molecular markers related to pigeon flipping behavior is limited. However, utilizing stress regulatory factors associated with pigeon flipping behavior holds potential value for genetic improvement and breeding. Therefore, accurately locating indels associated with pigeon flipping behavior and screening for DNA molecular markers that can identify individual flipping behavior can not only improve the accuracy of selection and the genetic progress of selection, but also significantly contribute to the effective selection of pigeons suitable for human aesthetic appreciation. Summary of the Invention
[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an indel molecular marker related to pigeon flipping behavior and its application. The indel molecular marker of this invention is located at positions 17,338,130~17,338,140 bp in NC_088609.1 of the pigeon assembled chromosome level reference genome (bColLiv1.pat.W.v2, https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_036013475.1 / ), containing an 11 bp insertion / deletion variant. This invention provides an effective molecular genetic marker for the breeding and improvement of pigeons.
[0005] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides an indel molecular marker associated with the flipping behavior of domestic pigeons, the nucleotide sequence of which is shown in SEQ ID NO: 1.
[0006] This invention also provides the application of the aforementioned indel molecular marker in identifying pigeon flipping behavior, screening pigeon breeds with flipping ability, and in the genetic breeding of pigeon flipping behavior.
[0007] The present invention also provides a primer pair for obtaining the aforementioned indel molecular marker, wherein the primer pair is: Upstream primer F: CTCCCACCACAGTAGCGG; Downstream primer R: CTGAGGACTGCGGAGAA.
[0008] Furthermore, when the primer pair amplifies a 280 bp band, the indel molecular marker is homozygous deletion; Alternatively, if the primer pair amplifies a 291 bp band, the indel molecular marker is a homozygous insertion; Alternatively, if the primer pair amplifies two bands of 280 bp and 291 bp, the indel molecular marker is heterozygous.
[0009] This invention also provides a method for detecting indel molecular markers related to pigeon flipping behavior, wherein the method uses the primer pair described above for amplification and sequencing comparison to complete the detection.
[0010] The present invention also provides the application of the primer pair described above in identifying pigeon flipping behavior, screening pigeon breeds with flipping ability, and genetic breeding of pigeon flipping behavior.
[0011] The present invention also provides a kit for detecting the indel molecular marker, the kit comprising the primer pair described above.
[0012] The present invention also provides a method for identifying pigeon flipping behavior using the aforementioned kit, comprising the following steps: Detect indel molecular markers associated with pigeon flipping behavior, and determine pigeon flipping behavior based on the insertion / deletion of indel molecular markers; When the indel molecular marker is homozygous deletion, pigeons have the ability to reverse; when the indel molecular marker is homozygous insertion or heterozygous, pigeons do not have the ability to reverse.
[0013] Furthermore, the detection method includes the following steps: (1) Extract DNA from the pigeons to be tested; (2) Perform PCR amplification on the extracted DNA using the primer pairs provided in the kit; (3) Sequencing analysis of the PCR amplification products to obtain sequencing results; (4) Based on the sequencing results, the insertion / deletion status of indel molecular markers is obtained. When the indel molecular marker is homozygous deletion, the pigeon has the ability to flip over. When the indel molecular marker is homozygous insertion or heterozygous, the pigeon does not have the ability to flip over. When the sequencing result is a 280bp band, the indel molecular marker is homozygous deletion; Alternatively, if the sequencing result is a 291 bp band, the indel molecular marker is a homozygous insertion; Alternatively, if the sequencing results show two bands at 280 bp and 291 bp, the indel molecular marker is heterozygous.
[0014] The present invention also provides the application of the kit described herein in identifying pigeon flipping behavior, screening pigeon breeds with flipping ability, and in the genetic breeding of pigeon flipping behavior.
