Method for detecting prokaryotic microorganisms in crop rhizosphere based on high-throughput sequencing

Abstract

The invention relates to a method for detecting prokaryotic microorganisms in rhizosphere soil of different crops based on 16S rDNA high-throughput sequencing. The method comprises the following stepsof 1, collecting shaking-off soil (as a system control) of different genotype crops outside root zones or at root systems and the rhizosphere soil; 2, extracting total microbial DNA from each samplesoil; 3, using a double-label primer method for PCR amplification of V5-V7 hypervariable region fragments of 16S rDNA in the total DNA to construct a library; 4, for a qualified library, simultaneously conducting synthesis and sequencing of a double-end 300 nucleotide pattern by using an Illumina Miseq platform; 5, splicing after pure paired READS is obtained, and generating 40,000 effective TAGsfrom each sample for clustering into operable classification units; 6, according to the system control, accurately determining the composition, structure, diversity and relative abundance saliency analysis of prokaryotic microbial communities in the rhizosphere soil of each sample, and comparing similarities and differences between different crops.

Description

technical field [0001] The invention belongs to the technical field of soil microbiology, and specifically relates to a method for detecting rhizosphere soil microorganisms of different crops based on deep high-throughput sequencing of 16SrRNA gene (V5-V7 hypervariable region) under field planting mode. Background technique [0002] The rhizosphere (Rhizosphere) is defined as the biologically active area closely surrounding the roots of viable plants, which contains a large number of microorganisms, including bacteria, fungi, and actinomycetes, as well as some algae and viruses, generally within 1cm from the root surface . Different plants or even different growth stages of the same plant, their root exudates will affect the growth and reproduction of rhizosphere microorganisms; and rhizosphere microorganisms participate in the decomposition of organic matter in the soil, the formation of humus, the transformation and circulation of nutrients, etc. Physiological and biochem...

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Problems solved by technology

[0006] The problem to be solved by the present invention is to overcome the shortcomings of the prior art, such as low detection throughput, small scale, and low coverage of non-cultivable microorganisms, and provide a method based on 16S rDNA (V5-V7) high-throughput sequencing to detect the rhizosphere of different crops. The soil microbial method provides a new method for the accurate assessment of the impact of different genotype crops, including transgenic crops and their recipient varieties, on the rhizosphere soil microbial community

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