Protocols used in this experiment
Ming-Dao Chia, Anna Simonsen, Andrew Almonte, Justin Borevitz
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Rhizobia (nitrogen-fixing bacterial plant symbionts) are a vital source of bioavailable nitrogen, particularly in poor soils. Nutrients from legumes (rhizobia host plants) are important for agriculture and ecological regeneration.
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Many rhizobia species are well studied in single strain inoculation experiments, in culture, and in relation to their plant hosts.
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Less is known about their ecology in a complex microbiome, with closely related strains and uncultivated microbes.
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Recent developments in metagenomic methods allows studying abundance of rhizobia strains in nodules and free living environments with minimal culture or amplification bias.
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Observing patterns of rhizobia strain abundance changes in legume nodules and associated soil.
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Observing patterns of rhizobia strain abundance changes in soil in the absence of a legume host.
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Identification of horizontal gene transfer events in known strains throughout the samples sequenced.
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Study species: legume Acacia acuminata with rhizobial symbiont Bradyrhizobium japonicum (4 strains).
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Plants are grown from surface sterilized seeds on autoclaved sand media for 8 weeks inside a growth chamber.
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A complex microbiome is inoculated from an external soil sample. Strains are inoculated at once as a balanced culture mix.
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DNA is extracted from sand and pots with a soil extraction kit.
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Long read sequencing (8-100 kb reads) is conducted with Oxford Nanopore MinION, allowing for strain-level identification and potential for observing horizontal gene transfer in single reads.
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Abundances of strains are measured through counting reads.
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Potential horizontal gene transfer events are identified with bioinformatics.
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Nanopore sequencing consistently yields some multigene metagenomic reads (>50 kb) with our samples.
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Combining long and short reads yields single contig reference genomes.
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Direct gDNA sequencing of sand and nodules produces reads that can be mapped to references.
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Link specific rhizobia genes or variants to abundance patterns
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Find potential co-inoculants to maintain stable free living rhizobia populations
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Create assays to identify stable inoculants for a variety of hosts and soil conditions
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Observe distribution between and within nodules of the same plant
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Correlate host genomic variance with abundance patterns
Contact: Ming-Dao.Chia@anu.edu.au