Identification and characterization of microsatellite markers in Penstemon scariosus (Plantaginaceae)1

Because of increasing efforts to recover hydrocarbon deposits found in this geological formation, P. scariosus var. albifl uvis is being considered for listing under the Endangered Species Act of 1973 (Ashe, 2013). Thus, there is an urgent need to understand genetic diversity within P. scariosus and especially within variety albifl uvis. Identifying robust and reliable P. scariosus simple sequence repeats (SSRs, i.e., micro-satellites) would prove useful in such diversity studies. Two previous reports of SSR markers for Penstemon Schmidel have been reported (Kramer and Fant, 2007 ; Dockter et al., 2013) in the literature. However, only two of the reported SSR markers from Kramer and Fant (2007) proved to be robust and produce reliable results, while the others were less dependable, without modifi cations, in our expanded survey of P. scariosus. Thus, the objective of this study was to develop additional extensively tested SSR markers for P. scariosus. METHODS AND RESULTS DNA was extracted from lyophilized leaf tissue collected, in situ, from up to four individual plants from each population described in Appendix 1 , using the method detailed by Todd and Vodkin (1996). To identify the SSRs, we used the genomic reduction protocol described by Maughan et al. (2009) , which has also been successfully used to develop SSRs for Utah agave (Agave utahensis Engelm.) and post oak (Quercus stellata Wangenh.) (Byers et al., 2014 ; Chatwin et al., 2014). The genomic reduction procedure used DNA samples from two (Pennell) N. H. Holmgren, and eight var. scariosus. The 454 pyrosequencing of those samples provided us with a total of 1,579,847 reads, representing over 877 Mb, equaling approximately 60,763 reads per sample or about 1,336,786 reads across the 22 P. scariosus samples. The average read and mode lengths were 556 bp and 594 bp, respectively. Using default parameters of the Roche Newbler assem-bler program (version 2.3; 454 Life Sciences, Branford, Connecticut, USA), we obtained a total of 46,628 contigs from those reads. Using the computer program MISA (Thiel et al., 2003), we identifi ed 1067 P. scariosus contigs with perfect di-, tri-, tetra-, and pentanucleotide motifs with fi ve to 15 repeat units. There were 45 sequences with two repeating motifs in a single contig meeting the above criteria , while 433, 357, 107, and 144 had single di-, tri-, tetra-, and pentanucleotide repeats within the sequences, respectively. The most common repeat motifs were AT/AT (258), AAT/ATT (150), AAAT/ATTT (38), …