Haplotype molecular marker detection reagent, kit, detection method related to meat pigeon breeding and application thereof

Abstract

The application discloses a detection reagent, a kit, a detection method and application of a haplotype molecular marker related to meat pigeon breeding. The application screens a haplotype molecular marker significantly related to meat pigeon reproduction and growth traits in an exon of a meat pigeon IGF2BP2 gene, and the haplotype molecular marker can be applied to early assisted selection of meat pigeon reproduction and growth traits. The haplotype molecular marker on the IGF2BP2 gene of the meat pigeon is detected by using the detection reagent, the kit and the detection method, so that the reproduction and growth performance of the meat pigeon can be quickly, effectively and accurately predicted, and the early breeding work of high-yield meat pigeons can be carried out, thereby breeding time is shortened and breeding progress is accelerated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular breeding, in particular to a detection reagent, a kit, a detection method of haplotype molecular markers related to meat pigeon breeding and application thereof. BACKGROUND

[0002] Pigeon meat is fresh and juicy, high in protein and low in fat, and has medicinal value, known as "animal ginseng", and is deeply loved by consumers, so the scale of meat pigeon breeding is expanding. However, the "2+3" and "2+4" production modes are often used in meat pigeon farms to ensure high production efficiency, but there is a lack of awareness of breeding work for breeding pigeons, resulting in degradation of breeding pigeons in the feeding process, such as smaller body size, longer egg production interval, fewer number of offspring per year, and smaller market weight.

[0003] With the development of molecular biology technology, great progress has been made in the research of molecular marker technology in livestock and poultry breeding. Combining molecular breeding with traditional breeding can carry out early breeding work, speed up genetic progress, and reduce breeding costs.

[0004] The IGF2BPs family includes IGF2BP1, IGF2BP2 and IGF2BP3 members, which are highly conserved post-transcriptional regulatory factors involved in RNA processing, localization, translation and stability. Most studies on IGF2BP2 have focused on the impact of polymorphisms in this gene on type 2 diabetes and different cancers, and there is no related report in meat pigeon breeding. SUMMARY

[0005] In order to overcome the above-mentioned defects and shortcomings in the prior art, the present application provides a detection reagent, a kit, a detection method of haplotype molecular markers related to meat pigeon breeding and application thereof.

[0006] The first object of the present application is to provide a haplotype molecular marker for meat pigeon breeding.

[0007] The second object of the present application is to provide a detection reagent for the haplotype molecular marker.

[0008] The third object of the present application is to provide the use of the haplotype molecular marker or the detection reagent in the preparation of a meat pigeon breeding product.

[0009] The fourth object of the present application is to provide a kit for meat pigeon breeding.

[0010] The fifth object of the present application is to provide a method for meat pigeon breeding.

[0011] Therefore, this invention claims protection for the following:

[0012] A haplotype molecular marker for use in pigeon breeding, the haplotype molecular marker consisting of five single nucleotide polymorphism sites SNP1, SNP2, SNP3, SNP4 and / or SNP5:

[0013] SNP1 is located at position 2306652 on the NW_004973255 gene. It is a C or T polymorphism and has two genotypes: CC and CT.

[0014] SNP2 is located at position 2306515 on the NW_004973255 gene. It is a G or T polymorphism and has three genotypes: GG, GT and TT.

[0015] SNP3 is located at position 2306459 of the NW_004973255 gene and is an A or G polymorphism, with two genotypes: AA and AG.

[0016] SNP4 is located at position 2306415 on the NW_004973255 gene. It is a C or T polymorphism and has three genotypes: CC, CT and TT.

[0017] SNP5 is located at position 2306381 on the NW_004973255 gene and is a G or A polymorphism, with two genotypes: GG and GA.

[0018] Individuals with the genotype CT for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GA for SNP5 had significantly higher body weight than individuals with the genotype CC for SNP1, GG for SNP2, AA for SNP3, CC for SNP4, and GG for SNP5.

[0019] Individuals with the genotype CT for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GA for SNP5 had significantly higher body weight than individuals with the genotype CC for SNP1, GT for SNP2, AA for SNP3, CT for SNP4, and GG for SNP5.

[0020] Individuals with the genotype CT for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GA for SNP5 had significantly higher body weight than individuals with the genotype CC for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GA for SNP5.

[0021] Individuals with the genotype CC for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GG for SNP5 had significantly higher body weight than individuals with the genotype CC for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GA for SNP5.

[0022] Individuals with the genotype CC for SNP1, GG for SNP2, AA for SNP3, CT for SNP4, and GG for SNP5 had significantly higher body weight than individuals with the genotype CC for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GA for SNP5.

[0023] Individuals with the genotype CC for SNP1, GG for SNP2, AA for SNP3, CT for SNP4, and GG for SNP5 had significantly greater chest depth than individuals with the genotype CC for SNP1, GT for SNP2, AA for SNP3, CT for SNP4, and GG for SNP5.

[0024] Individuals with the genotype CC for SNP1, GG for SNP2, AA for SNP3, CT for SNP4, and GG for SNP5 had significantly greater chest depth than individuals with the genotype CC for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GA for SNP5.

[0025] The above-mentioned haplotype molecular marker detection reagent is characterized in that the detection reagent is a primer with a nucleotide sequence as shown in SEQ ID NO: 19-20.

