CN118531135A - Detection reagent, kit and detection method of haplotype molecular marker related to breeding of meat pigeons and application of detection reagent and kit - Google Patents
Detection reagent, kit and detection method of haplotype molecular marker related to breeding of meat pigeons and application of detection reagent and kit Download PDFInfo
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Abstract
The invention discloses a detection reagent, a kit, a detection method and application of a haplotype molecular marker related to breeding of meat pigeons. The haplotype molecular marker remarkably related to the breeding and growth traits of the pigeons is obtained by screening the exons of the pigeon IGF2BP2 gene, and can be applied to early auxiliary selection of the breeding and growth traits of the pigeons. The detection reagent, the kit and the detection method are used for detecting the haplotype molecular marker on the IGF2BP2 gene of the meat pigeon, can rapidly, effectively and accurately predict the reproduction and growth performance of the meat pigeon, and can be used for early breeding work of the high-yield meat pigeon, thereby shortening the breeding time and accelerating the breeding process.
Description
Technical Field
The invention relates to the technical field of molecular breeding, in particular to a detection reagent, a kit, a detection method and application of a haplotype molecular marker related to breeding of meat pigeons.
Background
The pigeon meat is fresh, tender and succulent, has high protein and low fat, has medicinal value, is called as animal ginseng, and is deeply favored by consumers, so that the breeding scale of the pigeon meat is continuously enlarged. However, the production modes of 2+3 and 2+4 are often adopted in the production of the meat pigeon farm to ensure higher production efficiency of the meat pigeons, but the awareness of breeding work of the breeding pigeons is lacking, so that the breeds of the breeding pigeons are degraded in the breeding process, such as smaller size of the meat pigeons, longer egg laying interval time, smaller number of annual young pigeons, smaller weight of the young pigeons on the market and the like. Researches show that the interval difference between the eggs of the meat pigeons is larger by adopting different production modes, and the consumption of the breeding pigeons in the process of feeding offspring is large.
With the development of molecular biology technology, the research of molecular marking technology in livestock breeding has been greatly advanced. The molecular breeding is combined with the traditional breeding, so that the early breeding work of the variety can be developed, the genetic progress is quickened, and the breeding cost is reduced.
The IGF2BPs family, which includes IGF2BP1, IGF2BP2, and IGF2BP3 members, is a highly conserved post-transcriptional regulator involved in RNA processing, localization, translation, and stability. Among them, most of researches on IGF2BP2 mainly focus on the influence of polymorphism of the gene on type 2 diabetes and different cancers, and no relevant report is found in breeding of meat pigeons.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the invention provides a detection reagent, a kit, a detection method and application of a haplotype molecular marker related to breeding of meat pigeons.
The first object of the invention is to provide a haplotype molecular marker for breeding meat pigeons.
It is a second object of the present invention to provide a detection reagent for the above-mentioned haplotype molecular markers.
The third object of the invention is to provide the application of the haplotype molecular marker or the detection reagent in the preparation of breeding products of meat pigeons.
The fourth object of the invention is to provide a kit for breeding meat pigeons.
A fifth object of the invention is to provide a method for breeding meat pigeons.
Accordingly, the present invention claims the following:
a haplotype molecular marker for breeding meat pigeons, the haplotype molecular marker consisting of 5 single nucleotide polymorphic sites SNP1, SNP2, SNP3, SNP4 and/or SNP 5:
SNP1 is located at 2306652 on NW_004973255 gene, is C or T polymorphism, and exists in both CC and CT genotypes;
SNP2 is located at 2306515 on NW_004973255 gene, is a G or T polymorphism, and exists in three genotypes GG, GT and TT;
SNP3 is located at 2306459 on NW_004973255 gene, is A or G polymorphism, and exists in both AA and AG genotypes;
SNP4 is located at 2306415 on NW_004973255 gene, is C or T polymorphism, and exists in three genotypes of CC, CT and TT;
SNP5 is located at 2306381 on NW_004973255 gene, is a G or A polymorphism, and exists in both GG and GA genotypes;
Individuals with genotype CT for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4, and genotype GA haplotype for SNP5 are significantly heavier than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AA for SNP3, genotype CC for SNP4, and genotype GG haplotype for SNP 5;
individuals with genotype CT for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4, and genotype GA haplotype for SNP5 are significantly heavier than individuals with genotype CC for SNP1, genotype GT for SNP2, genotype AA for SNP3, genotype CT for SNP4, and genotype GG haplotype for SNP 5;
Individuals with genotype CT for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP5 have a significantly higher body weight than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP 5;
individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GG haplotype for SNP5 have significantly higher body weight than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP 5;
individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AA for SNP3, genotype CT for SNP4 and genotype GG haplotype for SNP5 weigh significantly higher than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP 5;
Individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AA for SNP3, genotype CT for SNP4 and genotype GG haplotype for SNP5 have significantly higher chest depth than individuals with genotype CC for SNP1, genotype GT for SNP2, genotype AA for SNP3, genotype CT for SNP4 and genotype GG haplotype for SNP 5;
Individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AA for SNP3, genotype CT for SNP4 and genotype GG haplotype for SNP5 have significantly higher chest depth than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP 5.
