A method for assessing the egg-laying interval of pigeons

Abstract

This invention discloses a method for assessing the egg-laying interval of pigeons. The invention identifies six SNP loci in the FOXO3 gene associated with egg-laying interval, as well as two haplotype combinations that significantly affect pigeon egg production traits. Furthermore, using these SNP loci and haplotype combinations, a method and kit for assessing pigeon egg-laying interval were established. Both the method and the kit are applied to molecular-assisted breeding of pigeons, playing a significant role in breeding pigeons with short egg-laying cycles.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, and more specifically, to a method for evaluating the egg-laying interval of pigeons. Background Technology

[0002] Currently, there are more than ten breeds of meat pigeons raised in Guangdong Province. Among them, Tianxiang No. 1 pigeon is a new breed jointly developed by breeding enterprises and research institutes. It is pure white all over. Its parent male pigeons have broad backs and deep chests with full breast muscles, while the female pigeons are plump and sturdy. It has the advantages of being able to tolerate roughage, having excellent meat quality, and being highly adaptable, making it one of the superior meat pigeon breeds in Guangdong Province.

[0003] Compared to common poultry, pigeons are among the few monogamous birds, possessing unique reproductive characteristics and egg-laying behavior. Under natural conditions, pigeons have a breeding cycle of nearly two months, including mating, egg laying, incubation, and brooding. Young pigeons begin pairing at 6-7 months of age, and begin laying eggs about 10 days after successful mating. The time from the start of continuous egg laying, through incubation and brooding, to the next egg-laying cycle is called the egg-laying cycle. A typical egg-laying cycle involves laying only two eggs consecutively, with the second egg laid 24-48 hours after the first, forming a clutch. In current pigeon farm production models, an important reference indicator for the breeding process of breeding pigeons is the egg-laying interval, which is further divided into the interval between the two eggs laid in one egg-laying cycle and the interval between the first eggs of two adjacent clutches. The latter interval represents the female pigeon's egg-laying cycle. With the continuous adjustment of commercial pigeon farm production models, parent pigeons can skip natural incubation and brooding time and focus solely on egg laying, shortening the egg-laying cycle to 10-15 days. However, pigeons still need to lay eggs every ten days or so, resulting in low egg production efficiency and hindering the development of the pigeon industry.

[0004] With the development of molecular biology techniques, early selection through molecular markers has a positive impact on the economic benefits of the aquaculture industry. DNA markers are the most commonly used molecular marker methods in genetic research. Single nucleotide polymorphism (SNP) is one type, referring to the polymorphism of DNA sequences caused by mutations in a single nucleotide base at the genomic level.

[0005] FOXO3, also known as forkhead box transcription factor 3a (FOXO3a), is an important member of the forkhead transcription factor (FOXO) family and a crucial transcription factor regulating apoptosis, cell cycle, oxidative stress, and other cellular functions. Research on FOXO3 gene polymorphism has primarily focused on humans, with relatively few studies on mammals and birds. There are no reports of it affecting egg-laying intervals in pigeons. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for evaluating the egg-laying interval of pigeons.

[0007] The first objective of this invention is to provide a method for assessing the egg-laying interval of pigeons.

[0008] A second objective of this invention is to provide the application of a product for detecting SNP sites or haplotype combinations in pigeons in the preparation of a kit for assessing pigeon egg-laying intervals or in the establishment of a method for assessing pigeon egg-laying intervals.

[0009] A third objective of this invention is to provide a kit for assessing the egg-laying interval of pigeons.

[0010] A fourth object of the present invention is to provide the application of any of the methods, products, or kits described herein in pigeon molecular breeding.

[0011] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0012] This invention claims a method for assessing the egg-laying interval of pigeons, detecting the genotype of one or more of the following SNP loci or haplotype combinations:

[0013] The SNP site is g.3149417G>A, located at the 3149417th base of NW_004973554.1. The egg-laying interval of individuals with the AA genotype is significantly lower than that of individuals with the GG genotype, and the egg-laying interval of individuals with the AA genotype is significantly lower than that of individuals with the GA genotype.

[0014] The SNP site is g.3149444C>T, located at the 3149444th base of NW_004973554.1. The egg-laying interval of individuals with the TT genotype is significantly lower than that of individuals with the CC genotype.

[0015] The SNP site is g.3149486A>G, ​​located at the 3149486th base of NW_004973554.1. The egg-laying interval of individuals with the GG genotype is significantly lower than that of individuals with the AA genotype.

[0016] The SNP site is g.3150038T>C, located at the 3150038th base of NW_004973554.1. The egg-laying interval of individuals with the TT genotype is significantly lower than that of individuals with the TC genotype, and the egg-laying interval of individuals with the CC genotype is significantly lower than that of individuals with the TC genotype.

[0017] The SNP site is g.3151252G>T, located at the 3151252nd base of NW_004973554.1. The egg-laying interval of individuals with the TT genotype is significantly lower than that of individuals with the GG genotype.

[0018] The SNP site is g.3152087A>G, located at the 3152087th base of NW_004973554.1. The egg-laying interval of individuals with the GG genotype is significantly lower than that of individuals with the AA genotype, and the egg-laying interval of individuals with the GG genotype is significantly lower than that of individuals with the AG genotype.