[0026] A haplotype molecular marker for use in pigeon breeding, the haplotype molecular marker consisting of two single nucleotide polymorphism sites, SNP6 and / or SNP7:

[0027] SNP6 is located at position 2314208 on the NW_004973255 gene. It is a T or C polymorphism and has three genotypes: TT, TC and CC.

[0028] SNP7 is located at position 2308546 on the NW_004973255 gene. It is a T or C polymorphism and has three genotypes: TT, TC and CC.

[0029] Individuals with the genotype CC for SNP6 and the genotype CC for SNP7 had significantly higher body weight than individuals with the genotype TT for SNP6 and the genotype CC for SNP7.

[0030] Individuals with the CC genotype for SNP6 and the CC genotype for SNP7 had significantly higher body weight than individuals with the CC genotype for SNP6 and the TT genotype for SNP7.

[0031] Individuals with the CC genotype for SNP6 and the CC genotype for SNP7 had significantly longer tibias than individuals with the CC genotype for SNP6 and the TT genotype for SNP7.

[0032] Individuals with SNP6 genotype TT and SNP7 genotype CT haplotype had significantly higher tibia circumference than individuals with SNP6 genotype CC and SNP7 genotype CC haplotype.

[0033] The keel length of individuals with genotype TT for SNP6 and CT for SNP7 was significantly higher than that of individuals with genotype TT for SNP6 and CC for SNP7.

[0034] Individuals with the TT genotype of SNP6 and the CT genotype of SNP7 had significantly higher body slant length than individuals with the TT genotype of SNP6 and the CC genotype of SNP7.

[0035] Individuals with SNP6 genotype TT and SNP7 genotype CT had significantly higher body slant length than individuals with SNP6 genotype TC and SNP7 genotype CT.

[0036] Individuals with the TT genotype of SNP6 and the CT genotype of SNP7 had significantly higher body slant lengths than individuals with the TC genotype of SNP6 and the CC genotype of SNP7.

[0037] Individuals with the TT genotype of SNP6 and the CT genotype of SNP7 had significantly longer body slant lengths than individuals with the CC genotype of SNP6 and the TT genotype of SNP7.

[0038] Individuals with the TT genotype of SNP6 and the CT genotype of SNP7 had significantly higher body slant lengths than individuals with the CC genotype of SNP6 and the CC genotype of SNP7.

[0039] Individuals with SNP6 genotype TC and SNP7 genotype CC had significantly higher egg production than individuals with SNP6 genotype CC and SNP7 genotype CC.

[0040] Individuals with genotype TT for SNP6 and CT for SNP7 had significantly higher egg production than individuals with genotype CC for SNP6 and CC for SNP7.

[0041] The above-mentioned detection reagent for haplotype molecular markers is characterized in that the detection reagent is a primer with a nucleotide sequence as shown in SEQ ID NO: 7-8 and / or a primer with a nucleotide sequence as shown in SEQ ID NO: 13-14.

[0042] The application of any of the above-mentioned haplotype molecular markers and / or detection reagents in the preparation of meat pigeon breeding products.

[0043] A kit for breeding meat pigeons, comprising any of the above-mentioned detection reagents.

[0044] Preferably, the kit further comprises Taq Plus MasterMix and / or ultrapure water.

[0045] A method for breeding meat pigeons, using the above-mentioned detection reagent to detect the above-mentioned haplotype molecular marker.

[0046] Preferably, it includes the following steps:

[0047] S1. Extract genomic DNA from the pigeons to be tested;

[0048] S2. Use the above detection reagents to perform PCR amplification on the genomic DNA obtained in step S1, and then sequence the PCR amplification products to obtain sequencing data;

[0049] S3. Analyze the sequencing data obtained in step S2, and determine the traits of the pigeons to be tested based on the haplotype molecular markers mentioned above.

[0050] As an implementable method, the sequencing described in step S2 is Sanger sequencing.

[0051] Preferably, the traits described in step S3 include body weight, chest depth, tibia length, tibia circumference, keel length, body oblique length, and / or egg production.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] This invention discloses detection reagents, kits, and methods for haplotype molecular markers related to pigeon breeding, as well as their applications. This invention screens exons of the pigeon IGF2BP2 gene to obtain haplotype molecular markers significantly associated with pigeon reproductive and growth traits, which can be applied to early auxiliary selection for these traits. Using the detection reagents, kits, and methods of this invention to detect haplotype molecular markers on the pigeon IGF2BP2 gene can rapidly, effectively, and accurately predict the reproductive and growth performance of pigeons, and can be used for early selection of high-yielding pigeons, thereby shortening breeding time and accelerating the breeding process. Attached Figure Description

[0054] Figure 1 Sequencing peak diagram of 15 SNP sites of the IGF2BP2 gene.

[0055] Figure 2 Linkage disequilibrium analysis of 15 SNPs in the IGF2BP2 gene; A: linkage diagram, B: haplotype analysis. Detailed Implementation

[0056] 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.

[0057] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0058] The pigeons used in this example are 28-month-old female Tianxiang No. 1 pigeons, which come from Guangdong Kangzheng Pigeon Industry Co., Ltd. The breeding pigeons of this factory adopt the production model of "2+4".

