OVERVIEW: Merlins were sampled at migratory bird banding stations (Alaska, Wisconsin, and Minnesota) or local rehabilitation facilities in southcentral Alaska. Peregrine Falcons and Cooper's Hawk samples were part of the USGS Alaska Science Center's genetic collection. Previously published microsatellites developed in closely-related falcon species were screened for variability in Merlins. Eight were selected and are presented here. Additional microsatellites were isolated and developed from two shotgun sequencing efforts. Sixteen novel microsatellites from these efforts were identified and presented here. Merlin-specific primers for one microsatellite located within a gene associated with circadian rhythms and polymorphism linked to juvenile dispersal and migratory behavior in other species were designed from conserved sequences identified in closely-related falcon species.
DNA EXTRACTION: DNAs were extracted using a modified salt extraction technique (Medrano et al. 1990; Sonsthagen et al. 2004), quantified using fluorometry, and diluted to 50 ng/ul working solutions.
MICROSATELLITE ISOLATION: Genomic DNA used to generate microsatellite sequences was extracted from blood tissue from a Merlin from Eagle River, Alaska, admitted for treatment at the Bird Treatment and Learning Center, a wild bird rehabilitation center, during August 2007 (USGS ASC: 07-393), and muscle tissue from a merlin collected in March 2007, in Anchorage, Alaska (USGS ASC: CPD1272). We performed two separate next generation shotgun sequencing runs using GS Junior Titanium rapid library preparation and sequencing chemistry (Roche, Branford CT). Briefly, DNA extracted from the specimens was nebulized and ligated with 454 sequencing primers. The prepared library for sample 07-393 was clonally amplified and sequenced in a half-panel Roche 454 GS Junior fragment run, resulting in 15,410 reads from the Merlin library, with an average length of 238 base pairs. A second Merlin library, from sample CPD1272, was clonally amplified and sequenced in a quarter-panel Roche 454 GS Junior fragment run, which produced 46,409 reads, with an average length of 443 base pairs.
NOVEL PRIMER DEVELOPMENT: Msatcommander 0.8.2 (Faircloth 2008) searched 182 contigs and 34,944 unassembled reads, combined from both Roche 454 GS Junior fragment runs, for microsatellite repeats with 8 or more repeat units, and designed primer sets. Designed primers were examined and modified when possible so that primers amplified a product of less than 200 bp, as recommended to decrease the likelihood of PCR artifacts, particularly for DNA extracted from low-quality or low-quantity DNA sources (Sefc et al. 2003). Of 934 reads with microsatellite motifs, we selected 23 loci containing dinucleotide motifs for screening. Among these, fifteen novel microsatellite loci were characterized as presented in the data file associated with this data release (merlin_genetics_sage_2020.csv). Complete primer sequences are provided in Hull et al (2020).
PREVIOUSLY PUBLISHED MICROSATELLITES: Microsatellites isolated from Peregrine and Gyrfalcon and previously published by Nesje et al (2000) and Nesje and Røed (2000) were screened, and those found to be variable in Merlins were screened and included in this data release (merlin_genetics_sage_2020.csv).
We also aligned sequences in Peregrine Falcon (GenBank No. NW_005940506) and Saker Falcon (F. cherrug; GenBank No. XM_014285770) that include a (GA)12 (A)n (GA)11 microsatellite found in the 3’UTR of the adenylate cyclase activating polypeptide 1 gene (ADCYAP1), and designed primers (Hull et al 2020) to amplify and screen for polymorphism in merlin. The ADCYAP1 gene is associated with endogenous circadian clock control, and polymorphism in the target microsatellite has been associated with juvenile dispersal behavior and migratory behavior in some avian species (Chakarov et al 2013 and Mueller et al 2011).
MICROSATELLITE VISUALIZATION: Polymerase chain reaction (PCR) amplification and scoring of microsatellites followed Hull et al.(2020) and Sonsthagen et al (2004). Microsatellite alleles were resolved on a 25 cm 6% polyacrylamide gel using a LI-COR (IR 4200) automated DNA sequencer. A small subset of individuals were run on an initial gel for each locus alongside an M13 sequence ladder of known size and allele sizes assigned using Gene Profiler 4.05 (Scanalytics, Inc.). Subsequent gels included these known size individuals on every gel from which relative sizes of unknown individuals were assigned, using Gene Profiler 4.05 (Scanalytics).