CN116397034A - Molecular marker related to chicken antler crown character and application thereof - Google Patents
Molecular marker related to chicken antler crown character and application thereof Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a molecular marker related to chicken antler crown character and application thereof, belonging to the fields of animal genetic breeding technology and molecular biology. The nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1. According to the molecular marker, a primer group with a nucleotide sequence shown as SEQ ID NO. 2-3 and a Taqman probe with a nucleotide sequence shown as SEQ ID NO. 4 are designed, and the isolation and identification of the chicken antler crown can be completed through real-time fluorescent quantitative nucleic acid amplification.
Description
Technical Field
The invention relates to the field of animal genetic breeding technology and molecular biology, in particular to a molecular marker related to chicken antler crown character and application thereof.
Background
With the issuance of market-prohibited live bird trade policies, slaughtered frozen products have become a major choice for consumers. The traditional means for subjectively judging the quality and the source of the products through the yellow degree of chicken feather color, feather color and length cannot be realized, and a new mark is urgently needed as a means for judging the ice fresh products of high-quality Jiangcun yellow chickens and other chickens.
The cockscomb is reserved in most whole chicken frozen fresh products, the deer antler crown is taken as a characteristic of the Jiangcun yellow chicken, is an effective mark for judging the whole chicken frozen fresh products, has distinct appearance with the characteristic of the deer antler crown and the single comb, and presents the appearance of one third of the bifurcation of the rear edge of the cockscomb. This bifurcation occurs in the area of the crown floor, and a second bifurcation occurs on the bifurcated teeth, and the overall form is like a "crotch" and a "deer", so it is called a deer-horn crown. However, the phenomenon of fork at the rear edge of the cockscomb also appears similarly in the character of the three-fork cockscomb, compared with the deer-horn cockscomb, the fork degree of the three-fork cockscomb is weaker, the number of fork cockscomb teeth is fewer (usually only 1-2 extra fork cockscomb teeth) but is often mixed into the deer-horn cockscomb individual, and the fork-horn cockscomb individual is difficult to distinguish only by phenotype, so that the breeding work is influenced. Furthermore, deer crown is a monogenic controlled trait that is dominant to the wild-type monocrown trait. Therefore, how to use an objective method to correctly select the character of the deer horn and eliminate heterozygotes with single crown alleles is an important problem for ensuring stable inheritance of the deer horn.
Real-time fluorescent quantitative nucleic acid amplification (qPCR) is a method that monitors the reaction process and products in real time while PCR is performed. The TaqMan real-time fluorescence quantitative PCR is based on the fluorescence resonance energy transfer principle, namely a specific fluorescent probe primer is added in PCR amplification, a reporter group and a quenching group are respectively marked at two ends of the probe primer, and the purpose of monitoring the amplification amount of a PCR product can be achieved by detecting the fluorescence intensity in a PCR reaction system. The method has the characteristics of strong specificity, high sensitivity and the like, and is most widely applied to domestic clinical diagnosis at present. In the TaqMan real-time fluorescent quantitative PCR procedure, if the initial copy number of the target nucleic acid sequence is higher, the fluorescence is observed to be remarkably increased faster. Thus, the method can be used to detect whether Copy Number Variation (CNV) occurs in a nucleic acid sequence at the same DNA concentration. Therefore, molecular markers of the 'deer horn crown' are identified based on TaqMan real-time fluorescence quantitative PCR, and the molecular markers are used for screening heterozygous 'deer horn crown' genotypes, and have important value for auxiliary selective breeding of 'deer horn crown' characters.
Disclosure of Invention
The invention aims to provide a molecular marker related to the character of the deer antler crown of a chicken and application thereof, so as to solve the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a molecular marker related to the character of chicken antler crown, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1.
The invention also provides a Taqman probe primer group for detecting the molecular marker, which comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 2, a reverse primer with a nucleotide sequence shown as SEQ ID NO. 3 and a Taqman probe with a nucleotide sequence shown as SEQ ID NO. 4.
The invention also provides a detection kit for the character of the deer horn crown of the chicken, which comprises the Taqman probe primer group.