The detection reagent of the haplotype molecular marker is characterized in that the detection reagent is a nucleotide sequence shown as SEQ ID NO:19 to 20.
A haplotype molecular marker for breeding meat pigeons, the haplotype molecular marker consisting of 2 single nucleotide polymorphic sites SNP6 and/or SNP 7:
SNP6 is located at 2314208 on NW_004973255 gene, is a T or C polymorphism, and exists in three genotypes of TT, TC and CC;
SNP7 is located at 2308546 on NW_004973255 gene, is a T or C polymorphism, and exists in three genotypes of TT, TC and CC;
individuals with genotype of SNP6 of CC and genotype of SNP7 of CC haplotype weigh significantly higher than individuals with genotype of SNP6 of TT and genotype of SNP7 of CC haplotype;
individuals with genotype of SNP6 of CC and genotype of SNP7 of CC haplotype weigh significantly higher than individuals with genotype of SNP6 of CC and genotype of SNP7 of TT haplotype;
the individual with genotype of SNP6 of CC and genotype of SNP7 of CC haplotype has a shank length significantly higher than the individual with genotype of SNP6 of CC and genotype of SNP7 of TT haplotype;
The shin circumference of the individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype is significantly higher than that of the individuals with genotype of SNP6 being CC and genotype of SNP7 being CC haplotype;
The keel length of the individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype is significantly higher than that of the individuals with genotype of SNP6 being TT and genotype of SNP7 being CC haplotype;
Individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype have significantly higher body diagonal length than individuals with genotype of SNP6 being TT and genotype of SNP7 being CC haplotype;
individuals with genotype TT for SNP6 and genotype CT haplotype for SNP7 have significantly higher body diagonal lengths than individuals with genotype TC for SNP6 and genotype CT haplotype for SNP 7;
individuals with genotype of SNP6 of TT and genotype of SNP7 of CT haplotype have significantly higher body diagonal length than individuals with genotype of SNP6 of TC and genotype of SNP7 of CC haplotype;
individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype have significantly higher body diagonal length than individuals with genotype of SNP6 being CC and genotype of SNP7 being TT haplotype;
Individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype have significantly higher body diagonal length than individuals with genotype of SNP6 being CC and genotype of SNP7 being CC haplotype;
The number of eggs laid by individuals with genotype TC of SNP6 and genotype of SNP7 of CC haplotype is significantly higher than those with genotype of SNP6 of CC and genotype of SNP7 of CC haplotype;
Individuals with genotype TT for SNP6 and genotype CT haplotype for SNP7 give significantly higher numbers of eggs than individuals with genotype CC for SNP6 and genotype CC haplotype for SNP 7.
The detection reagent of the haplotype molecular marker is characterized in that the detection reagent is a nucleotide sequence shown as SEQ ID NO: 7-8 and/or the nucleotide sequence of the primer is shown as SEQ ID NO:13 to 14.
The application of any one of the haplotype molecular markers and/or detection reagents in the preparation of meat pigeon breeding products.
A kit for breeding meat pigeons, comprising any one of the above detection reagents.
Preferably, the kit further comprises Taq Plus MasterMix and/or ultrapure water.
A method for breeding meat pigeons, wherein the haplotype molecular markers are detected by the detection reagent.
Preferably, the method comprises the following steps:
S1, extracting genomic DNA of a meat pigeon to be detected;
S2, carrying out PCR amplification on the genome DNA obtained in the step S1 by using the detection reagent, and then sequencing a PCR amplification product to obtain sequencing data;
s3, analyzing the sequencing data obtained in the step S2, and judging the characters of the meat pigeons to be tested according to the haplotype molecular markers.
