DNA Microsatellite and Sex Identification Marker Screening for Mexican Spotted Owl (Strix occidentalis lucida)

Feathers were collected opportunistically near known Mexican Spotted Owl nesting and roosting sites, opportunistically when found away from known roosting/nesting sites and five feathers were collected from a known adult mortality. Feathers were placed in dry envelopes and stored at ambient temperature. Additionally, 10 tissue samples were obtained from preserved mortalities to allow marker screening with higher quality DNA possible from tissue samples. Collection and sampling occurred between 2016 and 2019. These specimens were preserved in Longmire Buffer (Longmire et al. 1988). All sampling was provided by National Park Service, Walnut Canyon National Monument.

DNA Extraction and quantification (Talbot et al. 2011): DNA extractions from all tissue sources used a salt extraction procedure described in Medrano et al. (1990) and modified as described in Sonsthagen et al. (2004). DNA extractions from feathers used the same protocol, with the following exceptions: dithiotrhreitol (0.1 mg ml−1) was added to the lysis buffer, and 1% glycogen was added to the DNA precipitation step. DNA concentration for tissue samples was quantified using fluorometry, and diluted to 50 ng μl−1 working concentrations for subsequent analyses.

Except for locus specific modifications noted below, PCR amplifications were carried out in a final volume of 10 μL and contained 50 ng (or 1 uL) genomic DNA, 0.1 mM dNTPs, 0.5 µmole unlabeled primers, 0.1 µmole IRD-labeled primer, 1 μg BSA, 1× PCR buffer [Perkin Elmer Cetus I: 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris/HCl, pH 8.3, 0.01% gelatin (w/v)], and 3 Units GoTaq Flexi DNA polymerase (Promega). PCR reactions began at 94°C for 2 min and continued with 40 cycles each of 94°C for 30 sec; 50°C for 30 sec; 72°C for 1 min followed by a single 30-min extension at 72°C. Exceptions: For locus SneD202w-1 IRD-labeled primer was increased to 1.5 µmole. For loci Fepo5-1 and Bb111-1 IRD-labeled primer was decreased to 0.75 µmole. For loci Bb101-1 IRD-labeled primer was decreased to 0.50 µmole. For loci 8G11-1 and 15A6 1.00 uL of Betaine was added to enhance PCR product. Adding the same amount of betaine to locus 13D8-1 may or may not improve product. For locus SneA127w, thermocycler parameters were shortened to 94°C for 2 min and continued with 40 cycles each of 94°C for 15 sec; 50°C for 15 sec; 72°C for 30 sec; followed by a 30-min extension at 72°C. Primers originally published in Hsu et al. 2003 (for locus Oe128), Hull et al. 2008 (for loci SneD105, SneD202w, and SneA127), Proudfoot et al. 2005 (for locus Fepo5), and Thode et al. 2002 (for loci 6H8 and 8G11) were redesigned and are provided in the table "mexicanSpottedOwl_screen_pierson.csv" included with this data package. Primers originally published in Isaksson and Tegelstroem 2002 (for loci Bb101 and Bb111) were previously redesigned and published in Dial et al. 2012 and are provided in the same table.

PCR products were electrophoresed on a 48-well 25-cm 6% polyacrylamide gel on a LI-COR 4200LR automated sequencer (LI-COR, Inc.) (Sonsthagen et al. 2004)

Henke (2005) P2Owl and P8Owl primers were redesigned (see "mexicanSpottedOwl_screen_pierson.csv" P2MelOwl/P8MelOwl) after sequencing the CHD gene and reducing the size of the targeted marker from 387/393 bp to approximately 210 base pairs to reduce the chance of allelic dropout when using poor quality samples (e.g. feathers, degraded, opportunistic ground sampling). PCR amplifications were carried out in a final volume of 10 μL and contained 50 ng (or 1 uL) genomic DNA, 0.2 mM dNTPs, 0.4 µmole unlabeled primers, 0.03 µmole IRD-labeled primer, 1 μg BSA, 1× PCR buffer [Perkin Elmer Cetus I: 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris/HCl, pH 8.3, 0.01% gelatin (w/v)], and 0.5 Units GoTaq Flexi DNA polymerase (Promega). PCR reactions began at 94°C for 1:30 min and continued with 40 cycles each of 94°C for 30 sec; 48°C for 45 sec; 72°C for 45 sec; followed by a single cycle of 48°C for 1 min; 72°C for 5 min.