
streptavidin beads (DynaBeads M-280 Streptavidin, Invitrogen) and verifi ed with PCR as above. Approximately 20 ng/ μ L of amplifi ed DNA was used in transformation with the TOPO TA Cloning Kit (Invitrogen) and grown on Luria-Bertani plates with 50 ng/mL ampicillin. About 350 colonies were screened for microsatellites using fl uorescent DIG probes (Roche Diagnostics). For positive clones, insert sizes were estimated with PCR using M13 primers and verifi ed on 1% agarose gels. DNA was extracted from 115 positive clones with appropriate insert sizes and PCR products were sequenced at Genome Quebec (McGill University, Montreal, Quebec, Canada) or Robarts Research Institute (University of Western Ontario, London, Ontario, Canada). Ninety-three of these clones contained a total of 90 unique microsatellite regions. Primer pairs were designed for the 32 clones that had both linkers, suitable fl anking region at both ends, and a minimum of eight repeats. We used Primer3web version 4.0.0 ( Koressaar and Remm, 2007 ;  and 3 M sodium acetate (NaOAc ), and then resuspended in TE buffer (10 mM Tris [pH 8.0], 1 mM EDTA). DNA quality and size were evaluated on 1.5% agarose gels (fragments ranged from 200-1000 bp).

Digested DNA was ligated to 1 μ M of SNX double-stranded linkers using T4 DNA ligase (Invitrogen, Burlington, Ontario, Canada) and 20 units Xmn I (New England Biolabs, Whitby, Ontario, Canada) overnight at 16 ° C. Linker ligation was tested using PCR amplifi cation with SNX forward primer (5 ′ -CTA-AGGCCTTGCTAGCAGAAGC-3 ′ ) in a reaction with 1 × buffer, 2.0 mM MgCl 2 , 150 μ M dNTPs (Roche Diagnostics, Laval, Quebec, Canada), 0.5 μ M primer, 1 μ g/ μ L bovine serum albumin (BSA), and 1 unit Taq polymerase (reagents from Invitrogen except dNTPs).

Linker-ligated DNA was hybridized to 3 ′ biotinylated (AC) 13 and (AG) 13 probes for 4 h at 70 ° C after 10 min at 95 ° C. Enriched DNA was captured using Note : T a = annealing temperature. a Range of fragment sizes including the M13 tag (5 ′ -CACGACGTTGTAAAACGA-3 ′ ) attached to the forward primer. b For genotyping, we used single primer pair reactions for 11 loci, one triplex reaction (loci C135b+C49+C19), and fi ve duplex reactions (B11+E30, C110+B34, E70+C67, C133+E42, and B59+C89), adjusting the number of cycles in the PCR program for B59+C89 to 32 ( Table 1 ) . c For two loci (A31b and C135b), we developed two additional primer pairs (A31c and C135c; see text for details). http://www.bioone.org/loi/apps Untergasser et al., 2012 ) and Amplifi x 1.5.4 (http://crn2m.univ-mrs.fr/AmplifX) to design primer pairs optimized to contain 18-22 bases, 40-60% GC content, 50-60 ° C melting temperature, and yield 100-350 bp PCR products. The forward primer of each pair was labeled with a D4 red-labeled M13 tail (5 ′ -CACGACGTTGTAAAACGA-3 ′ ) (Sigma-Aldrich Canada, Oakville, Ontario, Canada). The number and the identity of samples used for an initial testing of each pair varied. We used one to seven DNA samples from fi ve to 42 (mean = 30.6) populations covering the entire geographic range of C. cheiranthifolia and one to six DNA samples from three to 12 (mean = 9.7) populations of C. bistorta (Appendix 1). Each sample was genotyped twice in single-locus 5-μ L PCR reactions containing 0.5 μ L of DNA template (10 μ g/ μ L), 2.5 μ L of Multiplex PCR Master Mix (QIAGEN, Toronto, Ontario, Canada), 0.1 μ L of each forward and reverse primers (10 μ M), 1.1 μ L of M13taq (1 μ M; Sigma-Aldrich Canada), 0.2 μ L of Q-Solution, and 0.5 μ L of sterile double-distilled water. PCR involved 15 min of denaturation at 94 ° C, followed by 35 cycles of 20 s at 94 ° C, 30 s at 55 ° C or 57 ° C, and 40 s at 72 ° C, with a 10-min fi nal extension at 72 ° C. PCR product was diluted with double-distilled water to a fi nal volume of 15 μ L, and fragments were sized using a GenomeLab GeXP with the CEQ 8000 Genetic Analysis System version 9.0 (Beckman Coulter, Mississauga, Ontario, Canada).

Of 32 primer pairs, 24 yielded variable fragments of expected size and two of these amplifi ed within two other loci ( Table 1 ) . For these two loci (A31b and C135b), a second primer pair (A31c and C135c) was redesigned to improve consistency of amplifi cation in some C. cheiranthifolia but mainly in C. bistorta populations. For each locus, we estimated the number of alleles ( A ), observed ( H o ) and expected ( H e ) heterozygosities in one LF-SI population, one LF-SC population, two SF-SC populations (southern and northern parts of the range), and one LF-SI C. bistorta population using GenAlEx version 6.5 ( Peakall and Smouse, 2012 ) . We did not test for deviations from Hardy -Weinberg equilibrium because all populations of C. cheiranthifolia , including LF-SI populations, can exhibit some self-fertilization , so that H o is less than H e in many cases reported below.

Within populations, A ranged from one to 12 across loci (mean = 4.3) and was highest in the LF-SI populations compared to the LF-SC population and the two SF-SC populations ( Table 2 ) . Using only 13 loci for which the same individuals were genotyped, we detected 130 alleles total, of which 56 were found only in C. cheiranthifolia (mean ± 1 SE = 4.30 ± 0.49 private alleles per locus) and 10 only in C. bistorta (0.77 ± 0.26 private alleles per locus), suggesting that these markers could be useful to detect hybridization between these species, although a broader sample is required to determined which are diagnostic. H o and H e were highly variable but predictable based on the mating system, as both were highest in the two LF-SI populations, lower in the mixed-mating LF-SC population, and lower still in the two SF-SC populations ( Table 2 ) , thereby verifying the potential of these markers for studying the genetic consequences of mating system differentiation. Although cross-amplifi cation often failed in samples from the eight related taxa, there were many loci at which amplifi cation was successful (Appendix 1, 
CONCLUSIONS
All 24 microsatellite loci were variable in C. cheiranthifolia and C. bistorta , and a number of them also amplifi ed in eight (Failed) and loci that were not tested in some species (NT) are indicated. c Data for the three species of Camissonia are from Dick et al. (2014) . d These primers amplifi ed two non-overlapping variable regions in the species indicated, so two fragment ranges are provided.
CONCLUSIONS
closely related taxa, providing opportunities to test a broad range of ecological and evolutionary questions within species and across taxa. These markers will facilitate our ongoing studies of mating system evolution and geographic range limits in C. cheiranthifolia , as well as the genetic and ecological consequences of hybridization between C. cheiranthifolia and C. bistorta . The high frequency of cross-amplifi cation in related taxa provides opportunities for comparative studies investigating the genetic consequences of variation in life history and mating system, and ongoing hybridization in this morphologically and ecologically variable group. 

{'paper_id': '35861704', 'title': 'Microsatellite Primers for Camissoniopsis cheiranthifolia (Onagraceae) and Cross-Amplification in Related Species', 's2_url': 'https://api.semanticscholar.org/CorpusID:35861704', 'source': 'S2ORC'}