Penstemon scariosus Pennell (Plantaginaceae) exhibits a broad and complex range of morphological variability (Holmgren, 1984; Neese and Atwood, 2008). Of the four penstemon varieties recognized by Neese and Atwood (2008), P. scariosus var. albifluvis (England) N. H. Holmgren (White River penstemon), native to the Green River Formation of the western United States, is considered the most distinct (England, 1982; Holmgren, 1984; Neese and Atwood, 2008). Because of increasing efforts to recover hydrocarbon deposits found in this geological formation, P. scariosus var. albifluvis 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 albifluvis. Identifying robust and reliable P. scariosus simple sequence repeats (SSRs, i.e., microsatellites) 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 modifications, 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 P. scariosus var. albifluvis, six var. cyanomontanus Neese, six var. garrettii (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 assembler 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 identified 1067 P. scariosus contigs with perfect di-, tri-, tetra-, and pentanucleotide motifs with five 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), and AAAAT/ATTTT (20).
We randomly selected 240 putative SSRs and designed flanking primers using Primer3 version 2.0 (Rozen and Skaletsky, 2000), with default parameters except for: product size = 120–250 bp, maximum melting temperature (T m) difference = 1°C, and maximum poly X = 3. These synthesized primer pairs (Integrated DNA Technologies, Iowa City, Iowa, USA) were first screened for PCR amplification and polymorphism efficacy on 3% Apex SFR Agarose Super Fine Resolution (Genesee Scientific, San Diego, California, USA) gels electrophoresed at 45 V for 20–24 h. PCR amplifications were performed in 12-µL reactions consisting of 3.0 µL (30 ng/µL) DNA, 0.5 µL of each 10 µM forward and reverse primer, 6.0 µL MyTaq HS Red Master Mix (Bioline, Taunton, Massachusetts, USA), and 2.0 µL ddH2O. PCR reactions were performed using a C1000 or a T100 thermal cycler (Bio-Rad, Applied Biosystems, Foster City, California, USA) with the following parameters : 95°C for 60 s; 35 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 10 s; and a single final extension cycle of 72°C for 60 s.
From the 240 primer pairs tested, we selected 17 for fluorescent labeling of one member of each pair with one of three dyes: NED (yellow [Life Technologies, Grand Island, New York, USA]), 6-FAM (blue [Integrated DNA Technologies]), or HEX (green [Integrated DNA Technologies]). These primers were selected based on robust repeatability and allele diversity across 30 samples. The samples included the broad geographic range of P. scariosus, P. comarrhenus A. Gray, P. compactus (D. D. Keck) Crosswh., P. cyananthus Hook. var. cyananthus, P. fremontii Torr. & Gray var. fremontii, P. fremontii var. glabrescens Dorn & Lichvar, P. gibbensii Dorn, P. strictus Benth., and P. subglaber Rydb. Labeled markers were amplified in 6-µL reactions containing 1.5 µL (30 ng/µL) DNA, 0.25 µL of each primer (10 µM each fluorescently labeled forward and unlabeled reverse), 3.0 µL MyTaq HS Red Master Mix (Bioline), and 1.0 µL ddH2O. PCR reactions were performed using the following parameters: 95°C for 60 s; 25 cycles of 95°C for 15 s, 57°C for 15 s, and 72°C for 10 s; and a single final extension cycle of 72°C for 60 s. PCR products were diluted 1:20, and 1 µL of each sample was vacuum dried at 45°C for approximately 30 min using an SPD1010 SpeedVac (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Samples were analyzed at the Brigham Young University DNA Sequencing Center (Provo, Utah, USA), utilizing the ABI 3730xl (Applied Biosystems) with GeneScan 500 ROX Size Standard (Applied Biosystems). Fragment length analysis was accomplished using Geneious version 8.0.5 (Kearse et al., 2012). Hardy–Weinberg equilibrium (HWE) for loci within a population was calculated with GenAlEx version 6.501 (Peakall and Smouse, 2012).
Sixteen of the 17 SSRs provided reliable products across the 95 samples listed in Appendix 1, while one marker could not be scored with precision (Table 1). Ten of these 16 markers have not been previously reported (PS077– PS086), and four of the previously reported (Dockter et al., 2013) markers (PS014, PS016, PS048, PS064) were redesigned for this study to optimize for reliability across the range of P. scariosus varieties. The two remaining markers, Pen04 and Pen23, were SSR markers found to be viable in this study using the same PCR primers reported by Kramer and Fant (2007). These 16 SSR markers produced 360 unique alleles, ranging from one to 21 per taxon, or an average of 22.5 alleles per locus across P. scariosus and P. fremontii var. glabrescens (Table 2). The mean observed and expected heterozygosity values for each taxon tested were as follows: P. scariosus var. albifluvis (0.549 and 0.705), P. scariosus var. cyanomontanus (0.586 and 0.686), P. scariosus var. garrettii (0.496 and 0.808), P. scariosus var. scariosus (0.394 and 0.655), and P. fremontii var. glabrescens (0.430 and 0.646) (Table 2). Within populations of varieties albifluvis and cyanomontanus, none of the loci deviated from HWE, while two of the loci significantly deviated from HWE in the var. scariosus population. Interestingly, 12 of the 16 loci in the var. garrettii population demonstrated significant deviation from HWE, which may reflect that our sampling of this variety was from across a large geographic range representing multiple populations (Appendix 1). Markers PS078 and PS048 were monomorphic in P. scariosus var. cyanomontanus and P. fremontii var. glabrescens, respectively (Table 2). We note, however, the limited numbers of samples tested for these two taxa, which may account for the lack of polymorphisms (Appendix 1). Fifteen of the 16 primer combinations produced robust, usually polymorphic, markers across P. comarrhenus, P. compactus, P. cyananthus var. cyananthus, P. fremontii var. fremontii, P. gibbensii, P. strictus, and P. subglaber. However, PS078 poorly amplified with multiple weak bands in P. compactus and P. gibbensii (Table 3).
Characteristics of 16 microsatellite markers developed for Penstemon scariosus.
The 16 markers presented here consistently produced robust data sets across the four P. scariosus varieties tested, and 15 were reliable across eight additional related taxa. The SSRs identified in this study will provide a reliable set of markers needed to conduct studies of the genetic diversity of P. scariosus.
Observed and expected heterozygosity values using 16 SSR markers of the four named varieties of Penstemon scariosus and P. fremontii var. glabrescens.a
Cross-amplification of the 16 microsatellite markers developed for Penstemon scariosus in each of eight related Penstemon taxa.a
 The authors gratefully acknowledge Dr. Robert L. Johnson for assisting in the taxonomic identification of the samples used in this study. Financial support for this study was provided by Brigham Young University and a Bureau of Land Management Vernal Field Office conservation agreement (no. L14AC00346).