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Molecular marker primer pair capable of predicting and identifying the natural length of sheep and wool and application thereof

A molecular marker, natural length technology, applied in the field of animal molecular genetics, can solve the problems of long time consumption, low efficiency of forced enzyme digestion, and complicated operation, and achieve the effect of high accuracy, simple primer design, and simple experimental operation.

Active Publication Date: 2019-01-11
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the incomplete match of the primers, the efficiency of forced digestion is relatively low
[0005] Publication No.: CN107619870A, Invention Name: Molecular markers capable of predicting and identifying the length of sheep wool and its specific primer pairs and applications. In the original invention, the PCR products were sequenced by DNA pool pooling, and the SNP detection technology of Fluidigm was used for typing According to the typing results, the individual sheep with longer natural wool length can be identified. The whole process requires four primers, and the operation is cumbersome and time-consuming

Method used

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  • Molecular marker primer pair capable of predicting and identifying the natural length of sheep and wool and application thereof
  • Molecular marker primer pair capable of predicting and identifying the natural length of sheep and wool and application thereof
  • Molecular marker primer pair capable of predicting and identifying the natural length of sheep and wool and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Obtaining Genomic DNA of Chinese Merino Sheep

[0036] 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:

[0037] (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;

[0038] (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;

[0039] (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;

[0040] (4) Take the supernatant, add the same amount of phe...

Embodiment 2

[0046] Obtaining the Digestion Product of Sheep TOMM70A Gene

[0047] According to the TOMM70A gene G163139235A site in the sheep genome, a pair of primers TOMM70AF and TOMM70AR were designed, and then the sheep genomic DNA was amplified by PCR to obtain the PCR amplification product, and then the PCR amplification product was digested with the endonuclease Bae I to obtain the enzyme cut product.

[0048] Such as figure 2 As shown, after the G163139235A site in the 7th intron region of TOMM70A gene is mutated, it can be completely recognized by BaeI endonuclease, so the primer sequence completely matches the template sequence, and no forced enzyme digestion is required.

[0049] The primer sequences are as follows:

[0050] TOMM70AF: 5'-ATGTGGCTTCTTGGAGTG-3'

[0051] TOMM70AR: 5'-CACAAATAAGCGCAGTTC-3'

[0052] The PCR amplification system is as follows:

[0053]

[0054] PCR amplification conditions were: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 53...

Embodiment 3

[0061] Determination of TOMM70A Genotype in Sheep

[0062] 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 313bp and 152bp, when the G163139235A site in the 7th intron region of the sheep TOMM70A gene is not mutated, the PCR amplification product can be completely cut by Bae I enzyme, and it is named GG genotype; (2) Electrophoresis shows a band, If the size is 498bp, when the G163139235A site in the 7th intron region of the sheep TOMM70A gene is mutated, the PCR amplification product cannot be digested by Bae I, and it will be named as the AA genotype; (3) Electrophoresis shows three bands with a size of 498bp , 313bp and 152bp, the G163139235A site in the 7th intron region of the sheep TOMM70A gene is in a heterozygous ...

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Abstract

The invention discloses a molecular marker primer pair capable of predicting and identifying the natural length of sheep and wool and application thereof, wherein the pair of primers is shown in sequence lists Seq ID No. 1 and Seq ID No. 2. The primer pairs were designed and the sheep genomic DNA was extracted for amplification, and then digested with enzyme to obtain digested products. Enzyme digestion product electrophoresis separation, according to the electrophoresis separation result carries on the genotype determination; The results showed that the natural wool length of GG genotype population was significantly higher than that of AA genotype and GA genotype population. Finally, the ewes with GG genotype were selected as breeding population. The invention can predict and identify thenatural length of sheep wool, provides a more effective molecular marker method for improving the quality traits of sheep wool, and is applied to the field of screening sheep molecular assisted breeding with long natural length of wool fiber. The invention is simple in operation and low in cost.

Description

technical field [0001] The invention relates to the field of animal molecular genetics, in particular to a pair of molecular marker primers capable of predicting and identifying the natural length of sheep wool and its application. Background technique [0002] Wool natural length is an important indicator of sheep wool quality traits. The natural length of wool is the length of wool in its natural state directly measured with a ruler. Different breeds of fine-wool sheep have different natural lengths of wool. For example, the natural length of wool of Australian Merino sheep is longer than that of many fine-wool sheep breeds in my country. In general, the natural length of wool is positively correlated with the average fiber diameter of wool, that is, the longer the natural length of wool, the larger the average fiber diameter of wool. The significance of the natural length of wool in textile technology is second only to the average fiber diameter of wool. It not only aff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 马广伟周怡婷杨嘉文张雪妍郭雅彬
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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