Kit for identifying genetype of silky fowl skin color by detecting EDN3 (endothelin 3) gene copy number difference

A gene copy number and kit technology, applied in the field of agricultural biological diagnosis, can solve problems such as high cost, complicated detection process, and low sensitivity, and achieve reliable germplasm guarantee, high detection efficiency, and good repeatability

Inactive Publication Date: 2014-05-07
HANGZHOU ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Traditional gene copy number detection techniques include western blotting, fluorescent probe hybridization, multiple amplifiable probe hybridization, multiple ligation probe hybridization, etc., but most of them have complex detection process, tedious and time-consuming, high cost, radioactive hazards, false positives, etc. Disadvantages such as positive and low sensitivity

Method used

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  • Kit for identifying genetype of silky fowl skin color by detecting EDN3 (endothelin 3) gene copy number difference
  • Kit for identifying genetype of silky fowl skin color by detecting EDN3 (endothelin 3) gene copy number difference
  • Kit for identifying genetype of silky fowl skin color by detecting EDN3 (endothelin 3) gene copy number difference

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 EDN3 gene copy number detection

[0031] 1 Experimental instruments and materials

[0032] Real-time fluorescent quantitative PCR instrument (Bio-Rad OPTICON2, USA); ultra-low temperature refrigerator (Thermo, USA); high-speed refrigerated centrifuge (Eppendorf5417R, Germany); gradient PCR instrument (ABI PCR system9700, USA); adjustable micropipette ( Eppendorf, Germany); GEL EQ electrophoresis gel imaging system (Bio-Rad, USA); UV-Vis spectrophotometer (BioSpec-nano, Japan).

[0033] 2 Genomic DNA extraction from chicken blood

[0034] Collection of Yisha laying hens B-series (brown feather, white skin, red crown, white shank), Xing'an Ma chicken (mammy feather, white skin, red crown, green shank), Dongxiang green shell hens (mainly black feather, black skin , black crown, black or green shank) and other chicken species blood, preserved in 75% ethanol. Genomic DNA from chicken blood was extracted using the AxyPrep Total Blood Genomic DNA Mini Kit (Axygen,...

Embodiment 2

[0055] The skin color Fm genotype analysis of embodiment 2 different chicken species individuals

[0056] According to the method of Example 1, Dongxiang green-shell hens (mainly black feather, black skin, black crown, black or green shank), Xing'an Ma chicken (maiyu, white skin, red crown, green shank), Yisha The copy numbers of EDN3 gene of layer B line (brown feather, white skin, red crown, white shank) and other chicken breeds are shown in Table 3. It can be seen from Table 3 that the EDN3 gene copy number of white-skinned Xing'an Ma chicken and Yisha layer B line is basically the same as that of the calibration sample, which is about 1.0; the EDN3 gene copy number of Dongxiang green-shell layer chicken has a fold difference from that of the calibration sample. According to this, the Fm genotype related to the skin color of the black-bone chicken to be tested is judged: when the EDN3 gene copy number is about 2.0, it is homozygous Fm / Fm; when the gene copy number is about ...

Embodiment 3

[0061] Embodiment 3 test cross verification test

[0062] Dongxiang green-shell layer roosters with known EDN3 gene copy number were bred with Isa layer B-series hens at a ratio of male to female of 1:6. If Dongxiang ♂ genotype is Fm / Fm homozygous, the skin color of F1 offspring male and female chickens should be black; if Dongxiang ♂ genotype is Fm / fm heterozygous, the F1 offspring male and female chicken skin color There will be white, and the proportion of white skin tends to be roughly 50%. The results of the testcross test are shown in Table 4, and the statistical data of the skin color separation confirmed the accuracy of the method for detecting the difference in the copy number of the EDN3 gene.

[0063] Table 4 Testcross Verification Results

[0064] Table3Cross test's results

[0065]

[0066] The operation of the kit does not require the construction of a standard curve, and is simple and fast, with good repeatability and high detection efficiency; the method ...

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Abstract

The invention relates to a kit for identifying the genetype of a silky fowl skin color by detecting EDN3 (endothelin 3) gene copy number difference. The kit comprises a reference gene GAPDH (reduced glyceraldehyde-phosphate dehydrogenase) amplification primer (1), a target gene EDN3 amplification primer (2), calibration sample DNA (deoxyribonucleic acid) (3), fluorescent quantitative PCR (Polymerase Chain Reaction) reaction liquid (4) and double distilled water (5). The kit is characterized in that real-time fluorescent quantitative PCR reaction is carried out on a target gene EDN3 and a reference gene GAPDH by using the DNA of a tested sample as a template, Ct values are respectively recorded by using the calibration sample DNA as a reference, the EDN3 gene copy numbers of the tested sample are obtained by calculation, and further the Fm genetype of the silky fowl skin color is judged according to the EDN3 gene copy number difference. The operation of the kit does not need the establishment of a standard curve; the kit is simple, convenient, fast, good in repeatability and high in detection efficiency; and through chicken breed test-crossing testing, the method can accurately reflect the Fm homozygosis and heterozygosis of the skin color of the individual silky fowl. The detection method is conductive to acceleration of screening of homozygosity of the silky fowl skin color, guides target breeding of high-quality silky fowl new variety lines and provides reliable germplasm guarantee for follow-up commercialization mating hybridization.

Description

technical field [0001] The invention relates to a molecular biology kit for detecting the copy number difference of EDN3 gene of black-bone chicken, belonging to the technical field of agricultural biological diagnosis. Background technique [0002] Copy number variation (CNVs) is a newly discovered genomic structural variation that is as important as single nucleotide polymorphisms (SNPs). Human studies have shown that CNVs can lead to single gene diseases and rare diseases with Mendelian inheritance, and at the same time Also associated with many multifactorial diseases and phenotypes such as height. Black skin, black bones, black meat and other black traits are one of the most characteristic appearances of black-bone chickens. This phenomenon of melanin deposition in the body is also called Dermal hyperpigmentation or Fibromelanosis (Fm). In production, effectively distinguishing the purity and purity of skin and black traits will help to speed up the breeding process of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2531/113C12Q2563/107C12Q2537/16
Inventor 章学东王欢欢张成先李庆海陈贤惠张国军楼立峰张雷
Owner HANGZHOU ACAD OF AGRI SCI
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