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).

补充文件1 详细实验方案：(A) 植物核DNA提取；(B) 核酸溶液透析；(C) 使用Misonix 3000型超声破碎仪将DNA剪切为450 bp片段；(D) 使用Chelex树脂去除DNA溶液中的金属离子；(E) 配制0.5 mol/L 磷酸钠缓冲液（SPB）；(F) 配制ASE缓冲液（PDF文件，744 KB）。

补充文件2 靶向基因列表（涵盖碳代谢、木材发育、转录因子、信号传导或抗病性方向）、相关参考文献及筛选所用的overgo探针（overgo probe）序列。本次共使用73条overgo探针筛选细菌人工染色体（Bacterial Artificial Chromosome, BAC）宏观阵列：首批筛选使用25条（标注为蓝色），第二批筛选使用剩余48条（标注为绿色）（XLS文件，41 KB）。

补充文件3 用于鉴定目标BAC克隆的筛选步骤示意图：(A) 筛选流程概述；(B) 样品混合模式（PPTX文件，46 KB）。

补充文件4 首批(A)与第二批(B)筛选所用的4×4宏观阵列筛选实例。宏观阵列上阳性克隆的坐标对应384孔板的孔位，双点的排布模式则对应克隆所在384孔板的板号。图C展示了与重复序列的杂交实验结果，图D则为对图C中鉴定出的低拷贝BAC克隆开展NotI酶切以验证插入片段存在性的结果（PPTX文件，876 KB）。

补充文件5 选定用于测序的BAC克隆列表，包含对应的GenBank登录号、基因索引、组装序列长度、BAC克隆类型、用于筛选目标BAC克隆的探针、备注信息、LP草图基因组支架ID以及查询序列覆盖百分比。本次共测序100个BAC克隆：50个靶向BAC克隆、25个低拷贝序列BAC克隆以及25个随机BAC克隆（XLS文件，48 KB）。