Brassica napus 300k Microarray Hybridization To assess the reproducibility of the microarray analysis, we repeated the experiment two or three times with independently prepared total RNA. For the synthesis of double strand cDNAs, Superscript Double-Stranded cDNA Synthesis Kit(Invitrigen, U.S.A.) was used. Briefly, 1㎕ of oligo dT primer(100 ㎛) and 10 ㎕ (10 ㎍) of total RNA were combined and denatured at 70 ℃ for 10 minutes and renatured by cooling the mixture in ice. First strand DNA was synthesized by adding 4 ㎕ of 5X First Strand Buffer, 2 ㎕ of 0.1M DTT, 1 ㎕ of 10mM dNTP mix and 2 ㎕ of SuperScript enzyme and by incubating at 42 ℃ for 1 hour. To synthesize the second strand, 91 ㎕ of DEPC-water, 30 ㎕ of 5X Second Strand Buffer, 3 ㎕ of 10mM dNTP mix, 1 ㎕ of 10U/㎕ DNA ligase, 4㎕ of 10U/㎕ DNA Polymerase I and 1 ㎕ of 2U/㎕ RNase H were added to the first strand reaction mixture and the reaction was proceeded at 16 ℃ for two hours. After RNA strand was removed by RNase A (Amresco, U.S.A.) reaction mixture was clarified by phenol/chloroform extraction and then cDNA was precipitated by centricfugation at 12,000 x g force after adding 16 ㎕ of 7.5 M Ammonium Acetate and 326 ㎕ of cold ethanol. For the synthesis of C3-labeled target DNA fragments, 1 ㎍ of double strand cDNA was mixed with 40 ㎕ (1OD) of Cy3-9mer primers(Sigma-Aldrich, U.S.A.) and denatured by heating at 98 ℃ for 10 minute. The reaction was further proceeded by adding 10 ㎕ of 50X dNTP mix (10mM each), 8 ㎕ of deionized water, 2 ㎕ of Klenow fragment(50 U/㎕, NEB, U.S.A.) and incubating at 37 ℃ for 2 hours. DNA was precipitated by centrifugation at 12,000 x g force after adding 11.5 ㎕ of 5M NaCl and adding 110 ㎕ of isopropanol. Precipitated samples were rehydrated with 25 ㎕ of water. The concentration of each sample was determined by using spectrophotometer. Thirteen ㎍ of DNA was used for microarray hybridization. The sample was mixed with 19.5㎕ of 2X hybridization buffer (Nimblegen, U.S.A.) and finalized to 39 ㎕ with deionized water. Hybridization was performed with MAUI chamber (Biomicro, U.S.A.) at 42℃ for 16 hours. After the hybridization, the microarray was removed from MAUI Hybridization Station and immediatly immersed in the shallow 250㎖ Wash I (Nimblegen, U.S.A.) at 42 ℃ for 10-15 seconds with gentle agitation and then transferred to the second dish of Wash I and incubated for 2 min with gentle agitation. The microarray was transferred into dish of Wash II and further washed in Wash III 15 seconds with agitation. The micrarray was dried in a centrifuge for 1 minute at 500g and scanned using GenePix scanner 4000B(Axon, U.S.A.) Themicroarray was scanned with Genepix 4000 B (Axon Instruments) preset with a 5 um resolution and for Cy3 signal. Signals were digitized and analyzed by Nimblescan (Nimblegen, U.S.A.). The grid was aligned to the image with a chip design file, NDF file. The alinment was checked by ensuring that the grid’s corners are overlaid on the images corners. This was further checked by uniformity scores in the program. An analysis was performed in a two-part process. First pair reports (.pair) files were generated in which sequence, probe, and signal intensity information for Cy3 channel were collected. Data-based background subtraction using a local background estimator was performed to improve fold change estimates on arrays with high background signal. The data was nomalized and processed with cubic spline normalization using quantiles to adjust signal variations between chips (Workman et al., 2002. Genome Biol. 3: research0048.1 - research0048.16 ). Probe-level summarization by Robust Multi-Chip Analysis (RMA) using a median polish algorithm implemented in NimbleScan was used producing calls files. The method identifies probes that are outliers in the overall behavior of the expression measured for a given gene and the contribution by those outliers is reduced in the reported gene expression level. This improves the sensitivity and reproducibility of microarray results (Irizarry et al., 2003. Nucleic Acids Research 31:e15).