As an example, the sequencing described in step S2 is Sanger sequencing.
Preferably, the traits described in step S3 include body weight, chest depth, shin length, shin circumference, keel length, body bias length and/or egg number.
Compared with the prior art, the invention has the following beneficial effects:
The invention discloses a detection reagent, a kit, a detection method and application of a haplotype molecular marker related to breeding of meat pigeons. The haplotype molecular marker remarkably related to the breeding and growth traits of the pigeons is obtained by screening the exons of the pigeon IGF2BP2 gene, and can be applied to early auxiliary selection of the breeding and growth traits of the pigeons. The detection reagent, the kit and the detection method are used for detecting the haplotype molecular marker on the IGF2BP2 gene of the meat pigeon, can rapidly, effectively and accurately predict the reproduction and growth performance of the meat pigeon, and can be used for early breeding work of the high-yield meat pigeon, thereby shortening the breeding time and accelerating the breeding process.
Drawings
FIG. 1 is a diagram showing the sequencing peaks of 15 SNP sites of IGF2BP2 gene.
FIG. 2 shows the analysis of 15 SNP linkage disequilibrium of IGF2BP2 gene; a: linkage map, B: haplotype analysis.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The meat pigeon used in the examples was 28 month old female Tianxiang No. 1 pigeon, from Guangdong Kang Zhengge, inc., and the production model adopted by the breeding pigeon in the factory was "2+4".
Example 1 amplification of the exon of the meat pigeon IGF2BP2 Gene
1. Experimental method
1. Extraction and purity detection of blood DNA
292 Female meat pigeons were randomly selected, DNA was extracted with a blood genomic DNA extraction kit, DNA integrity was checked by electrophoresis on a 1% agarose gel (w/v), OD values were determined with a spectrophotometer, and according to OD260: OD280 judges the purity and concentration of DNA to obtain 292 female meat pigeon DNA.
2. Primer design
The pigeon IGF2BP2 gene sequence (GenBank accession number: NW_ 004973255) in NCBI database is used as a reference sequenceThe specific primers of exons were designed by Viewer 2.3.4, the primer sequences are shown in Table 1, and the primers were synthesized by Shanghai biological engineering (Biotechnology) service Co., ltd, diluted to 10. Mu. Mol.L -1 with sterilized double distilled water, and placed in a refrigerator at-20℃for use.
TABLE 1 primer sequences
PCR amplification
The DNA samples of 292 female breeding pigeons were diluted to 40 ng/. Mu.L respectively, and 10 pools were prepared by pipetting 5. Mu.L to 1.5mL from each DNA sample into a centrifuge tube, each pool having 30 DNA samples (22 DNA samples were in the 10 th pool).
Taking the primer as a template to carry out PCR amplification reaction, wherein a PCR reaction system is as follows: 2 XTaq Plus Master mix 10.0. Mu.L, upstream primer 0.5. Mu.L, downstream primer 0.5. Mu.L, DNA 1.0. Mu.L, ddH 2 O8.0. Mu.L.
The PCR reaction conditions were: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 60℃for 45s (wherein the annealing temperatures for the primers when amplifying IGF2BP2-Exon 1 and IGF2BP2-Exon 13 were 72℃and 54℃respectively), extension at 72℃for 50s,32 cycles; extending at 72℃for 8min.
The PCR amplified product was subjected to 1.5% agarose gel electrophoresis (w/v), and the target band was confirmed to be clear and sent to the Bio-company for sequencing. After obtaining SNP sites by SeqMan software, PCR amplification and sequencing were performed on each DNA sample.
2. Experimental results
The extracted DNA was detected using a spectrophotometer, OD260: OD280 is between 1.8 and 2.0, which means that the quality and purity of the extracted DNA reach the standards. The agarose gel electrophoresis detection result shows that the DNA electrophoresis strip is neat, has no tailing and impurity bands, and can be used for subsequent experiments.
The exons of the meat pigeon IGF2BP2 gene are respectively amplified by PCR, and the amplified products are detected by 1.5% agarose gel electrophoresis (w/v), so that the electrophoresis bands are all the required target bands, and the specificity is good, and the meat pigeon IGF2BP2 gene can be used for subsequent sequencing.
