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SNP (Single Nucleotide Polymorphism) marker of dorper sheep as well as screening method and application thereof

A screening method and technology of Dorper sheep, applied in the field of Dorper sheep SNP markers and its screening, can solve the problems of difficult standardization and scale, and achieve the effect of high-throughput operation, high accuracy, and easy operation

Active Publication Date: 2017-09-08
内蒙古中科正标生物科技有限责任公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Microsatellite markers were first used for individual identification, but the disadvantage is that it is difficult to achieve standardization and scale due to the different conditions in different laboratories

Method used

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  • SNP (Single Nucleotide Polymorphism) marker of dorper sheep as well as screening method and application thereof
  • SNP (Single Nucleotide Polymorphism) marker of dorper sheep as well as screening method and application thereof
  • SNP (Single Nucleotide Polymorphism) marker of dorper sheep as well as screening method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Determination of Dorper sheep breeds by pyrophosphoric acid method

[0052] (1) Reagents: GoTaqMaster Mix solution produced by Promega Company; specific amplification primers and sequencing primers synthesized by Shanghai Sangon; Sepharose Bead produced by Biotage Company.

[0053] (2) Amplification reaction system and amplification program: the total volume of the amplification reaction is 50 μL, and its various components are: 2×GoTaqMaster Mix 25 μL, 10 μmol / L primers 1 μL each, DNA template 2 μL for the test variety and the control variety (Cucumber seed DNA), make up to 50 μL with sterilized deionized water; reaction program: pre-denaturation at 95°C for 10 minutes, denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, extension at 72°C for 45 seconds, 50 cycles, and finally extension at 72°C for 7 minutes , stored at 4°C.

[0054] (3) Sequencing reaction system: Add 47 μL Binding buffer and 3 μL Sepharosebeads to 50 μL PCR product, vort...

Embodiment 2

[0057] Embodiment 2PCR amplification method is measured the breed of Dorper sheep

[0058] Randomly select 24 Altay sheep, 24 Bayinbulak sheep and 29 Dorper sheep as sample individuals, collect the blood of these sheep, use heparin sodium anticoagulant, use Tiangen blood genome kit to extract genomic DNA, 0.8% agarose Gel electrophoresis detection, nucleic acid protein quantification instrument to measure its concentration and purity, dilute to 100ng / ul, save for future use. Use the primers listed in Table 2 to perform PCR amplification using the genomic DNA of the sample to be tested as a template. The PCR amplification reaction system is: add 0.4 μL of TaqDNA polymerase (5U / μL), 5 μL of 10×PCR Buffer, 4 μL of MgCl2 ( 25mmol / μL), 1μL of Forwardprimer (10pmol / μL), 1μL of Reverse primer (10pmol / μL), 4μL of dNTP (2.5mmol / μL), 2μL of template DNA and 32.6μL of ddH2O, a total of 50μL.

[0059] PCR products were recovered and purified using an agarose gel DNA recovery kit. Collec...

Embodiment 3

[0060] Example 3 Determination of Dorper sheep breeds by gene chip method

[0061] Step 1, taking experimental animals, taking blood from ear veins, and extracting genomic DNA from the blood samples.

[0062] Step 2. Take the genomic DNA obtained in step 1, and hybridize with the nucleic acid chip with 30 inherent probes (the 30 probes are the single-stranded DNA molecules shown in sequence 1 to sequence 30 in Sequence Table 2, respectively).

[0063] Step 3. After step 2 is completed, the ends of each point in the nucleic acid chip are extended, so as to obtain the genotypes of the 30 SNP sites in the genomic DNA.

[0064] Step 4, taking the genomic DNA obtained in step 1, performing whole-genome sequencing, and obtaining the genotypes of 1522 SNP sites in the genomic DNA. The result shows that the result of step 3 is exactly the same as the result of step 4. Among the 1522 SNP sites of 48 experimental animals of Dorper sheep, at least 861-998 sites could be matched. The m...

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Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) marker of a dorper sheep as well as a screening method and application thereof. According to the screening method, based on the specific SNP marker of the dorper sheep, the SNP typing analysis is carried out by adopting a pyrosequencing method, a sanger sequencing method, a gene chip method or time-of-flight mass spectrometry; whether a to-be-tested variety is the dorper sheep or not is authenticated. The authenticity authentication method of a variety, which is provided by the invention, compared with a conventional measurement method, is simple and convenient to operate, is free from the interference between sites, and is suitable for high-throughput operation. The SNP marker is based on a pyrosequencing technique and has quite high accuracy; the authentication work of the variety can be completed within 5h; the result is determined easily; an authentication result can be directly read out from the peak value of a visible light signal.

Description

technical field [0001] The invention belongs to the technical field of sheep breed identification, and in particular relates to a Dorper sheep SNP marker and a screening method and application thereof. Background technique [0002] Dorper sheep (Dorper) originated from meat-type sheep in the arid region of South Africa. It is a hybrid of Persian black-headed sheep and horned Dorset sheep. This breed has the advantages of fast growth, strong drought and disease resistance, and delicious meat. For the world's diamond class sheep. Dorper sheep have strong adaptability, are easy to feed, and have a high utilization rate of forage. They can grow healthily in arid and semi-tropical regions and have a high reproductive rate. Meat sheep production is a weak point in my country's animal husbandry. In recent years, my country has introduced many high-quality meat sheep from abroad to hybridize and improve local sheep, and has achieved certain results. [0003] Understanding the gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q2600/156
Inventor 姜雨周东珂侯雨楠杜多孙晨婧郑典郝志强王文袁圣钧
Owner 内蒙古中科正标生物科技有限责任公司
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