Use of Next Generation Sequencing for Transcriptomic Analysis of a Deletion Region Containing a Gene Responsible for Rust Resistance in Common Bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) is an important grain legume in many developing countries throughout the world and is most useful for its high protein and dietary fiber. The fungal rust pathogen Uromyces appendiculatus causes crop loss in susceptible cultivars of common bean. Of the 89 races of rust cultivated in the U.S., the Ur-3 locus confers resistance to 44 of those races with at least one other gene Crg (Complements resistance gene), a gene required for Ur-3-mediated rust resistance. With the advent of the full genome sequence of common bean cultivar G19833, we are now able to use a global approach for a better understanding of the molecular mechanisms responsible for rust resistance in common bean. In order to better understand this interaction, we inoculated several common bean genotypes with bean rust race 53, isolated leaf RNA at various time points, and sequenced their transcriptomes. Information generated from Illumina RNA-Seq data was then used to analyze two wild type cultivars, ‘Sierra’ (resistant) and ‘Olathe’ (susceptible), and three Sierra-derived susceptible mutants -crg, ur3-D2 and ur3-D3. RNA-Seq data was used to identify and characterize the deletion region in crg (which carries a deletion of the Crg locus) using comparative gene expression analysis against Sierra. Genomic PCR with candidate gene primers from this region indicates no amplification in crg, but shows amplification in Sierra. Subsequently, mock inoculated and inoculated samples of Sierra leaf RNA were compared for differential expression among candidate genes in the deletion region, which is approximately 250kb.