• Premise of the study: Development of genetic markers can be costly and time-consuming, especially when multiple primer pairs are fluorescently labeled. This step was streamlined by combining two techniques in the same PCR reaction: (1) custom-labeling of primers by the investigator and (2) multiplexing multiple primers together in the same reaction.• Methods and Results: This technique was successfully used to develop microsatellite markers in several plant species. Microsatellites amplified with this multiplexing process were identical to those generated from PCR using individual primer pairs and with traditional methods using a priori labeled fluorescent primers. Tests of PCR cycling programs revealed that conditions recommended for the commercial kit generated stronger fragment peaks than the previously recommended cycling protocol.• Conclusions: This technique is an efficient and economical way to fluorescently label multiple microsatellite primers in the same reaction. It is also applicable to other markers used in PCR amplification of genetic material.

During microsatellite marker development, researchers must choose from a pool of possible primer pairs to further test in their species of interest. In many cases, the goal is maximizing detectable levels of genetic variation. To guide researchers and determine which markers are associated with higher levels of genetic variation, we conducted a literature review based on 6782 genomic microsatellite markers published from 1997–2012. We examined relationships between heterozygosity (He or Ho) or allele number (A) with the following marker characteristics: repeat type, motif length, motif region, repeat frequency, and microsatellite size. Variation across taxonomic groups was also analyzed. There were significant differences between imperfect and perfect repeat types in A and He. Dinucleotide motifs exhibited significantly higher A, He, and Ho than most other motifs. Repeat frequency and motif region were positively correlated with A, He, and Ho, but correlations with microsatellite size were minimal. Higher taxonomic groups were disproportionately represented in the literature and showed little consistency. In conclusion, researchers should carefully consider marker characteristics so they can be tailored to the desired application. If researchers aim to target high genetic variation, dinucleotide motif lengths with large repeat frequencies may be best.

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Environmental change, accelerated by anthropogenic activities, threatens many species and can be especially challenging for rare species given their potentially limited capacity for migration and adaptation relative to more common species. The ability to acclimate via phenotypic plasticity could provide an important path to species persistence in the face of such change. We investigated the responses of an endangered plant species endemic to a highly dynamic riparian habitat in southeastern Tennessee, USA, and its most widespread congener to environmental change to elucidate their current statuses and future vulnerability. Specifically, we compared the population-and species-level plasticity of rare Pityopsis ruthii and common P. graminifolia to contrasting light, temperature, and water conditions in a growth chamber experiment to evaluate their potential to acclimate to environmental change. Contrary to our expectations, P. ruthii had greater phenotypic plasticity than its common congener in response to both altered light and water availability. But this plasticity was not associated with increased fitness, suggesting that it was not adaptive. Concurrently, we genotyped these individuals at nine putatively neutral microsatellite loci to contrast genetic diversity across the range of each species. As expected, P. ruthii exhibited reduced genetic diversity relative to its more common congener. Overall, our findings accord with the narrow range and current habitat specificity of P. ruthii, especially its tolerance of highly variable water, and highlight its potential vulnerability to future environmental change.

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