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A molecular marker method and its primer pair that can predict and identify the curl degree of sheep wool

A technology of curling and sheep, applied in the field of animal molecular genetics, achieves the effect of high precision, simple operation and low cost

Inactive Publication Date: 2018-08-28
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 SHCBP1 gene in livestock and poultry

Method used

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  • A molecular marker method and its primer pair that can predict and identify the curl degree of sheep wool
  • A molecular marker method and its primer pair that can predict and identify the curl degree of sheep wool
  • A molecular marker method and its primer pair that can predict and identify the curl degree of sheep wool

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Obtaining Genomic DNA of Chinese Merino Sheep

[0033] 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:

[0034] (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;

[0035] (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;

[0036] (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;

[0037] (4) Take the supernatant, add the same amount of phe...

Embodiment 2

[0043] Obtaining of Enzymatic Digestion Product of Sheep SHCBP1 Gene

[0044] According to the SHCBP1 gene G14408374T site in the sheep genome, a pair of primers SHCBP1-P1-F and SHCBP1-P1-R were designed, and then the sheep genome DNA was amplified by PCR to obtain PCR amplification products, and then the endonuclease Taq I enzyme Cut the PCR amplified product to obtain the digested product.

[0045] The primer sequences are as follows:

[0046] SHCBP1-P1-F: 5'-TGATTCAATGCGCAACTGGTA-3'

[0047]SHCBP1-P1-R: 5'-AGACATGAAGCAAAGTATGATAGCAG-3'.

[0048] The PCR amplification system is as follows:

[0049]

[0050] PCR amplification conditions were: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 53.8°C annealing for 25 s, 72°C extension for 30 s, a total of 40 cycles; 72°C final extension for 10 min.

[0051] The enzyme digestion reaction system is as follows:

[0052] 10×Buffer Taq I 1μL

[0053] Endonuclease Taq I 1 μL

[0054] PCR product 10μL

[0055] Th...

Embodiment 3

[0057] Determination of Sheep SHCBP1 Genotype

[0058] 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 309bp and 36bp, when the G14408374T site mutation in the 14th exon region of the sheep SHCBP1 gene is mutated, the PCR amplification product can be completely cut by Taq I enzyme, and it is named as the TT genotype; (2) Electrophoresis shows a band, the size If it is 345bp, when the G14408374T site in the 14th exon region of the sheep SHCBP1 gene is not mutated, the PCR amplification product cannot be digested by Taq I, and it is named GG genotype; (3) Electrophoresis shows three bands, the size is 345bp , 309bp and 36bp, the G14408374T site in the 14th exon region of the sheep SHCBP1 gene is in a heterozygous state, and...

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Abstract

The invention provides a molecular marker method capable of indicating and identifying curling degree of sheep wools and a primer pair for the molecular marker method. The primer pair comprises an upper primer SHCBP1-P1-F shown in the sequence chart Seq No.1 and a lower primer SHCBP1-P1-R shown in the sequence chart Seq No.2. The molecular marker method comprises the following steps: designing a primer pair, extracting a sheep genome DNA for amplification, and then performing digestion to obtain a digestion product; performing electrophoretic separation on the digestion product, and judging the genotypes according to the electrophoretic separation result; performing correlation analysis, estimating the least squares means of the character, wherein the result is that the wool fineness of a GG genotype group and a GT genotype group in three genotypes is obviously higher than that of a TT genotype group; dividing the test group into three types to complete the method. The molecular marker method can indicate and identify the curling degree of sheep wools, and can be effectively applied to the auxiliary molecular breeding field of high-curling-degree sheep.

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 curl degree of sheep wool and a primer pair thereof. Background technique [0002] With the improvement of people's living standards, the world's wool quality has undergone epoch-making changes, mainly in the fineness and curl 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. It needs to import a large number of high-quality wool with low fineness and high crimp from abroad every year. Wool has become the only bulk that my country relies on imports. livestock products. Therefore, it is imperative to rapidly breed high-curly sheep and expand the germplasm scale of high-curly sheep by using modern molecular breeding techniques. [0003] The breeding of Chinese Merino sheep (Xinjiang ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6888C12Q2600/124C12Q2600/156C12Q2531/113C12Q2521/301C12Q2565/125
Inventor 王宁马广伟张志威杨华杨涵羽璐张文建王宇祥李辉
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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