The invention also provides a detection method of the cockscomb shape genotype, which comprises the following steps:
(1) Extracting genome DNA of a chicken to be detected;
(2) Taking diluted chicken genome DNA to be detected as a template, and carrying out real-time fluorescent quantitative nucleic acid amplification by using the Taqman probe primer group to obtain a CT value;
(3) Calculating RQ value according to CT value, taking single crown character chicken as control group, passing
Identifying the genotype of the character of the deer horn crown of the chicken to be tested, if ∈10>
Judging the genotype of the individual to be detected as AA; if it is/>
Judging the genotype of the individual to be detected as Aa; if->
And judging the genotype of the individual to be detected as aa.
Further, in step (2), the reaction system for real-time fluorescent quantitative nucleic acid amplification comprises: 1. Mu.L of template DNA, 0.4. Mu.L of forward primer at 10. Mu.M concentration, 0.4. Mu.L of reverse primer at 10. Mu.M concentration, 0.1. Mu.L of Taqman probe primer at 10. Mu.M concentration, probe qPCRMaster mix. Mu.L of ddH 2 O 3.1μL。
Further, in step (2), the reaction procedure for real-time fluorescent quantitative nucleic acid amplification comprises: removing pollution at 50deg.C for 2min; pre-denaturation at 95℃for 10min; denaturation at 95℃for 15s, annealing and extension at 60℃for 60s,40 cycles.
Further, in the step (2), the concentration of the DNA in the template is 100 ng/. Mu.L.
The invention also provides application of the molecular marker, the Taqman probe primer set or the detection kit in detecting cockscomb traits, wherein the cockscomb traits comprise deer horn, single crown and trigeminal crown.
The invention also provides application of the molecular marker, the Taqman probe primer set or the detection kit in identifying the genotype of the deer horn crown of the chicken.
The invention also provides application of the molecular marker, the Taqman probe primer set or the detection kit in breeding of chicken antler crown characters.
The invention discloses the following technical effects:
the invention discovers about 15kb Copy Number Variation (CNV) on a chromosome 19 of a deer antler crown chicken genome, designs a primer and a Taqman probe on the sequence, screens the conditions of mismatch, hairpin structure, primer dimer and the like which occur by combining the primer and the Taqman probe, and finally obtains a primer group with good specificity, and can finish the separation and identification of the deer antler crown of the chicken through DNA extraction, DNA dilution and real-time fluorescence quantitative nucleic acid amplification.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a significance analysis of differences between RQ values of different chicken populations, where the differences are extremely significant (P < 0.001); * Is significant difference (P < 0.05); ns is the difference is not significant;
FIG. 2 is a diagram of individuals of different genotypes
Significance analysis of the differences between values, where x is the difference is extremely significant (P<0.001 A) is provided; * Is significant in difference (P<0.05 A) is provided; ns is the difference is not significant.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The invention discovers that the deer crown locus relates to Copy Number Variation (CNV) of about 15kb on chicken chromosome 19, and can effectively detect the locus by using a Taqman probe method to carry out real-time fluorescent quantitative nucleic acid amplification (qPCR) on chicken DNA. At the same DNA concentration, individuals possessing the antler crown trait will show significantly different RQ values (P<0.001 Differences in RQ values between heterozygous and homozygous crown individuals would then be due to
And reacting to obtain the product. The three-fork crown and the deer-horn crown are two characters with similar appearance but completely different loci, and the separation and identification of the deer-horn crown and the three-fork crown characters can be completed through DNA extraction, DNA dilution and real-time fluorescence quantitative nucleic acid amplification.
Example 1TaqMan real-time fluorescent quantitative PCR detection of the use of molecular markers of the deer crown trait in different populations
In this example, a total of 4 populations were used to verify whether the TaqMan real-time fluorescent quantitative PCR method could be used for detection of the deer crown trait loci. The total number of samples was 99, and the four groups were: jiang Cunhuang chicken flock (offered by the company limited, jiang Fengshui, guangzhou) mixed with single crown, trifoliate crown and deer crown; a river field chicken population (provided by river field chicken breeding farms) mixed with single crown and deer crown; a silk feather black-bone chicken population of rose crowns (supplied by the agricultural university of south China city-increasing base); a population of spotted-brown chickens (offered by the westerner food group inc.) mixed with trigeminal crowns and single crowns.