[0019] The haplotype combination contains three SNP sites: g.3149417G>A, g.3149444C>T, and g.3149486A>G, located at the 3149417th, 3149444th, and 3149444th bases of NW_004973554.1, respectively.

[0020] The egg-laying interval of individuals with genotypes AA, TT, and GG at the three SNP loci was significantly lower than that of individuals with genotypes GA, CC, and AA at the three SNP loci.

[0021] The egg-laying interval of individuals with genotypes AA, TT, and GG at the three SNP loci was significantly shorter than that of individuals with genotypes GG, CC, and AA at the three SNP loci.

[0022] The egg-laying interval of individuals with genotypes GG, CC, and AA at the three SNP loci was significantly lower than that of individuals with genotypes GA, CC, and AA at the three SNP loci.

[0023] The egg-laying interval of individuals with genotypes GA, CT, and AG at the three SNP loci was significantly lower than that of individuals with genotypes GA, CC, and AA at the three SNP loci.

[0024] The haplotype combination contains five SNP sites: g.3151252G>T, g.3151281G>T, g.3152087A>G, g.3152096G>T, and g.3152151G>A, located at the 3151252nd, 3151281st, 3152087th, 3152096th, and 3152151st bases of NW_004973554.1, respectively.

[0025] The egg-laying interval of individuals with genotypes TT, TT, GG, GG, and GG at the five SNP loci was significantly lower than that of individuals with genotypes GG, GG, AA, GG, and GG at the five SNP loci.

[0026] Preferably, primers are used to amplify the pigeon's DNA to detect the genotype of one or more of the SNP sites or haplotype combinations.

[0027] More preferably, the nucleotide sequences of the primers are shown in SEQ ID NO: 3-6 and 9-12.

[0028] Specifically, primers with nucleotide sequences as shown in SEQ ID NO: 3-4 are used for the detection of g.3149417G>A, g.3149444C>T, or g.3149486A>G; primers with nucleotide sequences as shown in SEQ ID NO: 5-6 are used for the detection of g.3150038T>C; primers with nucleotide sequences as shown in SEQ ID NO: 9-10 are used for the detection of g.3151252G>T or g.3151281G>T; and primers with nucleotide sequences as shown in SEQ ID NO: 11-12 are used for the detection of g.3152087A>G, g.3152096G>T, or g.3152151G>A.

[0029] This invention also claims the use of a product for detecting SNP loci or haplotype combinations in pigeons in the preparation of kits for assessing pigeon egg-laying intervals or in establishing methods for assessing pigeon egg-laying intervals, characterized in that the product detects one or more of the following SNP loci or haplotype combinations:

[0030] The SNP site is g.3149417G>A, located at the 3149417th base of NW_004973554.1. The egg-laying interval of individuals with the AA genotype is significantly lower than that of individuals with the GG genotype, and the egg-laying interval of individuals with the AA genotype is significantly lower than that of individuals with the GA genotype.

[0031] The SNP site is g.3149444C>T, located at the 3149444th base of NW_004973554.1. The egg-laying interval of individuals with the TT genotype is significantly lower than that of individuals with the CC genotype.

[0032] The SNP site is g.3149486A>G, ​​located at the 3149486th base of NW_004973554.1. The egg-laying interval of individuals with the GG genotype is significantly lower than that of individuals with the AA genotype.

[0033] The SNP site is g.3150038T>C, located at the 3150038th base of NW_004973554.1. The egg-laying interval of individuals with the TT genotype is significantly lower than that of individuals with the TC genotype, and the egg-laying interval of individuals with the CC genotype is significantly lower than that of individuals with the TC genotype.

[0034] The SNP site is g.3151252G>T, located at the 3151252nd base of NW_004973554.1. The egg-laying interval of individuals with the TT genotype is significantly lower than that of individuals with the GG genotype.

[0035] The SNP site is g.3152087A>G, located at the 3152087th base of NW_004973554.1. The egg-laying interval of individuals with the GG genotype is significantly lower than that of individuals with the AA genotype, and the egg-laying interval of individuals with the GG genotype is significantly lower than that of individuals with the AG genotype.

[0036] The haplotype combination contains three SNP sites: g.3149417G>A, g.3149444C>T, and g.3149486A>G, located at the 3149417th, 3149444th, and 3149444th bases of NW_004973554.1, respectively.

[0037] The egg-laying interval of individuals with genotypes AA, TT, and GG at the three SNP loci was significantly lower than that of individuals with genotypes GA, CC, and AA at the three SNP loci.

[0038] The egg-laying interval of individuals with genotypes AA, TT, and GG at the three SNP loci was significantly shorter than that of individuals with genotypes GG, CC, and AA at the three SNP loci.

[0039] The egg-laying interval of individuals with genotypes GG, CC, and AA at the three SNP loci was significantly lower than that of individuals with genotypes GA, CC, and AA at the three SNP loci.

[0040] The egg-laying interval of individuals with genotypes GA, CT, and AG at the three SNP loci was significantly lower than that of individuals with genotypes GA, CC, and AA at the three SNP loci.

