A Molecular Marker Related to Litter Size Trait in Sheep and Its Application

A technology of molecular markers and litter size, applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of slow progress in genetic improvement and low heritability, and achieve good technical means and reduce costs , the effect of broad application prospects

Active Publication Date: 2016-08-24
甘肃兰天同和农业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Reproductive traits are threshold traits, which are traits with low heritability (average 0.1), and the litter size is also affected to a certain extent under different environments and different feeding conditions. Understanding of genetic mechanisms of reproductive traits to facilitate genetic improvement of reproductive traits in sheep

Method used

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  • A Molecular Marker Related to Litter Size Trait in Sheep and Its Application
  • A Molecular Marker Related to Litter Size Trait in Sheep and Its Application
  • A Molecular Marker Related to Litter Size Trait in Sheep and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. Cloning of 5'-UTR sequence of FSHR gene

[0019] (1) Primer design

[0020] According to the sheep FSHR gene sequence (GenBank accession number: NC_019460.1), use Primer5.0 software to design the upstream and downstream primers M-F and M-R, the sequence is as follows

[0021] FHSR: M-F: 5'- CGTATCTTTCCACGCCCTCT-3',

[0022] M-R: 5'- CCATCCACCCGATTGCTT-3'.

[0023] (2) Cloning and sequencing of PCR products

[0024] Connect the purified PCR product to the pMD-18 T vector (purchased from Takara) overnight at 4°C in a water bath; aseptically take 100~120 μl of competent cells into a 1.5 ml Ependorff tube, and add 5 μl of the ligation product Mix well, place on ice for 30 min, heat shock at 42°C for 90 s, then ice bath for 3~4 min, add 400 μl of antibiotic-free LB liquid medium, culture with shaking at 37°C for 45 min. Spread 100 μl on the agar plate of isopropylthio-β-D-galactoside (IPTG) X-gal, place it flat at 37°C for 1 h, and then invert it. Pick a single colony on ...

Embodiment 2

[0027] Example 2. Establishment of PCR-RFLP diagnostic method

[0028] (1), primer sequence

[0029] FSHR:M-F: 5'- CGTATCTTTCCACGCCCTCT-3',

[0030] M-R: 5'- CCATCCACCCGATTGCTT-3'.

[0031] (2) PCR amplification conditions

[0032] The total volume of the PCR reaction is 20μl, including about 100 ng sheep genomic DNA, containing 1× buffer (purchased from Promega), 1.5 mmol / L MgCl 2 The final concentration of dNTP is 150 μmol / L, the final concentration of primer is 0.4 μmol / L, and 2U Taq DNA polymerase (Promega). The PCR amplification program is: 94℃ 3 min, 35 cycles of 94℃ 30 s, 58℃ 30 s, then 72℃ 25s, and finally 72℃ extension for 5 min. PCR reaction products were detected by 2% agarose gel electrophoresis. The result is figure 1 Shown. Amplified to obtain a 244bp specific fragment, which is located in the 5'-UTR. The result of sequencing revealed that there is a BsiE I restriction site (CGRY↓CG) in the 244bp fragment, and the 90th bp is a polymorphic cut site located in the 5'-UT...

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Abstract

The invention belongs to the technical field of preparation of molecular markers for livestock, and particularly relates to a preparation method and application of a molecular marker which is related to lambing number character of sheep and used for sheep marker assisted selection. The molecular marker is obtained by FSHR gene cloning, and the nucleotide sequence of the molecular marker is as shown in a sequence table SEQ ID NO:1. One T88 - C88 base substitution is generated at the 88 bp of the sequence table SEQ ID NO:1, thus resulting in BsiEI-RFLP digestion polymorphism. The invention also discloses a primer for amplifying a part of DNA (deoxyribonucleic acid) sequence of an FSHR gene and a method used for polymorphism detection, and provides a new molecular marker for selection assisted by the marker related to lambing number character of sheep.

Description

Technical field [0001] The invention belongs to the technical field of livestock molecular marker preparation, and specifically relates to a preparation method and application of a molecular marker related to sheep littering traits used as a sheep marker assisted selection application. Background technique [0002] According to statistics from FAO (2012), there are 187 million sheep in my country, which accounts for 16% of the world's breeding population (1.169 billion), ranking first in the world (http: / / www.fao.org / home / en / ), It is the largest producer and consumer of mutton. The genetic improvement of sheep is of great significance for ensuring the supply of mutton, promoting the development of the national economy and improving people's living standards. The reproductive traits of sheep, especially the number of litters, is one of the most important economic traits in modern sheep breeding. With the implementation of the national ecological protection strategy, the space for...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 李发弟王维民翁秀秀刘世佳潘香羽马友记唐德富李冲
Owner 甘肃兰天同和农业有限公司
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