[0059] Example 1: Amplification of exons of the IGF2BP2 gene in pigeons

[0060] I. Experimental Methods

[0061] 1. Extraction and purity testing of blood DNA

[0062] 292 female pigeons were randomly selected, and DNA was extracted using a blood genomic DNA extraction kit. DNA integrity was detected by 1% agarose gel electrophoresis (w / v), and the OD value was measured by spectrophotometer. The DNA purity and concentration were determined based on the OD260:OD280 ratio, and the DNA of 292 female pigeons was obtained.

[0063] 2. Primer design

[0064] Using the pigeon IGF2BP2 gene sequence (GenBank accession number: NW_004973255) from the NCBI database as a reference sequence, and utilizing... Viewer 2.3.4 Design of exon-specific primers, primer sequences are shown in Table 1, synthesized by Shanghai Bioengineering (Sangon) Technology Service Co., Ltd., and diluted with sterile double-distilled water to a concentration of 10 μmol·L⁻¹. -1 Store in a -20℃ refrigerator for later use.

[0065] Table 1 Primer sequences

[0066]

[0067]

[0068] 3. PCR amplification

[0069] DNA samples from 292 female breeding pigeons were diluted to 40 ng / μL. Then, 5 μL of each DNA sample was transferred to 1.5 mL centrifuge tubes to make 10 pools, each pool containing 30 DNA samples (the 10th pool contained 22 DNA samples).

[0070] Using this as a template, PCR amplification was performed. The PCR reaction system consisted of: 10.0 μL of 2×Taq Plus MasterMix, 0.5 μL of upstream primer, 0.5 μL of downstream primer, 1.0 μL of DNA, and 8.0 μL of ddH2O.

[0071] The PCR reaction conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 45 s (the annealing temperatures of the primers for amplifying IGF2BP2-Exon 1 and IGF2BP2-Exon 13 were 72℃ and 54℃, respectively), 72℃ extension for 50 s, 32 cycles; 72℃ extension for 8 min.

[0072] The PCR amplification products were detected by 1.5% agarose gel electrophoresis (w / v). After confirming the target bands were correct, the samples were sent to Sangon Biotech for sequencing. SNP sites were obtained using SeqMan software, and then each DNA sample underwent PCR amplification and sequencing.

[0073] II. Experimental Results

[0074] The extracted DNA was analyzed using a spectrophotometer, and its OD260:OD280 ratio was between 1.8 and 2.0, indicating that the extracted DNA quality and purity met the standards. Agarose gel electrophoresis results showed that the DNA bands were neat, without tails or stray bands, and could be used for subsequent experiments.

[0075] PCR amplification was performed on the exons of the IGF2BP2 gene in pigeons. The amplification products were detected by 1.5% agarose gel electrophoresis (w / v), and the electrophoretic bands were all the desired target bands with good specificity, which can be used for subsequent sequencing.

[0076] Example 2: DNA pooling sequencing and SNP site analysis

[0077] I. Experimental Methods

[0078] After comparing the sequencing sequence with the pigeon IGF2BP2 gene sequence on NCBI using SeqMan software, the peak diagram in the sequencing results was observed, and the genotypes of the SNP sites were recorded.

[0079] II. Experimental Results

[0080] Sanger sequencing peak diagram of SNP locus genotypes is shown below. Figure 1 Fifteen SNP sites were detected in exons 3, 5, 9, and 13 of the pigeon IGF2BP2 gene. Based on their specific locations on the chromosome, these SNPs are named as follows:

[0081] g.2320439C>A on exon 3;

[0082] g.2314208T>C and g.2314196G>A on exon 5;

[0083] g.2308546T>C on exon 9;

[0084] The following numbers are listed on exon 13: g.2306652C>T, g.2306515G>T, g.2306484G>A, g.2306459A>G, g.2306429A>C, g.2306425C>T, g.2306415C>T, g.2306411C>G, g.2306396C>G, g.2306381G>A, and g.2306306T>G;

[0085] The specific information of the 15 SNP sites is shown in Table 2. As shown in Table 2, 5 SNPs (g.2320439C>A, g.2314208T>C, g.2314196G>A, g.2308546T>C, g.2306652C>T) are located in the coding region of exons. Among them, one SNP (g.2320439C>A) is a missense mutation, replacing alanine with glutamic acid. This change affects the structure and function of the protein, thus affecting the heritable trait. The other 4 sites (g.2314208T>C, g.2314196G>A, g.2308546T>C, g.2306652C>T) are synonymous mutations, which do not change the amino acid type.

[0086] Table 2 SNP locus information

[0087]

[0088]

[0089] Example 3: SNP locus genetic characteristics analysis

[0090] I. Experimental Methods

[0091] Use Microsoft Excel to calculate allele frequency, genotype frequency, genetic heterozygosity, effective allele count, polymorphism information content, and χ². 2 Values, etc.

[0092] II. Experimental Results

[0093] The analysis results are shown in Tables 3 and 4. As shown in Table 3, among the 15 SNPs of the IGF2BP2 gene, only 3 SNPs (g.2320439C>A, g.2314208T>C and g.2308546T>C) have heterozygous genotypes, while the dominant genotypes at the other loci are homozygous.