[0041] The haplotype combination contains five SNP sites: g.3151252G>T, g.3151281G>T, g.3152087A>G, g.3152096G>T, and g.3152151G>A, located at the 3151252nd, 3151281st, 3152087th, 3152096th, and 3152151st bases of NW_004973554.1, respectively.

[0042] The egg-laying interval of individuals with genotypes TT, TT, GG, GG, and GG at the five SNP loci was significantly lower than that of individuals with genotypes GG, GG, AA, GG, and GG at the five SNP loci.

[0043] Preferably, the product is a primer.

[0044] More preferably, the nucleotide sequences of the primers are shown in SEQ ID NO: 3-6 and 9-12.

[0045] The present invention also claims a kit for assessing the egg-laying interval of pigeons, the kit containing the product described above.

[0046] Preferably, the kit contains the product described above.

[0047] More preferably, the kit further contains PCR amplification reagents.

[0048] As a specific example, 2×Easy PCR Mix

[0049] The present invention also claims protection for the application of any of the methods, products, and kits described herein in pigeon molecular breeding.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] This invention identified six SNP sites in the FOXO3 gene that are associated with egg-laying intervals, as well as two haplotype combinations that significantly affect pigeon egg-laying traits. Furthermore, it established a method and a kit for assessing pigeon egg-laying intervals using these SNP sites and haplotype combinations. Both the method and the kit have been applied to molecular-assisted breeding of pigeons, playing a significant role in breeding pigeons with short egg-laying cycles. Attached Figure Description

[0052] Figure 1The image shows the electrophoresis results of the PCR products; lane M is the DL 2000 Marker, lanes 1-10 are mixed pool DNA, and images A-H are the electrophoresis images of the PCR products FOXO3-Exon1, FOXO3-Exon2-1, FOXO3-Exon2-2, FOXO3-Exon2-3, FOXO3-Exon2-4, FOXO3-Exon2-5, FOXO3-Exon3-1, and FOXO3-Exon3-2, respectively.

[0053] Figure 2 Sanger sequencing peak diagram for SNP site g.3100281C>G genotype.

[0054] Figure 3 Sanger sequencing peak diagram for SNP locus g.3100380A>G genotype.

[0055] Figure 4 Sanger sequencing peak diagram for SNP site g.3149417G>A genotype.

[0056] Figure 5 Sanger sequencing peak diagram for the SNP site g.3149444C>T genotype.

[0057] Figure 6 Sanger sequencing peak diagram for SNP site g.3149486A>G genotype.

[0058] Figure 7 Sanger sequencing peak diagram for SNP site g.3149933G>C genotype.

[0059] Figure 8 Sanger sequencing peak diagram for SNP site g.3150038T>C genotype.

[0060] Figure 9 Sanger sequencing peak diagram for the SNP site g.3150194C>T genotype.

[0061] Figure 10 Sanger sequencing peak diagram for the SNP locus g.3150702T>G genotype.

[0062] Figure 11 Sanger sequencing peak diagram for SNP locus g.3150730A>G genotype.

[0063] Figure 12 Sanger sequencing peak diagram for SNP locus g.3150836A>G genotype.

[0064] Figure 13Sanger sequencing peak diagram for the SNP site g.3151162C>T genotype.

[0065] Figure 14 Sanger sequencing peak diagram for the SNP locus g.3151170C>T genotype.

[0066] Figure 15 Sanger sequencing peak diagram for the SNP locus g.3151207C>T genotype.

[0067] Figure 16 Sanger sequencing peak diagram for the SNP locus g.3151233A>T genotype.

[0068] Figure 17 Sanger sequencing peak diagram for the SNP locus g.3151252G>T genotype.

[0069] Figure 18 Sanger sequencing peak diagram for the SNP locus g.3151281G>T genotype.

[0070] Figure 19 Sanger sequencing peak diagram for SNP locus g.3152087A>G genotype.

[0071] Figure 20 Sanger sequencing peak diagram for the SNP locus g.3152096G>T genotype.

[0072] Figure 21 Sanger sequencing peak diagram for the SNP locus g.3152151G>A genotype.

[0073] Figure 22 This is a linkage disequilibrium plot of the FOXO3 gene SNP sites. The values ​​in the boxes are the D' value and r. 2 The value is obtained by multiplying it by 100, based on D′ and r. 2 To evaluate the linkage between SNPs, D′ is on top, r 2 I am. Detailed Implementation

[0074] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0075] Example 1: Amplification of the SNP site of the FOXO3 gene in Tianxiang No. 1 pigeon

[0076] I. Experimental Methods

[0077] 1. Data and Sample Collection in the Experiment

[0078] 250 Tianxiang No. 1 pigeons, aged 24 months and of similar weight, with normal egg production, were randomly selected from Guangdong Kangzheng Pigeon Industry Co., Ltd.

[0079] Under identical rearing conditions, male and female pigeons were caged together, and the dates on which the female pigeons did not incubate or feed the chicks but only laid eggs were recorded. The number of days between the first egg of two adjacent clutches of the female pigeon was recorded as one laying interval. A total of four laying intervals were recorded, and the average was taken.

[0080] Low-invasive wing vein blood collection was performed using medical disposable blood collection tubes containing EDTA-K2 (dipotassium EDTA salt).