Example 2DNA pool sequencing and SNP site analysis
1. Experimental method
After aligning the sequencing sequence with the pigeon IGF2BP2 gene sequence at NCBI using SeqMan software, peak patterns in the sequencing results were observed and the genotype of the SNP site was recorded.
2. Experimental results
The Sanger sequencing peak diagram of SNP locus genotype is shown in FIG. 1. 15 SNP sites were detected on exons 3, 5, 9 and 13 of the pigeon IGF2BP2 gene. SNPs are named according to their specific position on the chromosome, they are:
G.2320439c > a on exon 3;
G.2314208t > C, g.2314196g > a on exon 5;
G.2308546t > C on exon 9;
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 on exon 13;
The specific information of the 15 SNP loci is shown in Table 2. As can be seen from Table 2, 5 SNPs (g.2320439C > A, g.2314208T > C, g.2314196G > A, g.2308546T > C, g.2306652C > T) are located in the exon coding region, wherein 1 SNP (g.2320439C > A) is a missense mutation, a substitution from alanine to glutamate, which change affects the structure and function of the protein, and thus the genetic trait. The other 4 sites (g.2314208T > C, g.2314196G > A, g.2308546T > C, g.2306652C > T) are synonymous mutations, without changing the amino acid type.
TABLE 2SNP site information
Example 3SNP locus genetic characterization
1. Experimental method
Allele frequency, genotype frequency, genetic heterozygosity, effective allele factors, polymorphic information content, χ 2 values, etc. were calculated using Microsoft Excel software.
2. Experimental results
The analysis results are shown in tables 3 and 4. As can be seen from table 3: of the 15 SNPs in IGF2BP2 gene, only 3 SNPs (g.2320439C > A, g.2314208T > C and g.2308546T > C) have heterozygous dominant genotypes, and other loci have homozygous dominant genotypes.
TABLE 3 allele information of SNP of IGF2BP2 gene
Hardy-Weinberg equilibrium was examined by χ 2 (Table 4), and 3 SNPs (g.2320439C > A, g.2314208T > C, and g.2308546T > C) were deviated from Hardy-Weinberg equilibrium (P < 0.05) and the other sites were in Hardy-Weinberg equilibrium (P > 0.05). He and Ho results showed that the observed heterozygosity of the 4 SNPs (g.2314196 g > a, g.2306515g > T, g.2306425c > T, g.2306415c > T) was lower than the expected heterozygosity, but the other SNPs observed were higher than the expected heterozygosity. PIC calculation values show that the Polymorphism Information Content (PIC) of 4 SNPs (g.2320439C > A, g.2314208T > C, g.2314196G > A and g.2308546T > C) belongs to moderate polymorphism (0.25 < PIC < 0.5), and the Polymorphism Information Content (PIC) of each other site is lower than 0.25 and belongs to low polymorphism.
TABLE 4 genetic index of IGF2BP2 Gene SNP
Example 4 linkage disequilibrium and haplotype analysis of SNP in IGF2BP2 Gene
1. Experimental method
The linkage disequilibrium relationship of SNP loci was analyzed using Haploview 4.2 software.
2. Experimental results
When D' >0.8, R 2 >0.33, it was shown that there was a strong linkage disequilibrium between SNPs. According to this condition, 2 LD blocks were constructed at 15 SNP sites of IGF2BP2 gene, as shown in table 5 and fig. 2.
Block 1 consisted of g.2306652C>T(SNP1),g.2306515G>T(SNP2),g.2306459A>G(SNP3),g.2306415C>T(SNP4),g.2306381G>A(SNP5) with 6 haplotypes including H1, H2, H3, H4, H5 and H6.
Block 2 consisted of g.2314208T > C (SNP 6), g.2308546T > C (SNP 7), with H7, H8, H9 and H10 haplotypes. After haplotype combinations, haplotype combinations with individuals less than 3 did not participate in statistical analysis and comparison.