[0015] The beneficial effects of this invention are: This invention provides an indel molecular marker for detecting the flipping ability of pigeons, and provides conventional PCR (polymerase chain reaction) primers for detecting the genotype of this fragment, enabling rapid detection of the genotype of the individual being tested. When the fragment is homozygous deletion, it can be used as a dominant genotype for selecting pigeons based on their flipping ability. This provides an effective, simple, and inexpensive molecular genetic marker for marker-assisted breeding of pigeons with better flipping ability, and offers an effective molecular genetic marker for the breeding and improvement of pigeons. Attached Figure Description
[0016] Figure 1 A genomic region map showing the distribution of candidate genes; Figure 2 The image shows the deletion of 11 bp in exon 1 of the GLRX2 (glutathione reductase 2) gene and its predicted protein structure. Figure 3 Heatmap showing the significance of candidate gene expression in the cerebellum, cerebrum, and optic lobe; Figure 4 Genotype frequency distribution of mutations in flip-pigeon; Figure 5 This is a sequence diagram of the PCR (polymerase chain reaction) amplification product. Detailed Implementation
[0017] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.
[0018] Example 1 Candidate gene screening 1. A total of 171 blood samples from domestic pigeons were collected from private pigeon lofts in different geographical locations (including Xinjiang and Henan in China, Hungary, and Romania). Genomic DNA (deoxyribonucleic acid) was isolated from the 171 blood samples using the TIANamp Genomic DNA Extraction Kit (Tiangen Biotech, Beijing, China) for library construction.
[0019] 2. Download the genome data of 55 flip-flop pigeons (representing 23 breeds), 83 other breeds of domestic pigeons (representing 30 breeds), 29 rock pigeons and 1 mountain pigeon from the NCBI SRA database, as shown in Table 1, and merge them into the sample set of step 1 for subsequent analysis.
[0020] Table 1. Genomic information of pigeons 3. Trimmomatic v0.39 was used to remove adapter sequences and low-quality bases from both ends of the reads (short reads). The obtained reads (short reads) were then aligned to the pigeon reference genome (bColLiv1.pat.W.v2) using Burrows-Wheeler Aligner v0.7.17. Subsequently, Picard v1.16.1 was used to convert the alignment results from a Sequence Alignment / Mapping (SAM) file to Binary Alignment Mapping (BAM) format and sort them by genomic coordinates. Afterwards, PCR repetitive sequences were systematically removed, retaining only uniquely aligned reads (short reads) for subsequent analysis. Single nucleotide polymorphisms (SNPs) and small fragment insertions / deletions (indels) were detected using GATK v4.2.6.1.
[0021] 4. To identify genomic selection signals associated with flipping behavior, this example compares and analyzes ground-rolling pigeons and Xinjiang tumbling pigeons with good flipping abilities, respectively, with non-tumbling domestic pigeon breeds and wild rock pigeons. Three complementary selection signal detection methods were used: ① site-specific branch length (LSBL); ② nucleotide diversity (Pi) ratio; ③ cross-population complex likelihood ratio (XP-CLR). The results are as follows: Figure 1 As shown, at the 99th percentile cutoff value of the empirical distribution, the three methods jointly identified 75 positively selected genes (PSGs) distributed across 58 genomic regions.
[0022] 5. Further transcriptomic analysis was performed on the brain, cerebellum, and optic lobe of pigeons with good rolling ability (ground pigeons and Xinjiang rolling pigeons) and other pigeons. The results are as follows: Figure 3As shown, among all 75 positively selected genes, only GLRX2 showed significant and persistently low expression levels in all three brain regions of the tumbling pigeon, while the remaining candidate genes did not reach the significance threshold in at least one brain region.
[0023] GLRX2 (glutathione reductase 2) encodes a vertebrate-specific glutathione reductase that plays a crucial role in protecting proteins from oxidative damage and promoting axonal growth, neuronal cell survival, and the establishment of functional neuronal networks. Based on the selection signal analysis and transcriptome differential expression analysis results, GLRX2 was identified as a candidate gene associated with tumbling behavior in pigeons. Further analysis revealed an 11 bp deletion variant in exon 1 of this candidate gene (the mutation region is shown in the image). Figure 2 As shown in the figure, the 11 bp deletion variant is located at positions 17,338,130~17,338,140 bp in the pigeon assembled chromosome level reference genome (bColLiv1.pat.W.v2) NC_088609.1, and at positions 53~63 of the GLRX2 gene. This 11 bp is used as an indel molecular marker associated with pigeon flipping behavior and is named indel molecular marker indel-G11. The nucleotide sequence of indel molecular marker indel-G11 is shown in SEQ ID NO: 1.
