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Molecular marker method and primer pair for predicting and identifying sheep wool fineness

A technology of molecular markers and primer pairs, applied in the field of animal molecular genetics, to achieve the effects of low cost, accelerated breeding process, and high accuracy

Inactive Publication Date: 2018-04-03
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few reports on the study of ICT1 gene in livestock and poultry

Method used

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  • Molecular marker method and primer pair for predicting and identifying sheep wool fineness
  • Molecular marker method and primer pair for predicting and identifying sheep wool fineness
  • Molecular marker method and primer pair for predicting and identifying sheep wool fineness

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Obtaining Genomic DNA of Chinese Merino Sheep

[0034] The sheep ear tissues were collected and stored at -20°C for future use. The sheep genomic DNA was extracted by conventional phenol / chloroform method. The specific method is as follows:

[0035] (1) Take 5 g of ear tissue from Chinese Merino sheep (Xinjiang Army Reclamation type), remove the connective tissue, clean and disinfect the tissue block with 70% alcohol, put it in an Eppendorf tube, cut it with scissors, or grind it to pieces;

[0036] (2) After the alcohol in the Eppendorf tube is completely evaporated, add 700 μL of separation buffer, suspend the chopped tissue, add 5.0 μL of proteinase K (20 mg / mL), and act at 55 ° C for 8-12 hours until there are no tissue pieces;

[0037] (3) Take out the digested tissue fluid, add an equal amount of saturated phenol in the tissue fluid, mix for 10 minutes, centrifuge at 12,000 r / m at 4°C for 10 minutes;

[0038] (4) Take the supernatant, add the same amount of phe...

Embodiment 2

[0044] Obtaining of Digestion Product of Sheep ICT1 Gene

[0045] Design a pair of primers ICT1F and ICT1R according to the ICT1 gene C55420707T site in the sheep genome, and then perform PCR amplification on the sheep genome DNA to obtain the PCR amplification product, and then use the endonuclease Ban II to digest the PCR amplification product to obtain the enzyme cutting products, such as figure 1 shown.

[0046] The primer sequences are as follows:

[0047] ICT1F: 5'-AGTTCACACAGGACAGGAGCG-3'

[0048] ICT1R: 5'-CTGCAGAGAAGAGGTTTCAAAGAG-3'.

[0049] The PCR amplification system is as follows:

[0050]

[0051]The PCR amplification conditions were as follows: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 65°C annealing for 25 s, 72°C extension for 30 s, a total of 36 cycles, and 72°C final extension for 10 min.

[0052] The enzyme digestion reaction system is as follows:

[0053] 10×CutSmart Buffer 1μL

[0054] Endonuclease Ban II 1μL

[0055] PCR pr...

Embodiment 3

[0058] Determination of ICT1 Genotype in Sheep

[0059] Use agarose gel with a concentration of 2% to 3% to carry out electrophoresis separation of the digested products in Example 1, and determine the genotype according to the results of the electrophoresis separation. The criteria for determination: (1) electrophoresis presents two bands, the size of which is 249bp and 89bp, when the C55420707T site in the fifth exon region of the sheep ICT1 gene is not mutated, the PCR amplification product can be completely cut by Ban II enzyme, and it is named CC genotype; (2) Electrophoresis shows a band, If the size is 338bp, when the C55420707T site in the 5th exon region of the sheep ICT1 gene is mutated, the PCR amplification product cannot be digested by Ban II, and it is named as the TT genotype; (3) Electrophoresis shows three bands with a size of 338bp , 249bp and 89bp, the C55420707T site in the fifth exon region of the sheep ICT1 gene is in a heterozygous state, and the PCR amp...

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Abstract

The invention provides a molecular marking method and a primer pair for predicting and identifying the wool fineness of sheep. The primer pair includes a forward primer ICT1F as shown in Seq No.1 in a sequence table and a reverse primer ICT1R as shown in Seq No.2 in the sequence table. The molecular marking method concretely comprises the following steps of designing the primer pair, extracting sheep genome DNA to amplify, and carrying out enzyme digestion to obtain an enzyme-digested product; carrying out electrophoretic separation on the enzyme-digested product, and judging the genetype according to an electrophoretic separation result; carrying out correlation analysis, and estimating the least square average value of a character to obtain a result that the wool fineness of a colony with a CT genetype in three genetypes is remarkably lower than that of a colony with a TT genetype; and dividing experimental colonies into three kinds of types. By using the molecular marking method, the wool fineness of the sheep can be predicted and identified; a more effective molecular marking method is provided for quality character improvement of sheep wool and marker assisted selection; and the molecular marking method can be effectively applied to the field of molecular-assisted breeding of sheep with superfine wool.

Description

technical field [0001] The invention relates to the field of animal molecular genetics, in particular to a molecular marker method capable of predicting and identifying the fineness of sheep wool and a primer pair thereof. Background technique [0002] At present, wool spinning products at home and abroad have developed in the direction of lightness, softness, crispness, and high-end. The quality of wool in the world has also undergone epoch-making changes, mainly in the fineness of wool. Wool fineness is an important quality and economic trait of wool. The finer the wool (the smaller the diameter of the wool fiber), the better the softness of the wool and the higher the textile performance, which has an important impact on the price of wool. my country is the country that consumes the most wool in the world. The self-sufficiency rate of wool is only 1 / 3, and there is a huge gap between supply and demand. Every year, a large amount of fine wool needs to be imported from abro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6888C12Q2600/124C12Q2600/156C12Q2531/113C12Q2521/313C12Q2565/125C12Q2537/165
Inventor 王宁马广伟张文建杨华王宇祥李辉
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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