Isolates were grown in Potato Dextrose Broth (DifcoTM) for up to 5 days before the mycelia was harvested and lyophilized.
Extracted molecule
genomic DNA
Extraction protocol
Total genomic DNA was extracted from freeze-dried mycelia using either the Qiagen maxiprep kit (Qiagen, Valencia, CA) or the MasterPure Yeast DNA Purification Kit (Epicentre Technologies, Madison, WI) following the manufacturer’s protocol.
Label
biotin
Label protocol
Genomic DNA was labeled using the BioPrime DNA labeling System (Invitrogen Catalogue No. 18094-011) as follows: (1) fungal DNA in the amount of 300-350 ng was mixed with 60 μl of 2.5X random primers (BioPrime kit) and 132 μl of dH2O, (2) the reaction mixture was denatured in an Eppendorf thermocycler at 99°C for 10 min, and then cooled at 4°C for 15 min, (3) 15 μl of 10X dNTPs containing biotin dCTP and 3 μl Klenow polymerase were added and incubated at 25°C in a thermocycler for 18 h, (4) the reaction mixture was precipitated by adding 15 μl of 3M NaOAc and 400 μl of cold 95% ethanol, followed by incubation at 20°C for 15 min, (5) samples were centrifuged at high speed in a refrigerated microcentrifuge for 20 min, and (6) the pellets were washed with 500 μl ice-cold 70% ethanol, centrifuged, and then vacuum dried with heating for 10 min. DNA was resuspended in 100 μl of dH2O and 5 μl was used to check the quality of the labeled DNA on a gel.
Hybridization protocol
Each sample was analyzed by the Microarray Laboratories of Expression Analysis (Raleigh-Durham NC). Briefly, 300-350 ng of fragmented genomic DNA was diluted in hybridization buffer (MES, NaCl, EDTA, Tween 20, Herring Sperm DNA, Acetylated BSA) containing biotin-labeled OligoB2 and Eukaryotic Hybridization Controls (Affymetrix). The hybridization cocktail was denatured at 99°C for 5 minutes, incubated at 45°C for 5 minutes and then injected into a GeneChip cartridge. The GeneChip array was incubated at 42°C for at least 16 hours in a rotating oven at 60 rpm. GeneChips were washed with a series of nonstringent (25°C) and stringent (50°C) solutions containing variable amounts of MES, Tween20 and SSPE. The microarrays were then stained with streptavidin-phycoerythrin and the fluorescent signal was amplified using a biotinylated antibody solution.
Scan protocol
Fluorescent images were detected in a GeneChip® Scanner 3000 and array data was extracted using the GeneChip Operating System v 1.1 (Affymetrix). Arrays images were further inspected for defects or debris. Quality control was based on a comparison of the conformity of hybridization controls, scaling factor, and noise; percent detection metrics were analyzed to determine if any outliers were present.
Description
NRRL: 62358
Data processing
For the parent-offspring trio heat maps, array data for all strains examined using aCGH were imported into JMP genomics (SAS, Cary, NC, USA) for further transformation and normalization. Data were log2 transformed and normalized using Loess normalization. Because we are interested in detecting differences in hybridization intensities no background correction was applied. For the genome-wide parentage plots, the A. flavus probe data were normalized using Loess normalization but not log2 transformed.
