Special primer combination for quickly detecting polyploid of willow tree and detection method for polyploid

A detection method and primer combination technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of complex preparation process, increased difficulty, and the effect is not as good as that of animal cells, so as to improve detection The effect of high efficiency, high efficiency and simple operation

Active Publication Date: 2016-12-14
NANJING FORESTRY UNIV
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Problems solved by technology

However, plant cells contain cell walls, the sample preparation process is more complicated, the difficulty is increased, and the effect is not as good as that of animal cells, so flow cytometry is not suitable for large-scale screening and detection of polyploid willow (Vrána et al., 2014)

Method used

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  • Special primer combination for quickly detecting polyploid of willow tree and detection method for polyploid
  • Special primer combination for quickly detecting polyploid of willow tree and detection method for polyploid
  • Special primer combination for quickly detecting polyploid of willow tree and detection method for polyploid

Examples

Experimental program
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Effect test

Embodiment 1

[0025] The primers used in this method were developed from willow genome DNA sequence, and 192 pairs of primers were designed and developed by Primer Premier 5.0 software, and synthesized by Shanghai Jierui Biotechnology Co., Ltd.

[0026] Common willows are divided into arbor willows and shrub willows. In order to verify the versatility of the primers, this embodiment selected two common tree willows and two kinds of shrub willows, wherein the tree willows include weeping willow (Salix babylonica) and dry willow (S.matsudana), and the shrub willows include dustpan willow (S. suchowensis) and willow bark (S.integra). The collected cuttings were propagated by cuttings under greenhouse conditions, and the newly germinated young leaves were taken, and DNA was extracted by CTAB method. The concentration of the extracted DNA was detected with a micro-nucleic acid protein detector, and then the DNA concentration was diluted to 10 ng / μL, and stored at -20°C for later use. Each sele...

Embodiment 2

[0033] Weeping willow (Salix babylonica), dry willow (S.matsudana), dustpan willow (S.suchowensis) and willow willow (S.integra) preserved in the willow germplasm resources nursery of Nanjing Forestry University using the 10 pairs of primers screened in Example 1 A total of 48 samples of 4 species of willow were tested for genotype. The specific method is as follows:

[0034] The DNA of each sample was extracted from willow leaves according to the CTAB method. Afterwards, using this as a template, 10 pairs of primers obtained by screening were used for PCR amplification on an ABI-9700 thermal cycler. The total reaction system of PCR is 15 μL, which includes: template DNA 25ng, upstream and downstream primers 10ng each, concentration of 2.5mM dNTP 0.4μL, fluorescently labeled 1mM Fluorescein-12-dUTP (Roche Diagnostics) 0.01μL, Taq polymerase 0.5 U, 10×buffer (containing 100μM Tris-HCl pH 8.3, 500mM KCl, 20mM MgCl 2 and 10.0g / L BSA) 1.5 μL, add sterilized deionized water to s...

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Abstract

The invention discloses a special primer combination for quickly detecting polyploid of a willow tree and a detection method for the polyploidy. A primer provided by the invention is used for performing PCR amplification on a DNA template of the to-be-detected willow tree; by means of electrophoresis detection of an amplification product, if the number of alleles detected on amplification sites of any pair of primers of the detected sample is greater than 2, the sample is the polyploid. Compared with the existing willow tree polyploid detection method, the special primer combination which is provided by the invention and is used for willow tree polyploid screening has the advantages of simple and quick operation method and reliable result, can detect a large quantity of samples within short time, and supplies efficient and reliable technical measures to willow tree polyploid screening. An important technical support is supplied to willow tree polyploid cultivation; the special primer combination has extremely high actual application values, can achieve relatively high economic and social benefits, and has wide application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a special primer combination for rapid detection of willow polyploid and a detection method thereof. Background technique [0002] Willow generally refers to plants of the genus Salix, belonging to the phylum Magnoliophyta, the class Magnoliopsida, the subclass Dilleniidae, the order Salicales, and the family Salicaceae. There are about 500 species of willows in the world, many of which have excellent characteristics such as rapid growth, strong adaptability and easy vegetative reproduction, and play a very important role in my country's forestry production. Willow is widely used and is an important ornamental tree species in gardens. In recent years, it has also played an important role in phytoremediation and ecosystem restoration; in addition, because of its high biomass production, it is also used as an important biomass energy tree species (Tu Zhong Yu, 1989; Tang Guimei et al., ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/124C12Q2565/125
Inventor 尹佟明郭炜李淑娴
Owner NANJING FORESTRY UNIV
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