#METABOLOMICS WORKBENCH ramkhattri_20181220_061854 DATATRACK_ID:1588 STUDY_ID:ST001116 ANALYSIS_ID:AN001812 PROJECT_ID:PR000747 VERSION 1 CREATED_ON December 20, 2018, 12:10 pm #PROJECT PR:PROJECT_TITLE Variability in metabolomic profiles among unique genotypes of Acropora PR:PROJECT_TITLE cervicornis PR:PROJECT_TYPE intraspecific variability PR:PROJECT_SUMMARY This project aims to identify differences in metabolomic profiles among known, PR:PROJECT_SUMMARY unique genotypes of the threatened staghorn coral Acropora cervicornis. Previous PR:PROJECT_SUMMARY studies have shown that the three genotypes selected for study possess unique PR:PROJECT_SUMMARY phenotypes related to growth and thermotolerance. Improved understanding of PR:PROJECT_SUMMARY metabolomic differences could aid in selection of A. cervicornis genotypes for PR:PROJECT_SUMMARY use in restoration. PR:INSTITUTE University of Florida (This is where the analysis is being performed, i.e. UF) PR:DEPARTMENT Department of Fisheries and Aquatic Sciences PR:LAST_NAME Patterson PR:FIRST_NAME Joshua PR:ADDRESS Florida Aquarium Center for Conservation, 529 Estuary Shore Lane, Apollo Beach, PR:ADDRESS FL 33572 PR:EMAIL joshpatterson@ufl.edu PR:PHONE 8134194917 #STUDY ST:STUDY_TITLE Variability in metabolomic profiles among unique genotypes of Acropora ST:STUDY_TITLE cervicornis ST:STUDY_TYPE intraspecific variability ST:STUDY_SUMMARY We hypothesized that each of the three genotypes tested would have unique ST:STUDY_SUMMARY metabolomic profiles. These data increase our basic knowledge of the coral ST:STUDY_SUMMARY metabolome and represent an important step toward linking genotype, phenotype, ST:STUDY_SUMMARY and metabolome in reef-building corals. ST:INSTITUTE University of Florida ST:LAST_NAME Patterson ST:FIRST_NAME Joshua ST:ADDRESS Florida Aquarium Center for Conservation, 529 Estuary Shore Lane, Apollo Beach, ST:ADDRESS FL 33572 ST:EMAIL joshpatterson@ufl.edu ST:PHONE 8134194917 #SUBJECT SU:SUBJECT_TYPE Invertebrate SU:SUBJECT_SPECIES Acropora cervicornis SU:TAXONOMY_ID 6130 SU:GENOTYPE_STRAIN U25, U41, U44 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - U25_A1b Genotype:U25 SUBJECT_SAMPLE_FACTORS - U25_A2b Genotype:U25 SUBJECT_SAMPLE_FACTORS - U25_A3b Genotype:U25 SUBJECT_SAMPLE_FACTORS - U25_B1b Genotype:U25 SUBJECT_SAMPLE_FACTORS - U25_B2b Genotype:U25 SUBJECT_SAMPLE_FACTORS - U41_A1b Genotype:U41 SUBJECT_SAMPLE_FACTORS - U41_A2b Genotype:U41 SUBJECT_SAMPLE_FACTORS - U41_A3b Genotype:U41 SUBJECT_SAMPLE_FACTORS - U41_A4b Genotype:U41 SUBJECT_SAMPLE_FACTORS - U41_B1b Genotype:U41 SUBJECT_SAMPLE_FACTORS - U41_B2b Genotype:U41 SUBJECT_SAMPLE_FACTORS - U44_A1b Genotype:U44 SUBJECT_SAMPLE_FACTORS - U44_A2b Genotype:U44 SUBJECT_SAMPLE_FACTORS - U44_A3b Genotype:U44 SUBJECT_SAMPLE_FACTORS - U44_B1b Genotype:U44 SUBJECT_SAMPLE_FACTORS - U44_B2b Genotype:U44 #COLLECTION CO:COLLECTION_SUMMARY Coral colonies were brought to the surface intact, and ~3 cm nubbins were CO:COLLECTION_SUMMARY clipped from actively growing branch tips. Nubbins were placed in 20 mL CO:COLLECTION_SUMMARY scintillation vials containing 10 mL of 100% methanol spiked with a 0.005 mM CO:COLLECTION_SUMMARY aminoanthracene standard and immedately placed on ice. Samples were stored