Molecular marker method capable of indicating and identifying curling degree of sheep wools and primer pair for molecular marker method

A technology of molecular markers and curling degree, applied in the field of animal molecular genetics, achieves the effect of simple operation, low cost and accelerated breeding process

Inactive Publication Date: 2015-11-18
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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  • Molecular marker method capable of indicating and identifying curling degree of sheep wools and primer pair for molecular marker method
  • Molecular marker method capable of indicating and identifying curling degree of sheep wools and primer pair for molecular marker method
  • Molecular marker method capable of indicating and identifying curling degree of sheep wools and primer pair for molecular marker method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Acquisition of Chinese Merino Goat Genomic DNA

[0033] Collect sheep ear tissue and store at -20°C for later use. The conventional phenol / chloroform method was used to extract sheep genomic DNA. The specific method is as follows:

[0034] (1) Take 5g of ear tissue of Chinese Merino sheep (Xinjiang Army Reclamation type), remove connective tissue, wash and disinfect the tissue block with 70% alcohol, put it in Eppendorf tube, cut it with scissors, or grind it;

[0035] (2) After the alcohol in the Eppendorf tube is completely volatilized, add 700μL of separation buffer, suspend the cut tissue, add 5.0μL of proteinase K (20mg / mL), and act at 55°C for 8-12h until there is no tissue mass;

[0036] (3) Take out the digested tissue fluid, add the same amount of saturated phenol in the tissue fluid, mix for 10min, 4℃, 12000r / m, and centrifuge for 10min;

[0037] (4) Take the supernatant, add the same amount of phenol / chloroform, mix gently for 10min, 4℃, 12000r / m, and centrifuge for ...

Embodiment 2

[0043] Obtaining the product of sheep SHCBP1 gene digestion

[0044] Design a pair of primers SHCBP1-P1-F and SHCBP1-P1-R according to the G14408374T site of the SHCBP1 gene in the sheep genome, and then perform PCR amplification on the sheep genomic DNA to obtain the PCR amplification product, and then digest it with endonuclease TaqI PCR amplifies the product to obtain the digested product.

[0045] The primer sequence is 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 minutes; 94°C denaturation for 30s, 53.8°C annealing for 25s, 72°C extension for 30s, a total of 40 cycles; 72°C final extension for 10 minutes.

[0051] The digestion reaction system is as follows:

[0052] 10×BufferTaqI1μL

[0053] Endonuclease TaqI1μL

[0054] PCR product 10μL

[0055] Enzyme digestion conditions: 65°C di...

Embodiment 3

[0057] Judgment of sheep SHCBP1 genotype

[0058] Use agarose gel with a concentration of 2% to 3% to carry out electrophoretic separation of the digested products in Example 1, and determine the genotype according to the results of the electrophoretic separation. The criteria for determination are: (1) Two bands appear in electrophoresis, the size is 309bp and 36bp, when the G14408374T site in the 14th exon region of the sheep SHCBP1 gene is mutated, the PCR amplified product can be completely cleaved by TaqI enzyme and named TT genotype; (2) The electrophoresis shows a band with a size of 345bp, when the G14408374T site in the 14th exon region of the sheep SHCBP1 gene is not mutated, the PCR amplified product cannot be cleaved by TaqI, and it is named GG genotype; (3) Electrophoresis shows three bands with sizes of 345bp and 309bp And 36bp, the G14408374T site in the 14th exon region of the sheep SHCBP1 gene is in a heterozygous state, and the PCR amplified product cannot be co...

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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 marking method capable of predicting and identifying the curling degree of sheep wool and its primer pair. 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 crimp of the 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 the gap between supply and demand is very large. Every year, it needs to import large quantities of high-quality wool with low fineness and high curl from abroad. Wool has become the only bulk in my country that depends on imports. Livestock products. Therefore, it is imperative to use modern molecular breeding techniques to quickly breed high-curly sheep and expand the germplasm scale of high-curly wool sheep. [0003] The breeding of Chinese Merino sheep (Xi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
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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