Optimizing T-RFLPs for the Analysis of Rhizosphere Communities of Lactuca spp

The rhizosphere is the zone of soil influenced by plant roots where bacteria have close physical interactions.  Understanding how plants interact with bacteria in the rhizosphere has important implications for developing sustainable agriculture and effective bioremediation strategies.  Micallef et al. (2009) characterized the succession of rhizosphere bacteria communities associated Arabidopsis thaliana ecotypes Cvi and Ler during the plant’s lifecycle using terminal restriction fragment length polymorphisms (T-RFLPs).  The development of ecotype specific rhizosphere communities suggested that genes in the plant may drive changes in the bacterial communities.   T-RFLPs are generated using bacteria specific primers to amplify 16s ribosomal RNA genes extracted from rhizosphere soil.  The PCR amplicons are digested with a restriction enzyme and visualized to create a community fingerprint.  The presence/absence or relative abundance of different species within the fingerprint are used to determine differences in community composition and structure.  We extend the same approach to study the rhizosphere interactions of wild (Lactuca serriola and Lactuca saligna) and domesticated (Lactuca sativa) lettuce to determine when unique communities develop, and how they may change over time.   A member of the Composite family, lettuce is an ideal model to study because it is easy to cultivate and because it is both an important crop and a weedy/invasive species.  Here the goal is to characterize the bacteria specifically associated with wild and domesticated lettuce.  The association may be qualitative (present for one plant genotype and not the other) or quantitative (more abundant for one plant genotype and not the other).