TABLE 5 haplotype composition of linkage regions
Subsequently, the correlation between the haplotype combinations and the traits was analyzed, and the results are shown in Table 6. Block1 has 6 haplotype combinations of:
H1H1 (genotype of SNP1 is CC, genotype of SNP2 is GG, genotype of SNP3 is AA, genotype of SNP4 is CC and genotype of SNP5 is GG);
H1H2 (genotype of SNP1 CC, genotype of SNP2 GT, genotype of SNP3 AA, genotype of SNP4 CT and genotype of SNP5 GG);
H1H3 (genotype of SNP1 is CC, genotype of SNP2 is GG, genotype of SNP3 is AG, genotype of SNP4 is CC, and genotype of SNP5 is GG);
H1H4 (genotype of SNP1 is CC, genotype of SNP2 is GG, genotype of SNP3 is AG, genotype of SNP4 is CC and genotype of SNP5 is GA);
H1H5 (genotype of SNP1 is CT, genotype of SNP2 is GG, genotype of SNP3 is AG, genotype of SNP4 is CC and genotype of SNP5 is GA);
H1H6 (genotype of SNP1 is CC, genotype of SNP2 is GG, genotype of SNP3 is AA, genotype of SNP4 is CT, and genotype of SNP5 is GG);
Block 2 has 6 haplotype combinations of:
h7 (genotype of SNP6 is TT and genotype of SNP7 is CC);
H7H8 (genotype of SNP6 is TC and genotype of SNP7 is CT);
H7H9 (genotype of SNP6 is TC and genotype of SNP7 is CC);
H7H10 (genotype of SNP6 is TT and genotype of SNP7 is CT);
H8 (genotype of SNP6 is CC and genotype of SNP7 is TT);
H9H9 (genotype of SNP6 is CC and genotype of SNP7 is CC).
Correlation results show that the haplotype of Block 1 is significantly related to body weight and chest depth traits (P < 0.05); the haplotype of Block 2 was significantly related to body weight, shin circumference, shin length, keel length, body diagonal length and egg number traits (P < 0.05).
TABLE 6 correlation analysis of IGF2BP2 Gene haplotypes and Properties
Note that: the same lowercase letters and untagged letter values following the same column of values within a single trait in the table represent insignificant differences between treatments, and different lowercase letters represent significant differences between treatments.
EXAMPLE 5 analysis of correlation between different shapes of meat pigeons
1. Experimental method
The coefficient of variation is calculated by the following steps: coefficient of variation= (standard deviation/average value) ×100%; the Pearson correlation coefficient was calculated using IBM SPSS22.0 software.
2. Experimental results
The average value and the variation coefficient of the different shapes of the meat pigeons are shown in Table 7. As is clear from Table 7, the variation coefficients were smaller, namely, the body weight and the egg laying interval were 1.03% and 1.10%, respectively. Therefore, the weight and the egg laying interval are small in dispersity and relatively concentrated, which indicates that the meat production performance and the egg laying interval of pigeons are relatively stable.
TABLE 7 phenotype statistics of pigeons
The Pearson correlation coefficients are shown in table 8. As can be seen from table 8, there is no significant correlation between chest depth and shin circumference, but there is very significant positive correlation between other indices (P < 0.01). It follows that the variety can be selectively selected according to the correlation between different properties to improve the meat performance of the meat pigeon.
TABLE 8 Pearson correlation between traits
Note that: * Represent a significant correlation at the 0.01 level (two-tailed); * Representing a significant correlation at the 0.05 level (double tail).
Example 6 association analysis of IGF2BP2 Gene SNP with pigeon Properties
1. Experimental method
The correlation of the different genotypes of the 15 SNPs with body weight and body size, egg laying performance was analyzed using the model as follows:
Yijl＝u+Gi+eijl
wherein Y ijl is a property observation value; u is the population mean; g i is the effect of the ith genotype or haplotype of the SNP locus; e ijl denotes random errors, and the result is expressed in the form of "average ± standard error". The difference significance was tested according to the Duncan method, P <0.05 showed significant difference, and P <0.01 showed very significant difference. Multiple comparisons were made using the "tam black ni" method for variance-poor traits and independent sample T-tests were used for traits of only two genotypes.
2. Experimental results
In the correlation analysis of 15 SNP loci of IGF2BP2 gene and meat pigeon properties, the correlation results of 7 SNPs related to haplotype combination and meat pigeon properties are shown in Table 9.
The results showed that among 15 SNPs of IGF2BP2 gene:
there is a significant difference in the interval trait between 3 genotypes of SNP (g.2314208 t > C), the interval of TC genotypes is significantly higher than TT, CC genotypes (P < 0.05); the locus is not obvious with other characters;
3 genotypes of SNP (g.2306515G > T) have significant differences in body weight and chest width traits, the body weight of TT genotype is significantly higher than GT genotype, and the chest width trait of GG genotype is significantly greater than GT genotype (P < 0.05); the locus is not obvious with other characters;
There were significant differences in body weight traits for the 3 genotypes of SNP (g.2306415 c > T), the body weight of the TT genotype was significantly higher than the CT genotype (P < 0.05); the locus is not obvious with other characters;
SNP (g.2308546T > C), SNP (g.2306652C > T), SNP (g.2306459A > G) and SNP (g.2306381G > A) are not significant with the growth and reproduction traits of meat pigeons.
