In order to tie the observed adaptation of functional traits collected and the genetic architecture of those traits collected, to genomic and proteomic divergence, we will create the tools and improve computational pipelines necessary to complete the analysis.

We will develop EST libraries using leaf, flower, and root tissue from Dubautia reticulata and Dubautia menziesii at the Clemson University Genomics Institute (CUGI). An estimated 12,000 ESTs will be sequenced, assembled and annotated, for an expected 5-7,000 unigenes.  Unigene sequences will be screened for variation among field-collected samples representing the three targeted species pairs. Preference for further analyses in remaining Hawaiian silversword alliance species will be given to ~120 unigenes with apparent orthology to functional candidate genes, as identified by comparison to the Arabidopsis, Helianthus, and Lactuca genome studies.

Genomic large-insert (BAC) libraries will be developed for one representative species at eight-fold redundancy, requiring ~72,000 clones of 150 kb average size based on the estimated genome size of 1.35 x 109 bp (Friar, unpublished data). Libraries will be screened using probes derived from an electronic sequence database of EST sequences from our libraries and existing libraries of other species available at CUGI, using modifications of algorithms developed by collaborators. Regions of interest will be shotgun sequenced from positive BAC clones and completely annotated. These sequences will be used to identify corresponding genomic regions for analysis of gene/genome structure and regulatory elements related to key adaptive ecological traits.

We will also utilize an integrated system of shotgun proteomics and small molecule analysis that has proved useful in studying other plant processes at CUGI. Transcriptome profiles will be carefully compared to proteome profiles using software and analysis pipelines currently being developed in-house at CUGI. The comparison of the transcriptome and proteome networks, and analysis of small molecules will permit an unprecedented examination of the molecular differences which ultimately represent the cellular processes that are manifested as morphological and physiological features. Importantly, these analyses will serve as a bridge between the genetic/genomic patterns of diversity and divergence and the morphological and physiological responses to the environment. To our knowledge, no such integrative approach has been developed or implemented, especially in the framework of evolutionary diversification.