Additional Files for "Characterizing the loblolly pine (Pinus taeda L.) genome by BAC sequencing and Cot analysis"
Additional File 1. Detailed protocols: (A) Isolation of nuclear DNA from plants; (B) Dialysis of nucleic acid solutions; (C) Shearing DNA into 450 bp fragments using the Misonix sonicator 3000; (D) Removing metal ions from DNA solutions using Chelex; (E) Preparing 0.5 m sodium phosphate buffer (SPB); (F) Preparing ASE buffer (PDF 744 kb). Additional File 2. List of targeted genes (carbon metabolism, wood development, transcription factor, signaling, or disease resistance), their references, and the overgo probe sequences used in screening. A total of seventy-three overgo probes were used to screen BAC macroarrays: twenty-five (colored in blue) were included in a first batch of screening while the last forty-eight (colored in green) were utilized in a second screen (XLS 41 kb). Additional File 3. Schematic representation of the screening steps performed to identify the targeted BACs: (A) screening outline (B) Pooling pattern (PPTX 46 kb). Additional File 4. Examples of screened 4 x 4 macroarrays from the first (A) and the second (B) batches of screening. The coordinates of the positive clones on the macroarrays correspond to the well position in the 384-well plate while the configuration of the double spots corresponds to the plate number of the 384-well plate where the clones are located. Panel C shows hybridization with repetitive elements and D shows NotI digestion of the identified low-copy BACs from C to verify the presence of inserts (PPTX 876 kb). Additional File 5. List of the BAC clones that were selected for sequencing, the corresponding GenBank accession numbers, gene index, length of assembly, type of BAC clone, the probe used to screen the targeted BAC clones, notes, LP draft genome scaffold ID, and percent query coverage. A total of 100 BAC clones were sequenced: 50 targeted BAC clones, 25 low-copy sequence BAC clones, and 25 random BAC clones (XLS 48 kb).