1. Extraction of total DNA of chicken to be tested
And extracting DNA from the chicken to be tested. The method comprises the following specific steps:
(1) Fresh blood samples were taken at 50. Mu.L, placed in 1.5mL centrifuge tubes, and 2. Mu.L of proteinase K at 10mg/mL and shaken at 37℃for 12h.
(2) RNaseA was added to a final concentration of 20. Mu.g/mL and the mixture was water-bath for 30min.
(3) 700. Mu.L of phenol was added: chloroform: isoamyl alcohol (volume ratio 25:24:1), gently mixed upside down, and centrifuged at 14000rpm for 5min at room temperature.
(4) The supernatant was aspirated and an equal volume of chloroform was added: isoamyl alcohol (volume ratio 24:1) was centrifuged at 14000rpm for 5min.
(5) The supernatant was aspirated, twice the volume of absolute ethanol was added, and after inversion mixing, it was centrifuged at 14000rpm for 5min.
(6) Washing the precipitate twice with 75% ethanol solution, and air drying at room temperature.
(7) Add 50. Mu.L ddH 2 O, vortex mixing, and storing in a refrigerator at-20 ℃ for standby.
2. Dilution of total DNA of chickens to be tested
In the TaqMan real-time fluorescence quantitative PCR procedure, if the initial number of target nucleic acid sequences is higher, the fluorescence is observed to be remarkably increased faster, so that a smaller CT value is obtained. Therefore, in detecting genomic copy number variations of the target sequence, we need to ensure that the initial concentration of the template DNA used is the same. The invention uses TE Buffer to dilute the total DNA of the chicken to be tested, and comprises the following steps:
(1) The concentration of total DNA was detected by a spectrophotometer.
(2) The amount of TE Buffer to be added for dilution to 100 ng/. Mu.L was calculated from the total DNA concentration.
(3) TE Buffer of a corresponding volume was added and mixed by shaking with a vortex oscillator to obtain diluted DNA at a concentration of 100 ng/. Mu.L.
3. Primer synthesis and real-time fluorescent quantitative nucleic acid amplification
The beta-actin is used as an internal reference gene to amplify a target variant sequence, and the primer group comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 2, a reverse primer with a nucleotide sequence shown as SEQ ID NO. 3 and a Taqman probe primer with a nucleotide sequence shown as SEQ ID NO. 4:
SEQ ID NO:2：5’-CTCAGGGAAGTGTTTGGTGC-3’；
SEQ ID NO:3：5’-ACACTTTCACTGCTGTGTGTC-3’；
SEQ ID NO:4：5’-(6-FAM)-ATCCTCCACACTGCCCTCCCTGTGCT-(MGB NFQ)-3’；
the 5 'end and the 3' end of the Taqman probe primer are modified by adopting 6-FAM and MGB-NFQ labels respectively.
Taqman probe primer set is synthesized by biological company and comprises a forward primer shown as SEQ ID NO. 2, a reverse primer with a nucleotide sequence shown as SEQ ID NO. 3 and a modified probe with a nucleotide sequence shown as SEQ ID NO. 4.
Using the diluted DNA obtained in the dilution of the total DNA of the chicken to be detected as a template, and carrying out real-time fluorescence quantitative nucleic acid amplification on the DNA template by using the dissolved Taqman probe primer group:
the reaction system for real-time fluorescence quantitative nucleic acid amplification comprises: 1. Mu.L of template DNA, 0.4. Mu.L of forward primer at 10. Mu.M concentration, 0.4. Mu.L of reverse primer at 10. Mu.M concentration, 0.1. Mu.L of Taqman probe at 10. Mu.M concentration, probe qPCR Master mix. Mu.L, ddH 2 O 3.1μL。
The reaction procedure for real-time fluorescent quantitative nucleic acid amplification is: removing pollution at 50deg.C for 2min; pre-denaturation at 95℃for 10min; denaturation at 95℃for 15s, annealing and extension at 60℃for 60s,40 cycles.