[0094] Table 3 Allelic information of IGF2BP2 gene SNPs

[0095]

[0096]

[0097] Hardy-Weinberg equilibrium via χ 2 The test results (Table 4) showed that three SNPs (g.2320439C>A, g.2314208T>C, and g.2308546T>C) deviated from Hardy-Weinberg equilibrium (P<0.05), while the other sites were in Hardy-Weinberg equilibrium (P>0.05). He and Ho results indicated that the observed heterozygosity of four SNPs (g.2314196G>A, g.2306515G>T, g.2306425C>T, and g.2306415C>T) was lower than expected, but the observed heterozygosity of the other SNPs was higher than expected. The calculated PIC values ​​indicate that the polymorphic information content (PIC) of the four SNPs (g.2320439C>A, g.2314208T>C, g.2314196G>A, and g.2308546T>C) is moderate (0.25 < PIC < 0.5), while the polymorphic information content (PIC) of the remaining points is all below 0.25, which is low.

[0098] Table 4. Hereditary index of IGF2BP2 gene SNPs

[0099]

[0100] Example 4: Linkage disequilibrium and haplotype analysis of IGF2BP2 gene SNPs

[0101] I. Experimental Methods

[0102] The linkage disequilibrium of SNP sites was analyzed using Haploview 4.2 software.

[0103] II. Experimental Results

[0104] When D'>0.8, R 2 A value >0.33 indicates a strong linkage disequilibrium among SNPs. Based on this condition, two LD blocks were constructed from 15 SNP sites of the IGF2BP2 gene, as shown in Table 5. Figure 2 As shown.

[0105] Block 1 consists of g.2306652C>T (SNP1), g.2306515G>T (SNP2), g.2306459A>G (SNP3), g.2306415C>T (SNP4), and g.2306381G>A (SNP5), and has 6 haplotypes, including H1, H2, H3, H4, H5, and H6.

[0106] Block 2 consists of g.2314208T>C (SNP6) and g.2308546T>C (SNP7), and it has 4 haplotypes: H7, H8, H9, and H10. After haplotype combinations, haplotype combinations with fewer than 3 individuals are not included in statistical analysis and comparison.

[0107] Table 5 Haplotype Composition of Linked Zones

[0108]

[0109] Subsequently, the correlation between haplotype combinations and traits was analyzed, and the results are shown in Table 6. Block 1 has 6 haplotype combinations, namely:

[0110] H1H1 (SNP1 genotype is CC, SNP2 genotype is GG, SNP3 genotype is AA, SNP4 genotype is CC and SNP5 genotype is GG);

[0111] H1H2 (SNP1 genotype is CC, SNP2 genotype is GT, SNP3 genotype is AA, SNP4 genotype is CT and SNP5 genotype is GG);

[0112] H1H3 (SNP1 genotype is CC, SNP2 genotype is GG, SNP3 genotype is AG, SNP4 genotype is CC and SNP5 genotype is GG);

[0113] H1H4 (SNP1 genotype is CC, SNP2 genotype is GG, SNP3 genotype is AG, SNP4 genotype is CC and SNP5 genotype is GA);

[0114] H1H5 (SNP1 genotype is CT, SNP2 genotype is GG, SNP3 genotype is AG, SNP4 genotype is CC and SNP5 genotype is GA);

[0115] H1H6 (SNP1 genotype is CC, SNP2 genotype is GG, SNP3 genotype is AA, SNP4 genotype is CT and SNP5 genotype is GG).

[0116] Block 2 has 6 haplotype combinations, namely:

[0117] H7H7 (SNP6 genotype is TT and SNP7 genotype is CC);

[0118] H7H8 (SNP6 genotype is TC and SNP7 genotype is CT);

[0119] H7H9 (SNP6 genotype is TC and SNP7 genotype is CC);

[0120] H7H10 (SNP6 genotype is TT and SNP7 genotype is CT);

[0121] H8H8 (SNP6 genotype is CC and SNP7 genotype is TT);

[0122] H9H9 (SNP6 genotype is CC and SNP7 genotype is CC).

[0123] The association results showed that the haplotype of Block 1 was significantly associated with body weight and chest depth (P<0.05); the haplotype of Block 2 was significantly associated with body weight, tibia circumference, tibia length, keel length, body oblique length and egg production (P<0.05).

[0124] Table 6 Association analysis between IGF2BP2 gene haplotype and traits

[0125]

[0126]

[0127]

[0128] Note: In the table, the same lowercase letter after the value in the same column of a single trait and the unlabeled letter value indicate that the difference between treatments is not significant, while different lowercase letters indicate that the difference between treatments is significant.

[0129] Example 5: Correlation Analysis Among Different Traits of Pigeons

[0130] I. Experimental Methods

[0131] The coefficient of variation is calculated as follows: Coefficient of variation = (Standard deviation / Mean) × 100%; the Pearson correlation coefficient is calculated using IBM SPSS 22.0 software.

[0132] II. Experimental Results

[0133] The average values ​​and coefficients of variation for different traits of pigeons are shown in Table 7. Table 7 shows that the coefficients of variation for body weight and egg-laying interval are relatively small, at 1.03% and 1.10%, respectively. This indicates that the dispersion of body weight and egg-laying interval is small and relatively concentrated, suggesting that the pigeons' meat production performance and egg-laying interval are relatively stable.

[0134] Table 7. Phenotypic statistics of pigeons

[0135]

[0136] The Pearson correlation coefficients are shown in Table 8. Table 8 shows that there is no significant correlation between chest depth and shank circumference, but there are highly significant positive correlations between other indicators (P<0.01). Therefore, it is evident that breeds can be selectively chosen based on the correlations between different traits to improve the meat production performance of pigeons.