[0024] Example 2 Validation of the correlation between indel molecular markers and pigeon flipping behavior To further investigate whether the loss of the indel molecular marker indel-G11 is related to the flipping behavior in pigeons, this example selected three local breeds of pigeons: 22 ground-rolling pigeons (with flipping behavior), 52 Xinjiang flipping pigeons, and 20 flipping pigeons (without flipping behavior). Within each breed, samples were collected from multiple independent breeders to minimize genetic correlation and avoid pedigree-specific bias. Blood (0.2–0.5 mL) was drawn from the brachial vein (infraptional vein), temporarily stored at 4°C, and subsequently stored at -80°C for long-term preservation. After DNA quality control, the target fragment associated with flipping behavior was amplified. The upstream and downstream primers for sequence amplification were: Upstream primer F: CTCCCACCACAGTAGCGG (SEQ ID NO: 2); Downstream primer R: CTGAGGACTGCGGAGAA (SEQ ID NO: 3); Using the genomic DNA of the four pigeon breeds obtained in the examples as templates, PCR amplification was performed using the primers described above.
[0025] The amplification system is as follows: Genomic DNA: 1 µL 50 ng / µL DNA template; 1.1× Golden PCR mix: 22 µL; 10 µM primer mixture: 2 µL; The PCR reaction procedure was as follows: 98℃ for 2 min pre-deformation; 98℃ for 10 s denaturation, 60℃ for 15 s annealing, 72℃ for 10 s extension, 35 cycles; 72℃ for 5 min termination extension.
[0026] The amplified PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for first-generation bidirectional sequencing. The obtained sequences were compared with the reference genome (bColLiv1.pat.W.v2) to identify the corresponding indel molecular markers and deletion / insertion status.
[0027] Genotype determination principle: When the sequencing results show that the indel molecular marker indel-G11 is homozygous deletion, it is determined to be a genotype with flipping ability; when the sequencing results show that the indel molecular marker indel-G11 is homozygous insertion or heterozygous, it is determined to be a genotype without flipping ability.
[0028] To further explore whether the indel molecular marker indel-G11 is suitable for screening populations exhibiting flipping behavior, this embodiment re-collected and identified the genotypes of 41 Xinjiang flipping pigeons. The results are as follows: Figure 4 As shown, the indel molecular marker indel-G11 has an extremely high homozygous deletion genotype frequency in Xinjiang flip-pigeons, further illustrating that the indel molecular marker indel-G11 can be used as a key indicator for screening domestic pigeons with flip-pigeon ability.
[0029] Example 3 Detection of indel molecular markers associated with pigeon flipping behavior Using the genomic DNA of domestic pigeons (bColLiv1.pat.W.v2) as a template, PCR amplification was performed using the primer pairs from Example 2. The nucleotide sequences of the upstream primer F and the downstream primer R are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively. Upstream primer F: CTCCCACCACAGTAGCGG; Downstream primer R: CTGAGGACTGCGGAGAA.
[0030] The gene sequence of the PCR product was obtained, and the result is as follows: Figure 5As shown, when the PCR product is a 280 bp band, the indel molecular marker is homozygous deletion; when the PCR product is a 291 bp band, the indel molecular marker is homozygous insertion; and when the PCR product has two bands of 280 and 291 bp, the indel molecular marker is heterozygous.
[0031] Example 4 This embodiment provides a kit for detecting indel molecular markers associated with flipping behavior in pigeons, including the primer pair upstream primer F and downstream primer R from Example 3.
