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Research Project: Discovery of Plant Genetic Mechanisms Controlling Microbial Recruitment to the Root Microbiome

Location: Plant Gene Expression Center

Title: Exploring the root microbiome: extracting bacterial community data for the soil, rhizosphere, and root endosphere

Author
item Simmons, Tuesday - University Of California
item Caddell, Daniel - University Of California
item Deng, Siwen - University Of California
item Coleman-derr, Devin

Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/10/2018
Publication Date: 5/2/2018
Citation: Simmons, T., Caddell, D., Deng, S., Coleman-Derr, D.A. 2018. Exploring the root microbiome: extracting bacterial community data for the soil, rhizosphere, and root endosphere. Journal of Visualized Experiments. 135:1-10.

Interpretive Summary: The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples.

Technical Abstract: As with many experimental platform, amplicon-based profiling of microbial communities can introduce a number of potential biases which should be considered during the experimental design and data analysis. These include the methods of sample collection, DNA extraction, selection of PCR primers, and how library preparation is performed. Different methods can significantly impact the amount of usable data generated, and can also hinder the efforts to compare results between studies. Additionally amplicon-based profiling are often customized, making comparisons across studies can be challenging. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.