[0137] Table 8. Pearson correlations among traits

[0138]

[0139]

[0140] Note: ** indicates a significant correlation at the 0.01 level (two-tailed); * indicates a significant correlation at the 0.05 level (two-tailed).

[0141] Example 6: Association Analysis of IGF2BP2 Gene SNPs with Pigeon Traits

[0142] I. Experimental Methods

[0143] The correlation between different genotypes of 15 SNPs and body weight, body size, and egg production performance was analyzed using the following models:

[0144] Y ijl =u+G i +e ijl

[0145] In the formula, Y ijl G represents the observed trait value; u represents the population mean; G represents the population mean. i This refers to the effect of the i-th genotype or haplotype at the SNP locus; e ijl The result represents random error and is expressed as "mean ± standard error". Significance of differences was tested using Duncan's method; P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference. For traits with unequal variances, the Tammhenko test was used for multiple comparisons; for traits with only two genotypes, the independent samples t-test was used.

[0146] II. Experimental Results

[0147] In the association analysis of 15 SNP sites of the IGF2BP2 gene with traits of meat pigeons, the association results of 7 SNPs involved in haplotype combinations with traits of meat pigeons are shown in Table 9.

[0148] The results showed that among the 15 SNPs in the IGF2BP2 gene:

[0149] There were significant differences in the laying interval trait among the three genotypes of SNP (g.2314208T>C), with the TC genotype having a significantly higher laying interval than the TT and CC genotypes (P<0.05); this locus was not significant for any other trait.

[0150] The three genotypes of SNP (g.2306515G>T) showed significant differences in body weight and chest width traits. The body weight of the TT genotype was significantly higher than that of the GT genotype, and the chest width of the GG genotype was significantly greater than that of the GT genotype (P<0.05). This locus was not significant for any other trait.

[0151] The three genotypes of SNP (g.2306415C>T) showed significant differences in body weight trait, with the TT genotype having a significantly higher body weight than the CT genotype (P<0.05); this locus was not significant for any other trait.

[0152] SNPs (g.2308546T>C), (g.2306652C>T), (g.2306459A>G), and (g.2306381G>A) were not significantly associated with growth or reproductive traits in pigeons.

[0153] Table 9. Association analysis of IGF2BP2 gene SNPs with growth and reproductive traits in pigeons.

[0154]

[0155] 5. Note: In the table, the same lowercase letter after the values ​​in the same column indicates that the difference between treatments is not significant, and different lowercase letters indicate that the difference between treatments is significant.

[0156] Significant.

[0157] Example 7: A method for breeding meat pigeons

[0158] 1. Extract DNA from the sample to be tested using a blood genomic DNA extraction kit.

[0159] 2. Using the DNA extracted from the sample in step 1 as a template, perform PCR amplification:

[0160] The PCR reaction system consisted of: 10.0 μL of 2×Taq Plus MasterMix, 0.5 μL each of primers with nucleotide sequences as shown in SEQ ID NO: 7-8, primers with nucleotide sequences as shown in SEQ ID NO: 13-14, or primers with nucleotide sequences as shown in SEQ ID NO: 19-20, 1.0 μL of DNA, and 8.0 μL of ddH2O.

[0161] The PCR reaction conditions are as follows:

[0162] (1) When using primers with nucleotide sequences as shown in SEQ ID NO: 7-8 or primers with nucleotide sequences as shown in SEQ ID NO: 13-14 for amplification: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 45 s, 72℃ extension for 50 s, 32 cycles; 72℃ extension for 8 min.

[0163] (2) When using primers with nucleotide sequences as shown in SEQ ID NO: 19-20 for amplification: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 45 s, 72℃ extension for 50 s, 32 cycles; 72℃ extension for 8 min.

[0164] 3. Perform agarose gel electrophoresis on the PCR amplification products obtained in step 2. If the electrophoretic bands are the same size as the target bands and are clear and bright, then sequence the PCR amplification products obtained in step 2.

[0165] 4. Result Interpretation:

[0166] SNP1 is located at position 2306652 on the NW_004973255 gene. It is a C or T polymorphism and has two genotypes: CC and CT.

[0167] SNP2 is located at position 2306515 on the NW_004973255 gene. It is a G or T polymorphism and has three genotypes: GG, GT and TT.

[0168] SNP3 is located at position 2306459 of the NW_004973255 gene and is an A or G polymorphism, with two genotypes: AA and AG.

[0169] SNP4 is located at position 2306415 on the NW_004973255 gene. It is a C or T polymorphism and has three genotypes: CC, CT and TT.

[0170] SNP5 is located at position 2306381 on the NW_004973255 gene and is a G or A polymorphism, with two genotypes: GG and GA.

[0171] SNP6 is located at position 2314208 on the NW_004973255 gene. It is a T or C polymorphism and has three genotypes: TT, TC and CC.

[0172] SNP7 is located at position 2308546 on the NW_004973255 gene. It is a T or C polymorphism and has three genotypes: TT, TC and CC.

[0173] Individuals with the genotype CT for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GA for SNP5 had significantly higher body weight than individuals with the genotype CC for SNP1, GG for SNP2, AA for SNP3, CC for SNP4, and GG for SNP5.

[0174] Individuals with the genotype CT for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GA for SNP5 had significantly higher body weight than individuals with the genotype CC for SNP1, GT for SNP2, AA for SNP3, CT for SNP4, and GG for SNP5.

