normalization data transformation protocol
All arrays were inspected for possible artefacts (e.g. dust particles or bubbles). Small, suspicious areas were corrected by imputing the expression intensity of the given features using the k-nearest neighbour (KNN) averaging method implemented in the R package impute (Troyanskaya et al., 2001). Arrays were then processed with the robust multichip average (RMA) algorithm implemented in the R package oligo (Irizarry et al., 2003a; Irizarry et al., 2003b) from Bioconductor (https://www.bioconductor.org). This method corrects for background noise, and accomplishes quantile normalisation, followed by log2 transformation and median polish summarisation.
array scanning protocol
NimbleGen One-Color Array Scanning (Expression)
nucleic acid hybridization to array protocol
NimbleGen Hybridization & Washing (Expression)
nucleic acid labeling protocol
NimbleGen Sample Labeling (Expression)
Seeds were scarified with 100 grit sand paper, and sown in Petri dishes, on damp filter paper soaked with 1% Plant Preservative Mixture (Plant Cell Technology, Washington, DC, USA) to prevent fungal growth during germination. Petri dishes were then placed in a germination chamber set for 30 _C, 80% humidity and 16h daylight. Newly-emerged seedlings were transferred onto a 1:1 sand:soil mixture when root growth reached 1-2cm length, and moved to a growth chamber set for 20-22 _C, 40% humidity and 16h daylight. After 1-2 weeks acclimation in the growth chamber, when 1-2 pairs of true leaves were out, young plantlets were transferred into 3.5 inch pots filled with the same sand:soil mixture, and moved to a flood bench at the UBC Horticulture Greenhouse.
Five individuals from each family were randomly assigned to each of three growth conditions: (i) a ÔcontrolÕ, non-stressful condition, (ii) a nutrient stress, and (iii) a light stress (Fig. 1). All plants, with the exception of those assigned to the nutrient-stress treatment, received 1.5mL Osmocote 13-13-13 slow release fertiliser. The light stress was simulated by growing the plants in a shade box (ca. 4.5 x 3.5 x 1.8m) made of PVC pipes and covered by 121 Lee green filters (Andover, UK) and neutral density shade cloths, to mimic the spectral quality of light that is transmitted through the canopy of neighbouring plants (Bonser & Aarssen, 2003). The green filter reduced light transmittance by 73%, and the shade cloth further reduced transmittance to 92% of the original value (light intensities were reduced from an average of 873.3 to 66.1 _molám-2ás-1 based on an average of three measurements taken at noon on sunny days). Plants were randomly distributed within each treatment block on the bench, for a total of 250 plants per treatment. Three weeks following the last transplant, young, similar-sized leaves were randomly harvested, quick frozen in liquid nitrogen, and stored at -80ûC until RNA extraction. To minimise the effect of individual differences within populations, three average-sized plants from three different families were pooled as one biological sample for subsequent microarray experiments.
nucleic acid extraction protocol
Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA)/RNeasy (Qiagen, Valencia, CA, USA) approach described in Lai et al. (2006) and quantified by spectrophotometry (NanoDrop, Wilmington, DE, USA). Total RNA was further treated with RNase-free DNase I (Qiagen) to eliminate possible genomic contamination.