TABLE 9 correlation analysis of IGF2BP2 Gene SNP with pigeon growth trait and reproduction trait
5, Pouring: the same lowercase letters after the same column of values in the table represent insignificant differences between treatments and different lowercase letters represent differences between treatments
Is remarkable.
Example 7A method for meat pigeon breeding
1. And extracting DNA of the sample to be detected by using a blood genome DNA extraction kit.
2. Performing PCR amplification by taking the DNA of the sample to be detected extracted in the step 1 as a template:
The PCR reaction system is as follows: 2X Taq Plus MasterMix 10.0.0 mu L, the nucleotide sequence of which is shown in SEQ ID NO: 7-8, and the nucleotide sequence is shown as SEQ ID NO: 13-14, and the primer or nucleotide sequence shown in SEQ ID NO:19 to 20, 0.5. Mu.L of each of the primers, 1.0. Mu.L of DNA, and 8.0. Mu.L of ddH 2 O.
The PCR reaction conditions were:
(1) The nucleotide sequence shown in SEQ ID NO: 7-8, and the primer or nucleotide sequence shown in SEQ ID NO:13 to 14, when the primers shown in FIGS.: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 60℃for 45s, extension at 72℃for 50s,32 cycles; extending at 72 ℃ for 8min;
(2) The nucleotide sequence shown in SEQ ID NO:19 to 20, in the case of amplification of the primers shown in FIGS.: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 54℃for 45s, extension at 72℃for 50s,32 cycles; extending at 72℃for 8min.
3. And (3) detecting the PCR amplified product obtained in the step (2) by agarose gel electrophoresis, and sequencing the PCR amplified product obtained in the step (2) if the size of the electrophoresis band is consistent with that of the target band and the electrophoresis band is clear and bright.
4. And (3) judging results:
SNP1 is located at 2306652 on NW_004973255 gene, is C or T polymorphism, and exists in both CC and CT genotypes;
SNP2 is located at 2306515 on NW_004973255 gene, is a G or T polymorphism, and exists in three genotypes GG, GT and TT;
SNP3 is located at 2306459 on NW_004973255 gene, is A or G polymorphism, and exists in both AA and AG genotypes;
SNP4 is located at 2306415 on NW_004973255 gene, is C or T polymorphism, and exists in three genotypes of CC, CT and TT;
SNP5 is located at 2306381 on NW_004973255 gene, is a G or A polymorphism, and exists in both GG and GA genotypes;
SNP6 is located at 2314208 on NW_004973255 gene, is a T or C polymorphism, and exists in three genotypes of TT, TC and CC;
SNP7 is located at 2308546 on NW_004973255 gene, is a T or C polymorphism, and exists in three genotypes of TT, TC and CC;
Individuals with genotype CT for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4, and genotype GA haplotype for SNP5 are significantly heavier than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AA for SNP3, genotype CC for SNP4, and genotype GG haplotype for SNP 5;
individuals with genotype CT for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4, and genotype GA haplotype for SNP5 are significantly heavier than individuals with genotype CC for SNP1, genotype GT for SNP2, genotype AA for SNP3, genotype CT for SNP4, and genotype GG haplotype for SNP 5;
Individuals with genotype CT for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP5 have a significantly higher body weight than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP 5;
individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GG haplotype for SNP5 have significantly higher body weight than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP 5;
individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AA for SNP3, genotype CT for SNP4 and genotype GG haplotype for SNP5 weigh significantly higher than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP 5;
Individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AA for SNP3, genotype CT for SNP4 and genotype GG haplotype for SNP5 have significantly higher chest depth than individuals with genotype CC for SNP1, genotype GT for SNP2, genotype AA for SNP3, genotype CT for SNP4 and genotype GG haplotype for SNP 5;
Individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AA for SNP3, genotype CT for SNP4 and genotype GG haplotype for SNP5 have significantly higher chest depth than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP 5;
individuals with genotype of SNP6 of CC and genotype of SNP7 of CC haplotype weigh significantly higher than individuals with genotype of SNP6 of TT and genotype of SNP7 of CC haplotype;
individuals with genotype of SNP6 of CC and genotype of SNP7 of CC haplotype weigh significantly higher than individuals with genotype of SNP6 of CC and genotype of SNP7 of TT haplotype;
the individual with genotype of SNP6 of CC and genotype of SNP7 of CC haplotype has a shank length significantly higher than the individual with genotype of SNP6 of CC and genotype of SNP7 of TT haplotype;