in a CO:COLLECTION_SUMMARY -20°C freezer overnight, then transported back to the laboratory n ice and CO:COLLECTION_SUMMARY again stored in a 20°C freezer overnight prior to extraction. The next day, CO:COLLECTION_SUMMARY samples were agitated for 5 minutes, then allowed to settle for one hour in the CO:COLLECTION_SUMMARY -20°C freezer. One mL of the sample extract was transferred to clean 1.5 mL CO:COLLECTION_SUMMARY microcentrifuge tubes and centrifuged at 20,000 g for 5 minutes. The supernatant CO:COLLECTION_SUMMARY was then transferred to a new 1.5 mL microcentrifuge tube and stored in a -80°C CO:COLLECTION_SUMMARY freezer until processing. CO:SAMPLE_TYPE Tissue and skeleton CO:STORAGE_CONDITIONS -80? #TREATMENT TR:TREATMENT_SUMMARY No treatment was applied; study was conducted on natural metabolomic variation TR:TREATMENT_SUMMARY among genotypes #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Corol holobiont extract (in methonol) was added to double distilled water (1:2 SP:SAMPLEPREP_SUMMARY v/v of sample to water), then flash freeze lyophilized (Labconco) until dry. SP:SAMPLEPREP_SUMMARY Lyophilized dry powder was re-suspended in phosphate buffer in deuterium oxide SP:SAMPLEPREP_SUMMARY at pH 7. The final volume for the 1H-NMR samples was 60 ?L (in a 1.5 mm O.D. SP:SAMPLEPREP_SUMMARY tube) with 90 % (v/v) of deuterated 50 mM sodium phosphate buffer (pH 7) with 2 SP:SAMPLEPREP_SUMMARY mM of ethylene diamine tetra-acetic acid (EDTA). The remaining 10 % (v/v) was SP:SAMPLEPREP_SUMMARY occupied by an internal standard [5 mM D6-4,4-dimethyl-4-silapentane-1-sulfonic SP:SAMPLEPREP_SUMMARY acid (DSS-D6) and 0.2% sodium azide in deuterated environment; Chenomx, Inc.]. SP:PROCESSING_METHOD Lyophilization SP:PROCESSING_STORAGE_CONDITIONS -80? SP:EXTRACT_STORAGE -80? #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE - CH:INSTRUMENT_NAME - CH:COLUMN_NAME - #ANALYSIS AN:ANALYSIS_TYPE NMR AN:LABORATORY_NAME AMRIS AN:OPERATOR_NAME Ram Khattri AN:DETECTOR_TYPE NMR AN:SOFTWARE_VERSION Topspin 3.2 AN:ACQUISITION_DATE 6/27/2017 #NMR NM:INSTRUMENT_NAME Bruker 600 MHz NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:FIELD_FREQUENCY_LOCK Deuterium NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_PROBE CP TXI Cryoprobe NM:NMR_SOLVENT Phosphate buffer (pH 7) + 2 mM EDTA + 0.5 mM DSS + 0.2% of sodium azide in NM:NMR_SOLVENT deuterated environment NM:NMR_TUBE_SIZE 1.5 mm O.D. 7 inches NM:SHIMMING_METHOD Topshimgui NM:PULSE_SEQUENCE noesypr1d NM:WATER_SUPPRESSION presat NM:PULSE_WIDTH 5.51 microsecond NM:POWER_LEVEL 7.37 W NM:RECEIVER_GAIN 406 NM:OFFSET_FREQUENCY 2827.31 Hz NM:PRESATURATION_POWER_LEVEL 0.000031623 W NM:CHEMICAL_SHIFT_REF_CPD DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) NM:TEMPERATURE 25 NM:NUMBER_OF_SCANS 64 NM:DUMMY_SCANS 4 NM:ACQUISITION_TIME 4 s NM:RELAXATION_DELAY 1 s NM:SPECTRAL_WIDTH 7211.54 Hz NM:NUM_DATA_POINTS_ACQUIRED 57690 NM:REAL_DATA_POINTS 65536 NM:LINE_BROADENING 0.22 Hz NM:ZERO_FILLING 65,536 points NM:APODIZATION Exponential NM:BASELINE_CORRECTION_METHOD Spline NM:CHEMICAL_SHIFT_REF_STD DSS at 0 ppm NM:BINNED_INCREMENT 0.4 ppm NM:BINNED_DATA_EXCLUDED_RANGE greater than 9.5 ppm and below 0.5 ppm NM:NMR_RESULTS_FILE ST001116_AN001812_Results.txt UNITS:ppm #END