[0175] Individuals with the genotype CT for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GA for SNP5 had significantly higher body weight than individuals with the genotype CC for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GA for SNP5.

[0176] Individuals with the genotype CC for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GG for SNP5 had significantly higher body weight than individuals with the genotype CC for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GA for SNP5.

[0177] Individuals with the genotype CC for SNP1, GG for SNP2, AA for SNP3, CT for SNP4, and GG for SNP5 had significantly higher body weight than individuals with the genotype CC for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GA for SNP5.

[0178] Individuals with the genotype CC for SNP1, GG for SNP2, AA for SNP3, CT for SNP4, and GG for SNP5 had significantly greater chest depth than individuals with the genotype CC for SNP1, GT for SNP2, AA for SNP3, CT for SNP4, and GG for SNP5.

[0179] Individuals with the genotype CC for SNP1, GG for SNP2, AA for SNP3, CT for SNP4, and GG for SNP5 had significantly greater chest depth than individuals with the genotype CC for SNP1, GG for SNP2, AG for SNP3, CC for SNP4, and GA for SNP5.

[0180] Individuals with the genotype CC for SNP6 and the genotype CC for SNP7 had significantly higher body weight than individuals with the genotype TT for SNP6 and the genotype CC for SNP7.

[0181] Individuals with the CC genotype for SNP6 and the CC genotype for SNP7 had significantly higher body weight than individuals with the CC genotype for SNP6 and the TT genotype for SNP7.

[0182] Individuals with the CC genotype for SNP6 and the CC genotype for SNP7 had significantly longer tibias than individuals with the CC genotype for SNP6 and the TT genotype for SNP7.

[0183] Individuals with SNP6 genotype TT and SNP7 genotype CT haplotype had significantly higher tibia circumference than individuals with SNP6 genotype CC and SNP7 genotype CC haplotype.

[0184] The keel length of individuals with genotype TT for SNP6 and CT for SNP7 was significantly higher than that of individuals with genotype TT for SNP6 and CC for SNP7.

[0185] Individuals with the TT genotype of SNP6 and the CT genotype of SNP7 had significantly higher body slant length than individuals with the TT genotype of SNP6 and the CC genotype of SNP7.

[0186] Individuals with SNP6 genotype TT and SNP7 genotype CT had significantly higher body slant length than individuals with SNP6 genotype TC and SNP7 genotype CT.

[0187] Individuals with the TT genotype of SNP6 and the CT genotype of SNP7 had significantly higher body slant lengths than individuals with the TC genotype of SNP6 and the CC genotype of SNP7.

[0188] Individuals with the TT genotype of SNP6 and the CT genotype of SNP7 had significantly longer body slant lengths than individuals with the CC genotype of SNP6 and the TT genotype of SNP7.

[0189] Individuals with the TT genotype of SNP6 and the CT genotype of SNP7 had significantly higher body slant lengths than individuals with the CC genotype of SNP6 and the CC genotype of SNP7.

[0190] Individuals with SNP6 genotype TC and SNP7 genotype CC had significantly higher egg production than individuals with SNP6 genotype CC and SNP7 genotype CC.

[0191] Individuals with genotype TT for SNP6 and CT for SNP7 had significantly higher egg production than individuals with genotype CC for SNP6 and CC for SNP7.

[0192] Example 8: A kit for meat pigeon breeding

[0193] I. Composition

[0194] Primers with nucleotide sequences as shown in SEQ ID NO: 7-8, primers with nucleotide sequences as shown in SEQ ID NO: 13-14, primers with nucleotide sequences as shown in SEQ ID NO: 19-20, Taq Plus MasterMix, and ddH2O.

[0195] II. Instructions for Use

[0196] The detection and result interpretation were carried out in accordance with Example 7.

