Sagehen Creek Field Station

02 Jun, 01 AM - 03 Jun, 10 AM

Project Description The purpose of this field project is to collect fresh root-nodules from 2 genera of host plants that form these nitrogen-fixing symbioses with a particular Frankia sp, that has remarkable nodulation characteristics. DNA and RNA will be extracted from the nodules for high throughput sequencing. The host genera, Purshia and Ceanothus, are actinorhizal plants -- a group of mostly woody perennials belonging to 8 Angiosperm families, that form nitrogen-fixing root-nodule symbioses in association with the bacterial genus, Frankia. These root nodules are similar to the those formed by associations between legumes and rhizobia; indeed, all 8 actinorhizal families, and the Fabaceae, are phylogenetically grouped in a single subclade of the Eurosid I, dubbed the ?nitrogen-fixing clade? (Soltis et al. 1995 Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2647-2651, March 1995). We seek to better understand host responses to Frankia , using comparative metagenomics and metatranscriptomics of the root nodules of Purshia, and Ceanothus. The results will give insight into evolutionary mechanisms underlying root-nodule symbioses. It is crucial to collect fresh, active root nodule material that can be flash-frozen, to preserve the nucleic acids properly. Because of seasonal patterns of soil profile drying, it is imperative to time the collections of field material to coincide with the active vegetative growth period. Methods We visited Sagehen in 2011, and identified sites containing Ceanothus and Purshia tridentata. We will make a walking survey to identify individual plants of P. tridentata, and Ceanothus spp. (either velutinus, or integerrimus, or prostratus), that would be appropriate for partial root excavation to find root nodules. Plants will be selected based on vigorous aboveground growth, and whether they are located on sites with reasonable access to soil and root systems (i.e. not too rocky, or crowded with vegetation, and with reasonable soil moisture). Excavation is carried out slowly and carefully, with shovel to gain access to the roots, and then mainly with geologist?s pick. In this way, nodules can be uncovered without damage. Nodules will be excised from the subtending roots with razor blades, into sterile Falcon tubes, and the contents of the tubes will be flash-frozen in a mixture of 100% ethanol and dry ice, in a Dewar flask. We will transport the ethanol in sealed metal containers, and the dry ice separately, in styrofoam coolers, mixing them in the Dewar flask at the time when we excavate the nodules. Dry ice sublimates and 100% ethanol evaporates, so there will be no disposal involved. We will replace the soil around the excavated portion of the root system of the shrubs, after collecting the nodules. It will most likely not be necessary to excavate an entire shrub, but more likely a sector of the root system. Sampling will require partial excavation of something like 5-8 shrubs per species. We will transport the collected nodule material in dry ice/styro coolers back to UC Davis.

Visit #28836 @Sagehen Creek Field Station

Approved

Under Project # 23867 | Research

Collection of nitrogen-fixing root nodules of native actinorhizal plants for genomic analysis

faculty - University of California, Davis

Reservation Members(s)

Group of 2 Research Assistant (non-student/faculty/postdoc) Jun 2 - 3, 2012 (2 days)

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