Flying Through Microsatellite Primer Redesign of Southwestern Willow Flycatcher
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Authors
Michael Fox
Issue Date
2026
Type
Language
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Abstract
The Southwestern Willow Flycatcher (Empidonax traillii extimus) is an endangered subspecies of Willow Flycatcher that breeds in riparian habitats within isolated subpopulations of the southwestern United States. Southwestern Willow Flycatchers are neotropical migratory species, which makes it difficult to track population structure through demographic analysis alone; however, genetic monitoring has been employed successfully in previous studies. Specifically, DNA microsatellites are used to determine population structure, how migration impacts gene flow across subpopulations, and overall breeding success of returning groups. Microsatellite analysis of Southwestern Willow Flycatchers last occurred during 2009, and both microsatellite loci mutation and technology improvements have made older primers outdated for future work. The objective of this study was to redesign primers by comparing existing primer sequences to catalogued genomes of the Southwestern Willow Flycatcher. We modified existing forward and reverse primer sequences in accordance with paired melting temperature, similar base-pair length, and location in comparison to the targeted tandem repeats of (AAAG), (CTT), (ACG), and (GATA). Our next steps are to evaluate the compatibility of these primers with the targeted repeated sequences used for microsatellites. These new primers will be tested on DNA collected from 315 individuals sampled throughout Arizona and Nevada between 2019 and 2023. We will use housekeeping genes like mitochondrial alleles to determine the quality of the DNA sample library and conduct further PCR trials of both new and existing primer sets upon PCR quality template DNA. Updating older primer sets will allow for the reevaluation of population structure of recently sampled individuals, which would reinforce prior knowledge and potentially offer new information towards the genetic f low of returning breeding migrant populations.