The target sequence of the real-time fluorescent quantitative nucleic acid amplification is a section of marker sequence on the copy number variation of about 15kb, and the specific nucleotide sequence is shown as SEQ ID NO. 1:
CTCAGGGAAGTGTTTGGTGCATCCTCCACACTGCCCTCCCTGTGCTCTTTTCCAGCT CTCTTATTTTCTTATTGAGATGGAGAGTCCAAAAAGACACACAGCAGTGAAAGTGT。
4. deriving CT values and calculating RQ values by software
And (3) deriving the CT value of each sample to be detected through qPCR matched software. The RQ value was calculated using β -actin as reference gene and by the following equation:
ΔΔCt＝ΔCt(test)－ΔCt(reference)
RQ＝2 -ΔΔCt
wherein, test: a sample to be tested; reference: control samples (negative samples, here wild type with known single crown).
The RQ values of all samples tested are shown in Table 1. The differences between RQ values of different chicken populations were analyzed for significance using GraphPad mapping according to the results of Table 1, as shown in FIG. 1, where different chicken breeds (lines) and crowns were identified by different columns, respectively, using the known single crown spotted-brown chickens as control samples. RQ values of all deer crown populations can be seen to differ extremely significantly from RQ values of single and triple crown populations (x is extremely significant (P < 0.001); is significant (P < 0.05); ns is not significant).
TABLE 1 crown and RQ values for different chicken breeds (complete set)
The results show that the gene fragment with the sequence shown in SEQ ID NO. 1 is used as a molecular marker, the detection method is adopted to carry out real-time fluorescence quantitative nucleic acid amplification on a total of 99 samples of four chicken varieties (complete sets), and the results show that the RQ value of the deer horn character population is extremely obvious from the RQ value of the single crown and the three-crown character population, which indicates that the molecular marker sequence is used for carrying out Taqman real-time fluorescence quantitative amplification experiments on the sample to be detected, can effectively separate deer horn character individuals from the mixed population of the single crown, the three-crown and the deer horn, and can be used for identification and auxiliary breeding of deer horn character.
Example 2TaqMan real-time fluorescent quantitative PCR detection of the action of the molecular marker of the Corona Cervi trait in the purification of Corona Cervi alleles
In this example, a mating experiment was first performed, and an isotactic cell population with separated offspring appearance was obtained by mating the antler crown individuals (genotype AA or AA) in table 1 with the individual (genotype AA) in table 1 (both antler crown and individual crown are in Jiangcun Huang Ji), and then the genotype of the individual having the antler crown trait in the isotactic cell population was AA heterozygous, and the genotype of the individual having the wild type individual crown trait was AA. The offspring had no parent with separated traits, and the deer crown genotype was judged as AA.
In this example, 5 heterozygous antler crowns (genotype Aa) and 5 wild type single crowns (genotype Aa) were selected from the isotactic cells with separated appearance; a total of 15 chickens were selected from the parental deer horn crown (genotype AA) with no segregation of traits in 5 offspring.
1. Extraction of total DNA of chicken to be tested
And extracting DNA from the chicken to be tested. The method comprises the following specific steps:
(1) Fresh blood samples were taken at 50. Mu.L, placed in 1.5mL centrifuge tubes, and 2. Mu.L of proteinase K at 10mg/mL and shaken at 37℃for 12h.
(2) RNaseA was added to a final concentration of 20. Mu.g/mL and the mixture was water-bath for 30min.
(3) 700. Mu.L of phenol was added: chloroform: isoamyl alcohol (volume ratio 25:24:1), gently mixed upside down, and centrifuged at 14000rpm for 5min at room temperature.
(4) The supernatant was aspirated and an equal volume of chloroform was added: isoamyl alcohol (volume ratio 24:1) was centrifuged at 14000rpm for 5min.