[0197] 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 use of reagents for detecting haplotype SNP molecular marker combinations for meat pigeon breeding in the preparation of a meat pigeon breeding product, characterized in that, The haplotype SNP molecular marker combination consists of 5 single nucleotide polymorphism sites SNP1, SNP2, SNP3, SNP4 and SNP5 SNP1 is located at position 2306652 on NW_004973255, is a C or T polymorphism, and exists in two genotypes of CC and CT; SNP2 is located at position 2306515 on NW_004973255, is a G or T polymorphism, and exists in three genotypes of GG, GT and TT; SNP3 is located at position 2306459 on NW_004973255, is an A or G polymorphism, and exists in two genotypes of AA and AG; SNP4 is located at position 2306415 on NW_004973255, is a C or T polymorphism, and exists in three genotypes of CC, CT and TT; SNP5 is located at position 2306381 on NW_004973255, is a G or A polymorphism, and exists in two genotypes of GG and GA; The body weight of individuals with the haplotype of the genotype of SNP1 being CT, the genotype of SNP2 being GG, the genotype of SNP3 being AG, the genotype of SNP4 being CC and the genotype of SNP5 being GA is significantly higher than that of individuals with the haplotype of the genotype of SNP1 being CC, the genotype of SNP2 being GG, the genotype of SNP3 being AA, the genotype of SNP4 being CC and the genotype of SNP5 being GG; The body weight of individuals with the haplotype of the genotype of SNP1 being CT, the genotype of SNP2 being GG, the genotype of SNP3 being AG, the genotype of SNP4 being CC and the genotype of SNP5 being GA is significantly higher than that of individuals with the haplotype of the genotype of SNP1 being CC, the genotype of SNP2 being GT, the genotype of SNP3 being AA, the genotype of SNP4 being CT and the genotype of SNP5 being GG; The body weight of individuals with the haplotype of the genotype of SNP1 being CT, the genotype of SNP2 being GG, the genotype of SNP3 being AG, the genotype of SNP4 being CC and the genotype of SNP5 being GA is significantly higher than that of individuals with the haplotype of the genotype of SNP1 being CC, the genotype of SNP2 being GG, the genotype of SNP3 being AG, the genotype of SNP4 being CC and the genotype of SNP5 being GA; The body weight of individuals with the haplotype of the genotype of SNP1 being CC, the genotype of SNP2 being GG, the genotype of SNP3 being AG, the genotype of SNP4 being CC and the genotype of SNP5 being GG is significantly higher than that of individuals with the haplotype of the genotype of SNP1 being CC, the genotype of SNP2 being GG, the genotype of SNP3 being AG, the genotype of SNP4 being CC and the genotype of SNP5 being GA; The body weight of individuals with the haplotype of the genotype of SNP1 being CC, the genotype of SNP2 being GG, the genotype of SNP3 being AA, the genotype of SNP4 being CT and the genotype of SNP5 being GG is significantly higher than that of individuals with the haplotype of the genotype of SNP1 being CC, the genotype of SNP2 being GG, the genotype of SNP3 being AG, the genotype of SNP4 being CC and the genotype of SNP5 being GA; ​ The thoracic depth of the individual with the genotype CC of SNP1, the genotype GG of SNP2, the genotype AA of SNP3, the genotype CT of SNP4 and the genotype GG of SNP5 is significantly higher than that of the individual with the genotype CC of SNP1, the genotype GT of SNP2, the genotype AA of SNP3, the genotype CT of SNP4 and the genotype GG of SNP5; The thoracic depth of the individual with the genotype CC of SNP1, the genotype GG of SNP2, the genotype AA of SNP3, the genotype CT of SNP4 and the genotype GG of SNP5 is significantly higher than that of the individual with the genotype CC of SNP1, the genotype GG of SNP2, the genotype AG of SNP3, the genotype CC of SNP4 and the genotype GA of SNP5.

2. Use according to claim 1, characterized in that, The detection reagent is a primer with the nucleotide sequence shown in SEQ ID NO: 19-20.

3. Use of reagents for detecting haplotype SNP molecular marker combinations for meat pigeon breeding in the preparation of a meat pigeon breeding product, characterized in that, The haplotype SNP molecular marker combination consists of two single nucleotide polymorphism sites SNP6 and SNP7: SNP6 is located at position 2314208 on NW_004973255, and is a T or C polymorphism, which exists in three genotypes of TT, TC and CC; SNP7 is located at position 2308546 on NW_004973255, and is a T or C polymorphism, which exists in three genotypes of TT, TC and CC; The body weight of the individual with the genotype CC of SNP6 and the genotype CC of SNP7 is significantly higher than that of the individual with the genotype TT of SNP6 and the genotype CC of SNP7; The body weight of the individual with the genotype CC of SNP6 and the genotype CC of SNP7 is significantly higher than that of the individual with the genotype CC of SNP6 and the genotype TT of SNP7; The tibia length of the individual with the genotype CC of SNP6 and the genotype CC of SNP7 is significantly higher than that of the individual with the genotype CC of SNP6 and the genotype TT of SNP7; The tibia girth of the individual with the genotype TT of SNP6 and the genotype CT of SNP7 is significantly higher than that of the individual with the genotype CC of SNP6 and the genotype CC of SNP7; The tibia girth of the individual with the genotype TT of SNP6 and the genotype CT of SNP7 is significantly higher than that of the individual with the genotype TT of SNP6 and the genotype CC of SNP7; The tibia girth of the individual with the genotype TT of SNP6 and the genotype CT of SNP7 is significantly higher than that of the individual with the genotype TC of SNP6 and the genotype CT of SNP7; The tibia girth of the individual with the genotype TT of SNP6 and the genotype CT of SNP7 is significantly higher than that of the individual with the genotype TC of SNP6 and the genotype CC of SNP7; ​ The body length of the individual with genotype TT of SNP6 and genotype CT of SNP7 is significantly higher than that of the individual with genotype CC of SNP6 and genotype TT of SNP7; The body length of the individual with genotype TT of SNP6 and genotype CT of SNP7 is significantly higher than that of the individual with genotype CC of SNP6 and genotype CC of SNP7; The egg production of the individual with genotype TC of SNP6 and genotype CC of SNP7 is significantly higher than that of the individual with genotype CC of SNP6 and genotype CC of SNP7; The egg production of the individual with genotype TT of SNP6 and genotype CT of SNP7 is significantly higher than that of the individual with genotype CC of SNP6 and genotype CC of SNP7.

4. Use according to claim 3, characterized in that, The detection reagent is a primer with nucleotide sequence as shown in SEQ ID NO: 7-8 and a primer with nucleotide sequence as shown in SEQ ID NO: 13-14.