[0081] 2. Sample collection and DNA extraction

[0082] DNA was extracted from blood samples using a blood genomic DNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd.). Purity and concentration were determined by a combination of 1.5% agarose gel electrophoresis and a NanoDrop Lite spectrophotometer. All samples were stored at -20°C for later use.

[0083] 3. Primer design

[0084] Based on the pigeon (Columba livia) FOXO3 gene sequence (GenBank accession number: NW_004973554.1) provided in the NCBI database as a reference sequence, specific primers were designed. The primer sequences are shown in Table 1 below. They were synthesized by Shanghai Bioengineering (Sangon) Technology Service Co., Ltd., diluted with sterile double-distilled water to 10 μmol / L, and stored at -20℃ for later use.

[0085] Table 1 Primer sequences for the FOXO3 gene

[0086]

[0087] 4. PCR amplification and sequencing

[0088] Dilute the DNA of each sample to the same concentration, and take 5 μl of each sample into a 1.5 ml centrifuge tube to make a pool, which will be used as a template for PCR amplification.

[0089] The PCR reaction conditions are shown in Tables 2 and 3. The PCR products were stored at 4°C and used for subsequent sequencing. The PCR amplification product was detected by 1.5% agarose gel electrophoresis. If the electrophoretic band was the same size as the target band, the obtained PCR product was sent to Shanghai Jierui Biotechnology Co., Ltd. for direct sequencing.

[0090] Table 2 PCR amplification system

[0091]

[0092] Table 3 PCR Procedure

[0093]

[0094] II. Experimental Results

[0095] The mixed-cell DNA amplification products all contain the desired target bands, as shown in the electrophoresis diagram. Figure 1 As shown.

[0096] The amplification products of primers FOXO3-Exon1 were 304 bp in length, FOXO3-Exon2-1 were 706 bp in length, FOXO3-Exon2-2 were 779 bp in length, FOXO3-Exon2-3 were 754 bp in length, FOXO3-Exon2-4 were 863 bp in length, FOXO3-Exon2-5 were 796 bp in length, FOXO3-Exon3-1 were 643 bp in length, and FOXO3-Exon3-2 were 591 bp in length. Detection by 1.5% agarose gel electrophoresis showed that the bands were bright and clear, with no extraneous bands, and were suitable for subsequent sequencing.

[0097] The sequencing results were compared with the reference fragment on NCBI, and the peaks in the sequence results were viewed using SnapGene Viewer 2.3.4 software to screen for SNP sites. The results showed that a total of 20 SNP sites were detected in the amplified fragment of the FOXO3 gene of Tianxiang No. 1 pigeon, all of which had three genotypes. The information on the 20 mutation sites of the pigeon's FOXO3 gene is shown in Table 4, and the Sanger sequencing peaks of the SNP site genotypes are shown in Table 4. Figures 2 to 21 They are respectively:

[0098] The site g.3100281C>G on exon 1 has three genotypes: CC, CG, and GG. This site is a missense mutation between the charged polar amino acid arginine and the uncharged nonpolar amino acid glycine.

[0099] The genotypes of exon 1 fragment with intron site g.3100380A>G are AA, AG, and GG.

[0100] There are 6 SNP sites in the CDS region of exon 2, all of which are synonymous mutations: g.3149417G>A has 3 genotypes GG, GA, AA; g.3149444C>T has 3 genotypes CC, CT, TT; g.3149486A>G has 3 genotypes AA, AG, GG; g.3149933G>C has 3 genotypes GG, GC, CC; g.3150038T>C has 3 genotypes TT, TC, CC; and g.3150194C>T has 3 genotypes CC, CT, TT.

[0101] There are 12 SNP sites in the 3'UTR region of exon 2: g.3150702T>G genotypes are TT, TG, GG; g.3150730A>G, g.3150836A>G, and g.3152087A>G genotypes are AA, AG, and GG; g.3151162C>T, g.3151170C>T, and g.3151207C>T genotypes are CC, CT, and TT; g.3151233A>T genotypes are AA, AT, and TT; g.3151252G>T, g.3151281G>T, and g.3152096G>T genotypes are GG, GT, and TT; and g.3152151G>A genotypes are GG, GA, and AA.

[0102] Table 4. Information on FOXO3 gene mutation sites

[0103]

[0104]

[0105] Example 2: Genetic characteristics of the FOXO3 gene SNP locus in Tianxiang No. 1 pigeon

[0106] I. Experimental Methods

[0107] Following the method in Example 1, DNA samples from 250 Tianxiang No. 1 pigeons corresponding to Example 1 were subjected to PCR amplification and sequencing.

[0108] The sequencing results were compared with the reference fragment using DNASTAR.Lasergene.v7.1 SeqMan software. SnapGene Viewer 2.3.4 software was used to observe the peaks in the sequencing results, obtain the SNP sites of each sample, and perform genetic characteristic analysis.

[0109] Using Excel, calculate the allele frequency, genotype frequency, genetic heterozygosity (Expected Heterozygosity, He), effective number of alleles (Effective Number Of Alleles, Ne), polymorphism information content (Polymorphism Information Content, PIC), chi-square test (Chi-square Test, χ2), and P value according to the formula, and analyze whether the SNP locus is in Hardy-Weinberg (H-W) equilibrium state.