(5) The supernatant was aspirated, twice the volume of absolute ethanol was added, and after inversion mixing, it was centrifuged at 14000rpm for 5min.
(6) Washing the precipitate twice with 75% ethanol solution, and air drying at room temperature.
(7) Add 20. Mu.L ddH 2 O, vortex mixing, and storing in a refrigerator at-20 ℃ for standby.
2. Dilution of total DNA of chickens to be tested
In the TaqMan real-time fluorescence quantitative PCR procedure, if the initial number of target nucleic acid sequences is higher, the fluorescence is observed to be remarkably increased faster, so that a smaller CT value is obtained. Therefore, in detecting genomic copy number variations of the target sequence, we need to ensure that the initial concentration of the template DNA used is the same. Here, TE Buffer was used to dilute the total DNA of the chickens to be tested, as follows:
(1) The concentration of total DNA was detected by a spectrophotometer.
(2) The amount of TE Buffer to be added for dilution to 100 ng/. Mu.L was calculated from the total DNA concentration.
(3) TE Buffer of a corresponding volume was added and mixed by shaking with a vortex oscillator to obtain diluted DNA at a concentration of 100 ng/. Mu.L.
3. Primer synthesis and real-time fluorescent quantitative nucleic acid amplification
The beta-actin is used as an internal reference gene to amplify a target variant sequence, and the primer group comprises a forward primer with a nucleotide sequence shown as SEQ ID NO. 2, a reverse primer with a nucleotide sequence shown as SEQ ID NO. 3 and a Taqman probe primer with a nucleotide sequence shown as SEQ ID NO. 4:
SEQ ID NO:2：5’-CTCAGGGAAGTGTTTGGTGC-3’；
SEQ ID NO:3：5’-ACACTTTCACTGCTGTGTGTC-3’；
SEQ ID NO:4：5’-(6-FAM)-ATCCTCCACACTGCCCTCCCTGTGCT-(MGB NFQ)-3’；
the 5 'end and the 3' end of the Taqman probe primer are modified by adopting 6-FAM and MGB-NFQ labels respectively.
Taqman probe primer set is synthesized by biological company and comprises a forward primer shown as SEQ ID NO. 2, a reverse primer with a nucleotide sequence shown as SEQ ID NO. 3 and a modified probe with a nucleotide sequence shown as SEQ ID NO. 4.
Using the diluted DNA obtained in the dilution of the total DNA of the chicken to be detected as a template, and carrying out real-time fluorescence quantitative nucleic acid amplification on the DNA template by using the dissolved Taqman probe primer group:
the reaction system for real-time fluorescence quantitative nucleic acid amplification comprises: 1. Mu.L of template DNA, 0.4. Mu.L of forward primer at 10. Mu.M concentration, 0.4. Mu.L of reverse primer at 10. Mu.M concentration, 0.1. Mu.L of Taqman probe at 10. Mu.M concentration, probe qPCR Master mix. Mu.L, ddH 2 O 3.1μL。
The reaction procedure for real-time fluorescent quantitative nucleic acid amplification is: removing pollution at 50deg.C for 2min; pre-denaturation at 95℃for 10min; denaturation at 95℃for 15s, annealing and extension at 60℃for 60s,40 cycles.
The target sequence of the real-time fluorescent quantitative nucleic acid amplification is a section of marker sequence on the copy number variation of about 15kb, and the specific nucleotide sequence is shown as SEQ ID NO. 1:
CTCAGGGAAGTGTTTGGTGCATCCTCCACACTGCCCTCCCTGTGCTCTTTTCCAGCT CTCTTATTTTCTTATTGAGATGGAGAGTCCAAAAAGACACACAGCAGTGAAAGTGT。
4. deriving CT values and calculating RQ values by software
And (3) deriving the CT value of each sample to be detected through qPCR matched software. The RQ value was calculated using β -actin as reference gene and by the following equation:
ΔΔCt＝ΔCt(test)－ΔCt(reference)
RQ＝2 -ΔΔCt
wherein, test: a sample to be tested; reference: control samples (negative samples, here wild type with known single crown).
