plants were grown under normal growth conditions, no treatment applied.
Growth protocol
Arabidopsis halleri ssp. halleri (accession Langelsheim) was obtained from one cloned individual (W 504) and from 10 pooled F1 progeny of controlled, reciprocal crosses between two Langelsheim individuals (Lan3.1 and Lan5) grown from seeds .Arabidopsis lyrata ssp. petraea (accession Kubova Hut, kindly provided by Marc Macnair, University of Exeter) and Arabidopsis thaliana (accession Col-0) were grown from seed.A. thaliana was cultivated on standard soil, and the other two species were cultivated in hydroponics.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was isolated, fragmented and end-labeled with Bio-N6-ddATP. Total genomic DNA was isolated from 4 to 6 g fresh biomass of plant leaf tissue with CTAB buffer (0.8% (w/v) CTAB, 800 mM NaCl, 1% (w/v) N-lauryl sarcosine, 140 mM Sorbitol, 22 mM EDTA, 220 mM Tris pH 8). Frozen plant tissue was ground to a fine powder in liquid N2, transferred to a 50 ml tube containing 30 ml CTAB buffer and incubated at 65°C with occasional vigorous shaking for 20 min. After addition of 12 ml chloroform/isoamylalcohol (24:1) and vigorous mixing, the tubes were placed at RT on an inverter for 20 min. After centrifugation at 4,000 rpm for 5 min in a tabletop centrifuge, the aqueous phase was transferred to a fresh tube, 1 vol. isopropanol was added and nucleic acids were precipitated on ice for 30 min. After centrifugation at 9,500 rpm (rotor: JA25-50), 4°C for 8 min, the supernatant was drained and the pellets were resuspended in 6 ml ddH2O. 1 vol. 4 M LiAc was added, and the samples were incubated on ice for 20 min to precipitate RNA. After centrifugation at 9,500 rpm (rotor: JA25-50), 4°C for 11 min, the supernatant was transferred to a fresh tube, 2 vol. ethanol were added and the samples were placed at RT for a few seconds. To collect the precipitate, samples were centrifuged at 12,000 rpm (rotor: JA25-50), 4°C for 20 min. The pellets were resuspended in 1.35 ml ddH2O, followed by the addition of 150 µl 3 M sodium acetate. Phenol/chloroform (1 vol., composition see above) was added, samples were mixed by shaking, and centrifuged at 14,000 rpm for 5 min to resolve phases. The aqueous phase was collected, 2 vol. ethanol were added and samples were placed on ice for 5 min. The precipitate was collected by centrifugation at 14,000 rpm for 5 min. Each pellet was washed with 80% (v/v) ethanol and dried at 37°C for 10 min. Pellets were resuspended in 300 µl ddH2O by gentle pipetting. Nucleic acid concentration and purity were determined spectrophotometrically by measuring absorption at 260 and 280 nm and by subjecting 4 µg of each sample to agarose gel electrophoresis.
Label
biotin
Label protocol
The genomic DNA fragments were 3’-end labelled with biotin by adding 40 u terminal deoxynucletidyl transferase (Promega) and 2 µl of 1 mM Bio-N6-ddATP (Enzo Life Sciences, USA) to the remaining 32 µl of the fragmentation reaction and incubating the samples for 1 h at 37°C in the dark
Hybridization protocol
Hybridization was conducted according to the standard protocol for hybridizing fragmented cRNA [Weber et al 2004] using 20 µl of the fragmented and labelled genomic DNA instead of fragmented cRNA
Scan protocol
Hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450, and the fluorescent signals were measured with an Affymetrix GeneChip Scanner 3000.
Description
pooled root and shoot tissue, rep1 Cited publication for protocols: Weber M, Harada E, Vess C, V Roepenack-Lahaye E, Clemens S: Comparative microarray analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP transporter and other genes as potential metal hyperaccumulation factors. Plant J 2004, 37:269–281.
Data processing
The .CEL files were laoded into R and microarray data for each species was normalized separately using MAS5 background correction and vsn normaliztion using Bioconductor, affy and vsn packages. Prior to normalization, some compact errors in Aha_gDNA_02 sample were corrected using the Harshlight package in Bioconductor. The heterologous species hybridizations (A. halleri and A. lyrata) were scaled to bring the signal strength at par with A. thaliana hybridizations. A probe-level ANOVA test was conducted to test the strangth of differential hybridization signal using the lmfit and ebayes functions in Limma. The average of hybridization signals from all probes of a probeset is reported here as the summarized signal intensity.
