1. Seeds were sown on May 17, 2011 in the experimental station of Qinghai University (Xining, Qinghai Province, China). 2. Individual plants of each accession, which did not exhibit any obvious phenotypic differences, were reserved by eliminating potential false types at several developmental stages. 3. The development of the main inflorescence (MI) was observed under an anatomical microscope (OLympus, Japan). The inflorescence primordia of three to five plants for each accession was examined every three days under the microscope, beginning at the eighth leaf stage. 4. The main inflorescence primordia of five plants per line that were at the pistil-stamen primordia initiation stage of the first floral on the main inflorescence, were harvested in the morning, immediately frozen in nitrogen, and then kept at -80⁰С for total RNA isolation.
Growth protocol
1. Two elite Brassica napus L. lines, cv. Zhongshuang 11 (ZS11), which was used for genome sequencing, and 73290, which was used for re-sequencing, developed by the Rapeseed Biotechnology Breeding Unit, Oil Crops Research Institute of Chinese Academy of Agriculture Sciences (Wuhan, China), were used to generate an F2:3 population harboring 183 offspring for QTL mapping of yield traits. 2. Subsequently, an F6 inbred line (RIL) population was obtained from the F2:3 population by single-seed-descendant. Nine lines with an extremely high number of PMIs and Nine with an extreme number were selected from the above F6 RIL population. Afterwards, ZS11, 73290, and the 18 extreme RIL lines were used for gene expression profiling analysis in the main inflorescence.
Extracted molecule
total RNA
Extraction protocol
1. Using an RNeasy Mini Kit (Cat. 74124, Qiagen, Mississauga, ON) according to the manufacturer’s recommendations, total RNA of inflorescence primordia was extracted in biological triplicate, each consisting of three main inflorescence sections taken from three independent plants. 2. Trace amounts of DNA were removed using DNase I (Cat. 18068-015, Invitrogen, USA). 3. The RNA yield and purity were determined spectrophotometrically with a NanoDrop®1000 spectrophotometer (Thermo Fisher Scientific, USA), and the intactness of RNA was verified by electrophoresis on a 1% agarose gel with 1×TBE running buffer at 70 V for 40 min. 4. Purified total RNA was precipitated and re-suspended in DEPC-treated water to a final concentration of about 500 ng/µL. 5. For the extreme PMI lines, the total RNA of inflorescence primordia was extracted individually and pooled at equal concentrations within each extreme-PMI group. Nine lines were used for each biological replicate in the reverse transcription analysis.
Label
Cy5
Label protocol
1. About 3.0 µg of total RNA for each sample was reverse transcribed into amino allyl-modified aRNA using a Message™ II aRNA Amplification Kit (Cat: 1753, Ambion, Austin, TX, USA), according to the manufacturer's protocol. 2. The quality of amplification was identified using agarose gel electrophoresis, and the aRNA yield was quantified using a NanoDrop® 1000 spectrophotometer. 3. The aRNA was selected as the template for fluorescent target preparation for microarray experiments. 4. About 5.0 µg of aRNA was labeled with mono-reactive NHS esters of Cy5 in the dark for 30 min at room temperature, and the samples were then purified and used as targets for hybridization.
Hybridization protocol
1. The pre-hybridization and hybridization procedures were performed precisely according to the protocol of CombiMatrix (www.combimatrix.com). 2. Cy5 dye-labeled RNA populations from individual tissue samples were hybridized to the 90K array and three biological replicates were performed.
Scan protocol
1. Hybridized arrays were scanned using a LuxScan 10K Scanner (CapitalBio Corporation, Beijing, China) at 5 μm resolution, 100% laser power, and different PMT values to obtain a similar overall intensity between slides. 2. The scans were saved as TIF files.
Description
A Brassica genomics resource was developed at the Plant Biotechnology Institute, National Research Council Canada (PBI-NRC) and Agriculture and Agri-Food Canada (AAFC) in collaboration with other institutes. About 95,418 unique sequences were assembled using 781,826 EST sequences mainly from three species: B. napus, B. rapa, and B. oleracea. These unique sequences were submitted to CombiMatrix for the development of a 35-40mer oligonucleotide microarray, for which probes were synthesized in situ onto electrodes on a microchip. The customized Combimatrix Brassica array comprised 90,500 probes and was called as 90K array.
Data processing
1. Raw spot fluorescence intensities were collected using LuxScan version 2.0 (CapitalBio Corporation, Beijing, China) and saved as .txt file. 2. Samples that were originally negative after background subtraction and those with an overall signal intensity of less than 100 were filtered out. 3. Array features annotated as “Empty”, “Blank”, and internal controls were flagged and excluded from the analysis. 4. Twelve LSR files containing the raw probe intensity values were imported into R and the signal intensities were quantile normalization was performed to normalize between arrays using the R-package LIMMA [24] with a 5% false discovery rate (FDR<=0.05). 5. Probes with at least one missing value within triplicates were removed from subsequent analysis. 6. The background-corrected signal intensity of each probe on the array was combined by averaging three biological replicates. 7. A gene was considered to be differentially expressed when its fold-change in expression between two samples was larger than 1.5- or less than 0.67-fold.