[0110] II. Experimental Results

[0111] As shown in Table 5, the calculated PIC values indicate that the loci of g.3100380A>G, g.3149933G>C, g.3150702T>G, g.3151170C>T, and g.3151207C>T are of low polymorphism, and the remaining loci are of moderate polymorphism. The results of the Hardy-Weinberg equilibrium test show that g.3150038T>C, g.3150730A>G, g.3151162C>T, g.3151252G>T, and g.3151281G>T are significantly deviated from the Hardy-Weinberg equilibrium (P<0.05) in the Tianxiang No. 1 pigeon population, and the remaining loci are not deviated from the Hardy-Weinberg equilibrium (P>0.05).

[0112] Table 5 Genetic characteristics of SNP loci of FOXO3 gene

[0113]

[0114]

[0115] Note: PIC>0.5 is of high polymorphism, 0.25<PIC<0.5 is of moderate polymorphism, and PIC<0.25 is of low polymorphism; the Hardy-Weinberg equilibrium is tested with the χ2 value, df = 2, P<0.05 indicates significant difference; deviation from the Hardy-Weinberg equilibrium.

[0116] Example 3 Association between each SNP locus of FOXO3 gene and egg-laying interval in Tianxiang No. 1 pigeon

[0117] I. Experimental Method

[0118] The egg-laying interval data of each individual obtained in Example 1, combined with the genotypes of the SNP loci of each individual obtained in Example 2, were used for association analysis using IBM SPSS Statistics 26.0 software. The results are expressed as mean ± standard error. The LSD method and the Tammhenko method were used for multiple comparisons to determine the significance of differences in egg-laying intervals among different SNP loci genotypes. P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

[0119] II. Experimental Results

[0120] The results are shown in Table 6. Among the 20 SNP loci of the pigeon FOXO3 gene, the loci g.3149417G>A, g.3149444C>T, g.3149486A>G, ​​g.3150038T>C, g.3151252G>T, and g.3152087A>G were correlated with the egg-laying interval, while the remaining loci were not correlated. Specifically, at the g.3149417G>A locus, the AA genotype was significantly lower than the GG genotype (P<0.01), the AA genotype was significantly lower than the GA genotype (P<0.05), and there was no significant difference between the GG and GA genotypes (P>0.05). At the g.3149444C>T locus, the TT genotype was significantly lower than the CC genotype (P<0.05), and there was no significant difference between the CT genotype and the CC and TT genotypes (P>0.05). 0.05); at the g.3149486A>G locus, the GG genotype was significantly lower than the AA genotype (P<0.05), and the AG genotype was not significantly different from the AA and GG genotypes (P>0.05); at the g.3150038T>C locus, the TT genotype was extremely significantly lower than the TC genotype (P<0.01), the CC genotype was significantly lower than the TC genotype (P<0.05), and the TT and CC genotypes were not significantly different (P>0.05); at the g.3151252G>T locus, the TT genotype was significantly lower than the GG genotype (P<0.05); at the g.3152087A>G locus, the GG genotype was extremely significantly lower than the AA genotype (P<0.01), the GG genotype was significantly lower than the AG genotype (P<0.05), and the AA and AG genotypes were not significantly different (P>0.05).

[0121] Table 6 Egg Laying Intervals of FOXO3 Gene SNP Loci for Each Genotype

[0122]

[0123]

[0124] Note: In the table, lowercase superscript letters indicate significant differences (0.01 < P < 0.05), and uppercase letters indicate extremely significant differences (P < 0.01). The same applies to the following tables.

[0125] Example 4: Linkage disequilibrium analysis of SNP sites in the FOXO3 gene of Tianxiang No. 1 pigeon.

[0126] I. Experimental Methods

[0127] Linkage disequilibrium (LD) analysis was performed on the SNP sites of the FOXO3 gene using Haploview 4.2 software to estimate haplotype combination types.

[0128] II. Experimental Results

[0129] The results of the chain imbalance analysis are shown below. Figure 22 And Table 7. 2 >0.33 and D'>0.8 indicate strong linkage disequilibrium between SNP sites, which can be used for mapping.

[0130] Four linkage disequilibrium (LD) blocks (haplotype blocks) were constructed from 20 SNP sites of the FOXO3 gene in Tianxiang No. 1 pigeon:

[0131] Block1 is composed of g.3149417G>A, g.3149444C>T, and g.3149486A>G, with D' values ​​ranging from 0.98 to 1.0, and r 2 The values ​​ranged from 0.834 to 0.99, indicating a strong linkage disequilibrium among the three sites, resulting in three haplotypes: H1 (GCA), H2 (ATG), and H3 (ACA).

[0132] Block2 consists of g.3150038T>C and g.3150194C>T, exhibiting a strong linkage disequilibrium (D'=1, r 2 =0.583), resulting in three haplotypes: H4 (TC), H5 (CT), and H6 (CC);

[0133] Block 3 consists of g.3150702T>G and g.3150836A>G, ​​exhibiting a strong linkage disequilibrium (D'=0.96, r 2 =0.612), resulting in three haplotypes: H7 (GA), H8 (TG), and H9 (TA);

[0134] Block 4 is composed of g.3151252G>T, g.3151281G>T, g.3152087A>G, g.3152096G>T, and g.3152151G>A, with D' values ​​ranging from 0.96 to 0.82. 2The values ​​ranged from 0.605 to 0.881, indicating a strong linkage disequilibrium among the five loci, resulting in six haplotypes: H10 (TGAGG), H11 (GGAGG), H12 (TTTGTA), H13 (GGGGG), H14 (TTGGG), and H15 (TTTAGG).

