Molecular Marker Development Using 454-Pyrosequencing in Four Species of Penstemon

Penstemon represents a large number of drought tolerant, perennial species native to the Intermountain West and is considered to have great value as an ornamental plant.  Despite its problematic complexity and enormity as a genus, it is especially important in the application of environmentally wise landscaping practices, such as xeriscaping. 454 pyrosequencing, a relatively new high-throughput sequencing technology, is one of the fastest, most economical methods of genome sequencing.  Using a genome reduction technique, which reduced the Penstemon genome complexity by 104 fold, we sequenced four species of Penstemon (P. cyananthus, P. disectus, P. davidsonii, and P. fruticosus) with an expected 11.46 fold average coverage for each fragment and then aligned using Newbler de-novo assembler.  These species were specifically chosen because two are phylogenetically close (P. davidsonii and P. fruticosus), another is phylogenetically distant (P. cyananthus) and one has been difficult to classify (P. disectus).  From one half plate of the pyrosequencer, about 733,000 sequences were produced with an average length of 392 bases; about 287 million bases were sequenced in total.  Alignments produced contigs representing 0.545%, 0.578%, 0.499%, and 0.495% of the genomes of P. cyananthus, P. disectus, P. davidsonii, and P. fruticosus respectively.  SSRs and SNPs were mined from sequence data using MISA and custom Perl scripts.  An average of 2311 true SNPs were found between any two of the four species of Penstemon.  301 total SSRs were discovered from an alignment of all four species.  97, 113, 49 and 58 SSRs were found from the individual alignments of P. cyananthus, P. disectus, P. davidsonii, and P. fruticosus respectively.  Both SNP and SSR markers, derived from these four species, will be utilized to detect genetic differences between species across the genus in order to refine the complex Penstemon taxonomy which will aid in breeding programs and further genetic studies.