Dryas as a Model for Studying the Root Symbioses of the Rosaceae

The nitrogen-fixing root nodule symbiosis is restricted to four plant orders: Fabales (legumes), Fagales, Cucurbitales and Rosales (Elaeagnaceae, Rhamnaceae, and Rosaceae). Interestingly all of the Rosaceae genera confirmed to contain nodulating species (i.e., , , , and ) belong to a single subfamily, the Dryadoideae. The genus is particularly interesting from an evolutionary perspective because it contains closely related nodulating ( and non-nodulating species ( . The close phylogenetic relationship between these two species makes an ideal model genus to study the genetic basis of nodulation by whole genome comparison and classical genetics. Therefore, we established methods for plant cultivation, transformation and DNA extraction for these species. We optimized seed surface sterilization and germination methods and tested growth protocols ranging from pots and Petri dishes to a hydroponic system. Transgenic hairy roots were obtained by adapting -based transformation protocols for species. We compared several DNA extraction protocols for their suitability for subsequent molecular biological analysis. Using CTAB extraction, reproducible PCRs could be performed, but CsCl gradient purification was essential to obtain DNA in sufficient purity for high quality genome sequencing of both species. Altogether, we established a basic toolkit for the culture, transient transformation and genetic analysis of sp.