[0135] Table 7 Haplotype analysis of SNP sites in the FOXO3 gene

[0136]

[0137] Example 5: Association between FOXO3 gene SNP haplotypes and egg production interval

[0138] I. Experimental Methods

[0139] The egg-laying interval data of each individual obtained in Example 1, combined with the haplotypes obtained in Example 4, were used for correlation analysis using IBM SPSS Statistics 26.0 software. The results are expressed as mean ± standard error. The LSD method and the Tammhenko method were used for multiple comparisons to determine the significance of differences in egg-laying intervals among different haplotype genotypes. P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference.

[0140] II. Experimental Results

[0141] There are 15 haplotypes in the four blocks of the FOXO3 gene, and only 34 haplotype combinations were observed. The association analysis results of haplotype combinations with more than 10 individuals are shown in Table 8.

[0142] Table 8. Association between FOXO3 gene SNP site haplotype combinations and egg production interval.

[0143]

[0144]

[0145] Association analysis revealed that H2H2 in Block 1 had the shortest laying interval, significantly lower than H1H3 (P<0.01) and significantly lower than H1H1 (P<0.05); H1H1 and H1H2 were also significantly lower than H1H3 (P<0.01); H14H14 in Block 4 had the shortest laying interval, significantly lower than H11H11 (P<0.05). No haplotype combinations associated with laying interval were found in Block 2 and Block 3.

[0146] Example 6: A method for evaluating the egg-laying interval of Tianxiang No. 1 pigeons

[0147] 1. PCR amplification and sequencing

[0148] DNA from Tianxiang No. 1 pigeon was amplified using primers with nucleotide sequences shown in SEQ ID NO: 3-6 and 9-12.

[0149] The amplification system consisted of: 1 μL DNA, 0.5 μL upstream primer, 0.5 μL downstream primer, 8 μL ddH2O, and 2×Easy... PCR Mix 10 μL;

[0150] The amplification program was: 94℃ for 3 min, 94℃ for 30 s, 60℃ for 45 s, 72℃ for 50 s, 32 cycles, 72℃ for 8 min.

[0151] The PCR products were detected by 1.5% agarose gel electrophoresis. If the electrophoretic bands were the same size as the target bands, the obtained PCR products were sequenced.

[0152] 2. Result Interpretation

[0153] The SNP site is g.3149417G>A, located at the 3149417th base of NW_004973554.1. The egg-laying interval of individuals with the AA genotype is significantly lower than that of individuals with the GG genotype, and the egg-laying interval of individuals with the AA genotype is significantly lower than that of individuals with the GA genotype.

[0154] The SNP site is g.3149444C>T, located at the 3149444th base of NW_004973554.1. The egg-laying interval of individuals with the TT genotype is significantly lower than that of individuals with the CC genotype.

[0155] The SNP site is g.3149486A>G, ​​located at the 3149486th base of NW_004973554.1. The egg-laying interval of individuals with the GG genotype is significantly lower than that of individuals with the AA genotype.

[0156] The SNP site is g.3150038T>C, located at the 3150038th base of NW_004973554.1. The egg-laying interval of individuals with the TT genotype is significantly lower than that of individuals with the TC genotype, and the egg-laying interval of individuals with the CC genotype is significantly lower than that of individuals with the TC genotype.

[0157] The SNP site is g.3151252G>T, located at the 3151252nd base of NW_004973554.1. The egg-laying interval of individuals with the TT genotype is significantly lower than that of individuals with the GG genotype.

[0158] The SNP site is g.3152087A>G, located at the 3152087th base of NW_004973554.1. The egg-laying interval of individuals with the GG genotype is significantly lower than that of individuals with the AA genotype, and the egg-laying interval of individuals with the GG genotype is significantly lower than that of individuals with the AG genotype.

[0159] The haplotype contains three SNP sites: g.3149417G>A, g.3149444C>T, and g.3149486A>G, located at the 3149417th, 3149444th, and 3149444th bases of NW_004973554.1, respectively.

[0160] The egg-laying interval of individuals with genotypes AA, TT, and GG at the three SNP loci was significantly lower than that of individuals with genotypes GA, CC, and AA at the three SNP loci.

[0161] The egg-laying interval of individuals with genotypes AA, TT, and GG at the three SNP loci was significantly shorter than that of individuals with genotypes GG, CC, and AA at the three SNP loci.

[0162] The egg-laying interval of individuals with genotypes GG, CC, and AA at the three SNP loci was significantly lower than that of individuals with genotypes GA, CC, and AA at the three SNP loci.

[0163] The egg-laying interval of individuals with genotypes GA, CT, and AG at the three SNP loci was significantly lower than that of individuals with genotypes GA, CC, and AA at the three SNP loci.

