DNA microsatellite markers for Mustard Hill Coral (Porites astreoides) from the Florida Keys Reef Tract

Porites astreoides samples were obtained from four locations along the FKRT using a Global Positioning System (GPS): Fowey Rocks in Biscayne National Park (25.59047, -80.09560), Crocker Reef in the Upper Florida Keys (24.90908, -80.52665), Sombrero Reef in the Middle Florida Keys (24.62687, -81.10893), and Pulaski Shoal in the Dry Tortugas National Park (24.69355, -82.77280). The corals were collected within a 100 to 200 square meter area at each site, and colonies selected for sampling were no closer than approximately 20 meters (Lenz and others, 2021; compare with Kuffner and others, 2013). After collection, the corals were grown for two years (from 2015 to 2017) on cinderblocks installed on the reef as part of the U.S. Geological Survey calcification assessment project (Kuffner and others 2013; Morrison and others 2013). Coral colony condition was assessed, and growth rates were monitored every six months during the study using the methods outlined in Kuffner and others (2013; data presented in Lenz and others, 2021). At the end of the two-year period, several 4 millimeter thick slabs were cut from the center of the colonies. One slab was used for host tissue sampling in this study as well as symbiont sampling and histological examination in an associated study (Lenz and others, 2021). The rest of the colonies were outplanted onto the reefs.

DNA Extraction: Preparation of cetyltrimethylammonium bromide (CTAB) mixture (Baker and Cunning 2016).

Run electrophoresis gel using a 1.5% agarose gel suspension to visualize DNA extraction.

Amplified DNA using PCR in 10 microliter (μL) reactions with 10 nanograms (ng) DNA, 1 micromole (μM) fluorescently-labeled forward primer, 1 μM reverse primer, 0.2 millimole (mM) deoxynucleoside triphosphate (dNTP) (Promega), 2μL 5 times (5x) PCR GoTaq Flexi buffer (Promega), 0.25 units (0.05 μL) GoTaq Flexi polymerase (Promega), 2 mM MgCl2 (Promega). The PCR procedure was based on the protocols of Davies and others (2013), but was modified as follows: (1) increased to 1 μM fluorescently-labeled forward primer and 1 μM reverse primer, (2) modified to 2 μL 5X PCR GoTaq Flexi buffer (Promega). Increased 0.25 U GoTaq Flexi polymerase (Promega), (3) added 2 mM magnesium chloride (MgCl2) (Promega), and (4) thermocycler protocol modifications: Increased initial denaturing temperature 95 degrees Celsius (˚C). Increased number of cycles to 40 at (95˚C for 40 seconds, 58–60˚C for 60 seconds, 72˚C for 60 seconds).

Sent select samples to the University of Illinois Urbana-Champagne (UIUC) Core Sequencing Facility for fragment analysis of dilution sets to determine optimum dilution for each primer set.

Sent samples to the UIUC Core Sequencing Facility for fragment analysis using Applied Biosystems 3730x1 DNA Analyzer and ROX1000 standards.

USGS researchers worked with collaborators at University of South Florida (USF), Saint Petersburg to analyze microsatellites using GeneMapper 5.0 to determine microsatellite calls. Micro-Checker (version 2.2.3) was used to confirm that calls were accurate (for example, correct number of bases between calls). Allele bins were assigned by rounding observed size to the closest integer, except when Micro-Checker indicated that the bins should be adjusted due to errors with nucleotide repeats (for example, only 2 base pairs [bp] difference for trinucleotide repeats). In those cases, alleles were marked as unknown "?" instead of called.

Reran PCR for samples that failed to amplify well or indicated potential null alleles.

Sent rerun samples to the UIUC Core Sequencing Facility for fragment analysis using Applied Biosystems 3730x1 DNA Analyzer and ROX1000 standards. "GeneScan™ 1000 ROX™ Size Standard is an internal lane size standard developed for use with Applied Biosystems® fluorescence-based DNA electrophoresis systems. The use of an internal lane size standard during electrophoresis enables automated data analysis and precise DNA fragment size comparisons between electrophoresis runs. GeneScan™ 1000 ROX™ Size Standard is designed for sizing DNA fragments in the 47–946 bp range and provides labeled fragments of 47, 51, 55, 82, 85 93 99, 126, 136, 262, 293, 317, 439, 557, 692, 695 and 946 bases. Each of the DNA fragments is labeled with ROX™ fluorophore, which results in a single peak when run under non-denaturing conditions." Quotation is from the ThermoFisher Catalog # 401098