Synthesis and radiolabelling of DNA probes Oligonucleotide were designed to amplify 421 bp fragment in the 39 region of the publicly available N. caerulescens ecotype Prayon HMA4 cDNA sequence, GenBank accession ID AY486001.1, (http://www.ncbi.nlm.nih.gov/) using forward: 59-GCTAGG- GAATGCTTTGGATG-39, and reverse: 59-CTTCTCTCGCA- GAAGCAACA-39, primer sequences (MWG Biotech, Ebersberg, Germany). DNA probes (50 ng) for library hybridisations were labelled with dCTP a-32P (0.4 MBq ml21), by random priming using Ready-To-Go DNA Labelling Beads (-dCTP) kit (GE Healthcare, Buckinghamshire, UK) as described by the manufacturer. The labelled probe, dissolved in 50 ml of TE buffer, was separated from unincorporated nucleotides by passing through an Illustra Nick column (GE Healthcare, Buckinghamshire, UK) and heat denatured as described by the manufacturer. Radiolabelling and hybridisation of the HMA4 library probe Each pair of library filters were submerged for 4 h at 55uC in 250 ml prehybridisation solution, then incubated (55uC) over- night with the radiolabelled probe, before reducing radioactivity to 15–30 counts per minute through repeated washing in solutions of 2 X SSC +0.1% SDS [49]. Hybridised filters were sealed in plastic and exposed to autoradigraphy film (Kodak X- Omat AR Film XAR-5, Sigma-Aldrich GmbH, Steinheim, Germany) at 280uC for 4–5 d. Positive hybridisations were localised and corresponding fosmids, grown and their plasmid extracted. Identification and ‘fingerprinting’ fosmids of interest Fosmids containing genes of interest were confirmed initially by colony PCR using probe specific primers. Selected fosmids were then ‘fingerprinted’ through individual restriction digestion with EcoRI, HindIII and BamHI (4 h at 37uC) (Promega, Southampton, UK) before running 10 ml of each digest in a 1% (w/v) agarose/ TAE electrophoresis gel for 12 hrs at 0.5 V cm21. Gels were blotted onto a pre-cut nylon membrane (12 h) and hybridised with the HMA4 library probe [49]. Genomic DNA extracted from N. caerulescens Saint Laurent Le Minier was used as a positive control to compare all observed hybridisation patterns. Sequencing N. caerulescens fosmid clones of interest Pooled fosmid pyrosequencing and shotgun library preparation using a 454 Genome Sequencer FLX (454 GS-FLX) Next Generation (NextGen) platform with standard sequencing chem- istry (,250 bp read lengths; Roche Diagnostics GmbH) was carried out by Cogenics Genome Express (Cambridge, UK) while individual fosmid shotgun libraries and GS-FLX Titanium sequencing chemistry (350–450 bp read lengths, 20-fold coverage) with gap filling by Sanger dideoxy sequencing was performed by Eurofins Genetic Services Limited (82152 Martinsried, Germany). For all, sequencing and assembly of the shotgun data was performed using a standard whole genome sequencing assembly with the 454/Roche Newbler assembler V 1.1.02.15. [28,29]. Fosmids were extracted from bacterial suspension following the Maxiprep plasmid isolation protocol [49]. Contig alignments of N. caerulescens fosmid sequences Fosmid pCC1FOSTM vector sequences were isolated from Noccaea caerulescens inserts in silico, via the NCBI database Basic Local Alignment Search Tool (BLASTn) algorithm, against all available nucleotide sequences at default parameters (http://blast.ncbi.nlm. HMA4 Quadruplication in Noccaea caerulescens nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome). Inserts were aligned to A. thaliana orthologous regions and assembled into one large contiguous sequence via AlignX and ContigExpress software using default gap settings (Vector NTI 11; Invitrogen, Paisley, UK). Overlapping insert regions were identified between fosmid end- sequences which aligned to identical A. thaliana regions and shared .99% sequence identity. Consensus sequences were assigned to assemblies of repetitive regions and poly-A and poly-T stretches that showed variation between homologous fosmid sections. All protein and nucleotide sequence comparisons and percentage identities were calculated using Dot Matrix at a stringency of 30% and window of 5 (Vector NTI 11). Creating promoter::GUS fusion constructs Primers specific for the promoter regions of Arabidopsis thaliana (L.) Heynh. Colombia (Col-0) (AtHMA4), A. halleri (L.) O’Kane & Al-Shehbaz ssp. halleri (AhHMA4-3), and N. caerulescens (J.&C. Presl) F.K. Mey. Saint Laurent Le Minier (NcHMA4-2) were designed using Primer 3 Version 0.4.0 (http://frodo.wi.mit.edu/primer3). Promoter fragments were PCR amplified from plant DNA with PhusionH proofreading polymerase (Finnzymes, Finland) and ligated into the pCR8H/GW/TOPOH entry vector using the TA cloning system (Invitrogen, Paisley, UK.). Cloned promoter sequences were fused with b-glucuronidase (GUS) in pGWB3 Gateway-compatible destination vectors [50] via LR-mediated Gateway cloning technology as described by the manufacturer (GatewayH LR ClonaseH; Invitrogen, Paisley, UK). Bacterial transformations Presence and orientation of promoters were confirmed in all constructs through Sanger sequencing. Plasmid extractions, antibiotic selection and transformations for chemical- (E. coli DH5a) and electro- (Agrobacterium tumefaciens GV3101 [51]) competent bacterial cells were performed as described [49]. Analysis of GUS expression in T2 transgenic Arabidopsis thaliana Histochemical detection of GUS activity [52] was performed on T2 segregating transformed Arabidopsis thaliana Col-0 plants [53], selected on agar-based medium (10 g l21 sucrose, 8 g l21 agar and 2.1 g l21 Murashige and Skoog (MS) basal medium (M5524, Sigma-Aldrich, Poole, UK)), supplemented with 50 mg ml21 kanamycin sulphate. Seven DAS healthy, green, actively growing plants were transferred under axenic conditions to translucent polycarbonate growth boxes containing 75 ml of un-supplemented agar-based media and cultured for a further 14 days (21 DAS) at 2062uC, under 16 h photoperiod, at 50–80 mmol photons m22 s21 light intensity from 58 W white halophosphate fluorescent tubes (Cooper Lighting and Security, Doncaster, UK). A randomised block design comprising three replicates was em- ployed, with three independent transformed lines for each of the three promoter constructs and one wild type (WT) line allocated at random within each replicate (n = 30). For each replicate, all samples from each line, including WT control, were placed into individual sterile glass universals (3 plants per bottle) containing 10 ml of GUS assay solution [52] (5 mg of X-Gluc (5-bromo-4- chloro-3-indolyl-b-D-glucuronic acid; Melford Laboratories Ltd, Ipswich, UK) dissolved in 100 ml of dimethyl formamide (DMF), phosphate buffer (0.2 M NaH2PO4 plus 0.2 M Na2HPO4, pH 7.0), 0.5 M Na2EDTA, 10 mM K3Fe(CN)6, 10 mM K4Fe(CN)6.3H2O and 0.1% (v/v) Triton-X-100 (Sigma-Aldrich Co., Steinheim, Germany), and incubated in the dark at 37uC for 16 h. PLoS ONE | www.plosone.org 6 March 2011 | Volume 6 | Issue 3 | e17814