Design, amplification and sequencing method of four pairs of Floccularia luteovirens nuclear gene primers

Abstract

The invention relates to a design, amplification and sequencing method of four pairs of Floccularia luteovirens nuclear gene primers. The method comprises steps as follows: (1), universal primers matched with EF1-alpha, RPB1, RPB2 and Mcm7 genes are adopted to amplify Floccularia luteovirens, and amplified fragments of the EF1-alpha, RPB1, RPB2 and Mcm7 genes are obtained respectively; (2), the amplified fragments of the EF1-alpha, RPB1, RPB2 and Mcm7 genes are subjected to gel detection, target bands are cut and subjected to sequencing after purification and recovering by a SanPrep column type DNA gel recovery kit, and 521 bp EF1-alpha gene fragments, 751 bp RPB1 gene fragments, 782 of RPB2 gene fragments and 688 bp Mcm7 gene fragments are obtained; (3), 30 bp sequences of two ends of each of the EF1-alpha, RPB1, RPB2 and Mcm7 gene fragments obtained in the step (2) are removed, the primers are designed online, and a pair of primers with the highest score is selected; (4), the specificity of the primers in the step (3) is verified through amplification and sequencing of Floccularia luteovirens strains with different geographical distribution sources. The method is simple and easy, and the primers have excellent amplification performance.

Description

technical field [0001] The invention relates to the technical field of fungal phylogeny and phylogeography, in particular to four pairs of yellow-green curly sclerotia genes ( EF1– α, RPB1 , RPB2 , Mcm7 ) Primer design, amplification and sequencing methods. Background technique [0002] Chrysanthemum viridis ( Floccular luteovirens ) belonging to the family Agaricales (Agaricales) T richolomataceae), is a fruiting body-producing ectomycorrhizal fungus mainly distributed in the Qinghai-Tibet Plateau. Because of its bright yellow color, delicious taste and rich nutrition, it has been regarded as a special delicacy on the Qinghai-Tibet Plateau for a long time and has high economic value. However, in recent years, with the increasing trade of fruiting bodies, Armillaria chrysanthemum resources are facing the danger of over-harvesting, and at the same time, its taxonomic status has always been controversial. Therefore, it is very necessary to use molecular markers to s...

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Problems solved by technology

[0004] However, in the process of studying the genetic diversity and lineage geography of C. chrysanthemum, the general primers for the above genes were used to amplify the corresponding genes of the fungus, but no amplification results were obtained or multiple non-specific bands were amplified.

Therefore, although the above-mentioned genes have many advantages, they have shown limitations in the research of Viridans chrysanthemum, which limits the use of the above-mentioned genes for related research on Viridans chrysanthemum.

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