5. A method for breeding of meat pigeons, characterized in that, The haplotype SNP molecular marker combination as claimed in claim 1 is detected; The body weight of the individual with genotype CT of SNP1, genotype GG of SNP2, genotype AG of SNP3, genotype CC of SNP4 and genotype GA of SNP5 is significantly higher than that of the individual with genotype CC of SNP1, genotype GG of SNP2, genotype AA of SNP3, genotype CC of SNP4 and genotype GG of SNP5; The body weight of the individual with genotype CT of SNP1, genotype GG of SNP2, genotype AG of SNP3, genotype CC of SNP4 and genotype GA of SNP5 is significantly higher than that of the individual with genotype CC of SNP1, genotype GT of SNP2, genotype AA of SNP3, genotype CT of SNP4 and genotype GG of SNP5; The body weight of the individual with genotype CT of SNP1, genotype GG of SNP2, genotype AG of SNP3, genotype CC of SNP4 and genotype GA of SNP5 is significantly higher than that of the individual with genotype CC of SNP1, genotype GG of SNP2, genotype AG of SNP3, genotype CC of SNP4 and genotype GA of SNP5; The body weight of the individual with genotype CC of SNP1, genotype GG of SNP2, genotype AG of SNP3, genotype CC of SNP4 and genotype GG of SNP5 is significantly higher than that of the individual with genotype CC of SNP1, genotype GG of SNP2, genotype AG of SNP3, genotype CC of SNP4 and genotype GA of SNP5; The body weight of the individual with the genotype of CC of SNP1, the genotype of GG of SNP2, the genotype of AA of SNP3, the genotype of CT of SNP4 and the genotype of GG of SNP5 is significantly higher than the individual with the genotype of CC of SNP1, the genotype of GG of SNP2, the genotype of AG of SNP3, the genotype of CC of SNP4 and the genotype of GA of SNP5; The chest depth of the individual with the genotype of CC of SNP1, the genotype of GG of SNP2, the genotype of AA of SNP3, the genotype of CT of SNP4 and the genotype of GG of SNP5 is significantly higher than the individual with the genotype of CC of SNP1, the genotype of GT of SNP2, the genotype of AA of SNP3, the genotype of CT of SNP4 and the genotype of GG of SNP5; The chest depth of the individual with the genotype of CC of SNP1, the genotype of GG of SNP2, the genotype of AA of SNP3, the genotype of CT of SNP4 and the genotype of GG of SNP5 is significantly higher than the individual with the genotype of CC of SNP1, the genotype of GG of SNP2, the genotype of AG of SNP3, the genotype of CC of SNP4 and the genotype of GA of SNP5.

6. A method for breeding of meat pigeons, characterized in that, Detecting the haplotype SNP molecular marker combination in claim 3; The body weight of the individual with the genotype of CC of SNP6 and the genotype of CC of SNP7 is significantly higher than the individual with the genotype of TT of SNP6 and the genotype of CC of SNP7; The body weight of the individual with the genotype of CC of SNP6 and the genotype of CC of SNP7 is significantly higher than the individual with the genotype of CC of SNP6 and the genotype of TT of SNP7; The tibia length of the individual with the genotype of CC of SNP6 and the genotype of CC of SNP7 is significantly higher than the individual with the genotype of CC of SNP6 and the genotype of TT of SNP7; The tibia girth of the individual with the genotype of TT of SNP6 and the genotype of CT of SNP7 is significantly higher than the individual with the genotype of CC of SNP6 and the genotype of CC of SNP7; The metacarpus length of the individual with the genotype of TT of SNP6 and the genotype of CT of SNP7 is significantly higher than the individual with the genotype of TT of SNP6 and the genotype of CC of SNP7; The body oblique length of the individual with the genotype of TT of SNP6 and the genotype of CT of SNP7 is significantly higher than the individual with the genotype of TT of SNP6 and the genotype of CC of SNP7; The body oblique length of the individual with the genotype of TT of SNP6 and the genotype of CT of SNP7 is significantly higher than the individual with the genotype of TC of SNP6 and the genotype of CT of SNP7; The body oblique length of the individual with the genotype of TT of SNP6 and the genotype of CT of SNP7 is significantly higher than the individual with the genotype of TC of SNP6 and the genotype of CC of SNP7; The body girth of the individual with the genotype TT of SNP6 and the genotype CT of SNP7 haplotype is significantly higher than that of the individual with the genotype CC of SNP6 and the genotype TT of SNP7 haplotype; The body girth of the individual with the genotype TT of SNP6 and the genotype CT of SNP7 haplotype is significantly higher than that of the individual with the genotype CC of SNP6 and the genotype CC of SNP7 haplotype; The egg production of the individual with the genotype TC of SNP6 and the genotype CC of SNP7 haplotype is significantly higher than that of the individual with the genotype CC of SNP6 and the genotype CC of SNP7 haplotype; The egg production of the individual with the genotype TT of SNP6 and the genotype CT of SNP7 haplotype is significantly higher than that of the individual with the genotype CC of SNP6 and the genotype CC of SNP7 haplotype.

7. The method according to claim 5 or 6, characterized in that, The method comprises the following steps: S1. Extracting the genomic DNA of the meat pigeon to be tested; S2. performing PCR amplification on the genomic DNA obtained in step S1 by using reagents, and then performing sequencing on the PCR amplification product to obtain sequencing data; S3. analyzing the sequencing data obtained in step S2, and determining the traits of the meat pigeon to be tested according to the haplotype SNP molecular marker combination.

8. The method of claim 7, wherein, The sequencing in step S2 is Sanger sequencing.

9. The method of claim 7, wherein, The traits in step S3 are body weight, chest depth, shank length, shank circumference, keel length, body girth and / or egg production.

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