[0164] The haplotype combination contains five SNP sites: g.3151252G>T, g.3151281G>T, g.3152087A>G, g.3152096G>T, and g.3152151G>A, located at the 3151252nd, 3151281st, 3152087th, 3152096th, and 3152151st bases of NW_004973554.1, respectively.

[0165] The egg-laying interval of individuals with genotypes TT, TT, GG, GG, and GG at the five SNP loci was significantly lower than that of individuals with genotypes GG, GG, AA, GG, and GG at the five SNP loci.

[0166] Example 7: A kit for evaluating the egg-laying interval of Tianxiang No. 1 pigeons

[0167] I. Composition

[0168] Nucleotide sequences are shown in SEQ ID NO: 3-6 and 9-12. Primers, 2×Easy PCR Mix and ddH2O

[0169] II. Instructions for Use

[0170] Same as Example 6.

Claims

1. A method for assessing egg laying intervals in pigeons, characterized in that, detecting the genotype of one or more of the following SNP sites; or detecting the genotype of all SNP sites in any one of the following haplotype combinations: The SNP site is g.3149417 G>A, located at the 3149417th base of NW_004973554.1, the egg laying interval of AA genotype individuals is significantly lower than that of GG genotype individuals, and the egg laying interval of AA genotype individuals is significantly lower than that of GA genotype individuals; The SNP site is g.3149444 C>T, located at the 3149444th base of NW_004973554.1, the egg laying interval of TT genotype individuals is significantly lower than that of CC genotype individuals; The SNP site is g.3149486 A>G, located at the 3149486th base of NW_004973554.1, the egg laying interval of GG genotype individuals is significantly lower than that of AA genotype individuals; The SNP site is g.3150038 T>C, located at the 3150038th base of NW_004973554.1, the egg laying interval of TT genotype individuals is significantly lower than that of TC genotype individuals, and the egg laying interval of CC genotype individuals is significantly lower than that of TC genotype individuals; The SNP site is g.3151252 G>T, located at the 3151252th base of NW_004973554.1, the egg laying interval of TT genotype individuals is significantly lower than that of GG genotype individuals; The SNP site is g.3152087 A>G, located at the 3152087th base of NW_004973554.1, the egg laying interval of GG genotype individuals is significantly lower than that of AA genotype individuals, and the egg laying interval of GG genotype individuals is significantly lower than that of AG genotype individuals; The haplotype combination consists of three SNP sites: g.3149417 G>A, g.3149444 C>T and g.3149486 A>G, located at the 3149417th base, the 3149444th base and the 3149444th base of NW_004973554.1, respectively, The egg laying interval of individuals with genotypes of AA, TT and GG of the three SNP sites in turn is significantly lower than that of individuals with genotypes of GA, CC and AA of the three SNP sites in turn, The egg laying interval of individuals with genotypes of AA, TT and GG of the three SNP sites in turn is significantly lower than that of individuals with genotypes of GG, CC and AA of the three SNP sites in turn, The egg laying interval of individuals with genotypes of GG, CC and AA of the three SNP sites in turn is significantly lower than that of individuals with genotypes of GA, CC and AA of the three SNP sites in turn, The egg production interval of the individual whose genotypes of the three SNP sites are GA, CT and AG in sequence is significantly lower than that of the individual whose genotypes of the three SNP sites are GA, CC and AA in sequence; The haplotype combination is composed of five SNP sites: g.3151252 G>T, g.3151281 G>T, g.3152087 A>G, g.3152096 G>T and g.3152151 G>A, which are located at the 3151252th, 3151281th, 3152087th, 3152096th and 3152151th bases of NW_004973554.1 in sequence, The egg production interval of the individual whose genotypes of the five SNP sites are TT, TT, GG, GG and GG in sequence is significantly lower than that of the individual whose genotypes of the five SNP sites are GG, GG, AA, GG and GG in sequence; The pigeon is Tianxiang No.1 pigeon.

2. The method of claim 1, wherein, The DNA of the pigeon is amplified using primers to detect the genotype of one or several of the SNP sites or haplotype combinations.

