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Method for detecting slow-feathering mutation genotypes of cocks

A technology for mutant genes and roosters, applied in the field of detection of rooster slow-feather mutant genotypes, can solve the problems of inability to determine slow-feather alleles, complicated processes, time-consuming and labor-intensive, etc., and achieves the effect of short cycle, few links, and less error-prone effects.

Pending Publication Date: 2016-12-07
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Since the slow feather phenotype controlled by the slow feather allele (K) is dominant to the fast feather phenotype controlled by the fast feather allele (k), the slow feather phenotype of a rooster with a slow feather phenotype cannot be determined only by phenotype Genotype of allele (K)
Patent application number 200910062895.0 discloses a molecular identification method for fast and slow feathers in chickens; patent application number 201310502809.X discloses a method for identifying chicken fast and slow feather genotypes and a method for identifying male and female chickens; patent application number 201510680524.4 Disclosed is a molecular identification primer and identification method for the fast and slow feather phenotype of chickens. However, these molecular detection methods only stop at distinguishing whether the rooster is slow feather or not, and cannot determine the genotype of the slow feather allele (K) of the rooster individual
The patent application number 201310435980.3 discloses a rapid breeding method for slow-feather pure line chickens. This method can only determine the genotype of the slow-feather mutation through the test cross method, which is not only complicated, time-consuming and labor-intensive, but also error-prone

Method used

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  • Method for detecting slow-feathering mutation genotypes of cocks
  • Method for detecting slow-feathering mutation genotypes of cocks
  • Method for detecting slow-feathering mutation genotypes of cocks

Examples

Experimental program
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Effect test

Embodiment 1

[0016] Example 1 Effect of different concentrations of DNA on detection results

[0017] The animal materials used in this embodiment are 35 rooster individuals known to be homozygous for slow plumage, and 35 individual roosters known to be heterozygous for slow feather.

[0018] A method for detecting rooster slow feather mutation genotype, comprising the steps:

[0019] S1. DNA extraction: Use a disposable syringe to extract about 1 mL of blood from the vein under the chicken wing, inject it into a 1.5 mL centrifuge tube that has been sterilized by high pressure and filled with about 200 μL of 2% sterile EDTA anticoagulant, and shake it gently , record the wing number, and store at -20°C for future use.

[0020] Genomic DNA was extracted using phenol-chloroform extraction (Osper F et al., 1998):

[0021] (1) Take 30 μL of whole blood and place in a 1.5 mL centrifuge tube, add 470 μL of 1×SET buffer, 12.5 μL of 20% SDS and 6 μL of 10 mg / mL proteinase K, mix well and place i...

Embodiment 2

[0038] Example 2 The Influence of Two Different Types of Real-time Fluorescent Quantitative PCR Instruments on the Detection Results

[0039] The animal materials used in this example were 35 individual roosters known to be homozygous for slow plumage, and 35 individual roosters known to be heterozygous for slow feathering; the fluorescent quantitative PCR instrument models were ABI 7300 and Bio-rad CFX96.

[0040]A method for detecting rooster slow feather mutation genotype, comprising the steps:

[0041] S1. DNA extraction: Use a disposable syringe to extract about 1 mL of blood from the vein under the chicken wing, inject it into a 1.5 mL centrifuge tube that has been sterilized by high pressure and filled with about 200 μL of 2% sterile EDTA anticoagulant, and shake it gently , record the wing number, and store at -20°C for future use.

[0042] Genomic DNA was extracted using phenol-chloroform extraction (Osper F et al., 1998):

[0043] (1) Take 30 μL of whole blood and ...

Embodiment 3

[0058] Example 3 Influence of different real-time fluorescent quantitative reagents on detection results

[0059] The animal materials used in this example were 35 individual roosters known to be homozygous for slow plumage, and 35 individual roosters known to be heterozygous for slow feathering; the model of the fluorescent quantitative PCR instrument was Bio-rad CFX96.

[0060] A method for detecting the genotype of the rooster's slow-feather mutation, the specific steps are the same as those in Example 1, and the only different fluorescent quantitative reagents used are Bio-rad SYBR Green Fluorescent Quantitative PCR Mix or KAPA SYBR FAST qPCR Master Mix. It was found that the reagents of KAPA SYBR FAST qPCR Master Mix could not amplify the results at all.

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Abstract

The invention belongs to the technical field of animal breeding test and particularly discloses a method for detecting slow-feathering mutation genotypes of cocks. The method includes the following steps of S1, extracting DNA (deoxyribonucleic acid) of blood samples of detected individuals and diluting the DNA to be 40-100nmol / mu L; S2, performing real-time fluorescence quantitative PCR (polymerase chain reaction), wherein the type of a fluorescence quantitative PCR instrument is United States Bio-rad CFX96, a fluorescence quantitative reagent is Bio-rad SYBR Green fluorescence quantitative PCR Mix, a fluorescence quantitative primer sequence is as shown in SEQ ID NO: 1-4; S3, calculating delta Ct value of the detected individuals, and judging the genotypes of the individuals according to the delta Ct, where when the delta Ct is greater than 1.5, the genotypes are heterozygotes, when 0.3 is less than delta Ct which is less than 1.3, the genotypes are homozygotes, and when 1.5 is greater than or equal to delta Ct which is greater than or equal to 1.3, the genotypes are eliminated. The method has the advantages of short period, high speed, 100% of accuracy, less steps, less proneness to making mistakes and the like.

Description

technical field [0001] The invention relates to the technical field of animal breeding detection, in particular to a method for detecting the slow feather mutation genotype of roosters. Background technique [0002] Since the slow feather phenotype controlled by the slow feather allele (K) is dominant to the fast feather phenotype controlled by the fast feather allele (k), the slow feather phenotype of a rooster with a slow feather phenotype cannot be determined only by phenotype Genotype of allele (K). Patent application number 200910062895.0 discloses a molecular identification method for fast and slow feathers in chickens; patent application number 201310502809.X discloses a method for identifying chicken fast and slow feather genotypes and a method for identifying male and female chickens; patent application number 201510680524.4 Disclosed is a molecular identification primer and identification method for the fast and slow feather phenotype of chickens. However, these m...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q1/6827C12Q1/686C12Q2600/124C12Q2600/156C12Q2561/113C12Q2563/107C12Q2545/114C12Q2531/113
Inventor 许继国张细权聂庆华李健林希冉陈杰
Owner SOUTH CHINA AGRI UNIV
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