Method for distinguishing green shell egg laying chicken and chicken green shell egg genotype as well as special fragment and primers

A green-shell laying hen, genotyping technology, applied in biochemical equipment and methods, DNA/RNA fragments, microbial determination/inspection, etc., can solve the problem of reaction failure, high cost, high template quality, high amplification conditions, etc. problems, to achieve the effect of low cost, simple operation and short identification time

Active Publication Date: 2013-01-16
CHINA AGRI UNIV
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Problems solved by technology

This method can accurately distinguish green-shell laying hens from non-green-shell laying hens, and can determine whether the genotype of green-shell laying hens is dominant homozygous (O/O) or heterozygous (O/o), However, this method has three disadvantages: (1) Genotype detection takes a long time, because the length of the PCR amplified fragment is nearly 6kb including the sequences on both sides of the

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  • Method for distinguishing green shell egg laying chicken and chicken green shell egg genotype as well as special fragment and primers
  • Method for distinguishing green shell egg laying chicken and chicken green shell egg genotype as well as special fragment and primers
  • Method for distinguishing green shell egg laying chicken and chicken green shell egg genotype as well as special fragment and primers

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Embodiment 1

[0047] Example 1. Identification of green-shell laying hens and chicken green-shell egg genotype short fragments and multiple PCR primers

[0048] The green-shell egg trait of chickens is caused by a 4.2kb insertion sequence at the 5' end of the upstream SLC01B3 gene on chromosome 1 (the insertion sequence only appears in green-shell laying hens), so it is only necessary to detect whether the individual carries this insertion sequence. Individual phenotypes can be inferred.

[0049] After research, a short DNA molecule (425bp, partly 4.2kb insertion sequence) was found. The nucleotide sequence of the DNA molecule is sequence 4 in the sequence list; design relevant multiple PCR primers based on this DNA molecule; the specific design principle is as follows figure 1 As shown, two pairs of parallel lines represent two homologous chromosomes, the solid line represents the normal genome sequence, the dotted line represents the 4.2kb insertion sequence, and the arrows represent the ...

Embodiment 2

[0051] Embodiment 2, multiplex PCR distinguishes the genotype of producing green-shell layer hens and chicken green-shell eggs

[0052] 1. Using known genotype individuals to verify the accuracy of multiplex PCR to identify the genotype of green-shell laying hens

[0053] 1. Experimental animals

[0054] f 0 The generation individuals are 5 Dongxiang roosters (known genotype is O / O), 20 Dongxiang hens (known genotype is o / o), F 1 The generation individuals are 46 hens (known genotype is O / o) crossed from the above individuals. Dongxiang chickens were purchased from Jiangxi Dongxiang Hualu Breeding Poultry Co., Ltd.

[0055] 2. Extraction of DNA

[0056] respectively in F 0 and F 1 Take 1mL of blood from the subwing vein of the individual, anticoagulate with ACD (ACD: blood = 3:1), and extract the genomic DNA according to the standard phenol-imitation method:

[0057] (1) 30 μL anticoagulant blood + 300 μL PBS (1×, pH: 7.2) + 300 μL poultry blood lysate (120 g urea, 10 g...

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Abstract

The invention discloses a method for distinguishing green shell egg laying chicken and chicken green shell egg genotype as well as a special fragment and primers. The invention provides a method for identification or auxiliary identification of whether chicken to be detected is green shell egg chicken or not, in order to detect the genome DNA (deoxyribonucleic acid), if objective DNA molecules (sequence 4) are contained in the genome DNA, the chicken to be detected belongs to or is candidated as the green shell egg chicken, and when the objective DNA molecules are not contained in the genome DNA, the chicken to be detected does not belong to or is not candidated as the green shell egg chicken. Experiments prove that a 425bp short fragment is adopted, three primers are designed and synthesized according to the short fragment and are used for multiple PCR (polymerase chain reaction) amplification, whether the chicken to be detected lays green shell eggs or not can be distinguished through detecting whether amplification products contain the short fragment or not, and meanwhile, the chicken green shell egg genotype can be distinguished.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for identifying the genotype of green-shell laying hens and green-shell eggs, special fragments and primers. Background technique [0002] Eggshell color is one of the factors that affect egg quality. In the poultry egg market, eggs with smooth surface, beautiful color and uniformity are often favored by consumers. Green-shell eggs are popular in the poultry egg market for their unique eggshell color, and often appear in the high-end poultry egg market as "healthy eggs" and "healthy eggs". The traditional breeding of green-shell hens has the problems of impure selection and large variation in eggshell color. [0003] The green-shelled egg trait of chicken is a quality trait controlled by a dominant single gene (O), and both dominant homozygous (O / O) and heterozygous (O / o) individuals lay green-shelled eggs. This genetic characteristic determines that it is difficult to di...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 邓学梅王哲鹏吴常信
Owner CHINA AGRI UNIV
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