3. The method of claim 2, wherein, The nucleotide sequences of the primers are shown in SEQ ID NO: 3-6 and 9-12.

4. Use of a product for detecting a SNP locus or haplotype combination of a pigeon in the manufacture of a kit for assessing the laying interval of a pigeon or in the establishment of a method for assessing the laying interval of a pigeon, characterized in that, The product detects the genotype of one or several of the following SNP sites; or the product detects the genotype of all SNP sites in any one of the following haplotype combinations: The SNP site is g.3149417 G>A, located at the 3149417th base of NW_004973554.1, the egg production interval of the individual with AA genotype is significantly lower than that of the individual with GG genotype, and the egg production interval of the individual with AA genotype is significantly lower than that of the individual with GA genotype; The SNP site is g.3149444 C>T, located at the 3149444th base of NW_004973554.1, the egg production interval of the individual with TT genotype is significantly lower than that of the individual with CC genotype; The SNP site is g.3149486 A>G, located at the 3149486th base of NW_004973554.1, the egg production interval of the individual with GG genotype is significantly lower than that of the individual with AA genotype; The SNP site is g.3150038 T>C, located at the 3150038th base of NW_004973554.1, the egg production interval of the individual with TT genotype is significantly lower than that of the individual with TC genotype, and the egg production interval of the individual with CC genotype is significantly lower than that of the individual with TC genotype; The SNP site is g.3151252 G>T, located at the 3151252th base of NW_004973554.1, the egg production interval of the individual with TT genotype is significantly lower than that of the individual with GG genotype; The SNP site is g.3152087 A>G, located at the 3152087th base of NW_004973554.1, and the egg interval of the GG genotype individual is significantly lower than that of the AA genotype individual, and the egg interval of the GG genotype individual is significantly lower than that of the AG genotype individual; The haplotype combination consists of three SNP sites: g.3149417 G>A, g.3149444 C>T and g.3149486 A>G, located at the 3149417th base, the 3149444th base and the 3149444th base of NW_004973554.1 in turn, The egg interval of the individual with the genotype AA, TT and GG of the three SNP sites in turn is significantly lower than that of the individual with the genotype GA, CC and AA of the three SNP sites in turn, The egg interval of the individual with the genotype AA, TT and GG of the three SNP sites in turn is significantly lower than that of the individual with the genotype GG, CC and AA of the three SNP sites in turn, The egg interval of the individual with the genotype GG, CC and AA of the three SNP sites in turn is significantly lower than that of the individual with the genotype GA, CC and AA of the three SNP sites in turn, The egg interval of the individual with the genotype GA, CT and AG of the three SNP sites in turn is significantly lower than that of the individual with the genotype GA, CC and AA of the three SNP sites in turn; The haplotype group consists of five SNP sites: g.3151252 G>T, g.3151281 G>T, g.3152087 A>G, g.3152096 G>T and g.3152151 G>A, located at the 3151252th base, the 3151281th base, the 3152087th base, the 3152096th base and the 3152151th base of NW_004973554.1 in turn, The egg interval of the individual with the genotype TT, TT, GG, GG and GG of the five SNP sites in turn is significantly lower than that of the individual with the genotype GG, GG, AA, GG and GG of the five SNP sites in turn; The pigeon is Tianxiang No.1 pigeon.

5. Use according to claim 4, characterized in that, The product is a primer.

6. Use according to claim 5, characterized in that, The nucleotide sequence of the primer is shown in SEQ ID NO: 3-6 and 9-12.

7. The use of the method of any one of claims 1 to 3, the product of any one of claims 4-6, the kit containing the product of any one of claims 4-6 in the molecular breeding of the short egg interval of the pigeon, and the pigeon is Tianxiang No.1 pigeon, The SNP site is g.3149417 G>A, located at the 3149417th base of NW_004973554.1, and the egg interval of the AA genotype individual is significantly lower than that of the GG genotype individual, and the egg interval of the AA genotype individual is significantly lower than that of the GA genotype individual; The SNP site is g.3149444 C>T, located at the 3149444th base of NW_004973554.1, the egg production interval of the individual with TT genotype is significantly lower than that of the individual with CC genotype; The SNP site is g.3149486 A>G, located at the 3149486th base of NW_004973554.1, the egg production interval of the individual with GG genotype is significantly lower than that of the individual with AA genotype; The SNP site is g.3150038 T>C, located at the 3150038th base of NW_004973554.1, the egg production interval of the individual with TT genotype is extremely significantly lower than that of the individual with TC genotype, and the egg production interval of the individual with CC genotype is significantly lower than that of the individual with TC genotype; The SNP site is g.3151252 G>T, located at the 3151252th base of NW_004973554.1, the egg production interval of the individual with TT genotype is significantly lower than that of the individual with GG genotype; The SNP site is g.3152087 A>G, located at the 3152087th base of NW_004973554.1, the egg production interval of the individual with GG genotype is extremely significantly lower than that of the individual with AA genotype, and the egg production interval of the individual with GG genotype is significantly lower than that of the individual with AG genotype; The haplotype combination consists of three SNP sites: g.3149417 G>A, g.3149444 C>T and g.3149486 A>G, located at the 3149417th base, the 3149444th base and the 3149444th base of NW_004973554.1 respectively, The egg production interval of the individual with the genotype AA, TT and GG of the three SNP sites in turn is extremely significantly lower than that of the individual with the genotype GA, CC and AA of the three SNP sites in turn, The egg production interval of the individual with the genotype AA, TT and GG of the three SNP sites in turn is significantly lower than that of the individual with the genotype GG, CC and AA of the three SNP sites in turn, The egg production interval of the individual with the genotype GG, CC and AA of the three SNP sites in turn is extremely significantly lower than that of the individual with the genotype GA, CC and AA of the three SNP sites in turn, The egg production interval of the individual with the genotype GA, CT and AG of the three SNP sites in turn is extremely significantly lower than that of the individual with the genotype GA, CC and AA of the three SNP sites in turn; The haplotype group consists of five SNP sites: g.3151252 G>T, g.3151281 G>T, g.3152087 A>G, g.3152096 G>T and g.3152151 G>A, located at the 3151252th base, the 3151281th base, the 3152087th base, the 3152096th base and the 3152151th base of NW_004973554.1 respectively, The egg production interval of individuals with genotypes of TT, TT, GG, GG, and GG at the five SNP sites in order was significantly lower than that of individuals with genotypes of GG, GG, AA, GG, and GG at the five SNP sites in order.

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