The shin circumference of the individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype is significantly higher than that of the individuals with genotype of SNP6 being CC and genotype of SNP7 being CC haplotype;
The keel length of the individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype is significantly higher than that of the individuals with genotype of SNP6 being TT and genotype of SNP7 being CC haplotype;
Individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype have significantly higher body diagonal length than individuals with genotype of SNP6 being TT and genotype of SNP7 being CC haplotype;
individuals with genotype TT for SNP6 and genotype CT haplotype for SNP7 have significantly higher body diagonal lengths than individuals with genotype TC for SNP6 and genotype CT haplotype for SNP 7;
individuals with genotype of SNP6 of TT and genotype of SNP7 of CT haplotype have significantly higher body diagonal length than individuals with genotype of SNP6 of TC and genotype of SNP7 of CC haplotype;
individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype have significantly higher body diagonal length than individuals with genotype of SNP6 being CC and genotype of SNP7 being TT haplotype;
Individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype have significantly higher body diagonal length than individuals with genotype of SNP6 being CC and genotype of SNP7 being CC haplotype;
The number of eggs laid by individuals with genotype TC of SNP6 and genotype of SNP7 of CC haplotype is significantly higher than those with genotype of SNP6 of CC and genotype of SNP7 of CC haplotype;
Individuals with genotype TT for SNP6 and genotype CT haplotype for SNP7 give significantly higher numbers of eggs than individuals with genotype CC for SNP6 and genotype CC haplotype for SNP 7.
Example 8A kit for meat pigeon breeding
1. Composition of the composition
The nucleotide sequence is shown in SEQ ID NO: 7-8, and the nucleotide sequence is shown as SEQ ID NO: 13-14, and the nucleotide sequence is shown as SEQ ID NO:19 to 20, taq Plus MasterMix and ddH 2 O.
2. Application method
Detection and result interpretation were performed according to example 7.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A molecular marker of a haplotype for breeding meat pigeons, characterized in that the molecular marker of a haplotype consists of 5 single nucleotide polymorphism sites SNP1, SNP2, SNP3, SNP4 and/or SNP 5:
SNP1 is located at 2306652 on NW_004973255 gene, is C or T polymorphism, and exists in both CC and CT genotypes;
SNP2 is located at 2306515 on NW_004973255 gene, is a G or T polymorphism, and exists in three genotypes GG, GT and TT;
SNP3 is located at 2306459 on NW_004973255 gene, is A or G polymorphism, and exists in both AA and AG genotypes;
SNP4 is located at 2306415 on NW_004973255 gene, is C or T polymorphism, and exists in three genotypes of CC, CT and TT;
SNP5 is located at 2306381 on NW_004973255 gene, is a G or A polymorphism, and exists in both GG and GA genotypes;
Individuals with genotype CT for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4, and genotype GA haplotype for SNP5 are significantly heavier than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AA for SNP3, genotype CC for SNP4, and genotype GG haplotype for SNP 5;
individuals with genotype CT for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4, and genotype GA haplotype for SNP5 are significantly heavier than individuals with genotype CC for SNP1, genotype GT for SNP2, genotype AA for SNP3, genotype CT for SNP4, and genotype GG haplotype for SNP 5;
Individuals with genotype CT for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP5 have a significantly higher body weight than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP 5;
individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GG haplotype for SNP5 have significantly higher body weight than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP 5;
individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AA for SNP3, genotype CT for SNP4 and genotype GG haplotype for SNP5 weigh significantly higher than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP 5;
Individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AA for SNP3, genotype CT for SNP4 and genotype GG haplotype for SNP5 have significantly higher chest depth than individuals with genotype CC for SNP1, genotype GT for SNP2, genotype AA for SNP3, genotype CT for SNP4 and genotype GG haplotype for SNP 5;
Individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AA for SNP3, genotype CT for SNP4 and genotype GG haplotype for SNP5 have significantly higher chest depth than individuals with genotype CC for SNP1, genotype GG for SNP2, genotype AG for SNP3, genotype CC for SNP4 and genotype GA haplotype for SNP 5.