Taking herba Ephedrae chicken of known single crown as control sample by
Normalization was performed and the results are shown in table 2. The results are plotted using GraphPad according to the results of table 2, as shown in figure 2. In FIG. 2, the genotypes of the different cockscomb traits are marked with different columns, respectively, and it can be seen that +.>
The difference between each two was extremely remarkable (the difference was extremely remarkable (P<0.001 A) is provided; * Is significant in difference (P<0.05 A) is provided; ns is not significantly different).
The invention discovers that if
Judging the genotype of the deer crown individual to be detected as AA; if it is
Judging the genotype of the deer crown individual to be detected as Aa; if->
And judging the genotype of the deer crown individual to be detected as aa. The specific 15 genotyping results are shown in table 2:
TABLE 2
The results show that the Taqman real-time fluorescent quantitative amplification experimental result is the same as the actual cockscomb genotype judged by the hybridization experimental result, and the Taqman real-time fluorescent quantitative amplification experiment is carried out on a sample to be detected by taking a gene fragment with a sequence shown as SEQ ID NO. 1 as a molecular marker, so that the homozygous or heterozygous genotype of the cockscomb can be effectively distinguished, the method can be used for purifying and assisted breeding of the genotype of the cockscomb, and has important value for establishing a new chicken variety or mating line with the phenotype of the cockscomb.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. The molecular marker related to the chicken antler crown character is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1.
2. A Taqman probe primer set for detecting the molecular marker of claim 1, which is characterized by comprising a forward primer with a nucleotide sequence shown as SEQ ID NO. 2, a reverse primer with a nucleotide sequence shown as SEQ ID NO. 3 and a Taqman probe with a nucleotide sequence shown as SEQ ID NO. 4.
3. A kit for detecting the properties of chicken antler crowns, which is characterized by comprising the Taqman probe primer set according to claim 2.
4. The detection method of the cockscomb shape character genotype is characterized by comprising the following steps:
(1) Extracting genome DNA of a chicken to be detected;
(2) Carrying out real-time fluorescent quantitative nucleic acid amplification by using diluted chicken genome DNA to be detected as a template and using the Taqman probe primer set of claim 2 to obtain a CT value;
(3) Calculating RQ value according to CT value, taking single crown character chicken as control group, passing
Identifying the genotype of the character of the deer horn crown of the chicken to be tested, if ∈10>
Judging the genotype of the individual to be detected as AA; if->
Judging the genotype of the individual to be detected as Aa; if->
Judging the genotype of the individual to be detected asaa。
5. The method according to claim 4, wherein in the step (2), the reaction system for real-time fluorescent quantitative nucleic acid amplification comprises: 1. Mu.L of template DNA, 0.4. Mu.L of forward primer at 10. Mu.M concentration, 0.4. Mu.L of reverse primer at 10. Mu.M concentration, 0.1. Mu.L of Taqman probe primer at 10. Mu.M concentration, probe qPCRMaster mix. Mu.L of ddH 2 O 3.1μL。
6. The method according to claim 4, wherein in the step (2), the reaction procedure for real-time fluorescent quantitative nucleic acid amplification comprises: removing pollution at 50deg.C for 2min; pre-denaturation at 95℃for 10min; denaturation at 95℃for 15s, annealing and extension at 60℃for 60s,40 cycles.
7. The method according to claim 4, wherein in the step (2), the concentration of DNA in the template is 100 ng/. Mu.L.
8. Use of the molecular marker of claim 1, the Taqman probe primer set of claim 2 or the detection kit of claim 3 for detecting cockscomb traits, including deer horn, single crown and trigeminal crown.
9. Use of the molecular marker of claim 1, the Taqman probe primer set of claim 2 or the detection kit of claim 3 for identifying the genotype of chicken horn crown.
10. Use of the molecular marker of claim 1, the Taqman probe primer set of claim 2 or the detection kit of claim 3 in breeding of chicken antler crown traits.
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