[0032] Example 5 This embodiment provides a method for identifying pigeon flipping behavior using the kit from Example 4, comprising the following steps: Detect indel molecular markers associated with pigeon flipping behavior, and determine pigeon flipping behavior based on the insertion / deletion of indel molecular markers; The detection method includes the following steps: (1) Extract DNA from the pigeons to be tested; (2) Perform PCR amplification on the extracted DNA using the primer pairs provided in the kit; (3) Sequencing analysis of the PCR amplification products to obtain sequencing results; (4) Based on the sequencing results, the insertion / deletion status of indel molecular markers is obtained. When the indel molecular marker is homozygous deletion, the pigeon has the ability to flip over. When the indel molecular marker is homozygous insertion or heterozygous, the pigeon does not have the ability to flip over. When the sequencing result is a 280 bp band, the indel molecular marker is homozygous deletion; Alternatively, if the sequencing result is a 291 bp band, the indel molecular marker is a homozygous insertion; Alternatively, if the sequencing results show two bands at 280 bp and 291 bp, the indel molecular marker is heterozygous.
[0033] In summary, this invention provides an indel molecular marker associated with the flipping trait in pigeons, and a molecular detection method for this marker. In the selection of pigeons for ornamental purposes, individuals with a homozygous deletion genotype of this fragment can be selected using molecular detection, thereby identifying pigeons with better ornamental value and flipping ability. The identification method, mutation site, and detection method of this invention can serve as potential genetic markers for improving the flipping ability of pigeons and for selection, and can be used in the field of genetic breeding.
[0034] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. An indel molecular marker associated with flipping behavior in domestic pigeons, characterized in that: The nucleotide sequence of the indel molecular marker is shown in SEQ ID NO:
1.
2. The application of the indel molecular marker as described in claim 1 in identifying pigeon flipping behavior, screening pigeon breeds with flipping ability, and genetic breeding of pigeon flipping behavior.
3. A primer pair for obtaining the indel molecular marker of claim 1, characterized in that: The primer pair is: Upstream primer F: CTCCCACCACAGTAGCGG; Downstream primer R: CTGAGGACTGCGGAGAA.
4. The primer pair according to claim 3, characterized in that: When the primer pair amplifies a 280 bp band, the indel molecular marker is homozygous deletion; Alternatively, if the primer pair amplifies a 291 bp band, the indel molecular marker is a homozygous insertion. Alternatively, if the primer pair amplifies two bands of 280 bp and 291 bp, the indel molecular marker is heterozygous.
5. A method for detecting indel molecular markers related to the flipping behavior of domestic pigeons, characterized in that: The method uses the primer pair described in claim 3 for amplification, and sequencing comparison is used to complete the detection.
6. The application of the primer pair described in claim 3 in identifying pigeon flipping behavior, screening pigeon breeds with flipping ability, and genetic breeding of pigeon flipping behavior.
7. A kit for detecting the indel molecular marker of claim 1, characterized in that: The kit includes the primer pair as described in claim 3.
8. A method for identifying pigeon flipping behavior using the kit described in claim 7, characterized in that: Includes the following steps: Detect indel molecular markers associated with pigeon flipping behavior, and determine pigeon flipping behavior based on the insertion / deletion of indel molecular markers; When the indel molecular marker is homozygous deletion, pigeons have the ability to flip over; when the indel molecular marker is homozygous insertion or heterozygous, pigeons do not have the ability to flip over.
9. The method according to claim 8, characterized in that: The detection method includes the following steps: (1) Extract DNA from the pigeons to be tested; (2) Perform PCR amplification on the extracted DNA using the primer pairs provided in the kit; (3) Sequencing analysis of the PCR amplification products to obtain sequencing results; (4) Based on the sequencing results, the insertion / deletion status of indel molecular markers is obtained. When the indel molecular marker is homozygous deletion, the pigeon has the ability to flip over. When the indel molecular marker is homozygous insertion or heterozygous, the pigeon does not have the ability to flip over. When the sequencing result is a 280 bp band, the indel molecular marker is homozygous deletion; Alternatively, if the sequencing result is a 291 bp band, the indel molecular marker is a homozygous insertion; Alternatively, if the sequencing results show two bands at 280 bp and 291 bp, the indel molecular marker is heterozygous.
10. The application of the kit according to claim 7 in identifying pigeon flipping behavior, screening pigeon breeds with flipping ability, and genetic breeding of pigeon flipping behavior.