2. A molecular marker of a haplotype for breeding meat pigeons, characterized in that the molecular marker of a haplotype consists of 2 single nucleotide polymorphism sites SNP6 and/or SNP 7:
SNP6 is located at 2314208 on NW_004973255 gene, is a T or C polymorphism, and exists in three genotypes of TT, TC and CC;
SNP7 is located at 2308546 on NW_004973255 gene, is a T or C polymorphism, and exists in three genotypes of TT, TC and CC;
individuals with genotype of SNP6 of CC and genotype of SNP7 of CC haplotype weigh significantly higher than individuals with genotype of SNP6 of TT and genotype of SNP7 of CC haplotype;
individuals with genotype of SNP6 of CC and genotype of SNP7 of CC haplotype weigh significantly higher than individuals with genotype of SNP6 of CC and genotype of SNP7 of TT haplotype;
the individual with genotype of SNP6 of CC and genotype of SNP7 of CC haplotype has a shank length significantly higher than the individual with genotype of SNP6 of CC and genotype of SNP7 of TT haplotype;
The shin circumference of the individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype is significantly higher than that of the individuals with genotype of SNP6 being CC and genotype of SNP7 being CC haplotype;
The keel length of the individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype is significantly higher than that of the individuals with genotype of SNP6 being TT and genotype of SNP7 being CC haplotype;
Individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype have significantly higher body diagonal length than individuals with genotype of SNP6 being TT and genotype of SNP7 being CC haplotype;
individuals with genotype TT for SNP6 and genotype CT haplotype for SNP7 have significantly higher body diagonal lengths than individuals with genotype TC for SNP6 and genotype CT haplotype for SNP 7;
individuals with genotype of SNP6 of TT and genotype of SNP7 of CT haplotype have significantly higher body diagonal length than individuals with genotype of SNP6 of TC and genotype of SNP7 of CC haplotype;
individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype have significantly higher body diagonal length than individuals with genotype of SNP6 being CC and genotype of SNP7 being TT haplotype;
Individuals with genotype of SNP6 being TT and genotype of SNP7 being CT haplotype have significantly higher body diagonal length than individuals with genotype of SNP6 being CC and genotype of SNP7 being CC haplotype;
The number of eggs laid by individuals with genotype TC of SNP6 and genotype of SNP7 of CC haplotype is significantly higher than those with genotype of SNP6 of CC and genotype of SNP7 of CC haplotype;
Individuals with genotype TT for SNP6 and genotype CT haplotype for SNP7 give significantly higher numbers of eggs than individuals with genotype CC for SNP6 and genotype CC haplotype for SNP 7.
3. The detection reagent of the haplotype molecular marker according to claim 1, wherein the detection reagent is a nucleotide sequence shown in SEQ ID NO:19 to 20.
4. The detection reagent of the haplotype molecular marker as set forth in claim 2, wherein the detection reagent has a nucleotide sequence as set forth in SEQ ID NO: 7-8 and/or the nucleotide sequence of the primer is shown as SEQ ID NO:13 to 14.
5. Use of a molecular marker of a haplotype according to any one of claims 1-2 and/or a detection reagent according to any one of claims 3-4 in the preparation of a breeding product for meat pigeons.
6. A kit for breeding meat pigeons, comprising the detection reagent according to claim 3 or 4.
7. A method for breeding meat pigeons, characterized in that the haplotype molecular marker according to claim 1 is detected with the detection reagent according to claim 3 or the haplotype molecular marker according to claim 2 is detected with the detection reagent according to claim 4.
8. The method according to claim 7, comprising the steps of:
S1, extracting genomic DNA of a meat pigeon to be detected;
s2, carrying out PCR amplification on the genome DNA obtained in the step S1 by using the detection reagent in the claim 3 or 4, and then sequencing a PCR amplification product to obtain sequencing data;
S3, analyzing the sequencing data obtained in the step S2, and judging the characters of the meat pigeons to be tested according to the haplotype molecular markers in the claims 1 or 2.
9. The method of claim 8, wherein the sequencing of step S2 is Sanger sequencing.
10. The method of claim 8, wherein the trait of step S3 comprises body weight, chest depth, shin length, shin circumference, keel length, body diagonal length, and/or egg number.
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